Studies on the development of transformation in Phalaenopsis.

蝴蝶兰转化进展研究。

• 批准号: 07660038
• 负责人: TANAKA Michio
• 金额: $ 1.02万
• 依托单位: Kagawa 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-07660038/
• 关键词: Phalaenopsis; transformation; bar; particle gun; protocorm-like body; callus formation; PLB; エンドグルカナーゼ

Transgenic Phalaenopsis plants have been obtained by particle bombardment with a pneumatic particle gun device. All promoters of cauliflower mosaic virus (CaMV) 35S,maize ubiquitin and rice actin genes could be expressed in Phalaenopsis using transient assay of beta-glucuronidase (gus) gene as a reporter. Bialaphos resistance gene (bar) which confers tolerance to herbicide bialaphos, was used as a selectable marker. Protocorm-like bodies (PLBs) of Phalaenopsis derived from the leaf segment culture were bombarded by gold particles coated with pMSP38 and pWI-GUS DNA containing the bar gene and the gus gene respectively driven by 35S promoter. Newly-formed PLBs were selected on the medium including bialaphos. Finally seven transgenic plants resistant to bialaphos were obtained, and one of them contained and expressed gus gene. PCR analysis confirmed their transgenic nature and western analysis and histochemical GUS assay showed the expression of bar and gus gene respectively.New PLBs (T_1 … More ) were formed from the segments obtained by the division of the transgenic PLB (T_0). The bisected segments of T_1 PLBs formed T_2 PLBs on the segments. From the detection of amplified DNA by PCR,all plantlets of the T_1 and T_2 generation contained the bar gene. The western analysis showed that the bar gene expressed in all of the clonal progenies as well T_0 generation.To use the callus of Phalaenopsis for transformation, the callus formation and plant regeneration in Phalaenopsis Richard Shaffer 'Santa Cruz' were examined. PLB segments formed calli in Vacin and Went (1949) medium with sucrose. The optimal concentration of sucrose was 40 g・1^<-1>. Media containing 200 ml・1^<-1> coconut water were effective for the callus formation. The media solidified with gellan gum was suitable for the callus induction as compared with those with agar. The calli formed PLBs easily after transferring to media without sucrose. No variation was observed in the flowering plants regenerated through somatic embryogenesis.The calli bombarded the gus gene showed the expression of the gene through the histochemical assay, suggesting the availability of calli for the transformation of Phalaenopsis. Less

用气动粒子枪装置进行粒子轰击获得了转基因蝴蝶兰植物。以β-葡萄糖醛酸酶（gus）基因为报告基因，通过瞬时表达检测，花椰菜花叶病毒（CaMV）35 S、玉米泛素和水稻肌动蛋白基因的启动子在蝴蝶兰中均能表达。双丙氨膦抗性基因（bar）是一个对除草剂双丙氨膦具有抗性的基因，它被用作选择标记。以蝴蝶兰叶段培养的类原球茎（Protocorm-like bodies，PLBs）为材料，用35 S启动子驱动的含bar基因和gus基因的pMSP 38和pWI-GUS DNA包被金粒子轰击。在含有双丙氨膦的培养基上选择新形成的原球茎。最终获得了7株双丙氨膦抗性转基因植株，其中1株含有并表达gus基因。PCR分析证实了它们的转基因性质，Western分析和GUS组织化学分析分别证实了bar和gus基因的表达 ...更多信息 （T_0）的原球茎进行分裂，获得了转化子原球茎（T_0）。T_1 PLB的二等分段在段上形成T_2 PLB。经PCR检测，T_1和T_2代植株均含有bar基因。为了利用蝴蝶兰愈伤组织进行遗传转化，以蝴蝶兰“圣克鲁斯”为材料，研究了其愈伤组织的形成和植株再生。PLB片段在含有蔗糖的Vacin和Went（1949）培养基中形成愈伤组织。蔗糖的最佳浓度为40 g·l ^<-1>。含200 ml·l ~（-1）<-1>椰子水的培养基对愈伤组织的形成有较好的效果。结冷胶固化培养基较琼脂固化培养基更适合愈伤组织的诱导。愈伤组织转移到无蔗糖培养基上后，易形成原球茎。经体细胞胚胎发生再生的开花植株中未观察到任何变异，组织化学检测表明基因枪轰击的愈伤组织中有gus基因的表达，表明获得的愈伤组织可用于蝴蝶兰的遗传转化。少

项目成果

Anzai, H., Y.Ishii, K.Katsumata, M.Shichinohe, C.Nojiri, H.Morikawa, M.Tanaka: "Transformation of phalaenopsis by particle bombardment" Plant Tissuc Culture Letters. 13(3). 256-272 (1996)

Anzai，H.，Y.Ishii，K.Katsumata，M.Shichinohe，C.Nojiri，H.Morikawa，M.Tanaka：“粒子轰击蝴蝶兰的转化”植物组织文化快报。

H.Anzai,Y.Ishii,M.Shichinohe,K.Katsumata,C.Nojiri,H.Morikawa,M.Tanaka: "Transformation of phalaenopsis by particle bombardment" plant Tissue Culture Letters. 13(3). 265-272 (1996)

H.Anzai，Y.Ishii，M.Shichinohe，K.Katsumata，C.Nojiri，H.Morikawa，M.Tanaka：“粒子轰击蝴蝶兰的转化”植物组织培养快报。