Genetic transformation of Phalaenopsis using embryogenic calli

我们已经尝试从EC从粒子轰炸中再生的PLBs植物中获得转基因植物。委员会的Phalaenopsis Richard Shaffer 'Santa Cruz'从PLB段受到启发。欧盟委员会曾被用于粒子轰炸。Calli （fw. 500-600毫克）在塑料盘中放置30毫米的圆圈，直径在过滤器纸上。首先，在欧共体中，粒子枪对B-葡萄糖原化酶（GUS）基因瞬态表达的加速压力的影响是测试的。largest number of transient GUS expression was obtained at 140 kg/cm，D12 by plasmid DNA pB1221 and at 170 kg/cm，D12 by plasmid DNA pWI-GUS。使用pWI-GUS的瞬态GUS表达式的数目比使用pBI 221的数目大，在同一个加速压力下。The number of transient GUS expression using each plasmid were clearly increased at an accelerating pressure of 140 kg/cm的D12。然后，我们试着买转基因植物。等离子体DNA pWI-GUS含有GUS基因（gas）在欧共体中被捆绑 ... More plasmid DNA pMSP 38 containing herbicide Bialaphos-resistant gene （bar） as a selection marker at 140 kg/cm的D12。Approx。15，000 PLBs是从疫苗和进入介质上接种的EC形成的。PLBs在选择性介质中被转移，每两周含有0.5或1.0毫克/1双甲醚。Furthermore所有PLBs都已选定两次包含媒体的5.0 mg/1 bialaphos。在选择后，六个PLB和一个PLB集群保持绿色或黄色-绿色和被认为活着。Although Living PLBs Were Transferred onto media without bialaphos， five of those were dead without subsequent growth。最后，两个PLBs幸存下来，这些被分成了两块。2013年，PLBs的上部分已经形成了植物和较低的部分最近形成了8个或14个PLBs。基因组DNA从这些存活的植物中分离出来，并被用于PCR。不管是不是DNA片段，因为它是预期的气体还是酒吧都被检测到。没有产品在另一种PCR中被放大，因为它改变了热条件。瞬态气体分析在这些植物中也是阴性的。Less（低）

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