Copyright is owned by the Author of the thesis. Permission is given for a copy to be downloaded by an individual for the purpose of research and private study only. The thesis may not be reproduced elsewhere without the permission of the Author. DRY MATTER PARTITIONING IN Zantedeschia K. Spreng, AS INFLUENCED BY TE:MPERA TURE AND PHOTOSYNTHETIC PHOTON FLUX Keith Alien Funnell 1993 A thesis presented in partial ? fulfilment of the requirements for the degree of Doctorate of Philosophy in Horticultural Science at Massey University 11 ABSTRACT The in vivo and in vitro dry matter accumulation and partitioning in plants of the Zantedeschia pentlandii-like (Watson) Wittm. selection 'Best Gold' were described under a range of either temperature and photosynthetic photon flux (PPF) regimes, or sucrose concentrations, using plant growth analysis. The initiation of tuber growth, as denoted by increases in both structural and starch dry weights, did not require an obligative environmental trigger. Relative rates of dry matter accumulation (RGRw) increased linearly with increasing temperature up to ?a maximum of 28 C, with maximum final total and tuber dry weight occurring between 21 and 26 C both in vivo and in vitro. The linear relationship between the relative rate of dry matter accumulation of the tuber (RGRT) and temperature, indicated a PPF dependent base temperature for tuber growth between 4 .8 and 6. 1 C . By principally altering dry matter partitioning, total dry matter accumulation was highly adaptive to PPF regime. The ability to alter the photosynthetic rate and the partitioning of the daily increment of dry matter into leaf area (LWP), resulted in greater values of the estimated final total plant dry matter under the low PPF regime (348 l-'mol?m-2?s-1), at temperatures less than 22 C. At temperatures greater than 19 C the estimated maximum to? plant dry weight was either not influenced by PPF or was slightly greater under the high PPF regime (694 l-'mol?m-2?s-1). This ability to effectively utilize a low PPF regime indicates that this selection is shade tolerant. The optimum PPF for growth was found to be temperature dependent: estimated maximum total plant dry weight occurred under high PPF at 25 C, whereas the estimated maximum tuber dry weight occurred at 24.5 C under low PPF. RGRw was highly correlated with LWP. In contrast, only a poor correlation was determined between RGRw, and either the efficiency of these leaves to produce additional dry matter, i.e., net assimilation rate (NAR), or starch concentration or soluble carbohydrate concentration. Photosynthetic rate was correlated with RGRw, but not with RGRT. While the photosynthetic process must be involved in contributing photoassimilates for tuber growth, it was suggested that the plant's response to dry matter partitioning into the leaf, i.e., LWP, and the tuber, i.e., TWP, had a greater influence in determining tuber growth than could be accounted for by the photosynthetic rate. Mechanisms of acclimation under both PPF regime suggested that tuber growth was principally source limited. Source limitation was expressed either in terms of: 111 1) enhanced ?ntersink competition for assimilates, as occurred under the low PPF regime, where enhanced leaf area development (LWP) was in direct competition with enhanced tuber growth (RGRT). This was also confirmed in vitro where dry matter partitioning to the tuber was reduced under limited source strength. 2) efficiency of dry matter accumulation of leaf area present, as occurred under the high PPF regime, where large increases in RGRT were correlated with increased NAR. This was also confirmed in vitro where increased source strength increased tuber dry weight. However, in vitro experiments where source strength was controlled, illustrated that tuber growth was also potentially sink limited at temperatures both lower and higher than the optimum. At 3 1 C the sink limitation of tuber growth arose from more than the temperature-induced limitation on growth and respiration found at other sink limiting temperatures. At this temperature an additional form of sink limitation was evident where partitioning of dry matter towards the tuber was also restricted. It was suggested that this additional form of sink limitation may have arisen from high temperature inactivation of starch metabolising or sucrose unloading enzymes. Application of the dry matter partitioning term TWP, provided a more sensitive measure of short term changes in partitioning than the conventionally used term, harvest index. \ ; ' The optimum temperature range for growth was close to the average daily air temperature during the season for the sites of natural habitat of the suggested parent specie, Zantedeschia pentlandii. Similarly the shade tolerance_ status of this selection was paralleled by the diversity of PPF habitats it naturally occupies, as created by open grassland and forest margins. It was therefore suggested that Zantedesclzia 'Best Gold' is well adapted to optimise growth under the temperature and PPF regimes of its natural habitat. This study suggests that improvements in commercial yield of Zantedeschia tubers can be achieved in all regions of New Zealand through the use of protected cultivation with supplemental heating. However, unless using protected cultivation, the potential improvements in commercial tuber yields, through the application of shading, are only likely to be evident in warmer regions of N ew Zealand where growers utilize extended periods of cultivation and optimise leaf area duration. lY ACKNOWLEDGEMENTS The completion of this thesis is a piece of independent research, but so many individuals have contributed either directly or indirectly. In panicular, I would like to the thank the following: my supervisors Dr I. J. Warrington, Dr J.A. Plummer and Dr E. W. Hewett for their challenges, guidance and constructive criticism; Dr D.J. Chalmers for his leadership and foresight as Head of Depanment of the then Depanment of Honicultural Science, to initiate the unique opponunity for me to study towards my PhD,? the management and staff of HonResearch (formally DSIR Plant Physiology Division, and DSIR Fruit and Trees) especially Dr D. Cohen, and the Depanment of Plant Science (formally the Depanment of Honicultural Science) for the provision of facilities and technical advice; Massey University Research Fund (MURF), Massey University Agricultural Research Fund (MUARF), and The C. Alma Baker Trust who all provided various forms of financial contribution; Dr J. M. Wilson for an endless supply of tubers and seed, as well as thought provoking discussion; my family and friends who either provided moral suppon or without knowing it, suitable distractions that created some semblance of normality during a demanding few years of my life. Parts of Sections 1 , 3 , 4 and 5 of this thesis have been published in the following; Funnell, K.A. 1993. Zantedeschia, p. 683-739. In: A. De Hertogh and M. Le Nard (eds.). The physiology of flower bulbs. A comprehensive treatise on the physiology and utilization of ornamental flowering bulbous and tuberous plants. Elsevier Science Publishers, Amsterdam. Funnell, K.A., J.A. Plummer and I.J. Warrington. 1990. Temperature and light effects on tuber growth in calla lilies. Abstracts XXIII Intl. Hort. Congr., Firenze, Italy, 1990, no. 3261. V CO? PAGE ABSTR.Acr . . ? . ? ? . . ? ? . . . ? ? ? ? . ? . . ? . . . . . . . . ? . . . . . . . . . . . . . n ACKNO?GEMENTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. w co?s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v UST OF ABBREVIATIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xi NOTES ON CITATION FORMAT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xiii UST OF TABLES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xiv UST OF FIGURES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xvii LIST OF PLATES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xxiv 1 BOTANICAL, ECOLOGICAL, PHYSIOLOGICAL, AND HORTICULTURAL BACKGROUND OF THE GENUS Zantedeschia. 1 1.1 Introduction and overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 1.2 World production areas and volumes . . . . . . . . . . . . . . . . . . . . . . . . . 1 1.3 I \ I i Botanical classification, morphology, distribution and habitat 1. 3.1 Botanical classification and morphological description . . . . . . . . . . 2 2 1.3.2 Distribution and climate of origin . . . . . . . . . . . . . . . . . . . . . . 5 1.4 ' Breeding: goals and specific problems . . . . . . . . . . . . . . . . . . . . . . . . 9 1.5 Vegetative growth and development . . . . . . . .. . . . . . . . . . . . . . . . . 1 1 1. 5 .1 General overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 1 1.5.2 Influence of internal factors .................. : . . . . . . 1 1 1.5.2.1 Dormancy . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 1 1.5.3 Influence of external factors . . . . . . . . . . . . . . . . . . . . . . . . . 12 1.5.3.1 Temperature . . . . . . . . . . . . . . . . . . . . . . . . . 12 1.5.3.2 Light . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 1.5.3.3 Chemical growth regulators . . . . . . . . . . . . . . . . 13 1.5.3.4 Growing medium, irrigation, nutrition and weed control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 1.5.4 Commercial rhizome and tuber production . . . . . . . . . . . . . . . . 14 1.5.4.1 Goals and techniques . . . . . . . . . . . . . . . . . . . . 14 1.5.4.2 1.5.4.3 Planting to harvest requirements . . . . . . . . . . . . . 15 Postharvest storage and transport requirements . . . . 16 vi 1. 6 Control of flowering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 7 1. 6 . 1 General overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 7 1. 6 . 2 Flowering process and terminology . . . . . . . . . . . . . . . . . . . . 1 7 1. 6 . 3 Influence of internal factors . . . . . . . . . . . . . . . . . . . . . . . . . 2 0 1 . 6. 3 . 1 Rhizome and tuber size . ? . ? ? ? ? ? ? ? ? ? ? ? ? . . . . 2 0 1. 6 . 3 . 2 Dormancy and floral induction . . . . . . . . . . . . . . 2 0 1. 6 . 4 Influence of external factors . . . . . . . . . . . . . . . . . . . . . . . . . 21 1. 6. 4 . 1 Temperature . . . . . . . . . . . . . . . . . . . . . . . . . 21 1. 6 . 4 . 2 Light . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 1. 6. 4 . 3 Nutrition . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24 1. 6. 4 . 4 Chemical growth regulators . . . . . . . . . . . . . . . . 24 1. 6 . 4 . 5 Air pollutants . . . . . . . . . . . . . . . . . . . . . . . . . 26 1. 6 . 5 Commercial forcing for l>ot and cut flower production . . . . . . . . . 26 1. 6. 5 . 1 Rhizome and tuber storage . . . . . . . . . . . . . . . . . 26 1. 6 . 5 . 2 1. 6. 5 . 3 1. 6 . 5 . 4 1. 6 . 5 . 5 1 . 6 . 5 . 6 1 . 6 . 5 . 7 Pre-plant treatments . . . . . . . . . . . . . . . . . . . . . 26 Planting . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27 Height control . . . . . . . . . . . . . . . . . . . . . . . . 27 Forcing environment . . . . . . . . . . . . . . . . . . . . 28 Physiological disorders . . . . . . . . . . . . . . . . . . . 28 Post-greenhouse handling and marketing . . . . . . . . 29 1. 7 Diseases and insects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29 I \ I 1. 8 Miscellaneous physiological and biochemical studies . . . . . . . . . . . . . . 3 1 1 .8 . 1 Spathe regreening . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 1 1 .8 . 2 Tuber respiration . . . . . . . . . . . . . . . . -. . . . . . . . . . . . . . . 3 1 1.9 Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 1 1. 1 0 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33 2 INTRODUCTORY OVERVIEW AND AIM OF THE CURRENT STUDY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 1 2. 1 Overview of study . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 1 2. 2 Aim of this study . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 2 2. 3 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43 3 DRY MA TIER -ACCUMULATION AND LEAF LAMINA vii DEVELOPMENT OF Zantedeschia 'Best Gold' IN RESPONSE TO TEMPERATURE AND PHOTOSYNTHETIC PHOTON FLUX . . . . . . . 4 5 3.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 5 3.2 Materials and Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48 3.2.1 Cultural . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48 3.2.2 Environmental . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 8 3.2.3 Experimental . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . 49 3.3 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 4 3. 3.1 Overview and initial eStablishment . . . . . . . . . . . . . . . . . . . . . 5 4 3. 3. 2 Repetition of treatments over years . . . . . . . . . . . . . . . . . . . . 5 7 3.3.3 Curve fitting of total plant dry weight . . . . . . . . . . . . . . . . . . . 5 7 3.3.4 Curve fitting of leaf area and dry weight . . . . . . . . . . . . . . . . . 61 3.3.5 Relationships between derived parameters . . . . . . . . . . . . . . . . 68 3.4 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70 3.4.1 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75 3.5 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76 4 TUBER DRY MATTER ACCUMULATION OF Zantedeschia 'Best Gold' IN RESPONSE TO TEMPERATURE AND PHOTOSYNTHETIC PHOTON FLUX . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82 4 . 1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82 4 .2 Materials and Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86 4 .3 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89 4 .3.1 Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89 4 .3.2 Commencement of tuber growth . . . . . . . . . . . . . . . . . . . . . . 91 4 . 3. 3 Curve fitting of tuber growth . . . . . . . . . . . . . . . . . . . . . . . . 9 2 4 .3.4 Maximum tuber weight . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 6 4 .3.5 Base temperature for tuber growth . . . . . . . . . . . . . . . . . . . . . 9 6 viii 4. 3 . 6 Tuber and leaf weight partitioning . . . . . . . . . . . . . . . . . . . . . 9 7 4 . 3 . 7 Net assimilation rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 01 4 . 3. 8 Relationships between derived parameters . . . . . . . . . . . . . . . 1 02 4 . 4 Discussion . . . . . . . . . . . . . . ? . . . . . . . . . . . . . . . . . . . . . . . . . 1 07 4 . 4 . 1 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111 4.5 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 1 2 5 PHOTOSYNTHETIC ACTIVITY OF Zmztedeschia 'Best Gold' IN RESPONSE TO TEMPERATURE AND PHOTOSYNTHETIC PHOTON FLUX 116 5 .1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116 5.2 Materials and Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119 5 . 2.1 Experimental . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119 5.2.1.1 Photosynthesis as a function of leaf expansion . . . . 119 5.2.1. 2 Photosynthetic rate as a function of duration from commencement of daily lighting . . . . . . . . . . . . 119 5.2.1.3 5.2.1.4 Photosynthetic rate as a function of photosynthetic photon flux . . . . . . . . . . . . . . . . . . . . . . . . . 120 Photosynthesis during plant development . . . . . . . 1 21 5. 3 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122 5.4 5.3 . 1 Photosynthesis as a function of leaf expansion . . . . . . . . . . . . . 122 5.3.2 Photosynthesis as a function of duration from commencement of daily lighting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 124 5.3.3 Photosynthetic rate as a function of photosynthetic photon flux . . . 125 5.3.4 Photosynthesis during plant development . . . . . . . . . . . . . . . . 1 29 5.3 . 5 Photosynthesis as a predictor of growth and yield . . . . . . . . . . . 13 2 Discussion 133 5.5 References . . . . . . .... . ? . . . . . . . . . . . . . . . . . . . . . . . . . . . 139 6 CARBOHYDRATE CONCENTRATION OF Zantedeschia 'Best Gold' IN RESPONSE TO TEMPERATURE AND PHOTOSYNTHETIC PHOTON ix FLUX . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145 6.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145 6.2 Materials and Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 148 6.2.1 Cultural and environmental . . . . . . . . . . . . . . . . . . . . . . . . 148 6.2.2 Experimental . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 148 6.2.2.1 Determination of concentration of specific soluble carbohydrates . . . . . . . . . . . . . . . . . . . . . . . . 148 6.2.2.2 Starch and soluble carbohydrate concentration as a function of duration from commencement of daily lighting . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149 6.2.2.3 Starch and soluble carbohydrate concentration during plant development . . . . . . . . . . . . . . . . . 15 1 6.3 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 5 6.3.1 Leaf and tuber soluble carbohydrate composition . . . . . . . . . . . 15 5 6.3.2 Starch and soluble carbohydrate concentration as a function of duration from commencement of daily lighting . . . . . . . . . . . . 156 6.3.3 Starch and soluble carbohydrate concentration during plant development 6.3.3.1 6.3.3.2 6.3.3.3 6.3.3.4 Overview . . . . . . . . . . . . . . . . . . . . . . . . . . Tuber starch concentration . . . . . . . . . . . . . . . . Tuber soluble carbohydrate concentration . . . . . . . Leaf starch and soluble carbohydrate concentration . 6.3.4 Carbohydrate and structural dry weight concentration as predictors 15 7 15 7 158 161 163 of growth and yield . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 164 6.4 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 166 6.4.1 Specific soluble carbohydrates and sample preparation . . . . . . . . 166 6.4.2 Diurnal and developmental changes in starch and soluble carbohydrate concentration . . . . . . . . . . . . . . . . . . . . . . . . . 166 6.4.3 Tuber starch and structural dry weight changes with development . 169 6.4.4 Carbohydrate and structural dry weight concentration as predictors of growth and yield . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 171 6.4. 5 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 172 6.5 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ? 173 X 7 MANIPULATION OF IN VITRO SOURCE AND SINK STRENGTH, AND DRY MATI'ER PARTITIONING IN Zantedeschia 'Best Gold' . . . 18 0 7. 1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ? . . . . . . . 18 0 7. 2 Materials and Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 185 7. 2. 1 Germination media, media transfer and sucrose concentrations . . . 185 7. 2. 2 Manipulation of in vitro source and sink strengths . . . . . . . . . . 187 7. 3 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 189 7. 3 . 1 Germination media, media transfer and sucrose concentrations . . . 189 7. 3 . 1 . 1 Germination . . . . . . . . . . . . . . . . . . . . . . . . . 189 7. 3 . 1. 2 Dry matter accumulation and partitioning . . . . . . . 189 7. 3 . 2 Manipulation of source and sink strengths . . . . . . . . . . . . . . . 192 7. 4 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 199 7. 4 . 1 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 204 7. 5 References 8 ECOLOGICAL AND HORTICULTURAL RELEVANCE, AND MECHANISMS OF CONTROL OF DRY MATI'ER ACCUMULATION AND PARTITIONING IN Zantedeschia 'Best Gold' - AN 205 INTEGRATIVE DISCUSSION . . . . . . . . . . . . . . . . . . . . . . . . . . 212 8. 1 Ecological relevance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 212 8. 2 Mechanisms of control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 213 8 .3 Horticultural relevance and consequences . . . . . . . . . . . . . . . . . . . . 218 8. 4 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 221 xi UST OF ABBREVIATIONS a . . . . . . . . . . . . . . . . . . . . . . . . . . . . . apparent photosynthetic quantum yield aw,A,L,u,TocT? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? upper asymptote of factor under investigation A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . leaf area. flw ,A,r.,:u,T ? T? ? ? ? ? ? ? ? a measure of the starting size of the factor under investigation BA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . benzyl ( lH-purin- 6-yl) amine C ................................................ Celsius cm . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . centimetre 2 . cm . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . square centimetre CE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . controlled environment C02 ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? carbon dioxide D1'EMP . . . . . . . . . . . . . . . . ? . . . . . . . . . . . . . . . . . . . . . . day temperature g ................................................. grnm. GA3 ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? gibberellic acid G.A"+7 ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? gibberellin 4 and 7 h . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . hour ha ................................................ hectare HPLC . . . . . . . . . . . . . . . . . . . . . . . . . high performance liquid chromatograph i.e. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (id est) that is "w .A.L.u,T or T? ? ? ? ? ? ? ? rate constant of factor under investigation as a function of size kg . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . kilogrnm. L . i. . . . . . . . . . . . . . ? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .leaf weight LAP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .leaf area partitionmg L.AR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . leaf area ratio L WP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . , . . . . leaf weight partitioning L WR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . leaf weight ratio loge . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . natural logarithm Ls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . leaf starch dry weight LS . . . . . . . . . . . . . . . . . . . . . . . . . . . Linsmaier and Skoog organic additives m ................................................. metre m2 ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? square metre m3 ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? cubic metre . mg . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . milligrnm. min . . . . . . . . . . . . . . : . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . minute ml . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . millilitre M . . ? ................. .-............................. molar mm . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . millimetre MS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Murashige and Skoog medium Xll n ..... number of observations in a sample ng .. nm . NAR N.B. . ............................. nanogram . ....... nanometre . . net assimilation rate . (nota bene) note well NTEMP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . night temperature P . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . probability Pa .. pH . . Pmax . Pn . PPF pp m r R .. r .. RGR . ........................... Pascal . . . . . . . . . . . . measure of acidity or alkalinity . . maximum photosynthetic rate at saturating PPF . . . net photosynthetic rate . photosynthetic photon flux . . . . parts per million . . . . . . . . . . . partial correlation . . . . . . . . . . . . . respiration rate . . . . . . . . . . . . . . coefficient of determination ................... relative growth rate RLAER ....... . . ................ relative leaf area expansion rate RLSWR ....... . . . . . . . . . . . . . . . relative leaf starch weight rate RLWR ........ . . ....................... relative leaf weight rate RWP s .. ? . SAS I s.e . . SLA str . . . . . . . . . . . . . . . . . . . . . . . . ? . . root weight partitioning ............................... second .. Statistical Analysis System (statistical software) . . . . . . . . . . . . . . . . . . . . . . . . . . . standard error of the mean . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . specific leaf area . . . dry weight of structural material (i.e., minus soluble sugars and starch) t ................................................... time T .. T% . tanh Ts Tstr . TWP j.tl .. Jlm . j.tmOl . . . . . . . . . . . . . . . . . . . ? . . . . time to commencement of tuber growth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . tuber dry weight . . . percentage tuber weight loss at the commencement of tuber growth ..................................... hyperbolic tangent ................................. tuber starch dry weight . dry weight of tuber structural material (i.e., minus soluble sugars and starch) . . . . . . . tuber weight partitioning . . . . . . . . microlitre . . . . . . . micrometre . .......... micromole viz. . . . . . . . . . . . . . . . . . . . . . . . . . . . (videlicet) namely v/v ........................................ volume (mix ratio) xiii W . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . total plant dry weight %LA ................................ percentage maximum leaf area o 'S 0 .... . JlP .. . . . .. ... ns,, , angular distance on its meridian South of equator in degrees and minutes . . . . . . . . . . . . . . . . . . . . . . . mathematical notation for an interval . . . . . . . . difference between photosynthetic rate under saturating PPF and photosynthetic rate under the growth PPF unless otherwise stated, probability of a significant F value; nonsignificant or significant at P = 0. 10, 0.05 , or 0.01 , respectively NOTES ON CITATION FORMAT With a view to publishing this thesis as a series of scientific papers in journals such as those produced by the American Society for Horticultural Science (ASHS), the style of literature citation follows that recommended by ASHS. The citation system used therefore follows the Harvard system, and abbreviations for periodical titles are as suggested by ASHS. xiv LIST OF TABLFS Table 1 . 1 Table 1 .2 Table 1.3 Table 3.1 Table 3.2 I ' ' Table 3.3 Table 3.4 PAGE Descriptive features of the species and subspecies of Zantedeschia Spreng. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 Average daily maximum, minimum, mean air, and minimum soil (8.00 am, at 10 cm), temperatures (C) during the winter (June to July), for sites of natural habitat of Zantedeschia aethiopica, Zantedeschia pentlandii and Zantedeschia rehlnannii . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 Average daily maximum, minimum, mean air, and minimum soil (8.00 am, at 10 cm), temperatures (C) during the summer (October to February) for sites of natural habitat of Zantedeschia aethiopica, Zantedeschia pentlandii and Zantedeschia rehlnannii . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 Duration of growth (days) until attainment of 7 5% or more expansion of the first leaf of Zantedeschia 'Best Gold: at a range of temperatures, and high and low PPF regimes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 5 Total plant relative growth rates (RGRw ? standard error), and associated rl, for plants of Zantedeschia 'Best Gold' grown at day/night temperatures of 22/16 C, at high and low PPF, in two CE rooms over two years . 5 7 Nonlinear least-squares parameter estimates, associated asymptotic standard error (s.e.), and mean square error values, from fitting the Gompertz function to lo? transformed total plant dry weight data for Zantedeschia 'Best Gold' grown at a range of temperatures, and under high and low PPF regimes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 9 Nonlinear least-squares parameter estimates, associated asymptotic standard error (s.e.), and mean square error values, from fitting the logarithmic Gompertz function to lo? transformed total plant leaf area data for Zantedeschia 'Best Gold' grown at a range of temperatures, and under high and low PPF regimes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63 Table 3.6 Table 4. 1 Table 4.2 Table 4.3 Table 5. 1 Table 7.1 Table 7.2 Table 7.3 XV Nonlinear least-squares parameter estimates, associated asymptotic standard error (s.e.), and mean square error values, from fitting the logarithmic Gompertz function to lo? transformed total plant leaf dry weight data for Zantedeschia 'Best Gold' grown at a range of temperatures, and under high and low PPF regimes . . . . . . . ? . . . . . . ? . . . . . . . . . . . . . . . 64 Leaf area (cm? at inflection point of the fitted total plant dry weight Gompertz curve of Zantedeschia 'Best Gold; grown at a range of temperatures, and high and low PPF regimes . . . . . . . . . . . . . . . 65 Parameters examined in the development of a mechanistic multiple regression model . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88 Nonlinear least-squares parameter estimates, associated asymptotic standard error (s.e.), and mean square error values, from fitting the Gompertz function to loge transformed tuber dry weight data for Zantedeschia 'Best Gold' grown at a range of temperatures, under high and low PPF regimes ...................................... 94 Partial correlation matrix between TWP, L WP, NAR and RGRT at the inflection point of the Gompertz fit of tuber, dry weight curves of Zantedeschia 'Best Gold' grown under a range of environments . . . 103 Correlation between photosynthetic rate (Pn) and relative growth rate of total plant weight (RGRw) and tuber dry weight (RGRT) of Zantedeschia 'Best Gold' grown at six day/night temperatures and two PPF regimes, during two stages of development . . . . . . . . . . . . . . . . . . . . . 132 Dry matter accumulation and partitioning in seedlings of Zantedeschia 'Chromatella: as influenced by the presence of light or dark, and sucrose concentration in the growing medium . . . . . . . . . . . . . . . . . . . 191 Dry matter accumulation and partitioning in seedlings of Zantedeschia 'Best Gold: as influenced by temperature and sucrose concentration . . . . 193 Dry matter accumulation within the shoot of seedlings of Zantedeschia 'Best Gold: as influenced by temperature and sucrose concentration . . . . 194 xvi Signifi?ce of trend analyses of increasing temperature and sucrose concentration on dry matter accumulation and partitioning in seedlings of Zantedeschia 'Best Gold' . . . . . . . . . . . . . . . . . . . . . . . . . . . 195 .. Figure 1.1 ' Figure 1.2 Figure 1 .3 Figure 3.1 Figure 3.2 Figure 3.3 Figure 3.4 xvii PAGE Distribution of species in the genus Zantedeschia across the southern regions of Africa. (a) Z. odoraJa ?, Z. jucunda e, and Z. pentlandii IIl, (b) Z. rehmannii [], (c) Z. albomaculata [l], and (d) Z. aethiopica El. Adapted from Letty (1973); Anon (1989); Perry (1989) .......... 6 Diagrammatic illustration of sympodial growth habit of a single primary shoot of Zantedeschia . . ? . . . . . . . . . ? . . . . . . . . . . . . . . . . . 19 Diagrammatic illustration of primary shoot of Zantedeschia, with secondary shoots arising from leaf axils . . . . . . . . . . . . . . . . . . . . . . . . . 19 Examples of total plant dry weight (logJ as a function of time, for Zantedeschia 'Best Gold? ? indicates day of transfer to treatments . 54 Examples of relative growth rate (RGR.w) as a function of time, for Zantedeschia 'Best Gold? ? indicates day of transfer to treatments . 55 Maximum value of RGRw as a function of temperature, for plants of Zantedeschia 'Best Gold' grown under high and low PPF regimes . ? 56 Total plant dry weight (lo? fitted Gompertz curves) for Zantedeschia 'Best Gold' at a range of temperatures, under a high PPF regime. ? indicates day of transfer to treatments . . . . . . . . ? . . . . . . . . . . . . . . . . . 58 Figure 3.5 Total plant dry weight (log., fitted Gompertz curves) for Zantedeschia 'Best Gold' at a range of temperatures, under a low PPF regime. ? indicates day of transfer to treatments . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58 Figure 3.6 Lo? m?mum total plant dry weight (aw) as a function of temperature, for \ Zantedeschia 'Best Gold' under high and low PPF regimes. Fitted line for high PPF regime only . . . . . . . . . . . . . . . . . . . . . . . . . . ? . . . 60 Figure 3. 7 Interrelation between the rate of decline of RGRw as a function of plant size (Kw), and temperature, for Zantedeschia 'Best Gold' under high and low PPF regimes. Fitted line for low PPF only . . . . . . . . . . . . . . . . 61 xviii f2iii!e 3. 8 Fitted logarithmic Gompertz curves and mean data of total plant leaf area (log.) as a function of time, for Zantedeschia 'Best Gold' at a range of temperatures, under a high PPF regime . . . . . . . . . . . . . . . . . . . 6 2 -? 3. 9 Fitted logarithmic Gompertz curves and mean data of total plant leaf area (log.) as a function of time, for Zantedeschia 'Best Gold' at a range of temperatures, under a low PPF regime . . . . . . . . . . . . . . . . . . . 6 2 F.tgUI'e 3. 1 0 Lo? maximum total plant leaf area (a.J as a function of temperature, for Zantedeschia 'Best Gold' under high and low PPF regimes. Fitted line for high PPF regime only . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 5 igure 3. 11 Parameter {JA as a function of temperature, for Zantedeschia 'Best Gold' under high and low PPF regimes. Fitted line for low PPF only . . . 66 FigUre 3. 12 Interrelation between the rate of decline of RLAER8 (K.J and temperature, for Zantedeschia 'Best Gold' under high and low PPF regimes . . . . 67 Fjgure 3. 1 3 RGRw as a function of RLAER, for Zantedeschia 'Best Gold' grown under two PPF regimes and six temperatures . . . . . . . . . . . . . . . . . . . 69 ' . Figure 3: 1 4 RGRw as a function of NAR, for Zantedeschia 'Best Gold' grown under two PPF regimes and six temperatures . . . . . . . . . . . . . . . . . . . . . . 69 Figure 3. 1 5 RGRw as a function of LAP, for Zantedeschia 'Best Gold' grown under two PPF regimes and six temperatures . . . . . . . -. . . . . . . . . . . . . . . 69 Figure 3. 1 6 RGRw as a function of LWP, for Zantedeschia 'Best Gold' grown under two PPF regimes and six temperatures . . . . . . . . . . . . . . . . . . . . . . 69 Figure 4 . 1 Shoot, tuber, root and total dry weight per plant of Zantedeschia 'Best Gold' plants grown at 2 5 C under high PPF. n=6 or 1 2 . . . . . . . . . . . . 89 Figure 4 . 2 Tuber dry weight (expressed as logJ of Zantedeschia 'Best Gold' plants grown under high or low PPF at 1 6 or 2 8 C . . . . . . . . . . . . . . . . 90 figure 4 . 3 Time to commencement of tuber growth (tJ as a function of temperature for Zantedeschia 'Best Gold: grown under high and low PPF regimes . . 91 xix Figure 4.4 Extent of tuber weight loss (T ") at the time of commencement of tuber growth, as a function of temperature, for Zantedeschia 'Best Gold: grown under high and low PPF regimes . . . . . . . . . . . . . . . . . . . . . . . 92 Figure 4.5 Fitted Gompertz curves and mean data points of lo? tuber dry weight as a function of time, for Zantedeschia 'Best Gold' at a range of temperatures, under high PPF regime . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93 Figure 4.6 Fitted Gompertz curves and mean data points of loge tuber dry weight as a function of time, for Zantedeschia 'Best Gold' at a range of temperatures, under a low PPF regime . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93 Figure 4.7 Loge maximum tuber dry weight (aT) as a function of temperature, for Zantedeschia 'Best Gold' under high and low PPF regimes . . . . . . . 95 Figure 4.8 RGRT as a function of temperature, for plants of Zantedeschia 'Best Gold: grown under high and low PPF regimes. (N.B., line for low PPF excludes 28 C) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97 Figure 4.9 Proportion of daily increment in total weight partitioned to the tuber (TWP) as a function of time, for Zantedeschia ?'Best Gold: at a range of temperatures, under a high PPF regime . . . . . . . . . . . . . . . . . . . 98 Figure 4.10 Proportion of daily increment in total weight partitioned to the tuber (TWP) as a function of time, for Zantedeschia 'Best Gold: at a range of temperatures, under a low PPF regime . . . . . . . . . . . . . . . . . . . 98 Figure 4. 1 1 Proportion of daily increment in total weight partitioned to the tuber (TWP), as a function of time, for Zantedeschia 'Best Gold: grown under high and low PPF regimes at 13 or 28 C . . . . . . . . . . . . . . . . . . . . . . . . 99 Figure 4.12 Proportion of daily increment in total weight partitioned to the tuber (TWP), as a function of temperature, for zantedeSchia 'Best Go1d' undet high and low PPF regimes . . . . . . ? . . . . . . . . . . . . . . . . . . . . . . . . 100 Figure 4.13 Proportion of daily increment in total weight partitioned to leaf (LWP) as a function of temperature, for Zantedeschia 'Best Gold' under high and low PPF regimes. (N.B., line for high PPF excludes 13 C) . . . . . . . . 101 XX 14 Net assimilation rate (NAR) as a function of temperature, for plants of ?............ Zantedeschia 'Best Gold; grown under high and low PPF regimes. (N.B. , line for high PPF regime excludes 13 C) . . . . . . . . . . . . . . ? . . 102 lf!i? 4. 15 Relative growth rate of the tuber (RGRT) as a function of daily partitioning to the tuber (IWP) for Zantedeschia 'Best Gold' grown under high and low PPF regimes at all temperatures . . . . . . . . . . . . . . . . . . . . . . 104 ? 4. 16 Relative growth rate of the tuber (RGRT) as a function of daily partitioning to the leaf (L WP) under high and low PPF regimes, at all temperatures. Jjne is for low PPF regime at temperatures ? 16 C . . . . . . . . . . 104 FJgUie 4. 17 Relative growth rate of the tuber (RGRT) as a function of net assimilation rate (NAR) under high and low PPF regimes, at all temperatures. Line is for high PPF regime at temperatures ? 16 C . . . . . . . . . . . . . . 105 ?JgUie 5.1 Net photosynthetic rate (Pn) as a function of individual leaf area expansion, at selected temperature and PPF regimes. Mean values ? se. , n=6, cubic spline fit. Arrows represent 75% maximum leaf area . . . . . . . . . 122 tF"IgUre 5.2 Photosynthetic rate (Pn) as a function of duration from commencement of daily lighting, at selected temperature regimes and high PPF. Mean values ? se. , n=6. Broken lines indicate limits of diurnal temperature changeovers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 124 ? ?Figure 5.3 Photosynthetic rate (Pn) per unit leaf area (a)_ and (b) , and per unit leaf weight (c) and (d) , as a function of photosynthetic photon flux (PP F), for plants of Zantedeschia 'Best Gold' grown at day temperatures of 16, 22 and 28 C, under high (a) and (c), or low (b) and (d), PPF regimes. n=6 or 18, function = equation 5 . 1 . . . . . . . . . . . . . . . . . . . . . . . . . 125 Figure 5.4 Maximum photosynthetic rate (Pmax) per unit leaf area as a function of day temperature: for plants of Zantedeschia 'Best Gold' grown under high and low PPF. Vertical bars = 2 x standard error . . . . . . . . . . . . . 126 Figure 5.5 Maximum photosynthetic rate (Pmax) per unit leaf weight as a function of day temperature, for plants of Zantedeschia 'Best Gold' grown under high and low PPF. Vertical bars = 2 x standard error . . . . . . . . . . 126 xxi Quantum yield (ex) per unit leaf area as a function of day temperature, for plants of Zantedeschia 'Best Gold' grown under high and low PPF. Vertical bars = 2 x standard error . . . . . . . . . . . . . . . . . . . . 127 Figure 5.7 Quantum yield (ex) per unit leaf weight as a function of day temperature, for plants of Zantedeschia 'Best Gold' grown under high and low PPF. Vertical bars = 2 X standard error . . . ? . . . . . . . . . . . . . . . . 127 ?Figure 5 .8 Difference (MJ) between P JIMX and Par- per unit leaf area, for plants of Zantedeschia 'Best Gold' grown at a range of temperatures, under high and ? '? low PPF . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 128 Figure 5.9 Difference (MJ) between P JIMX and Par- per unit leaf weight, for plants of t Zantedeschia 'Best Gold' grown at a range of temperatures, under high and low PPF . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ! ? ? 128 ?Figure 5 . 10 Photosynthetic rate (Pn), per unit leaf area, of the most recently expanded ?L leaf as a function of time, for Zantedeschia 'Best Gold' grown at a range of day/night temperatures under high and low PPF regimes. (a) 16/10 C (b) 22/10 C (c) 22/16 C (d) 28/16 C (e) 28/22 C (f) 28/28 C. Vertical bars = 2 X standard error, arrows indicate commencement of tuber growth under high (H) and low (L) PPF . . . . . . . . . ? . . . . . . . . . . . . 130 Figure 5. 1 1 Photosynthetic rate (Pn), per unit leaf weight, of the most recently expanded leaf as a function of time, for Zantedeschia 'Best Gold' grown at a range of day/night temperatures under high and low_ PPF regimes. (a) 16/10 C (b) 22/10 C (c) 22/16 (d) 28/16 C (e) 28/22 C (f) 28/28 C. Vertical bars = 2 x standard error, arrows indicate commencement of tuber growth under high (H) and low (L) PPF . ? . . . . . . . . . . . . . . . . . . . . . . . . 131 Figure 6. 1 Soluble carbohydrate concentration of mature leaves of Zantedeschia 'Best Gold' determined either immediately after harvest (fresh) or after vacuum drying (dry). Mean values ? se., n=3 . . . . . . . . . . . . . . . . . 155 Figure 6.2 Solub!e carbohydrate concentration of tubers of Zantedeschia 'Best Gold' determined either immediately after harvest (fresh) or following vacuum drying (dry). Mean values ? se. , n=3 . . . . . . . . . . . . . . . . . 155 xxii Figure 6.3 Photosynthetic rate (Pn) and foliar carbohydrate concentration, as a function of duration of daily lighting, at 28/22 C under high PPF. Mean values ? se., n = 3. Broken lines indicate limits of diurnal environmental chan.geovers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 156 .;Figure 6. 4 Tuber carbohydrate concentration as a function of duration of daily lighting at 28/22 C under high PPF. Mean values ? se., n=3. Broken lines indicate limits of diurnal environmental chan.geovers . . . . . . . . . . 156 Figure 6.5 Total, structural and starch tuber dry weight QogJ, as a function of time, of Zantedeschia 'Best Gold' plants grown at 25 C under high PPF. ? indicates day of transfer to treatment environment . . . . . . . . . . 157 Figure 6.6 Minimum tuber starch concentration as a function of temperature, for plants of Zantedeschia 'Best Gold' grown under high and low PPF regimes. Mean values ? se., n =4 . . . . . . . . . . . . . . . . . . . . . . . . . . 158 Figure 6. 7 Carbohydrate concentration of the tuber as a function of time, for Zantedeschia 'Best Gold' grown at three temperatures under high and low PPF regimes. (a) 13 C, high PPF (b) 1 3 C, low PPF (c) 19 C, high PPF (d) 19 C, low PPF (e) 25 C, high PPF (f) 2 5 C, low PPF. Mean values ? se., n=4, ? indicates commencement of tuber growth . . . . . . . 159 Figure 6. 8 Tuber starch dry weight as a function of tuber structural dry weight. Low PPF before (-D-), and both high and low PPF after (-0-), the commencement of tuber growth, for plants grown at three temperatures and two PPF regimes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 160 Figure 6.9 Relative growth rate (RGR) of tuber starch and tuber structural dry weight as a function of time, for Zantedeschia 'Best Gold' grown at three temperatures under high and low PPF regimes. (a) 19 C, high PPF (b) 19 C, low PPF (c) 25 C, high PPF (d) 25 C, low PPF . . . . . . 162 Figure 6.10 Starch concentration of first leaf, as a function of temperature, for plants of Zantedeschia 'Best Gold' grown under high and low PPF regimes. Mean values ? se., n =4 ...... ................ . ?. . . . . . . 16 3 xxiii Figure 6.11 Soluble carbohydrate concentration ? of first leaf, as a function of temperature, for plants of Zantedeschia 'Best Gold' grown under high and low PPF regimes. Mean values ? se. , n=4 .............. 163 Figure 6.12 Carbohydrate concentration of mature leaves as a function of time, for Zantedeschia 'Best Gold' grown at three temperatures under high and low PPF regimes. (a) 13 C, high PPF (b) 13 C, low PPF (c) 19 C, high PPF (d) 19 C, low PPF (e) 2 5 C, high PPF (f) 2 5 C, low PPF. Mean values ? se. , n=4, ? indicates commencement of tuber growth . . . . . . . 165 Figure 7.1 Diagrammatic summary of germination and transfer treatment media used for seedlings of Zantedeschia 'Chromatella' . . . . . . . . . . . . . . . 18 7 Figure 7.2 Surface response curves illustrating the influence of increasing temperature and sucrose concentration on the dry matter accumulation and partitioning in seedlings of Zantedeschia 'Best Gold? (a) Total dry weight, (b) Tuber dry weight, (c) Shoot dry weight, and (d) Proportion of total dry weight in the tuber . . . . . . . . . . . . . . . . ? . . . . . . . . . . . . . . . . . . . . . 196 Figure 7.3 Surface response curves illustrating the influence of increasing temperature and sucrose concentration on the number of (a) shoots and (b) leaves in seedlings of Zantedeschia 'Best Gold' . . . . . . . . . . . . . . . . . . . 19 7 xxiv LIST OF PLATES Plate 1.1 Plate 1. 2 Plate 3. 1 Plate 7. 1 Plate 7. 2 Plate 7. 3 PAGES Dissection of spathe to reveal complete separation of (m) male and (f) female flowers on spadix of the group 2 selection 'Best Gold' (right), compared with being interspersed on lower part of spadix in the group 1 specie Z. aethiopica (left). (a) region of dark pigmentation at the base of the spathe of 'Best Gold' . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Flowering sized tuber of a group 2 selection indicating examples of (a) dominant bud, (b) developed axillary bud, and (c) undeveloped axillary bud . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 Harvested plant of Zantedeschia 'Best Gold' illustrating components measured. (a) shoot (sheath leaves, petioles and apex, (b) exposed leaves, (c) tuber, (d) roots . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50 Germinated seedling of Zantedeschia 'Chromatella' after being excised from the cotyledon . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18 5 Seedlings of Zantedeschia 'Chromatella' after 2 9 weeks of cultivation in vitro at a range of sucrose concentrations in either the dark (upper) or light (4 5 JLmoi-m-2?s-1 PPF) (lower) . . . . . . . . . . . . . . . . . . . . . . . . 19 0 Tubers from seedlings of Zantedeschia 'Best Gold' after 24 weeks of growth in vitro, at a range of temperatures and sucrose concentrations. N.B. shoots and roots removed . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 197 1 1 BOTANICAL, ECOWGICAL, PHYSIOLOGICAL, AND HORTICULTURAL BACKGROUND OF THE GENUS Zantedeschia. 1 .1 Introduction and overview Zantedeschia K. Spreng. species, and their hybrids, may be grown outdoors as garden plants and commercial cut flowers in temperate to sub-tropical climates. Forcing programmes are under development to provide cut flower and pot plant production year round. The range of available flower colours, foliage spotting (maculation) , and plant heights has created interest in their use as bedding plants. 1.2 World production areas and volumes Currently New Zealand produces the widest range of Zantedeschia, with more than 3 . 1 million flower stems of over 120 cultivars exported during 1990/91 (N.Z. Trade Development Board , personal communication) . Tuber exports from New Zealand were estimated to be in excess of 1 .4 million during 1988/89 (Kepner et al . , 1990) . While no data are available on the total area used for Zantedeschia production in New Zealand, i t is estimated to be in excess of 100 ha (unpublished observations) . Other countries with significant areas of production are The Netherlands (10 ha; 1987/88) and Israel ( 10 ha; 1987/88) (De Hertogh, personal communication) . Japan also produces cut flowers (30 ha; 199 1), predominantly Z. aethiopica (L.) Spreng. 'Childsiana; and cultivates a small area of pot plants (Hayashi, personal communication) . However, Japan is a net importer of Zantedeschia flowers and tubers. While the U.S.A. also produces a significant quantity, no production data have been published since the 1940's (3 . 5 million plants; Butterfield , 1948; Hayward, 1948). Emerging cut flower production industries are also located in Italy, South Africa, Kenya, Mexico, Columbia and Costa Rica. International production of true species is limited to Z. aethiopica, Z. albomaculata (Hook.) Baill. , Z. elliottiana (Watson) Engl . , and Z. rehmannii Engl. With ongoing breeding and selection programmes, New Zealand currently produces two species and over 120 cultivars. However, planting material of only 56 species/cultivars is currently available in commercial quantities. Together with supplies from The Netherlands and U .S .A. planting material of 6 1 species/cultivars are commercially available internationally. To avoid the continued use of the phrase species/cultivars, the term "Selection(s)" will be used hereafter. 1.3 Botanical classificatif!n, morphology, distribution and habitat 1.3.1 Botanical classification and morphological description 2 The name Zantedeschia was first established by Sprengel (18 26), but other genus names such as Arodes, Aroides, Calla and Richardia have also been used subsequent to the first recording in 16 8 7 (Letty, 19 7 3). The genus has been revised on a number of occasions (Engler, 1915; Traub, 194 8; Letty, 19 7 3). It is the most recent revision by Letty (19 7 3) which is now generally followed, in which the genus is presented as containing six species and two subspecies {Table 1. 1). A seventh species, Z. odorala (Perry), was proposed subsequent to that revision (Perry, 19 89). Horticulturally, two important groups have been recognised. (1) The first group is typified by Z. aethiopica, in which the foliage does not die down in winter in the native habitat (Letty, 19 7 3). It flowers from late winter to late spring with male and female flowers interspersed in the lower part of the spadix (Plate 1. 1) . The fruits turn an orange colour and become soft and mucilaginous with maturity. For completeness Z. odorala is included in this group, but as discussed later in this Section differences do exist. ( 2) The second group contains the five acknowledged remaining species that typically exhibit complete foliage senescence in winter and flower during the summer months (Letty, 19 7 3) . The male and female flowers are not intet:spersed on the spadix, being physically separated with female flowers at its base (Plate 1. 1). The fruits remain firm and grbn with maturity. I I \ Z. aethiopica plants grow to 12 0 cm tall, and comprise ovate-cordate or hastate shaped leaves up to 60 cm in length (Letty, 19 7 3). The leaves do not commonly exhibit the maculation present in some of the other species {Table 1. 1), and are, therefore, referred to as being immaculate. However, maculate forms of this species have been noted (Letty, 19 7 3). The other species are smaller in stature, not exceeding 8 0 cm in height, especially Z. rehmannii which does not exceed 60 cm. While leaf shape of these latter species is generally similar to that of Z. aethiopica, plants of Z. rehmannii have lanceolate leaves. The degree of leaf maculation also varies between these species in group 2 {Table 1. 1). The perennating storage organ of Z. aethiopica is a rhizome, whereas species in group 2 have what is best describ? .as a compact stem. While both forms may be branched, the latter is globular in shape. Classic horticultural texts describe all species of Zantedeschia as possessing a rhizome (Bailey, 19 3 0), but a satisfactory answer as to whether botanically the latter? of the two structures is a corm, tuber, or rhizome, has not been resolved. It is widely accepted that the storage organ of other members of the Araceae family, e.g., 3 Colocasia, is a corm (Coursey, 1968; Okonkwo, 1987) . While Traub (1948) described the storage organs of Zantedeschia as rhizomes or corms, Letty (1973) referred to rhizomes or tubers in her revision of the genus. While such debates are often referred to as being primarily of academic interest, the acknowledgement of the existence of the two forms is important when growing members of the two groups (refer Sections 1 .5 and 1 . 6) . In an effort to avoid confusion, at least during this thesis, the term rhizome will be used to refer to the storage organs of group 1 , while the term tuber will be used for those in group 2 . Plate 1.1 Dissection of spathe to reveal complete separation of (m) male and (f) female flowers on spadix of the group 2 selection 'Best Gold' (right) , compared with being interspersed on lower part of spadix in the group 1 specie Z. aethiopica (left) . (a) region of dark pigmentation at the base of the spathe of 'Best Gold! The preceding discussion of the two forms of storage organs provides strong evidence for supporting the recen?y reported seventh species (Perry, 1989) . While leaves and flowers of Z. odorata appear similar to those of Z. aethiopica, plants grow and flower later in the season, ? and they do not possess the typical rhizomatous storage organ (Perry, 1989) . The storage organ is similar to that of group 2. It is compact and has a pyriform shape. Unlike Z. aethiopica, the fruits of Z. odorata do not change colour with maturity but remain flrm and green like those of group 2. Table 1.1 Descriptive features of the species and subspecies of 7Antedeschia Spreng. --- Foliage Flower Species Group Duration Maculation Shape Duration Colour Dark throat Z. aethiopica 1 Evergreen, late Rare Ovate-cordate or Late winter to Milk-white, No winter to late spring hastate late spring pink z. rehmannii 2 Deciduous, spring to No Lanceolate Summer White, pink, No late autumn dark maroon Z. jucunda 2 Deciduous, spring to Yes, Triangular-hastate Summer Golden yellow Yes late autumn densely Z. elliottiana 2 Deciduous, spring to Yes Orbicular-ovate Summer Golden yellow No late autumn Z. pentlandii 2 Deciduous, spring to Oblong-elliptic to Lemon-chrome late autumn Seldom oblong-lanceolate Summer yellow Yes Z. albomaculata 2 Deciduous, spring to White, ivory, sub. albomaculata late autumn Rare Oblong-hastate Summer pale yellow, Yes coral pink Z. albomaculata 2 Deciduous, spring to Yes, Triangular-hastate Summer Straw yellow Yes sub. macrocarpa late autumn sparse Z. albomaculata 2 Deciduous, spring to Ovate-cordate to sub. valida late autumn No ovate-orbicular- Summer Ivory to cream Yes cordate Z. odorata 1 Deciduous, late No Ovate to cordate Late winter Milk white No winter to late spring 4 Reference: Letty, 1973; Perry, 1989. 5 Studies of the root systems have not been previously reported. In all species they are predominantly contractile and not extensively branched (unpublished observations). The inflorescence comprises a spadix, carrying the true male and female flowers, subtended by a coloured bract known as the spathe (Plate 1.1). The inflorescence and spathe are typically presented at or above foliage height on a fleshy peduncle. The spathe may vary in shape from regularly funnel or trumpet-shaped with minimal convolutions, e.g . , Z. pentlandii (Watson) Wittm. , to a tightly folded tube with a tapering tip, e.g. , Z. rehmannii, (Letty, 19 7 3). Spathe colour varies from the milk-white of Z. aethiopica, through yellow, orange, pink and dark maroon, with the latter colours being primarily derived from the other species (Table 1.1). The inside of the spathe may also exhibit a region of dark pigmentation at its base, i.e. , dark throat (Plate 1.1). The presence of this additional pigmentation is also dependent on species (Table 1.1). At initial emergence, the spathe is pigmented with chlorophyll. With subsequent unfurling the spathe develops its full colour at or near the commencement of pollen shed. In this thesis, the term "Flower" will refer to the combination of the spadix and spathe. 1.3.2 Distribution and climate of origin While other members of the Araceae are endemic to South America, Asia, and Africa, the genus Zlmtedeschia is confined to the African continent. It is most prevalent in the south (i.e. , ?ape Province, Orange Free State, Natal, Lesotho, Swaziland, Transvaal), but also extends into Zimbabwe, Malawi, Zambia, Angola, and Nigeria (Letty, 19 7 3). Z. i aethiopica is almost completely confmed to the south and eastern coastal belt of southern Africa, but is also found in the south-eastern mountainous regions at altitudes up to 1000 m (Figure 1.1) (Letty, 19 7 3; Anon. , 19 8 9). While Z. albo_maculata is widespread in its distribution across the coastal and mountainous regions of south-eastern Africa, Z. jucunda (Letty), Z. pentlandii, and Z. rehmannii are restricted to the eastern mountainous regions (Figure 1.1), at altitudes of 1200 to over 2000 m (Letty, 19 7 3; Anon. , 19 8 9). The distribution of Z. elliottiana has not been documented, since it has not been found in the wild (Letty, 19 7 3). ' While frequently found at "forest margins: the natural habitat of Zlmtedeschia species has generally been associated with open grasslands (Letty, 19 7 3). While exceptions are always evident, the natural distribution of perennial species has been shown to be critically dependent on the temperature minima during both the growing - seasOn and/or annual period (Korner and Larcher, 19 8 8 ; Woodward, 19 8 8). In addition, the heat sum during the season has also been shown to be influential. If like other genera, 6 the natural distribution of Zantedeschia species has been influenced by such temperature parameters, it would be exJ>ected that climatological data of the sites of origin would provide some indication of the possible temperature tolerance and growth response ranges. Figure 1.1 Distribution of species in the genus Zantedeschia across the southern regions of Africa. (a) Z. odorata ? , Z. jucunda e, and Z. pentlandii _m , (b) Z. rehmannii ill , (c) Z. albomaculata UJ , and (d) Z. aethiopica B . Adapted from Letty (1973); Anon (1989); Perry (1989) . Three species are discussed in an endeavour to more closely describe the climates of origin of those currently important to horticulture; (1) z. aethiopica (an example of a group 1 species) . ( 2) Z. pentlandii (an example of a group 2 species and presumed to be dominant in the parentage of_ ?e selection 'Best Gold'). (3) Z. rehmannii (an example of a group 2 species). Climatological data for 2 4 sites of natural habitat of Z. aethiopica, 10 sites of Z. pentlandii, and 1 4 sites of Z. rehmannii, as outlined by Letty (1973) and Anon. (1989), were analysed 7 from 2Q-year average climate statistics (Anon. , 1954). Due to the wide geographic distribution of Z. aethiopica (Figure 1 . 1), climatological data for this species were also analysed as two subgroups consisting of; (1) those sites located along the south and south-eastern coast (warm). (2) those sites located in inland mountainous regions (cool) , typically also occupied by Z. pentlandii and Z. rehmannii. This analysis by subdivision was not intended to infer the existence of ecotypes of Z. aethiopica, but to facilitate an appropriate focus on the climatological data of the regions where this species is most abundant. Climatological data from the natural habitats of the three species examined varied primarily in temperature during the winter (i.e. , June to July), and in the seasonal distribution of rainfall. During winter, the average mean daily air temperature of all sites of Z. aethiopica (12.2 C) was between 1 and 2 C warmer than that of Z. pentlandii (1 1 . 1 C) and Z. rehmannii (10.6 C, Table 1 .2). The average mean daily temperature in winter, for those sites of Z. aethiopica identified as being cool (10.8 C) , only varied from that of Z. pentlandii and Z. rehmannii by less than half a degree Celsius. However, the winter average for those sites of Z. aethiopica identified as being warm (14.3 C), was between 3 and 4 C warmer than that of Z. pentlandii and Z. rehmannii. Table 1.2 \Average daily maximum, minimum, mean air, and minimum soil (8.00 \am, at 10 cm) , temperatures (C) during the winter (June to July), for ' sites of natural habitat of Zantedeschia aethiopica, Zantedeschia pentlandii and Zantedeschia rehmannii. Z. aethiopica Air warmz coolY allx max. 20.0 19.2 19.5 min. 8.5 2.4 4.9 mean 14.3 \ 10.8 12.2 z data from 10 sites from coastal region. Z. pentlandii Air Soil,.. 20.2 2.9 1 1 . 1 8. 1 '1 data from 14 sites from inland mountainous regions z data from all -24 sites. ? average from 2 sites. v data from 1 site. Z. rehmannii Air Soilv 19.9 2.7 6 . 1 10.6 8 Average daily mean air temperature during summer (i.e. , October to February) for sites where each of the three species grow varied by only half a degree Celsius {Table 1 . 3) . While mean summer temperatures varied little, the diurnal range in temperature was greater for sites where Z. pentlandii, Z. rehmannii, and those of Z. aethiopica identified as being cool, were located (i.e. , up to 14 C). In contrast, those sites of Z. aethiopica identified as being warm exhibited a diurnal range in temperature of only 9.4 C in summer. Similarly, the diurnal range in temperature was also greater for these same sites in winter, with approximately a 17 C range for sites of Z. pentlandii, Z. rehmannii, and those of Z. aethiopica identified as being cool {Table 1 .2). This was in contrast to the 1 1 .5 C range for those sites of Z. aethiopica identified as being warm. Table 1.3 Average daily maximum, minimum? mean air, and minimum soil (8.00 am, at 10 cm), temperatures (C) during the summer (October to February) for sites of natural habitat of Zantedeschia aethiopica, Zantedeschia pentlandii and Zantedeschia rehmannii. Z. aethiopica Air warmz coolY allx max. 25.0 27.8 26.8 min . 15.6 13 .8 14.5 i 20.5 20.8 20.6 mean z data from 10 sites from coastal region. Z. pentlandii Air Soilw 25.4 14.3 20.0 21 1 data from 14 sites from inland mountainous region. "' data from all 24 sites. ? average from 2 sites. v data from 1 site. Z. rehmannii Air Soilv 27. 1 14. 8 17.9 20.4 Differences between species in daily minimum temperature were most evident during winter with sites of Z. pentlandii, Z. rehmannii, and those of Z. aethiopica identified as being cool, averaging 2.7 C {Table 1 .2). This was in comparison to 8.5 C for those sites of Z. aethiopica identified as being warm. Even the average of all sites of Z. aethiopica had a winter daily minimum air temperature at least 2 C warmer than the average for the sites of Z. pentlandii or Z. rehmannii. While data available on soil temperatures was limited to two sites of Z. pentlandii and one of Z. rehmannii, the results at least indicate the temperatures experienced by apical 9 meristems of these species during growth (20 C), and winter quiescence [7 . 1 C) (Table 1 .2 and Table 1 .3). Annual rainfall was predominantly distributed during the summer for sites of Z. pentlandii, Z. rehmannii, and those of Z. aethiopica identified as being cool, compared with being distributed either relatively evenly or predominantly during winter for those sites of Z. aethiopica identified as being warm (data not presented). In summary, Zantedeschia species in group 1 originate from a warm-temperate climatic zone, but also extend into cool-temperate zones. This warm-temperate zone is typified by rainfall in the winter months. In those cases where Z. aethiopica is endemic to regions with summer rainfall, it occupies the habitat of marshy depressions (Letty, 1973; Anon. , 1989). With relatively warm temperatures all year round, plus winter rainfall, it is not surprising that foliage of Z. aethiopica does not die down in winter. In fact, the main period of growth and flowering is during late winter through spring, with no flowering occurring during summer (Letty, 1973). It is suggested that the lack of any pronounced differences in summer air temperatures, between sites of origin of the three species examined, indicates Z. aethiopica exhibits an upper temperature limit in summer, for growth and flowering, which is lower than that of group 2 species. In contrast, species in group 2, such as Z. pentlandii and Z. r(!hmannii, are more restricted in their natural distribution to cool-temperate climates with associated summer rainfall. As wjth other temperate geophytes (Rees, 1972) it is not surprising that species/cultivars 1 in group 2 exhibit a seasonal periodicity which requires a period of endodormancy before ? growth can rec:Ommence (Corr and Widmer, 1988). Thus winter is concomitant with complete foliage senescence, while growth and flowerin? occur during the late spring through summer (Letty, 1973). 1.4 Breeding: goals and specific problems The incidence of bacterial soft-rot (Erwinia carotovora) is one of the most important factors limiting the future commercial viability of this crop. While disease avoidance production programmeS have proven successful, tolerance or resistance to bacterial soft-rot needs to be developed. Z. aethiopica is more tolerant to E. carotovora than selections from group 2 (Long et al. , 1988) . Interspecific hybrids between the two groups may, therefore, result in mmsference of this tolerance to the summer flowering group (group 2) . In addition to breeding for flower colour, high floral productivity must be a primary objective of breeders. Those selections that exhibit minimal apical dominance, and 10 therefore tend to carry a greater number of dominant buds, will be multibranching and produce a greater number of flowers (refer Section 1 .6.2). This multibranching growth habit is readily seen in selections with Z. rehmannii or Z. aethiopica as a parent. The potted plant requirement for "Fullness" is also facilitated by this multibranching growth habit, since those selections with increased shoot number result in an increased density of foliage. High floral productivity is also dependent on the proportion of shoots producing flowers (refer Section 1 .6.2). With regard to this factor, it is uncertain whether or not genetic differences exist, but breeding for a growth habit that ensured that a high proportion of shoots flowered, especially after prolonged storage (refer Section 1 .6.4. 1) would be beneficial. In addition to high floral productivity, the ability to schedule year? round flowering is desirable. With selections classified as group 1 flowering naturally in winter/spring, and those in group 2 flowering in summer, breeders are endeavouring to achieve interspecific hybrids that will produce a greater range of flowering times and durations. With reduced international acceptance of the use of agricultural chemicals, the development of selections with a natural dwarf growing habit has been given a high priority by breeders. In contrast, the current availability of a wide range of peduncle lengths from different selections does not present a problem for breeders focusing on cut flower or bedding plant production. Spathe reg?eening is a primary determinant limiting the display life of selections in group 2 (Funnell \and Downs, 1987). Selections vary in the rate of regreening (Funnell and I Downs, 1987), and breeders are now utilizing this knowledge in developing improved selections with slow rates of spathe regreening. While the spathe also regreens in selections of Z. aethiopica (Pais and Chaves das Neves, 1982/?3), the decline in quality in this specie is first seen as a wilting of the spathe (I'jia and Funnell, 1986; Plummer et al. , 1990). Little is known about market preferences for leaf and flower shape, or leaf maculation, but wide variations in these morphological features are possible (Shibuya, 1956; Harrison, 1972; Letty, 1973). Fragrance is another characteristic that, while not considered of high priority, may offer a new dimension to consumer acceptance of the product. Z. odorata (Perry, 1989) and selections of Z. aethiopica (Letty, 1973) are fragrant. The fragrance is somewhat like that of freesias, but subtle. Research is required to determine market preference for these characteristics before breeders can set new goals . . Interspeeific hybrids are readily achieved between species within group 2. However, because of incompatibility, no successful crosses have been achieved between species in 1 1 group 1 and 2 (Traubl_ 1948; Letty, 1973; Chi, 1990). While embryo culture techniques have enabled the fertilized embryos of these incompatible crosses to be grown in vitro, the resulting plants were albino (Chi, 1990; Jialong and Cohen, 1991). The techniques of embryo culture have also been utilized to produce triploid and tetraploid selections (Jialong and Cohen, 1991). The normal chromosome number of species of Zantedeschia is n = 16 (Earl, 1957). 1.5 Vegetative growth and development 1.5.1 General overview Following tuber planting, tuber dry weight initially declines while leaf development commences (Kobayashi et al. , 1978; Funnell and MacKay, 1987; Warrington and Southward, 1989). As further leaf development continues, tuber dry weight commences to increase following a sigmoidal pattern of growth. While it is uncertain what control mechanisms are exerted over vegetative growth in Zantedeschia, preliminary studies point to the importance of leaf area development and its duration (Funnell and MacKay, 1987; Warrington and Southward, 1989). 1.5.2 Influence or internal factors 1.5.2.1 I>ornmancy Whil? a popular viewpoint is that selections in group 1 do_ not exhibit endodormancy, no published reports are evident to substantiate this. Leaf production of the group 1 selection 'Childsiana' does cease in summer (Sakanishi, 1955), but buds on harvested rhizomes readily resume growth under controlled conditions (Welsh et al. , 1988; Plummer, 1990). Bud endodormancy during winter has been reported for selections in group 2 (Corr and Widmer, 1988). Just when bud endodormancy commences is unknown, but the cessation of leaf development occurred chronologically earlier as the temperature of growth increased up to 24 C (unpublished observations). The cessation of further leaf development was not as a result of the exhaustion of a limited number of preformed leaves present at the time of planting. Dissection of primary shoots of the group 2 selections 'Galaxy' and 'Pink Petticoat: once endodormant, revealed an average of 10 ? 1 primordial structures, with --. the most developed primordium carrying senesced leaf lamina. Few investigations into the influence of tuber storage temperatures have studied the phenomenon of bud endodormancy. Nearly all experiments have utilized tubers with buds 12 ? ?a non-dormant state, and have, therefore, often unknowingly investigated the amount of growth and development that has occurred while tubers were held dry. Changes in endogenous growth regulator concentration and activity, which might be expected to be associated with bud endodormancy, have not been reported. Endodorrnant buds on tubers of Z. rehmannii and Z. elliottiana have been induced to develop by either withholding water for 45 days from plants in leaf, or by lifting and storing tubers for 42 days at 22 C (Corr and Widmer, 1988). Plants replanted after. having their foliage mechanically removed did not emerge until they also were dry stored, confirming that the buds were endodorrnant at the commencement of the treatment. However, the induction of bud ecodorrnancy by withholding water for 56 days, commencing when shoots of the group 2 selection 'Chromatella' were still in rapid growth, did not result in rapid and uniform bud growth (Funnell and MacKay, 1989b). When subsequently stored at 10 C for 84 days bud growth was found to be rapid and uniform. In both of the aforementioned studies no developing shoots had flowered after 100 days following replanting. Clearly the topic of bud eco- and endodormancy requires further investigation to assist with the development of rapid programming of tubers for forcing. 1.5.3 Influence of external factors 1.5.3.1 Temperature I While detailed analyses of growth response to temperature have not been reported for Zantedeschia, growth of plants of the group 1 selection 'Childsiana' was severely restricted ? when grown at 12 C (Halligan and Warrington, personal communication) . While leaf differentiation continued at 28 C, leaf area expansion was restricted and the duration of leaf area was reduced, when compared to plants grown at 20 C. To extend the flowering season, cooling is typically required in summer (Sakanishi, 1955). Similarly for group 2 selections, a complete temperature response curve for growth has not previously been reported. Research carried out on group 2 selections primarily pertains to flowering and will therefore be dealt with in Section 1 .6.4 . 1 . However preliminary studies indicated that leaf area development and subsequent tuber growth commenced earlier and at greater rates with increasing temperature up to 25 C (Warrington and Southward, 1989). The time interval between planting and commencement of net tuber growth was approximately 60 days at 25 C compared with 140 days at 13 C. 1 3 1.5.3.2 Light Tuber growth occurs year-round and is, therefore, presumed to be independent of day length (personal observations). Detailed analyses of vegetative growth and development of selections of Zantedeschia in response to photosynthetic photon flux (PPF) regimes have not been reported. However a preliminary study using the group 1 selection 'Childsiana' indicated that a 12 h day at a PPF regime higher than 450 ?-tmol?m-2?s-1 resulted in chlorotic foliage regardless of temperature (White and Halligan, personal communication). In contrast, tuber growth commenced earlier and tuber size was greater when group 2 selections were grown under a 12 h day at higher (700 ?-tmol?m-2?s-1) compared with lower (350 ?-tmol?m-2?s-1) PPF (Warrington and Southward , 1989) . In addition, leaf area expansion and duration were greater under the low PPF regime. ? 1 .5.3.3 Chemical growth regulators While the preplant application of gibberellic acid (50 mg-litre-1 GA3) to tubers resulted in an increased proportion of buds emerging as shoots, total plant leaf area, leaf number and tuber size were reduced (Funnell and MacKay, 1987) . The subsequent removal of flowers did not result in any difference in final tuber size, indicating that flowers were not a significant competitive sink for photoassimilates. The preplant application of GA4+7 between 2 and 8 mg?li tre?? also resulted in reduced tuber size (Funnell and ?acKay, \ 1988?) . Neither the preplant appl ication o f benzyl ( lH-purin-6-yl) amine (BA) between 0 and 7 .2 mg?Iitre?? , nor ethylene (500 ?-tl?litre-1 at 20 C for 24_ h) , resulted in any alteration of vegetative growth or development (Funnell and MacKay, 1988a and 1988b) . Since the primary influence of chemical growth retardants is on height control, their influence will be discussed in Section 1 . 6.4 .4 . 1 .5.3.4 Growing medium, irrigation, nutrition and weed control Growth and development of Zantedeschia is believed to be optimal in a free draining growing medium with pH near 6.0. While tolerance ranges for growing medium moisture content and aeration have not been quantified, it is popularly believed that Z. aethiopica is tolerant of wet, poorly aerated. soils. However, while being tolerant, growth is not necessarily optimal under such conditions. Inadequate irrigation regimes can result in reduced leaf area development (Sakanishi, 1955) . With leaf area development being a 14 potentially important determinant of plant and tuber growth (refer Section 1 .5 .3), water stress must be avoided if growth is to be maximized. Nutrient requirements are closely correlated with the pattern of growth, with the greatest rates of uptake occurring between six to 12 weeks after field planting in summer (Clark and Boldingh, 1991) . In anticipation of this growth, initial recommendations for broadcast application rates are 300 kg?ha-1 N, 45 kg?ha-1 P and 400 kg?ha-1 K (Clark and Boldingh, 1991). Applications of the following residual herbicides, prior to crop emergence, have given the most satisfactory weed control while being tolerated by Zantedeschia: terbumeton/terbuthylazine (3 kg?ha-1) , simazine (2 kg?ha-1), and oxadiazon (1.5 kg?ha-1) (Ingle and Bussell, 1991). Methabenzthiazuron (2 kg?ha-1), metribuzin (0.6 kg?ha-1), and prometryn (1 .6 kg?ha-1) have been successfully applied after emergence without causing plant damage. 1.5.4 Commercial rhizome and tuber production 1.5.4.1 Goals and techniques The primary goal of rhizome and tuber growers is to produce, in as short a period of time as practical, rhizomes or tubers that will provide multiple flowered, quality plants, that can be scheduled for production for given market periods. \ ; ' Commercially, multiplication is achieved using three main systems, i.e. , seed, offsets/division, and tissue culture. Only five selections are known to be commercially produced from seed , viz. the species Z. rehmannii, Z. elliottiana, Z. albomaculata, and the group 2 selections 'Chromatella' and 'Best Gold? However only 'Best Gold' grows true-to-type from seed. Annual multiplication through offsets and division of flowering sized tubers (refer Section 1 .6.3 . 1) can increase the number of flowering sized tubers, and total tuber number by over 100% and 300% , respectively. While this may be adequate once stock numbers are high, tissue culture techniques have been developed to provide large scale bulk-up rates, especially of virus tested lines (Cohen, 1981 ; Rong et al. , 1989; Anon. , 1990) . Multiplication may also be achieved by scooping dominant buds with some tuber material attached, and subsequently treating this as a cutting (Clark et al. , 1987; Funnell and MacKay, 1989a). When non-flowering sized material is replanted for annual replacement, increases in tuber dry weight are frequently in excess of 400% per annum (Funnell and MacKay, 1987). Selections of Z. aethiopica normally produce between 25 to 30 rhizomatous offsets after 6 months' growth (Zizzo, 1987; Welsh et al. , 1988). 15 Under optimum growth conditions in controlled environment facilities, natural flowering plants of Z. aethiopica can be obtained in 9 months from seed (Cohen, personal communication) , but under normal field conditions the production of flowering sized tubers of all selections takes two to three years. Depending on selection, 90% of 1 to 2 cm diameter tubers, replanted for annual tuber replacement under optimum New Zealand field conditions, will reach a flowering size by the end of one growing season (Funnell and MacKay, 1987) . With tuber producers endeavouring to supply flowering sized tubers of new selections to the market place as quickly as possible, this two to three year period of cultivation is seen as being undesirably long. The need for repeated periods of cultivation, tuber lifting and storage are associated with increased costs of production which must be passed onto the market place. Hence investigations into means of reducing this time interval may provide tuber producers with the potential to reduce the costs of production. Environmental control of cultural factors such as temperature (refer Section 1 .5 .3 . 1) , light intensity (refer Section 1 .5 .3 .2), nutrition, and irrigation regimes (refer Section 1 .5 .3 .4) are used to maximise tuber yield. While the majority of tuber production utilizes natural mineral soils, the need to have greater control over principle environmental factors and disease (refer Section 1 . 7) has resulted in increasing production utilizing soilless growing media. However, as noted in the aforementioned sections, detailed analyses of vegetative growth and development in response to these environmental parameters have not been reported. \ 1.5.4.2 Planting to harvest requirements Seed, offsets/divisions, and tubers for .annual replacement are sown in spring in an insect, disease and weed free area. With growth potential varying among selections and environments a median spacing for seed is 5 cm square. Small non-flowering tubers, which will be lifted after one year, are spaced at 10 cm square. Tissue cultured plants are ex-flasked into containers of soilless growing media at a 3.5 cm square spacing. Under controlled environmental conditions (refer Section 1 .5 .3) , ex-flasking can be carried out year-round. Flowering sized tubers, which will be lifted after two or more years, are spaced at 30 cm square. When replanting for annual replacement, tubers of Z. elliottiana inverted at planting produced tubers of a distorted shape (Clark et al. , 1987) . Thus, if tuber shape is considered to be an important quality criterion, tubers should not be inverted at planting. Without any evident period of endodormancy (refer Section 1 .5 .2. 1 ) , it is believed that lifting and division of plants in group 1 can be carried out at any time of the year. 16 However, since the natural season of growth and flowering is during the winter and spring months, this activity is generally carried out during late summer/autumn. While plants from this group may be lifted and divided for immediate replanting, plants may also be dried for a month to facilitate handling as dry rhizomes (Welsh et al. , 1988). To achieve maximum tuber size, harvesting of tubers from selections in group 2 is not carried out until the foliage has senesced. Except where plants have been artificially dried down (refer Section 1 .5.2. 1), tubers at this stage of development will have had their endodormancy requirement satisfied. Under New Zealand conditions, buds on tubers remaining in the ground after endodormancy produce minimal growth because of the cool autumn/winter soil temperatures that prevail naturally. 1.5.4.3 Postharvest storage and transport requirements It is popularly believed that actively growing offsets/divisions of selections in group 1 do not require curing or dormancy-breakage treatments. Replanting can be carried out once foliage and roots have been trimmed and plants treated with fungicide. If not immediately replanted plants may be coolstored in a slightly moist, free-draining growing medium until planted. Alternatively, rhizomes lifted and divided from plants which have been dried down, can be graded, treated in a fungicide solution, and cured at 1 8 C until remaining roots and leaf bases senesce (Welsh et al. , 1988). Rhizomes are stored dry at 10 C until replanted (Sakanishi, 1955). Once lifted and treated in a fungicide/bactericide solution, tubers of selections in group 2 ' are cured at 20 to 30 C and high relative humidity (i.e. , 80% or greater), for a minimum of seven days, to enhance the rate of suberin and wound periderm deposition (Funnell et al. , 1987). The extent of tuber weight loss, and the weight of tissue lost due to ?chalking; was reduced under high (80%) compared with low (40%) humidity (Funnell and ?cKay, 1988c). This ?chalking: i.e. , the formation of a layer of white coloured dead tissue on cut tuber surfaces, was the result of desiccation and death of cells before an adequate barrier of suberin and wound periderm could be formed. To suppress shoot growth, non-dormant tubers of group 2 . selections must be stored at 7 ? 3 C (Funnell and MacKay, 1988b). Exposure to ethylene (500 l'llitre?1 at 20 C for 24 h) prior to storage or planting did not alter growth (Funnell and MacKay, 1988b), hence during transport mixed container consignments with ethylene-producing materials should not cause any .p roblems. 17 1.6 Control of flowering 1.6.1 General overview With control over tuber availability, and over both the storage and forcing environments, Zantedeschia may be flowered year-round. Floral differentiation occurs under conditions suitable for vegetative development (refer Section 1 . 6.2) . Therefore, the potential for flowering exists as long as development continues. Providing that a minimum growth temperature (refer Section 1 .6.4 . 1) is met by the use of protected cultivation, the primary limitation to successfully achieving year-round flowering of selections in group 1 appears to be the cessation of flowering during the heat of summer (refer Section 1 . 3 .2) . The growth and flowering of selections in group 2 are not limited by exposure to these same summer temperatures, and, therefore, are readily programmed for forcing. 1.6.2 Flowering process and terminology Flowering sized tubers (refer Section 1 .6 .3 . 1) have buds with a range of flowering potentials. Dominant buds are those that flower when placed under "normal" cultural conditions (refer Section 1 .6.4) . These dominant buds are physically swollen and encircled by a number of axillary buds arranged spirally on the tuber (Plate 1 .2) . Plate 1.2 Flowering sized tuber of a group 2 selection indicating examples of (a) dominant bud, (b) developed axillary bud, and (c) undeveloped axillary bud. ? 18 Axillary buds are smaller in size than dominant buds, and are not encircled by smaller buds. The growth and flowering potential of axillary buds may be further classlfied as; (1) Developed - Those axillary buds that are physically swollen and are readily induced to flower with the application of gibberellins. (2) Undeveloped - Those axillary buds that are not swollen and are not readily induced to grow and flower, even with application of gibberellins. The growth habit of Zantedeschia is sympodial, with dominant buds on tubers initially emerging to produce primary shoots (Figure 1 .2) (Funnell and MacKay, 1988a). Above ground, each of these primary shoots typically present two to three protective sheath leaves followed by two leaves below the flower (Kobayashi et al. , 1977; Funnell and MacKay, 1988a; Funnell et al. , 1992). Additional flowers may also develop from apical meristems of secondary shoots, stimulated into growth 'from primordia located in the leaf axils of the primary shoot (Figure 1 .3). This readily discernable developmental pattern, of secondary shoots arising from primary, may continue with tertiary shoots arising from the leaf axils of secondary shoots ad injinitwn until endodormancy ensues. Secondary and tertiary shoots carry one bract and between zero and two leaves below the flower. Floral differentiation commences once vegetative growth has commenced (l'akahashi et al. , 1957; Kobayashi et al. , 1978). Therefore, unless planting is delayed, flowers are typically not differentiated at planting. The sequential differentiation of the components of the flower has been described for selections in group 1 (l'akahashi et al. , 1957) and group 2 (Kobayashi et al. , 1978), with no differences being evident. Corr (1988) confirmed that the spathe is 1 ,?e first component of the flower to differentiate. Therefore, total flower productivity per tuber is a function of: (1) the number of buds that are stimulated to grow; (2) the number of primary shoots that subsequently flower; (3) the number of secondary and/or tertiary shoots that flower (refer Sections 1 .6.4. 1 and 1 .6.4.4) . Hence, flowering sized tubers of selections derived from Z. elliottiana, for example, typically carrying only one dominant bud, will have an inherently lower flowering potential than tubers of similar diameter derived from Z. rehmannii, which typically carry four or more dominant buds (Funnell et al. , 1988). primary shoot axillary bud primary ? flower leaf tuber F1gure 1.2 Diagrammatic illustration of sympodial growth habit of a single primary shoot of Zantedeschia. primary flower from primary shoot __. secondary flower fro? axillary shoot lower leaf? tuber---? secondary ? flower from axillary shoot Figure 1.3 Diagrammatic illustration of primary shoot of Zantedeschia, with secondary shoots arising from leaf axils. 19 1.6.3 Influence of internal factors 1.6.3.1 Rhizome and tuber size 20 Rhizome size of the group 1 selection 'Childsiana' influences the growth of flowers and foliage, with smaller rhizomes producing plants with reduced height, reduced number and size of leaves, and reduced flower size (Welsh et al. , 1988) . While a minimum rhizome size to achieve flowering for group 1 selections has not been reported, the commercial use of the saleable product dictates the size of rhizome selected. A maximum production of 18 flowers per plant was recorded for rhizomes of Z. aethiopica weighing between 201 to 250 g fresh weight (Zizzo, 1987) . Rhizomes of both smaller (50 to 200 g) , and larger (251 to 300 g) weight, produced fewer flowers. While varying between selections, a 2.5 cm tuber diameter is necessary to ensure 100% natural flowering of group 2 selections not exposed to extended periods of storage (Corr and Widmer, 1991). Tubers of a smaller size are readily induced to flower by the application of gibberellins (Cohen, 1981 ; Funnell and Go, 1993) , but foliage and flower size are smaller than that achieved with larger sized tubers. Again, the commercial use of the saleable product dictates the tuber size selected. 1.6.3.2 Dormancy and floral induction Dormant buds of group 2 selections induced to grow, either by withholding water (45 days) or manipulating temperature in storage (42 days at 22 C or 84 days at 10 C), did not ' flower (refer Section 1 .5.2. 1) (Corr and Widmer, 1988; Funnell and MacKay, 1989b; Corr and Widmer, 1991). In addition, buds on tubers of Z. rehmannii which were lifted from the field in California, U.S.A. during early September, emerged _within 41 days, but did not flower over a subsequent 150 day growing period (Corr, 1988) . However storage of these tubers at 15 C for a minimum of 21 days resulted in flowering, with the number of flowers per plant increasing with longer periods of storage. While the emergence of shoots from non-stored tubers indicated that the buds were not dormant, the inability of plants from these tubers to flower unless tubers were stored for 21 days, indicates that something other than the presence of vegetative growth is needed to achieve flowering. It is possible that the level of bud ecodormancy induced by artificially drying the plants may be very different from the endodormancy encountered under normal cultural conditions. Clearly the topics of bud eco- and endodormancy, and their association with floral induction, requires further investigation to assist with the development of programming tubers for forcing. 1.6.4 Innuence of external factors 1.6.4.1 Temperature 21 As noted in Section 1 .6.2, differentiation of flower parts does not commence until shoot growth has begun. Shoot growth occurring during tuber storage or forcing, at a range of temperatures, did not result in any alteration of the sequence of floral differentiation (Funnell and MacKay, 1988b; Corr, 1988). Therefore, it is assumed that as long as the requirements for minimum rhizome or tuber size (refer Section 1 .6.3. 1) and endodormancy (refer Section 1.6.3.2) have been satisfied, temperatures conducive to vegetative growth are also conducive to floral differentiation. However, while the growing temperature does not result in differences in flower productivity of selections in group 2 (Warrington and Southward, 1989), flowering of the group 1 selection 'Childsiana' was inhibited at 28 C (Halligan and Warrington, personal communication). Plant growth is faster at increasing temperatures, and flower development is also accelerated (Post, 1936). Plants of the group 2 selection 'Galaxy' grown in a controlled environment temperature regime of28/22 C day/night, reached first flowering 57 ? 1 days after planting, but this increased to 80 ? 2 days when grown at 22/16 C, and to 140 ? 3 days at 16/10 C (Warrington and Southward, 1989). When grown under controlled conditions in the greenhouse (minimum air temperature'" of 15 C and ventilation at 20 C), comparable results were obtained with an average 70 days to ilower for four selections in group. 2 (MacKay et al. , 1991). Selections in group 1 responded similarly, with plants of I 'Childsiana' taking 88 days to flower at 20 C (White and Halligan, personal comm?nication) . . Basal heating at temperatures above ambient air temperatures can reduce the time to flowering (Corr and Widmer, 1990). In addition to reducing time to flower, increasing temperatures increased total plant height and peduncle length. Warrington and Southward (1989) reported a 13% increase in peduncle length when the daily mean temperature was increased from 19 to 25 C. The use of night temperatures which were higher than day temperatures did not result in significant differences in total plant height or peduncle length compared with use of the reversed day/night regime, or with constant mean temperatures (Reiser and Langhans, 1993). Hence the application of the technology of programmed diurnal temperature control, i.e. , DIF (Karlsson et al. , 1989), may not provide a satisfactory means to reduce plant height. ?- The effects of storage temperature on non-dormant rhizomes of selections in group 1 have not been extensively studied. Sakanishi (1955) determined that if storage was required, subsequent growth and flowering of Z. aethiopica 'Childsiana' was optimal after storage 22 of plants in leaf at 10 C. 'Y!:elsh et al. (1988) reported increased leaf production of 'Childsiana' if rhizomes were dried, and cured at 18 C compared with immediate replanting of offsets/ divisions in leaf. However, no resultant increase in flowenng was reported. With selections in group 2 a storage temperature of 7 ? 3 C is recommended to inhibit shoot growth (refer Section 1 .5.4.3). Inhibition of shoot growth is important if physical damage during transport is to be minimized . However, a reduced time to flower can be achieved by storage at warmer temperatures and/ or with increasing duration of storage (Tjia, 1987a; Funnell and MacKay, 1988b). The earlier flowering was attributed to shoot growth and subsequent floral differentiation in storage. Further research is required to develop combinations of cool and warm storage into commercial scheduling programmes. In addition to influencing the time to flower, short-term storage ofnon-endodormant tubers at fixed temperatures ranging between 8 and 15 C, increased the proportion of buds emerging as shoots (Corr and Widmer, 1988; Funnell and MacKay, 1988b). In contrast, tubers stored at the higher temperatures of 22 or 25 C did not respond similarly. The rapid establishment of apical dominance (i.e. , paradormancy) at the high temperatures resulted in the reduced number of buds developing into shoots. The resultant proportion of buds at planting that subsequently flowered was also increased after storage at these cooler temperatures with the optimum proportion occurring after 10 weeks at 8 C or 4 weeks at 15 C. After consideration of the unknown comparative e?dodormancy status, this result is n?t too dissimilar to that reported by Corr (1988), where total flower number was greatest after 12 weeks storage at 15 C. \ \ While storage of non-endodormant tubers permits scheduled planting programs, the flowering potential of these tubers is reduced compared with non-endodormant tubers planted without any period of storage (Funnell et al. , 1988). The proportion of tubers of Z. elliottiana which flowered was progressively reduced with increased storage temperature up to 24 C, but not with the Z. rehmannii-like selection 'Pink Satin'. The maintenance of the proportion of tubers of 'Pink Satin' flowering, with increased storage temperature, reflects the greater number of dominant buds per tuber, and therefore greater potential floral initiation sites (refer Section 1 .6.2). Tubers of Z. elliottiana used in this experiment typically carried only one domiriant bud compared with the four or five dominant buds on tubers of 'Pink Satin' . It is unlikely that this decline in flowering resulted from chilling injury or abortion of the apex, since the reduction in flowering occurred regardless of storage temperature, viz. 5, 12, 18, or 24 C (Funnell et al. , 1988) . Also, dissection of buds did not reveal abortion of apices (Funnell and Go, 1993). While the actual m?hanisms involved in this reduction in flowering potential have not been determined, a preplant application of gibberellin (GA3 or GA,.+7) is able to compensate almost totally for 23 the reduction in flowering potential (refer Section 1 .6.4.4) (Funnell et al. , 1988; Funnell and Go, 1993). In addition, storage of tubers in moist media partially alleviated the reduction in flowering potential associated with storage, but was not as effective as the application of gibberellin (Funnell et al. , 1988). Not only did increased temperatures during tuber storage result in a reduction in flowering potential, but increasing duration of storage also resulted in a reduction in flower number (Funnell et al. , 1988; Funnell and MacKay, 1990). While the preplant application of gibberellins was able to maintain commercially acceptable floral productivity for up to 9 months storage, the rapid increase in plant death with storage beyond 5 months, identifies the need for further research into using extended periods of tuber storage to schedule flowering (Funnell and MacKay, 1990). 1.6.4.2 Light Flowering of Zantedeschia is not dependent on photoperiod (Greene et al. , 1932; Corr and Widmer, 1990). However, under an 8 h photoperiod, the addition of a 4 h night interruption of non photosynthetically active radiation, increased plant height of Z. elliottiana by 40% and flower peduncle length by 23% (Corr and Widmer, 1990). The influence of photoperiod on peduncle length was highly dependent on selection, since the same treatment resulted in a 95% increase with z. rehmannii, wa.rnAgton and Southward (1989) reported a 26% increase in peduncle length from plants I of the' Z. rehmannii-like selection 'Galaxy' with a . 50% reduction in PPF ? (700 to 350 #'molm?2?s-1) . These environments resulted in total daily integrals of light of 30 and 15 mol?m?Z, and are similar to that intercepted during cl? skies in summer and winter (Latitude 45 oS) , respectively. Other researchers have reported increases in peduncle length and total plant height, as a result of reduced PPF, ranging between 13% and 87% depending on growing environment and selection (MacKay et al. , 1987b; Corr and Widmer, 1990). While differential responses of selections may account for the preceding variation in results, a more pronounced increase in peduncle length with reduced PPF resulted when plants were grown under cooler temperatures (MacKay et al. , 1987b; Warrington and Southward, 1989). While Corr and Widll).?r (1990) reported no reduction in total flower production, as a result of a PPF reduction by 45% or 15% of normal summer conditions, Warrington and Southward (1989) reported a 40% reduction in flower number with a reduction in PPF from 700 to 350 #'moi-m?2?s?1? Although Corr and Widmer (1990) did not report total daily integrals of light under each treatment environment, their treatments would have 24 encompassed the range reported by Warrington and Southward (1989). Clearly, further research is required to clarify this apparent contradiction in response to PPF and/or daily light integral. The time to flowering was not influenced by PPF regime (MacKay et al. , 1987b; Warrington and Southward, 1989; Corr and Widmer, 1990). As noted in Section 1 .5.3.2, foliage of the group 1 selection 'Childsiana' became chlorotic when grown under a 12 h PPF regime greater than 450 pmolm-2?s?1 (White and Halligan, personal communication). 1.6.4.3 Nutrition The proportion of plants flowering was optimal when liquid fertilizer (15N-7P-14K) was applied twice weekly at 100 or 200 ppm N, but was reduced by 40% when increased to a rate of 400 ppm N (MacKay et al. , 1987a). As a result of salinity damage, the incidence of plant death was increased at the higher rate. Using a slow release fertilizer (19N-2.6P-10K) at rates of 2 g?litre?? and 4 g?litre?! provided optimal flowering in peat and bark-based growing mediums, respectively (Dennis, 1986). In view of this preceding information, ? crop nutrition during forcing does not appear to influence flowering unless fertilizing at excessively high rates, or if moisture is inadequate. \ 1.6.4.4 Chemical growth regulators Application of gibberellin (GA3 or GAH7) promotes flowering in Zantedeschia selections of both groups (Corr and Widmer, 1987; Plummer, 1990), and Z. odorata (unpublished observations) . Corr and Widmer (1987) reported optimal flowering after applying 500 mg?litre?? GA3 as a preplant tuber immersion. However Funnell and Go (1993) illustrated that maintenance of floral productivity after increasing periods of tuber storage required an increased concentration of gibberellin. This requirement reflects the decline in flowering potential with tuber storage duration (refer Section 1 . 6.4 . 1) . Further research is required to provide recommended rates of gibberellin application after removal from storage at progressively increasing periods. Treatment with gibberellins prior to storage at 9 C did not increase flower number beyond one months' storage (Funnell and MacKay, 1990) . It is important, thC??efore, if cool-storage is to be used, that tubers are treated after removal from storage. The duration of tuber immersion in gibberellin, ranging from 10 seconds to 30 minutes, did not result in differences in flower number (Funnell et al. , _1992). Therefore, it is not surprising that spray applications of gibberellins to the tuber have also been effective (Funnell et al. , 1988). Kobayashi (1980) showed that the 25 temperature of the gibberellin solution in which the tubers are immersed, viz. 5, 15, 25 and 35 C, did not influence flowering. Both GA3 and GA.., are effective, but GA.+7 was found to be more effective when applied at the same concentration as GA3 (Funnell and MacKay, 1988a). Both sources of gibberellin increase flower production through an increase in the number of buds emerging as primary shoots, in addition to an increase in the proportion of shoots flowering. In New ?land, a label recommendation exists for the pre-plant tuber immersion in GA.+7 (i.e. , Promalin: Abbott Lab., N. Chicago, n.. 60064) at a rate of 8 mg?litre-1 for 1 minute. However, while 100% of dominant buds flowered when following current label recommendations, only 30% of all buds (i.e. , dominant + axillary), and 60% of shoots flowered (Funnell and MacKay, 1988a). Clearly, this indicates that further potential flowering sites exist. The BA com?nent of Promalin did not influence flower production (Funnell and MacKay, 1988a). Foliar application of GA3 at rates of 50 and 150 mg?litre-1 increased flower production by three-and five-fold, respectively (Kobayashi, 1977a). The combination of a preplant tuber immersion (100 ppm GA3) and subsequent foliar application (100 mg?litre-1 GA3) resulted in a seven-fold increase in total flower number. In contrast, when only a preplant tuber immersion was used a five-fold increase was obtained (Kobayashi, 1977b & 1977c). Most . selections of Zantedeschia suitable for potted plant ' production require growth retar4tion to control plant height. Tjia (1987c) reported that the application of the chemi? growth retardant paclobutrazol, as a growing medium drench at 4 mg/pot, resulted in plants with aesthetic proportionality. Ancymidol was not found to be effective (Tjia, 1987c), but uniconazole at 4 mg/pot resulted in similar results to that of paclobutrazol (Reiser, 1990). The application of paclobutrazol as a preplant tuber immersion of 80 mglitre-1 for 24 h was more effective in reducing height than that achieved with the 4 mg/pot growing medium drench (MacKay et al. , 1986). When paclobutrazol was applied as a preplant tuber immersion, foliage height, flower peduncle and spathe length were reduced by 40% , 56% and 10% , respectively. Regardless of which growth retardant is used, the rate of application to achieve aesthetic proportionality will be dependent both on selection and on the forcing conditions (refer Sections 1 .6.4. 1 . and 1 .6.4.2) . The application of paclobutrazol or uniconazole also reduces the number of flowers produced, with the application as a preplant tuber immersion reducing flowers more than that if applied as a growing medium drench (Corr, 1988; Reiser, 1990). While the application of gibberellic acid partially alleviated the reduction in flowering, higher concentrations of gibberellic acid were required to achieve the same floral productivity when a growth retardant was applied (Corr, 1988). 26 1.6.4.5 Air pollutants In contrast to bulbous crops such as tulip and iris (Kamerbeek and De Munk, 1976), growth and flowering of Zantedeschia was not affected by exposure of non-dormant tubers to ethylene (500 ?Lllitre?? at 20 C for 24 h) either prior to storage or planting (Funnell and MacKay, 1988b). 1.6.5 Commercial forcing for pot and cut flower production Information presented in this Section covers aspects of commercial pot and cut flower forcing not already covered above. 1.6.5.1 Rhizome and tuber storage Optimum flower productivity is obtained when non-endodormant rhizomes or tubers are planted without storage (refer Section 1 .6.4. 1). While lifting of rhizomes of selections in group 1 may be carried out at any time of year, rhizomes are normally available from the southern hemisphere in December through February, and the northern hemisphere in June through August. Application of current technology allows tubers of selections in group 2 to be stored for up to 5 months after breakage of endodormancy and lifting (refer Section 1 .6.4. 1). In New Zealand, tubers are lifted in May through July and processed prior to shipping in June through December. In the northern hemisp?ere tubers are lifted in October tru;ough November and processed prior to shipping in January through April. Therefore, po? plant and cut flower forcers can expect to store tubers for a further one to four months, as dictated by lifting date. 1.6.5.2 Pre-plant treatments Between arrival and planting, tubers should be dipped in a fungicide/bactericide solution. Tubers should be allowed to air dry before further handling. The use of disease-free planting material and proper sanitation practices are of prime importance in disease control (refer Section 1 . 7) . Flowering of Zantedeschia may be improved by over 400% by the application of gibberellins (refer Section 1 .6.4.4). While a label recommendation exists in New Zealand for a pre-plant tuber immersion in GA-'+7 (i.e. , Promalin: Abbott Lab., N. Chicago, n.. 60064) at a rate of 8 mg?litre?? for 1 minute, application of higher concentrations of both GA3 and Promalin have increased flowering, and may be a requirement to maintain satisfactory floral productivity with increased periods of tuber storage (refer Section 1 .6.4. 1), or if chemical growth retardants are used (refer 1 .6.4.4). 27 1.6.5.3 Planting and nutrition Satisfactory rhizome or tuber size for flowering varies with selection and desired use (refer Section 1 .6.3 . 1). For pot plant usage of group 1 selections, generally only a single rhizome of a minimum diameter of 1 .5 cm and several offsets of 0.5 to 1 cm in diameter are placed in a 0. 7 litre pot. For selections in group 2 suitable for pot production, a single tuber measuring 4 to 5 cm in diameter with at least three dominant buds (refer Section 1 .6.2) is considered suitable for a 15 cm pot (approximately 1 .5 litre volume). For a 10 cm pot, a single tuber measuring 3 to 4 cm in diameter with three dominant buds is considered suitable. Since flower size varies with rhizome or tuber size (refer Section 1 .6.3. 1), the selection of rhizome or tuber size for cut flower production is dictated by market preferences. However, for group 2 selections, a minimum tuber diameter of 4 cm is generally preferred. Cut flower producers using tubers for forcing should also seek a high number of dominant buds per tuber (refer Section 1 .6.2). Unlike most other bulbous crops, roots of Zantedeschia emerge from around the emerging shoots at the top of the tuber. Therefore, it is important when planting that the top of the tuber is covered with 2 to 3 cm of medium to avoid roots drying out. Tubers should be planted upright with minimal damage to buds. Planting tubers upside down delays flowering and results in poor quality plants (Clark et al. , 1987). Media and nutrition requirements have been discussed above in Sections 1 .5.3.4 and 1 .6.4.3. \ ' 1.6.5.4 Height control Some form of height control is often required in the production of Zantedeschia as pot plants. Paclobutrazol has been found to be an effective-growth retardant. It can be applied as a growing medium drench at a rate of 2 to 4 mg/pot at shoot emergence. The precise rate depends on the selection and the forcing environment conditions (1 .7.4. 1 and 1 . 7 .4.2). For crops in 15 and 20 cm pots, the recommended volume of diluted paclobutrazol to apply is 120 and 180 ml, respectively. Root confinement appears to provide adequate height control of the group 1 selection 'Childsiana' obviating the need to use chemical growth retardants for pot production (unpublished observations). ? 28 1.6.5.5 Forcing environment A day/night temperature regime of 20/15 C to 25/1 8 C is suitable for growth (refer Section 1 . 6.4. 1) . An 88-day production period should be used when initially planning crop schedules for group 1 selections, while 70 days should be used for group 2 selections. Forcers may find that under their particular cultural conditions (refer Sections 1 .6.4 . 1 and 1 . 6.4 .2) and depending on prior storage history of the tubers ( 1 .6.4. 1) , modifications may be required to these schedules. While under controlled environment conditions plants of group 1 selection 'Childsiana' produce a higher quality plant if grown under a PPF near 450 J.Lmol"m?2?s?1 (daily light integral of 19 mol?m-2) , group 2 selections produce a higher quality pot plant if grown under a minimum PPF of 700 J.Lmol?m?2?s?1 (daily l ight integral of 30 mol?m-2) (refer Section 1 . 6.4 .2). Increases in peduncle length as a result of manipulation of the environment (refer Sections 1 .6 .4. 1 and 1 . 6.4 .2) may be desirable for cut flower production, but pot forcers must give consideration to minimum light intensities, photoperiod and temperature if excessive plant height is to be avoided. The use of chemical growth retardants to assist control of plant height was discussed above (refer Sections 1 . 6.4 .4 and 1 .6 .5 .4) . Both temperature and PPF can have an important effect on flower colour. Cool temperatures and high PPF enhance colour expression of some pink through dark maroon selections (MacKay et al . , 1987b; Corr, 1988) . Lowering greenhouse night temperatures to less than 15 C or moving potted plants to an outdoor environmen?t during spathe opening enhances c9lour expression without greatly increasing production time (personal observations) . Flower colour of those selections in the cream through yellow to gold range is generally not affected by growing environment. While with some selections the expression of colour may change with environment, some selections are naturally dynamic in their expression as the flower develops. 1 .6.5.6 Physiological disorders Some incidents of malformed flowers after gibberellin application have been reported (Corr and Widmer, 1987; Tjia, 1987b; Funnell et al. , 1992) . These malformations typically consist of fluted spathes, double spathes, and coloured leaves. While the number of malformed flowers increased with increasing rates of gibberellin, the proportion of the total number of flowers that were malformed was not increased (Funnell et al. , 1992) . In addition , the classification of malformation is highly subjective. In some cut flower markets, premium prices have been paid for malformed flowers such as those with double spathes. 29 The expression of flower colour (refer Section 1 .6.5.5), and the occurrence of ?chalking? on cut surfaces of tubers (refer Section 1 .5.4.3), have been discussed previously. 1.6.5. 7 Post-greenhouse handling and marketing Potted Zantedeschia in group 1 should be marketed when the first flowers are unfurling (Plummer et al. , 1990). However, as a result of the need to enhance flower colour, potted group 2 selections are often not marketed until the first flowers are showing colour. Standard procedures for shipping flowering pot plants should ensure maintenance of quality, i.e. , keep cool, at 10 C, and well ventilated. Harvesting of cut flowers is carried out one to two days prior to pollen shed. This coincides with the development of full spathe opening and colour. Flowers of Z. aethiopica may also be bud-harvested when the spathe is still cigar-shaped (unpublished results). Harvest at this stage of development can be used to facilitate packaging and anticipate long market periods, without reducing vaselife. Investigations into bud harvesting of flowers from group 2 selections have not been reported. Pulsing cut stems in a solution of 0. 1 M sucrose and 0.5 g?litre?1 iprodione successfully minimized the incidence of stem splitting and postharvest spathe spotting (refer Section 1 . 7) (Tjia and Funnell, 1985) . The use of commercial preserv?tives commonly available on the ?ket, or solutions cOntaining 3 g?litre?? sucrose, resulted in rapid dehydration and necrd?is of the spathes of Z. aethiopica (Bakker and Stephan, 1961). The vaselife of cut flowets of selections in group 2 was not influenced by exposure to ethylene (10 JLl?litre?1 at 20 C for up to 48 h) subsequent to harvest (Funnell and Downs, 1987). 1. 7 Diseases and insects The major diseases affecting Zantedeschia are: (1) Alternaria species (I'jia and Funnell, 1985). Symptoms are chlorotic/necrotic leaf margins, and black spots on the spathe. (2) Erwinia carotovora (Dosdall, 1955; Forsberg, 1963; Long et al. , 1988) . Bacterial soft-rot; symptOms are erratic shoot emergence, shoot collapse, and. tuber decay. This pathogen has also been associated with postharvest collapse of the peduncle of cut flowers. A pungent odour is associated with tissue collapse. (3) Penicillium species (unpublished observations). Symptoms are blue-green mould on ?surface of tubers during storage. Considered more unsightly than detrimental to growth. 30 (4) Phytophthora species (Do?, 1955; Forsberg, 1963). Tuber rot, root rot, leaf blight; symptoms are yellowing/necrotic leaf and spathe margins, shoot collapse, and/or tuber and root decay. (5) Pythium species (Forsberg, 1963; Long, 1988). Symptoms are translucent or pink coloured tissue on infected tubers and roots. Translucent lesions on tuber surface increase in size, but remain odourless. (6) Rhizoctonia species (unpublished results). Symptoms are shoot collapse, and root and tuber rot. (7) Xanthomonas campestris (Joubert and Truter, 1972). Symptoms are water-soaked lesions on leaves that result in leaf collapse under moist conditions. Under dry conditions lesions become chlorotic and necrotic. (8) Virus diseases reported (Rana et al. , 1983; Long, 1988; Anon. , 1990) include: (a) alfalfa mosaic virus; (b) arabis mosaic virus; (c) cucumber mosaic virus (CMV), with symptoms of mosaic foliage patteming; (d) dasheen mosaic virus (DMV), causing leaf distortion, ring-spotting of foliage, and colour break in flowers; (e) potato virus x; (f) tomato spotted wilt virus (fSW), causing white or yellow foliage spots, or spots on leaves and flowers. Generally ?edeschia are remarkably tolerant of most diseases. Apart from tuber soft? rot, disease outbreaks are rare. Latent tuber infection was found to be a major source of inoculum of the soft-rot causing bacterium Erwinia carotovora (Long et al. , 1988). While the pathogen did not sufvive in soil for more than five months, cross-contamination from plant material and equipment can provide a ready source of inoculum. The use of disease-free planting material in addition to sanitation practices that avoid disease should ensure minimal occurrence of diseases. Since dry tubers infected with soft-rot often appear firm and healthy, it is important for growers to ensure a clean health status at the time of tuber purchase. While laboratory tests have indicated streptomycin as the most effective chemical inhibiting growth of E. carotovora (Long et al. , 1988) , use of this chemical in the field has not been satisfactory. Insects of significance in field or greenhouse production of Zimtedeschia include thrips and aphids. These can be readily controlled by standard insecticides. Root-knot nematode has been reported as a problem in the U.S .A. (Hayward, 1948) resulting in the need for soil sterilization/nematocide treatments. 1.8 Miscellaneous physiological and biochemical studies 1.8.1 Spathe regreening ? 31 During the formation of fruit on Z. aethiopica, the cytokinins 6-(0-hydroxybenzylamino )-9- {J-D-ribofuranosy lpurine and 6-( 0-H ydroxybenzy !amino)-2-me thy lthio-9-{J-D? glucofuranosylpurine have been isolated (Chaves das Neves and Pais, 1980a and 1980b). These naturally occurring cytokinins, as well as the synthetic cytokinin 6-(0- hydroxybenzylamino )-purine, have been associated with initiating the onset of regreening in the spathe (Pais and Chaves das Neves, 1982/83). The regreening of the spathe has been associated with a 341 % increase in chlorophyll content (Pais and Chaves das Neves, 1982/83) as amyloplasts, in Z. aethiopica, or chromoplasts, in group 2 selections, convert to chloroplasts (Gronegress, 1974). While removal of the spadix prevented regreening in Z. aethiopica (Pais and Chaves das Neves, 1982/83), no significant response was evident in group 2 selections (I'jia, 1986). However, the possibility of different mechanisms initiating regreening in selections of the two groups has not been reported. 1 .8.2 Tuber respiration Non-endodormant tubers of the group 2 selection 'Pink Petticoat' respired at a rate of 20 mg?kg-t.hr-1 C02 at a temperature of 20 C (Funnell and MacKay, 1990). For the first three weeks of storage, respiration rates were higher for tubers removed from 25 C storage than . in those removed from either 15 C or 8 C. The rates of respiration were not influ?nced by previous storage temperatures subsequent to this initial three week period. Expo?ure of tubers to ethylene (500 I'Hitre?? at 20 C for 24 h) prior to storage at 25 C resulted in respiration rates being reduced by 50% for the first two weeks of storage, compared with non-ethylene treated tubers (Funnell and MacKay, 1990). Subsequent to this initial two week period, respiration rates of ethylene treated tubers returned to that of non-ethylene treated. No ethylene related responses were obtained in subsequent growth and flowering. 1.9 Concluding remarks The species and hybrids of Zantedeschia offer a wide diversity of uses for the ornamental industry. While a comparatively new crop, in recent years the amount of research and commercial attention has expanded rapidly. With distinct physiological differences being evident between the - two horticultural groups, it is evident that research must recognise these differences when developing industry orientated production programmes. 32 While the goals outlined under the section on breeding (refer Section 1 .4) form part of the future research requirements for this crop, more generic areas for future research are: (1) improved resistance and/or control of bacterial soft-rot (E. carotovora). (2) determining the factors controlling vegetative growth and tuber development. (3) determining the factors controlling bud eco- and endodormancy. (4) more precisely determining the factors that control floral initiation and development. (5) development of post-harvest treatments to enhance flower longevity, especially aimed at delaying spathe regreening of group 2 selections. 33 1.10 References Anon. 1954. Climate of South Africa. Part 1 - Climate statistics. Climatology Branch, Weather Bureau, Dept. Transport. Govt. Printer, Union of Sth. Africa, Pretoria. Anon. 1989. Zantedeschia. In: Pretoria National Herbarium Computerized Information System (PRECIS). Amalgamated Botanical Research Institute/National Botanic Gardens, Pretoria, South Africa. Anon. 1990. Virus-free cal1as - world first for NZ. Commercial Horticulture, N.Z. May, 1990, p. 16-19. Bailey, L.H. 1930. The Standard Cyclopedia of Horticulture, Vol m, The Macmillan Company, London. Bakker, J. and H. Stephan. 1961. Experiments on the vase life of cut flowers? Jversl. Proefst. Bloem. Aalsmeer 1: 133-135. Butterfield, H.M. 1948. The Zantedeschia (Calla) industry in California, p. 34-35. In: H.P. Traub and H.N. Moldenke (eds.). Plant Life, Vol. 4, Aroid Lily Edition. Amer. Plant Life Soc. , Stanford. Chaves das Neves, H.J. and M.S.S. Pais. 1980a. A new cytokinin from the fruits of \ Zantedeschia aethiopica. Tetrahedron Letters 21:4387-4390. i ' I Chaves das Neves, H.J. and M.S.S. Pais. 1980b. Identification of a spathe regreening factor in Zantedeschia aethiopica. Biochem. Biop?ys. Res. Comm. 95: 1387-1392. Chi, Y. 1990. A study of the incompatibility within the genus Zantedeschia. M.Hort.Sc. dissertation, Massey Univ. , Palmerston North, N.Z. Clark, C.J. and H.L. Boldingh. 1991 . Biomass and mineral nutrient partitioning in relation to seasonal growth of Zantedeschia. Scientia Hort. 47: 125-135 . Clark, H.K. , K.A. Funnell and B.R. MacKay. 1987. Influence of scooping and tuber orientation on shoot growth and multiplication of Zantedeschia. Tech. Rpt. 87/12, Dept. Hort. Sci. & N.Z. Nursery Research Centre, Massey Univ. , Palmerston ? North, N.Z. 34 Cohen, D. 1981 . Micropropagation of Zantedeschia hybrids. Proc. Int. Plant Prop. Soc. 31 :312-317. Corr, B.E. 1988. Factors influencing growth and flowering of Zantedeschia elliottiana and Z. rehmannii. PhD thesis, Univ. Minnesota, U.S.A. Corr, B.E. and R.E. Widmer. 1987. Gibberellic acid increases flower number in Zantedeschia elliottiana and Z. rehmannii. HortScience 22:605-607. Corr, B.E. and R.E. Widmer. 1988. Rhizome storage increases growth of Zantedeschia elliottiana and Z. rehmannii. HortScience 23: 1001-1002. Corr, B.E. and R.E. Widmer. 1990. Growth and flowering of Zantedeschia elliottiana and Z. rehmannii in response to environmental factors. HortScience 25:925-927. Corr, l3.E. and R.E. Widmer. 1991 . Paclobutrazol, gibberellic acid, and rhizome size affect growth and flowering of Zantedeschia. HortScience 26: 133-135. Coursey, D.G. 1968. The edible aroids. World Crops 20:25-30. Dennis, D.J. 1986. Pilot study on nutrition of Zantedeschia hybrids in two container media. Final Report, Levin Horticultural Research Centre, Ministry of Agriculture and Fish?ries, New Zealand. Dosdall, L.T. 1955. Calla rhizome treatments. Plant Dis. Rptr. 39:779-780. ) Earl, G. 1957. Chromosome numbers and speciation in the genus Zantedeschia. Plant Life 13: 140-146. Engler, A. 1915. Araceae. In: Das Pflanzenreich IV, 23 Dc:61 . Leipzig: Engelmann. (Cited by Letty, 1973). Forsberg, J.L. 1963. Diseases of ornamental plants. Univ. lllinois, College Agr. , Special Publ. 3 :31-34. Funnell, K.A. and e.G. DoWils. 1987. Effect of ethylene on spathe regreening in Zantedeschia hybrids. HortScience 22: 1333. 35 Funnell, K.A. and A.R. Go. 1993. Tuber storage, floral induction, and gibberellin in Zantedeschia. ISHS, 2nd lntl. Symp. Development New Floricultural Crops, Baltimore, U.S.A. , 1991 . (in press). Funnell, K.A. and B.R. MacKay. 1987. Effect of GA3 on growth of Zantedeschia. Tech. Rpt. 87/14, Dept. Hort. Sci. & N.Z. Nursery Research Centre, Palmerston North, Massey Univ. , N.Z. Funnell, K.A. and B.R. MacKay. 1988a. Identification of the active constituents of Promalin in optimising flowering in Zantedeschia. Tech. Rpt. 88/2, Dept. Hort. Sci. & N.Z. Nursery Research Centre, Massey Univ. , Palmerston North, N.Z. Funnell, K.A. and B.R. MacKay. 1988b. Effect of temperature and ethylene during storage on growth and flowering of Zantedeschia. Tech. Rpt. 88/3, Dept. Hort. Sci. & N.Z. Nursery Research Centre, Massey Univ. , Palmerston North, N.Z. Funnell, K.A. and B.R. MacKay. 1988c. Verification of in vitro curing regimes for Zantedeschia, using whole tubers. Tech. Rpt. 88/4, Dept. Hort. Sci. & N.Z. Nursery Research Centre, Massey Univ. , Palmerston North, N.Z. Funnell, K.A. and B.R. MacKay. 1989a. Scooping of Zantedeschia tubers as a means of \ vegetative propagation. Tech . . Rpt. 88/5 , Dept. Hort. Sci. & N.Z. Nursery \ Research Centre, Massey Univ. , Palmerston North, N.Z. ' Funnell, K.A. and B.R. MacKay. 1989b. Investigations into the practice of early lifting of Zantedeschia tubers. Tech. Rpt. 88/6, Dept. - Hort. Sci. & N.Z. Nursery Research Centre, Massey Univ. , Palmerston North, N.Z. Funnell, K.A. and B.R. MacKay. 1990. Alleviation of storage induced flowering decline, iii Zantedeschia, with Promalin. Tech. Rpt. 89/1, Dept. Hort. Sci. , Massey Univ. , Palmerston North, N.Z. Funnell, K.A. , B.R. MacKay and N.W. Comber. 1987. The effect of curing temperature, humidity and duration on the development of protective barriers in Zantedeschia tubers. ? Tech. Rpt. 8717, Dept. Hort. Sci. & N.Z. Nursery Research Centre, Massey Univ. , Palmerston North, N.Z. 36 Funnell, l(.A. , B.R. MacKay and C.R.O. Lawoko. 1992. Comparative effects of Promalin and GA3 on flowering and development of Zantedeschia 'Galaxy' . ISHS, Hort. Sci. in the Tropics, Malaysia, 1990. Acta Hort. 292: 173-179. Funnell, K.A. , B.O. Tjia, C.J. Stanley, D. Cohen and J.R. Sedcole. 1988. Effect of storage temperature, duration, and gibberellic acid on the flowering of Zantedeschia elliottiana and Z. 'Pink Satin' . J. Amer. Soc. Hort. Sci. 1 13(6):860-863. Greene, L. , R.B. Withrow and M. W. Richman. 1932. The response of greenhouse crops to electric light supplementing daylight. Purdue Univ. Agr. Expt. Sta. Bul. 366: 1-20. Gronegress, P. 1974. The structure of chromoplasts and their conversion to chloroplasts. J. Microscopie 19: 183-192. Harrison, R.E. 1972. Zantedeschia hybrids. J. Royal Hort. Soc. 97: 131-132. Hayward, W. 1948. The Zantedeschia (Calla) industry in Florida, p. 36-39. In: H.P. Traub and H.N. Moldenke (eds.). Plant Life, Vol. 4, Aroid Lily Edition. Amer. Plant Life Soc. , Stanford. Ingle, A. and W.T. Bussell. 1991 . Tolerance of ca11a lilies to herbicides. Proceedings of the 44th Weed and Pest Control Conference, New Zealand, 44: 138-142. i I Jialong, Y. and D. Cohen. 1991 . Zantedeschia breeding project. Ann. Rpt. 1990, N.Z. Nursery Research Centre, Massey Univ. , Palmerston North, N.Z. , p. 22-23 . Joubert, J.J. and S.J. Truter. 1972. A variety of Xanthomonas campestris pathogenic to Zantedeschia aethiopica. Neth. J. Plant Pathol. 78:212-217. Kamerbeek, G.A. and W.J. De Munk. 1976. A review of ethylene effects in bulbous plants. Scientia Hort. 4: 101-1 15. Karlsson, M.G. , R.D. Heins, J.E. Erwin, R.D. Berghage, W.H. Carlson and J. A. Biernbaum. 1989. Temperature and photosynthetic photon flux influence Chrysanthemum shoot development and flower initiation under short-day conditions. J. Amer? Soc. Hort. Sci. 1 14: 158-163. 37 Kepner, K.W. , T.E. Welsh and G.D. Steven. 1990. The future export potential for New Zealand calla flowers. Rpt. , N.Z. Trade Dev. Board, Wellington, N.Z. Kobayashi, T. 1977a. An examination of spraying gibberellic acid solution of differing strengths onto the stems and leaves of calla lily. Nagano Agr. Lab. Rpt. no. 2 1 , Japan. (Japanese) . Kobayashi , T. 1977b . An examination of spraying gibberellic acid solution onto stems and leaves of calla l i ly. Nagano Agr. Lab. Rpt. no. 20, Japan . (Japanese) . Kobayashi, T. 1977c. A study of spraying gibberellic acid on to stems and leaves of calla l i ly at several times. Nagano Agr. Lab. Rpt. no. 23 , Japan. (Japanese) . Kobayashi, T. 1980. Examination of temperature of calla tubers in gibberellic acid solution . Nagano Agr. Lab. Rpt. no. 95 , Japan. (Japanese) . Kobayashi, T. , T. Tsukaka and F. Otsuka. 1977. A study of the flower bud formation and the progression of growth in the golden calla lily (Zantedeschia e/liottiana Engl .) . Bul l . Nagano Veg. Ornamental Crops Expt. Sta. , Japan, 2 :63-68. (Japanese) . Kobayashi, T. , A. Tsukada, F. Otsuka and F. Yamanishi. 1978. Yellow calla \ (Zantedeschia elliottiana Engl.) Sun Light. Rpt. Hort. Assn. , Nagano Prefecture, : Japan, p. 57-58. (Japanese) . Korner, Ch. and W. Larcher. 1988. Plant life in cold climates, p. 25-58. In: S .P. Long and F.l . Woodward (eds.). Plants and Temperature. Symp. of the Soc. for Expt. Biol. XXXXII. The Company of Biologists, Cambridge. Letty, C . 1973 . The genus Zantedeschia. Bothalia 1 1 ( 1&2) :5-26. Long, P.G. 1988. New records of Pythium species in New Zealand. N.Z. J. Expt. Agr. 1 6: 165-166. Long, P.G. , S. Miller, K. Everett and L. Fisher. 1988. Bacterial softrot diseases of Zantedeschia. Rpt. to Int. Calla Prod. Assn . , Dept. Plant Health, Massey Univ. , Palmerston North, N.Z. 38 MacKay, B.R. , K.A. Funnell and H.K. Clark. 1987a. Nutrition of potted Zantedeschia 'Red Emperor' . Tech. Rpt. 87/3, Dept. Hort. Sci. & N.Z. Nursery Research Centre, Massey Univ. , Palmerston North, N.Z. MacKay, B.R. , K.A. Funnell and N.W. Comber. 1987b. The effect of shade and growing environment temperature on the growth and flower pigmentation development of Zantedeschia hybrids. Tech. Rpt. 87/4, Dept. Hort. Sci. & N.Z. Nursery Research Centre, Massey Univ. , Palmerston North, N.Z. MacKay, B.R. , K.A. Funnell, N.W. Comber and K.A. Bowers. 1986. The effects of PP333 concentrations and application methods on clones of Zantedeschia hybrids. Tech. Rpt. 87/2, Dept. Hort. and Plant Health, & N.Z. Nursery Research Centre, Massey Univ. , Palmerston North, N.Z . . MacKay, B.R. , K.A. Funnell and T.C.H. Tan. 1991 . Evaluation of the performance of export grade Zantedeschia cultivars. Tech. Rpt. 9111, Dept. Hort. Sci. , Massey Univ. , Palmerston North, N.Z. Okonkwo, C.A.C. 1987. Polymorphism in taro (Colocasia esculenta (L.) Schott) and its implication in taro breeding and production in Nigeria. J. Hort. Sci. 62:395-400. Pais, M.S. and JI.J. Chaves das Neves. 1982/83. Regreening of Zantedeschia aethiopica Spreng. kpathe induced by reapplied cytokinins. Plant Growth Regulat. 1 :233-242. ' ' Perry, P.L. 1989. A new species of Zantedeschia (Araceae) from the western Cape. South African J. Bot. 55 :447-451 . Plummer, J.A. 1990. The influence of rhizome storage and growth regulators on flower production in potted Zantedeschia aethiopica 'Childsiana' . Tech. Rpt. Dept. Hort. Sci. , Massey Univ. , Palmerston North, N.Z. Plummer, J.A. , T.E. Welsh and A.M. Armitage. 1990. Stages of flower development and postproduction longevity of potted Zantedeschia aethiopica 'Childsiana' . HortScience 25:675-676. Post, K. 1936. Further responses of miscellaneous plants to temperature. Proc. Amer. Soc. Hort. Sci. 34:627-629. 39 Rana, G.L. , C. Vovlas and F.W. Zettler. 1983 . Manual transmission of dasheen mosaic virus from Richardia to non-araceous hosts. Plant Dis. Rptr. 67: 1 121-1 122. Rees, A.R. 1972. The Growth of Bulbs. Academic Press, London. Reiser, R.A. 1990. Cultivation of Zmuedeschia species for potted plant production. M.S. thesis, Cornell, U.S .A. Reiser, R.A. and R.W. Langhans. 1993. Cultivation of Zlmtedeschia species for potted plant production. ISHS, 2nd Intl. Symp. Development New Floricultural Crops, Baltimore, U.S.A. , 1991 . (in press). Rong, L. , Wang, X. Qin, Wang, R. Zhen and Y. Jun. 1989. Tissue culture and rapid propagation of Zmuedeschia hybrid. Guihaia 9:97-102. Sakanishi, Y. 1955. Effects of various treatments during the summer months on growth and flower production of common calla (Zmuedeschia aethiopica var. Minor) . Inst. Floriculture, Dept. Agr. , Univ. Osaka Prefecture 24(3):63-68. Sprengel, C. 1826. Systema Vegetabilium 3:765. (Cited by Letty, 1973). Shibuya, R. 1956. Intercrossing among pink calla, white-spotted calla and yellow calla. I ? Monograph. Kasai Pub I. , Tokyo. Takahashi, I. , M. Kosugi and M. Yokoi. 1957. Flower habit and flower bud progression of calla (Zmuedeschia aethiopica var. Childsiana). Hort. Assn. Japan 14:288-289. (Japanese). Tjia, B. 1986. Keeping quality of Zmuedeschia. Commercial Horticulture, N.Z. , Feb. 1986, p. 20-21 . Tjia, B . 1987a. Calla lily tubers thrive in higher temperatures. Greenhouse Manager, July 1987, p. 106, 109, 1 10, 1 12. Tjia, B. 1987b. The acid test. Greenhouse Manager, March 1987, p. 128-134. Tjia, B. 1987c. Growth regulator effect on growth and flowering of Zantedeschia rehmannii hyb. HortScience 22:507-508. 40 Tjia, B. and K.A. Funnell. 1985. Control of stem split and altemaria flower spot following harvest. Commercial Horticulture, N.Z. , April 1985, p. 28-29. Tjia, B.O. and K.A. Funnell. 1986. Postharvest studies of cut Zantedeschia inflorescences. Acta Hort. 181:451-458. Traub, H.P. 1948. The genus Zantedeschia. Plant Life 4:9-32. Warrington, I.J. and R.C. Southward. 1989. Influence of temperature and light intensity on Zantedeschia growth and development. Commercial Rpt. No. 46, Plant Physiol. Div. , DSIR, Palmerston North, N.Z. Welsh, T.E. , J.A. Plummer and A.M. Armitage. 1988. Preliminary evaluation of the dwarf white calla lily as a potted plant. Proc. Int. Plant Prop. Soc. 38:384-388. Woodward, F.I. 1988. Temperature and the distribution of plant species, p. 59-75. In: S.P. Long and F.I. Woodward (eds.). Plants and Temperature. Symp. of the Soc. for Expt. Bioi. :xxxxn. The Company of Biologists, Cambridge. Zizzo, G.V. 1987. Influenza del peso dei rizomi di cal1a (Zantedeschia aethiopica Spreng) sulla produzione dei fiori, rizomi e tubercoli. [Influence of the weight of r?mes of arum lily (Zaniedeschia aethiopica Spreng.) on production of flowers, rhizd?es and nodules]. Annali dell'Istituto Sperimentale per la Floricoltura 18 : 1 17- 125. : (Italian). 41 2 INTRODUCTORY OVERVIEW AND AIM OF THE CURRENT STUDY 2.1 Overview of study With 90% or more of plant dry matter comprising carbon (Irvine, 1975), it is self-evident that the processes of carbon acquisition (i.e. , photosynthesis) and utilization (i.e. , dry matter accumulation and respiration) should be a principal focus of any investigation into plant growth. Temperature and photosynthetic photon flux (PPF) are just two of the environmental parameters influencing these processes of acquisition and utilization. The responses of plants to temperature are considered to be governed by both the direct effects of temperature on metabolic (i.e. , enzyme and membrane carrier) activity and indirect physical (i.e. , diffusion and viscosity) effects (Farrar and Williams, 1991). In addition to any indirect effects of PPF on photosynthesis through stomatal aperture (Dwelle et al. , 1981 ; Valenzuela et al. , 1990), as a source of energy used in the acquisition and utilization of carbon, the interception of PPF is an integral component of dry matter accumulation (Warrington and Norton, 1991). The importance of photosynthesis as a determinant of dry matter accumulation and how it is influenced by temperature and PPF, will be presented in Chapter 5 . Consideration of total plant dry matter can provide a quantitative description of the plants' response to temperature and PPF (refer Chapter 3). Since leaves are the primary site of carbon acquisition and the tuber is the organ associated with harvestable yield, the partitioning of dry matter between organs (refer Chapters 3, 4 and 7) not only provides quantitative \ description, but environment induced changes in this partitioning may be used to suggest possible control mechanisms (Konings, 1990; Poorter, 1990). However development of a greater understanding of the potential mechanisms of control, requires investigation into the various biochemical components of the entire plant and its various organs (Warren Wilson, 1972; Obata-Sasamoto and Suzuki, 1979). Being the primary products of photosynthesis, carbohydrates potentially provide a major biochemical component of dry weight with which to begin examining the mechanisms of partitioning (refer Chapter 6) . Commercial production of Zantedeschia in New Zealand primarily utilizes group 2 selections (refer Section 1 .2). Flower and tuber producers in New Zealand were therefore more likely to benefit from adopting the research findings resulting from investigations into selections within this group. The Zantedeschia pentlandii-lilre selection 'Best Gold' was chosen as the subject of this study for a number of reasons . . (1) Its classification as a group 2 selection. (2) Its current use as a commercial cut flower crop. (3) Local availability of large quantities of disease free plant material of an even grade and at no financial cost. 42 If, like other genera, the natural distribution of Zantedeschia species has been influenced by the temperature minima during both the growing season and/or annual period (Komer and Larcher, 1988; Woodward, 1988) , it would be expected that climatological data of the sites of origin (refer Section 1 .3.2) would provide some indication of the possible temperature tolerance range, and therefore those temperatures worthy of investigation. Similarly, the natural habitat of Zantedeschia species in open grassland or forest margins (refer Section 1 .3 .2) might be indicative of an ability to tolerate shade. Using the data collated in Section 1 .3.2, treatments in the current study were selected to reflect such a range of possible temperatures and PPF. 2.2 Aim of this study International demand exists for Zantedeschia tubers year-round. However, at present the natural supply of quality tubers from New Zealand is restricted to a few months of the year. Although it is known that tubers can be produced out of season, the optimal environmental conditions to ensure the induction and maintenance of rapid tuber growth have not been defined. While preliminary studies have been conducted on the environmental response of vegetative growth and development of Zantedeschia (refer Section 1 .5 .3), no detailed analysis has been conducted that would enable possible control mechanisms to be elucidated. Further, gaining an understanding of how the plant responds to these environmental parameters is the foundation of information required to subsequently d.evelop crop management strategies, crop models, and future breeding strategies. Tii? aim of this study therefore, was to describe and interpret the vegetative growth and development of a group 2 selection in response to the environmental parameters of temperature and PPF. 2.3 References 43 Dwelle, R.B. , G.E. Kleinkopf and J.J. Pavek. 1981 . Stomatal conductance and gross photosynthesis of potato (Solanwn tuberosum L.) as influenced by irradiance, temperature, and growth stage. Potato Res. 24:49-59. Farrar, J.F. and M.L. Williams. 1991 . The effects of increased atmospheric carbon ? dioxide and temperature on carbon partitioning, source-sink relations and respiration. Plant, Cell and Environ. 14:819-830. lrvine, J.E. 1975. Relations of photosynthetic rates and leaf and canopy characteristics to sugar cane yield. Crop Sci. 15:671-676. Konings, H. 1990. Physiological ?d morphological differences between plants with a high NAR or a high LAR as related to environmental conditions, p. 101-124. In: H. Lambers, M.L. Cambridge, H. Konings, and T.L. Pons (eds.). Causes and consequences of variation in growth rate and productivity of higher plants. SPB Academic Publishing bv. , The Hague. Komer, C. and W. Larcher. 1988. Plant life in cold climates, p. 25-58. In: S.P. Long and F .1. Woodward (eds.). Plants and Temperature. Symp. of the Soc. for Expt. Biol. XXXXII. The Company of Biologists, Cambridge. Obata?Sasamoto, H. and H. Suzuki. 1979. Activities of enzymes relating to starch \ synthesis and endogenous levels of growth regulators in potato stolon tips during ' tuberization. Physiol. Plant. 45:320-324. Poorter, H. 1990. Interspecific variation in relative growth rate: on ecological causes and physiological consequences, p.45-68. In: H. Lambers, M.L. Cambridge, H. Konings, and T.L. Pons (eds.). Causes and consequences of variation in growth rate and productivity of higher plants. SPB Academic Publishing bv. , The Hague. Valenzuela, H.R. , B. Schaffer and S.K. O'Hair. 1990. Shade and nitrogen influence gas exchange and growth ofCocoyam (Xanihosoma sagittifolium). I. Amer. Soc. Hort. Sci. 1 15: 1014-1018. Warren Wilson, J. 1972. Control of crop processes, p. 7-30. In: A.R. Rees , K.E. Cockshull, D.W. Hand, and _ R.G. Hurd (eds.). Crop processes in controlled - environments. Academic Press, London. 44 Warrington, I.J. and R.A. Norton. 1991 . An evaluation of plant growth and development under various daily quantum integrals. J. Amer. Soc. Hort. Sci. 1 16:544-551 . Woodward, F.I. 1988. Temperature and the distribution of plant species, p . 59-75. In: S.P. Long and F.I. Woodward (eds.). Plants and Temperature. Symp. of the Soc. for Expt. Bioi. XXXXII. The Company of Biologists, Cambridge. 45 3 DRY MATTER ACCUMULATION AND LEAF LAMINA DEVEWPMENT OF Zantedeschia 'Best Gold' IN RESPONSE TO TEMPERATURE AND PHOTOSYNTHETIC PHOTON FLUX 3.1 Introduction I Zantedeschia is a relatively new horticultural crop with an expanding world-wide market for both cut flowers and tubers (refer Section 1 .2) . While complications with endodormancy have restricted the commercial development of all year round cultivation of Zantedeschia, it is known that under protected cultivation non-endodormant tubers of Zantedeschia can be forced into growth at any time of the year (Funnell and MacKay, 1990) . However, the response of this genus to manipulation of environmental parameters such as temperature and photosynthetic photon flux (PPF) during the growing season have not been previously described. For the commercial horticulturist the relatively large diurnal and seasonal variation of these parameters results in the need to investigate what impact they will have on growth . Further, gaining an understanding of how the plant responds to these environmental parameters is the foundation of information required to subsequently develop crop management strategies, crop models, and future breeding strategies. Traditional plant growth analysis provides an empirical description of growth and development of the plant through the derivation of "derivf!d quantities " from "measured quantities " (Warren Wilson et al . , 1986) . Equations relating these derived quantities pres?nt the relative growth rate (RGR) as a product of two components (e.g. , \ equation (3 . 1) ; West et al. , 1920) . where; RGR - LAR x NAR RGR = relative growth rate LAR = leaf area ratio NAR = net assimilation rate (3.1) The first component is a measure of size, and the second a measure of the efficiency of dry-matter production in terms of a resource of assimilates (Warren Wilson et al. , 1986) . Hunt (1982) summarised this simplistic but fundamental equation describing the growth of plants as "the growt!z rate of the plant depends simultaneously upon the efficiency of its leaves as producers of new material and upon the leafiness of the plant itself: Therefore, this empirical description also has the beginnings of being a mechanistic model, as the relative importance of tlie various components may be assessed. 46 While early research utilizing growth analysis endeavoured to illustrate a causal relationship between RGR and NAR, because of a strong negative correlation between NAR and LAR (Konings, 1990; Poorter, 1990), many examples have been reported where this correlation is weak (Heath and Gregory, 1938; Watson, 1952; Lambers, 1987; Tollenaar, 1989b; Poorter, 1990). Hence, while NAR is recognised as an important component ofRGR, its direct contribution is frequently maskoo by concomitant changes in LAR. Such changes in LAR may result from changes in leaf thickness (i.e. , specific leaf area) and/or the proportion of total plant dry matter within the leaf (i.e. , leaf weight ratio) (Konings, 1990). In contrast to the poor correlation between plant growth and NAR, for a large number of species strong correlations between RGR and the various forms of expressing the components of LAR (equation (3.2)) have been reported (Poorter, 1990). where; LAR - SLA x LWR LAR = leaf area ratio SLA = specific leaf area L WR = leaf weight ratio (3.2) For a large number of species, grown under a range of environments, this strong correlation between RGR and the partitioning of dry matter to leaf area -has also been expressed in terms of relative rates of leaf area expansion (RLAER) (Watson, 1947a & I 1947b; El-S?kawy et al. , 1965; Muramoto et al. , 1965; Duncan and Hesketh, 1968; Slatyer, 1970; Hanson, 1971; Delaney and Dobrenz, 1974; Potter and Jones, 1977; Collins and Jones, 1988). However, it should be appreciated that this correlation is not necessarily causal. Even though Jackson (1963) illustrated a clear correlation between RGR and the instantaneous ratio in the rates of change with time of leaf area and total plant dry weight (LAP, equation (3.3)) , it was not until Potter and Jones (1977) highlighted this relationship, that the greater potentially-causal relationship was appreciated. where; LAP = leaf area partitioning W . = total plant dry weight A = total plant leaf area t = time o = mathematical notation for an interval (3.3) 47 Potter and I ones (1977) suggested that since the daily partitioning of growth into leaf area will determine the area on the next day, the daily growth on the next day will depend to a large extent on the value of LAP. Patterson et al. (1978) extended our understanding of LAP by illustrating that as the ratio of RGR and RLAER approaches unity, then LAP approaches LAR. The value of LAP as a component of growth analysis is that it takes into account differences in RGR and RLAER when they occur. The strong correlation between RGR and LAP has been illustrated using a number of species grown under a range of temperatures and PPF regimes (Potter and I ones, 1977; Patterson et al. , 1978; Hunt and Halligan, 1981; Tollenaar, 1989a). Over the range of annual temperatures prevailing over much of the earth's inhabited surface (0 to 30 C), and for a diversity of plant species, the rate of development increases linearly with increasing temperature (Grace, 1988). In contrast, the response of RGR and final yield to this temperature range has generally been shown to be parabolic (Rajan et al. , 1973; Porter and Delecolle, 1988), with the optimum temperature of this parabolic response being species dependent (Rajan et al. , 1973). While increased temperatures have been associated with increased partitioning of dry matter towards leaf growth and therefore increasing the LAR and LAP (Farrar, 1988) over the naturally occurring range of PPF (0 to 2000 JLmol?m?2?s-1) , increased PPF typically is associated with increased dry matter accumulation, but with reduced LAR and LAP (Konings, 1990; Poorter, 1990). In addition, both increased temperature and PPF have been associated with increased NAR (Ko?gs, 1990; Poorter, 1990). \ The ?bjective of this study was to use the technique of plant growth analysis to gain an understanding of how growth of Zantedeschia 'Best Gold' responds to variable temperature and PPF during development, and, in doing so, _define horticulturally-relevant environmental parameters and management strategies. 3.2 Materials and Methods 3.2.1 Cultural 48 Non-endodonnant tubers of the Zantedeschia pentlandii-like selection 'Best Gold' , were lifted from the field (Palmerston North, New Zealand; 40?20'S) immediately prior to two plantings; one in early August 1988 and the other in late July 1989. On both occasions tubers were graded for evenness of size (approx. 1 cm diameter, 0.4 g dry weight), and for the presence of a single dominant bud. Tubers were planted in 60 m1 cell trays, containing a 50 peat : 30 pumice (v/v) growing medium to which 3 kg?m?3 dolomite lime was added. Plants were subsequently grown for 18 days on a heated (20 C), closed, mist bench, under greenhouse conditions (minimum air temperature 15 C, ventilation at 24 C). A uniform grade of emerged plants were selected, on the basis of development of protective sheath leaves (1 cm in length), pOtted into 1 .2 litre pots containing the same growing medium, and transferred to treatment environments. The experiment was carried out in controlled environment (CE) facilities in the National Climate Laboratory at the Horticulture and Food Research Institute of N.Z. Ltd, at Palmerston North, New Zealand. Over the two year period, plants were randomly allocated to one of 12 temperature-PPF treatment combinations, with two of the 12 combinations being employed at both plantings. Each environmental treatment comprised 96 plants with each pot supplied with a complete nutrient solution (5 x 100 m1 daily, half-strength Hoagland's A modified with the use of chelated iron, type ONC; Brooking (1976)). The amounts used ensured drainage at each application and avoided any outward symptoms of plant moisture stress. The avoidance of moisture sdess was also validated through the measurement of water potential of leaves ' during destructive harvests using a pressure bomb apparatus. Throughout the experiment an open plant canopy was maintained by the regular sampling of individual plants, eliminating interplant shading effects. 3.2.2 IWnviroDWGental The day/night temperature treatments were: 16/10, 22/10, 22/16, 28/16, 28/22 and 28/28 C ? 0.5 C. These day/night regimes resulted in the following daily mean temperatures: 13, 16, 19, 22, 25 and 28 C. In all temperature treatments, the day/night vapour pressure deficit was maintained at 1 .0/0.4 k:Pa. Photoperiod was 12 h and day/night and night/day temperature and vapour pressure changeovers were each of 2 h duration, with the lights switching off and on at the midpoint of each respective changeover. Lighting was provided by four 1000 Watt high-pressure multivapour-lamps (Sylvania "Metalarc") and four 1000 Watt Philips quartz halogen lamps, separated from the plant 49 growth room by a plate glass, and water, thermal barrier. The PPF at pot surface height was 694 ? 20 #'mol?m?2?s?1 (high PPF). Within the same CE room a PPF of 348 ? 10 JLmot-m?2?s?1 (low PPF) was achieved for half the plants, by covering four of the eight trolleys per room with a spectrally-neutral woven polypropylene shade cloth of nominal 50% density. At the same time as relocating the position of trolleys within each room at weekly intervals, trolleys were rotated 180? about their own axis. This procedure was carried out to minimise any influence of environmental gradients within each room. Additional information on room and lighting design has been described (Warrington et al. , 1978). 3.2.3 Experimental To monitor growth, 6 or 12 plants :were destructively harvested from the time of planting in cell trays until growth rate declined. Once in the CE rooms harvests occurred at the appearance of each successive leaf. This was taken to be when a minimum of 75% expansion of that leaf had occurred on 90% of plants within a treatment. Once increases in tuber dry weight were detected, ?sampling occurred more frequently, until the rate of total plant dry weight accumulation declined. At each harvest the following were recorded: number of sheath leaves (i.e. , protective structures which envelop the shoot, but do not produce leaf blad?), number and area of exposed leaves (i.e. , leaf blades not enveloped by subtending petioles), and number of leavbs commencing senescence (i.e. , when a minimum of 5 to 10% of the leaf had I . seneSced). Senescence was defined as that stage when leaf colour had progressed to the Judd-Hunter colorimetric L?a1) values (Francis and Clydesdale, 1975) of L? = 75 ? 4, a? = 5 ? 1 , and b. = 50 ? 5 . Leaf area data (i.e. , leaf blade excluding petiole) were recorded using a LICOR model 3100 leaf area meter. Aii measurements were carried out within 4 h of removal from treatment, after which plants were vacuum dried at 0.3 kPa and 40 C for a minimum of 48 h and the following dry-weights recorded: exposed leaves, shoot (i.e. , petioles of exposed leaves, together with the apex, non-exposed leaves, and sheath leaves), petioles of senescing exposed leaves, senescing exposed leaf blades, tuber, and roots (Plate 3. 1). Within each temperature-PPF treatment, plants were arranged in a completely randomized design. The number of treatments included in this study, and the time required to complete each treatment, preeluded any possibility of totally replicating the experiment. To partially evaluate the variation d?e to time and to between CE room effects, two temperature-PPF combinations (22/16 C high PPF and 22/16 C low PPF) were employed in different CE rooms at the two planting times. h a c L------- -- Plate 3 .1 Harvested plant of Zantedeschia 'Best Gold ' illustrating components measured. (a) shoot (sheath leaves, petioles and apex) , (b) exposed leaves, (c) tuber, (d) roots. (N.B. , scale half actual size) 50 A loge transformation of the dry weight data was used to meet the statistical assumption of homogeneity of variance throughout the period of study (Causton and Venus , 198 1 ) . Loge transformed total plant dry weight data, collected subsequent to placement in the CE rooms, were fitted to the Gompertz function, equation (3 .4) (Causton and Venus, 198 1 ) , utilizing the non-linear regression parameter estimation procedure of the Statistical Analysis System (SAS; SAS Institute, Inc. , Cary, N.C . ) . A constant was added to all transformed data to eliminate negative values from the fitting process . Because the Gompertz function is asymptotic, the magnitude of the added constant influences the calculated value of f3w. Hence the addition of a constant to all values prior to curve fitting eliminated any value of subsequent interpretation of differences in the value of f3w (i.e . , a measure of the starting size of total plant dry weight) . The Gompertz function was chosen over other sigmoidal growth functions due to the derivation of more biologically relevant parameters, its wide acceptability by other researchers (Causton and Venus, 198 1 ; Hunt, 1982) , and its ability to fit the current data-set with small mean square error values. In addition , with data from 5 1 some treatments, preliminary attempts to apply the more flexible Richard's function indicated over-parameterization of the model (Causton and Venus, 1981). This over? parameterization may have resulted from an inadequate number of harvest dates near critical points of the curve, such as near the upper asymptote. where; log? W = log? transformed value of total plant dry weight (lo? g) aw = upper asymptote of total plant dry weight (lo? g) f3w = a measure of the starting size of total plant dry weight (lo? g) Kw = rate constant of total dry weight as a function of size (g?g-1 -g-1) t = time (days) e = the base of natural logarithms (3 .4) Loge transformed total plant leaf area, and leaf dry weight data, collected subsequent to placement in the CE rooms, were fitted to the logarithmic form of the Gompertz function , equations (3 .5) and (3 .6) respectively (Causton and Venus, 1981) , utilizing the non-linear ' regr?ssion parameter estimation procedure of SAS . (3 .5) (3.6) where; log?, logeL = loge transformed value of total plant leaf area and leaf weight, respectively (loge cm2 and loge g, respectively) aA, aL = asymptote of attribute under investigation (loge cm2 and loge g, respectively) (3 A' (JL = a measure of the starting size of attribute under investigation (loge cm2 and loge g, respectively) KA, KL = rate constant of attribute as a function of size (cm2-cm?2-cm?2 and g-g?1 ?g?1, respectively) t = .time (days) e = the base of natural logarithms 52 During this time period total plant relative growth rate (RGRw), as a function of time, was calculated using equation (3. 7), being the first differential of equation (3.4) (Causton and Venus, 1981). (p -K ?t) C'w-xw?l).. RGR - ex ?x ?e w w ?e < -e J w w w (3.7) Similarly, total plant relative leaf area expansion rate (RLAER), and relative leaf weight rate (RLWR), as functions of time, were calculated using equations (3 .8) and (3.9), being the first differentials of equations (3.5) and (3.6), respectively (Causton and Venus, 1981). RLAER - x ?e

A (3.8) (3.9) Net assimilation rates (NAR), as defined by West et al. (1920), equ,ation (3. 10), were calculated using equation (3 . 1 1) , utilizing derived formulae and data from equations (3 . 4), \ (3.5) and (3 .7) .?. aw 1 NAR - -x- t,t A NAR - RGR ? e (log.JJ(,Y W(t) (3.10) (3.11) Leaf area partitioning (LAP) and leaf weight partitioning (LWP), as defined by Jackson (1963), equations (3.3) and (3. 12) , were calculated using equations (3. 13) and (3. 14), utilizing derived formulae and data from equations (3.4) to (3.9), respectively. (3.12) LAP - LWP - RLAER. ? (log.A. c -c i ? ci:o.04 L 0 W P P F '--' // 0.00 / / D (r2 - 0.96;??) (3.17) (r2 - 0.90, ???) (3.18) 0 3 6 9 1 2 1 5 1 8 2 1 24 27 30 MEAN TEMPERATURE Figure 3.3 Maximum value of RGRw as a function of temperature, for plants of Zantedeschia 'Best Gold' grown under high -o- and low -0- PPF regimes. 57 3.3.2 Repetition of treatments over years Comparison of relative growth rates (RGRw) is recognised as providing ?a more informative comparison of the plants ' relative perfonnances ? (Hunt, 1982). For the purpose of comparison of treatments over the two years in two different CE rooms (i.e. , 22/16 C day/night, at both high and low PPF), values of RGRw were calculated as the slope of the line of lo? transformed data (Hunt, 1982), during the period of exponential growth (i.e. , between 40 to 90 days from planting, Table 3.2). Examination of the slopes of the linear regressions for homogeneity did not detect differences between years (P s 0.05). In light of this finding it was assumed that CE room, and time, effects were small. Subsequent interpretation of data therefore utilized the mean value of data collected from both years for these treatments. Table 3.2 Total plant relative growth rates (RGRw ? standard error), and associated r, for plants of Zantedeschia 'Best Gold' grown at day/night temperatures of 22/16 C, at high and low PPF, in two CE rooms over two years. PPF regime High Low Year RGRw ? s.e. -r RGRw ? s.e. r 1st 0.047 ? 0.002 0.89 0.050 ? 0.002 0.93 2nd 0.050 ? 0.002 0.89 0.053 ? 0.003 0.89 signif.' n.s. n.s. 1 n.s. = years not significantly different (P < 0.05) (Zar, 1984). 3.3.3 Curve fitting or total plant dry weight Under all treatment regimes total plant dry weight followed a sigmoidal pattern of growth, with a family of Gompertz functions adequately describing the progression of total plant dry weight (lo? transformed) subsequent to placement in the treatments (Figure 3.4 and Figure 3.5, Table 3.3). 4 3 1 3 C -2 "' 1 0 30 50 70 90 1 1 0 1 30 1 50 1 70 1 90 2 1 0 DURATION OF GROWTH (DAYS) FigUre 3.4 Total plant dry weight (lo? fitted Gompertz curves) for Zantedeschia 'Best Gold' at a range of temperatures, under a high PPF regime. ? indicates day of transfer to treatments. 4 3 25 c ,...... ? 1- I 2 (.!) w :if; ? 0 1 ?--"-6 13 C ....J ? 0 0 1- "' (.!) 0 ....J - 1 - 2 "' 1 0 30 50 70 90 1 1 0 1 30 1 50 1 70 1 90 2 1 0 DURATION OF GROWTH (DAYS) Figure 3.5 Total plant dry weight (lo? fitted Gompertz curves) for Zantedeschia 'Best Gold' at a range of temperatures, under a low PPF regime. ? indicates day of transfer to treatments. 58 59 Table 3.3 Nonlinear least-squares parameter estimates, associated asymptotic standard error (s.e.), and mean square error values, from fitting the Gompertz function to lo? transformed total plant dry weight data for Zantedeschia 'Best Gold' grown at a range of temperatures, and under high and low PPF regimes. The PPF treatments at 19 C were repeated in two plantings, i .e. , (1st) and (2nd). Mean mean temperature PPF Ci.w' s.e. f3w s.e. Kw s.e. square (C) error 13 High 2.06 0.08 4.88 1 .33 0.057 0.014 0.20 Low 2.33 0.13 2.69 0.69 0.032 0.008 0.37 16 High 2.98 0. 1 1 2.22 0.24 0.040 0.005 0.10 Low 3 .90 0.24 2 . 15 0.21 0.030 0.004 0 . 15 19 ( I st) High 3.25 0.07 2.64 0.22 0.053 0.004 0.09 Low 3 .80 0.09 2.40 0. 17 0.042 0.003 0.08 19 (2nd) High 2.88 0.07 3 .05 0.31 0.058 0.007 0. 12 Low 3 .90 0 . 13 2.34 0. 17 0.038 0.003 0.12 22 High 3 .66 0.08 2.72 0. 18 0.054 0.004 0.07 Low 3.92 0. 10 2.65 0. 19 0.048 0.004 0.09 25 High 4.26 0.07 2.34 0.10 0.05 1 0.002 0.05 Low 4.19 0.12 2.34 0.15 0.04 1 0.003 0.08 28 High 4.07 0.09 2.75 0. 17 0.060 0.004 0.07 Low 3 .79 0.08 3 .03 0. 18 0.06 1 0.004 0.06 I Values of Ci.w presented are those resulting from the addition of a constant to avoid negative values of lo? transformed data. Values of the estimated maximum total plant dry weight (aw) were highly dependent on treatment. Under the high PPF regime aw increased linearly with increasing temperature (C) , up to 25 C (P < 0.00 1 , equation (3 . 19) , Figure 3 .6) . However, in contrast to the high PPF regime, values of aw under the low PPF regime were not influenced by temperatures above 13 C. Above 13 C, under the low PPF regime, aw averaged 3.92 (i.e. , 13 .64 g) ? 0. 13 , while at 13 C aw was depressed by 45% to 2 .33 (i .e. , 3 .28 g) + 0. 13 . aw = 0. 17 ? C - 1 .2 (3.19) This interaction between temperature and PPF on the estimated maximum total plant dry weight, resulted in greater values of aw under the low PPF regime than under the high PPF I 60 regime, at temperatures less than 22 C (Figure 3 .6) . At temperatures greater than 19 C the estimated maximum total plant dry weight was either not influenced by PPF (e.g . 22 C) or was greater under the high PPF regime. The maximum value of aw was attained at 25 C, reaching an estimated 4.26 (i. e. , 24.0 g) + 0 .07 under the high PPF regime, and 4. 19 (i. e. , 19 .9 g) + 0 . 12 under low PPF. A depression in the value of aw was noted at temperatures above this, i .e. , at 28 C, under both PPF regimes (Figure 3 .6) . 4 3 D a: w 2 1 3 1 6 LOW PPF J. D 1 9 22 MEAN TEMPERATURE (C) 0 D 25 28 Figure 3.6 Loge maximum total plant dry weight (aw) as a function of temperature, for Zantedeschia 'Best Gold' under high and low PPF regimes. Fitted line for high PPF regime only. I ' i The value of' parameter f:Jw of the fitted Gompertz curves, was not influenced by temperature nor PPF regime (P ? 0. 10). Across all treatments f:Jw averaged 2. 7 ? 0.2. In contrast to parameter f:Jw, the rate at which RGRw declined as a function of size, i .e. , parameter Kw, was generally greater under the high PPF regime than under the low PPF regime (P < 0. 1) . Only at a temperature of 28 C was no difference in the value of Kw detected between PPF regimes . Under the low PPF regime Kw increased linearly with increasing temperature (C) (P ? 0.05 , equation (3.20) , Figure 3 .7) . However, in contrast to the low PPF regime, values of Kw under the high PPF regime were not influenced by temperature. Across all temperatures under the high PPF regime, Kw averaged 0.053 + 0.003. Kw = 0.018 ? C + 0.006 (3.20) 0.07 0.06 0 .05 IC w 0.04 0.03 0.02 1 3 HIGH PPF -+ 0 0 D 1 6 0 LOW PPF 1 9 22 MEAN TEMPERATURE (C) D 25 28 Figure 3. 7 Interrelation between the rate of decline of RGRw as a function of plant size (Kw), and temperature, for Zantedeschia 'Best Gold' under high and low PPF regimes. Fitted line for low PPF only. 3.3.4. Curve fitting of leaf area and dry weight 61 Under all treatments total plant leaf area followed a sigmoidal pattern of growth subsequent to expansion of the first leaf and until the onset of declining total leaf area. A family of logarithmic Gompertz functions adequately described this progression of total plant leaf area (lo? transformed) (Figure 3.8, Figure 3.9, Table 3.4) . The progression of total plant leaf dry weight (lo? transformed) with time was similarly fitted to logarithmic Gompertz functions (Table 3 .5). Due to the similarity of the fitted curves for leaf area and dry weight, only figures depicting leaf area are presented. 7 6 :;---5 E 0 ........ ? 4 c( u.. LS _J G> 3 0 0 _J 2 1 0 ' ' : : , ,/ o6 30 50 70 90 1 1 0 1 30 1 50 1 70 1 90 2 1 0 DURATION OF GROWTH (DAYS) W?N4 TEIM'. (C) l:rl:rlJ. 13 8-&El 18 ...._.. 11 ??? 22 ....... 25 ??e 28 Figure 3.8 Fitted logarithmic Gompertz curves and mean data of total plant leaf area (logJ as a function of time, for Zantedeschia 'Best Gold' at a range of temperatures, under a high PPF regime. 7 6 :;---5 E ? ? 4 c( u.. LS _J G> 3 0 0 _J 2 1 0 ,'6 _JT-? / 9-/ / a/ !i e ,? . o -? ?-:;p 0 o..--0 0 o_g.---- o 0 ---- _.(5 _______ _ 30 50 70 90 1 1 0 1 30 1 50 1 70 1 90 2 1 0 DURATION OF GROWTH (DAYS) W?N4TEIM'. (c) l:rl:rlJ. 13 B-ETEl 18 ...._.. 11 ??? 22 ._..... 25 ??e 28 Figure 3.9 Fitted logarithmic Gompertz curves and mean data of total plant leaf area (logJ as a function of time, for Zantedeschia 'Best Gold' at a range of temperatures, under a low PPF regime. 62 63 Table 3 .4 Nonlinear Jea?t-squares parameter estimates, associated asymptotic standard error (s.e.), and mean square error values, from fitting the logarithmic Gompertz function to Jog. transformed total plant leaf area data for Zantedeschia 'Best Gold' grown at a range of temperatures, and under high and low PPF regimes. The PPF treatments at 19 C were repeated in two plantings, i .e . , ( 1 st) and (2nd). Mean temperature PPF a A s.e. {3A s.e. KA (C) 13 High 4.87 0.62 2.07 0.53 0.022 Low 4.87 0.88 1 .22 0.47 0.012 16 High 5.3 1 0 . 12 1 . 84 0.23 0.030 Low 6.07 0.28 1 .78 0. 15 0.019 19 ( 1st) High 5.68 0.09 2.34 0.25 0.004 Low 6.09 0.08 2.37 0.20 0.040 19 (2nd) High 5. 12 0 . 13 2.68 0.34 0.054 Low 5.74 0.08 2.29 0. 1 8 0.038 22 High 5.78 0.05 2.37 0.08 0.044 Low 6.06 0.08 2.25 0. 1 1 0.036 25 High 6.22 0.06 2 .36 0. 1 1 0.047 Low 6 .05 0.06 2.34 0 . 12 0.044 28 High 5 . 82 0.09 2 .26 0.21 0.049 Low 5 . 83 0.06 2 .59 0 . 13 0.055 s.e. 0.013 0.014 0.006 0.005 0.007 0.005 - .. 0.010 0.005 0.003 0.003 0.004 0.004 0.007 0.004 mean square error 0.35 0.72 0. 10 0.17 0. 1 1 0. 10 0.16 0. 1 1 0.05 0.08 0.06 0.07 0.09 0.07 As with total plant dry weight, the estimated maximum leaf area of plants grown under the high PPF regime, i . e . , a A of the fitted Gompertz curves, increased lin?ly with increasing temperature (C) up to 25 C (P < 0.05 , equation (3 .21) , Figure 3 . 10) . However, in contrast to the high PPF regime, values of aA under the low PPF regime were not influenced by temperatures above 13 C (P ? 0 . 10) . Above 13 C , under the low PPF regime, aA averaged 5 . 98 (i. e. , 395 .4 cm2) ? 0.06, while at 13 C aA was depressed by 67% to 4 . 87 (i.e. , 130.3 cm2) + 0 .88 . (N.B. cm2 as units of comparison) . a. A = 0. 1 1 ? C + 3.5 (3.21) The estimated maximum leaf dry weight (aJ responded similarly to aA, with maximum leaf dry weight increasing linearly with temperature (C) up to 25 C, under the high PPF regime (P ? 0.01 , equation (3 .22)) . As occurred with aA under the low PPF regime, temperatures above 13 C did not influence the value of aL (P ? 0. 10) , averaging 0.73 (2.08 g) ? 0.07 . At 1 3 C aL was depressed I by 78% to -0.77 (i .e . , 0 .46 g) + 0.27. (N.B. g as units of comparison) . ? L - 0. 14 ? C - 2.3 Table 3.5 64 (3.22) Nonlinear least-squares parameter estimates, associated a.?ymptotic standard error (s.e.), and mean square error values, from fitting the logarithmic Gompertz function to log. transformed total plant leaf dry weight data for Zantedeschia 'Best Gold' grown at a range of temperatures, and under high and low PPF regimes. Mean mean temperature PPF al s.e. {3L s.e. KL s.e. square (C) error 13 High -0.43 0.49 2.53 0.66 0.027 0.014 0.46 Low -0.77 0.27 2.28 0.97 0.028 0.016 0.83 16 High -0.09 0. 10 2.39 0.28 0.039 0.007 0. 14 Low 0.77 0.30 2.04 0.16 0.021 0.005 0.24 19 (1st) High 0.28 0.08 2.99 0.33 0.058 0.009 0. 15 Low 0.74 0.08 2.62 0.20 0.042 0.005 0. 14 19 (2nd) High -0.28 0 .12 3 . 1 8 0.3 1 0.062 0.009 0. 18 Low 0.37 0.09 2.45 0. 17 0.037 0.005 0.15 22 High 0.64 0.06 2.62 0.08 0.045 0.003 0.07 Low 0.92 0. 13 2.32 0.10 0.03 1 0.003 0 .14 25 High 1 .23 0.08 2.45 0.09 0.043 0.003 0.07 Low 0.93 0.09 2.33 0.09 0.035 0.003 0.09 28 High 0.78 0. 1 1 2.44 0.16 0.044 0.005 0. 1 1 Low 0.69 0.09 2.57 0.09 0.044 0.003 0.09 The greatest values of a A, i .e . , 6.22 (502. 7 cm2) ? 0.06, and of au i .e . , 1 .23 (3 .42 g) ? 0.08, were attained at a temperature of 25 C under high PPF. Both maximum leaf area (Fig . 4 .9) and leaf dry weight were reduced at 28 C. ,t ' .. .. ?, 7 LOW PPF J. 6 0 0: A 5 1 3 1 6 0 HIGH PPF 1 9 22 MEAN TEMPERATURE (C) 0 c 25 28 Figure 3.10 Lo? maximum total plant leaf area (a.J as a function of temperature, for Zantedeschia 'Best Gold' under high and low PPF regimes. Fitted line for high PPF regime only. 65 Treatment differences in the development of leaf area and dry weight were evident throughout the period of study. At the time RGRw was maximum, i.e. , inflection point of the fitted total plant dry weight Gompertz curve, total plant leaf area was greater under the low PPF regime compared with high PPF, at temperatures less than 22 C (Table 3.6) . In contrast, at thi? stage of development at temperatur? greater than 19 C, leaf area was greater under the high PPF regime. \ i ' Table 3.6 Leaf area (cm? at inflection point of the fitted total plant dry weight Gompertz curve of Zantedeschia 'Best Gold; grown at a range of temperatures, and high and low PPF regimes. Mean temperature (C) PPF 13 16 19 22 25 28 (cm? High 33.3 70. 1 82.5 73.9 99.9 87.4 Low 38.8 77. 1 97.6 65.5 71. 1 77.7 66 ?1be value of fJ A of the fitted Gompertz curves was influenced by an interaction between temperature and PPF regime (P ? 0.08). Under the low PPF regime there was a hyperbolic increase of fJA with increasing temperature (C) (equation (3.23), Figure 3. 1 1) . I P - 12.S?tanh( 1.4?C) -9.9 A 12.S 3 HIGH PPF -+ 0 ?" 2 D +- LOW PPF (=e - 0.02] e"or D !! 0 1??--??--???--??--???--? . 13 1 6 1 9 22 MEAN TEMPERATURE (C) 25 28 Figure 3.11 Parameter {:JA as a function of temperature, for Zantedeschia 'Best Gold' under high and low PPF regimes. Fitted line for low PPF only. (3.23) There was no effect of temperature on fJA under the high PPF regime, the mean value being 2.27 ? 0. 10, across all temperatures. In contrast to fJ A' the value of fJL was not influenced by temperature. However, across all temperature treatments the value of fJL was greater under the high PPF regime (i.e. , 2.66 ? 0. 12) than under the low PPF regime (i.e. , 2.40 ? 0.07; P ? 0.07). The rate of decline of the relative leaf area expansion rate, as a function of size (RLAERs), i.e. , parameter KA, of the fitted Gompertz cirrves, increased with increasing temperature (P ? 0.001). Under the low PPF regime this increase with temperature (C) was linear (equation (3.24), Figure 3 . 12)_. _ However, under the high PPF regime the increase in rate of decline of RLAERs, with increasing temperature, was hyperbolic (equation (3.25), Figure 3. 12) . K A - 0.0027 ? C - 0.022 K A - 0.3 ?tanh( 0.03 ? C) -0.24 0.3 67 (3.24) (3.25) At each temperature the rate of decline of RLAER8 was generally greater under the high PPF regime than the low PPF regime, except at a mean temperature of 28 C (Figure 3 . 12) . 0.06 0.05 0.04 IC A 0.03 0.02 0.01 1 3 1 6 1 9 22 MEAN TEMPERATURE (C) 25 28 Figure 3.12 Interrelation between the rate of decline of RLAER8 (KAl and temperature, for Zantedeschia 'Best Gold' under high and low PPF regimes. As found with the parameter KA, for plants grown under the low PPF regime the rate of decline of relative leaf weight expansion rate, as a function of size (RLWRs) i.e. , parameter KL of the fitted Gompertz curves, increased linearly with increasing temperature (equation (3.26)). KL - 0.0012?C + 0.01 (r1 - 0.62;?) (3.26) In contrast to the rate of decline of RLA.E.Rs under high PPF, no response of the rate of decline of RL WR8 to temperature was evident under the high PPF regime. Across all temperatures under the high PPF regime, the mean value of the rate of decline of RLWRs was 0.045 ? 0.004. However, as with the rate of decline of RLA.E.Rs, the rate of decline of RL WRs was generally greater under 68 .the high PPF regime than the low PPF regime (P < 0.07) . 3.3.5 Relationships between derived parameters During the exponential phase of total plant dry weight accumulation, a positive linear relationship :iexisted between total plant relative growth rate (RGRw) and both relative leaf area expansion rate ?(RLAER) and relative leaf weight rate (RLWR), regardless of PPF regime and temperature .(P ? 0.0001) (equations (3 .27) and (3 .28) , Figure 3 . 13). RGRw -= 0.82?RLAER + 0.015 (3 .27) RGRw = 0.75 ?RLWR + 0.01 (3.28) In contrast, no relationship between RGRw and net assimilation rate (NAR) was detected for the entire data set, nor for separate PPF regimes (P :S 0. 10, Figure 3 . 14). While NAR was typically 1 greater under the high PPF regime compared with the low PPF regime (P ? 0.01) , no influence of temperature was detected (P < 0. 10) . Independent, positive, l inear relationships were determined between RGRw and leaf area partitioning 1(LAP) under the high and low PPF regimes (equations (3 .29) and (3 .30) resp?tively, Figure 3 . 15). ?The slope of the line des?ribing the relationship between RGRw and LAP under the high PPF regime ?was greater than that from the low PPF regime (P :::; 0.05) . . ' (High PPF) RGRw = 0.0008 ?LAP + 0.003 (Low PP F) RGRw = 0.0003 ?LAP + 0.023 (r2 - 0.12;?) (3 .29) (r2 = 0.93;??) (3.30) In contrast, a single, positive, linear relationship between RGRw and the leaf weight partitioning (LWP) encompassed the response regardless of PPF regime and temperature (equation (3 .31) , Figure 3 . 1 6) . RGRw = 0. 156 ?LWP (r2 = 0.98, ? ? ?) (3 .31) 0.10 0.08 ?0.06 ;? "0 ;?0.04 -2 )0 50.02 "" o.oo'r-----,..---..-----.....-------.------,. 0.00 0.02 0.04 0.06 2 -J _, RUER (cm.em. day ) 0.08 0.10 Figure 3.13 RGRw as a function of RLAER, for Zantedeschia 'Best Gold' grown under two PPF regimes and six temperatures. 0. 10 0.08 0 0 ?0.06 ;,.. 0 ?- ?0.04 11: 0 ? 0.02 0.00 0 20 40 60 80 100 120 140 1 60 2 _, _, lN' (cm .day /g.daj ) Figure 3.15 RGRw as a function of LAP, for Zantedeschia 'Best Gold' grown under two PPF regimes and six temperatures. 0.1 0 0.08 ?0.06 ;,., 0 ? _;o.o4 )0 ? 0.02 "" 0.00 4 0 0 0 0 LOW Pf'f"_. 0 5 69 0 0 0 0 o._ HIGH PPF 6 7 8 9 1 0 Figure 3.14 RGRw as a function of NAR, for Zantedeschia 'Best Gold' grown under two PPF regimes and six temperatures. 0.10 0.08 ?0.06 ;? "0 j? ?0.04 )0 ? 0.02 "" 0.00 0.0 0.1 0.2 0.3 0.4 LWP (g.doy -fg.daj-) 0.5 0.6 Figure 3.16 RGRw as a function of LWP, for Zantedeschia 'Best Gold' grown under two PPF regimes and six temperatures. 3 .4 Discussion 70 The strong correlation between relative rate of leaf area expansion and growth during the exponential phase (Figure 3 . 13) has been illustrated with a number of plant species, grown under a range of environments (Watson, 1947a & 1947b; El-Sharkawy et al. , 1965 ; Muramoto et al. , 1965 ; Duncan and Hesketh, 1968; Slatyer, 1 970; Hanson, 1971 ; Delaney and Dobrenz, 1974; Potter and Jones, 1977). I t should be appreciated however, that this correlation is not necessarily causal. In fact, extrapolation of this equation (equation (3 .27)) infers that at a RLAER of 0 cm2-cm-2-day-1 , i .e. , no expansion of leaf area, a RGRw of 0 .015 ? 0.005 g -g-1 -day-1 would be expected. Therefore clearly RLAER can not be considered solely causal of growth. However, since the daily partitioning of growth into leaf area will determine the area on the next day, the daily growth on the next day will depend to a large extent on the value of the LAP (Potter and Jones, 1977) . While a strong correlation between RGRw and LAP was shown to exist for plants of Zantedeschia 'Best Gold' grown at a range of temperatures, the magnitude of the relationship, i . e. , slope of line, was more than doubled when plants were grown under high PPF compared with under low PPF (Figure 3 . 15) . In contrast a single, positive, linear correlation between RGRw and LWP was determined, which accounted for the differing temperature and PPF induced responses (Figure 3 . 16) . In developing the potentially more causal relationship between RGRw and LAP, Potter and Jones (1977) u tilized data derived from a number of species grown at a range of temperatures without variation in PPF regime. If the current experiment had used only one PPF regime ,the same conclusion would have been reached as that of Potter and Jones (1977) . It is therefore evident that the LAP may ';pe limited in its application in inferring a more direct relationship with RGRw to situations where a single PPF regime is utilized. The improvement in the ability to predict RGRw via a single relationship using L WP rather than two independent relationships using LAP, was as a result of L WP accounting for differences in specific leaf weight. Changes in daily quantum integral of light have been shown to account for differences in specific leaf weight of a number of plant species (Warrington and Norton, 1991) . In addition, differences in specific leaf weight have accounted for variation in LAP in perennial ryegrass and maize (Hunt and Halligan, 1981 ; Tollenaar, 1989a) , thereby supporting the logic of utilizing L WP under variable PPF regimes. The theory that LWP may be more robust in its use for determining plant growth in Zantedeschia 'Best Gold' is further strengthened when consideration is given to extrapolation of equation (3 . 3 1) to where a LWP of 0 g-day-1/g-day-1 would result in a RGRw of 0 g ?g-1 -day-1 ? This outcome is more logical than that achieved when using RLAER as a determinant of plant growth , as discussed above. Since in the natural enviro?ment PPF levels are not constant (i.e. , seasonal changes as well as changes with latitude) it is suggested that LWP will provide a more robust determinant of growth of 71 Zantedeschia as well ? other plant genera. The poor correlation between NAR and growth (Figure 3. 14) has been noted with other species (Heath and Gregory, 1938; Watson, 1952; Hunt and Parsons, 1974; Lambers, ? 1987; Poorter, 1990). Equally however, examples of increased RGRw as a result of increased NAR have been reported (Raj an et al. , 1973; Patterson et al. , 1978; Lambers, 1987; Tollenaar, 1989b; Poorter, 1990). When one considers that a primary component of NAR is derived from the photosynthetic rate per unit area, coupled with the frequently poor success of correlating unit photosynthetic rates with growth (Lambers, 1987), it is not surprising that no relationship between RGRw and NAR was determined in the current experiment. However, it should not be overlooked that Poorter (1990) indicated that the poor correlation was primarily attributable to strong negative correlation between NAR and LAR. Therefore, the poor correlation determined here does not necessarily negate the possible dominance of NAR determining growth when the growing environment more directly influences photosynthesis. This has been clearly illustrated with ? gaseous pollutants whose presence resulted in a direct influence on photosynthesis, and thereby resulted in a strong correlation between growth and NAR (Jensen, 1981). Further discussion of any possible relationship between photosynthetic rate and plant growth in Zantedeschia 'Best Gold' will be presented in Chapter 5. In addition to the aforementioned variation between species ? the degree of correlation betw?n growth and NAR, variation in both the presence and strength of any correlation betw?n growth and L WP and/or LAP, between species, has also been reported (Potter and Jones, \1977; Patterson et al. , 1978; lbrahim and Buxton, 1981; Sage and Pearcy, 1987). It is evident that species vary in their mechanisms of acclimating to their growing environment. While exceptions exist for any attempt to classify plants into definitive categories of sun and shade species/ecotypes, shade-tolerant species/ecotypes often exhibit an ability to acclimate to alteration in PPF through an enhanced alteration of LAR and thereby LWP and LAP (Bjorkman and Holmgren, 1966; Gauhl, 1976; Boardman, 1977; Pons, 1977; Bjorkman, 1981; Kuiper and Smid, 1985). In contrast sun-specieslecotypes frequently possess a greater capacity to alter NAR under conditions of increased PPF. While consideration of other factors contributing to the classification of Zantedeschia 'Best Gold' as a typical shade-tolerant or sun species will be discussed in Chapter 5, i.e. , adaptability of photosynthetic rate, from the data presented here it appears that Zantedeschia 'Best Gold' does exhibit the phenotypic plasticity of a shade-tolerant species. -? In terms of Hunt's (1982) simplistic description of growth, differences in Zantedeschia plarit dry weight that occur during the exponential phase of total plant dry weight accumulation, appear to be primarily attributable to changes in ?Ieajiness ? of plants. This 72 change in leafiness is clearly illustrated by the fact that increasing temperature resulted in increased leaf development (Figure 3 .8 , Figure 3 .9 , Table 3 .4 , and Table 3 .5) . By way of an example of this relationship, at the mid point of the exponential phase of total plant dry weight accumulation, at intermediate temperatures, leaf areas under the low PPF regime, were greater than under high PPF (fable 3 . 6) . This increased leaf development under low PPF was able to almost fully compensate for any reduction in NAR, and maintain RGRw at rates similar to those achieved under high PPF (Figure 3 .3) . Similarly, at intermediate temperatures the prediction that maximum total plant dry weight would be greater under the low PPF regime than under high PPF (Figure 3 .6) , even though photosynthetic rate was found to be greater under the latter (refer Chapter 5) , can primarily be explained by greater leaf area development under the low PPF regime (Figure 3 .8 to Figure 3 . 10) . As reported for other plant species, leaf area duration may also have contributed to the prediction of greater maximum total plant dry weight under the low PPF regime than under the high PPF regime (Radley, 1963; Rees, 1972 ; Alien and Scott, 1980) . However, the duration of the current study did not permit collection of data to the cessation of leaf development to investigate this possibility. Certainly the greater rate of decline of RLAER and RLWR, as a function of size, under the high PPF regime (Figure 3 . 12) , in conjunction with the earlier onset of leaf senescence under the high PPF regime, are indicative of a reduced leaf area duration. Differences in ?stimated maximum total plant dry weight between PPF regimes were greatest at intermediate temperatures, with little or no difference being evident between PPF regimes at extreme temperatures, i .e. , mean temperatures of 13 C and 28 C (Figure 3 .6) . This variation in response of dry matter accumulation to PPF regime, with temperature, is equally explained by variation in LAP and LWP (Figure 3 . 15 and Figure 3 . 1 6) . Therefore it is apparent, that Zantedeschia 'Best Gold' is readily able to acclimate and compensate for changes in PPF, via alteration in LAP and L WP, at temperatures greater than 13 C and less than 28 C. A growth temperature of 28 C, while initially stimulating very rapid growth (Figure 3 . 3) , resulted in a reduction of maximum total plant dry weight compared with that attained at 25 C (Figure 3 .6) . With reference to the potentially causal relationship between RGRw and LAP and LWP, values of all these parameters at 28 C were similarly reduced relative to those at 25 C, hence supporting the theory of the dominance of Hunt's (1982) "leajiness of the plant " correlating to total plant growth. While in theory parameter f3w of the fitted Gompertz curves is an estimate of the total plant dry weight at day 0, this interpretation is based on the assumption that substrate supply is non-limiting (fhomley and Johnson, 1990) . Clearly during the initial establishment stage, 73 i.e. , until functional leaf area was present, this was not the case as total plant dry weight declined (Figure 3. 1). Under normal circumstances the magnitude of differences in the value of fJw between treatments, might be indicative of the relative magnitude of weight loss that occurred until the establishment of functional leaf area, i.e. , higher values of fJw inferring less loss of total plant dry weight during this phase. However, due to the need to add a constant to all transformed data to eliminate negative values being submitted for curve fitting, no treatment differences between the values of fJw were evident. Hence in this experiment the magnitude of values of fJw do not carry any biological significance. As would be expected from the increase in specific leaf weight at increased PPF, the value of fJL was generally greater under the high PPF regime than the low PPF regime. Being estimates of the leaf area and leaf dry weight at day 0, fJA and fJL should be considered as being merely important parameters for curve fitting, as . their subsequent influence on growth is better explained in terms of LAP and L WP, as discussed above. The pooled predicted base temperature for growth of 2 . 1 ? 2.7 C is within the range predicted for germination of many plant species utilized as winter crops, such as wheat and turnips, i.e. , generally between 2 and 5 C. In contrast, warm season crops originating from temperate/warm-temperate climes, such as cotton, sorghum, rice, and corn, have base temperatures between 8 and 10 C (Angus et al. , 1981; Singh and Dhaliwal, 1972; Warrington and Kanemasu, 1983). Just how accurate such estimations of base temperature are is open to debate, as base temperatures have been noted to change with development in crops such as wheat (Porter and Delecolle, 1988). A positive linear response of growth rate with temperature has generally been found between 5 and 20 C (Grace, 1988). While I this linear response has been used to predict the base temperature (Figure 3.3) , without actual examination of growth at such low temperatures, the predicted base temperature may have to be taken as merely an estimation for mathematical crop modelling purposes only. The low base temperature for Zantedeschia 'Best Gold' is also somewhat surprising considering climatological data for the sites of natural habitat indicate a warm-temperate climate for this crop (refer Section 1 .3.2). While exceptions are always evident, the natural distribution of perennial species has been shown to be critically dependent on the temperature minima during both the growing season and/or annual period (Komer and Larcher, 1988; Woodward, 1988). With a predicted base temperature of2. 1 C compared with minimum soil temperatures in its natural habitat reaching 8. 1 C (fable 1 .2), it is suggested that either ecodormancy, resulting from soil moisture stress, and/or endodormancy, prevent growth during the winter period of its natural habitat. The minimal rainfall during winter was outlined in Section 1 .3.2, as was the existence of endodormancy in summer flowering Zantedeschia (Corr and Widmer, 1988). Since derivation of the base temperature used data collected when plant leaf area was well established, an alternative explanation for the low predicted base temperature could be that 74 rates of photoassimilate accumulation decline less, with decreases in temperature, than do rates at which assimilates are used (Warren-Wilson, 1966; Verkleij and Challa, ? 1988; Acock et al. , 1990). Hence, while growth and development of a recently planted tuber may be restricted at temperatures below 7 ? 3 C (refer Section 1 .5 .4.3), dry matter accumulation would be able to continue on a plant with developed leaf area due to continued photosynthesis at these temperatures. Maximum RGRw values of 0.080 g-g-1-day-1 at a temperature of 25 C and at high PPF (Figure 3.3) is well within the range of 0.02 to 0.4 g-g?1uy?1 reported for other plant species, but is between typical values for sun species (e.g. , 0. 1 to 0.4 g-g-1-day-1) on the one hand, and shade species (e.g. , 0.02 to 0.04 g-g?1uy-1) on the other (Briggs et al. , 1920; Blackman and Black, 1959; Warren Wilson, 1972). In reviewing the linear temperature response of RGRw in ? crop species, Grace (1988) indicated an average 7% increase in RGRw per degree Celsius. In the current experiment RGRw also increased by 7% per degree Celsius (Figure 3.3). In addition to the distribution of perennial species being determined by the temperature minima of the natural habitat, the heat sum during the growing season has also been shown to be critical (Woodward, 1988). While no attempt has been made to determine the equivalent heat sum for the natural habitat of Zantedeschia pentlandii, the mean temperature for maximal growth of the Zantedeschia pentlandii-like selection 'Best Gold: i.e. , between 2t and 25 C, was greater than the average daily mean air temperature during the growing season for the sites of natural habitat, i.e. , 20 C (refer Section 1 .3.2). In addition, with daily maximum air temperatures during the growing season averaging 25.4 C, it is apparent that Zantedeschia 'Best Gold' is well adapted to take advantage of the temperature regimes of its natural habitat. The linear relationship between RGRw and . temperature (Figure 3 .3) indicates that horticulturally relevant temperatures can be identified, i.e. , base temperature (2. 1 ? 2.7 C), the linear range of mean temperature response (2. 1 to 28 C), and mean temperatures resulting in maximum growth (i.e. , 25 C). During the initial stages of plant development no leaves were present, and therefore it is acknowledged that the ?resource ? of assimilates referred to by Warren Wilson et al. (1986), refers to the tuber at this stage of development. The change with development in role of organs as net assimilate source or sink, has previously been noted in geophytic crops (Rees, 1972). During this initial establishment stage no importation of recently? produced assimilates is possible, thus respiratory losses associated with the development of leaf and root tissue results in a net loss in total plant dry weight (Figure 3. 1). Since 75 leaves of plants on other species are found to be net exporters of assimilates once they have attained 50% or more of their full expansion (Giaquinta, 1978), the increments in total plant dry weight at the time of the first harvest within treatments, i.e. , when 75% or more expansion of the first leaf had occurred, was not unexpected (Figure 3. 1). The rate and extent of recovery of this lost dry weight was subsequently dependent on those factors associated with equation (3. 1) , in particular partitioning of assimilates into leaf growth. Plant responses to temperature are considered to be governed by both the direct effects of temperature on metabolic activity (i.e., enzyme and membrane carrier) and indirect physical effects (i.e. , diffusion and viscosity) (Farrar and Williams, 1991). While PPF can indirectly affect photosynthesis through stomatal aperature (Dwelle et al. , 1981 ; Valenzuela et al. , 1990), as a source of energy utilized to drive the acquisition and utilization of carbon, the interception of PPF is an integral component of plant growth . . (Warrington and Norton, 1991). While in the current study the focus of the environmental influences on plant growth has been presented in terms of mean temperature and PPF regimes within a controlled environment, it is appreciated that the concepts of heat units (Warrington and Kanemasu, 1983) and daily quantum integrals of light (Warrington and Norton, 1991) will need to be developed before a suitably robust crop model can be developed. In addition, between plant competition did not exist within the current study, eliminating the need to consider the concept of leaf area index. Considering the ability of Zantedeschia 'Best Gold' to com?nsate for reduced PPF via increased partitioning to the development of leaf, the I attain?ent of increased total plant dry weight under low PPF may not result in the field once competition between plants exists. 3.4.1 Conclusions In response to variable temperature and PPF, dry matter accumulation of Zantedeschia 'Best Gold' was highly correlated with the partitioning of dry matter into leaf development. In contrast, the correlation between dry matter accumulation and net assimilation rate was poor. Dry matter accumulation of Zantedeschia 'Best Gold' was highly adaptive to the PPF regime. While the optimum PPF under which to grow Zantedeschia 'Best Gold' was dependent on temperature, maximum dry matter accumulation occurred under the high PPF regime at a mean temperature of 25 C. Hence the horticultural management consequences of these findings are that the establishment and maintenance of an effective leaf area will be critical if growth is to be maximised. 3.5 References 76 Acock, B. , M.C. Acock and D. Pastemak:. 1990. Interactions of C? enrichment and temperature on carbohydrate production and accumulation in muskmelon leaves. I. Amer. Soc. Hort. Sci. 115:525-529. Allen, E.I. and R.K. Scott. 1980. An analysis of growth of the potato crop. I. Agr. Sci. (Cambridge) 94:583-606. Angus, I.F. , R.B. Cunningham, M.W. Moncur and D.H. MacKenzie. 1981 . Phasic development in field crops. I. Thermal response in the seedling phase. Field Crops Res. 3:365-378. Bjorkman, 0. 1981 . Responses to different quantum flux densities, p. 57-107. In: O.L. Lange, P.S. Nobel, C.B. Osmond, and H. Ziegler (eds.). Encyclopedia of Plant Physiology, vol. 12 A, Physiological plant ecology. Springer Verlag, Berlin. Bjorkman, 0. and P. Holmgren. 1966. 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Comparative physiological studies on the growth of field crops. TI. The effect of varying nutrient supply on net assimilation rate and leaf area. Ann. Bot. 1 1 :375-407. Watson, D.J. 1952. Physiological basis of variation in yield. Adv. Agron. 4: 101-145. West, C . , G.E. Briggs and F. Kidd. 1920. Methods and significant relations in the quantitative analysis of plant growth. New Phytol. 19:200-207. Woodward, F .I. 1988. Temperature and the distribution of plant species, p. 59-75. In: S.P. Long and F.I. Woodward (eds.). Plants and Temperature. Symp. of the Soc. for Expt. Bioi. XXXXII. The Company of Biologists, Cambridge. Zar, J.H. 1984. Biostatistical analysis. 2nd ed. Prentice Hall, New Jersey. 82 4 TUBER DRY MATI'ER ACCUMULATION OF 7Antedeschia 'Best Gold' IN RESPONSE TO TEMPERATURE AND PHOTOSYNTHETIC PHOTON FLUX 4.1 Introduction Zantedeschia is a relatively new horticultural crop with a rapidly expanding world-wide market for tubers (refer Section 1 .2). Although Zantedeschia may be grown at any time of the year under protected cultivation (refer Sections 1 .5 and 1.6), responses of this genus to manipulation of environmental parameters, such as temperature and photosynthetic photon flux (PPF), have not been described in detail. Since the above environmental parameters are frequently under the control of the horticulturist, gaining an understanding of how the plant responds to their modification would provide information required to develop assimilate partitioning models, crop management and plant breeding strategies. Improvements in the partitioning of photoassimilates between the economic yield component and the remainder of the plant have generally been responsible for the improved yields obtained from many field crops over the last century (Gifford et al. , 1984). When this partitioning is expressed at a single point in time, a measure of the cumulative partitioning to that date is derived (i.e. , harvest index). The harvest index therefore provides a simple but coarse measure of assimilate partitioning. With partitioning frequently changing with plant development, the use of harvest indices is generally restricted to crops with a single, readily determined point of harvest, e.g. grain crops, and ' therefpre is less applicable to crops such as Zantedeschia, cassava (Manihot esculenta Cranb:. ; Boerboom, 1978), potato (Solanum tuberosum L. ; Menzel, 1985), taro (Colocasia ? esculenta (L.) Schott) and tannia (xanthosoma sagittifolium (L.) Schott; Caesar, 1980), which possess geophytic storage organs with indeterminate storage organ capacity. In situations where a number of harvests are carried out over time, the proportion of the daily increment in total dry weight gain partitioned into tuber growth, i.e. , tuber weight partitioning (TWP, equation (4. 1)) may be calculated. While known as different terms by different authors, such as ?efficiency of storage root production? , and ?distribution ratio: this method of calculation of partitioning provides a more sensitive measure of short term changes in assimilate partitioning than does the harvest index (Boerboom, 1978; Keating et al., 1982). The importance of TWP is that it can be used as an improved indicator of the mechanisms of assimilate partitioning and to account for the way in which partitioning might be manipUlated by environmental influences. 1WP _ &T/&t &W/&t (4.1) where; TWP = tuber weight partitioning T = tuber dry weight W = total plant dry weight t = time o = mathematical notation for an interval 83 In its simplest form, two possible situations can be envisaged in the control of assimilate accumulation within a plant: either the system is source limited, or the system is sink limited (Wareing and Patrick, 1975). Which one is limiting under any particular circumstance will be dependent on both genotype and environment, and their interaction (Gifford et al. , 1973; Farrar, 1992). In proposing this model where either source or sink limitation can exist, it is assumed that assimilate movement between source and sink is non? limiting. While in most cases examined this assumption is valid (Wareing and Patrick, 1975; Minchin and Thorpe, 1992), it was not the intention of the current experiment to investigate this aspect. If sinks control partitioning of dry weight, then the corollary is that it is those factors which determine the initiation and relative activity of sinks which determine the pattern of assimilate distribution. The initiation of the storage organ in commercial selections of onion (Allium \ upa L.), potato, and tulip (Tulipa gesneriana L.) has an obligative requirement fo! specific photoperiods and/or temperatures (Garner and Allard, 1923; Niimi, 1978; EWing, 1987; Taeb and Alderson, 1990). In contrast, the initiation of the storage organ of other genera (e.g. , Beta), does not appear to be under obligative environmental control. Thickening of the storage organ in sugar beet (Beta vulgaris L.), comprised of the swollen hypocotyl and tap root, commenced soon after germination, and resulted from the successive initiation of cambia and associated production of parenchymatous tissues (Rapoport and Loomis, 1986). Although partitioning of assimilates between shoot and storage root of crops such as sugar beet followed an allometric relationship for the majority of the growth cycle, this was not applicable during the early stages of growth (McLaren, 1984). Milford et al. (1988) illustrated that, during early stages of growth, partitioning of assimilate gradually changed towards the storage root, and was not a sudden change as proposed by Green et al. (1986). Therefore the data of Milford et al. (1988) provided further support for the theory that initiation of storage organ growth in sugar beet was not under obligative environmental control. Initiation and activity of sinks is also dependent on the supply of photoassimilates at an earlier ontogenetic stage (Gifford and Evans, 1981). Increased sink initiation and activity through increased supply of photoassimilates can result from environmental manipulation 84 of photosynthetic activity (Gifford, 1977) , or removal of competitive sinks for assimilates (B iran et al., 1974) . While tuber initiation in potato is primarily dependent on photoperiod, this response was ameliorated by both the temperature and irradiance (B orah and Milthorpe, 1963; Menzel, 1985) . The inhibitory effects of long days or high temperatures were exaggerated by the reduced assimilate supply at low irradiance, resulting in a greater proportion of dry weight being allocated to the shoot. During active growth, partitioning of assimilates to storage organs may be influenced by environmental parameters such as PPF and temperature. Reduced partitioning of assimilates to storage organs under low irradiance (presumably PPF) has been reported with cassava (B oerboom, 1978) , potato (Menzel, 1985), taro and tannia (Caesar, 1980) . However, this partitioning was not influenced by irradiance where the storage organ consisted of a tap root (e.g. , sugar beet; Terry, 1968) , but appeared to be primarily a function of a time-dependent allometric relationship between the shoot and storage root (B arnes, 1979) . For most of the growing season an allometric relationship was also determined between total plant growth and storage root growth in cassava (B oerboom, 1978) . While in most cases the reduced partitioning to the storage organ under low PPF regimes resulted in reduced final yield (B oerboom, 1978; Menzel, 1985 ; Caesar, 1980) , members of the Araceae have been reported to be shade tolerant, achieving greater storage organ yield under low PPF regimes. From field experiments Valenzuela et al. (1991) reported increased corm yield in tannia when the daily int?grated PPF was reduced from 35 mpl?m?2?day?1 (i.e. , full sun) to 17.5 or 10.5 mol?m?2?day?1 (i.e. , 50% and 70% shading, respectively). Under the lower PPF regimes the increased total plant and corm yield was attributed to more light interception through increased leaf canopy development, and hence increased leaf area index. This occurred even though net photosynthetic rate was less under the lower PPF regimes than under the high PPF regime (Valenzuela et al. , 199 1). In contrast Caesar (1980) reported increased total plant dry weight but reduced corm yield when plants of taro and tannia were grown under 80% shade (daily integrated PPF = 12 mol?m?2?day-1) compared with full sun. Since total plant dry weight was greater under the low PPF regime, it is unlikely that this PPF regime seriously limited growth, as postulated by Valenzuela et al. (199 1). However, as Valenzuela et al. (199 1) did not detect differences in corm weight until 210 days after planting, it is possible the contradicting result of Caesar (1980) , reported after 146 days from planting, merely reflects some temporal function, i.e., delayed corm growth but subsequent increased leaf area duration under low PPF. While the objective of using varying PPF regimes has been to manipulate source strength, low irradiance, which has been shown to inhibit tuber growth in potato, also greatly increased the gibberellin-like substances in leaves (Woolley and Wareing , 1972) . Since application of gibberellic acid to potato plants inhibited tuber growth (Menzel, 1985) , and 85 application of the chemical growth regulators gibberellic acid, indoleacetic acid and cytokinin enhanced storage root growth in sugar beet (Das Gupta, 1972), the assumption that alteration of PPF only influences source strength may not be valid. While total plant dry weight generally increases with increasing temperature up to some optima, partitioning of assimilates towards the storage organ declines over that same temperature range (Ulrich, 1952 & 1955; Terry, 1968; Boerboom, 1978; Keating et al. , 1982). However, as a result of increased total plant dry weight at higher temperature, final storage organ yield was maximum between 17 and 24 C for sugar beet and at 24 C for cassava. Without any evidence to support the existence of an obligative environmental trigger for initiation of tuber growth in Zantedeschia (refer Section 1 .5.3), it is suggested that the growth and development of sugar beet may provide an adequate preliminary model for comparison. However, in contrast to the tap root of sugar beet, the tuber in Zantedeschia is a stem morphologically (refer Section 1 .3 .1) . Since a functional equilibrium of assimilate partitioning exists between the morphological shoot and root of plants (Richards and Rowe, 1977; Bames, 1979), the application of a dry matter partitioning simulation model for sugar beet, such as that developed and ?te:d b.r_ Fick et al. (1973), would therefore have to take cognizance of this potential difference in assimilate partitioning. With this in mind it is pertinent to summarise the hypotheses upon which the model of Fick et al. (1973) is based, i.e. ; \ ? (1) that storage beet growth and sugar accumulation requires an excess of photoassimilates beyond the needs for respiration and for growth of tops and fibrous roots (Ulrich, 1952 & 1955). (2) that the relative effects of reserves on sink activity differs for various sinks as a function of reserve level, i.e. , intersink competition increases as the reserve supply decreases , and the development of the storage structure, as well as the filling of the structure with sugars, all receive a lower priority than foliage development (Terry, 1968) . . (3) that a functional equilibrium exists between partitioning of assimilates and relative gro? rates .of the shoot and root (Barnes, 1979). (4) that respiration rates, growth and activity of sinks are functions of the amount of tissue capable of growth and of solar radiation (presumably PPF and temperature; Terry, 1968). 86 Preliminary studies on Zantedeschia suggested that leaf area development and duration are important in determining tuber growth (Funnell and MacKay, 1987; Warring ton and Southward, 1989) , but no detailed analysis of dry matter partitioning and how it might be influenced by temperature and PPF have been made. Therefore the objective of this study was to examine the influence of temperature and PPF on growth of tubers of Zantedeschia 'Best Gold ; in terms of timing and extent of assimilate partitioning. 4.2 Materials and Methods Data presented in this chapter were derived from the experiment described previously (refer Section 3 .2) . Cultural procedures (refer Section 3 .2 . 1) and environmental conditions (refer Section 3.2 .2) were the same. Tubers from each treatment were harvested throughout development and dried. to constant weight. A loge transformation of the dry weight data was used to meet the statistical assumption of homogeneity of variance throughout the period of study (Causton and Venus, 198 1) . Transformed data, collected during the period of increasing tuber dry weight, were fitted to the Gompertz function , equation (4.2) (Causton and Venus, 1981) , utilizing the non-linear regression parameter estimation procedure of the Statistical Analysis System, i .e. , SAS (SAS Institute Inc. , Cary , N.C.) . The Gompertz function was chosen over other sigmoidal growth functions due to the derivation of more biologically relevant parameters, its wide acceptability by other researchers (Causton and Venus, 198 1 ; Hunt, 1982) , and its ability to fit the current data-set with smal l mean square error values. A constant was added to all transformed data'. prior to curve fitting to eliminate negative values. Because the Gompertz function is asymptotic, the magnitude of the added constant influences the calculated value of {3T. Hence the addition of a constant to all values prior to curve fitting eliminated any value of subsequent interpretation of differences in the value of {3T. log eT where; logeT = loge transformed value of tuber dry weight (loge g) aT = upper asymptote of tuber dry weight (loge g) {3T = a measure of the starting size of tuber dry weight (loge g) KT = rate constant of tuber dry weight as a function of size (g?g?1 -g.1) t = time (days) e = the base of natural logarithms (4.2) 87 During the period of increasing tuber dry weight, the tuber relative growth rate (RGRT), as a function of time, was calculated using the first differential of equation (4.2) i.e., equation (4. 3) (Causton and Venus, 1981) . RGRr (4.3) Tuber weight partitioning (TWP; equation (4 . 1)) , i.e., the proportion of the daily increment in total plant dry weight partitioned into tuber growth, was calculated using equation (4.4) utilizing derived formulae from equations (3 .4) , (3 . 7) , (4.2) and (4. 3). TWP - where; RGR-r X e (log e T(t)) (t) W ? total plant dry weight (g) \ ' (4.4) The value of t used in the calculation of TWP was the time (days from planting) of maximum RGRT, which corresponds to the inflection point of the Gompertz curve, i.e., equation (4.5) (Causton and Venus, 1981) . t - (4.5) The time of commencement of tuber growth was estimated as the minimum of the fitted quadratic function describing changes in logverlielf 89 Changes in the dry weights of various components of the plant followed a sigmoidal pattern, with the commencement of net tuber growth occurring later than that of the other plant components (Figure 4. 1). Higher temperatures and higher PPF resulted in the chronologically earlier commencement of tuber growth, but the extent of the initial decline in tuber weight was greater at higher temperatures and at low PPF (Figure 4.2). In contrast, commencement of tuber growth was delayed morphologically at higher temperature; tuber growth commenced when only one fully expanded leaf was present on plants grown at 13 C compared with four expanded leaves on plants grown at 28 C (data not presented). Different PPF regimes had no effect on the morphological timing of commencement of tuber growth. While maximum tuber weight was not attained during the period of study, the estimated maximum tuber weight was greater under low PPF than high PPF regimes irrespective of temperature. 30 ?20 0> ........ 1-:r: (!) w 3: ? 0. 1 0 l ' ' ' 0 20 40 60 80 1 00 DURATION OF GROWTH (Days) USER 1 20 1 40 Figure 4.1 Shoot, tuber, root and total dry weight per plant of Zantedeschia 'Best Gold' plants grown at 25 C under high PPF. n=6 or 12. 3 2 28 C HIGH PPF 1 6 C HIGH PPF -3?--,----r--?--?----?--,----r--?--? 0 ? ? ? M 1 00 1 ? 1 ? 1 ? 1 M DURATION OF GROWTH (DAYS) Figure 4.2 Tuber dry weight (expressed as logJ of Zantedeschia 'Best Gold' plants grown under high or low PPF at 16 or 28 C. ' indicates day of transfer to treatment environments. 90 4.3.2 Commencement of tuber growth 91 Tuber weight declined after transfer to treatment environments. Duration of this decline was longer under low PPF than under high PPF for all temperatures (P :S 0.05, Figure 4.3). A negative curvilinear relationship existed between time to commencement of tuber growth (tJ and increasing mean temperature (C) under the low PPF regime (P :S 0.01 , equation (4.6)). While a negative curvilinear relationship also existed under the high PPF regime between 13 and 19 C, no difference in the time to commencement of tuber growth was evident between 19 and 28 C (P :S 0.0001 , Figure 4.3). Plants grown under high PPF at temperatures of 19 C or greater commenced tuber growth 36 days from planting. In contrast, plants at 13 C and low PPF commenced tuber growth 52 days after planting (Figure 4.3) . tc - 0.05?C2 - 2.7 ?C + 79 -01 >. 60 ?SO ?--........ LOW PPF ? ;, . ?---c?-... ?-?. -----?-.. ?-?-----.. -<. ----. [] ? 40 ----- --- ----------0 ---c I-W :::::< i= 1 3 HIGH PPF 1 6 1 9 22 MEAN TEMPERATURE (C) 25 28 Figure 4.3 Time to commencement of tuber growth (tc) as a function of temperature for Zantedeschia 'Best Gold: grown under high and low PPF regimes. (4.6) 92 There was a greater loss in tuber weight prior to the commencement of tuber growth in plants grown under low PPF than under high PPF at all temperatures (P s 0.05, Figure 4.4). A positive curvilinear relationship existed between the proportion (%) tuber weight loss at the commencement of tuber growth (T ") and increasing temperature (C) under low (P s 0. 10, equation (4.7)) and high (P s 0.01 , equation (4.8)) PPF regimes. (Low PPF) (High PPF) 1 00 ........ 80 ? (/) (/) 0 ...J ..... I (!) G:i 3: 0:: w CD ::J 40 ..... 1 3 T? - -0.09?C2 + S?C + 9 T? - -0.03?C2 + 3 ?C + 15 LOW PPF HIGH PPF 1 6 1 9 22 MEAN TEMPERATURE (C) 0 25 (r2 - 0.79;) (4.7) (r2 - 0.95, ? ?) (4.8) 0 28 Figure 4.4 Extent of tuber weight loss (T ") at the time of commencement of tuber growth, as a function of temperature, for Zantedeschia 'Best Gold: grown under high and low PPF regimes. 4.3.3 Curve fitting of tuber growth Under all treatment regimes lo? tuber weight followed a sigmoidal pattern of growth, with a family of Gompertz functions adequately describing the progression of tuber weight (lo? transformed) d?? the period of its increase (Figure 4.5 and Figure 4.6, Table 4.2). 3 ? c 2 ? -3 ?------.---.----r---r---.---.---.---.---. 1 0 30 50 70 90 1 1 0 1 30 1 50 1 70 1 90 2 1 0 DURATION OF GROWTH (DAYS) Figure 4.5 Fitted Gompertz curves and mean data points of lo? tuber dry weight (minus constant) as a function of time, for Zantedeschia 'Best Gold' at a range of temperatures, under high PPF regime. :? I? I 0 w ? >- 3 2 ? 0 0::: w CO ? -1 Cl> 0 0 ...J -2 25 C I J C 1 0 30 . 50 70 90 1 1 0 1 30 1 50 1 70 1 90 2 1 0 DURATION OF GROWTH (DAYS) Figure?4.6 Fitted Gompertz curves and mean data points of lo? tuber dry weight (minus constant) as a function of time, for Zantedeschia 'Best Gold' at a range of temperatures, under a low PPF regime. 93 94 Table 4.2 Nonlinear least-aquares parameter estimate&, IUIIIOCiated uymptotic ltandard error (1.e.), and mean square error values, from fitting the Gompertz function to loa. transformed tuber dry weight data for Zantedeschi4 'Best Gold' grown at a range of temperatures, under high and low PPF regimes. The PPF treabnenta at 19 C were repeated in two plantings, i.e., (ht) and (2nd). Mean mean temperature PPF a/ 1.e. fJT 1.e. ICT 1.e. square (C) error 13 High 2. 12 0.09 4.52 1 . 18 0.048 0.012 0.17 Low 2.55 0.17 3 . 13 0.70 0.031 0.007 0.29 16 High 2.79 0.25 2.80 0.41 0.037 0.007 0.19 Low 4.49 0.61 2.79 0.40 0.029 0.006 0.29 19 (1st) High 3.24 0.09 3.23 0.28 0.059 0.005 0.09 Low 3.97 0.16 4.07 0.42 0.051 0.006 0. 13 19 (2nd) High 3.40 0.20 2.89 0.32 0.040 0.005 0. 15 Low 4.54 0.21 3.32 0.34 0.041 0.005 0. 18 22 High 4.64 0.37 2.66 0.22 0.035 0.004 0. 13 Low 5.89 1 .05 2.93 0.40 0.032 0.007 0.25 25 High 4.60 0.20 3.28 0.24 0.048 0.004 0.1 1 Low 5.82 0.47 3.72 0.38 0.043 0.005 0. 16 28 High 4.30 0.20 3 .66 0.34 0.058 0.006 0.10 Low 5.14 0.43 3.03 0.30 0.042 0.006 0. 12 Values of aT presented are those resulting from addition of a constant to avoid negative values of log. transformed data. 95 Differences between the fitted curves of tuber weight were principally the result of differences in the estimated maximum tuber dry weight attained (aT) . There was a cubic increase of estimated maximum tuber weight (aT) with increasing temperature (C) for both PPF regimes (P < 0.001). Under the high PPF regime this relationship with temperature was highly significant (P s 0.00 1 , equation (4.9) , Figure 4 .7) , but the relationship was significant under the low PPF regime only after exclusion of the value estimated for 16 C (P < 0.05 , equation (4. 10) , Figure 4.7) . (High PPF) a T - -0.0035 ?C3 + 0.02 ?C 2 - 3.7 ? C + 22 (Low PPF) a T = -0.003?C 3 + 0. 19 ? C 2 - 3 ?C + 19 4 3 ex T 2 1 3 1 6 1 9 22 MEAN TEMPERATURE (C) 25 28 Figure 4. 7 Loge maximum tuber dry weight (aT) as a function of temperature, for Zantedeschia 'Best Gold' under high and low PPF regimes. (nb . , value of added constant removed) (4.9) (4.10) The value of the parameters {3T (an estimate of tuber dry weight at day 0) and KT (rate at which RGRT declined as a function of tuber size) were not influenced by temperature nor PPF regime. Mean values for {3T and KT across all environments were 3 . 3 + 0. 1 and 0.042 + 0.002 , respectively. However, as noted in Section 3.2, the need to add a 96 constant to all values prior to curve fitting eliminated any value of subsequent interpretation Of differenCeS in the ValUe Of {3T? 4.3.4 Maximum tuber weight Estimated maximum tuber weight was greater under the low PPF regime than under the high PPF regime at all temperatures (P < 0.00 1 , Figure 4.7) . From equation (4. 10) maximum aT [3 .29 (i.e. , 26.83 g) + 0. 1] was attained under the low PPF regime at 24.5 ? 0. 1 C. This was more than 1. 7 times that attained under the high PPF regime [2. 72 (i.e. , 15 . 17 g) ? 0.08] at the same temperature. The optimal temperature under the high PPF regime was estimated to be 24.9 + 0. 1 C (equation (4.9)) . While the optimal temperatures under each PPF regime were significantly different, the magnitude of the differences can not be considered to be of biological significance. aT decreased at 28 C under both PPF regimes (Figure 4.7) . 4.3.5 Base temperature for tuber growth At the time tuber relative growth rate (RGRT) was maximal, i .e. , inflection point of the fitted tuber dry weight Gompertz curve, a positive linear relationship existed between RGRT and temperature up to 28 C under the high PPF regime, and up to 25 C under the low PPF regime (Figure 4 . 8). A reduction in the value ofRGRT was noted at 28 C under the lQw PPF regime and this data was therefore not included in the determination of the linear\ regressions. Examination of the slopes of the linear regressions for homogeneity (Za.r, '1984) did not detect differences between PPF regimes (P ? 0.05) , but the point of interception of the Y -axis of the two functions were different (P < 0.05). Extrapolation of the two linear functions allowed an estimation of the base temperature for tuber growth of 4 . 8 + 2.7 C under high PPF (P < 0.00 1 , equation (4. 1 1)) , and 6 . 1 ? 2 . 6 C under low PPF (P < 0.01 , equation (4. 12)) . Pooling data from both PPF regimes resulted in an estimated base temperature for tuber growth of 4.6 ? 1 . 1 C. (High PPF) RGR.r = 0.0039?C - 0.018 (r2 = 0.93, ? ? ?) ?- (Low PPF) RGR.r ... 0.0049?C - 0.03 (r2 - 0.90, * *) (4. 11) (4. 12) ? '>-0 ;J:J I ? 0. 1 0 0.08 0.06 ?0.04 0\ ....... a:: ? 0.02 0 c HIGH PPF 4 7 1 0 1 3 1 6 1 9 22 25 28 ?EAN TE?PERATURE (C) Figure 4.8 RGRT as a function of temperature, for plants of Zantedeschia 'Best Gold: grown under high and low PPF regimes. (N.B. , line for low PPF excludes 28 C) 4.3.6 Tuber and leaf weight partitioning 97 From the time of commencement of tuber growth, the proportion of the daily increment in total plant weight that was partitioned to the tuber, i.e. , tuber weight partitioning (1WP), increased throughout the period of observation (Figure 4.9 and ?Figure 4. 10). Use of TWP proved more sensitive in detecting the early stages of partitioning dry weight to I the tuber than ?e frequently used harvest index. Harvest index, i.e. tuber dry weight as a fraction of total plant dry weight, continued to decline until well after the commencement of tuber growth, e.g. at 28 C under high PPF the harvest index did not increase until more than 45 days after planting (data not presented). In contrast, under these environmental conditions an increase in tuber dry weight was clearly detected after 36 days from planting (Figure 4.3). 4 1 0 30 50 70 90 1 1 0 1 30 1 50 1 70 1 90 2 1 0 Dl.JRATION OF GROWTH (Days) Figure 4.9 Proportion of daily increment in total weight partitioned to the tuber (fWP) as a function of time, for Zantedeschia 'Best Gold; at a range of temperatures, under a high PPF regime. 4 3 ...... '>- ? 2 1;7> :;--.. >, 0 ? 1;7> ........ 1 a. ? 0 1 0 28 c I t ! d ,// 22 c 1 3 c 30 . 50 70 90 1 1 0 1 30 1 50 1 70 1 90 21 0 DURATION OF GROWTH (Days) Figure 4.10 Proportion of daily increment in total weight partitioned to the tuber (fWP) as a function of time, for Zantedeschia 'Best Gold; at a range of temperatures, under a low PPF regime. 98 99 The magnitude of 1WP increased earlier, both chronologically and morphologically (i . e . , leaf number; data not presented) , under the high PPF regime than under the low PPF regime at all temperatures (Figure 4. 1 1) . However, at both 25 C and 28 C this earlier onset of increased 1WP under high PPF was subsequently surpassed, with the TWP under the low PPF regime exceeding that achieved under high PPF after approximately 90 and 1 10 days from planting , respectively. ......... I>, 0 4 3 ? 2 0> :::-... '>-0 " -5!; 28 C LOW PPF 1 3 C HIGH PPF 1 0 30 50 70 90 1 1 0 1 30 1 50 1 70 1 90 21 0 DURATION OF GROWTH (DAYS) Figure 4.11 Proportion of daily increment in total weight partitioned to tuber (fWP) , as a function of time, for Zantedeschia 'Best Gold; grown under high and low PPF regimes at 13 or 28 C. 100 At the time tuber relative growth rate (RGRy} was maximal, i.e. , inflection point of the fitted tuber dry weight Gompertz curve, a curvilinear decline occurred in the value of the TWP with increasing temperature (P s 0.0001 , equation (4. 13) , Figure 4. 12) , with no difference being detected between PPF regimes. Similarly, a curvilinear decline in the proportion of the daily increment in total plant weight that was partitioned to leaf area, i.e. , leaf weight partitioning (L WP), with increasing temperatures of 16 C and above was determined under the high PPF regime (P s 0. 1 , equation (4. 14), Figure 4. 13). A reduction in the value ofLWP was noted at 13 C under the high PPF regime. In contrast to the high PPF regime, under the low PPF regime there was a linear decline in the value of the LWP with increasing temperature (P s 0. 1 , equation (4. 15}, Figure 4. 13). 1WP - 0.0016?C2 - 0.08?C + 1.2 "' '>. 0.5 0 .4 ? O.J 01 ::-... ? "0 01 ...... o.2 1 3 0 c c 0 1 6 1 9 22 25 MEAN TEMPERAT'JRE (C) c 28 FigUre 4.12 Proportion of daily increment in total weight partitioned to the tuber (TWP), as a function of temperature, for Zantedeschia 'Best Gold' under high (-0-) and low (-0-) PPF regimes. (4.13) (High PPF) LWP - 0.00l ?C2 - 0.06?C + 0.9 (r2 - 0.78,-) (4.14) (Low PPF) LWP - -0.002?C + 0.19 (r2 - 0.47/) (4.15) 0.22 0 0.20 HIGH PPF ? '>. 0 0. 1 8 ? 0 . 1 6 [] :(?:. -------- 0 ? ------? 0. 1 4 LOW PPF -?----? [] 0 ? 0. 1 2 ...J +- 1 3 c [] [] 0. 1 OT-??.--?--.-??----.??--.-??-. 1 3 1 6 1 9 22 25 MEAN TEMPERATURE (C) 28 Figure 4.13 Proportion of daily increment in weight partitioned to leaf (LWP) as a function of temperature, for Zantedeschia 'Best Gold' under high (-0-) and low (-0-) PPF regimes. (N.B. , line for high PPF excludes 13 C) 4.3. 7 Net assimilation rate 101 A positive linear relationship existed between NAR and increasing temperature at both PPF regimes, but the magnitude of the slope under high PPF was more than' four times greater (P s 0.01 , equation (4. 16), Figure 4. 14) than that under low PPF (P s 0.05 , equation ( 4 . 17) , \Figure 4. 14). The relationship between NAR and temperature under high PPF was only linear at temperatures of 16 C and greater. (High PPF) NAR - 0.6?C - 4 (Low PPF) NAR - 0.14?C + 3.8 (4.16) er - o.6s,?> (4.17) 1 3 1 1 ? 7 z +- 1 3 C _____ -a--? __ .-- 0 0 HIGH PPF 0 0 0 -----? ---------- ----- ___ .?------ - ---- LOW PPF 8 0 0 ST-??.-????????-.??-. 1 3 1 6 1 9 22 25 28 MEAN TEMPERATURE (C) Figure 4.14 Net assimilation rate (NAR) as a function of temperature, for plants of Zantedeschia 'Best Gold; grown under high (-0-) and low (-0-) PPF regimes. (N.B . , line for high PPF regime excludes 1 3 C) 4.3.8 Relationships between derived parameters 102 Examination of the partial correlations between the parameters (fable 4 . 1) , at the time tuber relative growth rate (RGRT) was maximal, i . e. , inflection point of the fitted tuber dry weight Gompertz curve, indicated multicolinearity between parameters. A reduced range of parameters (fable 4 .3) were subsequently used for development of a possible model. Across all treatments RGRT was negatively correlated with TWP and LWP, and positively correlated with NAR (Table 4.3) . Standardized multiple regression coefficients (beta coefficients) for each of the three parameters indicated the relative contribution made by each in determining RGRT, independent of the others and unitless, i . e. , TWP = -0.70, LWP = -0. 3 1 , and NAR = 0. 19. However, NAR did not meet the 0. 15 significance level for entry into the final model (equation (4. 18)) . RGRT = -0. 16 ?TWP - 0.3 ?LWP + 0. 14 (4. 18) Table 4.3 Partial correlation matrix between TWP, L WP, NAR and RGRT at the inflection point of the Gompertz fit of tuber dry weight curves of Zantedeschia 'Best Gold' grown under a range of environments. RGRT TWP LWP NAR RG? 1 .00 -0.82 -{).52 0.33 TWP -0.82 1 .00 -{).45 0. 1 1 LWP -0.52 -0.45 1.00 -0. 17 NAR 0.33 0. 1 1 -{). 17 1 .00 103 A negative curvilinear relationship existed between RGRT and TWP across both PPF regimes (P S 0.01, equation (4. 19) , Figure 4. 15). However, under the low PPF regime there was a high partial correlation between TWP and LWP (r = -0.96). In light of this, two possible models were subsequently developed under the low PPF regime. Firstly, at temperatures of 16 C and greater a negative linear relationship was determined between RGRT and LWP (P s 0.001 , equation (4.20), Figure 4. 16). The further addition ofNAR to this function did not improve the model. RG? - 0.5 ?TWP2 - O.S ?TWP + 0.16 \ ' ' RG? - -0.69 ?LWP + 0. 167 (4.19) (4.20) 0. 1 0 0.08 ?0.06 ;,... 0 ;p I . ?0.04 ? a:: ? 0.02 OD 0 0 0 o.oo,__?---,---?--r--?----.--?-..,. 0.1 0 .2 0 .3 -1 -1 . TWP (g.day /g.doy ) 0.4 0.5 F.agure 4.15 Relative growth rate of the tuber (RGRT) as a function of daily partitioning to the tuber (TWP) for Zantedeschia 'Best Gold' grown under high (-0-) and low (-0-) PPF regimes at all temperatures. 0 . 1 0 0.08 ?0.06 ;,... 0 ;p ??0.04 ? a:: ? 0.02 0 0 0 0 0 t 1 3 c o.oo,_?--.--?-.-?-.-?-.-?-,..--?--.- o. 1o 0. 1 2 0. 1 4 0.1 6 0. 1 8 _, _, LWP {g.day /g.day } 0.20 0.22 Figure 4.16 Relative growth rate of the tuber (RGRT) as a function of daily partitioning to the leaf (LWP) under high (-0-) and low (-0-) PPF regimes, at all temperatures. Line is for low PPF regime at temperatures ? 16 C. 104 105 Secondly as an alternative model a negative linear relationship was determined between RGRT and TWP under the low PPF regime at temperatures of 16 C and greater (P s 0.01 , equation (4.21)). As with the previous model the addition of NAR did not significantly improve the model. RG? ? -0.17 ?TWP + 0. 11 (rl - 0.73;?) (4.21) In contrast, under the high PPF regime, there was no significant correlation between RGRT and LWP (Figure 4.16) , but a positive linear relationship was determined between RGRT and NAR at temperatures of 16 C and greater (P s 0.001 , equation (4.22), Figure 4. 17) . No relationship was detected between RGRT and NAR under the low PPF regime treatments. 0 . 10 0.08 0 0 0 0.06 ,..... i>. 0 ;SJ I . ?0.04 ? 0 0::: ? 0.02 \ 0.00 5 0 7 0 0 t 1 3 c 9 -2 -1 NAR (g.m. day ) 0 1 1 1 3 Figure 4.17 Relative growth rate of the tuber (RGRT) as a function of net assimilation rate (NAR) under high (-0-) and low (-0-) PPF regimes, at all temperatures. Line is for high PPF regime at temperatures ? 16 C. RG? - 0.0073?NAR + 0.001 (4.22) The further addition of TWP to the model improved the fit and accounted for the additional variability of RGRT at 13 C under high PPF (P s 0.01, equation (4.23)). However, the addition of LWP or RLWR did not meet the 0. 15 significance level for entry into the model. RG? - 0.006-NAR - O.l l ?TWP + 0.04 (4.23) 106 Under the high PPF regime the beta coefficients for each of the parameters in the model indicated the relative Contribution made by each in determining RGRT, independent of the other, i.e. , NAR = 0.56 and TWP = -0.53. 4.4 Discussion 107 Tuber growth occurred under all environmental regimes tested (Figure 4 . 5 and Figure 4.6) . Even though at planting all plants possessed tubers, and not all possible environmental parameters were examined, the data presented here did not support the hypothesis of an obligative environmental trigger for the commencement of tuber growth. The proportion of the daily increase in total plant dry weight partitioned to the tuber (IWP) followed a gradual increase with time (Figure 4.9 and Figure 4 . 10) . In contrast, an abrupt change would have been expected if environmentally determined induction was involved (Milford et al., 1988) . With increasing temperature the onset of tuber growth was chronologically earlier (equation (4 . 6), Figure 4 . 3) but delayed morphologically. The additional delay and extent of tuber weight loss prior to tuber growth under the low PPF regime, suggested intersink competition rather than the existence of an obligative environmental trigger for the commencement of tuber growth. With some crops, where evidence exists for environmental triggers, e.g. short photoperiod promotion of tuberization in dahlia, tuberization occurred regardless of photoperiod once competing sinks were removed (Biran et al. , 1974) . In Zantedeschia, respiration, root growth and leaf canopy development, can be viewed as potential competing sinks for assimilates. This scenario of a non? obligative environmental trigger for the commencement of tuber growth in Zantedeschia is, therefore, similar to that found in storage organ development of sugar beet (Fick et al. , 1973 ; Rapoport and Loomis, 1986) , and is in contrast to the obligative environmental requirements that exist with crops such as potato, onion, and tulip (Gamer and Allard, 1 923 ; Niimi, 1978 ; Ewing, 1987) . The strong negative relationship between RGRT and TWP under both PPF regimes at the time of maximum RGRT (equation (4. 19) , Figure 4 . 15) is indicative of the tuber, as a sink, receiving a higher priority for assimilates than other organs. This interpretation is illustrated by the increased partitioning of the daily increment in total dry weight to the tuber, as conditions for growth became less optimal (e.g. cooler temperature) (Figure 4 . 1 2) . This change in partitioning to the storage organ with temperature is similar to that reported for cassava, being a negative curvilinear decline with increasing temperature, to the maximum tested, i.e., 24 C (Keating et al. , 1982) . Assimilate partitioning toward the storage organ in sugar beet was also increased as temperatures departed from the optimum of 24 C (Ulrich, 1952 & 1955 ; Terry, 1968) . While at this stage of development a negative relationship also existed between LWP and increasing temperature (equations (4. 14) and (4. 15) , Figure 4 . 1 3) , the magnitude of the TWP was always greater than that of the LWP, and therefore indicates greater sink strength of the tuber over that of the leaf canopy. This increase in TWP with declining temperature was nbf a- direct result of increased source nor sink activity as both photosynthetic rate (refer Chapter 5) and RGRT (Figure 4 . 8) also declined with declining temperature. Clearly this priority of tuber growth over leaf development, at the time of maximum RGRT, was not 108 evident in the early stages of tuber growth. Just as the magnitude of the 1WP increased over time, so the magnitude of the L WP decreased over time. At the commencement of tuber growth the magnitude of the TWP was small, while L WP was at least 5 times greater. The time dependent nature of the TWP exhibited here with Zantedeschia is different from the allometric relationship found between growth rates of the shoot and root, in root crops such as sugar beet and carrot (Bames, 1 979), and total plant growth and storage root growth in cassava (Boerboom, 1978) . In light of this difference, any attempts to create a predictive, deterministic model of tuber growth in Zantedeschia will have to include this dynamic aspect of assimilate partitioning between organs. The strong correlation between RGRT and LWP under the low PPF regime (equation (4. 20) , Figure 4 . 1 6) is indicative of the mechanism of acclimation to the low PPF regime and resultant intersink competition for limited assimilates. The mechanism of acclimation, i .e. , enhanced leaf canopy development, has been introduced previously (refer Chapter 3) , and is a response recorded for other crops (Boerboom, 1978; Caesar, 1980; Menzel, 1985; Valenzuela et al. , 1991) . Any enhancement in the relative partitioning of dry matter to one organ, e.g. leaves, will be at the expense of other organs within the same system. Hence under this low PPF regime temperatures conducive to greater tuber growth were associated with reduced daily partitioning to leaves, with an increased proportion of assimilates having already been allocated to leaf canopy development, (Figure 4 . 1 6) . Interpretation of this relationship and its effect on actual tuber dry weight must also take into account the dynamic nature of the partitioning of assimilate distribution. At the time of maximum RGRT plants grown under the low PPF regime had already established a I great?r leaf area than under the high PPF regime (refer Chapter 3). Therefore, while ' temperatures conducive to greater tuber growth were associated with reduced partitioning to leaves, the photosynthetic activity of the enhanced leaf area was able to more than compensate for the low PPF conditions. As a net result greater values of RGRT were attained under the low PPF rather than high PPF regimes at most temperatures (equations (4. 1 1) and (4 . 12) , Figure 4 . 8) . The reduced RGRT at 28 C under the low PPF regime (Figure 4 . 8) was associated with an increase in the L WP from that obtained at 25 C. Since a reduction in RGRT at 28 C did not occur under the high PPF regime, it is suggested that source limitation was the primary determinant of this reduction in RGRT under the low PPF regime. This source limitation may have resulted from respiration having a greater priority for available assimilates than either tuber or leaf growth (Ulrich, 1952 & 1955) . The higher priority of respiration has been suggested and utilized in assimilate partitioning simulation models (Fick et al. , 1973). The failure of the 13 C environment to illustrate this correlation between RGRT and LWP was presumably not as a result of reduced photosynthetic capacity or leaf canopy development, as neither the addition of NAR nor RLAER significantly improved the 109 model. At 13 C the value of the TWP diverged from the negative linear relationship between RGRT and TWP that existed at temperatures of 16 C and greater (i.e. , equation (4. 19) compared with equation (4.21)) . At the same time the value of the LWP attained was less than that predicted from equation (4.20). While the presence of multicolinearity between RGRT, TWP and L WP, under the low PPF regime, negates the value of interpreting a model comprising all three parameters (Zar, 1984), it is suggested that a low temperature-mediated response in assimilate partitioning may explain the aberrant result. Such an enhancement in the value of TWP might be expected at low temperatures as a survival mechanism in warm season perennial crops originating from temperate/warm? temperate climes, as it supports the theories of evolution of the geophytic storage organ as a means of avoiding periods of adverse climate (Aoba, 1976; Rees, 1984). The strong correlation between RGRT and NAR (equation (4.22), Figure 4. 17) and the poor correlation between RGRT and LWP (Figure 4. 16), under the high PPF regime, is in contrast to the complete lack of correlation between these parameters under the low PPF regime. This contrast indicates differences in the primary mechanism of acclimation to the two PPF regimes. Under the low PPF regime tuber growth was primarily determined by increased intersink competition for assimilates resulting from the enhanced leaf canopy development. However, under the high PPF regime increased tuber growth was primarily determined by the enhanced assimilatory capacity of the leaf area present, i.e. , NAR. Unit increases in NAR with increases in temperature were four times greater under the high PPF regime than under the low PPF regime, reflecting the ability of the leaf area to i more effectively utilize the incoming PPF (refer Chapter 5). In addition to NAR, I differences in TWP contributed a similar, albeit negative, level of determination in the final model under the high PPF regime (equation (4.23)). The poor correlation of RGRT with L WP under the high PPF regime should not be interpreted ? suggesting that the partitioning of dry matter into leaf canopy development is not important, but rather the greater response of the unit increase of NAR outweighs any significant contribution of L WP in determining tuber growth under these conditions. The frequent breakdown of the above correlations at 13 C does not justify extrapolation of the proposed models to explain the lower predicted base temperature for tuber growth of 4.8 ? 2.7 C under the high PPF regime than the 6. 1 ? 2.6 C under the low PPF regime. The predicted base temperature from pooling data from both PPF regimes did not result in significant differences between that estimated from RGRw against temperature (refer Chapter 3). The similarity of the optimal temperatures for growth rates of the storage organ and total plant growth is in contrast to sugar beet where the optimal temperature for growth rateS of the storage root was slightly lower than that for total plant growth (Terry, 1968). 1 10 While at the inflection point of the fitted dry weight curve increasing temperature was generally associated with a positive linear increase in RGRT (Figure 4.8), final tuber weight followed a cubic function with temperature (Figure 4.7). As reported for other species, leaf area duration may also have contributed to the prediction of greater maximum tuber dry weight under the low PPF regime (Radley, 1963; Rees, 1972; Allen and Scott, 1980). However, the contribution of leaf area duration was not investigated as the duration of the current study did not permit collection of data to the cessation of leaf development. As noted in Chapter 3 under the high PPF regime, the greater rate of decline of the leaf area and leaf weight expansion rates, as a function of size, in conjunction with the earlier onset of leaf senescence, are indicative of a reduced leaf area duration compared with under the low PPF regime. The maximum leaf area attained also influences leaf area duration. Under the high PPF regime the reduced maximum leaf area estimated at 28 C from that at 25 C (refer Chapter 3, Figure 3. 10) may therefore explain the reduced maximum tuber weight at 28 C (Figure 4.7). Leaf canopy development and storage organ growth were also restricted in sugar beet at a temperature of 31 C compared with 24 C (Terry, 1968). Although the response at temperatures intermediate to these have not been reported for sugar beet, it is suggested that the results presented in the current study indicate the existence of an optimal temperature for tuber growth similar to that for sugar beet storage root growth. The greater final tuber weight under the low PPF regime may nor be of relevance under all ?mmercial horticultural situations. With financial pressure growers seek crops and/or I prod?ction systems that require a short cropping period. Hence while tubers may have ' attained a larger size if grown at 25 C under low PPF, tuber weight under the high PPF regime would be greater than that under the low PPF regime until day 140 (i.e. , 5 months of growth) . Hence growers wishing to maximise tuber -weight, but requiring a shorter production period, would be more likely to meet these goals by utilizing a high PPF regime. This time dependent aspect of tuber weight being greater under low PPF regimes may also explain the difference in results reported for other members of the A.raceae e.g. , taro and tannia (Caesar, 1980; Valenzuela et al. , 1991). When taro was cultivated under a comparable temperature regime (22 to 32 C day) and PPF (shaded to 350 Jtmoi-m-2?s-1) Caesar (1980) reported reduced storage organ weight under the low PPF regime after 146 days from planting. Hence, if the final harvest had been carried out later, storage organ yield may have been greater under the low PPF regime, as presented here and as also reported by Valenzuela et ai.. (1991). 1 1 1 4.4.1 Conclusions Tuber growth in Zantedeschia appears to have been source limited, at least to some extent. Source limitation was expressed either in terms of: 1) enhanced intersink competition for assimilates, as occurred under the low PPF regime through enhanced leaf canopy development, 2) assimilatory capacity of the leaf canopy, i.e. , NAR, as occurred under the high PPF regime. However, at the same time, the correlation between RGRT and TWP cannot be ignored. Whether the calculation of TWP merely provides an empirical description of dry matter distribution under source control, as is one interpretation of the correlation between TWP and LWP under low PPF regimes, or whether situations where true sink limitation exist, cannot be determined from the data presented here. Certainly with storage root crops such as sugar beet, carrot and cassava, a constant functional equilibrium exists between source and sink (Boerboom, 1978; Barnes, 1979). While such an equilibrium does not exist in Zantedeschia, the priority of the tuber as a sink, over other organs, indicates that situations of sink limitation may also exist. Examples of sink limited systems have been reported for other crops, e.g. ovule growth in peas where pod warming increased ovule growth and assimilate transport to the developing sinks (Williams and Williams, 1978). Similarly warming of the tap root of sugar beet increased the importation of assimilates (Geiger and I Fondy, 1985)?? In addition, the possibility of both source and sink limitation within the same crop, but under differing environmental situations, has previously been acknowledged ? (Gifford et al. , 1973). Since the manipulation of both temperature and PPF may potentially alter both source and sink activity, further experiments will be required to elucidate which is more limiting. 1 12 4.5 References -- Allen, E.J. and R.K. Scott. 1980. An analysis of growth of the potato crop. J. Agr. Sci. (Cambridge) 94:583-606. Aoba, T. 1976. Effect of temperature on the seasonal periodicity of bulb formation in plants. Environ. Control in Bioi. 14:81-90. Barnes, A. 1979. Vegetable plant part relationships. TI. A quantitative hypothesis for shoot/storage root development. Ann. Bot. 43:487-499. Biran, I. , B. Leshem, I. Gur and A. H. Halevy. 1974. Further studies on the relationship between growth regulators and tuberization of Dahlias. Physiol. Plant. 31 :23-28. Boerboom, B.W.J. 1978. A model of dry matter distribution in cassava (Manihot esculenta Crantz). Neth. J. Agr. Sci. 26:267-277. Borah, M.N. and F.L. Milthorpe. 1963. Growth of the potato as influenced by temperature. Indian J. Plant Physiol. 5:53-72. Caesar, K. 1980. Growth and development of Xanthosoma and Colocasia under different light and water supply conditions. Field Crops Res. 3:235-244. i Ca?ston, D.R. and J.C. Venus. 1981 . Single leaf growth and the Richards function: methodology, p. 87-143. In: The biometry of plant growth. Edward Amold, London. Das Gupta, D.K. 1972. Developmental physiology of sugar-beet. IV. Effects of growth substances and differential root and shoot temperatures on subsequent growth. J. Expt. Bot. 23: 103-1 13. Ewing, E. E. 1987. The role of hormones in potato (Solanum tuberosum L.) tuberization, p. 515-538. In. P.J. Davies (e(i.). Plant hormones and their role in plant growth and development. Martinus Nijhoff Publishers, Dordrecht. Farrar, J.F. 1992:-The whole plant: carbon partitioning during development. p. 163-179. In: C.J. Pollock, J.F. Farrar and A.J. Gordon (eds.). Carbon partitioning within and between organisms. Bios Scientific Publishers, Oxford. 1 13 Funnell, K.A. and B.R. MacKay. 1987. Effect of GA3 on growth of Zantedeschia. Tech. Rpt. 87/14, Dept. Hort. Sci. and N.Z. Nursery Res . Centre, Massey Univ. , Palmerston North, N.Z. Fick, G.W. , R.S . Loomis and W.A. Williams. 1973. Computer simulation of dry matter distribution during sugar beet growth. Crop Sci. 13:413-417. Gamer, W.W. and H.A. Allard. 1923. Further studies in photoperiodism. The response of the plant to relative length of day and night. I. Agr. Res . 23:871-920. Geiger, D.R. and B.R. Fondy. 1985. Responses of export and partitioning to internal and environmental factors. British Plant Growth Regulator Group, Monograph 12: 177-194. Gifford, R.M. 1977. Growth pattern, carbon dioxide exchange and dry weight distribution in wheat growing under differing photosynthetic environments. Australian I. Plant Physiol. 4:99-1 10. Gifford, R.M. , P.M. Bremner and D.B. Iones. 1973. Assessing photosynthetic limitation to grain yield in a field crop. Australian I. Agr. Res. 24:297-307. Gifford, R.M. and L.T. Evans. 1981. Photosynthesis, carbon partitioning, and yield. Annu. R?v. Plant Physiol. 32:485-509. I Gifford, R.M. , J.H. Thome, W.D. Hitz and R.T. Giaquinta. 1984. Crop productivity and photoassimilate partitioning. Science 225:801-808. Green, C.F. , L.V. Vaidyanathan and J.D. lvins. 1986. Growth of sugar-beet crops including the influence of synthetic plant growth regulators. I. Agr. Sci. 107:285- 297. Hunt, R. 1982. Plant growth curves. The functional approach to plant growth analysis. Edward Amold, London. Keating, B.A. , J.P. Evenson and S. Fukai. 1982. Environmental effects on growth and development of cassava (Manihot esculenta Crantz.) m. Assimilate distribution and storage organ yield. Field Crops Res. 5:293-303. McLaren, J.S. 1984. Investigation of quantitative relationships in dry matter distribution between tops and storage roots in sugarbeet. Ann. Bot. 54:383-390. 1 14 Menzel, B.M. 1985. _Tuberi.zation in potato at high temperatures: Interaction between temperature and irradiance. Ann. Bot. 55:35-39. Milford, G.F.J. , K.Z. Travis, T.O. Pocock, K.W. Jaggard and W. Day. 1988. Growth and dry-matter partitioning in sugar beet. J. Agr. Sci. 1 10:301-308. Minchin, P .E. H. and M.R. Thorpe. 1992. Carbon-11 in the study of phloem translocation. p. 225-247. In: C.J. Pollock, J.F. Farrar and A.J. Gordon (eds.). Carbon partitioning within and between organisms. Bios Scientific Publishers, Oxford. Niimi, Y. 1978. Influence of low and high temperatures on the initiation and the development of a bulb primordium in isolated tulip embryos. Scienti.a Hort. 9:61- 69. Radley, R.W. 1963. The effect of season on growth and development of the potato, p. 211-220. In: J.D. lvins and F.L. Milthorpe (eds.). The growth of the potato. Butterworths, London. Rapoport, H.F. and R.S. Loomis. 1986. Structural aspects of root thickening of Beta vulgaris L. : Comparative thickening in sugarbee? and . . chard. Bot. Gaz. ' 147:270-277. \ Rees, \A.R. 1972. The Growth of Bulbs. Academic Press , London. Rees, A.R. 1984. Dormancy, flowering and periodicity in ornamental bulbs. The Plantsman 6(1):33-44. Richards, D. and R.N. Rowe. 1977. Effects of root restriction, root pruning and 6- benzylaminopurine on the growth of peach seedlings. Ann. Bot. 41:729-740. Taeb, A.G. and P.G. Alderson. 1990. Effect of low temperature and sucrose on bulb development and on the carbohydrate status of bulbing shoots of tulip in vitro. I. Hort. Sci. 65: 193-197. Terry, N. 1968. ?velopmental physiology of sugar beet. I. The influence of light and ?temperature on growth. I. Expt. Bot. 19:795-811 . 1 15 Ulrich, A. 1952. The influence of temperature and light factors on the growth and development of sugar beets in controlled climatic environments. Agron. 1. 44:66-73. Ulrich, A. 1955. Influence of night temperature and nitrogen nutrition on the growth, sucrose accumulation and leaf minerals of sugar beet plants. Plant Physiol. 30:250- 257. Valenzuela, H.R. , S.K. O'Hair and B. Schaffer. 1991. Shading, growth, and dry-matter partitioning of cocoyam [Xanthosoma sagittifolium (L.) Schott]. J. Amer. Soc. Hort. Sci. 1 16: 1 1 17-1 121 . Wareing, P.F. and J. Patrick. 1975. Source-sink relations and the partition of assimilates in the plant, p. 481-499. In. J.P. Cooper (ed.). Photosynthesis and productivity in different environments. Cambridge Univ. Press, Cambridge, U.K. Warrington, I.J. and R.C. Southward. 1989. Influence of temperature and light intensity on Zantedeschia growth and development. Commercial Rpt. No. 46, Plant Physiol. Div. , DSIR, Palmerston North, N.Z. Williams, A.M. and K.R. Williams. 1978. Regulation of movement of assimilate into ovules of Pisum sativum cv. Greenfeast. Australian J. Plant Physiol. 5:295-300. \ \ ' Woolley, D.J. and P.F. Wareing. 1972. Environmental effects on endogenous cytokinins and gibberellin levels in Solanum tuberosum. New Phytol. 71: 1015-1025. Zar, J.H. 1984. Biostatistical analysis. 2nd ed. Prentice Hall, New Jersey. 1 16 5 PHOTOSYNTHETIC ACTIVITY OF Zantedeschia 'Best Gold' IN RESPONSE TO TEMPERATURE AND PHOTOSYNTHETIC PHOTON FLUX 5.1 Introduction The importance of photosynthesis to plant growth is self-evident since 90-95% of the dry weight of plants is derived from photosynthesis (e.g. lrvine, 1975). However, while photosynthesis is accepted as being of fundamental importance, the relationship between harvestable yield and net photosynthesis is not always clear, with variations in growth rate and yield not always being shown to relate to variation in net photosynthesis (Lambers, 1987). Assuming the importance of photosynthesis in determining plant growth and harvestable yield in Zantedeschia, modelling of such processes will require description of photosynthetic characteristics and how these might change with environment and development. Use of the term photosynthetic rate has generally referred to rates obtained on a sub-sample of leaves on a plant, and often fails to take account of leaf age, arrangement or angle. In many cases estimates of photosynthetic rate are limited to defining rate per unit area on a recently expanded leaf. However, a close relationship exists between the increasing rate of photosynthesis as the leaf expands, and the change of leaf activity from a net importer to a net exporter of assimilates as the leaf develops and expands. During this period of development the photosynthetic rate increased, reaching a m?mum at between 70% and 80% . of final leaf area with sugar beet (Beta vulgaris L. ; Giaquinta, 1978), 95% ?with soybean (Glycine max L. Merrill; Woodward, 1976), and closer to 100% final leaf area with taro (Colocasia esculenta Schott; Sato et al. , 1978) and cocoy? (Xanthosoma sagittifoliwn Schott; Schaffer and O'Hair, 1987). Following the attainment of maximum photosynthetic rate, the rate declines with age. Any experiments to define the photosynthetic characteristics of Zantedeschia would therefore need to be conducted with consideration of the influence of leaf expansion and maturation. Leaves within the upper canopy typically contribute a proportionately larger quantity of the total plant photoassimilates that are accumulated than those within the lower canopy (Acock et al. , 1978; Hodanova, 1979). While the potential photosynthetic activity of leaves is reduced with age, mutual shading of leaves within the canopy is also a contributing factor to this disproportionate contribution (Hodanova, 1979). The photosynthetic photon flux (PPF) at which photosynthetic rate becomes light saturated varies between species and growing environment; with photosynthetic activity acclimating to the environment in which the leaves have grown (Bowes et al. , 1972; Pallas and Samish, 1974; Bjorkman, 1981). _ Shade tolerance, where the photosynthetic rate is greater when leaves have been grown under shade compared with full sunlight, is a feature of a number of members of the A.raceae (O'Hair and Asokan, 1986; Schaffer and O'Hair, 1987). Bjorkman (1981) 1 17 suggested that obligate shade plants have an intrinsically low potential for PPF acclimation and particularly a low ability to increase their capacity for effective utilization of high PPF. However, at the same time obligate shade plants are more efficient at utilizing PPF when PPF is low than plants adapted to sun habitats. Even within the same species clones which have adapted to shade habitats exhibit differing acclimation ability than clones from sun habitats, as might normally be expected between sun and shade species (Bjorkman and Holmgren, 1966). With several tropical tree seedlings grown at a range of PPF, the increase in photosynthetic rate at increased PPF was associated with increased leaf thickness (Kwesiga et al. , 1986). The effect of low PPF on many species is to cause a reduction in respiratory loss and to decrease specific leaf weight (Boardman, 1977, Kwesiga et al. , 1986). Photosynthetic rate is dependent on both PPF and temperature. Photosynthetic rate increases with rising temperature up to an optimum which depends on growing environment and species (Warren-Wilson, 1966a). Both short-term and long-term preconditioning effects of temperature on photosynthetic rate of plants have been noted in many species (Bennett et al. , 1982; Bunce, 1985; Laing, 1985; Caulfield and Bunce, 1988). However, while some minor differences exist between plant species in the relationship between photosynthetic rate and day and night temperatures, the temperature at the time of measurement had the greatest single effect on photosynthetic rate (Bennett et al. , 1982). With most species, leaf thickness and therefore specific leaf weight values decrease with an increase in temperature (Laing, 1985). Such changes usually result in changes in photosynthetic d.te per unit leaf area. I The photosynthetic rate can be altered significantly by manipulating sink strength. In a number of crop species, once new sinks have been initiated or sink strength increased, photosynthetic rate has increased several fold (Moorby, 1968; Chatterton et al. , 1972; Habeshaw, 1973; Thome and Koller, 1974). In addition, such increases in photosynthe_tic rate have been correlated with increased assimilate export from the leaf to the developing sink. Conversely the removal of major sinks or reduction in sink activity has resulted in a lowered demand for assimilate, and a reduction in net photosynthesis (Habeshaw, 1973; Thome and Koller, 1974; Wang and Breen, 1986). In contrast Geiger (1976) found that manipulation of sink demand, or of export rate, in beans (Phaseolus vulgaris L.) resulted in no observed adjustment in the rate of net photosynthesis. Obviously a simple, negative feed-back mechanism does not seem to be adequate to bring about the control between sink demand and net photosynthesis in all cases. If changes in sink activity of Zantedeschia were to result in changes in photosynthetic rate, then it would be most likely that those leaves with the greatest potential photosynthetic rate (i.e. , at or near full expansion and without mutual shading) would exhibit the greatest measurable change in this parameter. 1 18 Under controlled environmental conditions several plant species show a decrease in photosynthetic rate during the day (Pallas, 1973; Warrington et al. , 1977). In contrast constant photosynthetic rates during the entire photoperiod have been reported for other species (Pallas, 1973; Massimino et al. , 1980; Mortensen and Moe, 1983). A negative correlation between assimilate level and photosynthetic rate during the photoperiod has been determined in a number of the cases describing decreased photosynthetic rates, being suggestive of feed-back inhibition (Chatterton et al. , 1972; Warrington et al. , 1977). However this correlation has not been found to be universally applicable, varying between species and with leaf maturity (Potter and Breen, 1980; Correia et al. , 1990). Since it is generally agreed that differences in PPF result in differences in photosynthetic rate, and therefore production of photoassimilates, the ability to detect differences between treatments in the experiments outlined in the current study, may be dependant on the time within the photoperiod at which photosynthetic rate is measured. The rates of those processes in which assimilates are utilized (i.e. , respiration and new growth) fall more rapidly with decrease in temperature than do the rates at which assimilates are produced (Warren-Wilson, 1966b; Verkleij and Challa, 1988; Acock et al. , 1990). At lower temperatures, therefore, assimilates such as starch will tend to accumulate. However, this may not happen with all species, especially those adapted to cooler temperatures (Paul et al. , 1990). Hence, if changes in PPF result in changes in photoassimilate production, and differing temperature regimes influence assimilate parti?oning, then measurement of assimilate concentrations and photosynthetic rates would be pc)?ntial tools to determine the mechanisms of assimilate acquisition and partitioning in Zaiuedeschia. There are no previously reported studies of the photosynth?tic characteristics of the genus Zantedeschia. Assuming the potential importance of photosynthesis in determining growth in Zantedeschia, such basic information and its possible correlation with total growth and harvestable yield will be required for determination of optimum growing environments, and for future development of crop simulation models. 5.2 Materials and Methods -- 1 19 Data presented in this chapter were primarily derived from the experiment described previously (refer Section 3 .2). Cultural procedures (refer Section 3 .2 . 1) and environmental conditions (refer Section 3.2.2) were as described in those sections. 5.2.1 ?rinlental 5.2.1.1 Photosynthesis as a function of leaf expansion Net photosynthetic rate and leaf area were monitored during expansion of the first leaf produced from plants grown under three treatments. Treatments used consisted of a day/night temperature regime of 22/16 C under both high and low PPF regimes, and 28/22 C under the high PPF regime. Photosynthetic rates were measured using an open system infra-red gas analyser (LCA-2, ADC Ltd. , Hoddesdon, England), with air obtained from outside the building for the inlet supply (350 ? 10 J.Lmol C02 per mol), calibrated against Wiscoff pumps. All photosynthetic measurements were carried out at the relevant treatment day temperature and PPF. The air supply was dried to a constant 20% relative humidity and pumped into the assimilation cuvette (Parkinson leaf chamber) at a rate ? of 0.625 litre?min?1 ? Net photosynthetic rates were calculated from the difference between inlet and outlet C02 concentrations as outlined in the LCA-2 user manual. Leaf area expansion over time was determined nondestructively by tracing the outline of selected leaves on paper following each photosynthetic measurement, and subsequently measuring I the area of ea?h tracing using a UCOR model 3 100 leaf area meter. Within each temperature x light treatment, plants were arranged in a completely randomized design. Net photosynthesis data were recorded from a random sample of 6 leaves at two day intervals, 8 h after the commencement of daily lighting, beginning from when the leaf lamina was free from enveloping protective sheath leaves, and continuing until further increases in leaf expansion were minimal. Partial correlations between; net photosynthetic rate, day temperature, night temperature, PPF, and proportion (%) maximum leaf area, were examined in the formulation of predictive models. Models were determined by regression analysis using the stepwise selection method of the REG procedure within the Statistical Analysis System (SAS; SAS Institute, Inc. , Cary, N.C.) 5.2.1.2 Photosynthetic rate as a function of duration from commencement of daily- lighting Photosynthetic rates of recently-mature leaves were monitored during a daily period of lighting, from plants grown under four treatments. Recently-mature was defined as the 120 stage when a leaf had attained a minimum of 75% expansion, and the experiment was carried out on the first such leaf on selected plants. Treatments consisted of day/night temper?ture regimes 16/10, 221 16 and 28/22 C under the high PPF regime, and 22/16 C under the low PPF regime. Photosynthetic rates were measured as outlined above except air flow rates were sometimes reduced to 0 .356 litre?min-1 to account for negative or minimal photosynthetic rates encountered. Data were recorded from a random sample of 6 plants, initially at 30 min intervals commencing 15 min prior to the commencement of lighting, and subsequently at more infrequent intervals across the 12 h period of lighting. Within each temperature X light treatment combination , plants were arranged in a completely randomized design. To avoid potential inherent problems arising from repeated measures, a different sample of 6 leaves was used from each treatment population at each time of measurement. Rates of change in net photosynthetic rate with time were determined by regression analysis using the REG procedure of SAS. 5.2.1.3 Photosynthetic rate as a function of photosynthetic photon flux At the time when the first leaf had reached maturity (as defined above) response curves for photosynthesis as a function of PPF were derived from a sample of 6 plants per treatment. Photosynthetic rates were measured 8 h after the commencement of daily lighting. PPF was measured using the quantum sensor on the LCA-2 apparatus. Measured leaves were seal? in the assimilation cuvette at the day temperature at which the plants were grown. The ?.rPF was then adjusted stepwise using spectrally neutral mesh screens to reduce PPF, and raising the height of the plant closer to the light source to increase PPF. Respiration was measured ? in the dark using black cloth to eliminate light from the cuvette, with reduced air flow rates as previously discussed. The photosynthetic rate and PPF data were fitted to a hyperbolic tangent function of the form: where; Pn = net photosynthetic rate (JLmol?m-2-s-1 or J.Lg ?g-1-s-1) Pmu = maximum photosynthetic rate at saturating PPF (JLmolm-2-s-1 or ?g ?g-1-s-1) Tanlz = hyperbolic tangent (5. 1) a = apparent quantum yield (i.e. , the initial slope, mol C02 per mol PPF or g C02 per mol PPF) PPF = PPF (JLmol?m-2 -s-1) R = respiration rate (JLmol-m-2?s-1) 121 This function was chosen above others reported in frequent use, such as the rectangular hyperbola, as the former offers parameters which are both mutually independent and biologically meaningful (Jassby and Platt, 1976). Specific leaf weights were calculated from destructive harvests carried out subsequent to recording photosynthetic rates. The data, both on a leaf area and leaf weight basis, were fitted by nonlinear regression analysis using the NLIN procedure of SAS. A11 6 replicates from a given treatment were fitted individually. Derived parameters from the curve fitting procedure were subjected to analysis of variance using the general linear models procedure ? of SAS. In addition to measuring actual rates of dark respiration as part of determining the PPF response curves, rates of dark respiration were also measured commencing 2 h after the termination of the lighting period. Plants used were derived from treatments consisting of day/night temperature regimes 22/10, 22/16, 28/16 and 28/22 C, under both high and low PPF. Respiration rates were measured as outlined above for photosynthetic rates, during the established night temperature and using the reduced air flow rate. Data were recorded from a random sample of 6 mature leaves. Rates of change in respiration rate as a function of actual leaf temperature, were determined by regression analysis using the REG procedure of SAS. In addition, rates were subjected to analysis of variance using the general linear models procedure of SAS. \ I ' 5.2.1.4 Photosynthesis during plant development Prior to the destructive harvesting of 6 or 12 plants used to monitor growth (refer Section 3.2.3) , the photosynthetic activity of the most recently-mature leaf was measured. Initial measurements were recorded at the time of recent-maturation of successive leaves. Once increases in tuber dry weight were detected, sampling occurred more frequently, but photosynthetic rate was always monitored on the most recently-mature leaf of plants sampled, 8 h after the commencement of daily lighting. Monitoring continued until the rate of total plant dry weight accumulation declined (refer Section 3 .2.3). At the time total plant relative growth rate (RGRw) and tuber relative growth rate (RGRT) were maximum, coefficients of correlation between net photosynthetic rates and both RGRw and RGRT were determined by correlation analysis using the CORR procedure of SAS. 5.3 Results 5.3.1 Photosynthesis as a function of leaf expansion 122 Net photosynthetic rate (Pn) increased with expansion of the leaf under all treatment environments (Figure 5. 1). Maximum photosynthetic rates did not occur until leaves had reached approximately 90% expansion regardless of temperature and PPF regime. At the stage of expansion subsequently defined as mature (i.e. , 75% expansion) , leaves had achieved 81% of the maximum photosynthetic rate. While described in more detail under Section 5.3.3, in brief, at maximum leaf expansion photosynthetic rates were greater at higher temperatures and under high PPF. 20 1 8 1 6 28/22 C HIGH PPF 1 4 1 2 1 0 i? .. 8 I E 0 6 22/1 6 C. LOW PPF E ..:; 4 d: 2 0 20 30 40 50 60 70 LEAF AREA (cm1) FigUre 5.1 Net photosynthetic rate (Pn) as a function of individual leaf area expansion, at selected temperature and PPF regimes. Mean values ? se. , n=6, cubic spline fit. ' represent 75% maximum leaf area. 123 Across all treatments Pn was positively correlated with: day temperature (DTEMP), night temperature (NTEMP), PPF, and percentage maximum leaf area (%LA). The magnitude of the standardized multiple regression coefficients (beta coefficients) were indicative of the relative contribution made by each in determining Pn over the entire period of expansion, i.e. , %LA = 0.56, PPF = 0.37, and DTEMP = 0.34. However, the addition of NTEMP did not meet the 0. 15 significance level for entry into the model. A single predictive model including the above parameters was developed (equation (5.2)) which accounted for 78% of the variation in Pn, under the environments examined and throughout the entire period of expansion. Pn - 0.122?%LA + O.Ol l?PPF + 0.53?DTEMP - 18 (r2 - 0.78;??) (5.2) Over the entire period of leaf expansion a strong partial correlation existed between Pn and %LA (r = 0.76) . However, once leaves had reached 75% or more of their maximum leaf area this partial correlation was reduced to 0.46. In contrast, during this later stage of expansion the partial correlation between Pn and PPF was 0.72, and with DTEMP was 0.66. As a result, beta coefficients for the prediction of Pn were: %LA = 0.22, PPF = 0.55, and DTEMP = 0.45. As noted earlier, the addition of NTEMP to this model did not meet the 0. 15 significance level for entry. A single predictive model for this later ' stage of expansion was developed (equation (5.3)) which accounted for 81% of the variation in Pn under the environments examined. Pn - 0.06?%LA + 0.013?PPF + 0.55-DTEMP - 14 (r2 - 0.8 1, ? ? ?) (5.3) 124 5.3.2 Photosynthesis as a function of duration from commencement of daily lighting Net photosynthetic rate increased rapidly during the first hour of lighting (Figure 5.2; low PPF regime data not presented). Subsequent changes in net photosynthetic rates were minor for the remaining period of stable, temperature, vapour pressure deficit, and PPF conditions. Over this time period, the magnitude of the greatest difference in photosynthetic rate, i.e. , 1 .0 #Lmoi-m?2?s?1 , was only slightly greater than the estimated accuracy of the equipment, i.e. , 0. 7 #Lmol?m?2?s?1? Considering the potential for a decline in net photosynthetic rate purely as a result of repeated measures on the same leaf (Biddington, 1986) , it was felt that further interpretation of these data was not warranted. 1 2 28/22 c 1 0 8 6 i"! 4 .. I 1 6/ 1 0 c E 0 2 E ..:; &! 0 -2 - 1 0 1 2 J 4 5 6 7 8 9 1 0 1 1 1 2 DURATION FROM COMMENCEMENT OF LIGHT (h) Figure 5.2 Photosynthetic rate (Pn) as a function of duration from commencement of daily lighting, at selected temperature regimes and high PPF. Mean values ? se. , n=6. Broken lines indicate limits of diurnal temperature changeovers. 125 5.3.3 Photosynthetic rate as a function of photosynthetic photon flux Under all treatments the increasing rate of net photosynthesis with increasing PPF was adequately described by a family of Tanh functions on a basis of both leaf area (Figure 5.3a and Figure 5.3b) and leaf weight (Figure 5.3c and Figure 5 .3d). Data from plants grown at a common PPF and day temperature were pooled, as differences in the magnitude of the fitted parameters (as per equation (5. 1)), between treatments ?with the same day temperature were not significant (P < 0.05). (a) (c) 1 2 1 0 8 4 2 -- ---- 21 C / --:. ?? --------------------? 22 c 18 c /......---?_,;..,.?? -???? /,-?' u/ ;/ 1/ 0 ;l -2.'-----.---.----.--..------,---. 12 10 I! 4 0 200 400 800 800 1000 1200 PPF 4-tmol.m-?.s-1) ---- ---28 C /-_-:::.-::'------------------------- 22 c // 1 6C / I 0 200 400 800 800 1 000 1200 PPF 4-tmol.m-?.s-') (b) (d) i'! ' "' j l 1 2 1 0 8 a v--s==-=-=-=-=-=-=-? ?: ? 4 . ne . 2 0 12 10 I! 0 200 400 800 1300 1000 1200 PPF 4-tmol.m-?.s-') -- ---------2a c / --------------------------?--------- 22 c /_..-? 1 8 C a 1/ ' 4 lu" :/ -2'-----r---.----.--..,.--?..------.- 0 200 400 800 800 1000 1200 PPF 4-tmol.m-?.s-1) Figure 5.3 Photosynthetic rate (Pn) per unit leaf area (a) and (b), and per unit leaf weight (c) and (d) , as a function of photosynthetic photon flux (PPF), for plants of Zantedeschia 'Best Gold' grown at day temperatures of 16, 22 and 28 C, under high (a) and (c) , or low (b) and (d) , PPF regimes. n=6 or 18, function = equation 5. 1 . 126 Across all temperatures maximum photosynthetic rate (P maJ was greater under the high PPF regime than under the low PPF regime (P ? 0.001 , Figure 5 .4, Figure 5 .5) . Under the high PPF regime P m?x per unit leaf area increased linearly with increasing temperature, resulting in a 40% increase across the temperature range examined (P < 0.001). In contrast, under the low PPF regime, differences among temperatures in P mu. per unit leaf area were minimal, amounting to less than 1 pmoi-m?2?s?1 , i .e . , only 12% . When expressed on the basis of leaf weight, P mu. increased linearly with increasing temperature under both PPF regimes (P < 0.001 , Figure 5 . 5) . This response of PfllAX with temperature resulted in a maximum 53% and 3 1% increase across the temperature range, under the high and low PPF regimes respectively . ;., ?E 0 E 2> X 0 et 1 2 1 6 LOW PPF 1 9 22 25 DAY TEMPERATURE (C) 28 Figure 5.4 Maximum photosynthetic rate .(Pmax) per unit leaf area as a function of day temperature, for plants of Zantedeschia 'Best Gold' grown under high and low PPF. Vertical bars = 2 X standard error. i? ' "' j )( 0 6 E ll., 4 16 LOW PPF 19 22 25 DAY TEMPERATURE (C) 28 Figure 5.5 Maximum photosynthetic rate (Pmax) per unit leaf weight as a function of day temperature, for plants of Zantedeschia 'Best Gold' grown under high and low PPF. Vertical bars = 2 x standard ?error. 127 When expressed on the basis of both leaf area and weight, apparent quantum yield (a) was greater under the low PPF regime than under the high PPF regime (P ? 0.001 , Figure 5.6, Figure 5.7). Under the high PPF regime, the reduced value of a per unit leaf area at 16 C resulted in a quadratic relationship between apparent quantum yield and increasing day temperature (P ? 0.01). However, under the low PPF regime, a declined linearly with increasing day temperature (P < 0.05). On a leaf weight basis under the high PPF regime the quadratic relationship between a and temperature was even more pronounced, while under the low PPF regime a was independent of temperature (Figure 5. 7). 0.05 co.o -4 Cl. Cl. 0 LOW PPF ? O.OJh r----------?? --------? N > ! 0 r -02 % ? HIGH PPF l:S 16 19 22 DAY TDIPERATURE (C) 25 28 Figure 5.6 \ Quantum yield (a) per unit leaf area as a 'function of day temperature, for plants ared by diluting 0, 2 , 4, 6, 8 , and 10 ml of a 2 mgml-1 sucrose stock solution, to 10 ml with 62.5% methanol. Aliquots (4 ml) were removed and treated in the same manner as the unknowns to give standards equivalent to 0, 4, 8 , 12, 16, and 20% soluble carbohydrates on a dry weight basis, respectively. After 10 min. standing with occasional shaking, 5 ml chloroform was added, and the tubes capped and vortex mixed. Tubes were then briefly centrifuged to aid phase separation. Aliquots (50 JLl) were removed from the upper, aqueous phase and added to 1 ml of 5% (w/v) phenol and 4 ml 98% sulphuric acid (specific gravity 1 .84). Samples were stood to cool for 60 min. and absorbance read at 490 nm using a Hitachi, model 101 , spectrophotometer. Detennination of starch concentration The residual plant material following soluble carbohydrate extraction, and solution aspiration, ?as treated with 3 ml 100% methanol at 100 C for 5 min. This was repeated I and the wa.Shings discarded after centrifugation. Starch standards were prepared by suspending soluble starch (100 mg, AR; British Drug Houses; corrected for moisture) in 10 m1 water and placed in a boiling water bath until the suspension became translucent. This was diluted to 50 ml with water. Aliquots (0, 2 and 4 ml) of this stock solution were diluted to 4 m1 with water to give standards equivalent to 0, 4 and 8 mg starch. The culture tubes of starch standards and 4 ml aliquots of unknowns were capped firmly and heated for 60 min at 100 C to gelatinise the starch. After cooling, 2 ml sodium acetate buffer (0.25 M, pH 4.5) and 0. 1 ml amyloglucosidase (Boehringer Mannheim NZ Ltd; 2.5 mg?mi-1 protein in 25 mM sodium citrate, pH 6.0) were added and the samples vortex mixed and incubated at 55 C for 60 min. Samples were finally diluted to 10 ml with water, vortex mixed and centrifuged. Glucose concentration was determined by incubating 0.2 ml aliquots of the diluted hydrolysate in a final volume of 1 ml water with 2 ml glucose oxidase reagent at 37 C for 60 ? ? . A blank of water together with standards containing 25, 50, 75 and 100 JLg?mi-1 glucose were treated similarly. If the colour of the unknown samples was darker than the glucose standard of highest concentration, a 1 in 10 dilution was carried out. Hydrochloric acid (5 ml, 5 M) was added, samples vortex mixed and absorbances read at 151 540 nm using a Hitachi, model 101 , spectrophotometer. Glucose oxidase reagent was prepared by dissolving o-dianisidine hydrochloride (120 mg) in 600 ml tris-glycerol buffer, and dissolving in this 1 80 mg glucose oxidase (Sigma Chemical Company) and 1 8 mg peroxidase (Horseradish; Sigma Chemical Company). Tris-glycerol buffer was prepared by dissolving 30.2 g Tris in 500 ml water (pH 7 .0) and adding 330 ml glycerol (AR). 6.2.2.3 Starch and soluble carbohydrate concentration during plant development During plant development, total soluble carbohydrate and starch concentration of leaf and tuber tissue were determined using plants sampled from CE rooms (refer Chapters 3 and 4). Samples were taken following? grinding of the entire non-senesced leaf or tuber. Dried tissue was ground so as to pass a 0.5 mm screen. Due to restrictions on time, soluble carbohydrate and starch concentration data could only be collec_ted from three of the day/night temperature treatments (16/10, 22116, and 28/22 C) under both high and low PPF regimes. In addition, not all harvest dates were able to be analyzed for carbohydrates. Four individual plant replicates from each destructive harvest were analyzed for both soluble carbohydrates and starch using the methods outlined above. A lo? transformation of the dry weight data was used to meet the statistical assumption of homogeneity of variance throughout the period of study (Causton and Venus, 1981). Transformed tuber starch data, collected during the period of increasing tuber dry weight, were fitted to the Gompertz function, equation (6. 1) (Causton and Venus, 1981), utilizing the non-linear regression parameter estimation procedure of SAS. Justification for the use of this type of sigmoidal curve was as discussed previously (refer Section 3.2.3). A constant was added to all transformed data prior to curve fitting to eliminate negative values. Because the Gompertz function is asymptotic, the magnitude of the added constant influences the calculated value of {JT?? Hence the addition of a constant to all values prior to curve fitting eliminated any value of subsequent interpretation of differences in the value of {JT?? where; log.,Ts = log., transformed value of tuber starch dry weight ar. = upper asymptote of tuber starch dry weight Pr. = a measure of the starting size of tuber starch dry weight Kr. = rate constant of tuber starch dry weight as a function of size t = time e = the base of natural logarithms 152 (6.1) During this time period tuber starch relative growth rate (RGRr.) , as a function of time, was calculated using equation (6.2), being the first differential of equation (6. 1) (Causton and Venus, 198 1). RG?s (6.2) - Having determined tuber starch and soluble carbohydrate concentration, the non- carbohydrate residual, or tuber structural dry weight (T *) was calculated using equation (6.3). Log? transformed tuber structural dry weight data over the same time period were subsequently fitted to the Gompertz equation as described for tuber starch dry weight above. logeTs where; logi!Ts = log- a:: 0 ffi -1 (IJ ? ou -2 (.;) 0 _I -J -4 0 ,J. 20 40 60 80 o S T R U C T U R A L a S T A R C H 100 1 20 140 DURATION OF GROWTH (DAYS) 160 180 Figure 6.5 _ _ Total, structural and starch tuber dry weight (logJ, as a function of time, of Zantedeschia 'Best Gold' plants grown at 25 C under high PPF. ' indic_ates day of transfer to treatment environment. 6.3.3.2 Tuber starch concentration 158 During plant development, changes in tuber starch dry weight (e.g. Figure 6.5), and tuber starch concentration (Figure 6.7) , followed a pattern similar to that of total tuber dry weight (refer Chapter 4) . As with tuber dry weight, tuber starch concentration initially declined and subsequently followed a sigmoidal pattern of increase. The extent of the initial decline in tuber starch concentration was greater at higher temperatures and under low PPF (Figure 6.6) . Under the high PPF regime the minimum tuber starch concentration declined linearly with increasing temperature (P s 0.001), compared with a quadratic decline under the low PPF regime (P ? 0. 10) . Under the high PPF regime an approximate doubling of the temperature of cultivation, i.e. , 13 C compared with 25 C, resulted in more than a two-fold reduction in the minimum tuber starch concentration at the higher temperature. In contrast, under the low PPF regime, this same increment in cultivation temperature resulted in more than a six-fold reduction in minimum tuber starch concentration. While cultivation under the low PPF regime compared with under the high PPF regime resulted in less than a one-fold reduction in the minimum tuber starch concentration at 13 C, this reduction was increased to two-fold at 19 C, and three? fold at 25 C. '" 30 H I I H P P F ? .... .s::: 0'? ?o; ? 5'JD ? :I: (.) ?? a:: ;5 Vl ? 10 L O W P P F z ? ? 0 1 3 1 9 25 MEAN TEMPERATURE (C) Figure 6.6 Minimum tuber starch concentration as a function of temperature, for plants of Zantedeschia 'Best Gold' grown under high (-0 -) and low (-0-) PPF regimes. Mean values ? se., n =4. 110 (a) 70 .!!.. w40 ? ?30 ? 20 10 110 70 ?60 -: ?50 0 .!!.. ..,40 ? ?30 g 20 ? 1 0 0 0 (c) 0 so (e) 70 0 I T A R C H ? I O L U I L E 40 110 1 20 160 DURATION OF GROWTH (DAYS) I T A R C H ? I O L U I L E 40 110 1 20 160 DURATION OF GROWll-i (DAYS) I T A II C I I O L U I L E 40 60 1 20 160 DURATIOI< OF GROWTH (DAYS) 110 70 .!!.. 1::! 40 ? ?30 ? 20 10 0 200 0 110 70 .!!.. w40 ? ?30 g 20 ? 10 0 200 0 110 70 200 0 (b) (d) (f) I T A R C H I O L U I L E 40 110 120 160 DURATION Of GROWTH (DAYS) I T A II C H . I O L U I L E 200 40 110 120 160 200 DURATION OF GROWll-i (DAYS) I T A II C H I O L U I L E 40 110 120 160 DURATION OF GROWll-i (DAYS) 200 159 Figure 6. 7 Carbohydrate concentration of the tuber as a function of time, for Zaiuedeschia 'Best Gold' grown at three temperatures under high and low PPF regimes. (a) 13 C, high PPF (b) 13 C, low PPF (c) 19 C, high PPF (d) 19 C, low PPF (e) 25 C, high PPF (f) 25 C, low PPF. Mean values ? se. , n=4, ? indicates commencement of tuber growth. 160 Under all temperature and PPF regimes changes in tuber starch dry weight were correlated with changes in tuber structural dry weight. Under the high PPF regime this correlation was seen as a positive, linear relationship between lo? tuber starch dry weight and lo? tuber structural dry weight (equation (6.6), P s 0.001, Figure 6.8). Under the low PPF regime, once tuber growth had commenced, the same functional relationship was evident (P < 0.05). However, under the low PPF regime prior to the commencement of any increase in tuber dry weight, the relationship indicated proportionately larger reductions in tuber starch relative to structural dry weight (equation (6.7), P s 0.001). Within each of the two phases of development, examination of the slopes for homogeneity did not detect differences between temperature regimes (P s 0.05). Similarly, differences between temperature and PPF regimes were not evident once tuber growth had recommenced (P s 0.05). \ 3 ? ........... 2 ' C\ - I- 1 :J: (.!) w 0 3:: >-a::: -1 0 :J: U -2 a::: ? V) -3 a::: ? -4 :::> I- "' -s (.!) 0 ...J -s -7 -4 (,-2 - 0.86, ? ? ? ) log,Y - log,X3..s + 3.9 (,-2 - 0.74, ? ? ? ) AFTER TUBER INCREASE Y = 1 .36X - 0.06 0 0 0 BEFORE TUBER INCREASE Y = 3 .5X + 3.9 -3 -2 -1 0 . LOGe TUBER STRUCTURAL DRY WEIGHT (g) 2 FigUre 6.8 Tuber starch dry weight as a function of tuber structural dry weight Low PPF before ( -0-), and both high and low PPF after ( -0-) , - the -commencement of tuber growth, for plants grown at three temperatures and two PPF regimes. (6.6) (6.7) 161 The timing of initial increases in tuber starch weight and concentration coincided with the commencement of total tuber dry weight increments, with both weight and concentration subsequently increasing with duration (Figure 6. 7). While both tuber starch weight and concentration were greater under the high PPF regime at the commencement of tuber growth, by the termination of the experiment they were greater under the low PPF regime at both 19 and 25 C. This difference between PPF regimes, in final tuber starch weight and concentration, primarily resulted from a slower decline in the relative growth rate of starch accumulation relative to structural dry weight (Figure 6.9). Since carbohydrate determinations of tubers grown at 13 C were not carried out beyond 120 days; it can not be definitely stated that the same result would have occurred at this temperature regime at the final harvest after 200 days. However, if it is assumed that the linear relationship between tuber starch concentration and tuber dry weight was still applicable over this time period, this greater tuber starch weight and concentration under the low PPF regime would also have occurred at this temperature. 6.3.3.3 Tuber soluble carbohydrate concentration In contrast to tuber starch concentration, tuber soluble carbohydrate concentration increased slightly over the first few weeks from planting. However, over the entire period of growth tuber soluble carbohydrate concentrations remained relatively constant (Figure 6. 7). At mean temperatures of 19 and 25 C, brief periods of marked increases in concentration of soluble carbohydrate were detected. These increases in tuber soluble carbohydrate I concen?tion occurred at or soon after the commencement of increased tuber growth (Figure 6. 7) . Tuber soluble carbohydrate concentration was not influenced by PPF regime (P < 0.05), but tubers grown at 13 C contained less than half the concentration of soluble carbohydrates than those at 19 or 25 C (P :S 0.05). Tubers derived from 13 C contained an average 1 . 7 ? 0.5% soluble carbohydrate. In contrast tubers derived from 19 or 25 C contained an average 3.9 ? 0.4% . 0.12 (a) 0.10 0.08 a: !E 0.02 I T II U C T U II A L 0.00\----?--?--..----..----? 0 0.12 (c) 0 40 10 120 110 DURATION OF GROWTH (DAYS) 40 110 120 110 i I DURATION OF GROWTH (DAYS) 200 200 0.12 (b) 0.10 0.08 a: !E 0.02 0.12 (d) 0.10 0.08 T!i'0.06 ? "' .. ?0.04 a: !E 0.02 o.oo 0 40 10 120 110 DURATION OF GROWTH (DAYS) I T A II C M I T II U C T U II A L 40 110 120 110 DURATION OF GROWTH (DAYS) 162 200 200 Figure 6.9 \ Relative growth rate (RGR) of tuber starch and tuber structural dry weight as a function of time, for Zantedeschia 'Best Gold' grown at three temperatures under high and low PPF regimes. (a) 19 C, high PPF (b) 19 C, low PPF (c) 25 C, high PPF (d) 25 C, low PPF. 163 6.3.3.4 Leaf starch and soluble carbohydrate concentration Since minimal determinations of foliar starch concentrations were carried out on plant material grown at 13 C, description of the results will primarily pertain to data eollected from the 19 and 25 C environments. As with total plant leaf area and leaf dry weight (refer Chapter 3), the total quantity of leaf starch followed a logarithmic Gompertz pattern of increase with duration of growth. In later stages of growth, once leaf senescence commenced, total plant foliar starch declined. To avoid potential problems arising from mutual shading and ontogenic variation, but to enable some comparison between all treatments, leaf starch and soluble carbohydrate concentrations were compared at the first mature leaf development stage. Under both high and low PPF regimes a negative quadratic trend was determined between leaf starch concentration and increasing temperature (Figure 6. 10; P ? 0.05 and P ? 0.01, respectively), but the magnitude of these differences under the low PPF regime were small compared with under the high PPF regime. As a result of this interaction between temperature and PPF, leaf starch concentration tended to be greater under the high PPF regime, and this difference increased with increasing temperature. In contrast, while under the high PPF regime temperature did not influence leaf soluble carbohydrate concentration (P ? 0. 10), under the low PPF regime soluble carbohydrate concentration declined linearly with increasing temperature (Figure 6. 1 1 , P ? 0.01). 10 \ I \ a ' 13 HIGH PPf 18 WEAN l?WPERATUR? (C) Figure 6.10 Starch cqncentration of first leaf, as a function of temperature, for plants of Zantedeschia 'Best Gold' grown under high and low PPF regimes. Mean values ? se. , n=4. 10 a LOWPPF :lE 2 . ? e ? ? ? ? HQ! PPF 2 0 13 18 WEAN l?WPERATUR? (C) Figure 6.11 Soluble carbohydrate concentration of first leaf, as a function of temperature, for plants of Zantedeschia 'Best Gold' grown under high and low PPF regimes. Mean values ? se. , n =4. __ - Under the high PPF regime no trend was detected between total leaf carbohydrate concentration, i.e. , starch + soluble, and increasing temperature (P ? 0. 10), whereas a negative quadratic trend was detected under the low PPF regime (P ? 0.05). The 164 existence of this trend resulted from the reduced total carbohydrate concentration at 25 C compared with either 13 or 19 C (P s 0.05). With the exception of the incomplete data at 13 C, leaf starch concentration changed dramatically during plant development and with environment (Figure 6. 12) . Regardless of treatment environment the foliar starch concentration declined with duration of growth. With the exception of plants grown at 25 C under the low PPF regime, by the termination of the experiment foliar starch concentrations declined by more than 70% from that recorded at their maximum. For plants grown at 25 C under the low PPF regime foliar starch concentration had declined by only 33 % over that same period. With the exception of plants grown at 19 C under low PPF, soluble carbohydrate concentration in the leaf followed no consistent pattern of change (Figure 6. 12). In contrast, for plants grown at 19 C under low PPF, the foliar concentration of soluble carbohydrate declined abruptly after 80 days by approximately 50% . Considering the highly variable nature of the soluble carbohydrate data with duration of growth, it was felt that further interpretation of these data was not warranted. 6.3.4 Carbohydrate and structural dry weight concentration as predictors of growth and yield At the time of the first mature leaf, leaf starch concentration was positively correlated to increased photosynthetic rate per unit weight. While being significant, the magnitude of this correlation was small (r = 0.56), and when examined over the entire period of study, the correlation reduced even further (r = 0.22) . Under all treatment regimes the relative growth rate of total tuber dry weight (RGRT) was positively correlated with the relative growth rate of both tuber starch (RGRTJ and tuber structural weight (RGRTm). At the time RGRT was maximum, i.e. , the inflection point of the fitted total tuber dry weight curve, correlations attained were r = 0.97 and r = 0. 78, respectively. At the time RGRT was maximum, no significant correlations were detected between RGRT and neither the relative leaf starch weight rate (RLSWR), nor RGRT., RGRTm' or NAR. Similarly no significant correlations were detected between total plant relative growth rate (RGRw) and these growth parameters. ,. (a) .. :c -r,. ? ? ? ? I ? I O L U I L ? p :il ? I T A II C I ? .. .? (c) ?? .. .. (e) ?? .. .. . .. ... DURATION Of GROWTH (DAYS) .. ... I O L U I L I I T A II C H . .. DURATION Of GROWTH (DAYS) I O L U I L I I T A II C I .. ... ... DURATION Of GRoWTM (DAYS) - ... ... ?? (b) ?? I O L U I L ? I T A II C I .. ?? (d) ?? "' , .. t . .!!. ? ? . i . ? .. . . (f) ?? .. .. ... . .. DURATION OF GROWTH (DAYS) ' 10 . 1JO I O L U I L E I T A II C I . .. DURATION Of GROWTH (DAYS) I O L U I L E I T A II C I .. ... ... DURATION Of GROWTH (DAYS) 165 - ... ... Figure 6.12 Carbohydrate concentration of mature leaves as a function of time, for Zantedeschia 'Best Gold' grown at three temperatures under high and low PPF regimes. - (a) 13 C, high PPF (b) 13 C, low PPF (c) 19 C, high PPF (d) 19 C, low PPF (e) 25 C, high PPF (f) 25 C, low PPF. Mean values ? se. , n =4, ' indicates commencement of tuber growth. 6.4 Discussion 6.4.1 Specific soluble carbohydrates and sample preparation 166 The primary products of photosynthesis in higher plants are partitioned between two major metabolic pathways, one leading to starch synthesis within the chloroplast, and the other, to cytoplasmic sucrose synthesis and extracellular translocation (Wardlaw, 1968; Chen, 1 969; Ho and Rees, 1975; Giaquinta, 1978). Monosaccharides such as glucose and fructose are frequently found in plant tissue, being intermediaries in the metabolism of sucrose and starch. Sucrose, glucose and fructose were found to be present in both leaf and tuber tissue of Zantedeschia 'Best Gold' (Figure 6. 1 and Figure 6.2) . No attempt was made to determine the existence of additional soluble carbohydrates, following vacuum drying of tuber material, but the sum of the three determined by HPLC (4. 1 ? 0.2%) was not significantly different from that determined by non-specific chemical analysis (3.9 ? 0.4 %). This result is similar to that reported for two cultivars of taro, i.e. , 3.5 - 3 .9% carbohydrate, using other biochemical assays (Hashad et al. , 1956). It is therefore concluded that sucrose, glucose and fructose comprise the major forms of soluble carbohydrate in Zantedeschia 'Best Gold! Reduction in leaf sucrose concentration, as a result of vacuum drying, was associated with smaller than expected increases in glucose and fructose concentrations (Figure 6. 1). Therefore total soluble carbohydrate concentrations of the leaf can J:>e expected to be 29 % lower than ?f determined using fresh material. In contrast, no significant changes in soluble carb6hydrate concentration were detected with tuber tissue following vacuum drying I (Figure 6.2): The method of sample preparation can influence the resultant concentration of soluble carbohydrates and starch, the extent being dependent on the method used and plant genera (Haslemore et al. , 1980). Haslemore et al. (1980) noted that vacuum drying generally resulted in only marginally inferior recoveries of soluble sugars and starch, compared with immediate determination. As vacuum drying of samples was used throughout this study, being consistent across all treatments and harvests, it is assumed that any reductions in soluble carbohydrate concentration should not affect interpretation of results. 6.4.2 Diurnal and developmental changes in starch and soluble carbohydrate concentration Changes in starch and soluble carbohydrate concentrations, both during the daily lighting period and with plant development, indicate that carbohydrate concentrations within Zantedeschia 'Best Gold' result from highly controlled processes. As an indication of this control, soluble carbohydrates are some of the first derivatives of photosynthesis, but leaf 167 soluble carbohydrate concentration remained relatively unchanged, both during the daily period of lighting (Figure 6.3) and with duration of growth and development (Figure 6. 12). Similarly, diurnal changes in soluble carbohydrate concentration occurred in tuber tissue, but the relative constancy of concentration within tubers during growth and development (Figure 6. 7), is also indicative of concentrations being controlled. Regulation of both foliar and storage organ soluble carbohydrate concentrations has been associated with the activity of various enzymes (Giaquinta, 1978; Silvius et al. , 1979; Preiss, 1982; Morrell and ap Rees, 1986; Rao, et al. , 1990), and it is suggested that similar mechanisms of control may be operative within Zantedeschia 'Best Gold! Accumulation of foliar starch during the daily lighting period (Figure 6.3) , while concentrations declined with development (Figure 6. 12) , is indicative of the foliage being used as a temporary store of photoassimilates during the light period. Similar cases of temporary storage of photoassimilates have been noted with taro (Hashad et al. , 1956), barley (Hordewn vulgare L. ; Gordon et al. , 1980), sugar beet (Fondy and Geiger, 1982), lettuce (Lactuca sativa L. ; Fomey and Austin, 1988) and celery (Apiwn graveolens L. ; Davis and Loescher, 1991). While accumulating during the daily lighting period, export of stored photoassimilates toward the developing sinks recommenced during the night (Gordon et al. , 1980; Fondy and Geiger, 1982). Without the use of techniques such as radioactive labelling of photoassimilates, it can not be determined whether the increase in tuber soluble carbohydrate concentration during the light period (Figure 6.4) resulted from th? continued export of soluble carbohydrates from leaves and/or transitory interconversion of . starch to soluble carbohydrate within the tuber itself. The decline in tuber starch concentration, concomitant with the increase in concentration of soluble carbohydrate (Figure 6.4) is supportive of the latter hypothesis. Accumulation of foliar carbohydrates during the light period also suggests that the processes involved in the utilization of photoassimilates were proceeding at a rate less than the rate of production of photoassimilate, i.e. , sink limitation. In potato (Werner, 1935) and sugar beet (Rapoport and Loomis, 1985) conditions which restricted sink activity also resulted in the accumulation of foliar carbohydrates. Manipulation of source:sink ratios have not always resulted in accumulation of carbohydrate (Farrar and Farrar, 1987); in Zantedeschia 'Best Gold' the decline in concentration of foliar starch with growth, indicates non-limiting sink activity. However leaf age and mutual shading influence the ability of individual leaves to accumulate carbohydrates in other crops (Davis and Loescher, 1991), and therefore treating all leaves as having equal potential, as done in the current experiment, may invalidate this? interpretation. Foliar starch concentration increased by 65 % over the light period with constant 168 environment, but no change . in photosynthetic rate was detected (Figure 6.3) . At the completion of this time starch accounted for only 8 % of the leaf dry weight. Warrington et al . (1977) reported a progressive reduction in photosynthetic rate (21 % maximum reduction) with increasing foliar starch (maximum concentration 1 1 % of dry weight) . However, others have either found no correlation, or have reported starch concentrations above 20 to 30% before reductions in photosynthetic rate were detectable (Forde et al. , 1975 ; Geiger, 1976; Nafziger and Koller, 1976; Potter and Breen, 1980; Cao and Tibbitts, 1991) . In addition to variation in response between species, variation in response also occurred with leaf maturity (Potter and Breen, 1980). Hence the failure to detect a diurnal reduction in photosynthetic rate with increased foliar starch concentration in Zantedeschia 'Best Gold; may be attributed to the comparatively low starch concentrations, the unresponsive nature of Zantedeschia 'Best Gold' itself, and/or the stage of leaf maturity chosen. While it has been established that under any single treatment, diurnal increases in leaf starch concentrations were not associated with changes in photosynthetic rate, when treatments were pooled a positive correlation was found between these two parameters. This correlation was highest at the time of the first mature leaf, presumably due to the elimination of the confounding influence of variable leaf age (Davis and Loescher, 199 1). In addition, increased photosynthetic rates, resulting from increased PPF, C02, or temperature, have been associated with a proportionate increase in translocation of carbohydrates out of the leaf (Williams and Williams, 1978; Ho, 1979; Borchers-Zampini I et al. , 1980,; Geiger and Fondy, 1985 ; Farrar and Farrar, 1987) . Hence the increased photosynthetic rates, as a result of increased temperature and PPF, presumably resulted in both increased accumulation of photoassimilates within the leaf and increased translocation rates. In light of this, the relatively low correlation should not be interpreted as indicating any lack of association between photosynthetic rate and production of carbohydrates in the leaf, but rather the inability to quantify parameters such as the flux of carbohydrates using the current data. At a daily mean temperature of 13 C compared with higher temperatures, the low leaf starch and high soluble carbohydrate concentrations (Figure 6. 10 and Figure 6. 1 1) are supportive of the hypothesis that the rates of those processes in which assimilates are utilized (i .e. , respiration and growth) fall more rapidly with decrease in temperature than does the rate of photosynthesis (Warren-Wilson, 1966; Verkleij and Challa, 1988; Acock et al. , 1990) . However, with regard to the concentration of total leaf carbohydrate, only _ p?a?ts grown at 25 C under the low PPF regime had a significantly lower carbohydrate concentration. Hence in addition to increased translocation rates at higher temperatures (Williams and Williams, 1978; Geiger and Fondy, 1985) , the primary influence of 169 temperature on carbohydrate composition was the partitioning between forms, i.e. , soluble carbohydrates and starch (Wardlaw, 1968; Chatterton et al. , 1972; Yelenosk:y and Guy, 1977). Processes mediating the partitioning between soluble carbohydrate and starch are unknown, but enzymes such as fructose 2,6-bisphosphate have been implicated (Cseke et al. , 1984; Stitt et al. , 1984). Under most of the temperatures studied the insignificant differences between PPF regimes in total foliar carbohydrate concentration is similar to that reported for soybean (Glycine max L. ; Silvius et al. 1979), but different from that reported for other genera, where higher concentrations are generally associated with increased PPF (Graper and Healy, 1992). Differences between genera may be the result of differential ability for translocation of assimilates out of the leaf. The low leaf starch concentration at 25 C under the low PPF regime may reflect a low concentration of photoassimilates remaining after utilization for new growth and respiration (Grange, 1985). Under this treatment the extended period before the commencement of tuber growth (Figure 6. 7) also supports the hypothesis of that photoassimilate supply was limiting at this stage of growth. 6.4.3 Tuber starch and structural dry weight changes with development The fact that starch accounted for approximately 55% of tuber dry weight at planting (Figure 6. 7), and the correlation of subsequent changes with growth of the tuber, indicates that starch is the primary form of storage carbohydrate in Zantedeschia 'Best Gold? This result was similar to that reported for other members of the A.raceae, i.e. , taro and tannia (Xarithosoma sagittifolium Schott) (Hashad et al. , 1956; Brouk, 1975; Onwueme, 1978). I Once tuber growth had recommenced, the existence of a constant relationship between tuber starch and structural dry weights, under all treatment regimes (equation (6.6)) , indicates that starch deposition within the tuber was a controlled process. However, prior to tuber growth the alteration of this relationship under the low PPF regime (equation (6. 7)) indicates that while this process was controlled, in situations where the availability of newly-produced photoassimilate is unable to meet sink demand, the rate of remobilization of stored starch can be increased. A similar situation has been reported in soybean where only under conditions of limited assimilate availability was starch from the pod wall remobilized to enable continued seed growth (Fader and Koller, 1985). Also Davies (1984) was able to manipulate starch concentration of sprouting potato tubers by manipulating sink activity by varying shoot number. Both storage organ starch degradation and synthesis have been correlated with enzyme activity and orthophosphate concentration (Mares et al. , 1981 ; Preiss, 1982; Ou-Lee and Setter, 1985). Future examination of changes in these components may provide new insights to the mechanisms controlling growth and development in Zantedeschia, in particular changes in the relationship between starch concentration and tuber growth. 170 Under the low PPF regime the bi-phaseal relationship between tuber starch concentration and tuber structural dry weight (Figure 6.8) , is indicative of the tuber's transition from a source to a sink. However, there is no biological relevance to determining the point of intersection of the bi-phaseal relationship as a region of overlap of data points exists. Clearly, while separating the data into two phases of development has allowed the determination of two independent relationships, the extent of overlap of data points coupled with the inability to detect the bi-phaseal relationship under the high PPF regime, indicates that it would be inappropriate to infer the existence of any abrupt switch in the onset of tuber growth being related to the attainment of a specific starch concentration and/or vice versa. Rather than looking to the tuber for the point of control, it is more likely that the onset of tuber growth, reflected by increases in both structural and starch dry weights, was dictated by the establishment of a photosynthetic leaf area large enough to provide an adequate supply/flux of photoassimilates . to meet the demands for continued leaf and root development, and respiration. The sink-source transition of leaves commences at 40% to 50% leaf expansion (Wardlaw, 1968; Giaquinta, 1978) . However, tuber growth in Zantedesclzia 'Best Gold' did not commence until one or more leaves had reached maturity. In addition, plants grown at low PPF and increasing temperature carried a greater leaf number and associated leaf area, at the time of onset of tuber growth, than plants grown at high PPF and cooler temperatures. This illustrates the greater demand for assimilates by organs other than the tuber. In addition the negative relationship between RGRT and LWP (refer Chapter 4) also supports the hypothesis that tuber, growth receives a lower priority for photoassimilates than foliage growth at this early stage (Loomls et al. , 1979; \ Ho, 1988). i I During tuber growth the increase in the proportion of starch dry weight relative to structural dry weight and/or total tuber dry weight, was simil? to that reported for potato tubers (Plaisted, 1957) , tulip bulbs (Aung et al. , 1973) and corms of other members of the Araceae (Hashad et al . , 1956; Ching, 1970) . However, this change in partitioning with development was different from that reported for sugar beet where no change in the distribution of assimilates between storage root growth and storage carbohydrate (sucrose) occurred (Das Gupta, 1969; Watson et al. , 1972; Milford and Thorne, 1973 ; Milford et al . , 1988) . Watson et al. (1972) suggested this constancy in distribution disputed the earlier proposed hypothesis (Ulrich , 1952, 1955) that carbohydrate stored in the tap root is photosynthate in excess of that which can be used for the growth of the rest of the plant. This constancy in distribution was not found with Zantedeschia 'Best Gold; and while not necessarily supporting Ulrich' s hypothesis, the data presented here also does not dispute the validity of this hypothesis. The additional finding that the relationship can be manipulated under periods of high competitive sink demand (Figure 6. 8), adds some validity to the application of Ulrich 's hypothesis to Zantedeschia. 171 In reviewing the literature on starch storing organs, Jenner (1982) confirmed the intimate linkage between growth of storage organs and their capacity for storing starch. As found in the current experiment, Jenner concluded ?that for any given genotype the proportion of dry matter contributed by starch does not vary with the size of the organ as much as does the size of the organ itself'; i.e. , large organs contain more starch than small ones. If correct, this would suggest that cell number will be a major determinant of tuber growth and the amount of starch stored in Zantedeschia 'Best Gold! With phytohormones being implicated in the control of cell division in storage organs such as potato tubers (Lovell and Booth, 1967; Mares and Marschner, 1980), future investigations may develop a greater understanding of the mechanisms controlling tuber growth in Zantedeschia if consideration is also given to rates of cell division and hormonal status. 6.4.4 Carbohydrate and structural dry weight concentration as predictors of growth and yield The inability to detect significant correlations between source activity, as quantified by NAR and RLSWR, and the relative rates of growth of the entire plant and/or its subcomponents (RGRw, RGRy, RGRTotn RGRyJ was not surprising. Warren Wilson (1972) suggested that more accurate determination of sink activity was gained by examining the relative growth rate of structural material, but with Zantedeschia 'Best Gold' no correlation was evident between the structural material - of the tuber and the various measures of source activity. This inability to detect a correlation may have arisen from I tw? sources. Firstly, time did not permit the structural dry weights of all organs of the plants in the current experiment to be quantified. Since roots, petioles and unexpanded leaves are likely to contribute towards the total sink activity, it is possible that their inclusion as the measure of sink activity was critical. - Secondly, while leaves were the primary sites of synthesis of new carbohydrates, quantification of carbohydrate concentrations within the leaf and other organs, fails to account for respiratory losses and the rates of assimilate flux. Both have been shown to be important determinants of growth of the total plant and/or organs, and not just the actual concentration of carbohydrates and/or their rates of change (Borchers-Zampini et al. , 1980; Fader and Koller, 1985; Ho, 1988, Farrar, 1990). Hence while not negating the importance of photosynthetic rate, photosynthetic area, and total photoassimilate supply, the methods of quantification/description of assimilate used here do not describe the rate of flux, nor respiratory losses once initially fixed . . The future development of mechanistic metabolic models to explain growth and development of Zantedeschia will therefore require the additional definition of the structural/carbohydrate concentrations of all organs, in addition to carbohydrate flux data 172 and respiratory losses. 6.4.5 Conclusions As with many plant genera, sucrose, fructose, and glucose, accounted for the majority of soluble carbohydrates in Zantedeschia 'Best Gold? The primary form of storage carbohydrate was starch, and changes in storage organ growth occurred together with changes in starch. It is suggested that the inability to correlate growth with carbohydrate concentration is as a result of the inability to quantify carbohydrate fluxes. The maintenance of concentrations of both soluble carbohydrate and starch was highly controlled, presumably involving enzymatic activity associated with storage and flux. While Morrell and ap Rees (1986) suggested that control of the carbohydrate concentration of developing potato tubers was achieved via fine control, i.e. , enzymatic synthesis and activity, Farrar (1990) suggested a modified form of coarse control, i.e. , regulation of enzymatic synthesis and activity via supply of carbohydrate. Future examination of changes in these components may provide new insights to the mechanisms controlling growth and development in Zantedeschia. In the absence of evidence suggesting the existence of a trigger for tuberization, rather than looking to the tuber for the point of control it is more likely that the onset of tuber growth, as increases in both structural and starch dry weights, is dictated by the establishment of a photos?thetic leaf area large enough to provide an adequate supply/flux of photoassimilates beyond the demands for continued leaf and root development, and respiration. 6.5 References 173 Acock, B. , M.C. Ac:Oek and D. Pastemak. 1990. Interactions of C? enrichment and temperature on carbohydrate production and accumulation in muskmelon leaves. 1. Amer. Soc. Hort. Sci. 1 15:525-529. Aung, L.H. , F. Tognoni and A. A. De Hertogh. 1973. Changes in the Carbohydrates of tulip bulbs during development. HortScience 8:207-208. Borchers-Zampini, C. , A.B. Glam, J. Hoddinott and C.A. Swanson. 1980. Alterations in source-sink patterns by modifications of source strength. Plant Physiol. 65: 1 1 16- 1 120. 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Prentice Hall, Englewood Cliffs, New Jersey. \ 1 I I 180 7 MANIPULATION OF IN VITRO SOURCE AND SINK STRENGTH, AND DRY MATTER PARTITIONING IN Zantedeschia 'Best Gold' 7.1 Introduction In the preceding experiments classic growth analysis was used to describe the growth and tuber development of Zantedeschia 'Best Gold' under a range of environmental conditions (refer Chapters 3 and 4). The growth analysis results, together with measurements of photoassimilate capacity (refer Chapter 5) and tissue carbohydrate status (refer Chapter 6), were used to hypothesize the possible mechanisms controlling dry matter accumulation and its partitioning towards the tuber. In its simplest form two possible situations can be envisaged in the control of dry matter accumulation within the plant, i.e. , either the system is source limited, or the system is sink limited (Wareing and Patrick, 1975). In the experiments described earlier, the range of environmental conditions used were chosen to offer a range of potential source and sink strengths, and thereby allow determination of controlling mechanisms. However, it was evident from these experiments that source and sink strengths may not have been manipulated independently when temperature and/or photosynthetic photon flux (PPF) regimes were altered. As an example, while cultivation under increased temperatures generally increased photosynthetic rate (refer Chapter 5) , and therefore source activity, it was uncertain how much of the concomitant increase in tuber growth (refer Chapter 4) resulted from the increased source activity, compared with the direct effect of temperature on sink activity itself. In addition, source size also increased wi? increased temperature (refer Chapter 3) , and together with the resultant intersink I coi:npetition between leaves and tuber, accurate description of source and sink strengths ' were confounded. Similarly, while cultivation under low PPF conditions reduced the photosynthetic rate (refer Chapter 5) and therefore potential source activity, leaf area and therefore source size increased (refer Chapter 3), again preventing any clear interpretation of results. It was therefore evident that if a greater understanding of possible mechanisms of dry matter accumulation was to be gained, some means of independently manipulating the source and sink strengths was required. In vitro systems have previously been used to examine the specific requirements for storage organ initiation, and/or investigation of source and sink limitations of growth and dry matter accumulation by storage organs (Gregory, 1956; Loomis and Torrey, 1964; Heath and Hollies, 1965; Palmer and Smith, 1970; Peterson, 1973; Gifford and Evans, 1981 ; Jones et al. , 1981 ; Cobb and Hannah, 1986; Tovar et al. , 1987; Cobb et al. , 1988; Taeb and Alderson, 1990; Chow et al. , 1992). In general the experimental unit utilized in such research has been the isolated storage organ, such as maize (Zea mays L.) kernels and sugar cane (Saccharum officinarum L.) stem segments (Jones et al. , 1981 ; Gifford and Evans, 1981), or an in vitro cutting, as with potato (Solanum tuberosum L.) and yam 181 (Dioscorea rotundata Poir.) (Gregory, 1956; Ng, 1988). Only infrequently have established, whole plants been utilized, such as seffiHngs of sugar beet (Beta vulgaris L.), radish (Raphanw; sativus L.) and turnip (Brassica rapa L.) (Peterson, 1973; Ting and Wren, 1980; Vreugdenhil and Bouwmeester, 1989), or vegetative explants as with tulip (Tulipa gesneriana L.) and narcissus (Narcissus sp. L.) (Taeb and Alderson, 1990; Chow et al. , 1992). Although use of isolated organs has enabled examination of the requirements of dry matter accumulation, use of whole plants is likely to permit a more accurate investigation of dry matter partitioning between competing organs. In this way an integrated appraisal of the physiological mechanisms involved in source and sink relationships would be permitted, in contrast to the potentially unrestrained development of isolated organs. Since the great majority of information pertains therefore to isolated organs and/or cuttings, this information is presented and discussed with the understanding that the responses of whole plants may be different. Zantedeschia plants have been successfully propagated in vitro from vegetative bud explants, using a Murashige and Skoog (1962) medium supplemented with various phytohormones to stimulate differentiation, and with between 2 and 3% sucrose as a source of carbohydrates (Cohen, 1981 ; Rong et al. , 1989). Although in vitro germination of seeds of other members of the Araceae has been achieved on non-amended agar, seedlings were not reported as having been grown until the onset of tuber growth (Kikuta et al. , 1937), as was proposed in these experiments. i Within th?. diversity of plant organs studied in vitro, generally sucrose has been found to I be the preferred respirable substrate for uptake and short-term growth or . storage, ? correlating well with the most common form of carbohydrate translocated (Lawrence and Barker, 1963; Heath and Hollies, 1965; Gifford and Evans, _1981 ; Cobb and Hannah, 1986) . The development of storage organs in vitro has been found to increase with increasing sucrose concentration, i.e. with increasing source strength, and generally has been found to be optimal in the range of 8 to 12% , with higher concentrations reducing storage organ growth (Gregory, 1956; Lawrence and Barker, 1963; Heath and Hollies, 1965; Ting and Wren, 1980; Koda and Okazawa, 1983; Cobb and Hannah, 1986; Tovar et al. , 1987; Mielke and Anderson, 1989; Taeb and Alderson, 1990; Chow et al. , 1992). However, lower optimal cOncentrations between 3 and 6% sucrose for storage organ growth have been reported for some species (van Aartrijk and Blom-Barnhoom, 1980; Rice et al. , 1983 ; Ng, 1988). -- Although the exogenous supply of carbohydrates should theoretically bypass the need for - . photosynthesis, and therefore the need to supply light in vitro, the presence of light may be required to stimulate morphogenic development (Kato, 1978; Hughes, 1981). The light 182 induced accumulation of starch in vitro has been implicated as a factor in morphogenesis of tobacco (Nicotiana tabacum L. ; Thorpe and Murashige, 1970). In addition, the assimilation of nitrogen and, therefore, the consequent synthesis of amino acids and enzymes are associated with the photosynthetic process and therefore light (Gibbs and Latzko, 1979). In in vitro systems, the utilization of very low light fluxes (e.g. , 20 - 50 l'mol?m?2?s?1 PPF), limited gas exchange, and the supply of exogenous sugars, were shown to be sub-optimal for any significant photosynthetic development (Grout and Aston, 1978; Hughes, 1981 ; Donnelly and Vidaver, 1984). Although photosynthesis does occur in vitro, limited gas exchange between the container and the outside environment results in significant recycling of respired C02 (Abbott and Belcher, 1982) and presumably, therefore, minimal net gain in photoassimilates. In fact, efforts to achieve in vitro photoautotrophism by the omission of a respirable carbohydrate source, so that plants might sustain their own growth, typically have not been successful unless supplied with high light intensities coupled with C? enrichment (Lakso et al. , 1986; Langford and Wainwright, 1987; Kozai, 1989). Hence it can be concluded that should light be required to optimise tuber growth in vitro, the use of conventional low PPF coupled with the limited gas exchange achieved under normal cultural conditions, should eliminate any significant contribution of photosynthesis to source strength. The Control of assimilate allocation among competing sinks is not well understood, but curreht research suggests factors regulating sink metabolism play a dominant role (Gifford \ . and Evans, 1981 ; Wardlaw, 1990; Farrar and Williams, 1991). Relevant sink metabolism involves phloem unloading, cell wall invertase activity, plasma membrane and tonoplast membrane transport, and conversion of assimilates to storage products. These metabolic activities all influence the steepness of the osmotic gradient within the phloem which has been suggested as a factor controlling assimilate flux from source to sink. For any given genotype, the proportion of dry matter in storage organs that is contributed by carbohydrates does not vary with the size of the organ as much as does the size of the organ itself (Jenner, 1982). In general, large specimens contain more storage carbohydrates than small ones. Thus the growth of storage organs and their capacity for storing carbohydrates are intimately linked. The total quantity of storage carbohydrate accommodated by any given organ is a product of the amount of storage carbohydrate per cell and the total number of eells. Although there may be substantial regional differences in storage carbohydrate content per cell within the organ, it follows nevertheless that cell number is a major determinant of the amount stored within that organ (Reeve et al. , 1973; Rapoport and Loomis, 1986). 183 Using tomato (Lycopersicon esculentum L.), Ho (1979) demonstrated that although assimilate export in vivo is primarily determined by the rate of assimilation in leaves, the utilization of assimilate in the sink organ (tomato fruit) can determine the rate of carbon import. This concept of sink activity being integral to growth of the storage organ was also exemplified through the reciprocal grafting of the large storage root forming sugar beet and the small storage root forming chard (Beta vulgaris L. ; Rapoport and Loomis, 1985). This reciprocal grafting clearly illustrated that the hypocotyl-root determines cell division within the storage organ and therefore sink strength, i.e., it is a genetic characteristic. Increases in temperature have been shown to increase metabolic activity and thereby sink activity (Wardlaw, 1968; Ho, 1979; Jones et al. , 1985) , with differences in storage organ growth at any temperature being dependent on the in vitro source strength (Dinar and Stevens, 1982; Koda and Okazawa, 1983). In vivo data to date have been interpreted as indicating that both total plant and tuber dry weights, and growth rates (i.e. , sink activity) of Zantedeschia 'Best Gold: increase with increasing temperature to an optimum near 25 C (refer Chapters 3 and 4). The ability to manipulate source strength in vitro, via sucrose concentration, independent of manipulating sink activity, via temperature, would hopefully permit determination of the relative importance of source and sink limitation under similar temperatures in vivo. Tissue water relations may play an important role in regulating so?ute transport processes within the . plant. In particular, turgor pressure emerges as a potential regulatory component \of sucrose fluxes in developing sinks. The lowering of cell turgor enhanced active sucr?se uptake in discs of sugar beet root and potato tuber (Wyse et al. , 1986; Oparka and Wright, 1988). In addition, the subsequent partitioning of sucrose to starch in discs of potato tuber was enhanced by reduced turgor pressure (Oparka and Wright, 1988). Although some response was evident as a result of increased osmotic potential, reduced turgor pressure was primarily responsible (Oparka and Wright, 1988). However, tuber formation of potato stolons in vitro, at concentrations of sucrose up to 12% was not a result of the osmotic concentration, as similar osmotic concentrations of the non? respirable carbohydrate mannitol, did not result in tuber formation (Lo et al. , 1972). In light of these findings investigations examining a range of in vitro source strengths must recognise the potential influence of changes in turgor and osmotic potential. Although exceptions exist, plants which are able to metabolize mannitol naturally contain mannitol (Trip et al. , 1964). With sucrose, fructose and glucose comprising the primary forms of soluble carbohydrate in Zdntedeschia (refer Chapter 6) it is assumed that mannitol would not be metabolized by Zantedeschia, and could therefore be used to regulate osmotic _ potential and turgor in vitro, without altering source strength. 184 The ready uptake ?of sugars from the bathing media by organs in vitro suggests that in vivo they may take up assimila? by the phloem from the free space, and that this apoplastic route is most commonly favoured. In roots, where water flow through the free space occurs in the opposite direction, symplastic transport of sucrose from stele to cortex and apical cells seems more likely and is supported experimentally (Hampson et al. , 1978; Gifford and Evans, 1981). Just how these hypotheses of assimilate transport apply to entire plants of Zantedeschia in vitro is uncertain. However, considering the aforementioned apoplastic movement of assimilates in in vitro systems, and the close proximity of the source to the sink in vitro, it is assumed that the significance of the assimilate transport pathway will be minimal. The objectives of this research were two-fold; \ ' ' 1) To develop in vitro techniques applicable to the growth of seedlings of Zantedeschia, which would permit tuber formation. 2) To use these techniques to independently manipulate source and sink strength, so as to facilitate the formation of a hypothesis on the control mechanisms of dry matter accumulation and partitioning in Zantedeschia in vivo. 7.2 Materials and Methods 7 .2.1 Germination media, media transfer and sucrose concentrration? 185 Seeds of Zantedeschia 'Chromatella' had been harvested at the end of the previous season and stored at 20 C for approximately 12 months. Seeds were surface sterilized in a solution of 20% sodium hypochlorite and 0.5 mUitre?1 Multifilm X-77 (Ivon Watkins-Dow Ltd . , N.Z.) wetting agent for 30 min, and subsequently rinsed in sterile distilled water. To determine the influence of sucrose during germination the use of two media was examined. These media consisted of either 5 g?litre?1 agar (Davis) and half-strength Murashige and Skoog's (1962) medium (MS), or the same medium plus 2 % sucrose. The pH was adjusted to 5 . 7 ? 0. 1 prior to adding agar and sterilization. Sterilization was carried out at 121 C and a pressure of 0. 1 MPa for 15 min. Each 200 ml jar contained 50 ml of medium with 20 seeds, and in total 120 seeds were sown onto each of the two germination media (i.e. , 12 jars total) . Jars with translucent polypropylene lids containing the seeds were incubated in the dark at 20 C for a period of 3 to 4 weeks. Plate 7.1 Germinated seedling of Zantedeschia 'Chromatella' after being excised from the cotyledon. Scale divisions = 1 mm. Once seedlings had germinated to the stage where the point of emergence of the plumule was visible, seedlings were excised from the cotyledons (Plate 7 . 1) and transferred to the treatment media. At the time of transfer, roots were trimmed to 5 mm length and plants graded for even size. Seedlings were soaked in a solution of 0.25 % sodium hypochlorite for 15 min and subsequently rinsed in a solution of 0.05 % sodium hypochlorite prior to 186 transfer to the treatment media. From each of the two germination media 10 seedlings, in individual jars, were allocated to each of 3 treatment media. Treatment media consisted of 20 m1 of 5 g-Iitre-1 agar and MS medium, plus either 2, 4, or 6% sucrose. A further 30 seedlings were transferred to an intermediary medium containing 2% sucrose until a second subsequent transfer after a three week period of establishment. At the time of the second transfer, 10 individual seedlings were transferred from each of the germination media prehistory groups, onto media containing 20 m1 of 5 g?Iitre-1 agar and MS medium, plus either 2, 4, or 6% sucrose. Subsequent to germination all treatments were also divided between being incubated in the dark or light (45 pmol?m-2?s-1 PPF). Figure 7. 1 diagrammatically summarizes treatments and relevant treatment transfers. Individual jars were covered with a 5 J.Lm, high density, translucent, polyethylene film, attached by rubber bands, and incubated at 25 C. During germination, development was monitored daily, but once transferred to subsequent media, development was monitored weekly. After 29 weeks, leaf number per plant was recorded prior to the entire plant being vacuum dried at 0.3 KPa for 48 h at 40 C. Measurements of shoot (leaf + petiole) , tuber, and root dry weights were subsequently recorded. Seedlings were arranged in a completely randomized design within each treatment environment. Leaf number, dry weight and dry weight partitioning data were subjected to analysis of variance using the general linear models procedure of SAS. Plants with fungal or bacterial contamination were excluded from the analysis. Gennination media non-sucrose germination media sucrose amended media Transfer 1 Transfer2 (Dark) % suer? % sucrose :?: (Light) % sucrose % sucrose 2? 2 4 4 6 6 (Dark) % sucrose % sucrose 2? 2 4 4 6 6 (Light) % sucrose % sucrose 2 - '> 2 4 ?;... 4 6 ? 6 187 Figure 7.1 Diagrammatic summary of germination and transfer treatment media used for seedlings of Zantedeschia 'Chromatella? 7 .2.2 Manipulation of in vitro source and sink strengths Seeds of the Zantedeschia pentlandii-like selection 'Best Gold' used in this experiment were harvested at the end of the previous season and stored at 25 C for approximately 12 months. Seed were surface sterilized and germinated as outlined in Section 7 .2. 1 , using a germination medium consisting of 5 g1itre?1 agar amended with MS. The pH was adjusted to 5 . 7 ? 0. 1 prior to adding agar and sterilization. During germination, 35 200 m1 jars, each containing 50 m1 of medium and 20 seeds (700 seeds in total) were incubated in the dark at 20 C. 1 88 Four weeks after sowing, germinated seedlings were excised from the cotyledon and transferred to treatment media. Excised seedlings were handled and sterilized as outlined in Section 7.2. 1 . Twenty seedlings in individual jars were allocated to 20 m l each o f 4 treatment media. Treatment media comprised the germination medium amended with Linsmaier and Skoog's (1965) organic additives (LS) plus either 1 , 2, 4 , or 6% sucrose. Mannitol was added to each treatment medium to result in an osmotic potential equivalent to that of 6% sucrose as outlined below. 1 % sucrose + 2.7% (0. 15 M) mannitol 2% sucrose + 2 .2% (0. 12 M) mannitol 4 % sucrose + 1 . 1 % (0.06 M) mannitol 6% sucrose + 0.0% (0.00 M) mannitol Final osmotic potential of each treatment medium was approximately 441 . 7 kPa. Individual jars were covered with a 5 p.m, high density , translucent, polyethylene film, attached by rubber bands, and placed under a 12 h diurnal light period of 45 p.mol?m-2?s-1 PPF, at one of 4 temperatures, i . e. , 19 , 25, 28, or 3 1 ? 1 C. In addition to the above treatments, the ability of Zantedeschia 'Best Gold' to metabolize mannitol was evaluated by the inclusion of additional treatments consisting of 20 ml of 5 g?litre-1 agar, MS , LS, plus either 0, 1 . 1 , 2 .2, or 2. 7% mannitol. The pH was adjusted to 5 . 7 + 0 . 1 prior to adding agar and sterilization. Plants were treated as outlined above, but due to limited plant material , seedlings were grown at 25 C only. Seedlings were evaluated after 24 weeks. Shoot and leaf number per plant were recorded prior to the entire plant being vacuum dried at a pressure of 0 .3 kPa for 48 h at 40 C. Measurements of shoot (leaf + petiole) , tuber, and root dry weights were subsequently recorded. Seedlings were arranged in a completely randomized design within each treatment environment. Shoot and leaf number, dry weight and dry weight partitioning data were subjected to analysis of variance using the general linear models procedure of SAS . Plants with fungal or bacterial contamination were excluded from the analysis. 7.3 Results 7 .3.1 Gennination media, media transfer and sucrose concentrations 7 .3.1.1 Gennination 189 Within two weeks of sowing, radicles had developed to between 2 and 3 mm in length, and by three weeks seedlings on the non-sucrose amended media had germinated to the stage where the point of emergence of the plumule was visible. Seedlings on the sucrose amended media did not reach this stage of development until 4 weeks after sowing. 7 .3.1.2 Dry matter accumulation and partitioning In the absence of any significant influence of the germination medium (P < 0. 10) or transfer onto an intermediary sucrose medium (P s 0. 10) , data presented here refer to those pooled across the aforementioned treatments. The most influential factor on growth and dry matter accumulation was the presence or absence of light (P :S 0.001, Table 7. 1 , Plate 7.2). Total plant dry weight was between two and six fold greater from seedlings grown under light compared with in the dark. Similarly tuber and shoot dry weights, and leaf production, were greater when seedlings were incubated under light compared with dark. When incubated under light, a positive linear trend of increasing total plant dry weight with increasing sucrose concentration was determined (P < 0.05, Table 7. 1). Total plant dry weight of seedlings grown on the 6% sucrose medium was more than 50% greater than that achieved on the 2% sucrose medium. However, no relationship between total plant dry weight and sucrose concentration, could be detected for those seedlings incubated in the dark. Positive linear trends were evident between sucrose concentration and tuber dry weight under both dark (P :S 0.05) and light (P ::S 0.001) regimes (Table 7. 1). At the time of final harvest, tuber dry weight of seedlings grown on the 6% sucrose medium was more than eight fold greater than that achieved on the 2% sucrose medium when incubated in the dark, and approximately four fold greater when incubated in the light. While under the dark regime a significant positive linear trend was determined between sucrose concentration and the proportion of the total plant dry weight in the tuber (P ::S 0.001), under the light regime this relationship was quadratic (P ::S 0. 001, Table 7. 1). .... __ , , ' / ---- - --- ------- - . --------??-- ?-- -- -------- 2 Ol : -:4 01"0 ;:?:..;:, ;? 6 OiO ? ? ? -???- ? .e ? ? - s COl \ 1 l i I I 1 1 I I 1 1 1 1 I I I I I I I I I o ? 2 3 4 5 6 7 8 9 1 Plate 7.2 Seedlings of Zantedeschia 'Chromatella' after 29 weeks of cultivation in vitro at a range of sucrose concentrations in either the dark (upper) or light (45 l'molm-2?s?1 PPF) (lower). 190 Under both dark and light regimes increasing sucrose concentration resulted in a reduced number of leaves at the time of final harvest (P s 0.001 , Table 7. 1). While under the light regime this negative trend was linear (P s 0.001), under the dark regime the trend was quadratic (P s 0.05). In contrast, while shoot dry weight was between two and three fold greater under the light regime than under the dark, no relationship between sucrose concentration and shoot dry weight was detected (P < 0. 10). Table 7.1 Dry matter accumulation and partitioning in seedlings of Zantedeschia 'Chromatella? as influenced by the presence of light or dark, and sucrose concentration in the growing medium. Light Sucrose Total dry Tuber dry Proportion Shoot dry /Dark cone. weight weight in tuber weight (%) (mg) se? (m g) se (%) se (m g) se Dark 2 41.4 42.1 2 .1 23.3 5.2 4.3 37.9 21.2 4 31 .4 42.1 5.1 23.3 18 .1 4.3 21.9 21.2 6 57:6 38.4 19.3 21.2 29.5 4.0 33.8 19.4 Light 2 144.5 23.5 27.6 13.0 22.0 2.4 97.2 11 .9 4 208.4 27.2 106.9 15.0 49.5 2.8 75. 1 13.7 6 224.8 24.3 128.9 13.5 54.3 2.5 71.2 12.2 SourCe' Light/Dark *** *** *** *** Sucrose * *** *** ns Light x Sucrose ns * ns ns Contrast (Dark) Linear ns ** *** ns Quadratic ns ns ns ns Contrast (Light) Linear ** *** *** ns Quadratic ns ns *** ns 'Standard error of least squares means, i.e., adjusted for variable replication resulting from contamination. Leaf number se 5.2 0.5 2.2 0.5 2.7 0.5 6 .8 0.3 5.5 0.3 5.1 0.3 *** *** ns ** ** *** ns 'Probability of a significant F value; ns,*,**,*** = nonsignificant or significant at P = 0.10, 0.05, or 0.001, respectively. 191 192 7.3.2 Manipulation of source and sink strengths At final harvest, all seedlings incubated in media amended with mannitol alone, were dead and showed little indication of previous growth. In contrast, plant loss in treatment media containing sucrose and mannitol was minimal, and attributable to fungal and/or bacterial contamination. In the absence of the plant' s ability to assimilate mannitol , data presented here refer to those derived from media consisting of sucrose treatments amended with mannitol, excluding those exhibiting evidence of fungal and/or bacterial contamination. With the exception of shoot dry weight and shoot number, significant interactions were determined between temperature and sucrose concentration with all parameters measured (Table 7.2 and Table 7 .3) . While the data in Table 7.2 through Table 7.4 are presented to allow determination of statistical validity, Figure 7.2(a) to (d) and Figure 7.3(a) and (b) present a more readily interpreted graphical display of the responses to increasing temperature and sucrose concentration. As determined earlier (refer Section 7.3 . 1) , a positive linear trend of increasing total plant dry weight with increasing sucrose concentration was determined at intermediate temperatures (P ? 0.00 1 , Table 7.4) , but the magnitude of this response was minimal at the temperature extremes of 16 and 3 1 C (Figure 7.2a) . At these temperature extremes increases in total plant dry weight were restricted compared with those at intermediate temperatures, but only at 16 C was a significant quadratic trend detected. Maximum total plant dry weight (0.4 g) occurred at a sucrose concentration of 6% , and between 2 1 and 26 C. These temperatures also resulted in maximum total plant dry weight at a sucrose concentration of 4% , but no differences in total plant dry weight were detected between temperatures at either 1 or 2% sucrose where growth was minimal (P ? 0. 10) . As with total plant dry weight, under all temperatures a positive linear trend was determined between tuber dry weight and sucrose concentration (Table 7 .4, Figure 7 .2b, Plate 7.3). Maximum differences between sucrose concentrations occurred between 21 and 26 C, with tuber weight being more than two hundred fold greater at 6% compared with 1 % sucrose. As found for total plant dry weight, a quadratic trend of tuber dry weight, with increasing temperature, was detected at both 4 and 6% sucrose concentrations (P < 0.00 1 , Table 7.4) . However at both 1 and 2% sucrose, where growth was restricted, no trend of tuber weight with increasing temperature was detected (P < 0. 10) . Table 7.3 Dry matter accumulation within the shoot of seedlings of Zantedeschia 'Best Gold: as influenced by temperature and sucrose concentration. Temp. Sucrose Shoot dry Shoot number Leaf number (C) cone. weight (%) (m g) se se 16 1 7.8 10.0 1 . 1 0.8 2.5 2 63.5 12.9 6.2 1.0 23.3 4 30.3 12.9 1 .7 1.0 5.5 6 28.2 14.1 1 .4 1 .0 4.6 21 1 8.6 12.0 1 .6 0.9 2.4 2 84.7 12.9 3.7 1 .0 16.5 4 64.8 12.0 1.3 0.9 6.4 6 61 .9 15.8 1.0 1 .2 4.3 26 1 9.8 12.0 1 . 1 0.9 3 . 1 2 96.3 1 1.2 5.5 0.9 16.8 4 84.2 12.9 1 .5 1 .0 6.7 6 74.7 1 1 .2 1 .6 0.9 5.6 3 1 1 8 .3 10.0 1 .0 0.8 2.0 2 66.0 10.6 4.0 0.8 5.6 4 56. 1 10.0 3 .7 0.8 5.8 6 36.5 12.0 1 .4 0.9 5.4 Source" Temperature *** ns Sucrose *** *** Temp x Sucrose ns ns ZStandaid error of least squares mean, 1.e., adjusted for vanabie replication resulting from contamination. se 1 . 8 2.4 2.4 2.6 2.2 2.4 2.2 2.9 2.2 2.0 2.4 2.0 1 .8 1 .9 1 .8 2.2 ** *** ** YJirobability of a significant F value; ns,*,**,*** = nonsignificant or significant at P = 0. 10, 0.05, or 0.001, respectively. 194 195 Under all temperatures shoot dry weight was maximal at 2% sucrose, resulting in a significant quadratic trend of shoot dry weight with increasing sucrose concentration (Table 7.4, Figure 7.2c). With minimal shoot dry weight occurring at 1 % and a maximum at 2% , under all temperatures the maximum difference in shoot dry weight, between these sucrose concentrations, averaged ten fold. Table 7.4 Significance of trend analyses of increasing temperature and sucrose concentration on dry matter accumulation and partitioning in seedlings of Zantedeschia 'Best Gold: Source Total dry Tuber dry Proportion Shoot dry Shoot Leaf weight weight in tuber weight number number L? Q L Q L Q L Q L Q L Q Increasing temperature Sucrose cone. (%) 1 rui' ns ns ns ns ns ns ns ns ns ns ns 2 ns ns ns ns ns ns ns ns ns ns ns ns 4 ** * ** * ns ns ns ns ns ns ns ns 6 ** * ** * ns ns ns ns ns ns ns ns Increasing sucrose concentration Temperature (C) 16 *** ** *** ns *** *** ** *** ns *** ns *** 21 *** ns *** ns *** *** *** *** ns *** *** *** 26 *** ns *** * *** *** *** *** ns *** ns *** 31 *** ns ** ns ** ns ** * * ns ** ns 'Linear (L) and Quadratic (Q) contrasts/trends. >'Probability of a significant F value; ns, *, **, *** = nonsignificant or significant at P = 0. 10, 0.05, or 0.001, respectively. ? "' 0.5 ;.:- 0.<4 6 w 31: O.J ?i 0 _J 0.2 ? g 0.1 0.5 t;: o .... Q w 3: 0.3 ? b 0.2 ? VJ 0.1 (a) (c) 0.5 $ 5: 0.4 C> ? O.J ? Ei 0.2 ,.... "' o.t :il eo :::> ,.... z :z oo 0 ;::: :5 ..a a.. I@ C.. 2Q 196 Figure 7.2 Surface response curves illustrating the influence of increasing temperature and sucrose concentration on the dry matter accumulation and partitioning in seedlings of Za.ntedeschia 'Best Gold: (a) Total dry weight, (b) Tuber dry weight, (c) Shoot dry weight, and (d) Proportion of total dry weight in the tuber. N.B. reversal of temperature scale. ? '>1-------r // ::f ?4+---77 (b) ?J----------? ID ::li :::> z ??ru----, 197 Figure 7.3 Surface response curves illustrating the influence of increasing temperatur' and sucrose concentration on the number of (a) shoots and (b) leaves in seedlings o Zantedeschia 'Best Gold: N.B. reversal of temperature scale. r------i r - , ? ? 2 SUCROSE ( o Plate 7.3 Tubers from seedlings of Zantedeschia 'Best Gold' after 24 weeks of growth in vitro, at a range of temperatures and sucrose concentrations. N.B. shoots and roots removed. 198 Treatment induced differences in shoot dry weight were paralleled by similar trends with shoot number (Figure 7. 3a) and leaf number (Table 7.4, Figure 7. 3b). With the exception of 3 1 C maximum shoot and leaf number were attained at 2% sucrose regardless of temperature. At 31 C the quadratic trend of shoot and leaf numbers with increasing sucrose concentration was not detected as at lower temperatures. Leaf number was only slightly greater at 2% sucrose than at higher concentrations at 31 C (Table 7.3), resulting in the detection of a positive linear trend at this temperature (P s 0.05). With the exception of 6% sucrose, the proportion of total plant dry weight partitioned to the tuber differed little between temperatures (P s 0. 10, Table 7.2, Figure 7.2d). At 6% sucrose this proportion was reduced by 34% at 31 C compared with all other temperatures (P s 0.01). A quadratic trend of an increased proportion of the dry weight partitioned to the tuber with increased sucrose concentration was detected at all temperatures below 31 C (P < 0.001, Table 7.4, Figure 7.2d). At 31 C a positive linear trend of increased partitioning to the tuber with sucrose concentration was detected (P s 0.05), but the greatest difference in magnitude between sucrose concentrations was always less than two fold. At 31 C the maximum proportion of dry weight partitioned to the tuber was 41.2% compared with an average of 75.6% across all other temperatures and at a sucrose concentration of 6%. 7.4 Discussion 199 Initiation of tuber growth in Zantedeschia 'Best Gold' appears to require no obligative environmental trigger. Whether incubation took place in the light or dark, and at a range of temperatures, tuber growth was noted in all treatments that sustained growth. ? To discuss the current results in terms of source and sink, a number of assumptions are required. Firstly, it is assumed that sucrose concentration within the growing medium in vitro was equivalent to source strength in vivo. This seems a reasonable assumption since the primary derivatives of source activity are carbohydrates, and sucrose was found to be the principal soluble carbohydrate in tubers of Zantedeschia (refer Chapter 6). Secondly, it is assumed that manipulation of the incubation temperature was equivalent to manipulation of sink activity (Ho, 1979; I ones et al. , 1985). With the exclusion of any influence of temperature on source strength, and a third assumption of the relative un? importance of the assimilate transport pathway (Minchin and Thorpe, 1992), the assumption that manipulation of temperature in vitro would principally alter sink activity and thereby sink strength, appears valid. Interpretation of in vitro dry weight data is also potentially confounded by respiratory losses and intersink competition (Wareing and Patrick, 1975). As the respiratory losses of imported assimilates in the sink organ can be substantial (Farrar, 1985), it should be acknowledged that in the current experiments only a measure of 'net sink strength' was possible. Similarly, the potential for inter-sink competitio11: to alter the calculated sink strength was illustrated by Ho (1978), and therefore it must be Ipferred that only a measure of 'apparent' or 'net sink strength' was obtained in the current experiment. Finally, it must be assumed that growth and development was not abnormal from that achieved in vivo, with no other factors limiting growth and development. While this last assumption is less readily proven _without further research, the use of in vitro techniques to repeatably produce plants of Zantedeschia for commercial use (Cohen, 1981) suggests that this too is a reasonable assumption. The possibility that after 24 or 29 weeks in vitro, plant growth may have become limited by the depletion of essential compounds (Morel and Wetmore, 1950; Loomis and Torrey, 1964) was not investigated. In vitro storage organ growth of other species has continued over similar time periods, and while repeated media renewal was not able to increase the duration of grain filling in maize kernels, it did increase the rate and extent of grain filling (Cobb et al. , 1988; Mielke and Anderson, 1989). While it can not be stated that depletion of essential compounds did n_ot occur, any occurrence of such an event should not have influenced the relative order of the treatment effects. -While assimilate partitioning in Zantedeschia seedlings must to some extent be determined genetically (Brooking and Kirby, 1981 ; Rapoport and Loomis, 1985; Ho, 1988; Chow et al. , 1992), within the bounds of the temperature regimes used in the current experiment, 200 source strength appears most limiting to the Partitioning of assimilates towards tuber growth (Figure 7.2d). However, at the same time, under conditions of controlled source strength it was evident that sink strength can limit final tuber yield (Figure 7.2b) . The response of both total and tuber dry weights to increases in sink strength, via increased temperatures, were either not evident (e.g. 1 and 2% sucrose) or were quadratic in nature (e.g. 4 and 6% sucrose, Table 7.4). This quadratic response indicates a progressive decline in response to incremental sink strength, and hence minimal sink strength limitation. Similarly, therefore, the lack of any detectable response to increased sink strength at 1 and 2% sucrose, suggests strong source strength limitation at these concentrations. In contrast, the generally linear response of both total and tuber dry weights to increased source strength, via sucrose concentration, suggests that further potential exists for incremental source strength to increase both total and tuber weights. In addition, the lack of any detectable trend of dry matter partitioning to the tuber to increased sink strength, compared with the generally quadratic response to increased source strength (fable 7.4), also supports the hypothesis of source strength being most limiting. Under conditions of minimal source limitation, e.g. a sucrose concentration of 6% , limitation of sink strength at the temperature extremes of 16 and 3 1 C, may have resulted from differing causes. As found in the in vivo experiments (refer Chapters 3 and 4), it is readily accepted that cooler temperatures, such as 16 C, might limit sink strength through limitations on growth and respiration (Wardlaw, 1968; Ho, 1979; Dinar and Stevens, 1982; Jones et al. , 1985; Farrar and Williams, 1991). Such limitation is most likely: to involve regulation of enzyme activity (Walker et al. , 1978; Ou-Lee and Setter, 1985) and possibly either growth regulators (Melis and van Staden, 1984) or the plants' sensitivity to growth regulators (frewavas, 1982; Fim, 1986). In contrast, at 3 1 C, not only was total dry matter accumulation restricted (Figure 7 .2a), reflecting the same broad? spectrum limitations on growth, but also partitioning to the tuber was reduced (Figure 7.2d). This reduction in partitioning of assimilates to the tuber did not occur at 1 6 C, and infers the involvement of additional limitation(s) associated with assimilate utilization and/or partitioning at 3 1 C. Since the tuber of Zantedeschia is a starch accumulating sink (refer Chapter 6) those processes involved in starch synthesis are likely to be integral in determining sink strength. While not inhibiting the accumulation of soluble carbohydrates, temperatures of 30 C or higher inhibited starch synthesis in potato tubers, maize kernels, and endosperm tissue of wheat (Triticum aestivum L.) and barley (Hordeum vulgare L.) (Krauss and Marschner, 1984; Hanft and I ones, 1986; Rijven, 1986; MacLeod and Duffus, 1988) . These examples of high temperature inhibition of starch s?thesis were all associated with? a reduction in the activity of starch metabolising - enzymes, and it is suggested that the same situation may apply at 3 1 C in the current experiment. Since the shoot and ?oot of the Zantedeschia plant can be considered to be 201 utilization sinks, i .e. , sinks where assimilates are principally used for growth and respiration (Ho, 1988; Ho et al. , 1989) , high temperature inhibition of starch synthesis would not be expected to inhibit their respective sink strengths as much. Hence, while growth of all organs was restricted at 3 1 C (Figure 7.2a, b and c) , tuber growth was restricted to a greater degree (Figure 7.2d) . In addition to changes in enzyme activity, the inhibition of tuberization at high temperatures has also been associated with increased concentration of gibberellic acid (Krauss, 1978) . Given the ability of exogenously applied phytohormones to influence storage organ development (Loomis and Torrey, 1964; Palmer and Smith, 1970; Peterson, 1973; Ting and Wren, 1980; Beruter, 1983; Koda and Okazawa, 1983 ; Rice et al. , 1983; Mielke and Anderson, 1989; Vreugdenhil and Bouwmeester, 1989; Chow et al . , 1992) , phytohormones might also be involved in the interaction between temperature and the carbohydrate metabolism of Zantedeschia. A change in the plant' s sensitivity to phytohormones under such temperature extremes (Trewavas, 1982; Firn, 1986) could also be involved in this interaction. The reduction in tuber growth in Zantedeschia following the application of GA3 (Funnell and MacKay, 1987) , has previously been discussed in Section 1 .5 .3 .3 , highlighting the relevance of considering the involvement of phytohormones. The removal of the tuber as a competitive sink at 3 1 C resulted in the consequent increased partitioning to other organs. However, shoot dry weight at 3 1 C was no greater than at other temperatures. In addition, even with an increased source strength, this removal of the tuber as.; a competitive sink did not result in increased shoot dry weight accumulation. I Hence in its simplest form it could be concluded that the peak in shoot dry weight accumulation at 2 % sucrose (Figure 7 .2c) reflected the upper limit of sink strength for this organ. Such an interpretation may be too simple however. The fact that a source strength above 2% sucrose resulted in reduced shoot growth and leaf number does not comply with such a simple hypothesis. Farrar (1990) and Farrar and Williams (1991) suggested that assimilate concentration can control growth and respiration rates. While not controlled directly through mass-action on pre-existing enzyme systems, they suggested that the concentration of enzymes is regulated by the supply of assimilate. It is therefore possible that in the current experiments, increased source strength above 2% sucrose, resulted in manipulation of the phytohormone and/or enzymatically regulated assimilate partitioning, with preference to tuber growth. If examined in terms of growth analysis such preferential partitioning of dry matter towards the tuber would be seen as an ever increasing enhancement o{ihe magnitude of the TWP (refer Chapter 4) for as long as the availability of assimilates were in excess of the demands of competing sinks. Such a resultant increase in tuber sink strength would therefore be manifest as a reduction in shoot growth at higher concentrations as reported here. The enhanced partitioning towards tuber growth may be related to enhanced sink activity within the tuber over that of the shoot, or 202 simply that when grown in vitro, sucrose must pass through the tuber to gain access to the shoot. Hence in the latter case, proximity of competing sinks to the source and/or the probable use of symplastic assimilate transport to the shoot may be important (Wardlaw, 1990) . While the current experiment does not eliminate the potential involvement of osmotic potential and turgor pressure as a contributor to the control of assimilate accumulation, the use of mannitol to achieve similar osmotic potentials within all treatments indicated that the results obtained here, were independent of their influence. Although it is recognised that the osmotic potential of any one treatment medium would increase with increasing temperature, a maximum differential in osmotic potential of 3 .5 % would have resulted from a 2% sucrose medium held at 16 versus 31 C. This is in contrast to a 50% difference in osmotic potential as a result of increasing the main sucrose treatment concentration from 4 to 6% . After amendment with mannitol, differences in plant response were therefore more readily attributed to changes in sucrose concentration than to minor temperature induced osmotic changes. The lack of any ability to metabolise mannitol for growth and development in Zantedeschia was similar to that found for onions, potatoes and tulip (Heath and Hollies, 1965 ; Lo et al . , 1972; Tovar et al. , 1987; Taeb and Alderson, 1990) . The presence of light clearly has a role in stimulating growth and development in Zantedeschia 'Best Gold! Although the exogenous supply of carbohydrates should \ theoretically have bypassed any need for development of photosynthesis, and therefore any I need to supply light in vitro, the presence of light clearly enhanced dry matter accumulation and partitioning to the tuber (fable 7 . 1) . As discussed in the introduction, the use of very low fluxes (45 f'mol?m?2?s?1 PPF), together with limited gas exchange and the supply of exogenous sugars, should have been sub-optimal for any significant photosynthetic development in vitro (Grout and Aston, 1978; Hughes, 198 1 ; Donnelly and Vidaver, 1984) . At best, without increased gas exchange with the outside environment, recycling of C02 would only achieve compensation for respiratory losses (Abbott and Belcher, 1982) . Hence it is unlikely that the presence of light in the current experiment was contributing to enhanced growth through photosynthetic carbohydrate production. Examples of the requirement of light to stimulate morphogenic development have been noted (fhorpe and Murashige, 1970; Kato, 1978; Hughes, 1981) , but equally examples of nil or enhanced storage organ development in the dark have also been reported for hyacinth (Hyacinthus orientalis L.), lilium (?ilium longiflorum Hort.) and iris (Pierik and Ruibing, 1973; Stimart and Asher, 198 1 ; Mielke and Anderson, 1989) . Low irradiance, which tends to inhibit tuberization in potatoes, greatly increased the gibberellin-like substances in leaves (Woolley and Wareing, 1972) . In addition, the joint application of sucrose and phytohormones stimulated growth in dark grown storage roots of turnip, radish and sugar 203 beet more than either sucrose or phytohormones alone (Loomis and Torrey, 1964; Peterson, 1973; Saftner and Wyse, 1984). Given the potential involvement of phytohormones in tuberization, it is possible that they, or changes in the plant's sensitivity to them, may be involved in the photomorphogenic response noted in the current experiment. In addition, it can not be ignored that synthesis of carbohydrates is not the only assimilatory process closely associated with photosynthesis e.g. , assimilation of nitrogen, and the consequent synthesis of amino acids (Gibbs and Latzko, 1979) and subsequently of enzymes, also occurs. Given the involvement of enzymes in the active process of assimilate uptake and utilization (Oparka et al. , 1992), limited synthesis of enzymes may also have contributed to the reduced dry matter accumulation and partitioning noted in plants incubated in the dark. In the in vivo experiments, while diunial changes were noted in the concentration of total soluble carbohydrates in the tuber, concentrations fluctuated little with development (refer Chapter 6). Without the ability to quantify rates of assimilate translocation, it is difficult to determine where the rate limiting steps for tuber growth exist. The constancy of tuber soluble carbohydrate concentrations could infer that their utilization for respiration, structural growth, and starch synthesis was not limited by enzyme activity within the tuber. Under such circumstances source strength and/or the assimilate translocation pathway would be limiting (Mares and Marschner, 1980; Jenner, 1982). However, if assimilate translocation occurs via a simple concentration gradient (Gifford and Evans, 1981 ; Jenner, 1982) ibis could also infer that the rate of utilization within the tuber is a rate limiting step I (Ho, 1?79; Dinar and Stevens, 1982). Clearly the increase of source strength in vitro incr? tuber growth, indicating the likelihood of source limitation. However, due to the close proximity of the source to the sink, and the probable use of an apoplastic pathway of assimilate movement to the tuber (Gifford and Evans, 1981), any possible influence of the assimilate translocation pathway was virtually eliminated in the current . in vitro experiments. Hence any direct comparison to the in vivo situation is hampered. Results from the current in vitro experiments compare well with those of the previous in vivo experiments. Using similar temperature ranges, in both the in vitro and in vivo experiments the temperature response for final total and tuber dry weights was curvilinear. In the in vitro experiment the inability to detect a difference between the optimum temperatures of 21 and 26 C (Table 7.2), was similar to no difference being detected between 22 and 25 C in vivo (refer Chapters 3 and 4). Direct comparison between in vitro and in vivo experiments on the basis of source strength is more difficult as in vivo source size and activity was a function of PPF, temperature, ontogeny (refer Chapters 3 and 5), and carbohydrate flux. However, it can be concluded that the in vitro system described here provides a useful research tool to independently manipulate both source and sink strength for in vivo comparison. 204 7 .4 .1 Conclusions Initiation of tuber growth in Zantedeschia does not require an obligate environmental trigger. The optimal conditions for in vitro tuber growth of Zantedeschia 'Best Gold' were between 21 and 26 C, and at a sucrose concentration of 6% . The similarity of the plant's response to temperature both in vivo, and in vitro, provides a useful research tool to independently manipulate both source and sink strength for in vivo comparison. Both source strength and sink strength may limit tuber growth in Zantedeschia 'Best Gold! At temperatures commonly encountered during the growing season, in warm-temperate climes, source strength would appear most limiting to tuber growth. While sink strength was found to be most limiting at temperatures above and below this optimum, this was under conditions of controlled source strength. Since in vivo such temperatures would potentially inhibit both source and sink strength, it is possible that both may be limiting at the same time. Further research will be required to quantify the actual sink activity and rates of translocation to determine exactly where the rate limiting step for tuber growth in Zantedeschia is located. It is still uncertain whether increases in source strength beyond supra-optimal directly inhibit shoot growth and leaf development, or whether there is a direct effect of source strength on the sink activity which subsequently becomes a greater competitor for assimilates. Future investigations into concentrations and activity of ?hydrate metabolizing enzymes and phytohormones, and/or changes in the plant's sensitivity to them, should help identify and explain the biochemical basis of such rate I limitffig steps. 7.5 References 205 Abbott A.J. and A. Belcher. 1982. Analysis of gases in culture flasks. I..ong Ashton Rpt. , 1980, p. 79. Beruter, J. 1983. Effect of abscisic acid on sorbitol uptake in growing apple fruits. J. Expt. Bot. 34:737-743. Brooking, I.R. and E.J.M. Kirby. 1981 . Interrelationships between stem and ear development in winter wheat: the effects of a Norin 10 dwarfing gene, Gai/Rht2. J. Agr. Sci. , Cambridge 97:373-381. Chow, Y.N. , C . Selby and B.M.R. Harvey. 1992. Stimulation by sucrose of Narcissus bulbil formation in vitro. J. Hort. Sci. 67:289-293. Cobb, B.G. and L.C. Hannah. 1986. Sugar utilization by developing wild type and shrunken-2 maize kernels. Plant Physiol. 80:609-611 . Cobb, B.G. , D.J. Hole, J.D. Smith and M.W. 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Plant Physiol. 81 :478-481. . ....... 212 8 ECOLOGICAL AND HORTICULTURAL RELEVANCE, AND MECHANISMS OF CONTROL, OF DRY MATTER ACCUMULATION AND PARTITIONING IN Zantedeschia 'Best Gold' - AN INTEGRATIVE DISCUSSION In addition to discussing the possible control mechanisms of dry matter partitioning in Zantedeschia, each of the preceding chapters has included discussion of the ecological and horticultural relevance of the results. However, each of these discussions has been presented in isolation, each focusing on a narrow range of subject areas. This final chapter integrates the findings contained in those preceding, presenting a broad overview of the relevance and consequences of this research. In addition , proposals for further experiments are presented to indicate possible avenues of research that should improve our understanding of the control mechanisms of dry matter partitioning in Zantedeschia. 8.1 Ecological relevance Relative rates of growth and development of Zantedeschia 'Best Gold ' increased with increasing temperature, with maximum total and tuber dry matter accumulation occurring between 21 and 26 C both in vivo and in vitro. This optimum temperature range for growth was close to the average daily air temperature during the growing season , i .e . ' 20 C, for the sites of natural habitat of the suggested parent specie, Zantedeschia pentlqndii. With daily maximum air temperatures during the growing season averaging 25 .4 C, i t is apparent that Zantedeschia 'Best Gold: as a representative selection of this specie, is well adapted to optimise growth under the temperature regimes of its natural habitat. The ability of Zantedesclzia 'Best Gold ' to alter the photosynthetic rate and the partitioning of the daily increment of dry matter into leaf area (LWP) in response to the photosynthetic photon flux (PPF) under . which it was cultivated , indicates that this selection is shade tolerant. This tolerance of shade, rather than being an obligative response, has its parallel in the recorded sites of natural habitat of the suggested parent specie, Zantedeschia pentlandii, i . e. , "open grassland" and "forest margins " (Letty, 1973) . Such habitats, i .e . ,? full sun and partial shade, would result in daily integrals of PPF similar to those used in the current study. While the photosynthetic acclimation to PPF was found to be less than complete, when considered in conjunction with changes in LWP, Zantedeschia 'Best Gold' can be regarded as being well adapted to optimise growth under the diversity of PPF regimes of its natural habitat. 213 The evolution of the geophytic growth habit has been suggested as arising from the selective pressures of the growing environment, as plants migrated or climates changed (Rees, 1981 & 1984) . In conjunction with the evolution of eco- and/or endodormancy and the resultant differing forms of cyclic periodicity of growth and development, the geophytic growth habit provides an excellent survival strategy for a number of plant species. As a survival strategy it would therefore be advantageous for geophytic plants to partition a greater proportion of the daily increment of dry matter toward the storage organ (i.e. , tuber weight partitioning; TWP) than to .leaf development, when placed in a sub-optimal environment. The magnit?.de of TWP in Zantedeschia increased at progressively lower temperatures, supporting the theory of the tuber evolving as a survival strategy. Increased partitioning of dry matter to the storage organ at cooler temperatures has been noted in a number of geophytic species (Aoba, 1976) . ln addition, enhanced dry matter partitioning to the storage organ in response to other environmental pressures such as declining soil moisture status has been observed (Boeken and Gutterman, 1991). However, while total plant dry matter accumulation in Zantedeschia was limited under low PPF, TWP was sometimes lower under the low PPF regime than under the high PPF regime. This apparent contradiction suggests that either PPF has not been a primary factor in its evolution or other forms of adaptation to PPF have taken precedence. The ability of Zantedeschia 'Best Gold ' to acclimate to a diversity of PPF regimes through photosynthetic rate, L WP, and leaf area duration suggests that the latter is more likely to be true . . ' 8.2 Mechimisms of control In reviewing :60 publications from the last three decades which utilized plant growth analysis, Poorter ( 1990) indicated that the partitioning of dry matter into leaf area was found to be "a crucial attribute determining the potential rate of dry matter accwnulation (RGRw) of a species:' ln contrast, unless grown under conditions which result in large changes in the efficiency of leaves to produce dry matter (i.e. , NAR, Konings, 1990) , NAR "seems only of secondary importance: Data presented in this thesis also indicate the importance of partitioning of dry matter into leaf area as a primary determinant of RGRw in Zantedeschia 'Best Gold: Across a 15 C range in daily mean temperature and a single-fold difference in PPF, the rate of dry matter accumulation (RGRw) of Zantedeschia 'Best Gold ' was highly correlated with the partitioning of dry matter into leaf development (LWP) . ln contrast, only a weak correlation was determined between RGRw and the efficiency of these leaves to produce additional dry matter, i . e. , net assimilation rate (NAR) . While many examples have been reported where this correlation is weak, there is evidence to suggest that this weak correlation results from a strong negative correlation between NAR and leaf area ratio (LAR), or as presented here LWP (Poorter, 1990) . Hence, while NAR is recognised as an important component of RGRw, its direct I ' 214 contribution is frequently masked by concomitant changes in LAR. With photosynthetic rate being a primary component of NAR, the strong correlation between photosynthetic rate and RGRw in Zantedeschia 'Best Gold' supports the interpretation that NAR is also an important determinant of plant growth. While photosynthetic rate was correlated with RGRw, it did not correlate with the dry matter accumulation rate of the tuber (RGRT) . Clearly the photosynthetic process must be involved in contributing photoassimilates for tuber growth, but it was suggested that the plant's response to dry matter partitioning into the leaf, i .e. , LWP, and the tuber, i .e. , TWP, had a greater influence on the relative growth rate of the tuber than could be accounted for by photosynthetic rate alone. In terms of RGRT it was evident that the mechanism of acclimation to temperature under the low PPF regime was different from that under the high PPF regime. However, the mechanisms of acclimation under both PPF regimes suggested that tuber growth was source limited. Source limitation was expressed either in terms of: 1) enhanced intersink competition for assimilates, as occurred under the low PPF regime, where enhanced leaf area development (L WP) was in direct competition with enhanced tuber growth (RGRT) . This was also confirmed in vitro where dry matter partitioning to the tuber and tuber dry weight was reduced under limited source strength. 2) efficiency of dry matter accumulation of leaf area present, as occurred under the high PPF regime, where large increases in RGRT were highly correlated with increased NAR. This was also confirmed in vitro where increased source strength increased partitioning to the tuber and tuber dry weight. The possibility of sink limitation of growth, as well as source limitation, was clearly portrayed in the in vitro studies. However, at temperatures commonly encountered during the growing season in warm-temperate climates, source strength would appear most limiting to tuber dry matter accumulation. While sink strength was found to be most limiting at temperatures above and below this range, i . e. , 31 and 1 6 C, this was under conditions of controlled source strength in vitro. Since under in vivo conditions such temperatures would potentially inhibit both source and sink strength, it is possible that an increase in either source or sink strength would have the potential to increase dry matter accumulation. ? Hence, while source and sink strengths must be in balance, in most cases dry matter accumulation can be considered to be simultaneously source and sink limited. Under in vitro conditions of minimal source limitation, it was suggested that the sink limitation at the temperature extremes of 16 and 3 1 C resulted from temperature-induced 215 limitations on growth and respiration. While this sink limitation of tuber growth occurred at both 1 6 and 3 1 C, an additional form of sink limitation was evident at 3 1 C. At 31 C partitioning of dry matter towards the tuber was restricted, reflecting the possibility of high temperature inactivation of starch metabolising or sucrose unloading enzymes (Krauss and Marschner, 1984; Hanft and Jones, 1986; Rijven, 1986; MacLeod and Duffus, 1988) . While it is possible that the reduced maximum total and tuber dry weights in vivo at 28 C, compared with 25 C, may have resulted from high temperature inactivation of starch metabolizing or sucrose unloading enzymes, 28 C was also associated with reduced values for LAP, LWP, and leaf area duration. Hence in vivo, at the temperature extreme of 28 C, a primary determinant of the reduced maximum total and tuber dry weights was also high temperature source limitation, through inhibition of leaf area expansion and duration . The depression in activity of the starch metabolising enzymes, ADPG-pyrophosphorylase and starch phosphorylase, in potatoes (Solanum tuberosum L.) at these high temperatures, could be used as the basis of a testable hypothesis to determine the rate limiting step of high temperature reduction of tuber growth in Zantedeschia. Similarly, as found in maize (Zea mays L.) kernels, examination of the activity of sucrose unloading enzymes such as acid invertase, would determine if this process was limiting. If conducted in vitro with controlled source strength, examination of the activity of these enzymes in Zantedeschia at temperatures inhibiting tuber growth, compared with those promoting tuber growth, would determine if in fact these enzymes and their activity might be associated with the rate limiting step for tuber growth. Conducting such an experiment at controlled source strength would enable the hypothesized control of enzyme concentration via assimilate supply (Farrar, 1990; Farrar and Williams, 1991) to be eliminated. Since the flux of soluble carbohydrates into the tubers of potato continued at temperatures which inhibited starch synthesis (Krauss and Marschner, 1984) it is suggested thCJ.t with Zantedeschia any influence of assimilate supply on enzyme concentration would be eliminated. However, quantification of carbohydrate fluxes, through use of radioactive labelling , would be required to validate this. Both C14 and C11 have been successfully used to investigate carbohydrate fluxes, and their relationship to source and sink strength, in a range of plant species (Ho 1979; Minchin and Thorpe, 1992) . In addition, conducting this experiment in vitro with controlled source strength would also eliminate the high temperature influence on inhibition of leaf area expansion and duration, and presumably therefore reduced source strength if conducted in vivo. The examination of cell numbers and their rates of division and enlargement, concomitant with the above proposed experiment, would indicate whether these processes might also be a primary determinant of growth of the storage organ. Across a diversity of storage organs, e.g. fruit, stolon-tuber or hypocotyl-root, the production of cells and their enlargement have been shown to be integral to the final size and the amount of assimilates stored (Reeve et al . , 1973; Rapoport and Loomis, 1986; 216 Cheng and Breen, 1992) . These processes of cell division and enlargement have been suggested as contributing to the high temperature inhibition of tuber growth in potato (K.rauss and Marschner, 1984) . The thickening of the hypocotyl-root of sugar beet (Beta vulgaris L.) can be regarded as a continuation from germination of successive initiation and enlargement of cambia and associated parenchymatous tissues (Rapoport and Loomis, 1986) . In contrast, the stolon? tuber of the potato arises from the summation of two separate processes, i .e . , stolon development and tuberization at the stolon tip. Therefore in contrast to sugar beet, the definite cellular differentiation that occurs in the formation of the potato tuber is more likely to involve a physiological trigger mechanism such as that involving phytohormones (Booth, 1963) . There is an obvious paucity of information on the anatomical structure of the compacted stem (tuber) of Zantedeschia species in group 2. However, since this storage organ is derived from the stem and its initiation required no obligate environmental trigger, its development is most likely similar to the continuation from germination of cellular division and enlargement, as reported for sugar beet. Therefore it remains to be seen just how important cell division and enlargement are in contributing to the rate limiting steps of tuber formation in Zantedeschia. The initiation of tuber growth, as denoted by increases in both structural and starch dry weights, did not require an obligate environmental trigger . . Hence, rather than looking to the tuber for the point of control of the onset of tuber growth, it is more likely that this was dictated by the establishment of a photosynthetic leaf area large enough to provide a carbohydrate flux beyond the demands for continued leaf and root development, and respiration. A future experiment which is able to differentially manipulate the competitive sink strengths of leaf development and tuber developme.nt would test the validity of this hypothesis. Differential manipulation of competitive sink strength could be achieved by removing a variable number of dominant buds from tubers prior to planting. Through the influence of apical dominance, the application of such a treatment would create a range of potential leaf area development rates and carbohydrate fluxes. It is suggested that those treatments with a greater number of dominant buds will take longer to reach a stage where the sink strength of continued leaf and root development, and respiration, is less than the sink strength of the tuber. As a result of this delay, the timing of the onset of tuber growth would also be delayed, hence supporting the hypothesis. In addition, radioactive labelling could be Wied in conjunction with the above experiment to test the hypothesis that differing sink strengths were associated with differing carbohydrate fluxes. During the last decade the frequently accepted theory that plant development is regulated by the concentration of phytohormones has been challenged (Trewavas, 1982; Fim, 1986) . 217 Such ? challenges have not denounced the involvement of phytohormones, but have alerted the scientific community to consider an alternative interpretation of their involvement. Stated simply, these authors have suggested that it is the plant' s sensitivity to phytohormones that changes rather than the concentration of phytohormones per se. ? Regardless of the potential for changes in sensitivity with development, phytohormone activity will involve interaction with some form of receptor within the plant, presumably a protein. While it is acknowledged that changes in sensitivity may also involve changes in affinity of receptors and the capacity of the cells to produce a response, techniques are available to determine receptor concentrations and their activities, as well as phytohormone concentrations. While it is unlikely that a phytohormone switch is involved in the tuber growth in Zantedeschia, the application of immunoassay techniques such as enzyme linked immunosorbent assay (ELISA), to determine their concentrations and activities, may prove beneficial in determining the potential role of phytohormones as a control mechanism for tuber development in Zantedeschia. Similarly, the synthesis of DNA and RNA associated with the synthesis of proteins can now be readily detected using DNA finger-printing techniques. Proteins such as patatin which have been associated with tuberization in potato, have been identified using immunoassays (Paiva et al. , 1983 ; Park et al. , 1983) . The accumulation of patatin in potato plants was found to be inversely related to the concentration of exogenously applied gibberellic acid (GA3) , and therefore supports the hypothesis of phytohormone regulation of development (Hannapel et al. , 1985) . However, the greater sensitivity of patatin accumulation to GA3 than starch, accumulation may be indicative of the lack of a direct causal link between these events, rather than being a "surprising " \result reported by Hannapel et al. (1985) . Re-examination of the role of proteins in tJberization, which encompasses the hypothesis of altered plant sensitivity as well as phytohormone "regulation, may provide a more effective avenue of research to determine the control mechanisms of storage organ development in crops such as Zantedeschia. Since publication of the paper most frequently cited as first interrelating the derived quantities of plant growth analysis (equation (3 . 1) , West et al. , 1920) , it might appear that our understanding of plant growth has not progressed to any degree. However, when considered in terms of the necessity for scientific hypotheses to be presented with complete unambiguity, the fact that the scientific community still utilizes this notational expression to describe plant growth can be viewed as an indication of its scientific rigour. Attempts to present some of the components of growth analysis in alternative forms (e.g . LAP and LWP), as used in the current experiments, and proposed by Jackson ( 1963) and Potter and Jones (1977) , can still be related back to the concept that "the growth rate of the plant depends simultaneously upon the efficiency of its leaves as producers of new material and upon the Ieafiness vf the plallt itself" (Hunt, 1982). However, in contrast to the ? ......... 218 conventionally used harvest index, application of this alternative expression of plant growth to partitioning of dry matter to the tuber (TWP), has provided a more sensitive measure of short term changes in partitioning as well as information on how it is influenced by growing environment (Boerboom, 1978; Keating et al. , 1982) , i .e. , equation (4. 1) . In the current study the superior sensitivity of TWP over that of harvest index was evident when the harvest index continued to decline well after the commencement of tuber growth , compared with the TWP which increased immediately. In this thesis plant growth analysis has been used to provide an empirical description of growth and dry matter partitioning, with at least the beginnings of a mechanistic model being developed, i .e. , equations (4. 18) to (4.23) . In addition to further investigating the mechanisms and rate limiting steps of tuber growth, as outlined above, future research could also focus on developing this empirical data into an assimilate partitioning model for Zantedeschia. Such models have been developed for a number of species (Fick et al . , 1973; Rees, and Thomley, 1973 ; Thomley and Johnson, 1990) and, since their robustness will be highly dependent on an understanding of the mechanisms of control, both forms of research will need to be developed concurrently. 8.3 Horticultural relevance and consequences The temperature treatments employed in the current study inciuded a range of mean tem?ratures (13 to 28 C) frequently encountered by N.Z. producers of Zantedeschia I during the growing season, under both protected and non-protected cultivation. Similarly, the two PPF regimes employed equated to a daily integral PPF received by plants in Palmerston North, N.Z. under peak, unshaded, mid-summer (high PPF) , and winter (low PPF) conditions. While it is appreciated that both temperature and PPF regimes vary with diurnal and seasonal progression, plant growth and dry matter accumula_tion are readily related to temperature and light integrals (Tollenaar et al. , 1979; Johnson and Thomley, 1985; Warrington and Norton, 1991), and therefore the findings of this study should have direct relevance to commercial horticulturalists. Since dry matter accumulation of Zaruedeschia 'Best Gold' was found to be highly adaptive to PPF regimes, an important horticultural management consequence of this study is that the establishment and maintenance of an effective leaf area will be critical if growth is to be maximised. The optimum PPF under which to grow Zaruedeschia 'Best Gold' was also dependent on temperature, with a maximum total plant dry weight occurring under the high PPF regime at 25 C . While the estimated maximum tuber dry matter accumulation occurred under the low PPF 219 regime, the commercial relevance of such a fmding must be interpreted with care. With financial pressure growers frequently seek crops and/or production systems that allow a short cropping period. Hence, while tubers of a greater weight may be attained eventually if plants are grown at 25 C under low PPF, tuber weight under the high PPF regime would be greater than that under the low PPF regime until 140 days (i. e. , 4 . 7 months) of growth. Therefore producers wishing to maximise tuber weight, but requiring a shorter production period, would be more likely to meet these goals by utilizing a high PPF regime. These findings are directly applicable to growers involved in the first season's growth from tissue cultured material, where protected greenhouse cultivation is typically used. In contrast, beyond the first season' s growth much of New Zealand's Zantedeschia production is from unprotected cultivation, and is therefore exposed to the cooler temperatures typical of a temperate to warm-temperate climate. Utilizing such a comparatively low cost production system, there is frequently less pressure to ensure a short annual cropping period. Using climate data from Palmerston North, New Zealand (40?23 'S) , at least 4 months would exist during the growing season where daily "normal " temperatures ([maximum + minimum]/2) average 16 C or greater (Shearer, 1973) . Interpolation of data presented in Figure 4.2 clearly indicates that after 120 days at this temperature tuber dry weight would be greater under the low PPF regime than under the high PPF regime. Considering the additional early and late periods of the growing season where growth continues to occur, it is suggested that as long as plant spacing was appropriate and leaf area duration was maintained, shading would result in a greater final tuber dry weight compared with non? shaded crops. Following back-transformation of the data presented in Figure 4.2 after 140 days growth,1 , tuber dry weight would be more than 60% greater under the low PPF regime than under the high PPF regime. A similar interpretation of results is attained for Kerikeri, New Zealand (35 ? 14'S), where more than 5 months would exist during the growing season where daily "normal " temperatures average 16 C or greater (Shearer, 1973). Hence shading of Zantedeschia crops in this region also has the potential to result in a greater final tuber dry weight compared with non-shaded crops. In contrast, in Invercargill, New Zealand (46?25 'S) , "rwrmal " temperatures during the growing season do not reach 16 C or greater, and only 2 months of the year receive a "nonnal " temperature of 13 C or greater. In the current study at 13 C, tubers of a greater weight were not attained under the low PPF regime compared with the high PPF regime until 160 days (i.e. , 5 . 3 months) . Hence use of shading in Invercargill can not be recommended. Even considering the lower daily integrated PPF in Invercargill than either Palmerston North or Kerikeri (De Lisle, 1966) , the natural growing season would appear to be too short to take advantage of the use of shading. Tuber producers in Invercargill would therefore need to examine the cost effectiveness of providing heating before considering the application of additional shading. 220 The paucity of any quantified data for Zantedeschia on just what is an appropriate plant spacing to optimise plant productivity makes it near impossible to give grower recommendations at this time. The current inability of commercial producers to offer any guarantee of adequate leaf area duration, places serious doubt on the commercial viability of making a general recommendation for the use of shade for all tuber producers in regions with climates similar to Palmerston North and Kerikeri. Leaf area duration will not only be influenced by temperature and PPF regime, but also irrigation, nutrition and weed control practices (refer Section 1 .5 .3) . In addition, the cost effectiveness of erecting shade structures for open-ground production areas will need to be evaluated before final commercial recommendations can be made. The linear relationship between temperature and RGRT indicated a PPF dependent base temperature for tuber growth between 4 .8 and 6. 1 C. This generally linear response of RGRT with temperature, between the base temperature and 28 C, can be used as the basis of a crop model (e.g . Karlsson et al. , 1989) to monitor and predict tuber growth. The development of a crop model into a grower useable system of graphical tracking (e.g. Heins et al. , 1987) would require further development of nondestructive measurements of leaf area and/or weight, but would eventually provide a much needed improvement in predicting and potentially improving tuber yield. A more immediate horticultural management consequence of the response of final tuber weight to temperature is that tuber producers must compare the cost effectiveness of growing under protected greenhouse cultivation with supplemental heating in the optimal range of 21 to 26 C, with the cost of utilizing unprotected cultivation in regions with daily "normal " temperatures near this optimal temperature range. Even warmer regions of New Zealand such as Kerikeri have "normal " temperatures during the growing season that only range between 17 and 19 C. Hence improvements in tuber yield should be obtainable in all regions of New Zealand through the use of supplemental heating. Clearly, the potential tuber yield from unprotected cultivation in warmer regions such as Kerikeri is greater than that of cooler regions such as Palmerston North or Invercargill. 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