Development of Agrobacterium-mediated Transformation System in Pear Cultivars with Low-regeneration Frequency

Abstract

The transformation of pears such as `Conference', `Doyenne du Comice' and `Passe-Crassane' has been attributed to the high regeneration frequency from leaf discs (71% to 97%; Leblay et al. 1991). However, it has been difficult to transfer desirable genes into cultivars with low-regeneration frequency such as `Silver bell' (35.4%) and `La France' (10.7%), which are the two major pear cultivars in Japan. In this study, we developed an Agrobacterium-mediated transformation system for `Silver bell' and `La France'. For `Silver bell', leaf discs derived from in vitro shoots were used as explants. The antibiotics for selection of transformants and elimination of Agrobacteria were investigated. In the most optimum condition, which is 30 mg·L-1 Kanamycin and 500 mg·L-1 Sulbenicillin, a 3.2 % transformation efficiency was obtained. However, no success was recorded in an effort to transform `La France' using leaf disc explants because of very poor regeneration frequency. Therefore, axillary shoot meristems were used as explants for transformation of `La France'. The conditions for antibiotic selection and elimination of Agrobacteria were also investigated. In 5 mg·L-1 Kanamycin and 375 mg·L-1 Carbenicillin, transformed shoots were produced at 4.8% efficiency. No chimera was observed in the transgenic shoots during a 2-year subculture period. Since the inoculated explants developed into multiple shoots during selection, it was thought that the problem of chimera might have been overcome. Therefore, this transformation method using axillary shoot meristem may be applicable to pear cultivars recalcitrant to regeneration from leaf disc. To the best of our knowledge, this is the first report of a transformation system in pear cultivars with low regeneration efficiency.