Genetic transformation of Phalaenopsis using embryogenic calli

利用胚性愈伤组织进行蝴蝶兰的遗传转化

• 批准号: 10660028
• 负责人: TANAKA Michio
• 金额: $ 1.54万
• 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: 1998
• 资助国家: 日本
• 起止时间: 1998 至 1999
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10660028/
• 关键词: Phalaenopsis; embryogenic; genetic transformation; particle-bombardment

We tried to obtain transgenic plantlets from PLBs regenerated from EC followed by the particle bombardment. EC of Phalaenopsis Richard Shaffer 'Santa Cruz' were induced from PLB segments. The EC were used for particle bombardment. Calli (fw. 500-600 mg) were placed in a circle of 30 mm in diameter on a filter paper in a plastic dish. First, the effect of accelerating pressure of particle gun on the transient expression of B-glucronidase (GUS) gene in EC was examined. The largest number of transient GUS expression was obtained at 140kg/cmィイD12ィエD1 by plasmid DNA pB1221 and at 170 kg/cmィイD12ィエD1 by plasmid DNA pWI-GUS. The number of transient GUS expression using pWI-GUS was larger than those using pBI221, at a same accelerating pressure. The number of transient GUS expression using each plasmid were clearly increased at an accelerating pressure of 140kg/cmィイD12ィエD1. Then, we tried to obtain the transgenic plantlets. Plasmid DNA pWI-GUS containing GUS gene (gus) were bombarded in EC with … More plasmid DNA pMSP38 containing herbicide Bialaphos-resistant gene (bar) as a selection marker at 140kg/cmィイD12ィエD1. Approx. 15,000 PLBs Were formed from bombarded EC on Vacin and Went medium. PLBs were transferred onto selection media containing 0.5 or 1.0 mg/1 bialaphos every two weeks. Furthermore all the PLBs Were selected twice on media containing 5.0mg/1 bialaphos. After selection, six PLBs and a PLB-cluster were kept green or yellowish-green and were considered to be alive. Although living PLBs Were transferred onto media without bialaphos, five of those were dead without subsequent growth. Finally two PLBs Were survived and these were divided into two pieces. The upper segments of the PLBs Were formed a plantlet and the lower ones were newly formed eight or fourteen PLBs. Genome DNA were isolated from these surviving plantlets and were used for the PCR. However neither DNA segment which were expected for gus or bar were detected. No products were amplified in an another PCR which was changed the thermal condition. The transient GUS assay were also negative in these plantlets. Less

我们尝试从EC再生的原球茎中获得转基因植株。以蝴蝶兰'圣克鲁斯'原球茎切段为外植体诱导出EC。EC用于粒子轰击。愈伤组织（fw. 500 - 600 mg）置于塑料皿中滤纸上直径为30 mm的圆中。首先，研究了粒子枪加速压力对B-葡萄糖醛酸酶（GUS）基因在EC中瞬时表达的影响。质粒DNA pB1221和质粒DNA pWI-GUS分别在140kg/cm接种量D12接种量D1和170kg/cm接种量D12接种量D1下获得最大瞬时GUS表达量。在相同的加速压力下，pWI-GUS瞬时表达的GUS数目比pBI221多。在140kg/cm × D12 × D1的加速压力下，各质粒的瞬时GUS表达量明显增加。然后，我们尝试获得转基因植株。将含GUS基因的质粒pWI-GUS转化EC，用 ...更多信息 质粒DNA pMSP 38含有除草剂双丙磷抗性基因（bar）作为选择标记，在140kg/cm的浓度下，D12和D13，约轰击EC在Vacin和Went培养基上形成15，000个PLBs。每两周将PLB转移到含有0.5或1.0 mg/l双丙氨磷的选择培养基上。在含5.0mg/l双丙氨磷的培养基上，所有的原球茎都经过两次筛选。选择后，6个PLB和1个PLB簇保持绿色或黄-绿色，并被认为是活的。虽然活的PLBs被转移到没有双丙氨膦的培养基上，但其中5个没有随后的生长而死亡。最后，两辆公共小巴幸存下来，这些被分成两部分。原球茎的上部形成一个小植株，下部形成8~14个新原球茎。从这些存活的小植株中分离基因组DNA并用于PCR。然而，没有检测到gus或bar预期的DNA片段。在改变热条件的另一PCR中没有扩增产物。瞬时GUS检测也为阴性。少