Transformation of Rice Using Choline Oxidase Gene (Glycine Betaine Synthesizer) to enhance salt tolerance.

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 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. Putative transformed T2 plants were analyzed in PCR with specific primers for choline oxidase gene. The results showed that 65 plants revealed CodA gene and 30 plants 101 weren't. The X2 test showed that this frequency do not differ from 3:1 Mendelian segregation ratio. Reverse Transcriptation-PCR (RT-PCR) of PCR positive plants revealed that CodA gene is transcripted in all plants. The germination test for T2 and control seeds in presence of 0, 100, 200 and 300 mM NaCl shoed that transformed seeds had more germination than control seeds in all salinity condition. Physiological experiment in hydroponic condition in presence of 0, 40, 80 and 120 mM NaCl showed that the best leaf growth dynamics was belonged to control plants. This result did not mean necessarily that CodA gene did not affect on increase in salinity tolerance. It seems that limitation in choline resources as a substrate for CodA gene may affect on its ability and decreased leaf growth dynamics. So, isolation of genes related to choline synthesis pathways and their transfer to plants may compensate the deficiency of subestrate in transformed plants.