Development of the Genetic Transformation System in Phalaenopsis by Pollen Incubated with Agrobacterium

• Main Authors: Hui-min Ho, 何慧敏
• Other Authors: Hsin-Sheng Tsay
• Format: Others
• Language: zh-TW
• Published: 2006
• Online Access: http://ndltd.ncl.edu.tw/handle/26y4bx

碩士 === 朝陽科技大學 === 生物技術研究所 === 94 === Phalaenopsis is one of the most important orchids grown for commercial production of cut flowers and potted plants. Genetic improvement of Phalaenopsis through sexual hybridization is however, restricted by long growth and reproductive cycles. Genetic transformation has been used to create mutants with attractive or new flower patterns. In the present study, a novel plant transformation method has been developed by co-cultivation of pollen with Agrobacterium tumefaciens strains EHA105 (pCAMBIA1301 or pCAMBIA1302) and EHA105 with an activation vector (pTAG-8). The construct pCAMBIA1302 had GFP and pigment biosynthetic gene DFR (dihydroflavonol 4-reductase) or CHI (chalcone isomerase), while pCAMBIA1301 had GUS and F3′H (flavanone 3′-hydroxylase) genes. Using the hygromycin-selection system, it was found that the optimum conditions for affecting transformation in Phalaenopsis were: A. tumefaciens (pTAG-8) concentration of 5×10P P cfu/ml, a supplement of 0.1mM acetosyringone (AS) in the medium and a co-culture period with pollen for 12 h. Under these conditions, 6 transformants were obtained that from one capsule under the selection pressure of 5 mg/l hygromycin. In another Phalaenopsis variety, Doritaenopsis, 68 transformants could be obtained that from one capsule under 5 mg/l hygromycin selection medium using A. tumefaciens ( pTAG-8 ) concentration of 1×10PP cfu /ml and a co-culture with pollen for 12 h without AS. Presence of GUS gene in transformants of Phalaenopsis and Doritaenopsis was confirmed by PCR and Southern blotting analysis. The transformed Phalaenopsis showed change in leaf pigments from green to pink or red. In Doritaenopsis, 4.89% of the transformants were found to have the GFP activity. The presence of DFR, CHI and F3′H genes were also confirmed by Southern blot analysis. Thus, in this thesis, for the first time, a new transformation technology for orchid Phalaenopsis has been reported.