| Abstract |
In the core of this Master dissertation is the description of a procedure for Greek cotton cultivars, that uses transformation-competent, pre-existing meristematic cells from the isolated shoot apex of aseptically germinating seedlings and from the excised embryonic axes of mature seeds. This clonal propagation system allows regeneration of cotton to be plant-driven and therefore, genotype independent. Induction of shoot development was achieved on a simple MS-basal Medium supplemented with 0,1 mg/l Kin in the case of shoot apices and with 3 mg/l BAP in the case of embryo axes. Elongation of shoots was obtained on agar-solidified, MS-basal Medium supplemented with 0,01 mg/l Kin. In vitro shoots were rooted on half-strength MS-basal Medium with 0,3 % AC for shoot apices and on Mc.Stewart and Hsu Medium with 0,01 mg/l NAA for embryo axes, within 6-10 weeks from isolation, depending on the cotton cultivar. All shoots regenerated directly without a callus phase. The procedure was found to be applicable to four different Greek cotton varieties (M1, M2, M3, ΦΩΤΕΙΝΗ), giving an average of 5-10 % regeneration efficiency. If shoot multiplication is desired the use of TDZ is proposed at the concentrations of 0,022-2,2 mg/l, though in so far experiments of ours multiple shoot production was not observed after 2 weeks in culture for the four cultivars. Also presented here are, preliminary tests to determine the optimum kanamycin concentrations for selection of transformed meristematic tissues as well as molecular analysis methods such as DNA extraction, genomic template PCR and GUS-assaying methods optimized for cotton tissues in order to be used for the future detection of cotton transformants. Finally, some preliminary results are presented on the development of the Agrobacterium-based transient expression assays based on the use of avirulence (avr) genes from Pseudomonas syringae pv. phaseolicola as facile indicators of Hypersensitive Reaction and non-host resistance of Greek cotton cultivars.
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Χρησιμοποίηση εμβρυακών αξόνων και μεριστωμάτων βαμβακιού (Gossypium hirsutum L.) για αναγέννηση γενετικά τροποποιημένων φυτών και ανάπτυξη μεθόδου παροδικής γονιδιακής έκφρασης.
