Development of efficient protocols for plant regeneration and agrobacterium-mediated transformation in okra (Abelmoschus esculentus (L.) Moench

Arora, Apoorva

Development of efficient protocols for plant regeneration and agrobacterium-mediated transformation in okra (Abelmoschus esculentus (L.) Moench

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CCSHAU-291789-Arora, Apoorva.pdf (734.36 KB)

Date

2009

Authors

Arora, Apoorva

Publisher

CCSHAU

Abstract

Present study was undertaken to evaluate the most suitable concentration of growth regulators for plant regeneration of okra cultivars Varsha Uphar and Hisar Unnat and to attempt to optimize conditions for Agrobacterium-mediated transformation. After optimization efforts were made to introduce cry 1A(c) gene of Bt in one of the okra cv. Varsha Uphar. Experiments were conducted to develop an efficient and reproducible plant regeneration system in two cultivars. Explants (hypocotyls, cotyledon and cotyledonary node) were excised from aseptically grown 5-15 days old okra (Abelmoschus esculentus) seedlings. No shoot regeneration was obtained from cotyledon and hypocotyl explants on any of the media combinations. Highest percentage (100%) of callus production was obtained on MS medium supplemented with Zeatin (0.5mg/l or 1.0mg/l) in both the cultivars from both the explants . Percent plant regeneration was achieved from cotyledonary node explants cultured on MS medium supplemented with NAA (2mg/l) and Kinetin (0.1 mg/l) in both the cultivars. The highest percent (98.89%) regeneration was in cv. Varsha Uphar. Agrobacterium mediated transformation conditions were optimized using EHA 105 strain carrying pTOK233 plasmid which had GUS as a reporter gene for both the cultivars of okra, Varha Uphar and Hisar Unnat. Different factors were worked out to optimize Agrobacterium mediated transformation viz. Inoculation time (5, 10 mins), co-cultivation period (48, 72 hours), bacterial concentration (OD600 2.0, 0.5) and pre-culture of explants (freshly cut, 3days). Best conditions were found to be 10 mins of inoculation time, 72 hours of co-cultivation, bacterial concentration with OD600 2.0 and freshly cut explants. Efforts were made to introduce Bt gene in one of the okra cultivars, Varsha Uphar.