School of Botany - Theses
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ItemImmunodissection of pollen antigens of Brassica and Lilium( 1993)Monoclonal antibodies (MAbs) were raised against a sperm cell enriched fraction from germinated Brassica napus pollen. Thirty-five cell lines secreted antibodies to components of the sperm cell and pollen grain, and four were selected for further characterisation. Western blot analysis, immunofluorescence and immunogold labelling showed that two of the MAbs correspond to nuclear antigens, including MAb BNS 1, labelled a 14 kDa antigen located within the nucleus of B. napus sperm cells and also the nuclei of other tissue and genera. Two other MAbs bound to high and polydisperse molecular weight antigens in the cytoplasm, inline and wall of B. napus pollen grains that may be involved in pollen germination. The plasma membrane surface proteins of intact somatic and reproductive protoplasts of Gillum longiflorum and Brassica napus were compared after probing with N-hydroxysuccinimido- (NHS) or sulfo-NHS- biotin. In lily, six proteins specific to the surface membrane of leaf protoplasts were identified, three proteins to pollen protoplasts and two proteins to generative cell protoplasts. In rapeseed leaf protoplasts, seven proteins were detected, while in the sperm enriched fraction five proteins were present. Protein bands of 65 and 67 kDa appeared to be unique to the reproductive cells of B. napus and L. longiflorum respectively. The proteins identified as membrane specific for generative cell protoplasts of lily were isolated and used as immunogens for monoclonal antibody production. Pre-fixed generative cells of L. longiflorum were also used to raise monoclonal antibodies to surface antigens. Monoclonal antibody, MAb 69-139-19, raised against sheep sperm, cross-reacted with anther proteins from four Brassica species tested. During development, the antigen was present as a low molecular weight polypeptide of ~42 kDa in anthers up to the late vacuolate stage. At the mid-maturation stage the MAb bound to the 42 kDa band as well as a high and polydisperse band of Mr ~160-230 kDa. In protein extracts from mature pollen the lower (42 kDa) antigen was no longer present and MAb 69-139-19 labelled the polydisperse region of 160-230 kDa as well as a faint but distinct band of 125 kDa. Immunofluorescence and immunogold labelling showed that the antigen was deposited from the tapetum onto the pollen grain wall, which at pollen maturity was strongly labelled by MAb 69-139-19. The antigen was detected at lower levels in the cytoplasm of immature and mature pollen. The level of labelling within the cytoplasm increased during germination and the sperm cell cytoplasm and the sperm and tube nuclei were also labelled. MAbs were raised against glutaraldehyde-fixed generative cells of Lilium longiflorum. MAb LLM1 recognised an antigen with a molecular weight >43 kDa, common to the plasma membrane and organelle membrane of somatic and reproductive cells. MAb LLG2 recognised a cytoplasmic antigen of 40 kDa, unique to the generative and sperm cells of L. longiflorum. The antigen was also detected in proteins of Lilium lancifolia but not in pollen proteins of other genera. [35S]-methionine incorporation by isolated generative cells of Lilium longiflorum has indicated that they possess their own set of mRNA and are capable of synthesising proteins independently from the vegetative cell. Approximately ten proteins were synthesised by the isolated generative cells, of which six appeared to be unique to the generative cells. Isolation of generative cells from pollen grains after [35S]-methionine labelling resulted in an identical protein profile, so that synthesis of these proteins was not due to isolation shock. Poly(A)+RNA was isolated from L. longiflorum generative cells and used to generate a PCR amplified cDNA library. Three clones, CP2, CP19 and CP23 (insert sizes 2200, 2300 and 2300 respectively), were isolated by differential screening. The site of expression was determined by in situ hybridisation and all three clones hybridised preferentially with the generative cell cytoplasm.
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ItemAspects of the reproductive biology, breeding system and horticultural improvement of the genus pandorea( 1992)The Australian flora represent a potential genetic resource for the production of new cultivars for the local and international ornamental plant industry. The genus Pandorea contains four species in Australia. All are climbers with a range of flower colour and size. This study aimed initially to collect a range of genotypes from the Australian species of Pandorea. It then sought to identify and study aspects of the reproductive biology and breeding system of the genus Pandorea. It also aimed to cross species using the basic techniques of plant breeding to provide unique genetic combinations as a basis for new horticultural varieties of Pandorea. Floral dimensions were measured and compared for all species. The reproductive biology was indicative of a genus comprising obligate outcrossing individuals. Strong self-incompatibility was found in three species. The fourth was not tested. The stigmas were receptive prior to anthesis. Pollen viability deteriorated over a five day period. Interspecific hybrid seedlings were obtained for some crosses through embryo culture. Embryos aborted if left on the parent plant apparently due to endosperm failure. The success of rescuing interspecific hybrid embryos was related to the stage at which embryo development was arrested. Well-developed cotyledonary embryos were readily grown in aseptic culture and were acclimatized with ease from tissue culture. Less mature embryos germinated precociously and failed to continue through the normal embryo development. Isozyme analyses were useful for corroborating the hybrid status of some putative hybrid seedlings produced in this study. One hundred and seventeen confirmed interspecific hybrids and seven unconfirmed hybrids have been acclimatized to standard nursery conditions. They are anticipated to flower for the first time in the 1994 flowering season when their floral characteristics can be assessed for horticultural potential.