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標題: 建立蜜糖文心蘭體胚繁殖系統及ZmCCD1基因轉殖至南西文心蘭之探討
Somatic embryogenesis of Oncidium Sweet Sugar and transformation of maize carotenoid cleavage dioxygenase 1 gene into Oncidium Gower Ramsey
作者: 鄭郁琪
Cheng, yu-chi
關鍵字: 文心蘭
Carotnoid Cleavage Dioxygenase 1 (CCD1)
Agrobacterium transformation of Oncidium NDM medium
protocorm like bodies (PLBs)
出版社: 生命科學系所
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摘要: 文心蘭Oncidium Sweet Sugar及Gower Ramsey商業價值高,但前者無法快速量產,後者缺乏香氣。本研究初步評估農桿菌轉殖技術對Gower Ramsey花香及Sweet Sugar產量之改善效果,以期增加此兩者之產業效益。 文心蘭Oncidium Sweet Sugar屬於盆花品系,目前商業為成熟株側芽增殖方式量產。本實驗建立擬原球體(protocorm-like bodies , PLBs)繁殖系統,期望能在幼年期即可提高其產率。以細胞分裂素BA (benzoic acid)、TDZ (thidiazuron)以及生長素NAA (naphthaleneacetic acid)搭配不同的培養基與醣類,誘導培植體由癒合組織分化成PLB。在BA /NAA=10的比例,BA為1 mg/L時,最適合誘導莖頂癒合組織。液態的誘導系統雖然褐變率高,但是誘導成功的培植體可快速增殖出大量的癒合組織。單獨使用TDZ易使植株褐變;但0.5 mg/L 的TDZ搭配0.5 mg/L的BA適合誘導癒合組織。 單獨添加1 mg/L BA或以0.5 mg/L BA搭配 1 mg/L TDZ適合PLBs分化。 0.5 mg/L 的TDZ添加在NAA則易使植株不經由PLBs途徑直接分化成植株。基本培養基NDM (New Dogashima Medium) 添加蔗糖的基本培養基最適合Sweet Sugar原球體增殖。 Sweet Sugar農桿菌轉殖系統測試實驗,在液態MS培養基中將農桿菌及癒合組織共培三天後除菌,經過一個月以上的復原期後,克服農桿菌引起之過敏反應 (hypersensitive response),再以非hygromycin的抗生素篩選二個月以上,才能初步篩選轉殖株。 目前已知文心蘭Gower Ramsey唇瓣含有豐富的類胡蘿蔔素,且類胡蘿蔔素和玉米的carotenoid cleavage dioxygenase 1 (ZmCCD1)作用可產生香氣,我們假設藉由農桿菌送入的ZmCCD1可和Gower Ramsey內的類胡蘿蔔素結合並產生花香,並藉由PCR與GUS染色法鑑定轉殖成功,初步確認外源基因已確實進入轉殖株。未來觀察將著重於HPLC或GC-MS分析ZmCCD1 參與文心蘭花瓣中何種類胡蘿蔔素代謝路徑,影響胡蘿蔔素的代謝,改變下游文心蘭的花色,產生帶有白花並帶有香氣的文心蘭新品系。
Oncidium ‘Sweet Sugar'' has economical importance in pot-plant industries recently but its micropropagation has not been well-studied. This study aims to investigate Agrobacterium tumefaciens mediated gene transformation of ‘Sweet Sugar'. Recently, studies on Oncidium' Gower Ranmsey' floral color which composed of carotenoid have been revised. However, there are rare research articles have been published as synchronal remodeling floral color and aromatic compounds of Oncidium cultivars. In this study, in vitro callus and protocorm-like bodies (PLBs) propagation system had been established. Shoot tip cultured in NDM medium was supplied with 1mg/L BA and 0.1 mg/L NAA which was the best formula for somatic embryogenesis. Only TDZ showed not suitable for somatic embryogenesis induction on NDM medium. For embryogenesis differentiation, adding 0.5 mg/L BA on NDM medium supplied with TDZ preferred to PLBs formation. Subculture 0.5 mg/L BA with 1mg/L TDZ proliferated the best quantity of PLBs. In this research, 0.5 mg/L NAA supplied with TDZ in NDM medium induced embryonic callus differentiation to shoot buds directly. This study developed Oncidium' Sweet Sugar' with Agrobacterium mediated gene transformation system preliminarily. In transformation step, co-cultured Agrobacterium with Oncidium 'Sweet Sugar” callus in liquid MS medium was better than in solid MS medium. After transformation, over 1-month recovering time is required to overcome the hypersensitive response, which was triggered by bacterial infection. It is necessary to transfer the survival explants and placed on selection medium for at least 2 months, in order to confirm the transgenosis plants. This research transformed Oncidium 'Gower Ramsey' PLBs with Agrobacterium which carrying maize carotenoid cleavage dioxygenase genes (ZmCCD1). The transgenic PLBs were confirmed by PCR and GUS histochemical staining. These results demonstrated that ZmCCD1 can be transferred in Orchid successfully. It is anticipated that transformed ZmCCD1 in Oncidium will elevate flower aroma and change the color of Orchid. We need to wait for transgenic plants to bloom, in order to accomplish further study as analyzing aroma volatiles and colors of flowers by using HPLC and GC-MS.
其他識別: U0005-0802201019545400
Appears in Collections:生命科學系所



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