Development of Simple and Efficient in Planta Transformation Method for Wheat (Triticum aestivum L.) Using Agrobacterium tumefaciens

摘要

Wheat (Triticum aestivum L.var.Shiranekomugi)seeds were soaked in water at 22 deg C for 1 d.Thereafter,the embryo of the soaked seeds was inoculated with Agrobacterium tumefaciens by piercing a region of the embryonic apical meristem with a needle that had been dipped in an A.tumefaciens inoculum.The inoculated seeds were incubated at 22deg C for 2 d and sterilized by cefo-taxime (Claforan)(1000 ppm water solution)treatment and then vernalized at 5 deg C for 25 d.Finally,the seedlings were grown to maturation (To plants)and allowed to pollinate naturally for seed setting (T_1 plants)in pots under nonsterile condition.To examine the transformation by various means,four different strains of A.tumefaciens were used for transformation.The following five lines of evidence proved the transformation:altered phenotype and its transmittance to the next generation,resistance of T_1 seed germination to geneticin or hygromycin B,the detection of a transgene in T_1 plants by PCR analysis and Southern hybridization and the rescue of the plasmid consisting of the integrated T-DNA and flanking wheat genome DNA from T_1 plants.The transformation efficiency of T_1 plants,which were transformed using different A.tumefaciens strains,was estimated to be 33% by PCR analysis,75% by Southern hybridization and 40% by plasmid rescue.