Studies on Organogenesis from explants of Begonia Erythrophylla
Degree GrantorUniversity of Canterbury
Degree NameDoctor of Philosophy
Caulogenesis and rhizogenesis were studied in cultured explants of Begonia erythrophylla. The patterns of development were compared to each other and to explants cultured on non-organogenic media. Initially morphological and histological observations were carried out using both light and scanning electron microscopy. Shoots and roots were formed from cells of epidermal origin and were often associated with glandular hairs. Deposition of starch was correlated to organogenesis, with starch grains localized in the superficial layers of the explant, prior to the formation of meristematic regions. Once formed, meristematic regions showed high levels of enzymatic activity compared to the surrounding tissues. Media transfer experiments were conducted between root inducing medium (RIM) and shoot inducing medium (SIM), shoot inducing medium and a basal medium containing no growth regulators (BM), and RIM and BM. It was found that explants became determined for shoot production after 7 days, while they were determined for root production after 3 days on RIM before transfer to BM. Explants were found to be weakly canalized for rhizogenesis for the first 2 days after determination, thereafter they became strongly canalized. Explants were strongly canalized for caulogenesis once determined. Culture on BM prior to exposure to an organogenic medium resulted in the gradual loss of competence with time, but exposure for 2 days to either SIM or RIM resulted in explants becoming competent to respond to opposite inductive medium. Using one and two-dimensional gel electrophoresis, both unlabelled protein and protein labelled in vivo with [35S]-methionine were extracted from organogenic and non-organogenic explants. Silver-stained gels and fluorographs were analyzed to identify different sets of polypeptides associated with development in vitro. Overall the levels of 59 silver-stained and 29 labelled polypeptides were altered. It was shown that rhizogenesis and caulogenesis in petiole sections of B. erythrophylla are associated with both quantitative and qualitative changes in the expression of polypeptides. Most of these changes occur at, or after, the point of organ determination. Some of those associated with caulogenesis were also observed when leaf discs were cultured on SIM. Tissues of mature plants were examined for the presence of these polypeptides. The process of caulogenesis in petiole explants can be further divided by the use of chemical inhibitors of shoot formation. Additions of sorbitol, ribose, acetylsalicylic acid or tri-iodobenzoic acid to SIM, respectively, inhibits shoot formation without killing the explants. These inhibitors are only effective if explants are exposed prior to certain stages of development, after which they fail to inhibit shoot formation. The effects of these inhibitors on the pattern polypeptide changes associated with culture on SIM was determined.