蝴蝶蘭之基因轉殖及芭菲爾拖鞋蘭癒合組織之誘導與植株再生

Abstract

蝴蝶蘭為台灣重要外銷花卉，為提升蝴蝶蘭植株抗軟腐病能力，本研究將蝴蝶蘭軟腐病原之果膠分解酶（pectate lyase）基因PelE-1，以農桿菌為媒介轉殖入蝴蝶蘭癒合組織。轉殖後經抗生素G418篩選存活之癒合組織，持續篩選及純化後，以聚合酶連鎖反應及南方氏雜交分析，確定12個轉殖細胞系含有PelE-1 基因；進一步培養長大之擬轉殖株，經南方氏雜交分析，確定其中8株含有PelE-1 基因。另一方面，為了以生物技術達到延緩花朵老化的目的，將苦瓜乙烯訊息傳導負向調控基因CTR1，以農桿菌媒介法轉殖至蝴蝶蘭癒合組織，經由GUS活性組織化學染色分析，目前已確定11個CTR1擬轉殖細胞系。 在芭菲爾拖鞋蘭（Paphiopedilum rothschildianum）癒合組織之誘導及再生系統建立方面，以含有1 mg/l 2,4-D及1 mg/l TDZ之1/2 MS培養基，為芭菲薾拖鞋蘭根尖誘導癒合組織產生之最適培養基，亦可作為芽體誘導癒合組織之培養基。誘導自根尖之癒合組織以蔗糖、麥芽糖及海藻糖不同醣類進行再生試驗，結果顯示麥芽糖及海藻糖均可促進類原球體生成；蔗糖濃度從3 ％降低到1.5 ％，則有助於黑暗培養下，自芽體誘導之癒合組織分化成類原球體（protocorm-like body）。

To enhance resistance to a soft-rot disease, pectate lyase gene PelE-1 isolated from Erwinia chrysanthemi was transferred into calli of Phalaenopsis using Agrobacterium-mediated transformation. Twelve putative transgenic cell lines and eight putative transgenic plantlets were confirmed for existence of PelE-1 gene via polymerase chain reaction (PCR) and Southern analysis. On the other hand, in order to prolong longevity of Phalaenopsis flowers, ethylene signal transduction involved gene CTR1 from Mormordica charantia L. was transferred into calli of Phalaenopsis using Agrobacterium-mediated transformation. Eleven CTR1 transgenic lines showed positive GUS activity after histochemical staining analysis. Totipotent calli of Paphiopedilum rothschildianum, induced from root tips on a 1/2 strength Murashige-Skoog medium plus 1 mg/l 2,4-D and 1 mg/l TDZ, were used for regeneration test. Both maltose and trehalose enhanced the formation of PLB from calli. A decrease in the concentration of sucrose from 3 % to 1.5 % promoted PLB formation from sprouts-derived calli in the dark.