蝴蝶蘭為商業市場上最重要的蘭花，可供生產盆花及切花。目前蘭花品種的改良仍依賴傳統的雜交育種，但因雜交育種的有性世代過長而限制了改良的空間，近來發展的基因轉殖方式，可有效改善此缺點。本研究利用花粉與農桿菌共培養的方式，發展出一種創新的基因轉殖技術，試驗中使用農桿菌EHA105 品系，構築質體pCAMBIA1302、pCAMBIA1301 與pTAG-8。
利用pCAMBIA1302 質體內帶GFP 基因，分別構築花色基因DFR 、CHI；pCAMBIA1301 質體內帶GUS 基因，及花色基因F3′H 基因；以pTAG-8 質體內帶有GUS 報導基因與4 套的加強子(enchancers)能使基因大量表現。
轉殖後的蝴蝶蘭植株利用hygromycin 篩選系統，發現使用含有pTAG-8質體構築的農桿菌與花粉共培養，以 5×10PP cfu /ml農桿菌濃度搭配 0.1 mM AS共培養12hr，每個果莢平均可獲得6棵疑轉植株，為蝴蝶蘭最適轉殖條件。另外以 1×10P
P cfu /ml農桿菌搭配 0.1 mM AS濃度共培養12hr，每個果莢平均可獲得68棵疑轉植株，為朵麗蝶蘭最適轉殖條件。最適條件下所獲得之轉植株，經PCR檢測確實帶有GUS報導基因，在蝴蝶蘭外觀型態上觀察到轉入F3′H基因的植株葉片，其顏色產生由綠色轉變為粉紅色或紅色的現象。另外於朵麗蝶蘭的種子中則觀察到，有4.89％的GFP螢光蛋白表現，以PCR-Southern檢測確實帶有DFR, CHI, F3′H等花色基因。本研究為第一個成功的使用農桿菌配合花粉進行蝴蝶蘭基因轉殖。

Phalaenopsis is one of the most important orchids grown for commercial production of cut flowers and potted plants. Genetic improvement of
Phalaenopsis through sexual hybridization is however, restricted by long growth
and reproductive cycles. Genetic transformation has been used to create mutants
with attractive or new flower patterns. In the present study, a novel plant
transformation method has been developed by co-cultivation of pollen with
Agrobacterium tumefaciens strains EHA105 (pCAMBIA1301 or
pCAMBIA1302) and EHA105 with an activation vector (pTAG-8). The
construct pCAMBIA1302 had GFP and pigment biosynthetic gene DFR
(dihydroflavonol 4-reductase) or CHI (chalcone isomerase), while
pCAMBIA1301 had GUS and F3′H (flavanone 3′-hydroxylase) genes.
Using the hygromycin-selection system, it was found that the optimum
conditions for affecting transformation in Phalaenopsis were: A. tumefaciens
(pTAG-8) concentration of 5×10P
P cfu/ml, a supplement of 0.1mM
acetosyringone (AS) in the medium and a co-culture period with pollen for 12 h.
Under these conditions, 6 transformants were obtained that from one capsule
under the selection pressure of 5 mg/l hygromycin. In another Phalaenopsis
variety, Doritaenopsis, 68 transformants could be obtained that from one capsule
under 5 mg/l hygromycin selection medium using A. tumefaciens ( pTAG-8 )
concentration of 1×10PP cfu /ml and a co-culture with pollen for 12 h without AS.
Presence of GUS gene in transformants of Phalaenopsis and Doritaenopsis was
confirmed by PCR and Southern blotting analysis. The transformed Phalaenopsis showed change in leaf pigments from green to pink or red. In
Doritaenopsis, 4.89% of the transformants were found to have the GFP activity.
The presence of DFR, CHI and F3′H genes were also confirmed by Southern
blot analysis. Thus, in this thesis, for the first time, a new transformation
technology for orchid Phalaenopsis has been reported.

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