蝴蝶蘭轉殖株雜交後代分析之研究

Abstract

蝴蝶蘭是臺灣重要的花卉，然而在栽培過程中，經常受到病毒害感染，造成植株生長不良、喪失商品價值，常見的病毒病害以蕙蘭嵌紋病毒(Cymbidium mosaic virus; CymMV)和齒舌蘭輪斑病毒 (Odontoglossum ringspot virus; ORSV)感染最為普遍。本論文所分析的雜交後代，母本為含CymmV外鞘蛋白基因默化構築之蝴蝶蘭轉殖株，或含CymMV外鞘蛋白基因默化構築之癒傷組織再轉殖ORSV外鞘蛋白基因默化構築所得之轉殖株，父本為未轉殖的商業栽培品種，期望透過短干擾RNA (small interfering RNA, siRNA)之產生，降解病毒外鞘蛋白基因mRNA，達到抗病效果。 GUS (β—glucuronidase)活性組織化學染色結果顯示，兩個轉殖系之雜交後代GUS呈現正反應者占50%，符合遺傳分離率。經由聚合酶連鎖反應 (polymerase chain reaction; PCR)和南方氏雜交分析，確認CymMV和ORSV外鞘蛋白基因雙重默化轉殖株之雜交後代，約35%植株同時具有CymMV和ORSV外鞘蛋白默化轉殖基因。部分雜交後代經病毒接種試驗顯示具有抗病性。

The Phalaenopsis orchids are important flowers in Taiwan. However, Phalaenopsis are usually infected by virus during cultivation and become growth retardation and loss of commercial vaule. The most common virus diseases are Cymbidium mosaic virus (CymMV) and Odontoglossum ringspot virus (ORSV). To utilize biotechnology on virus resistance for Phalaenopsis, RNA interference (RNAi) construct against coat proteim gene of CymMV was transformed into Phalaenopsis callus, and continued transformed with ORSV coat protein RNAi construct in previous studies. Phalaenopsis transformants used as maternal parent were cross-hybridized with untransformed commercial variety and progeny obtained were analyzed in this study. This construction can produce small interfering RNA (siRNA), make virus mRNA degradation then against virus infection. The β—glucuronidase (GUS) staing results showed that, about 50% of hybrid progeny expressed GUS activity, which was followed Mendelian segregation ratio. Transgene inheritance in GUS positive hybrid progeny was confirmed by both polymerase chain reaction and Southern analysis. After virus inoculation on several individuals of hybrid progeny by RNAi construct and accumulation of virus coat protein was inhibited plantets showed resistance to virus.