Development of genetic transformation of Japanese pear by inhibition of calcium ion influx

用5 μ m NAA + 10 μ m或25 μ m BA的培养基对16个品种的梨子叶不定芽的再生率均较高。采用含有绿色荧光蛋白基因的根癌农杆菌（Agrobacterium tumefaciens）共培养日本梨品种子叶。采用EGTA（一种钙螯合剂）处理和超声处理，分别可以防止植物防御反应和产生跨组织物理损伤。EGTA处理对GFP荧光的表达没有积极影响，而超声处理则显著增加。从Agenosho Shinanashi再生的一株植物显示出稳定的GFP荧光，证实是一种转化。另外三个myb基因转化的再生芽呈现红色，这是另一个实验中获得的Imamuraaki。在我们的研究中，转化系统被证明是可重复的。

High rates of regeneration of adventitious shoots were obtained from the cotyledons of Japanese pears Imamuraaki and Agenosho Shinanashi from 16 Japanese pear cultivars, when the media contained 5 uM NAA combined with 10 or 25 uM BA were used. Cotyledons of Japanese pear cultivars were co-cultivated with Agrobacterium tumefaciens which contained a green fluorescent protein (GFP) gene. EGTA (a chelator of calcium) treatments and sonication were applied, which could prevent plant defense reaction and produce physical wounds across the tissue respectively. EGTA treatment did not show a positive effect on expression of GFP fluorescence whereas sonication significantly increased. One plant regenerated from Agenosho Shinanashi showed stable GFP fluorescence and confirmed as a transformant. Three other transformed regenerated shoots by myb gene showed red color, which were derived from Imamuraaki in another experiment. Transformation system in our study was shown to be reproducible.