A study of the plant regeneration and gene transformation of leaf-inducedcallus in Oncidium Gower Ramsey (Orchidaceae)

• Main Authors: Po-Hung Lin, 林柏宏
• Other Authors: Yu-Chung Chiang
• Format: Others
• Language: zh-TW
• Published: 2010
• Online Access: http://ndltd.ncl.edu.tw/handle/75601845759455348942

碩士 === 國立屏東科技大學 === 生物科技研究所 === 98 === This study was to investigate the suitable culture condition for Oncidium callus regeneration and the optimal condition of gene transformation by particle bombardment. The callus of Oncidium Gower Ramsey were cultured on the medium supplemented with the combinations of different concentrations of BA (6-benzylaminopurine) and TDZ(thidiazuron) as well as BA and BR (brassinolide). The results showed that the combination of 1 mg/L BA and 1 mg/L BR was the optimal condition for callus regeneration. The percentage of plant regeneration and the number of plant per callus were 73.3% and 24.3. The resistance of Oncidium Gower Ramsey callus to hygromycin showed that to add 5-10 mg/L hygromycin in the media was suitable for regeneration and selection for transformed callus. pCAMBIA 1305.1 with the GUS reporter gene was used to investigate the optimal transient expression condition and pCAMBIA 1302 with the GFP reporter gene was used to select, observe and analyze. Histochemical GUS analysis was conducted to reveal that the highest transient expression was obtained at 1100 psi pressure and 6 cm flight distance. When callus pretreated with 0.6 M sorbitol for 3h, it could enhance the transient expression of callus. Amplification of GFP and hptII genes by polymerase chain reaction showed five putative transgenic plants were positive for both GFP and hptII genes.