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Cellular Biology

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Browsing Cellular Biology by Subject "Theses--Biological and conservation sciences."

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    Development of in vitro culture and gene transfer techniques in sugarcane (Saccharum species hybrids).
    (1992) Snyman, Sandra Jane.; Huckett, Barbara Isobel.; Watt, Maria Paula Mousaco Deoliveira.
    In vitro cell and tissue culture systems were developed for sugarcane in order to utilise current transformation techniques to introduce genes to South African sugarcane varieties, which would be difficult, if not impossible to achieve in conventional breeding programmes. Embryogenic calli were initiated in the dark from stem explants of sugarcane varieties NCo376 and N13, on a MS medium containing sucrose (20-50 g/l), 2,4-D (2-4 mg/l), casein (1 g/l), inositol (100 mg/l) and agar (9g/l). After 2 months the somatic embryos were cultured in a light/dark photoperiod for a further 2 months. The best combination of sucrose and 2,4-D for callus initiation, and subsequent plant regeneration, was 20 g/l and 2 mg/l, respectively. Plant yields ranged from 16 to 36 plants per gram fresh weight callus, and the yields were not significantly increased by the addition of activated charcoal to the regeneration medium. When plantlets reached a height of 10 cm, they were transferred to autoclaved soil in pots, hardened-off and placed in the glasshouse. Suspension cultures were initiated from friable NCo376 calli in liquid MS medium shaken at 100 rev/min in the dark at 27°C, and were subcultured every 3-7 days. Protoplasts from various sources (leaf, calli and suspension cultures) were obtained after enzymatic digestion in cellulase (20-30 g/l), macerozyme (0,2 g/l), hemicellulase (5 g/l), and sorbitol (0,55 M) in a calcium and magnesium salt solution. Protoplasts cultured for 48 h resulted in a loss in viability of 84%. The potential of the seed as a recipient for direct gene uptake was investigated, as this eliminated the need for in vitro culture and plant regeneration. Uptake of [3H] pBR322 DNA by seeds was demonstrated, and seeds with the testa removed exhibited higher initial uptake rates than those with intact seed coats. However, transient expression, using the GUS reporter gene (coding for bacterial B-glucuronidase) carried on plasmid pBI221, could not be conclusively shown using the histochemical GUS assay, due to GUS activity generated by either microbial contamination or endogenous plant GUS activity. Neither microwaving to eradicate contaminants nor the addition of methanol (20%) to the GUS incubation buffer were successful in overcoming positive results observed in control seeds. An alternative approach to sugarcane transformation, using PEG-mediated DNA uptake and subsequent transient expression of GUS by protoplasts was investigated, but microbial contamination was a persistant problem and no positive results were observed. Further examination and elimination of endogenous contamination is required before transformation studies can be continued.
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    The use of laryngeal and buccopharyngeal morphology in anuran taxonomy.
    (1991) Lambiris, Angelo John Leonis.; Poynton, John Charles.
    The taxonomic value of adult laryngeal and larval buccopharyngeal characters, not previously used in studies of southern African frogs, was investigated. The amphibians of Natal, recently examined using more traditional characters (Lambiris 1989), were used both for assessing the value of these characters and for testing the earlier taxonomic conclusions. The results indicate that: (a) Laryngeal and buccopharyngeal characters appear to be of value in anuran taxonomy; (b) Most of the taxonomic conclusions of Poynton (1964) and of Lambiris (1989) appear to be justified; (c) Taxonomic problems in certain of the genera and species-groups examined appear to be more easily resolved by the use of these characters than by those more generally used. The status of six taxa has been redefined; these are Bufo pusillus, Bufo poweri, Bufo cruciger, Breviceps maculatus, Rana umbraculata and Cacosternu striatum. Five undescribed species and one new genus have been identified and await description.