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    Gene expression in the precocious germination of late maturation Phaseolus vulgaris L. seeds: a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Plant Biology at Massey University
    (Massey University, 1994) Smith, Howard Russell
    Ethylene induces precocious germination in late maturation embryos (32-40 days after anthesis) of Phaseolus vulgaris L. cv. Seminole, thus overriding the endogenous controls which normally maintain quiescence. The possibility that ethylene exerts its effects at the gene expression level was investigated by in vitro translation of RNA extracted from embryo axis tissue of seeds induced to germinate precociously by incubation with ethylene. 35 S-labelled products so produced were analyzed by electrophoresis, fluorography, and scanning densitometry. Results were compared with normally germinating seeds and with embryos incubated in the absence of ethylene. Ethylene was found to induce a qualitative and quantitative change in gene expression in late maturation embryos detectable within 6 hours of ethylene exposure. Two products (37-38kD and 27kD) were up-regulated within 24 hours in both ethylene-induced precocious germination and normal germination. Four products (71kD, 67-68kD, 65-66kD, and 41-42kD) which appeared in normal germination were evidently not required for ethylene-induced precocious germination. In contrast with the findings of Misra & Bewley (1985;1986) for maize(Zea mays L.) no products could be identified as being unique to the developmental phase, however two products (38-39kD and 28kD) were strongly present in development but disappeared shortly after germination. A product of 22-23kD was apparently unique to the ethylene-induced precocious germination treatment and may represent a gene regulated by ethylene. This product was not seen until 24 hours after ethylene introduction. An attempt was made using SDS-PAGE to identify the major storage proteins of P.vulgaris to use as markers of the developmental phase, however this was only partially successful. Suggestions are made as to approaches and methods for future research.
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    Effects of plant density on seed yield and quality in different common beans (Phaseolus vulgaris L.) : a thesis presented in partial fulfilment of the requirements for the degree of Master of Agricultural Science in Plant Science in Seed Technology at Massey University, New Zealand
    (Massey University, 1996) Mesquita, Filipe Jose Almeida
    Common beans (Phaseolus vulgaris L.) are an annual legume used for human consumption. While many cultivars/genotypes have long been a feature of New Zealand home gardens and the frozen food market, there has recently been an interest in the production of new genotypes of this crop legume suitable for use particularly in fresh and canned salads, as well as for other commercial purposes. In New Zealand, little is known of the growth and performance of many genotypes of this plant as the agro-climatological conditions are different from the original native South American habitat. Therefore this study covered an evaluation of five unnamed but different seed coloured bean genotypes obtained originally from the CIAT collection by New Zealand Seed Bank Ltd. The objectives of this research were to: determine plant growth habit of the genotypes; describe plant growth habit of the genotypes; and assess the effect of plant density on vegetative and reproductive growth, seed yield and cooking quality. To facilitate the recognition of each genotype, they were named white, mottled brown, mottled black, black and brown according to their seed colour, after a visual selection of seeds at the beginning of this study. Plant morphological characteristics were assessed in a trial conducted in a glasshouse at the Seed Technology Centre, Massey University, from September to November 1994. A field trial from November 1994 to March 1995 was aimed at determining the effects of plant density and genotype on seed production and quality for sowing and eating purposes. The minimum and maximum temperatures in the glasshouse were 16°C and 25°C respectively. The daylength in September was around 11 h and gradually increased to about 14.5 h at the end of November. No supplementary illumination and no pesticides and insecticides were used in this trial. For this study, five plants of each colour group were used to determine plant morphological characteristics which included: leaf length and width for the 1st, 3rd and 8th trifoliolate leaves, recorded from the terminal leaf; length of pedicellate bracts; flower (standard and wing) colour; pod colour, length and width; plant height and branch number; main stem internode number and internode length; pods per plant; and seeds per pod. Trifoliolate leaf length was around 22 cm for all genotypes irrespective of leaf position, but leaf width increased from the 1st to the 8th trifoliolate leaf and differed with genotype. For example the 8th trifoliolate leaf width ranged from 11.0 cm in the mottled brown genotype to 14.6 cm in the brown genotype. Pedicellate bract length, main stem internode number and maximum internode length all varied with genotype, with the result that average plant height ranged from 166 cm for the brown genotype to 362 cm for the white genotype. None of the genotypes produced branches in the glasshouse. Flower colour was assessed using the Dictionary of Colour Standards and the Horticultural Colour Chart from the British Colour Council. The standard and wing were white in the white, mottled brown and brown genotypes, mauve in the mottled black genotype, and were either white or mauve to rose purple in the black genotype. The colour of the wing was mauve in the mottled black genotype and was either white or mauve in the black genotype. Pod colour for the white genotype was mimosa yellow to naples yellow, or mottled with either aster violet or hyacinth blue, while in the mottled brown genotype pod colour was predominantly naples yellow, mottled with china rose or also with chrysanthemum crimson. Pods from the mottled black genotype were mimosa yellow to amber yellow in colour, and sometimes mottled with purple brown. Pods from the black genotype were mimosa yellow or naples yellow and were either slightly mottled with lilac purple or with pansy violet, while pod colour from the brown genotype was erythrite red. Dried pod length varied from 9.3 to 12.1 cm in the brown and white genotypes respectively, while dried pod width ranged from 11.8 mm in the mottled black to 12.8 mm in the white genotype. The number of pods per plant varied from 13 in the mottled brown to 16 in the brown genotype, while seeds per pod varied from 4.4 in the brown genotype to 5.8 in the while genotype. Daylength for the field trial ranged from 14.5 h (November) to around 12.3 h (March), with a maximum daylength of about 15 h in December. Seeds from the same seed colour groups used for the glasshouse studies were used in the field trial which was located at the Frewen's block, Massey University. Seeds were sown at three different rates (2.8, 5.6 and 8.4 g/m2) by cone seeder on 28 November 1994 to obtain densities of 6.6, 13.3 and 20.0 plants/m2 at row spacings of 60, 30 and 20 cm respectively. Within the rows a uniform space of 25 cm was maintained. Each treatment (plant density x genotype) was replicated four times in a split plot design. For seed development studies, a total of 450 - 460 flowers per genotype (from the 13.3 plants/m2 density) were randomly selected and labelled at anthesis, and 60 pods per individual genotype were harvested manually at 14, 20, 26, 32, 40 and 50 days after labelling for the determination of seed moisture content, fresh weight, dry weight and percentage seed germination. Seed yield and seed yield components (number of pods per plant and seeds per pod) were recorded after hand harvesting of 10 sample plants/plot. The quality of seed for sowing purposes was assessed by germination, conductivity and accelerated ageing (AA) tests, while for cooking quality, seeds were assessed for their imbibition rate, seed texture and seed integrity after cooking. All the data acquired from this study were analyzed with the statistical analysis system of SAS with least significant differences at the 5% level. The white and black bean genotypes produced 11 and 17% plants with indeterminate climbing characteristics respectively, while the other genotypes each produced 1 - 3% of plants with indeterminate climbing characteristics. All other plants were bush-indeterminate. Plant height in all bean genotypes at all the densities measured between 50 - 60 cm with a min./max. height of 40/85 cm. The onset, peak and duration of flowering in all genotypes were not affected by plant density. The typical three phase sequence of seed development was recorded and physiological maturity, or the attainment of maximum dry weight, occurred at around 40 days after anthesis (d.a.a.) at more or less the same time for all genotypes. Seed germination started around 20 d.a.a. and reached a maximum (of 100%) about the same time as the attainment of maximum seed dry weight at 40 d.a.a. However differences in seed coat permeability influenced the rate of seed desiccation and caused differences in seed moisture content (smc) among genotypes. The number of branches per plant differed significantly from 4.6 in the brown genotype to 5.2 - 5.8 in other genotypes. At the 6.6 plants/m2 density the number of branches per plant was 7.0 and decreased to 3.8 at the 20.0 plants/m2 density. Flowers per plant varied from 46.9 to 63.9 in the brown and mottled brown genotypes respectively but did not differ with density. Pods per plant were similar for all genotypes, and reached 32.2 at the 6.6 plants/m2 density but decreased to 19.0 at the 20.0 plants/m2 density. Seeds per pod varied slightly from 4.1 in the brown genotype to 4.6 in the mottled black genotype, but did not differ with density. Seed weight/100 seeds varied from 35.7 g in the mottled black genotype to 45.2 g in the black genotype, and was similar for all densities. The black genotype produced an average seed yield of 5,705 kg/ha (the highest), while the brown genotype had an average of 4,723 kg/ha (the lowest) at 10% smc. The average seed yield from the white, mottled brown and mottled black genotypes did not differ from that of the black genotype. There was no genotype x density interaction and the average seed yield for all genotypes was 3,800 kg/ha at the 6.6 plants/m2 density, 5,366 kg/ha at the 13.3 plams/m2 density and 6,715 kg/ha at the 20.0 plants/m2 density. The conductivity test result varied from 4.7 μS cm-1 g-1 in the brown genotype to 15.5 μS cm-1 g-1 in the white genotype, which demonstrated the differences in seed coat characteristics among genotypes. The germination before AA was 100% for all genotypes, and did not differ after AA (between 97 and 99%). The conductivity test result, as well as the percentage germination before and after AA did not differ with density. The brown genotype produced an average of 53.5% of seeds with 'delayed permeable' seed coats, while this property varied from 3.5 to 10.0% in the white and mottled brown genotypes respectively. Seeds from all genotypes became soft after cooking for 20 min. However the force required to cut the seeds after cooking varied from 6.83 Newton in the white genotype to 15.23 Newton in the brown genotype. The high seed coat permeability in the white genotype caused 19.3% seed coat/cotyledonary damage, while the brown genotype had only 8.9%. The white, mottled brown, mottled black and black genotypes produced a high yield (between 5,136 and 5,705 kg/ha) of good quality seed for both sowing and eating purposes. The brown genotype had a lower seed yield (4,723 kg/ha), but the seed was also of good quality for sowing and eating purposes. Differences in the seed coat characteristic of different bean genotypes may mean a requirement for different lengths of cooking time to attain the same level of seed softness without seed coat splitting as required for human consumption.
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    A study of nitrogen fixation, nitrogen distribution and seed yield of selected legumes with two different growth types : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy at Massey University, Palmerston North, New Zealand
    (Massey University, 1986) Laohasiriwong, Suwit
    Plant growth types of the determinate and indeterminate growth forms are commonly distinguished in many legume species. However, there do not appear to be many studies where direct comparisons have been made of the two growth types in relation to nitrogen fixation and nitrogen distribution. Furthermore, there are disagreements in the literature about the yield advantage of these two growth types. This study was initiated to identify the influence of different growth types of selected grain legumes on seed yield, nitrogen fixation, and nitrogen distribution. In addition, the emphasis was also put on finding amongst the measured parameters, one that had the greatest influence on the differences observed. Initially determinate and indeterminate growth types of bean (Phaseolus vulgaris) and soybean (Glycinemax), were studied in glasshouse conditions. The indeterminate cultivar of both species had higher leaf area and nodule dry weight, more root growth, accumulated more total dry weight and had higher yield than that of the determinate cultivar. In both species, the indeterminate cultivar accumulated more total plant nitrogen than the determinate cultivar. However, only the indeterminate soybean cultivar showed significantly more nitrogen fixation (Acetylene reduction) than that of the determinate cultivar. Subsequently the same soybean cultivars ('Matara' =determinate and 'Amsoy' =indeterminate) were studied in controlled environment conditions. The indeterminate cultivar produced higher vegetative dry-matter and seed yield than that of the determinate cultivar. The higher acetylene reduction activity of the indeterminate cultivar came primarily from a greater nodule mass. About 30-40% of seed nitrogen of both cultivar came from redistribution from vegetative parts, but the stem of the indeterminate cultivar re-distributed a higher proportion of nitrogen to the seed than that of the determinate cultivar. Among several plant characters measured (viz. the dry-weights of the roots, nodules, stems, leaves, and pods, the leaf area, acetylene reduction activity and the total plant nitrogen) leaf area was identified as the key factor in determining the difference between the two growth types. In order to determine the relative importance of leaf area as a factor influencing seed yield, nitrogen fixation and nitrogen distribution the leaf area of the indeterminate cultivar 'Amsoy' was manipulated by imposing different levels of partial leaf removal starting at the flowering stage. For one treatment, partial pod removal was also applied to induce a reduced demand of assimilate. Partial defoliation of the indeterminate cultivar reduced markedly the root growth and the number of branches, but nodule growth, acetylene reduction activity and nitrogen distribution was reduced to a lesser extent. Partial pod removal did not change the overall pattern of response. When about 6 0 % o f the leaves o f t he indeterminate cultivar were removed, seed yield was reduced by about 1 7 % and it was still significantly higher than the undefoliated determinate cultivar . There was no significant difference between the rates o f nitrogen accumulation in the pods under each treatment . The final seed nitrogen concentration was not affected by defoliation treatments nor was the partitioning of nitrogen to seed. I t was concluded that there were differences between the two growth types o f soybean for seed yield, nitrogen fixation, and nitrogen distribution. Leaf area was the most important parameter in determining these difference [sic]. The greater overlapping of vegetative and reproductive growth in the indeterminate cultivar seemed to be advantageous rather than disadvantageous. This longer period of vegetative growth enabled the indeterminate cultivar to produce a bigger source capacity which consequently supported more nitrogen fixation activity and produced higher seed yield. The possible implications to tropical agriculture were discussed and some future research topic s were also suggested .
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    Studies on abscission cell differentiation in Sambucus nigra and Phaseolus vulgaris : submitted in partial fulfilment of the degree of Doctor of Philosophy in Plant Biology, Institute of Molecular Biosciences, Massey University, Palmerston North, New Zealand
    (Massey University, 2002) Flight, Simone
    This thesis examines aspects of abscission cell differentiation in Sambucus nigra and Phaseolus vulgaris. The experimentation is divided into two sections; an in vivo study examining the cell wall proteins from the leaf rachis abscission zones of S. nigra, to identify proteins that denote the abscission zone as a fully differentiated cell type, and an in vitro study examining aspects of secondary or adventitious abscission zone formation in petiole explants of P. vulgaris. As an initial approach to identify abscission cell-specific proteins, a survey of the total cell wall bound proteins in four tissues, leaf mid-rachis (MR), ethylene-treated leaf mid-rachis (MRE), 0 h, or freshly excised leaf rachis abscission zone (OZ) and ethylene-treated abscission zone (ZONE) was undertaken. The study also involved surveying these tissues over the vegetative seasons (spring, summer, autumn). Separation of these protein extracts using SDS-PAGE revealed proteins that were putatively uniquely expressed in each of the tissues. Moreover, the expression of some proteins changed from spring through to autumn. Further fractionation of the extracts using hydrophobic interaction chromatography (HIC), and separation of the fractions using SDS-PAGE, illustrated there were many more proteins that had not been resolved in the initial survey of wall extracts. In total, four proteins of ca. 10, 28, 38 and 43 kDa were identified in the UZ tissue only and six proteins (ca. 10, 34, 36, 40, 74 and 75 kDa) were detectable in the OZ and ZONE tissues. Three of the putative OZ-specific proteins (designated OZ10, OZ28 and OZ43) were trypsin-digested and some initial amino acid sequence data obtained. The OZ10 tryptic fragment had closest identity to a lipid transfer protein (LTP) from spinach, and the OZ43 fragment had closest identity to an aldose-1-epimerase-like protein expressed in tobacco. Two peptides were sequenced from the OZ28 protein; one had highest identity to a superoxide dismutase and the second had identity to a ribonuclease. Two of these, OZ10 and OZ43 were characterised further. Antibodies raised to LTPs protein from Daucus carota and Arabidopsis thaliana recognized a protein of 10 kDa that was expressed in both the rachis and abscission zone tissues of S. nigra before and after ethylene treatment. Moreover, the LTP antibodies detected a ca. 10 kDa protein in freshly excised and ethylene-treated distal pulvinus, primary abscission zone and petiole tissues of P. vulgaris with highest expression in ethylene-treated petiole tissue. The second protein, to be characterised further was most similar in sequence to a nuclear pore membrane protein identified in tobacco suspension cells and designated gp40. This protein appears to be an aldose-1-epimerase-like enzyme (otherwise known as mutarotase) from its homology to bacterial forms of mutarotase. An antibody to gp40 recognized a ca. 43kDa protein in the non-ethylene treated rachis and zone cell wall extracts of S. nigra, the putative OZ43. This same antibody did not recognize any proteins in the protein extract from porcine and lamb kidney, tissues that have mutarotase activity. A coupled enzyme assay was developed to measure the mutarotase activity in the plant samples. Although mutarotase activity was measured in both the soluble and cell wall bound fractions of the rachis cells, purification of the OZ43 protein using column chromatography or through cell fractionation revealed that the ca. 43 kDa protein recognised by the gp40 antibody did not appear to be responsible for this activity. For the second part of this thesis, the in vitro study, the aim was to measure the levels of IAA, ethylene and ACC oxidase enzyme activity in bean petioles explants during IAA-induced secondary abscission zone formation. In the bean explant system, the secondary zone forms at a site along the petiole which is removed from the primary zone and governed by the concentration of IAA added. The petiole tissue that links the primary zone with the secondary zone (the distal segment) remains green and, in this thesis, is designated as G1. The petiole tissue proximal to the zone senesces and yellows, and is divided into Y2 (immediately proximal to the secondary zone), Y3 (mid way) and Y4 (the most proximal petiole tissue). To measure changes in IAA concentration during secondary zone formation, an immunoassay (ELISA) was developed, initially using polyclonal antibodies to IAA, but the titre of these antibodies was not sufficient and so monoclonal antibodies were used. During secondary zone formation, the concentration of free IAA in the petiole tissue changed dramatically, with measurements ranging between 6 and 2608 pmol/g fresh weight (FW) of tissue. The IAA concentration in the petioles at separation at the primary zone before IAA was added was lower in the G1 and Y2 sections (ca. 30 pmol/g FW) when compared with the Y3 and Y4 sections (166 and 271 pmol/g FW respectively). At 6 h after the application of IAA, the concentration of IAA had increased to 146 pmol/g FW in the G1 section, remained the same in the Y2 section and increased to 208 and 423 pmol/g FW in the Y3 and Y4 sections, respectively. At 26 h after the application of IAA, and approximately the time of initiation of differentiation of the secondary zone, the IAA concentration was similar to the petioles after 6 h (179 and 21 pmol/g FW for G1 and Y2 respectively) and significantly lower in the Y3 and Y4 sections (35 and 69 pmol/g FW respectively). At the first point at which the green:yellow tissue can be ascertained (at 52 h) the IAA concentration was dramatically higher in the G1 and Y2 tissues (1125 and 1090 pmol/g FW respectively) compared to measurements in the Y3 and Y4 sections at 52 h of 17 and 107 pmol/g FW respectively. At separation at the secondary abscission zone, the IAA measurements in the G1, Y2 and Y4 sections were 405, 315 and 1198 pmol/g FW respectively. The ethylene produced from freshly excised pulvinus and petiole tissue was ca. 0.20 nmol/h/g FW and increased to 1.7 nmol/h/g FW in the pulvinus, 0.39 nmol/h/g FW in the G1 petiole section and 0.67 nmol/h/g FW in the Y2/Y3/Y4 pooled petiole sections at separation at the primary zone. At separation of the secondary zone, ethylene evolution measurements of 1.73 nmol/h/g FW in G1 and 4.37 nmol/h/g FW in the Y2/Y3/Y4 tissue were observed. However, the activity and expression of ACC oxidase was higher in the fresh tissues and non-senescent petiole region (G1), but was lowest in the senescent (Y2,Y3 and Y4) tissue at the formation of the secondary zone.