Nakamura Y, Kobayashi S, Nakajima I
Persimmon and Grape Research Center, National Institute of Fruit Tree Science, Akitsu, Hiroshima 729-24, Japan, , , , , , JP.
Plant Cell Rep. 1998 Apr;17(6-7):435-440. doi: 10.1007/s002990050421.
Hypocotyl segments from the seeds of Japanese persimmon (Diospyros kaki Thunb) were cultured on a modified Murashige and Skoog medium supplemented with N-(2-chloro-4-pyridyl)-N'-phenylurea, zeatin or 6-benzylaminopurine. The highest frequency of shoot regeneration was observed when the segments were cultured on medium containing 2 mg/l of zeatin. This culture system was adapted to Agrobacterium-mediated transformation. The hypocotyl segments were inoculated with Agrobacterium tumefaciens strains harboring binary vectors, which contained the neomycin phosphotransferase II gene and the β-glucuronidase gene. Regenerated shoots were selected on a medium containing kanamycin. Histochemical GUS assay showed that the shoots regenerated from the segments inoculated with EHA101/pSMAK251 expressed the gus gene. The presence and integration of the gus gene was confirmed by polymerase chain reaction (PCR) and Southern blot analysis. The regeneration frequency of transformed shoot was 11.1%. The transgenic shoots were rooted and developed into whole plants within 4-5 months.
将日本柿子（柿属柿树）种子的下胚轴切段接种在添加了N-（2-氯-4-吡啶基）-N'-苯基脲、玉米素或6-苄基腺嘌呤的改良Murashige和Skoog培养基上。当切段接种在含有2毫克/升玉米素的培养基上时，观察到最高的芽再生频率。该培养系统适用于农杆菌介导的转化。用携带二元载体的根癌农杆菌菌株接种下胚轴切段，这些二元载体包含新霉素磷酸转移酶II基因和β-葡萄糖醛酸酶基因。在含有卡那霉素的培养基上筛选再生芽。组织化学GUS分析表明，从接种了EHA101/pSMAK251的切段再生的芽表达了gus基因。通过聚合酶链反应（PCR）和Southern印迹分析证实了gus基因的存在和整合。转化芽的再生频率为11.1%。转基因芽生根并在4至5个月内发育成完整植株。
