PhD

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Browsing PhD by Subject "Agrobacterium mediated transfer"

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    Agrobacterium Mediated Transfer Of Bacillus Thuringiensis 6E Cry Gene To Ixora Odorata
    (University of Peradeniya, 2003) Bamunuarachchige, T. C.
    Ixora species are quite popular among landscape architects and gardeners as hedge plants and potted ornamentals. However, infestations by pests such as Ixora leaf webber, flower webber, green horned caterpillar and looper caterpillar have become a threat to the commercial exporter, especially with strict quarantine regulations under which presence of a single pest egg may spell disaster. Most chemical methods adopted have hazardous effects on the environment and other beneficial insects and therefore, are unpopular among the buyers. As an alternative Ixora odorata var. vulcan was transformed with the cry gene of Bacillus thuringiensis strain 6e by Agrobacterium mediated gene transfer. In the process, an effective plant regeneration procedure and a gene transfer system were developed. The cry gene of Bacillus thuringiensis strain 6e was isolated, purified and cloned into the Xbal site of the T-DNA region of the Agrobacterium vector pLG121Hm via an adapter. Recombinant pLG121Hm was transferred to Agrobacterium strain LBA4404 by electroporation. Positive cry clones were confirmed by Dot blot analysis of the plasmid DNA (extracted from the electroporated strains) with Dig labeled cry probe. In vitro grown Ixora odorata shoot tips (2cm) were co-cultivated with LBA4404, harboring the Bt 6e cry cloned binary vector pLG121Hm. Co-cultivated explants were transferred to shoot multiplication medium ('/2 MS with 2mg/l BAP and 500mg/l cefotaxime) and incubated at 25001ux with a 16h photoperiod. After 4 weeks the axillary shoots were screened on selective medium (Vz MS supplemented with 200mg/l hygromycin). Hygromycin positive plants were used for the PCR analysis and Southern blot analysis. PCR analysis of the putative transformants, carried out with gits specific primers, produced the expected 1.680kb fragment in all the hygromycin positive transformants. Southern blot analysis of these with the Dig labeled Bt 6e cry probe produced positive results. The positive results of both, the PCR analysis and the Southern blot confirmed genomic integration of the Bt 6e cry gene. The transformation efficiency was 20% for the shoot tips whereas it was 40% and 30% for callus and leaf disk transformations respectively but the inability of proper regeneration made both these techniques redundant.