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. The physiological basis of vigour control by apple rootstocks – an unresolved paradigm A thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Plant Physiology at Massey University, Palmerston North, New Zealand. Benedict Michael van Hooijdonk 2009 Abstract i Abstract For millennia, scions have been grafted onto dwarfing apple rootstocks to reduce final tree size. However, it is unclear how scion architecture is first modified by the dwarfing apple rootstock, the time from grafting when this occurs and the endogenous hormonal signalling mechanisms that may cause the initial modifications in growth that then define the future architecture of the scion. In this study, the dwarfing (M.9) rootstock significantly decreased the mean total shoot length and node number of ‘Royal Gala’ apple scions by the end of the first year of growth from grafting when compared with rootstock(s) of greater vigour (MM.106, M.793 and a ‘Royal Gala’ rootstock control). Similarly, the auxin transport inhibitor 1-N-naphthylphthalamic acid (NPA) applied to the stem of vigorous rootstocks significantly decreased mean total shoot length and node number of the scion, and the architectural changes imposed were generally similar to those imposed by M.9. For example, both treatments decreased the mean length and node number of the primary shoot, reduced the formation of secondary axes on the primary shoot and caused a greater proportion of primary and secondary shoots (if present) to terminate growth early. Decreased formation of secondary axes imposed by both treatments was reversed by applying the cytokinin benzylaminopurine (BAP) repeatedly to the scion, whilst applications of gibberellins (GA4+7) reduced the proportion of primary and secondary shoots that terminated growth early, therefore increasing the final mean length and node number of these shoot types. Both M.9 and NPA also significantly decreased the final mean dry mass and length of the root system. Given these general similarities, it is proposed that the basipetal IAA signal is of central importance in rootstock-induced scion dwarfing, and that a shoot/root/shoot signalling mechanism may exist whereby the stem tissue of the M.9 rootstock decreases the basipetal transport of IAA to the root during summer, thereby decreasing root growth and the amount of root- produced cytokinin and gibberellin transported to scion. Reduced amounts of cytokinin transported to the scion may decrease branching, whilst reduced amounts of gibberellins may decrease the duration for which a large proportion of primary and secondary shoots grow. Analysis of endogenous hormones for newly grafted composite ‘Royal Gala’ apple trees on rootstocks of different vigour provided some additional support for these ideas. It is recommended that future studies elucidate what unique properties of the M.9 bark act to restrict IAA transport, whilst it is concluded that gene(s) regulating rootstock-induced Abstract ii scion dwarfing are likely to control processes within the rootstock that modify the metabolism of IAA, its basipetal transport and the subsequent synthesis of root-produced vigour-inducing hormones including cytokinins and gibberellins. Acknowledgements iii Acknowledgements To my chief supervisor Dr David Woolley, thank you for your effort, advice, support and enthusiasm given throughout this study. I have appreciated, enjoyed and learnt from our many challenging discussions. Your passion for science made the whole experience very enjoyable. To Professor Ian Warrington, thank you for co-supervising this thesis given your demanding workload – your advice was the voice of reason, particularly when it came to deciding how much work was enough for this qualification. Your efficiency at returning chapters was legendary. To Dr Stuart Tustin, thank you also for co-supervising this thesis and for your advice given. Your enthusiasm for your work and encouragement was very instrumental in my decision to develop a project in the area of rootstock physiology. Your have always provided me with positive feedback, which was very motivational. I would also like to thank Chris Rawlingson for his friendship, technical assistance and many enjoyable and competitive fishing trips. I would also like to thank the technical staff of the Plant Growth Unit for their friendship and help given. To my parents, thank you for your help in planting my massive field trials – without this help I would not have completed this research. I am blessed to have such great parents and I love you both. I would like to thank my wonderful wife Rebecca. You were so supportive and made many sacrifices in order that I could complete this Degree. I will always be indebted to you. Thank you for putting up with me when I came home tired and grumpy. I love you very much. Lastly, I would also like to acknowledge the financial support of Pipfruit New Zealand Incorporated, the Tertiary Education Commission and Horticulture New Zealand. Extended thesis summary iv Extended thesis summary Recent research has identified a dwarfing locus (DW1) involved in the dwarfing mechanism of the M.9 rootstock (Rusholme-Pilcher et al., 2008) and a new genetic map has been constructed of apple rootstock progeny derived from a cross between M.9 and ‘Robusta 5’ (Celton et al., 2009). These are important scientific advancements in the attempt to breed new and improved dwarfing apple rootstocks. For example, the identification of genetic markers linked to genes involved in rootstock-induced scion dwarfing, and other important traits like pest and disease resistance, will enable desirable progeny to be selected from large populations of tree material at a very young age. Therefore, efficiency and effectiveness of rootstock breeding programmes will be increased. Genetic maps constructed for rootstock progeny derived from a M.9 x ‘Robusta 5’ cross (Celton et al., 2009) also have potential application in further elucidating the genetic control of rootstock-induced dwarfing of the scion. For the dwarfing apple rootstock, the fundamental biological processes that dwarfing gene(s) control are poorly understood, particularly the underlying physiological mechanism(s) that are first modified within the composite apple tree growing on a dwarfing rootstock, and their consequent expression in scion architecture during early tree phenology. Important physiological mechanisms by which dwarfing apple rootstocks decrease scion vigour may involve restricting the endogenous transport of nutrients, water and hormones. The most plausible of these is the modification of shoot/root/shoot signalling of endogenous plant hormones because alterations in their transport appear to explain some architectural changes imposed on the scion by the dwarfing apple rootstock. However, it is poorly understood how the modification of hormonal signals by a dwarfing apple rootstock may change scion architecture soon after grafting of the composite tree. Understanding this is essential to clearly identify those signals and processes that are the first physiological causes of scion dwarfing from those that are subsequent developmental effects. Therefore, ‘Royal Gala’ apple scions were grafted onto rootstocks of M.9 (dwarf), MM.106 (semi-vigorous), M.793 (vigorous) and ‘Royal Gala’ (self-rooted, very vigorous; control) to determine how each rootstock type initially modified scion architecture after propagation of the composite tree. These modifications were also Extended thesis summary v compared with those of root restriction and plant growth regulators applied to either the scion (± gibberellins (GA4+7), ± benzylaminopurine (BAP)) or an auxin transport inhibitor (± 1-N-naphthylphthalamic acid (NPA)) applied to the stem of the rootstock. In four different experiments, M.9 decreased the mean total shoot length and node number of ‘Royal Gala’ apple scions by the end of the first year of growth from grafting when compared with rootstocks of greater vigour (Chapters 3, 4, 5, and 6 and see Figure B for a typical example). The mean cumulative length and node number of the primary shoot was initially similar amongst rootstocks prior to December. Thereafter, cumulative growth of the primary shoot on M.9 was decreased compared with rootstocks of greater vigour. This occurred because a greater proportion of primary shoots either underwent a bicyclic pattern of growth in December (Chapter 4) or February (Chapter 5) and then terminated growth earlier in April (Chapter 4), or grew from a continuous season-long growth flush before terminating growth earlier in April (Chapter 3). The general effect of these growth patterns imposed by M.9 was to decrease the final mean length and node number of the primary shoot (Figure A) by complete growth cessation (Chapters, 3, 4 and 5). The mean internode length of the primary shoot was unaffected by rootstock type, hence the primary shoot on M.9 was shorter (i.e., Chapters 3, 4 and 5) because of fewer neoformed nodes. In Chapter 6, however, cumulative growth of the primary shoot on M.9 was similar to rootstocks of greater vigour, and the dwarfing effect mostly resulted from the development of fewer secondary shoots. The M.9 rootstock also decreased the formation of secondary axes on the primary shoot during the first year of growth from grafting, particularly secondary shoots (Figure B), and this was an important architectural change that also contributed to reduced total shoot growth of the ‘Royal Gala’ scion growing on M.9 (Chapters 3, 5, and 6 and see Figure B). In Chapter 4, however, the scion on M.9 produced a greater mean number of secondary axes compared with that on MM.106, although this was not a typical effect of M.9, and may have been imposed by transplanting the tree material into the field in December. In each experiment, M.9 formed proportionally more secondary spurs (i.e., < 25 mm) and short secondary shoots (i.e., ≥ 25 mm but with ≤ 10 nodes) compared with rootstocks of greater vigour. Both of these shoot types may have formed solely from preformed primordia within the vegetative axillary bud and terminated very soon after Extended thesis summary vi their outgrowth. The M.9 rootstock also decreased the proportion of secondary shoots with more than 10 nodes, or those that had produced neoformed nodes, particularly very long secondary shoots with more than 20 nodes. This was a likely result of proportionally more secondary shoots completely terminating growth early in February and March when the scion was grown on M.9. Regardless of rootstock type, secondary shoots with the same node number were of almost identical length. Hence, M.9 did not affect internode length of ‘Royal Gala’ shoots, and this result supports the findings of other previous studies (Selezynova et al., 2003, 2008). The application of NPA to the rootstock stem of MM.106, M.793 and ‘Royal Gala’ significantly decreased the final mean total length and node number of the scion, and the architectural modifications were most similar to those that occurred for untreated ‘Royal Gala’ trees growing on M.9 (compare Figures B and F). For example, following an application of NPA, the shoot apical meristem (SAM) on the primary shoot slowed, and/or, terminated its growth temporarily, thereby decreasing the final mean length and node number of the primary shoot. For both M.9 and NPA-treated trees, reduced cumulative growth of the primary shoot was reversed with GA4+7 applied repeatedly to the scion (Figure A), however few additional secondary and tertiary axes developed on the scion without applications of BAP (Figure B, D, F and H). The NPA treatment also decreased the formation of secondary axes on the primary shoot, and like M.9, the formation of secondary axes on the primary shoot was reinstated with exogenous BAP (Figures B, C, F and G). However, new secondary axes that formed for the BAP-treated scion on M.9 or NPA-treated rootstocks generally terminated without exogenous GA4+7 (compare C and E or G and I for M.9 or NPA, respectively). Sequential applications of BAP followed by GA4+7 to the scion on M.9 increased branch formation and decreased the proportion of primary and secondary shoots that terminated growth early during summer. Consequently, the dwarfing effect of M.9 was reversed to some extent, particularly for the BAP x GA4+7-treated scion on M.9 and MM.106 that developed very similar total shoot growth (Figure E). In addition, total shoot length and node number of the BAP x GA4+7-treated scion on M.9 was much greater than that of the untreated tree grown on the ‘Royal Gala’ rootstock. However, the BAP x GA4+7- treated scion on M.9 was still markedly smaller than the BAP x GA4+7-treated scion on M.793 or ‘Royal Gala’ (Figure E). Similarly, BAP x GA4+7 applied to the scion on Extended thesis summary vii NPA-treated rootstocks of MM.106, M.793 and ‘Royal Gala’ increased total shoot growth of the scion (Figures F and I). However, BAP x GA4+7 applied to the scion on NPA-treated rootstocks of MM.106, M.793 and ‘Royal Gala’ stimulated markedly less total shoot extension growth when compared with the BAP x GA4+7-treated scion on the same rootstock type that was not treated with NPA (compare Figures E and I). Hence, BAP x GA4+7 could not fully reverse reductions in total scion growth whilst IAA transport from shoot to root was impaired by the NPA treatment, and this may explain why the BAP x GA4+7-treated scion on M.9 was smaller than the BAP x GA4+7-treated scion on M.793 and ‘Royal Gala’ (Figure E). Treatments that decreased the size of the root system, such as M.9, NPA and root restriction, also decreased the total shoot growth or size of the scion. This indicated that a functional relationship existed between the size of the root and shoot, and that part of the dwarfing effect imposed by M.9 may be explained because of its smaller root system. However, some physiological mechanisms regulating scion vigour for M.9 and root restriction differ because, unlike M.9, decreased formation of axillary axes imposed by root restriction was not fully reversed with BAP applied repeatedly to the scion, and root restriction tended to decrease the size of leaves. Results from this study, based on both application of plant growth regulators and analysis of endogenous hormones, have led to the conclusion that the basipetal IAA signal is of central importance in rootstock-induced scion dwarfing. A shoot/root/shoot signalling mechanism may exist whereby the stem tissue of the M.9 rootstock decreases the basipetal transport of IAA to the root during the summer, thereby decreasing root growth and the amount of root-produced cytokinin and gibberellin transported to scion, which consequently decreases either branch formation or the duration for which primary and secondary shoots grow, respectively. In partial support of this hypothesis, the M.9 rootstock had a significantly lower concentration of GA19 in the xylem sap during March (Chapter 6). However, further research would be required to show more convincingly that the above model of growth regulation is reflected in the endogenous transport of hormones. In particular, it would be important to demonstrate that decreased shoot/root basipetal transport of IAA by the M.9 rootstock reduces root/shoot transport of either cytokinins or gibberellins, and that decreased root/shoot transport of cytokinins precede the period(s) in the growing season when axillary bud outgrowth Extended thesis summary viii occurs, whilst decreased root/shoot transport of gibberellins precedes the predominant time when shoot termination first occurs on the scion. It is also recommended that future studies elucidate what unique properties of the M.9 bark act to restrict IAA transport because it is likely that primary gene(s) regulating rootstock-induced scion dwarfing control processes within the rootstock that affect the transport and metabolism of IAA. Extended thesis summary ix 1/11 1/12 1/1 1/2 1/3 1/4 1/5 1/6 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 iii b ab b a c c c d M ea n le ng th o f p ri m ar y sh oo t ( m ) Day / month (2005-2006) 1/11 1/12 1/1 1/2 1/3 1/4 1/5 1/6 15 20 25 30 35 40 45 50 55 60 iv ab ab b a c c c d M.9 M.9 + GA4+7 MM.106 MM.106 + GA4+7 M.793 M.793 + GA4+7 'R. Gala' 'R. Gala' + GA4+7 M ea n no de n um be r of p ri m ar y sh oo t Day / month (2005-2006) Figure A. Gibberellin (GA4+7) x 1-N-naphthylphthalamic acid (NPA) (i and ii) and rootstock x GA4+7 interactions (iii and iv) for the mean cumulative length and node number of ‘Royal Gala’ primary shoots during their first growing season from grafting. Arrows on ii or iv with a dotted line denotes the timing of GA4+7 treatments to the scion, whilst arrows with a solid line on ii denote the timing of NPA treatment to the rootstock stem. On a single graph, means sharing the same letter are not significantly different. Mean separation in May is at P≤0.11 for i and P≤0.05 for ii, iii and iv (lsmeans tests with Tukey’s adjustment, SAS). Data for i and ii are averaged over BAP and rootstock treatments, whilst iii and iv are averaged over BAP and NPA treatments. 1/11 1/12 1/1 1/2 1/3 1/4 1/5 1/6 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 d c b a Day / month (2005 -2006) i M ea n le ng th o f p ri m ar y sh oo t ( m ) 1/11 1/12 1/1 1/2 1/3 1/4 1/5 1/6 15 20 25 30 35 40 45 50 55 60 ii -GA4+7 -NPA -GA4+7 +NPA +GA4+7 -NPA +GA4+7 +NPA Day / month (2005-2006) d c b a M ea n no de n um be r of p ri m ar y sh oo t Ex te nd ed th es is su m m ar y x Fi gu re B . E ffe ct o f M .9 (d w ar fin g) , M M .1 06 (s em i-v ig or ou s) , M .7 93 (v ig or ou s) a nd ‘R oy al G al a’ (v er y vi go ro us c on tro l) ro ot sto ck s (fr om le ft to ri gh t, re sp ec tiv el y) o n th e ar ch ite ct ur e of ‘R oy al G al a’ a pp le sc io ns b y th e en d of th ei r f irs t g ro w in g se as on fr om g ra fti ng . Y el lo w ru le is 1 m . Th e M .9 r oo ts to ck ty pi ca lly im po se d sc io n dw ar fin g by d ec re as in g br an ch fo rm at io n an d re du ci ng th e fin al le ng th o f t he p ri m ar y an d se co nd ar y sh oo ts b y in cr ea sin g th e pr op or tio n of th es e sh oo ts th at te rm in at ed g ro w th e ar ly . Ex te nd ed th es is su m m ar y xi Fi gu re C . E ffe ct o f be nz yl am in op ur in e (B A P) a pp lie d re pe at ed ly to ‘ Ro ya l G al a’ a pp le s ci on s on s ci on a rc hi te ct ur e by th e en d of th e fir st gr ow in g se as on a fte r g ra fti ng th e sc io n on to ro ot sto ck s o f M .9 , M M .1 06 , M .7 93 a nd ‘R oy al G al a’ (l ef t t o rig ht , r es pe ct iv el y) . Y el lo w ru le is 1 m . D ec re as ed b ra nc hi ng im po se d by M .9 (s ee F ig ur e B) w as r ev er se d by a pp ly in g BA P to th e sc io n, th er ef or e in di ca tin g th e dw ar fin g ap pl e ro ot st oc k m ay d ec re as e th e tr an sp or t o f e nd og en ou s c yt ok in in s t o th e sc io n, w hi ch r ed uc es b ra nc hi ng . Ex te nd ed th es is su m m ar y xi i Fi gu re D . E ffe ct o f g ib be re lli ns (G A 4+ 7) ap pl ie d re pe at ed ly to ‘R oy al G al a’ a pp le s ci on s on s ci on a rc hi te ct ur e by th e en d of th e fir st gr ow in g se as on a fte r g ra fti ng th e sc io n on to ro ot sto ck s o f M .9 , M M .1 06 , M .7 93 a nd ‘R oy al G al a’ (l ef t t o rig ht , r es pe ct iv el y) . Y el lo w ru le is 1 m . E ar lie r sh oo t te rm in at io n im po se d by M .9 w as p re ve nt ed b y ap pl yi ng G A 4+ 7 to t he s ci on , th er ef or e in di ca tin g th at t he d w ar fin g ro ot st oc k de cr ea se s t he tr an sp or t o f e nd og en ou s r oo t-p ro du ce d gi bb er el lin s t o th e sc io n, w hi ch in cr ea se s s ho ot te rm in at io n. Ex te nd ed th es is su m m ar y xi ii Fi gu re E . E ffe ct o f s eq ue nt ia l a pp lic at io ns o f B A P fo llo w ed b y G A 4+ 7 o n sc io n ar ch ite ct ur e of ‘R oy al G al a’ a pp le sc io ns b y th e en d of th e fir st gr ow in g se as on a fte r g ra fti ng th e sc io n on to ro ot sto ck s o f M .9 , M M .1 06 , M .7 93 a nd ‘R oy al G al a’ (l ef t t o rig ht , r es pe ct iv el y) . Y el lo w ru le is 1 m . A pp ly in g cy to ki ni n in cr ea se d br an ch in g w hi lst g ib be re lli n re du ce d th e pr op or tio n of s ho ot s th at t er m in at ed g ro w th e ar ly , b ut s ci on dw ar fin g st ill o cc ur re d on M .9 w he n co m pa re d w ith th e BA P x G A 4+ 7-t re at ed s ci on o n M .7 93 a nd ‘R oy al G al a’ . A li ke ly d iff er en ce th at st ill e xi st ed a m on gs t th es e tr ee s w as t he ir c ap ac ity t o tr an sp or t au xi n to t he r oo ts , w ith t he s te m t iss ue o f th e M .9 r oo ts to ck h av in g a re du ce d ca pa ci ty to d o so . T hi s re su lt su gg es ts th at a ux in tr an sp or t d iff er en ce s am on gs t t he se r oo ts to ck s m ay b e th e pr im ar y ca us e of sc io n dw ar fin g. Ex te nd ed th es is su m m ar y xi v Fi gu re F . E ffe ct o f t he a ux in tr an sp or t i nh ib ito r 1 -N -n ap ht hy lp ht ha la m ic a ci d (N PA ) a pp lie d to th e ste m o f M .9 , M M .1 06 , M .7 93 a nd ‘R oy al G al a’ ro ot sto ck s ( le ft to ri gh t, re sp ec tiv el y) o n sc io n ar ch ite ct ur e of c om po sit e ‘R oy al G al a’ a pp le tr ee s b y th e en d of th ei r f irs t g ro w in g se as on fro m g ra fti ng . Y el lo w ru le is 1 m . T he ‘R oy al G al a’ s ci on o n th e vi go ro us r oo ts to ck s co ul d be m ad e to b eh av e lik e th e sc io n gr af te d on to M .9 (c om pa re w ith F ig ur e B) , a nd th e ar ch ite ct ur al c ha ng es w er e ge ne ra lly si m ila r to th os e im po se d by M .9 (i .e ., re du ce d br an ch in g an d in cr ea se d sh oo t te rm in at io n) , th er eb y in di ca tin g th e ba sip et al t ra ns po rt o f au xi n is an i m po rt an t ph ys io lo gi ca l sig na l re gu la tin g ro ot st oc k- in du ce d sc io n dw ar fin g. Ex te nd ed th es is su m m ar y xv Fi gu re G . E ffe ct o f 1 -N -n ap ht hy lp ht ha la m ic a ci d (N PA ) a pp lie d to M .9 , M M .1 06 , M .7 93 a nd ‘R oy al G al a’ ro ot sto ck s ( le ft to ri gh t, re sp ec tiv el y) pl us e xo ge no us b en zy la m in op ur in e (B A P) a pp lie d to th e sc io n on th e ar ch ite ct ur e of ‘R oy al G al a’ a pp le sc io ns b y th e en d of th ei r f irs t g ro w in g se as on fr om g ra fti ng . Y el lo w ru le is 1 m . D ec re as ed b ra nc hi ng in r es po ns e to t re at m en t of t he r oo ts to ck s te m w ith t he a ux in t ra ns po rt in hi bi to r ‘N PA ’ co ul d be r ei ns ta te d by a pp ly in g cy to ki ni n to th e sc io n, a nd th e sc io n ph en ot yp e on M M .1 06 , M .7 93 a nd ‘R oy al G al a’ w as si m ila r to th e BA P- tr ea te d sc io n gr af te d on to th e M .9 r oo ts to ck th at w as n ot tr ea te d w ith N PA (s ee F ig ur e C ). Th er ef or e, d ec re as ed sh oo t/r oo t t ra ns po rt o f I A A a nd r ed uc ed r oo t/s ho ot tr an sp or t o f c yt ok in in m ay c au se d ec re as ed b ra nc h fo rm at io n of th e sc io n on M .9 . Ex te nd ed th es is su m m ar y xv i Fi gu re H . E ffe ct o f 1 -N -n ap ht hy lp ht ha la m ic a ci d (N PA ) a pp lie d to M .9 , M M .1 06 , M .7 93 a nd ‘R oy al G al a’ ro ot sto ck s ( le ft to ri gh t, re sp ec tiv el y) pl us e xo ge no us g ib be re lli n (G A 4+ 7) ap pl ie d to th e sc io n on th e ar ch ite ct ur e of ‘R oy al G al a’ a pp le sc io ns b y th e en d of th ei r f irs t g ro w in g se as on fro m g ra fti ng . Y el lo w ru le is 1 m . E xo ge no us G A 4+ 7 pr ev en te d th e pr im ar y sh oo t f ro m te rm in at in g gr ow th in r es po ns e to N PA tr ea tm en t of t he r oo ts to ck s te m , t he re by a lle vi at in g th e ef fe ct s of p ro ba bl e im pa ir ed a ux in t ra ns po rt , a nd i nd ic at in g th at d ec re as ed s ho ot /r oo t tr an sp or t o f a ux in a nd r ed uc ed r oo t/s ho ot tr an sp or t o f e nd og en ou s gi bb er el lin m ay c au se e ar lie r sh oo t t er m in at io n of th e sc io n on th e M .9 r oo ts to ck . Ex te nd ed th es is su m m ar y xv ii Fi gu re I. E ffe ct o f 1 -N -n ap ht hy lp ht ha la m ic a ci d (N PA ) a pp lie d to M .9 , M M .1 06 , M .7 93 a nd ‘R oy al G al a’ ro ot sto ck s ( le ft to ri gh t, re sp ec tiv el y) pl us b en zy la m in op ur in e (B A P) fo llo w ed b y gi bb er el lin (G A 4+ 7) ap pl ie d se qu en tia lly to th e sc io n on th e ar ch ite ct ur e of ‘R oy al G al a’ a pp le sc io ns by th e en d of th ei r f irs t g ro w in g se as on fr om g ra fti ng . Y el lo w ru le is 1 m . E xo ge no us B A P re in st at ed b ra nc hi ng w hi lst G A 4+ 7 pr ev en te d th e pr im ar y an d se co nd ar y sh oo ts f ro m t er m in at in g gr ow th e ar ly , t he re by in di ca tin g th e ba sip et al t ra ns po rt o f IA A in te ra ct s w ith b ot h ro ot -p ro du ce d cy to ki ni n an d gi bb er el lin . Table of contents xviii Table of contents Abstract .......................................................................................................................................... i Acknowledgements...................................................................................................................... iii Extended thesis summary ............................................................................................................ iv Table of contents...................................................................................................................... xviii List of figures.............................................................................................................................xxv List of tables........................................................................................................................... xxxvi List of appendices ................................................................................................................ xxxviii List of abbreviations .............................................................................................................. xxxix 1. General introduction ................................................................................................................. 1 1.1 Taxonomy of apple ............................................................................................................. 1 1.2 History of apple cultivation ................................................................................................ 1 1.3 Present utilisation of apple rootstocks in New Zealand...................................................... 3 1.4 The need to improve dwarfing apple rootstocks through breeding..................................... 5 1.4.1 Difficulties associated with breeding new dwarfing apple rootstocks......................... 5 1.5 Propagation of the composite tree in the nursery................................................................ 6 1.5.1 Budding........................................................................................................................ 7 1.5.2 Grafting ........................................................................................................................ 7 1.5.3 Extension growth of the scion in the first year after propagation and general terminology to describe growth ............................................................................................ 9 1.5.4 Propagation methods may modify early growth of the composite tree...................... 10 1.6 Alteration of scion architecture by dwarfing apple rootstocks ......................................... 12 1.6.1 Types of dwarfism in apple........................................................................................ 12 1.6.2 The initial modification of scion architecture by dwarfing apple rootstocks............. 13 1.7 Physiology of rootstock-induced dwarfing of the scion ................................................... 16 1.7.1 Nutrients..................................................................................................................... 16 Table of contents xix 1.7.2 Altered plant water status........................................................................................... 17 1.7.3 Alterations in shoot/root/shoot transport of endogenous plant hormones.................. 19 1.8 Summary, rationale and thesis objectives ......................................................................... 24 1.8.1 Summary of major objectives .................................................................................... 26 2. General materials and methods ............................................................................................... 27 2.1 Propagation of self-rooted ‘Royal Gala’ rootstocks.......................................................... 27 2.1.1 Hardwood cuttings ..................................................................................................... 27 2.1.2 Aerial layering ........................................................................................................... 27 2.1.3 Stool bed system ........................................................................................................ 28 2.2 Preparation and management of experimental tree material............................................. 30 2.3 Extraction procedures for hormone quantification ........................................................... 32 2.3.1 Collection of xylem sap from ‘Royal Gala’ apple scions for the analysis of cytokinins and gibberellins................................................................................................................... 32 2.3.2 Diffusion of indole-3-acetic acid from the primary shoot apex of ‘Royal Gala’ apple scions................................................................................................................................... 32 2.4 Chromatography of endogenous plant hormones ............................................................. 33 2.4.1 Development of a preliminary purification method for cytokinins and gibberellins within the xylem sap of apple ............................................................................................. 33 2.4.2 Pre-purification of cytokinins from xylem sap using Sep-Pak® C18 columns before separation of cytokinins by high performance liquid chromatography (HPLC)................. 34 2.4.3 Development of a purification method for indole-3-acetic acid diffused from the shoot apices......................................................................................................................... 35 2.4.4 Synthesis of carboxyl-labelled indole-3-acetic acid methyl ester.............................. 36 2.4.5 Routine procedure for pre-purification of indole-3-acetic acid using Sep-Pak® C18 columns before HPLC separation ....................................................................................... 38 2.4.6 High performance liquid chromatography of plant hormones ................................... 41 2.5 Hormone quantification .................................................................................................... 46 2.5.1 Repurification of cytokinin trialcohols by High Performance Liquid Chromatography ............................................................................................................................................ 46 2.5.2 Radioimmunoassay of cytokinins .............................................................................. 47 Table of contents xx 2.5.3 Quantification of indole-3-acetic acid by HPLC-spectrofluorimetry ........................ 49 2.5.4 Confirmation of indole-3-acetic acid concentrations determined by HPLC- fluorescence with quantification by gas chromatography-mass spectrometry.................... 51 2.5.5 Quantification of gibberellins in the xylem sap of apple by gas chromatography-mass spectrometry........................................................................................................................ 57 2.6 Synthesis of the auxin transport inhibitor 1-N-naphthylphthalamic acid ......................... 62 2.6.1. Synthesis of 1-N-naphthylphthalamic acid ............................................................... 62 2.6.2 Testing of the biological efficacy of synthesised 1-N-naphthylphthalamic acid using a lettuce root growth bioassay ............................................................................................... 62 2.6.3 Determination of a suitable physiological concentration of applied 1-N- naphthylphthalamic acid and 2,3,5,-Triiodobenzoic acid to reduce the growth of apple scions................................................................................................................................... 65 2.7 Measurement of scion growth and architecture ................................................................ 68 2.7.1 Measurement of vegetative shoot growth .................................................................. 68 2.8 Statistical analysis............................................................................................................. 72 3. Architectural responses of ‘Royal Gala’ apple scions to the influences of rootstock, root- restriction and benzylaminopurine.............................................................................................. 74 3.1 Introduction....................................................................................................................... 74 3.2 Materials and methods ...................................................................................................... 78 3.2.1 Site and establishment of experimental tree material ................................................ 78 3.2.2 Growing medium and irrigation................................................................................. 78 3.2.3 Application of the cytokinin benzylaminopurine....................................................... 79 3.2.4 Measurements of scion growth .................................................................................. 79 3.2.5 Statistical analysis ...................................................................................................... 79 3.3 Results............................................................................................................................... 81 3.3.1 Irrigation scheduling .................................................................................................. 81 3.3.2 Seasonal extension growth of the primary shoot for the main effects of rootstock, benzylaminopurine and root volume................................................................................... 81 3.3.3 Final leaf area of the primary shoot for the main effects of rootstock, benzylaminopurine and root volume................................................................................... 82 Table of contents xxi 3.3.4 Final extension growth of the primary shoot ............................................................. 82 3.3.5 Treatment effects on the formation of secondary axes on the primary shoot and tertiary spurs on the secondary shoots ................................................................................ 92 3.3.6 Leaf area of the secondary shoots ............................................................................ 105 3.3.7 Final growth measurements of the secondary shoots............................................... 106 3.4. Discussion...................................................................................................................... 115 3.4.1 Effectiveness of irrigation scheduling...................................................................... 115 3.4.2 Effect of rootstock type on vegetative growth ......................................................... 115 3.4.3 Effects of root volume on vegetative growth of the scion ....................................... 117 3.4.4 Effects of exogenous cytokinin on vegetative growth of the scion.......................... 119 3.5 Summary......................................................................................................................... 124 4. Effect of rootstock and root restriction on scion architecture and the first occurrence of flowering for field-grown ‘Royal Gala’ apple trees.................................................................. 126 4.1 Introduction..................................................................................................................... 126 4.2 Materials and methods .................................................................................................... 127 4.2.1 Experimental site...................................................................................................... 127 4.2.2 Tree establishment and planting .............................................................................. 127 4.2.3 Irrigation and cultural management ......................................................................... 128 4.2.4 Measurements of scion growth ................................................................................ 128 4.2.5 Statistical analysis .................................................................................................... 128 4.3 Results............................................................................................................................. 130 4.3.1 Irrigation scheduling ................................................................................................ 130 4.3.2 Seasonal growth of the primary shoot...................................................................... 130 4.3.3 Final leaf area of the primary shoot ......................................................................... 134 4.3.4 Final growth measurements of the primary shoot.................................................... 134 4.3.5 Seasonal growth of the secondary shoots................................................................. 137 4.3.6 Final measurements of extension growth for the secondary shoots......................... 143 Table of contents xxii 4.3.7 Treatment effects on the first occurrence of flowering in the spring of year two (2005)................................................................................................................................ 148 4.4 Discussion....................................................................................................................... 153 4.4.1 Effects of rootstocks on vegetative growth.............................................................. 153 4.4.2 Effects of root restriction on vegetative growth....................................................... 156 4.4.3 Effect of rootstocks on the first occurrence of flowering on ‘Royal Gala’ scions... 160 4.4.4 Similarities between rootstock and root restriction on the first occurrence of scion flowering........................................................................................................................... 162 4.5 Summary......................................................................................................................... 163 5. Initial alteration of scion architecture by dwarfing apple rootstocks may involve shoot/root/shoot signalling sequences by auxin, gibberellin and cytokinin.............................. 166 5.1 Introduction..................................................................................................................... 166 5.2 Materials and methods .................................................................................................... 169 5.2.1 Site preparation and management of tree material................................................... 169 5.2.2 Application of plant growth regulators .................................................................... 169 5.2.3 Measurements of tree growth................................................................................... 170 5.2.4 Statistical analysis .................................................................................................... 170 5.3 Results............................................................................................................................. 171 5.3.1 Irrigation scheduling ................................................................................................ 171 5.3.2 Growth of the primary shoot.................................................................................... 171 5.3.3 Treatment effects on the formation of secondary and tertiary axes ......................... 187 5.3.4 Final growth measurements of the scion.................................................................. 204 5.3.5 Final dry mass and length of the root system........................................................... 228 5.4 Discussion....................................................................................................................... 232 5.4.1 Effect of rootstocks on scion vigour and architecture.............................................. 232 5.4.2 Effect of rootstock, NPA, BAP and GA4+7 on meristematic activity ....................... 233 5.4.3 Effect of treatments on the formation of secondary axes......................................... 239 5.4.4 Effect of treatments on total scion growth ............................................................... 242 Table of contents xxiii 5.4.5 Effect of rootstock and NPA on root growth ........................................................... 245 5.5 Summary......................................................................................................................... 248 6. Effect of rootstock type on scion architecture, root growth and transport of some endogenous hormones in newly grafted ‘Royal Gala’ apple trees................................................................ 252 6.1 Introduction..................................................................................................................... 252 6.2 Materials and methods .................................................................................................... 254 6.2.1 Experimental site, establishment and management of tree material ........................ 254 6.2.2 Scion extension growth............................................................................................ 254 6.2.3 Leaf area................................................................................................................... 254 6.2.4 Diffusion of indole-3-acetic acid from the primary shoot apex ............................... 254 6.2.5 Extraction of xylem sap from the primary shoot ..................................................... 254 6.2.6 Total dry mass of stems and leaves.......................................................................... 255 6.2.7 Total length and dry mass of the root system .......................................................... 255 6.2.8 Statistical analysis .................................................................................................... 255 6.3 Results............................................................................................................................. 256 6.3.1 Growth of the primary shoot.................................................................................... 256 6.3.2 Growth of the secondary shoots............................................................................... 257 6.3.3 Effect of rootstock type on the total shoot length and node number per scion ........ 263 6.3.4 Effect of rootstock type on leaf area of the scion..................................................... 263 6.3.5 Effect of rootstock type on the mean total length of the root system....................... 264 6.3.6 Effect of rootstock type on mean total dry mass of stems, leaves and roots............ 265 6.3.7 Effect of rootstock type on endogenous transport of IAA, cytokinins and gibberellins .......................................................................................................................................... 279 6.3.8 Relationships between IAA, cytokinin and the formation of secondary axes.......... 289 6.3.9 Relationships between IAA and gibberellin on shoot apical meristem activity....... 290 6.4 Discussion....................................................................................................................... 293 6.4.1 Influence of rootstock type on growth of the scion.................................................. 293 Table of contents xxiv 6.4.2 Effect of rootstock type on scion and root growth and the transport of endogenous IAA, cytokinins and gibberellins ...................................................................................... 298 6.5 Summary......................................................................................................................... 307 7. General discussion and conclusions...................................................................................... 310 7.1 Effect rootstock type on the initial development of dwarfing scion architecture ........... 310 7.1.1 Extension growth of the primary shoot.................................................................... 311 7.1.2 Formation of secondary axes and extension growth of the secondary shoots.......... 313 7.2 Effect rootstock and root-restriction on scion architecture ............................................. 317 7.2.1 Growth of the primary shoot.................................................................................... 318 7.2.2 Growth of the secondary shoots............................................................................... 319 7.3 Endogenous hormonal control of the initial modifications in scion architecture on the M.9 rootstock................................................................................................................................ 321 7.3.1 Shoot/root/shoot signalling of IAA, cytokinin and gibberellin................................ 321 8. Appendices............................................................................................................................ 331 9. References............................................................................................................................. 334 List of figures xxv List of figures Figure 1.1. Example of a cleft cut into a one-year-old rooted stool of MM.106 and the matching wedge cut into a one-year-old ‘Royal Gala’ scion prior to (A) and after positioning of the scion into the cleft cut into the rootstock (B). .................................................................................................................................. 9 Figure 2.1. Aerial layering of a ‘Royal Gala’ shoot in November 2004 resulted in good root development by April, 2005. ........................................................................................................................................... 29 Figure 2.2. An example of a high-quality ‘Royal Gala’ stool produced in a stool bed system. ................. 29 Figure 2.3. Grafted tree material establishing in a tunnel house in mid September, 2004. ........................ 31 Figure 2.4. Tree material hardening off outside in November, 2004. ........................................................ 31 Figure 2.5. Mean concentration of zeatin measured by radioimmunoassay within fractions eluted off a Sep-Pak® C18 column. Zeatin (100 ng) was adsorbed onto the column before eluting with 5 mL of H20:0.5% formic acid (fraction 1), followed by three 2 mL washes (fractions 2 to 4) of 100% methanol. Vertical bars represent the standard error, n=2. ......................................................................................... 34 Figure 2.6. Recovery of carboxyl-labelled indole-3-acetic acid (14C-IAA) in fractions eluted off a Sep- Pak® C18 column. Following the adsorption of 14C-IAA onto the C18, the column was eluted with three 2 mL fractions of 15% methanol:0.5% formic acid (fractions 1, 2 and 3) followed by three 2 mL fractions of 100% methanol (fractions 4, 5 and 6). ................................................................................................... 36 Figure 2.7. Response of UV (top) and fluorescence (bottom) detectors to 500 ng of indole-3-acetic acid (IAA) after methylation with diluted diazomethane (1:5 dilution in ether) and separation by HPLC. The absence of a peak for IAA at 14.20 min indicates all of the IAA was methylated to indole-3-acetic acid methyl ester (IAA-Me) (24.30 min). .......................................................................................................... 39 Figure 2.8. Response of UV (top) and radioactivity (bottom) detectors to carboxyl-labelled indole-3- acetic acid methyl ester (14C-IAA-Me) after carboxyl-labelled indole-3-acetic acid (14C-IAA) was methylated with diazomethane. The peak of radioactivity (bottom) was delayed by approximately 30 sec because the radioactivity detector was downstream of the UV detector. ................................................... 40 Figure 2.9. (Top) Chromatogram of authentic cytokinin standards separated by HPLC and detected by UV at 268 nm. (Bottom) An immuno-histogram of putative cytokinins within the xylem sap of apple that were separated by HPLC and quantified by radioimmunoassay (see Section 2.5). ................................... 44 Figure 2.10. Retention times for indole-3-acetic acid (IAA) and indole-3-acetic acid methyl ester (IAA- Me) established with a UV (top) and fluorescence (bottom) detector using an HPLC elution gradient (Table 2.2). ................................................................................................................................................. 45 Figure 2.11. Radioactive peaks identified using a radioactivity detector (βRam) for unpurified 3H-ZR stock (top) and a sub-sample of the putative 3H-ZR peak after repurification by HPLC (bottom). ........... 47 Figure 2.12. Validation curves produced for radioimmunoassay (RIA) of zeatin-riboside (ZR) and isopentenyladenosine (IPA). Parallel lines between the cytokinin standard and cytokinin standard plus purified sap fraction indicate no interference to the RIA from the sample. Along the y-axis, the distance between the lines reflects cytokinin concentration within the sample........................................................ 49 List of figures xxvi Figure 2.13. Standard curve of indole-3-acetic acid (IAA) injected into the HPLC and quantified by fluorescence detection. ............................................................................................................................... 50 Figure 2.14. Response of fluorescence detector to standards of indole-3-acetic acid (IAA) and indole-3- acetic acid methyl ester (IAA-Me) (below, 100 ng injection) and endogenous IAA within a sample of diffusate from the shoot apices of ‘Royal Gala’ apple scions (top) after injection into an HPLC. The sample was quantified by fluorescence as containing 88 ng of IAA. Subsequent quantification of the collected HPLC fraction by GC-MS confirmed a similar concentration of 92 ng of IAA (see M.9 January, Table 2.3). .................................................................................................................................................. 50 Figure 2.15. Mass spectra of methyl ester trimethylsilyl ethers of [2H5]indole-3-acetic acid ([2H5]IAA- MeTMSi) (top), indole-3-acetic acid (IAA-MeTMSi) standard (middle) and indole-3-acetic acid (IAA- MeTMSi) within the sample (bottom) corresponding to the retention times of 20.63, 20.67, and 20.67 min, respectively. ....................................................................................................................................... 53 Figure 2.16. Single ion monitoring of ions 207/266 and 202/261 for methyl ester trimethylsilyl ethers of [2H5]indole-3-acetic acid ([2H5]IAA-MeTMSi) and indole-3-acetic acid (IAA-MeTMSi), respectively. The [2H5]IAA-MeTMSi internal standard chromatographed close to the IAA-MeTMSi standard (top) and endogenous IAA (IAA-MeTMSi) diffused from the shoot apices of ‘Royal Gala’ scions (bottom). Peak area ratios for ions 266:207 or 261:202 were similar between the standard (top) and sample (bottom).... 55 Figure 2.17. Standard curve of indole-3-acetic acid methyl ester trimethylsilyl ether (IAA-MeTMSi) (m/z 202) standards injected into the gas chromatograph (GC) and quantified by single ion monitoring using mass spectrometry. From left to right, data points represent a single injection of 0, 0.2, 0.4 and 1 ng of IAA-MeTMSi into the GC. Each standard was spiked with 50 ng of [2H5]indole-3-acetic acid methyl ester trimethylsilyl ether (m/z 207) as an internal standard (IS). ........................................................................ 56 Figure 2.18. Mass spectra of methyl ester trimethylsilyl ethers of the [2H2]GA19 internal standard (top), GA19 standard (middle) and the GA20 standard corresponding to the retention times of 24.68, 24.70 and 22.75 min, respectively. ............................................................................................................................. 59 Figure 2.19. Single ion monitoring of ions 436/376 and 434/374 for methyl ester trimethylsilyl ethers of [2H2]GA19 ([2H2]GA19-MeTMSi) and GA19 (GA19-MeTMSi), respectively. The [2H2]GA19-MeTMSi internal standard co-chromatographed with the GA19-MeTMSi standard (top) and with GA19 (GA19- MeTMSi) within the xylem sap of apple (bottom). Peak area ratios between ions 376:436 or 374:434 were similar between the standard (top) and sample (bottom). .......................................................................... 60 Figure 2.20. Chromatogram for single ion monitoring of ions 418 and 375 for methyl ester trimethylsilyl ethers of the GA20 standard (top) and GA20 within the xylem sap of apple (bottom). Peak area ratios between ions 375:418 were similar between the standard (top) and sample (bottom). .............................. 61 Figure 2.21. Mass spectra of 1-N-naphthylphthalamic acid synthesised using the methods of Thomson et al., (1973). .................................................................................................................................................. 63 Figure 2.22. Effect of different concentrations of 1-N-naphthylphthalamic acid (NPA) on the mean length of lettuce roots expressed as a percentage of mean root length for control seedlings (grown in distilled water only). After radical emergence from the seed coat, seeds were placed into the test solutions and grown for 72 hr........................................................................................................................................... 63 Figure 2.23. (top) Lettuce seedlings germinated in water (control) showing normal root development, (below) lettuce seeds germinated in 10-6 mol L-1 of 1-N-naphthylphthalamic acid showing roots growing upwards because of loss of gravitropism due to probable inhibition of auxin transport. ........................... 64 List of figures xxvii Figure 2.24. Epinasty of ‘Royal Gala’ apple scions developed within two days of applying a single application of 1-N-naphthylphthalamic acid (NPA) or 2,3,5,-Triiodobenzoic acid (TIBA) to the graft union of MM.106 rootstocks. From left to right, trees were treated with 0, 1, 5 and 25 mg of NPA in lanolin......................................................................................................................................................... 66 Figure 2.25. Mean photosynthetic rate (Pn) of ‘Royal Gala’ apple scions seven and nine days after application of 1-N-naphthylphthalamic acid (NPA) or 2,3,5,-Triiodobenzoic acid (TIBA) to the graft union of MM.106 rootstocks. A single leaf was measured per scion (n=3 trees). Vertical bars represent the SEM...................................................................................................................................................... 67 Figure 2.26. Axillary branching on the rootstock stem 28 days after a single application of 1-N- naphthylphthalamic acid was applied to the graft union. The first visible signs of axillary bud outgrowth were seen after 10 to 14 days. .................................................................................................................... 67 Figure 2.27. Mean cumulative length of the primary shoot of ‘Royal Gala’ apple scions in response to a single application of 1-N-naphthylphthalamic acid (NPA) applied to the graft union of M.9 (top) and MM.106 (bottom) rootstocks. Vertical bars represent the SEM, n=3. ....................................................... 68 Figure 2.28. Example of a shoot apical meristem on the primary shoot that had fully terminated (left) or that was actively extending (right). ............................................................................................................ 69 Figure 2.29. Example of possible axes and types of growth units measured on ‘Royal Gala’ apple scions at the end of the first year from grafting. Primary, secondary and tertiary vegetative shoots are colour- coded brown, blue and red, respectively. ‘I’ and its index correspond to the node at which an axis or growth unit formed, and/or, ended. Secondary spurs and shoots (i.e., excluding trace spurs) are collectively called secondary axes, whereas tertiary spurs and shoots are collectively called tertiary axes. .................................................................................................................................................................... 71 Figure 3.1. Main effect of rootstock (A and B), ± exogenous benzylaminopurine (BAP) (C and D) and root volumes (E and F) on the mean cumulative length (left) and node number (right) of ‘Royal Gala’ primary shoots during their first year of growth after grafting. Vertical bars represent the minimum significant difference (MSD) at P=0.05 using the Tukey’s test. The vertical dotted line along the x-axis of ‘A’ denotes the time after which shoot termination first began for all treatments. Data for the main effects of rootstock, BAP or root volume are averaged over BAP and root volume, rootstock and root volume or rootstock and BAP, respectively. ............................................................................................................... 86 Figure 3.2. Main effect of rootstock (A and B), ± exogenous benzylaminopurine (BAP) (C and D) and root volumes (E and F) on the daily mean growth rate (left) and rate of node production (right) of ‘Royal Gala’ primary shoots during their first year of growth after grafting. Vertical bars represent the MSD at P=0.05 using the Tukey’s test. The vertical dotted line along the x-axis of ‘A’ denotes the time after which shoot termination first began for all treatments. Data for the main effects of rootstock, BAP or root volume are averaged over BAP and root volume, rootstock and root volume or rootstock and BAP, respectively. ............................................................................................................................................... 87 Figure 3.3. Effect of ± exogenous benzylaminopurine (BAP) on the final mean node number of the primary shoot of ‘Royal Gala’ apple scions on rootstocks of M.9, MM.106 and M.793 at growth cessation in June, 2005. Means sharing the same letter are not significantly different at P≤0.07 (lsmeans tests with Tukey’s adjustment, SAS). Data are averaged over root volume treatments. ............................................ 91 Figure 3.4. Rootstock x root volume x benzylaminopurine (BAP) interaction (P=0.10) on the final mean shoot cross-sectional area (SCA) of ‘Royal Gala’ primary shoots at growth cessation in June, 2005....... 91 List of figures xxviii Figure 3.5. Effect of November and December applications of benzylaminopurine (BAP) on the number of secondary shoots formed by early January 2005 (right, + BAP) for ‘Royal Gala’ apple scions that were grafted onto M.9 during August, 2004. Natural feathering of the scion without BAP (left, - BAP) did not begin until late January. ............................................................................................................................. 92 Figure 3.6. Effect of root volumes and rootstock type on the mean number of secondary shoots on ‘Royal Gala’ apple scions at the end of their first growing season (June, 2005) from grafting. Means sharing the same letter are not significantly different at P≤0.05 (lsmeans tests with Tukey’s adjustment, SAS). Data are averaged over benzylaminopurine treatments. ..................................................................................... 96 Figure 3.7. Mean spur and shoot numbers formed on ‘Royal Gala’ apple scions by June 2005 as modified by rootstock x root volume interactions plus (C and D) or minus (A and B) exogenous benzylaminopurine (BAP) and rootstock x BAP interactions plus (A and C) or minus (B and D) root restriction. The rootstock x root volume interactions plus or minus BAP were significant only for tertiary spurs (P=0.02) and secondary shoots (P=0.006), respectively (compare means of C and D or A and B for tertiary spurs or secondary shoots, respectively). Rootstock x BAP interactions were significant only for secondary shoots of the 45 L root volume (P=0.02, compare means of secondary shoots between B and D only). Within a single interaction, means sharing the same letter are not significantly different at P≤0.05 (lsmeans tests with Tukey’s adjustment, SAS). ................................................................................................................ 99 Figure 3.8. Effect of 8 L and 45 L root volumes and ± exogenous benzylaminopurine (BAP) on the mean total number of axillary axes on ‘Royal Gala’ apple scions at the end of their first year of growth after grafting. The mean total number of axillary axes is the sum of trace spurs, secondary spurs, secondary shoots and tertiary spurs formed per scion. Data are averaged over rootstocks. ...................................... 100 Figure 3.9. Effect of rootstock type and plus (A) or minus (B) benzylaminopurine (BAP) applied to ‘Royal Gala’ apple scions on the angle of elevation for secondary shoots relative to their nodal position on the primary shoot. Angles of elevation were measured as acute angles with 0 and 90o elevation representing a shoot that was horizontal with the ground or vertical like the primary shoot, respectively. .................................................................................................................................................................. 102 Figure 3.10. Mean angle of elevation for secondary shoots formed along the primary shoot in response to ± benzylaminopurine (BAP) applied to ‘Royal Gala’ apple scions grafted onto rootstocks of M.9, MM.106 and M.793. Means sharing the same letter are not significantly different at P≤0.05 (lsmeans tests with Tukey’s adjustment, SAS). Means in parenthesis are transformed. Data are averaged over root volume treatments. ................................................................................................................................... 103 Figure 3.11. Effect of ± benzylaminopurine (BAP) applied to the scion and rootstock type on the mean proportion (%) of terminated secondary shoots on ‘Royal Gala’ apple scions in March, 2005. Means sharing the same letter are not significantly different at P≤0.05 (lsmeans tests with Tukey’s adjustment, SAS). Means in parenthesis are transformed. Data are averaged over root volume treatments. .............. 103 Figure 3.12. Effect of rootstock type and ± benzylaminopurine (BAP) applied to the scion on the mean proportion (%) of secondary shoots on ‘Royal Gala’ apple scions that had formed from two growth units (GU). At growth cessation in June 2005, the morphological marker of a ring of bud scale scars, and/or, compressed internodes was used to identify which shoots had temporarily ceased growing at some point in the growing season............................................................................................................................... 105 Figure 3.13. Effect of rootstock type and root volumes on the relationship between secondary shoot length and node number (A, C and E) for ‘Royal Gala’ apple scions without benzylaminopurine (BAP). B, D and F represent the node number distributions of the total secondary shoot population for each treatment. .................................................................................................................................................................. 112 List of figures xxix Figure 3.14. Effect of rootstock type and root volumes on the relationship between secondary shoot length and node number for ‘Royal Gala’ apple scions treated with benzylaminopurine (BAP) (A, C and E). B, D and F represent the node number distributions of the total secondary shoot population for each treatment................................................................................................................................................... 114 Figure 4.1. Example of under-row trickle irrigation and the growing system used for experimental apple trees of ‘Royal Gala’ on M.9 or MM.106 rootstocks grown with or without root restriction. A bare herbicide strip was maintained beneath the trees to prevent competition from weeds and the sward. Photo was taken during autumn (late April, 2005) when trees were nearing the end of their first growing season from grafting. ........................................................................................................................................... 129 Figure 4.2. Main effects of rootstocks (A and B) and ± root restriction (C and D) on the mean cumulative length (left) and node number (right) of ‘Royal Gala’ primary shoots during their first year of growth after grafting. Vertical bars are the minimum significant difference (MSD) at P=0.05 (Tukey’s test). Data for rootstock or root restriction main effects are averaged over root restriction or rootstock treatments, respectively. ............................................................................................................................................. 132 Figure 4.3. Seasonal patterns of rootstocks (A and B) and ± root restriction (C and D) effects on the daily mean rate of growth (left) and node production (right) by the shoot apical meristem on the primary shoot of ‘Royal Gala’ apple scions during their first year of growth after grafting. Vertical bars are the MSD at P=0.05 (Tukey’s test). Data for rootstock or root restriction main effects are averaged over root restriction or rootstock treatments, respectively. On graph A, * denotes measurement dates where 100% of shoot apical meristems were actively growing for all treatments within the experiment. ................................. 133 Figure 4.4. Rootstock x root restriction interaction (P=0.12) for the mean proportion (%) of shoot apical meristems (SAMs) that had terminated on ‘Royal Gala’ primary shoots in April (22/4/05). Means sharing the same letter are not significantly different at P≤0.12 (lsmeans tests with Tukey’s adjustment, SAS).134 Figure 4.5. Effect of field-grown M.9 (right) and MM.106 (left) rootstocks on the growth and architecture of ‘Royal Gala’ apple scions nearing the end of their first season of growth (May, 2005) from grafting. .................................................................................................................................................................. 135 Figure 4.6. Effect of rootstocks grown with or without root restriction (RR) on the distribution of secondary shoots formed along the primary shoot of ‘Royal Gala’ apple scions. Proportion (%) was calculated as the total number of secondary shoots formed at a given nodal position on the primary shoot divided by the total number of nodes at each position (between nodes 1 and 46 there were 48 nodes per position arising from 48 scions per treatment). ........................................................................................ 140 Figure 4.7. Rootstock x root restriction (RR) interactions for the mean length (A, P=0.02) and mean proportion (%) of secondary shoots (SS) that had terminated (B, P=0.24) on ‘Royal Gala’ apple scions in early March, 2005. For graph A, means sharing the same letter are not significantly different at P≤0.05 (lsmeans tests with Tukey’s adjustment, SAS). ....................................................................................... 143 Figure 4.8. Rootstock x root restriction (RR) interactions for the final mean length (A), node number (B) and internode length (C) of secondary shoots on ‘Royal Gala’ apple scions at the end of their first growing season (June, 2005) from grafting. For a single interaction only, means sharing the same letter are not significantly different at P≤0.05 (lsmeans tests with Tukey’s adjustment, SAS)......................... 146 Figure 4.9. Effect of rootstock and root restriction (RR) on the relationship between secondary shoot length and node number (A and C) for ‘Royal Gala’ apple scions at the end of their first season of growth from grafting. B and D represent the node number distribution of the secondary shoots. Data represents the total population of secondary shoots for each treatment. ................................................................... 147 List of figures xxx Figure 4.10. Rootstock x root restriction (RR) interaction for mean bud break (%) on ‘Royal Gala’ apple scions during early spring in year two (29/9/05) of growth from grafting. Bud break (%) was calculated as the total number of buds that had broken per scion divided by the total number of buds per scion x 100. Means sharing the same letter are not significantly different at P≤0.05 (lsmeans tests with Tukey’s adjustment, SAS)...................................................................................................................................... 150 Figure 4.11. Effect of M.9 (left) and MM.106 (right) rootstocks on the number of buds broken on ‘Royal Gala’ apple scions in the early spring of year two (5/10/05) after grafting. As flower buds broke before vegetative buds, the scion on M.9 had a greater number of buds broken in early spring......................... 150 Figure 4.12. Rootstock x root restriction (RR) interactions for the mean total number of flower clusters (A) and the mean proportion (%) of total buds per scion that were floral (B) as divided into component bud types formed on ‘Royal Gala’ apple trees. The total height of the columns represent the interactions for the mean total flower cluster number per scion (A, P=0.06) and the mean proportion (%) of total buds per scion that were floral (B, P=0.24). For A and B, the grey portion of chart columns represent the rootstock x root restriction interaction for the mean flower cluster number per scion formed on apical buds of the secondary shoots (A, P=0.07) or the mean proportion (%) of apical buds on the secondary shoots that flowered relative to the total bud number per scion (B, P=0.06), respectively. Within a single graph and interaction only, transformed means (in parenthesis) sharing the same letter across a single row are not significantly different (lsmeans tests, SAS). y mean separation at P≤0.07................................... 152 Figure 5.1. Main effects of rootstocks (A and B), ± exogenous benzylaminopurine (BAP) (C and D) or gibberellin (GA4+7) applied to the scion (E and F) and ± 1-N-naphthylphthalamic acid (NPA) (G and H) applied to the rootstock stem on the mean cumulative length and node number for primary shoots of ‘Royal Gala’ apple scions during their first year of growth from grafting. Vertical bars denote the minimum significant difference (MSD) at P=0.05 using the Tukey’s test. Arrows on D, F and H denote the timing of application for each growth regulator. ................................................................................ 173 Figure 5.2. Interactions between gibberellin (GA4+7) applied to the scion and 1-N-naphthylphthalamic acid (NPA) applied to the rootstock stem on the mean cumulative length (A) and node number (B) for primary shoots of ‘Royal Gala’ apple scions during their first growing season after grafting. Arrows on ‘B’ with a solid or dotted line denote the timing of NPA or GA4+7, respectively. On a single graph, means sharing the same letter are not significantly different. On the 20/5/06, mean separation is at P≤0.11 or P≤0.05 for graph A or B, respectively (lsmeans tests with Tukey’s adjustment, SAS). Data are averaged over BAP and rootstock treatments.......................................................................................................... 174 Figure 5.3. Rootstock x gibberellin (GA4+7) interactions on the mean cumulative length (A) and node number (B) for primary shoots of ‘Royal Gala’ apple scions during their first growing season after grafting. Arrows on ‘B’ denote the timing of GA4+7. On the 20/5/06, means sharing the same letter are not significantly different at P≤0.05 (lsmeans tests with Tukey’s adjustment, SAS). Data are averaged over BAP and NPA treatments......................................................................................................................... 175 Figure 5.4. Rootstock x benzylaminopurine (BAP) x gibberellin (GA4+7) interactions on the mean cumulative length (A, B, C and D) and node number (E, F, G and H) for primary shoots of ‘Royal Gala’ apple scions during their first year of growth after grafting. Arrows with a solid or dotted line on H denote the timings of exogenous BAP or GA4+7, respectively. Data are averaged over NPA treatments. ........... 177 Figure 5.5. Effect of apple rootstocks (M.9, MM.106, M.793 and ‘Royal Gala’ (R.G)), ± benzylaminopurine (BAP), ± gibberellin (GA) and ± 1-N-naphthylphthalamic acid (NPA) treatments on the final mean length (A), node number (B), internode length (C) and shoot cross-sectional area (SCA) (D) for primary shoots of ‘Royal Gala’ at the end of their first growing season after grafting................ 180 List of figures xxxi Figure 5.6. Effect of rootstock, benzylaminopurine (BAP), gibberellin (GA4+7) and 1-N- naphthylphthalamic acid (NPA) treatments on the relationship between the final length and node number of ‘Royal Gala’ primary shoots at the end of their first growing season after grafting. ........................... 181 Figure 5.7. Effect of 1-N-naphthylphthalamic acid (NPA) applied to the rootstock stem on activity of the apical meristem on the primary shoot of ‘Royal Gala’ apple scions. Photo ‘A’ shows the apex of the primary shoot actively growing before NPA application one (12/12/05), B shows transient epinasty developed 2 to 3 days after NPA treatment, C shows the termination of growth 14 to 21 days after NPA application one and D shows growth resumption of the shoot apex approximately 28 to 35 days after application one of NPA. ........................................................................................................................... 185 Figure 5.8. Effect of rootstocks, ± benzylaminopurine (BAP), ± gibberellin (GA) and ± 1-N- naphthylphthalamic acid (NPA) on the proportion of ‘Royal Gala’ primary (A) and secondary shoots (B) that were comprised of one, two or three growth units (GU) at the end of the first season of growth after grafting. Growth units were identified by the presence of bud scale scars, and/or, compressed internodes along each shoot type. .............................................................................................................................. 185 Figure 5.9. Interactions between benzylaminopurine (BAP) and gibberellin (GA4+7 (GA)) applied to ‘Royal Gala’ apple scions (A) or applications of BAP applied to the scion and 1-N-naphthylphthalamic acid (NPA) applied to the rootstock stem (B) on the mean number of secondary spurs formed on the primary shoot. For a single graph, means in parenthesis sharing the same letter are not significantly different at P≤0.05 (lsmeans tests with Tukey’s adjustment, SAS). Data are averaged over rootstocks and NPA or rootstocks and GA4+7 for graph A or B, respectively. ................................................................. 190 Figure 5.10. 1-N-naphthylphthalamic acid (NPA) x benzylaminopurine (BAP) x gibberellin (GA4+7 (GA)) interactions for the mean proportion (%) of axillary buds on the primary shoot that formed a secondary shoot (A) and the mean number of secondary shoots formed per ‘Royal Gala’ scion (B). For a single graph, means sharing the same letter are not significantly different at P≤0.05 (lsmeans tests with Tukey’s adjustment, SAS). Data are averaged over rootstocks.............................................................................. 192 Figure 5.11. Effect of rootstocks and treatment combinations of benzylaminopurine (BAP), gibberellin (GA4+7 (GA)) and 1-N-naphthylphthalamic acid (NPA) on the mean proportion (%) of axillary buds on the primary shoot that formed a secondary shoot (A) and the mean number of secondary shoots (B) on ‘Royal Gala’ apple scions at the end of their first growing season from grafting. ................................... 194 Figure 5.12. 1-N-naphthylphthalamic acid (NPA) x benzylaminopurine (BAP) x gibberellin (GA4+7 (GA)) interactions for the mean proportion (%) of axillary buds on the primary shoot that formed secondary axes (spurs + secondary shoots) (A) and the mean number of secondary axes formed per ‘Royal Gala’ scion (B). The interaction is for the total height of the columns and A and B were significant at P=0.16 and P=0.002, respectively. Within graph B, means sharing the same letter are not significantly different at P≤0.05 (lsmeans tests with Tukey’s adjustment, SAS). Data are averaged over rootstocks.................... 196 Figure 5.13. Effect of rootstock type and treatment combinations of benzylaminopurine (BAP) gibberellin (GA4+7 (GA)) and 1-N-naphthylphthalamic acid (NPA) on the mean proportion (%) of axillary buds on the primary shoot that formed secondary axes (A), the mean number of secondary axes (B) and the mean total number of axillary axes formed per scion (C) shown as the component parts comprising of tertiary spurs, secondary shoots, tertiary shoots or secondary spurs. The SEOD on A, B and C are for the total height of the columns. .............................................................................................................................. 199 Figure 5.14. Rootstock x 1-N-naphthylphthalamic acid (NPA) interaction for the mean number of tertiary spurs (A) and shoots (B) formed on secondary shoots of ‘Royal Gala’ apple scions treated with benzylaminopurine (BAP). Transformed means in parenthesis on ‘A’ sharing the same letter are not significantly different at P≤0.05 (lsmeans tests with Tukey’s adjustment, SAS). Data are averaged over List of figures xxxii gibberellin treatments and excludes data for the minus BAP treatment. Data for ‘B’ could not be transformed appropriately for ANOVA. .................................................................................................. 201 Figure 5.15. 1-N-naphthylphthalamic acid (NPA) x benzylaminopurine (BAP) x gibberellin (GA4+7 (GA)) interaction for the mean total number of axillary axes per ‘Royal Gala’ scion at the end of the first year of growth after grafting. The interaction for the mean total number of axillary axes formed per scion is equal to the total height of the columns. Means sharing the same letter are not significantly different at P≤0.05 (lsmeans tests with Tukey’s adjustment, SAS). Data are averaged over rootstocks. ............................... 203 Figure 5.16. 1-N-naphthylphthalamic acid (NPA) x benzylaminopurine (BAP) x gibberellin (GA4+7 (GA)) interactions for the mean total length (A) and node number (B) of ‘Royal Gala’ secondary shoots at the end of the first year of growth after grafting of composite ‘Royal Gala’ apple trees. Transformed means in parenthesis sharing the same letter are not significantly different at P≤0.05 (lsmeans tests with Tukey’s adjustment, SAS). Data are averaged over rootstocks.............................................................................. 208 Figure 5.17. Rootstock x 1-N-naphthylphthalamic acid (NPA) x benzylaminopurine (BAP) interaction for the mean total length (A) and node number (B) of secondary shoots on ‘Royal Gala’ apple scions at the end of their first year of growth after grafting. On a single graph, transformed means in parenthesis sharing the same letter are not significantly different at P≤0.05 (lsmeans tests with Tukey’s adjustment, SAS). Data are averaged over gibberellin treatments............................................................................... 212 Figure 5.18. Rootstock x benzylaminopurine (BAP) x gibberellin (GA) interaction for the mean total length (A, P=0.06) and node number (B, P=0.11) of secondary shoots on ‘Royal Gala’ apple scions at the end of their first year of growth after grafting. On graph A, transformed means in parenthesis sharing the same letter are not significantly different at P≤0.06 (lsmeans tests with Tukey’s adjustment, SAS). Data are averaged over NPA treatments. .......................................................................................................... 213 Figure 5.19. Effect of rootstock, benzylaminopurine (BAP), gibberellin (GA4+7 (GA)) and 1-N- naphthylphthalamic acid (NPA) treatments on the mean total length (A) and node number (B) of ‘Royal Gala’ apple scions at the end of their first year of growth from grafting. Total shoot length (A) or node number per scion (B) is shown as the component parts comprising of tertiary shoots, secondary shoots, primary shoot and is equivalent to the total height of a column. Error bars adjacent M.9 plus BAP are the SEOD for that shoot type calculated from the raw data. .......................................................................... 214 Figure 5.20. Effect of rootstock type, benzylaminopurine (BAP), gibberellin (GA4+7) and 1-N- naphthylphthalamic acid (NPA) treatments on the relationship between the final length and node number of secondary shoots on ‘Royal Gala’ apple scions at the end of their first year of growth from grafting.215 Figure 5.21. Effect of rootstock type, benzylaminopurine (BAP), gibberellin (GA4+7) and 1-N- naphthylphthalamic acid (NPA) treatments on the node number distributions of secondary shoots that had formed on the primary shoot of ‘Royal Gala’ apple scions by the end of their first year of growth from grafting. Node number distributions are of the total shoot population for each treatment. ...................... 216 Figure 5.22. Effect of rootstock type, benzylaminopurine (BAP), gibberellin (GA4+7 (GA)) and ± 1-N- naphthylphthalamic acid (NPA) treatments on the mean length (A) and node number (B) of ‘Royal Gala’ secondary shoots at the end of the first year of growth from grafting. Error bars are the SEOD. * on graph B denotes very few shoots per treatment mean in graph A or B. ............................................................. 217 Figure 5.23. Rootstock x 1-N-naphthylphthalamic acid (NPA) interactions on the mean total length (A, P=0.001) and node number (B, P=0.16) of tertiary shoots formed on secondary shoots of ‘Royal Gala’ apple scions in response to exogenous benzylaminopurine (BAP). For graph A, means sharing the same letter are not significantly different at P≤0.05 (lsmeans tests with Tukey’s adjustment, SAS). Data are averaged over gibberellin treatments. ...................................................................................................... 219 List of figures xxxiii Figure 5.24. 1-N-naphthylphthalamic acid (NPA) x benzylaminopurine (BAP) x gibberellin (GA4+7 (GA)) interactions for the mean total shoot length (A) and node number (B) formed per ‘Royal Gala’ scion. The total shoot length or node number per scion is equivalent to the total height of the columns of A and B, respectively. For a single graph, transformed means in parenthesis sharing the same letter are not significantly different at P≤0.05 (lsmeans tests with Tukey’s adjustment, SAS). Data are averaged over rootstocks. ................................................................................................................................................ 221 Figure 5.25. Rootstock x 1-N-naphthylphthalamic acid (NPA) interactions on the final mean total length (A) and node number (B) formed on ‘Royal Gala’ apple scions at the end of their first year of growth from grafting. For a single graph, means within parenthesis sharing the same letter are not significantly different at P≤0.05 (lsmeans tests with Tukey’s adjustment, SAS). Data are averaged over BAP and gibberellin treatments. .............................................................................................................................. 224 Figure 5.26. Rootstock x benzylaminopurine (BAP) x gibberellin (GA) interactions for the mean total length (A, P=0.02) and node number (B, P=0.12) of ‘Royal Gala’ apple scions. The interaction for the total length or node number per scion is equivalent to the total height of the columns on A or B, respectively. On graph A, means within parenthesis sharing the same letter are not significantly different at P≤0.05 (lsmeans tests with Tukey’s adjustment, SAS). Data are averaged over NPA treatments. ...... 225 Figure 5.27. Effect of rootstocks (minus NPA), benzylaminopurine (BAP) and gibberellin (GA4+7) treatments on the architecture of ‘Royal Gala’ apple scions at the end of their first year of growth (April, 2006) from grafting. Yellow rule is 1 m. ................................................................................................. 226 Figure 5.28. Effect of rootstocks (plus NPA), benzylaminopurine (BAP) and gibberellin (GA4+7) treatments on the architecture of ‘Royal Gala’ apple scions at the end of their first year of growth (April, 2006) from grafting. Yellow rule is 1 m. ................................................................................................. 227 Figure 5.29. Rootstock x 1-N-naphthylphthalamic acid (NPA) interactions on the final mean total dry mass (A) and length (B) of the root system for M.9, MM.106, M.793 and ‘Royal Gala’ rootstocks grafted with ‘Royal Gala’ apple scions. Root growth was measured at the end of the first growing season from grafting. For a single interaction only, means within parenthesis sharing the same letter are not significantly different at P≤0.05 (lsmeans tests with Tukey’s adjustment, SAS). Data are averaged over BAP and gibberellin treatments. .............................................................................................................. 230 Figure 5.30. Effect of rootstock, benzylaminopurine (BAP), gibberellin (GA4+7 (GA)) and ± 1-N- naphthylphthalamic acid (NPA) treatments on the mean total length (A) and dry mass of the root system for composite ‘Royal Gala’ apple trees at the end of their first growing season from grafting. Error bars are the SEOD of the raw data. .................................................................................................................. 231 Figure 6.1. Effect of rootstock type on the mean cumulative length, node number and leaf area of the primary shoot (A, D and G), the mean total length and node number of the secondary shoots (B and E), the mean total leaf area of the secondary axes (i.e., spurs plus shoots) (H) and the mean total shoot length, node number and leaf area (C, F and I) of ‘Royal Gala’ apple scions during their first growing season from grafting. x, *, **, *** significant ANOVA at P≤0.12, P≤0.05, P≤0.01, P≤0.001, respectively. Bars denote the LSD at P=0.05. ....................................................................................................................... 258 Figure 6.2. Effect of rootstock type on the mean internode length (A) and cumulative shoot cross- sectional area (SCA) (B) for primary shoots of ‘Royal Gala’ apple during their first growing season from grafting. *, **, *** significant ANOVA at P≤0.05, P≤0.01, P≤0.001, respectively. Bars denote the LSD at P=0.05.................................................................................................................................................. 259 Figure 6.3. Effect of rootstock type on the mean number of secondary spurs (A), shoots (B) and secondary axes (spurs plus shoots) (C) formed on the primary shoot of ‘Royal Gala’ apple scions during List of figures xxxiv their first growing season from grafting. D is the mean number of secondary spurs, shoots and their total produced by each rootstock type over the entire growing season (n=30 trees). x, *, **, *** significant ANOVA at P≤0.10, P≤0.05, P≤0.01, P≤0.001, respectively. Bars denote the LSD at P=0.05. .............. 261 Figure 6.4. Effect of rootstock type on the relationship between the length and node number of secondary shoots on ‘Royal Gala’ apple scions (A, C, E and G) measured in February, March and April, 2006. B, D, F and H are the node number distributions of secondary shoots for each rootstock type and month....... 262 Figure 6.5. Effect of rootstock type on the mean cumulative root length (A) and mean specific root mass (B) of composite ‘Royal Gala’ apple trees during their first year of growth from grafting. x, *, **, *** significant ANOVA at P≤0.10, P≤0.05, P≤0.01, P≤0.001, respectively. Bars denote the LSD at P=0.05. .................................................................................................................................................................. 265 Figure 6.6. Effect of rootstock type on the mean cumulative dry mass (DM) of the scion (A to I) and rootstock (J, K and L) of composite ‘Royal Gala’ apple trees during their first season of growth from grafting. x, *, **, *** significant ANOVA at P≤0.12, P≤0.05, P≤0.01, P≤0.001, respectively. Bars denote the LSD at P=0.05. .................................................................................................................................. 272 Figure 6.7. Effect of rootstock type on the mean cumulative total dry mass (A) and mean root:scion dry mass ratio (B) of ‘Royal Gala’ apple trees during their first season of growth from grafting. *, **, *** significant ANOVA at P≤0.05, P≤0.01, P≤0.001, respectively. Bars denote the LSD at P=0.05........... 274 Figure 6.8. Summary of component tree parts and the mean total dry mass (A) of composite ‘Royal Gala’ apple trees on M.9, MM.106, M.793 and ‘Royal Gala’ rootstocks (control) by the end of the first year of growth (6/4/06) after grafting (1/9/05). For graph A, total dry mass per tree is equivalent to the total height of a column. Graph B denotes the mean proportion (%) of total dry mass per tree allocated into stems, leaves and roots. For a single tree part and individual graph only, means sharing the same letter across the columns are not significantly different. LSD bars on A and B are for the rootstock (root plus rootstock stem) and scion (leaves plus stems).......................................................................................... 274 Figure 6.9. Root and scion dry mass allometry of composite ‘Royal Gala’ apple trees grafted onto rootstocks of M.9 (A), MM.106 (B) and M.793 (C) and compared with a ‘Royal Gala’ rootstock (control) during their first year of growth from grafting. An increase in slope (i.e., allometric coefficient between root and scion) from February onwards indicates a large shift in biomass allocation to root growth. Each data point is the mean of six trees and is natural log transformed. Mean root dry mass excludes the rootstock stem, while mean scion dry mass includes stems and leaves. .................................................. 278 Figure 6.10. Mean concentration of endogenous indole-3-acetic acid (IAA) diffusing from the primary shoot apex of ‘Royal Gala’ apple scions on rootstocks of M.9, MM.106, M.793 and ‘Royal Gala’ (control) during the first year of growth from grafting. For the measurement of IAA, the primary shoot apex from two scions were pooled providing three replicates per rootstock at each harvest date. .......... 279 Figure 6.11. Relationships between the mean concentration of endogenous indole-3-acetic acid (IAA) diffusing from the primary shoot apex of ‘Royal Gala’ apple scions and the mean root dry mass of M.9 (A), MM.106 (B) and M.793 (C) rootstocks compared with the ‘Royal Gala’ rootstock control during the first year of growth from grafting............................................................................................................. 281 Figure 6.12. Mean concentration of endogenous gibberellin A19 (GA19) in the xylem sap of ‘Royal Gala’ primary shoots on rootstocks of M.9, MM.106, M.793 and ‘Royal Gala’ (control) during the first year of growth from grafting. Data for graph A are expressed as ng mL-1 of GA19 in the xylem sap, whereas data for graph B are the estimated mass of GA19 transported in the xylem sap to ‘Royal Gala’ primary shoots per hour as calculated for differences in leaf area and transpiration imposed on the scion by each List of figures xxxv rootstock type in February, March and April (see Section 6.3.7.4 for calculations). x *, **, *** significant ANOVA at P≤0.10 P≤0.05, P≤0.01, P≤0.001, respectively. Bars denote the LSD at P=0.05. ............... 283 Figure 6.13. Mean concentration of zeatin (Z), zeatin riboside (ZR), isopentenyladenosine (IPA) and isopentenyladenine (2iP) in the xylem sap of ‘Royal Gala’ primary shoots on different apple rootstocks during the first year of growth from grafting. Data for A, C, E and G are expressed as ng mL-1 of cytokinin in the xylem sap, whereas data for B, D, F and H are estimated masses of cytokinins transported to the scion per hour (ng hr-1) calculated for differences in scion leaf area and transpiration on each rootstock type (see Section 6.3.7.4). x *, **, *** significant ANOVA at P≤0.10, P≤0.05, P≤0.01, P≤0.001, respectively. Bars denote the LSD at P=0.05. .......................................................................... 287 Figure 6.14. Relationship between the mean rate of indole-3-acetic acid (IAA) diffusing from the primary shoot apex and the mean concentration of isopentenyladenine (2iP) (A), isopentenyladenosine (IPA) (B) and zeatin riboside (ZR) (C) in the xylem sap of ‘Royal Gala’ primary shoots during their first growing season after grafting onto different size-controlling apple rootstocks...................................................... 288 Figure 6.15. Relationship between mean dry mass of roots and the mean number of secondary axes (spurs plus shoots) per scion (A), mean dry mass of roots and the mean concentration of zeatin riboside (ZR) in the xylem sap of the primary shoot (B) and mean concentration of ZR in the xylem sap of the primary shoot and the mean number of secondary axes formed per scion (C) for composite ‘Royal Gala’ apple trees on different size-controlling rootstocks during the first growing season from grafting................... 292 List of tables xxxvi List of tables Table 2.1. HPLC solvent gradient used for the separation of cytokinins and gibberellins. Curves 6 and 7 are linear and concave, respectively (Waters, 1985). ..........................................................................................................................42 Table 2.2. HPLC solvent gradient used for the separation of indole-3-acetic acid. ......................................................42 Table 2.3. Comparison of indole-3-acetic acid concentrations quantified by HPLC-fluorescence and gas chromatography-mass spectrometry for a single replicate of meristem diffusate collected during January and March from ‘Royal Gala’ apple scions grafted onto M.9 and ‘Royal Gala’ rootstocks. ..........................................................56 Table 2.4. Code file for Figure 2.29. Primary, secondary and tertiary axes are abbreviated as PA, SA and TA, respectively. Their index corresponds to the growth unit number. Sp (spur) differentiates spurs from vegetative shoots. A node along the primary shoot containing a secondary shoot may also contain a trace spur arising from basilary buds borne in the axils of bud scales (trace spur or t sp, see I 15 below and Figure 2.29 ). ............................72 Table 3.1. Main effect of rootstocks on the growth attributes of the primary shoot, secondary shoots and total growth of ‘Royal Gala’ apple scions at the end of their first season of growth (June, 2005) after grafting. .............................88 Table 3.2. Main effect of root volumes on the growth attributes of the primary shoot, secondary shoots and total growth of ‘Royal Gala’ apple scions at the end of their first season of growth (June, 2005) after grafting. .................89 Table 3.3. Main effect of exogenous benzylaminopurine (BAP) on the growth attributes of the primary shoot, secondary shoots and total scion growth of ‘Royal Gala’ apple scions at the end of their first season of growth (June, 2005) after grafting. ......................................................................................................................................................90 Table 3.4. Main effect of benzylaminopurine (BAP) on the mean proportion (%) of axillary buds on the primary shoot that formed a trace spur, secondary spur or secondary shoot, the mean proportion (%) of axillary buds on the secondary shoots that formed a tertiary spur and the mean number of secondary, tertiary and total axillary axes that formed on ‘Royal Gala’ apple scions. Trees were measured at the end of their first year of growth (June, 2005) from grafting. ........................................................................................................................................................................94 Table 3.5. Effect of ± exogenous benzylaminopurine (BAP) on the mean proportion (%) of axillary buds on the primary shoot that formed a trace spur, secondary spur or secondary shoot and the mean proportion (%) of axillary buds on the secondary shoots that formed a tertiary spur for ‘Royal Gala’ apple scions grafted onto rootstocks of M.9, MM.106 and M.793. Trees were measured at the end of their first year of growth (June, 2005) from grafting. ..........94 Table 3.6. Effect of ± exogenous benzylaminopurine (BAP) on the mean number of trace spurs, secondary spurs and secondary shoots formed on the primary shoot, the mean number of tertiary spurs formed on secondary shoots and the total number of axillary axes formed on ‘Royal Gala’ apple scions grafted onto rootstocks of M.9, MM.106 and M.793. Trees were measured at the end of their first year of growth (June, 2005) from grafting.................................95 Table 3.7. Main effect of root volume on the mean number of trace spurs, secondary spurs, secondary shoots, tertiary spurs and their total on ‘Royal Gala’ apple scions at growth cessation in June, 2005. .................................................96 Table 3.8. Rootstock x root volume interactions for the final growth attributes of the primary shoot, secondary shoots and their total for ‘Royal Gala’ apple scions at the end of their first year of growth (June, 2005) from grafting. ......108 Table 3.9. Effect of rootstocks grown in 8 L or 45 L root volumes on the mean length, node number and internode length of secondary shoots on ‘Royal Gala’ apple scions at growth cessation in June, 2005. ....................................109 Table 4.1. Main effects of rootstock and root restriction on growth attributes of the primary shoot, secondary shoots and total growth of ‘Royal Gala’ apple scions at the end of their first season of growth (June, 2005) after grafting. 136 List of tables xxxvii Table 4.2. Main effects of rootstock and root restriction on the mean proportion (%) of axillary buds on the primary shoot that formed secondary axes and the mean number of secondary axes that had formed on ‘Royal Gala’ apple scions by the end of their first year of growth after grafting.......................................................................................138 Table 4.3. Main effects of rootstock and root restriction on the first occurrence of flowering for different bud types on ‘Royal Gala’ apple scions at full bloom (24/10/05). Mean proportion (%) of bud type(s) differentiated into floral buds was calculated by dividing the total number of flower clusters per bud type or types on each scion by the total number of that bud type or types on that scion x 100. .............................................................................................................151 Table 5.1. Effect of rootstock, ± benzylaminopurine (BAP), ± gibberellin (GA4+7) and ± 1-N-naphthylphthalamic acid (NPA) treatments on the mean proportion (%) of primary and secondary shoots that were fully terminated in February, March and April for ‘Royal Gala’ apple scions during their first growing season after grafting................186 Table 5.2. Main effects of rootstock, benzylaminopurine (BAP), gibberellin (GA4+7) and 1-N-naphthylphthalamic acid (NPA) on the mean proportion (%) of axillary buds on the primary shoot that broke to form an axillary structure (spur or shoot) and the mean number of shoot types on ‘Royal Gala’ apple scions by the end of their first season of growth after grafting. ..................................................................................................................................................189 Table 5.3. Main effects of rootstock, benzylaminopurine (BAP), gibberellin (GA4+7) and 1-N-naphthylphthalamic acid (NPA) on the mean total growth of the primary shoot, secondary shoots, tertiary shoots and the total growth of ‘Royal Gala’ apple scions at the end of their first growing season after grafting. ......................................................206 Table 5.4. Main effects of rootstock, gibberellin (GA4+7) and 1-N-naphthylphthalamic acid (NPA) on the mean total length and node number of tertiary shoots that formed on secondary shoots of ‘Royal Gala’ apple scions in response to exogenous benzylaminopurine (BAP). ...................................................................................................................219 Table 5.5. Main effects of rootstock, benzylaminopurine (BAP), gibberellin (GA4+7) and 1-N-naphthylphthalamic acid (NPA) on the final mean dry mass and length of root systems on rootstocks of M.9, MM.106, M.793 and ‘Royal Gala’ at the end of the first growing season from grafting of composite ‘Royal Gala’ apple trees. ...........................228 Table 6.1. Estimated mass of gibberellin A19 (GA19) transported in the xylem sap per hour to the ‘Royal Gala’ primary shoot grafted onto rootstocks of M.9, MM.106, M.793 and ‘Royal Gala’ as calculated for actual differences in scion leaf area and probable differences in transpiration (T) imposed on the scion by each rootstock type during February, March and April. ........................................................................................................................................285 List of appendices xxxviii List of appendices Appendix 1. The irrigation schedule maintained volumetric water content of the growing medium close to field capacity (0.30 m3 m-3) over the 2004-2005 growing season for composite ‘Royal Gala’ apple trees grafted onto M.9, MM.106 and M.793 rootstocks. Trees were grown in two root volumes (8 L and 45 L) and treated with or without benzylaminopurine (BAP). Horizontal dotted lines represent field capacity of the medium (0.30 ± 0.01 m3 m-3). Vertical bars are ± SEM. ............................................................................................................................................331 Appendix 2. The irrigation schedule maintained soil volumetric water content close to field capacity (0.30 m3 m-3) over the 2004-2005 growing season for ‘Royal Gala’ apple trees grafted onto M.9 and MM.106 rootstocks grown with or without root restriction (RR). The horizontal dotted line indicates field capacity. Vertical bars are ± SEM..332 Appendix 3. The irrigation schedule maintained volumetric water content of the growing medium close to field capacity over the growing season for composite ‘Royal Gala’ apple trees grafted onto rootstocks of M.9, MM.106, M.793 and ‘Royal Gala’ (RG) and treated with or without benzylaminopurine (BAP), GA4+7 (GA) and 1-N- naphthylphthalamic acid (NPA). Horizontal dotted lines represent field capacity of the medium (0.30 ± 0.01 m3 m-3). Vertical bars represent ± SEM....................................................................................................................................333 List of abbreviations xxxix List of abbreviations AMU Atomic mass unit ANOVA Analysis of variance BAP Benzylaminopurine cv. Cultivar 14C-IAA Carboxyl-labelled indole-3-acetic acid 14C-IAA-Me Carboxyl-labelled indole-3-acetic acid methyl ester DPM Disintegrations per minute LSD Least significant difference lsmeans Least square means HPLC High performance liquid chromatography GAn Gibberellin n – denotes the number [2H2]GAn-MeTMSi Deuterium gibberellin methyl ester trimethylsilylether GA19-MeTMSi Gibberellin A19 methyl ester trimethylsilylether GC-MS Gas chromatography-mass spectrometry GLM General linear model MES 2-(N-morpholino)ethanesulphonic acid M.9 Malling 9 MM.106 Malling Merton 106 M.793 Merton 793 MPa Mega pascal(s) (1 MPa = 10 bars) MSD Tukey’s minimum significant difference MSTFA N-Methyl-N(trimethyl-silyl) trifluoroacetamide n Number NPA 1-N-naphthylphthalamic acid IBA Indole butyric acid List of abbreviations xl IAA Indole-3-acetic acid [2H5]IAA Pentodeuterium indole-3-acetic acid IAA-Me Indole-3-acetic acid methyl ester [2H5]IAA-MeTMSi Pentodeuterium indole-3-acetic acid methyl ester trimethylsilylether IAA-MeTMSi Indole-3-acetic acid methyl ester trimethylsilylether IPA Isopentenyladenosine 3H-IPA Tritiated isopentenyladenosine 2iP Isopentenyladenine ODS Octadecyl Silica RIA Radioimmunoassay SAM Shoot apical meristem SARD Specific apple replant disorder SAS SAS system for statistical analysis SCA Shoot cross-sectional area SEM Standard error of the mean SEOD Standard error of the difference TDR Time domain reflectometry TEA Acetic acid (40 mmol L-1) adjusted to pH 3.38 with triethylamine TIBA 2,3,5,-Triiodobenzoic acid T Transpiration 3H-ZR Tritiated zeatin riboside UV Ultraviolet θ Volumetric water content (m3 m-3) Z Zeatin ZR Zeatin riboside