Development of simple and efficient in Planta transformation method for wheat ( Triticum aestivum L.) using Agrobacterium tumefaciens

Wheat ( Triticum aestivum L. var. Shiranekomugi) seeds were soaked in water at 22°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 22°C for 2 d and sterilized by cefotaxime (Claforan) (1000 ppm water solution) treatment and then vernalized at 5°C for 25 d. Finally, the seedlings were grown to maturation (T 0 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.