Bioecology and management of pod bugs infesting vegetable cowpea (Vigna unguiculata ssp. sesquipedalis (L.) Verdcourt
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Date
2007
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Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara
Abstract
The study entitled ‘genetic transformation of chilli (Capsicum annuum L.) with osmotin gene’ was carried out at the Centre for Plant Biotechnology and Molecular Biology (CPBMB), College of Horticulture, Vellanikkara, from December 2003 to September 2005. The study was undertaken to standardize in vitro regeneration and Agrobacterium mediated transformation of chilli with osmotin gene.
Different explants, media and hormonal combinations (auxin and cytokinin) were tried in order to standardize in vitro regeneration in chilli var.Ujwala. The best explant for in vitro regeneration was hypocotyl. Only the buds originated from hypocotyl explants showed elongation. Shoot buds formed from cotyledon and leaf segments did not elongate in any of the media tested. Based on the percentage regeneration in cultures and no. of shoot-buds per explant, MS medium containing the hormonal combination; BA (5.0 mg l-1) + IAA (0.3 mg l-1) was found optimum. The regenerated plantlets were transferred to pots for acclimatization so that they can sustain and survive in the natural conditions. The hardened plantlets were planted out.
Agrobacterium mediated transformation protocol was optimized considering all the factors for successful transformation. Optimum inhibitory concentration of selectable marker (Kanamycin: 100mg l-1) was established. The antibiotic cefotaxime (200 mg l-1) was selected for killing the bacteria. Agrobacterium strain EHA 105 harbouring the gus reporter gene was used for the standardization of transformation. Hypocotyl explants of chilli were co-cultivated with Agrobacterium strain (EHA 105). The inoculum density-0.1 OD600 nm, infection time-5minutes and co-cultivation period-2 days were found optimum based on GUS assay and survival rate 60 days after co-cultivation.
The hypocotyl explants of chilli were co-cultivated with Agrobacterium strain (GV2260) harbouring osmotin gene in the plasmid pGV2260 tagged with 35S CaMV promoter. The transformed explants were regenerated on the selection medium optimized for regeneration of chilli. However, none of the transformed buds were elongated in the elongation medium containing selection agent. Hence the transformed plants were transferred to elongation medium containing no antibiotics. Even in this medium, no elongation of shoots was observed even after 60 days. So, further refinement of transformation protocol using an optimal selectable marker is needed for the production of transgenic chilli. After selection of the transformants, the putative transgenics were characterized employing molecular biology techniques viz. PCR-utilizing the gene specific primers of nptII. The presence of transgene was confirmed in the transformed plants.
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