The development of a pollen-mediated gene transformation system for Spathoglottis plicata (Orchidaceae)

• Main Authors: Po-Hung Pan, 潘柏宏
• Other Authors: Yu-Chung Chiang
• Format: Others
• Language: zh-TW
• Published: 2008
• Online Access: http://ndltd.ncl.edu.tw/handle/59616600861509919992

碩士 === 國立屏東科技大學 === 生物科技研究所 === 96 === Abstract Traditionally, developing a plant regeneration system is a prerequisite for most of plant gene transformation. In present study, we show that the gene transformation of Spathoglottis plicata (Orchidaceae) can be achieved by pollen-mediated transformation which does not need to develop a regeneration system. Agrobacterium tumefaciens strain EHA 105 harboring a plasmid pCAMBIA 1302 which contains a reporter gene green fluorescent protein (GFP) and a hygromycin resistance gene controlling by CaMV 35S promoter and NOS terminator. Spathoglottis plicata pollinia were cultivated in pollen germination medium to germinate for 16 hours and germinated pollens were cocultivated with EHA 105. The infected pollens were pollinated on the stigma of Spathoglottis plicata. After about two weeks later, those developing capsules were harvested. Developing embryos were observed under fluorescent microscope to detect the expression of GFP protein. About one month after pollination, matured capsules were cultured on seed germination medium. The plantlets were transplanted on selection medium containing hygromycin to obtain transgenic plantlets. Those transgenic plants were confirmed by PCR and RT-PCR for GFP gene. These results show that gene transfer by pollen-mediated is an easy and efficient method for Spathoglottis plicata. Keyword: pollen-mediated transformation, Spathoglottis plicata, Agrobacterium, green fluorescent protein.