Rice Transformation Using Choline Oxidase Gene and Verifying Putative Transformed Plants

In order to evaluate salt tolerance of four Iranian rice cultivars named as "Zayandeh Roud", "Tarom Molaie", "Neamat" and "Charam 2", and to select a tolerant cultivar so that does not use organic osmoregulators to transform it with the choline oxidase gene which covert choline to glycine betaine, a study was conducted in Agricultural Biotechnology Research Institute of Iran. In the first step, in vitro salt tolerance of the cultivars were evaluated using a few experiment in which 6 levels of NaCl comprising as 0, 20 , 40, 60, 80 and 100 mM were added to MS Media. Callus initiation and growth and regeneration of plantlets from callus as well as Na+, K+, Ca++, Mg++, Cl- ions 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 "Zayandeh Roud" and "Tarom Molaie" cvs have the best in-vitro salt tolerance and use organic osmoregulatores less than other cvs. Moreover, these cvs have the best callus growth and regeneration, and these characters dictate them as the best candidate for gene transformation. Seed-drived embryogenic calli of these cvs were targeted in biolistics method with two plasmids, pChlCOD and pCytCOD, contained choline Oxidase and nptII genes. The later gene was used as selectable marker. The first plasmid uses a transit peptide 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. Derived putative transformed calli that had been selected on media contained 50 mg/l hygromycin, developed into putative transformed plantlets. PCR analyses revealed that all six plantlets achieved from pChlCOD and five plantlets resulted from calli targeted with pCytCOD are PCR-positive. Southern analysis on four putative transformed plantlets targrted with pChlCOD showed that at least one copy of choline Oxidase gene was integrated into genome of the plantlets. RT-PCR analysis revealed that three plantlets can transcribe the choline Oxidase gene. Although these putative transformed plant showed semi sterility, but also a few seed achieved from plants no. 3, 5 and 6. Some of these T1 seeds were germinated and grew into T1 plantlets. PCR analysis for these plantlets showed the expected band and this resultes means that the plants achieved at least one copy of transgene from parental line. More molecular and functional analysis are needed in T1 and in the next generations to calculate copy number of the gene and to confirm transcription and translation of choline Oxidase and its effect on enhancement of salt tolerance.