Multi-gene transformation evaluation of a serine/threonine protein kinase with a gene from the cytokinin oxidase/dehydrogenase family and a transcription factor induced under stress from the NAM-ATAF-CUC family to rice

Production of drought tolerant crop is an important strategy for avoiding water scarce crisis. Improvement of the root structure leading to the higher yield and seed quality. In this study, three genes affecting root structure, drought tolerance and phosphorous absorbance are used in producing hybrid constructs used for the rice transformation. Three genes: a serine/threonine protein kinase (PSTOL1), a gene from the cytokinin oxidase/dehydrogenase family (OsCKX4) and a transcription factor induced under stress from the NAM-ATAF-CUC family (OsNAC5) isolated from the rice wild cultivars are cloned under separate regulatory elements in the T-DNA region of the Agrobacterium binary vector. OsNAC5 gene was cloned under RCc3 root specific promoter and PSTOL1 gene under ubiquitin promoter. Also, OsCKX4 gene was cloned once under ubiquitin promoter and once under RCc3 promoter. Two hybrid multi-gene constructs named pUhrN5CkPstol and pUhrCkPstol harboring multiple genes are synthetized and used for the gene transformation into the Hashemi cultivar. Gene transfer was done to callus obtained from mature rice seeds. Transgenic plants were confirmed using PCR analysis. From the number of 107 regenerated plants in which the presence of transgenes was proved, 14 transgenic events were finally obtained. Root structure of the T0 plants showed drastic phenotypic difference in comparison to the non-transgenic ones. By now, one transgenic event harboring CKX4 and PSTOL1 is confirmed to have a homozygous line in T2 generation. It is hoped that genetic engineering of rice for enhanced root structure lead to drought tolerance, reduce water consumption and improve yield under stress conditions.