Development of Simple and Efficient in Planta Transformation Method for Rice (Oryza sativa L.) Using Agrobacterium tumefaciens(PLANT BIOTECHNOLOGY)

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Seeds of rice (Oryza sativa L. var. Koshihikari) were soaked in water for 2d. Thereafter, the embryo containing an apical meristem was inoculated with Agrobacterium tumefaciens by piercing a site of the husk overlying the embryonic apical meristem with a needle that had been dipped in an A. tumefaciens inoculum. The inoculated seeds were then grown to maturation (T_0 plants) and allowed to pollinate naturally to set seeds (T_1 plants) in pots under nonsterile conditions. To examine the transformation by various means, three different strains of A. tumefaciens were used for transformation : an M-21 mutant, which is an avirulent mutant with a Tn5 insertion in the iaaM gene, and two LBA4404 strains each with a different binary vector. Five different lines of evidence were demonstrated the transformation : the altered phenotype and its inheritance by the next generation, histochemical detection of β-glucuronidase, resistance to hygromycin B, detection of the transgene by PCR and rescue of a plasmid consisting of the integrated T-DNA and the flanking rice genome DNA. Transformation efficiency of T_1 plants was estimated to be 40% and 43% by PCR and a histochemical assay of β-glucuronidase, respectively.
2005-10-25