Development Of Efficient Regeneration System In Different Recalcitrant Rice Cultivars And Expression Analysis Of Putative Transgenic Plants

Establishment of an efficient and reproducible regeneration system in three different rice cultivars, IRRI-6, IRRI-9 and KSK-282 remained obscured. In the present study regeneration system was developed for all three recalcitrant cultivars and resultant putative transgenic plants were analyzed for expression by reverse transcriptase polymerase chain reaction (RT-PCR). Cultivars responded to a particular concentration of 2, 4-D ranging from 2 to 4mg L(-1)for calli initiation and induction. Two types of calli with different frequencies were observed on both N6 and N6 supplemented with proline, glycine and MES. N6 media produced more number of type II calli while N6 supplemented favored type I calli in all three cultivars. Type I calli were found as yellowish, smaller in size and globular shape and better explants for transformation. GUS histochemical assay showed that 11, 13 and 14 days older type I calli of KSK-282, IRRI-6 and IRRI-9were suitable for transformation respectively. Fifteen minutes infection time and 2 days co-cultivation period showed the highest GUS positive expression in calli of all three cultivars. KSK-282 and IRRI-9 showed best regeneration response on 0.5 mg L-1 kinetin, 1 mg L(-1)NAA and 3 mg L-1 BAP, while the IRRI-6 showed the highest regeneration at 0.5 mg L-1 kinetin, 1mg L-1 NAA and 2 mg L-1 BAP. Using optimized protocol for each cultivar GUS reporter gene has been successfully incorporated in the genome of all three cultivars. Transgene expression in resultant lines was confirmed at transcriptional level through reverse transcriptase polymerase chain reaction (RT-PCR). (C) 2014 Friends Science Publishers