A modified in-planta transformation technique to generate stable gain-in function transformants in a recalcitrant indica rice genotype

A tissue culture independent in-planta transformation technique is an alternate option to generate transgenics in crop cultivars recalcitrant for regeneration. The T-0 plants generated by in-planta technique are chimeric hence very large number of T-1 seeds needs to be screened to identify the putative transformants. Besides, low transformation efficiency and stable integration is another lacuna, because the T-0 seedling explants are not subjected for selection. To overcome these constraints, a modified in-planta technique was developed in this study to generate gain-in-function activation tagged mutants in the background of a recalcitrant rice genotype, KMP-153. A binary vector was developed with three glyphosate selectable marker genes and 4X CaMV 35S enhancer elements for developing rice plants. Simultaneous expression of Glyphosate insensitive CP4-EPSPS with two other glyphosate degrading enzymes, PsAKR1 (Aldo-Keto reductases) and GO (Glycine Oxidase) substantially improved tolerance up to 100 ppm of glyphosate in the seedling bioassay system. Selecting the seedling explants and T-1 generation seeds at higher concentration (100 ppm) of glyphosate resulted in recovering fewer transformants but with stable integration and expression of all the transgenes compared to those screened at lower levels of glyphosate. Further, T2 generation plants from these transgenic lines grown in contained nethouse conditions were tolerant when sprayed up to 1500 ppm concentrations of glyphosate. These transgenics showed substantial lower levels of shikimic acid compared to wild type plants treated with glyphosate signifies the expression of the transgenes and less inhibition of shikimic acid pathway by glyphosate. The proteins extracted from the transgenic plants showed significant degradation of glyphosate. The stable integration was confirmed through flanking sequence information and event specific PCR information. Our results clearly demonstrate that the modified in-planta technique is a potential transformation protocol to generate stable transformants in rice cultivars.