| 摘要: | 台灣楊(Populus sp.)是楊柳科落葉性喬木，生長在溫帶地區。當它長為3年樹幹可為頂級合板的原料；5年樹幹可為高級的長纖紙漿原料和環保合板；10年樹幹可為高級建材，為全球製造紙幣之重要原料。楊柳科樹種富含生物活性之多酚類化合物，其中內含水楊酸，經醫學證實有效溶解角質層，而達到肌膚新生的功效，同時兼具保濕功能。儘管台灣楊具有許多優點，但是還是趕不及世界上土壤鹽化和沙漠化的問題，此問題也嚴重影響農林業的生產，所以必須培育出具抗鹽的新品種樹種，也由於現在基因轉殖技術日趨成熟，農桿菌轉殖法(Agrobacterium-mediated transformation)的興起，因此本研究遂利用農桿腫瘤菌(Agrobacterium tumefaciens)對台灣楊進行抗鹽基因轉殖，提高臺灣楊的抗鹽性，期能獲得更多經濟效益，目前尚未有文獻資料成功利用農桿菌轉殖出台灣楊抗鹽基因。本試驗先將台灣楊進行微體繁殖，用LBA 4404農桿菌株、pCAMBIA 1302載體，載體上有Camv35S啟動子、GFP螢光基因、抗kanamycin和hygromycin之基因。微體繁殖系統結果顯示，callus率以78.89%為佳；發芽率以38.73%為佳；發根率以57.14%為佳。農桿菌活化培養於LB固體培養基，之後取單一菌落於液態LB(Luria- Bertani)培養液震盪培養。待OD（optical density）值0.4~0.8將其離心，沉澱菌體加入含Acetosyringone(AS)的MS 液體培養基重新懸浮。在28℃黑暗下，培殖體以不同時間與菌液進行共培後取出，放入含AS100uM的MS固態培養基48hr後，再以無菌水清洗，接著移至含有抗生素cefotaxime和植物生長調節劑之培養基，每2周繼代1次。之後藉改變AS、菌液濃度、抗生素濃度及感染時間等因子，探討其不同因素對培植體褐化之影響。 試驗結果葉柄和中肋部位，較適用MS+BA1.2 mg/L+NAA0.1；葉肉部分較適合 MS+BA0.5 mg/L+NAA0.1之培養基；在callus誘導率和不定芽誘導率皆以TDZ1.0 mg/L效果為佳，但考慮其價格因素，因此用BA取代TDZ，兩者數據在統計方面無顯著差異性。農桿菌試驗方面，OD600=0.5效果為佳(36.67%)；感染時間以8min為佳(31.11%)；AS以100μM為佳；抗生素濃度以300ppm為佳。
Populus sp. is Salicaceae deciduous trees , growing temperate zone plants . When it grow 3-year , the trunk can be top plywood raw materials；grow 5-year , the trunk can be advanced long-fiber pulp raw material and environmental plywood；grow 10-year, the trunk can be for building materials , and it is an important paper money raw materials in the world . Salicaceae species have many biological activity of phenolic compounds , which contain salicylic acid , by medical confirmed effectively dissolved stratum corneum to achieve the effect of new born skin and moisturizing function . Although Populus sp. have many advantages , but still can not keep up soil salination and desertification , this problem also seriously affect agriculture and forestry production , so it must culture salt-resistant new varieties of species , also because transgenic technology is mature , the rise of Agrobacterium transgenic method , therefore in the study transfer Agrobacterium tumefaciens in Populus sp. for salt-resistant gene , improve the salt-resistant tolerance of Populus sp. , hoping to get more economic benefits. Currently , literature is not yet successful use salt-resistant gene transformation of Populus sp. with Agrobacterium tumefaciens. In this study , use Agrobacterium strain LBA 4404 harbouring the vector pCAMBIA 1302 having Camv35S promoter , mGFP5 , kanamycin and hygromycin-phosphotransferase (hpt) genes were used for gene transfer. The micropropagation system results show that the callus rate 78.89% is better than others; germination rate of 38.73%; rooting medium is 57.14 better than others. Agrobacterium tumefaciens activated cultured on LB medium, take single colony was cultured in a liquid LB (Luria-Bertani) and shake culture. Waiting OD (optical density) value of 0.4 to 0.8, it was centrifuged to bacterial. And contained MS liquid medium with Acetosyringone (AS). In the dark 28℃, explant culture at different time and OD value, removed in MS medium containing AS100uM and culture 48hr, then washed with sterile water, and moved containing cefotaxime and plant growth regulator, two weeks subculture once. And then change OD value, infection time, AS, cefotaxime factor, discuss the different factors on explant browning. Future work will continue to conduct the salt stress tests of transgenic plants. |
