Sutdy on Agrobacterium-mediated gene transformation of Phalaenopsis orchid

• Main Authors: Chen Hui-Chen, 陳慧真
• Other Authors: Liao Li Jen
• Format: Others
• Language: zh-TW
• Published: 2003
• Online Access: http://ndltd.ncl.edu.tw/handle/20209015522492803135

碩士 === 國立高雄師範大學 === 生物科學研究所 === 91 === Abstract Phalaenopsis is a monopodial epiphytic orchid with flowers gracefully borne on arching inflorescene. The flowers are long-lasting both on the plants and as cut flowers, and posses a high potential for flower markets in Taiwan. Researchers have been developed new species of Phalaenopsis with traditional hybridization method. However, it had some difficulties in breeding new orchid species. In this study, Agrobacterium — mediated pflp gene and hsf gene transformation of 10 -14 days old PLBs of Phalaenopsis orchid is expectative. As the results, the sterile explants were TS 97k for pflp gene and S86-39, TS448 and TS97k for hsf gene after the selection with GFP microscope. In this research, antibiotics are used for Agrobacterium inhibition. Nevertheless, the species and the concentration of antibiotics affected the PLBs’ growth and development. The results showed the M medium supplied with 200 ppm timentin doesn’t obviously affect the growth and development of PLBs. In the other hand, the M medium with 500 ppm timentin and 500 ppm cefotaxime, respectively, cause the growth and development inhibition of PLBs and their regeneration. During the transformation processing, the over growth of Agrobacterium will make some damages and reduce the survival PLBs numbers from the co-cultivation and regeneration. Therefore, it is important to select the suitable concentration of antibiotics in M medium and washing process. We find the best condition to remove Agrobacterium from the surface of PLBs is washing them with 500 ppm timentin solution and 500 ppm cefotaxime solution, and plating PLBs on sucrose-free M medium supplied with 250 ppm cefotaxime, the Agrobacterium on the PLBs can be more effective inhibited within the six days. It suggests that the concentration of antibiotics is 200 ppm timentin or 250ppm cefotaxime. The best processing to eliminating the Agrobacterium on the PLBs surface is washing the PLBs with 70 % alcohol for one minute and then treated with 1 % NaOCl for twenty minutes. However, suspension cell of Agrobacterium on PLBs’ infection step had better to incubate from the -70℃conserved colony. It can reduce the contamination and mutation of Agrobacterium. Among the three species of Phalaenopsis- S86-39, TS97k and TS448, the regeneration and development of PLBs of TS448 was less than the others. On the other hand, the best condition was S86-39. It suggests that Agrobacterium-mediated gene transformation has better to use the PLBs of S86-39 for subject.