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Inefficiency of the 'Agrobacterium'-mediated transformation in indica rice cultivar has been widely reported in the literature. Till date, mature seeds, primary and secondary calli have been the mostly used explants for transformation in indica rice. Therefore, the present investigation was undertaken to compare the Agrobacterium mediated transformation using primary and secondary callus as explants in the indica rice and testing a herbicide phosphinothricin (PPT) based bar gene selection system. Four different constructs containing the bar gene expression cassette were made. The constructs I and II were transformed with the secondary calli based protocol by three independent experiments. Similarly, constructs III and IV were transformed with the primary calli based protocol by 9 and 20 independent experiments, respectively. The transformation efficiency for the primary and secondary calli based transformation under PPT selection were calculated in the range of 0.13 - 0.26% and 2.0 - 2.66%, respectively. The transgenic escapes were detected by genomic DNA PCR amplification, Southern blot analysis and basta leaf painting assay. It was found that the primary calli based transformation has 46-50% transgenic escapes, whereas secondary calli based transformation have only 20-29% transgenic escapes. The present study reports that secondary calli as explants perform better than primary calli under PPT selection in the Agrobacterium-mediated indica rice transformation.

australian journal of crop science

Comparative analysis of the Agrobacterium mediated transformation using primary and secondary callus of indica rice (Oryza sativa L.) using phosphinothricin as selecting medium