Abstract
BackgroundCereal crops are the major targets for transformation mediated crop improvement and IR36 is an early maturing, high yielding, insect and disease resistant rice variety however, it is abiotic stress sensitive. Hence, development of an efficient and reproducible micropropagation system via somatic embryogenesis and Agrobacterium tumefaciens mediated transformation is prerequisite to develop abiotic stress tolerant IR36. Further, Genetic stability of analysis of plantlets through RAPD and ISSR and Ploidy level through Flow cytometry (FCM) measurement of 2C DNA content is necessary for future application of transformed IR36.ResultsIn this study, Mature seeds inoculated on (Murashige and Skoog) MS medium with 11.31 μM 2, 4-dichlorophenoxyacetic acid (2, 4-D) and 0.3 μM Kinetin (Kn) had highest callus induction frequency (98%). The highest regeneration frequency (80%) was observed in MS + 13.28 μM Benzyladenine (BA) with 8.06 μM α-naphthalene acetic acid (NAA). Randomly Amplified Polymorphic DNA (RAPD), Inter Simple Sequence Repeat (ISSR) and Flow Cytometry (FCM) analysis showed no significant variation in the 2C DNA (0.81 pg/2C) content and Ploidy level between wild type IR36 and in vitro maintained rice lines. Of the various OD bacterial culture, an optimum OD of 0.4 and inoculation duration of 10 min resulted in efficient Agrobacterium-mediated transformation. β-glucuronidase activity was maximum in callus (99.05%).ConclusionsThese results described here confirm the reliability of this protocol for micropropagation and delivery of desirable gene using A. tumefaciens into indica rice.Electronic supplementary materialThe online version of this article (doi:10.1186/1999-3110-54-47) contains supplementary material, which is available to authorized users.
Highlights
Cereal crops are the major targets for transformation mediated crop improvement and IR36 is an early maturing, high yielding, insect and disease resistant rice variety it is abiotic stress sensitive
The genetic stability of tissue culture derived plantlets could be analysed through Randomly Amplified Polymorphic DNA (RAPD) and Inter Simple Sequence Repeat (ISSR) markers (Priya et al 2011, Shilpha et al 2013)
D with Kn had resulted in maximum callus induction of 98% which is significantly higher than the maximum callus induction at 11.31 μM 2,4-D (93.75%) alone or in combination with 0.44 μM BAP (85%) (Table 1)
Summary
Cereal crops are the major targets for transformation mediated crop improvement and IR36 is an early maturing, high yielding, insect and disease resistant rice variety it is abiotic stress sensitive. Genetic stability of analysis of plantlets through RAPD and ISSR and Ploidy level through Flow cytometry (FCM) measurement of 2C DNA content is necessary for future application of transformed IR36. There is an urgent need to improve global rice production to meet the demand of ever increasing population. It necessitates manipulation and successful implementation of tissue culture and molecular biological approaches. The genetic stability of tissue culture derived plantlets could be analysed through RAPD and ISSR markers (Priya et al 2011, Shilpha et al 2013). Further the Ploidy level and the 2C DNA content (Swift 1950; Bennett et al 2000) can be analysed by flow cytometry
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