Genetic characterization and biosafety assessment of transgenic Oncidesa Gower Ramsey “Honey Snow”

台灣貴為蘭花王國，外銷切花又以文心蘭為大宗，由於外銷切花品系過於單一，要長期符合市場需求著實困難，有鑑於此，本實驗室將文心蘭phytoene synthase (OgPSY)-RNAi構築，透過農桿菌轉殖方式，孕育出了轉殖白花文心蘭。在此試驗中我們獲取了三個獨立轉殖株，分別為MF-1、MF-14以及MF-17。依照台灣現行針對基因改造作物 (GMOs) 的相關法規，必須通過基因轉殖植物「遺傳性狀調查」以及「環境生物安全評估」兩項檢測，方能申請該基因改造作物之品種權。透過本論文之研究顯示，由於選用花瓣專一表達啟動子 (Pchrc) 驅動OgPSY-RNAi構築，僅專一地降解文心蘭花辦中OgPSY之表現；HPLC結果亦顯示轉殖文心蘭花辦中類胡蘿蔔色素明顯下降；另外分析轉殖文心蘭母本兩次開花以及組培繁殖子代之花瓣，結果顯示OgPSY-RNAi構築能穩定地表現其功能；由於MF-1在組培的子代族群 (P1) 中出現了許多變異株，故進一步利用此獨立轉殖系 (MF-1) 探討光線與溫度對Pchrc活性的影響，發現兩者環境因子都可能影響OgPSY之表現，造成花色的差異。而經由Southern blot以及inverse PCR分析，瞭解基因轉殖文心蘭皆僅具有單一拷貝數，且其T-DNA兩翼之序列皆屬於非編碼DNA。透過環境生物安全相關試驗，相較於非轉殖株，轉殖文心蘭並無顯著生物相剋作用的現象發生，且並不會影響其根圈微生物相之生長。最後，本論文之研究亦可作為日後台灣基因轉殖花卉評估的先驅及準則，建立台灣基因轉殖花卉管理之完善制度。Taiwan has a reputation in the agricultural modification for Oncidesa, of the orchid Kingdom, which is mainly for export as cut flowers. One of the major crisis faced by marketing Oncidesa is its dull appearance of florets and it has remained unchanged for a very long time in any aspect of colors. Currently, the Oncidesa cut flower market is facing a new demand for a new spectrum of colors. With the aid of our previous study, we have devloped a white floret Oncidesa through RNA interference technology. However, one of the major concern deemed by the government of Taiwan is that it required a genetic characterization and environmental biosafety assessment on surrounding biosphere for any genetically modificated organisms (GMOs). Therefore in this study, our plant materials were all grown in a special netted house. Additionally, we were able to characterize this transgenic white Oncidesa and define its transgenic RNAi allele (OgPSY) through genetic characterization. Furthermore, we showed that its phenotype could be sustained within two repetitive vegetative regenerations and that the transformants showed no sign of detrimental and environmental impact to the biosphere as compared with its non-transformant counterparts. Moreover, we investigated the effects of light and temperature on the transgenic Pchrc activity and found that these two environmental factors affected the potency of OgPSY RNAi, resulting in floret color variation on independent generation line of transgenic Oncidesa. Thus, this study holds valuable information in the aspect of the viability of sustaining a stable transgenic line and successfully introducing transgenic Oncidesa in the biosphere