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

摘要

Seeds of rice (Oryza sativa L.var.Koshihikari) were soaked in water for 2 d.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 beta-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 beta-glucuronidase,respectively.