MSc

Investigation on ‘Development of in vitro regeneration and genetic transformation systems in Pumpkin (Cucurbita moschata Poir.)’ was carried out at the Centre for Plant Biotechnology and Molecular Biology (CPBMB), College of Horticulture, Vellanikkara, from October 2005 to July 2008. The study was undertaken to standardize a viable in vitro regeneration system and to develop protocol for genetic transformation in Pumpkin.

Axenic seedlings of Pumpkin variety Saras were raised under in vitro conditions to generate explants with reduced contamination for transformation. MS basal medium was found to be better compared to half MS basal with respect to the percentage of germination. Mercuric chloride (0.1%) treatment for 6 min was found to be effective in sterilizing the seeds.

In order to standardize a regeneration protocol, MS medium supplemented with varying concentrations of auxin and cytokinin were tried on different explants. Different explants such as cotyledonary leaf segments, hypocotyl segments, cotyledonary node segments from axenic seedlings and cotyledons from intact seeds.

Cotyledonary node explants produced multiple shoots in MS medium supplemented with 1 mg l-1 BA and 1 mg l-1 Kinetin. Shoot buds produced from cotyledonary node explants showed good multiplication in the same media. The shoots were successfully rooted in MS + 1 mg l-1 IBA. The plants were successfully hardened and planted out.

Embryogenic calli was induced from cotyledon explants cultured in MS medium containing 5 mg l-1 2,4 – D and 0.1 mg l-1 TDZ.

Agrobacterium mediated transformation protocol was optimized considering all the factors for successful transformation. EHA 105 p35S GUSINT was used for standardizing optimum conditions by comparing the levels of transient GUS expression in calli. The cotyledon explants were used for genetic transformation.

Optimum inhibitory concentration of selectable marker (Kanamycin: 200mg l-1) was established. The antibiotic cefotaxime (250 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. Cotyledon explants of pumpkin were co-cultivated with Agrobacterium strain (EHA 105).

The influence of different parameters such as bacterial inoculum, co-cultivation periods and infection time effects on transformation frequency were studied. The inoculum density-0.6 OD600 nm, infection time-10 minutes and co-cultivation period-2 days were found optimum based on GUS assay and survival rate 30 days after co-cultivation.

Histochemical GUS assays were performed to study and compare the transient GUS expression from transformed tissues. Transient GUS assay revealed faint blue staining on the surface of the infected cotyledon explants. When these blue coloured areas were examined under microscope, clear blue specks were observed indicating transformation.

The explants on transfer to selection medium containing 200 mg l-1 Kanamycin and 250 mg l-1 Cefotaxime produced callus 4 weeks after co-cultivation. Normal explants turned brown when transferred to selection medium.

After selection of the transformants, the transformed cotyledon tissues were characterized employing molecular biology techniques viz. PCR-utilizing the gene specific primers of npt II. The presence of transgene was confirmed in the transformed tissues.