In order to evaluate salt tolerance of four Iranian rice cultivars “Zayandeh Roud”, “Tarom Molaie”, “Neamat” and “Charam 2”, and to select a cultivar for transformation using choline oxidase gene, which converts choline to glycine betaine, a study was conducted in Agricultural Biotechnology Research Institute of Iran and College of Agriculture, Isfahan University of Technology. In the first step, in vitro salt tolerance of the cultivars was evaluated using a few experiments in which 6 levels of NaCl comprising as 0, 20, 40, 60, 80 and 100 mM were added to MS Media. Callus induction and growth and regeneration of plantlets from callus as well as Na + , K + , Ca ++ , Mg ++ , Cl - ion content, total sugars, total soluble proteins and total proline content of dried callus and cell sap of callus were measured and analyzed. Results revealed that calli derived from cultivars “Zayandeh Roud” and “Tarom Moline” have the best in-vitro salt tolerance and use organic osmoregulatores less than other cultivars. Moreover, these cultivars have the best callus growth and regeneration, making them good candidates for genetic transformation. Seed-derived embryogenic calli of these cultivars were targeted in biolistics method with plasmids pChlCOD and pCytCOD, which contain choline Oxidase gene and nptII gene as selectable marker. The first plasmid uses a transit peptide sequence to send gene product to chloroplast resulting to accumulation of glycine betaine in chloroplast, and other plasmid lacked this sequence resulting in sytosolic accumulation of glycine betaine. Calli that had been selected on media containing 50 mgl -1 hygromycin, developed into putative transgenic seedlings. All six plantlets transformed from pChlCOD and five plantlets bombarded with pCytCOD were PCR-positive. Southern analysis on four putative transgenic plantlets showed that at least a copy of choline Oxidase gene (pChlCOD) were integrated into genome of the plantlets. RT-PCR analysis revealed that the gene was transcripted in at least three. These putative transformed plant showed partial sterility, but a few seeds achieved from plants no. 3, 5 and 6. Some of these T 1 seeds were germinated and grown. PCR analysis for these plantlets showed that the gene was inherited to the next generation. More molecular and functional analyses are needed in T 1 and in the next generations to estimate the copy number of the gene transferred and to confirm transcription and translation of choline Oxidase and its effect in enhancement of salt tolerance.
