Positional cloning of the nitrite reductase gene associated with good growth and regeneration ability of calli and establishment of a new selection system for Agrobacterium-mediated transformation in rice (Oryza sativa L.)

Rice varieties vary in their capacity for callus induction, growth, and regeneration. We identified the locus and candidate gene which conferred good callus growth and regenerative ability in the rice variety Koshihikari, a notorious poor rice line for genetic transformation. In addition, we succeeded in establishment of a new selectable marker system using the NiR gene for Agrobacterium-mediated transformation of rice, c.v. “Koshihikari.” The locus was mapped onto chromosome 1, and the nearest RFLP marker was C0178. A total of 500 segregating individuals (BC6F2 seeds) were screened for recombination by PCR-based screening and its location narrowed to a 540-kb region that had been sequenced by the International Rice Genome Sequencing Project. One ORF encoded a putative ferredoxin-nitrite reductase (NiR), which has been suggested to be required for callus induction and growth. The growth and regeneration ability of the calli initiated from Koshihikari was improved through integration of the NiR gene from Konansou. Analysis of ORFs and the promoter region of NiR indicated that the promoter region of NiR gene is responsible for growth and regeneration ability of calli in rice, c.v. Koshihikari. We established a NiR selection system for Agrobacterium-mediated transformation in rice, c.v. Koshihikari, by integration of the NiR gene isolated from rice c.v. Konansou. Transgenic rice plants regenerated from selected calli exhibited β-glucuronidase (GUS) activity. A transformation frequency of 9% was obtained. The results indicated that the NiR selection system is devoid of the disadvantages and concerns of using foreign genes (antibiotics and herbicide resistant) for selection.