Genetic transformation of Phalaenopsis using embryogenic calli

1999 Fiscal Year Final Research Report Summary

• Project/Area Number: 10660028
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: 園芸・造園学
• Research Institution: KAGAWA UNIVERSITY
• Principal Investigator: TANAKA Michio Faculty of Agriculture, Kagawa University, Professor, 農学部, 教授 (10115975)
• Project Period (FY): 1998 – 1999
• Keywords: Phalaenopsis / embryogenic / genetic transformation / particle-bombardment

Research Abstract

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 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.