TRANSFORMATION SYSTEM INFRUIT TREES USING T-DNA REGION OF AGROBACTERIUM FOR REGENERATION EFFICIENCY

利用农杆菌 T-DNA 区域提高果树再生效率的转化系统

• 批准号: 07806002
• 负责人: NAKANISHI Tetsu
• 金额: $ 1.34万
• 依托单位: KOBE UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1995
• 资助国家: 日本
• 起止时间: 1995 至 1996
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07806002/
• 关键词: JAPANESE PEAR; TRANSFORMATION; TISSUE CULTURE; AGROBACTERIUM TUMEFACIENS; アグロバクテリウム; T-DNA; トランスジェニック; 再分化

To develop the method of genetic transformation in fruit trees, Agrobacterim binary system was studied using the Japanese pear. Among the several cultivers and the wild species of the Japanese pear, one of the strain of the wild species was found to have a rapid multiplification suitable for subjecting the shoot and leave for transformation materials. Gerlite as a supot medium for tissue culture was effective when it was used with microfilter in adjusting the relative humidy of the culture bottle. Tidiazuron was also effective in place of benzyladenine and also required the microfilter attached to the cap of the culture bottle. Seed coleoptiles were sujected for highly shoot regreneration materials. Transformation was carrid with Agrobacterim EHA101 strain with pIG121 as a binary transformation system. Gus expression was observed to confirm the transformation. In eary stage of the transfomation, pre-culture of the shoots for four days after discetion of the stem end was effective. Co-cultivation days were infuence for the infection of cotyledon. Leaf disk was infected with bacteria at the disceted part and the gus spots were observed on the disk surface where the stomata cells were aboundant. Concentration of the bacteria

为探索果树遗传转化的方法，以日本梨为试材，对农杆菌双元体系进行了研究。在日本梨的几个栽培种和野生种中，发现野生种的一个菌株具有适合于将芽和叶用于转化材料的快速增殖。用锗石作为组织培养的基质，配合微滤器调节培养瓶内的相对湿度，效果较好。替氮隆也有效地代替苄基腺嘌呤，并且还需要将微过滤器连接到培养瓶的盖上。种子胚芽鞘可作为高再生材料。以农杆菌EHA 101菌株和pIG 121作为二元转化系统进行转化。观察GUS表达以确认转化。试管苗增殖初期，切取茎尖后预培养4天效果较好。共培养天数对子叶侵染率有影响。叶盘在盘部被细菌侵染，在气孔细胞丰富的叶盘表面观察到gus斑点。菌体浓度