應用基因槍法於蝴蝶蘭基因轉殖之研究

Studies on Genetic Transformation of Phalaenopsis by Microprojectile Bombardment

本研究主要目的是以基因槍法進行蝴蝶蘭之基因轉殖，嘗試建立具有較高效率以及穩定性高之轉殖方式，以應用在蝴蝶蘭的育種。在kanamycin抗性試驗中，得知蝴蝶蘭種子播於含kanamycin 10 mg/l的1/4MS培養基即無法發芽：若播於G7培養基中，則需提高kanamycin濃度至100 mg/l，方能抑制種子發芽。至於已見葉形的植株，培養在T2培養基中，於kanamycin 50和100 mg/l濃度下，其生長均有明顯的抑制現象。在基因槍法方面，以PDS1 OOO/He型基因槍650psi壓力，處理蝴蝶蘭之幼胚，於三天後進行GUS活性分析，可得到約34%的暫時表現轉殖比率。經處理後30天，以南方氏核酸雜交分析，顯示外來質體DNA可以整合進入蝴蝶蘭細胞的染色體內。

The purpose of this study was to establish an efficient and stable gene transfer system for Phalaenopsis. In kanamycin tolerance test, results indicated that Phalaenopsis seeds sown on 1/4 MS medium with 10 mg/l kanamycin or sown on G7 medium with 100 mg/l kanamycin did not germinate. The growth inhibition for three- month-old seedlings planted on T2 medium with 50 or 100mg/l kanamycin was obserbed. A transformation protocol using germinated seedlings as target tissue was. developed by using PDS 1000/He particle gun with a gas pressure of 650 psi. The ratio in a population of seedlings expressing GUS activity when assayed 3 days after bombardment was as high as 34%. Southern hybridization analysis of genomic DNA isolated from seedlings 30 days after bombardment showed positive signals. This study demonstrated that transgenic Phalaenopsis plants could be produced using particle bombardment.