利用農桿菌配合花粉進行玉米及百合之基因轉殖

Abstract

本試驗之目的為：1.利用二元載體(binary vector)之農桿菌品系EHA105以及pCAMBIA1302質體建立農桿菌花粉轉殖之條件：探討農桿菌濃度、acetosyringon (AS)濃度及花粉和農桿菌菌液共培養時間等三個因子；2.轉殖篩選植株之分子鑑定；3.轉殖植株GFP螢光表現之觀察；4.進行轉殖百合類黃酮含量之分析。藉以探討玉米及百合農桿菌花粉轉殖系統之可行性。試驗結果摘錄如下： (1) 玉米、台灣原生（Lilium formosanum Wall.）及鐵砲百合(Lilium longiflorum)等作物之農桿菌花粉轉殖條件：在玉米方面：農桿菌液濃度為0.45×108 CFU/ml、AS濃度為1 mM及農桿菌與花粉共培養6小時的轉殖條件下，每次授粉處理可得到0.66棵篩選株。在鐵砲百合方面：農桿菌液濃度為9×108 CFU/ml、AS濃度為1 mM及農桿菌與花粉共培養0小時的轉殖條件下，每次授粉處理可得2.67棵篩選株。在台灣原生百合方面：農桿菌液濃度為0.45×108 CFU/ml、AS 濃度為1 mM及農桿菌與花粉共培養24小時轉殖條件下，每次授粉處理可得2.33棵篩選株。 (2) 轉殖株之分子鑑定：授粉轉殖之玉米種子經一個月的篩選後總共得到23棵篩選株，挑選13棵進行PCR反應，共有11棵具有gfp與hptII基因的條帶，9棵在PCR-Southern分析時具有訊號產生，其中3棵進行Southern blot分析時只有一棵具有gfp與hptII基因訊號。台灣原生百合種子經二個月的篩選後總共得到30棵篩選株，挑選13棵經由PCR反應，共有10棵具有gfp基因條帶，且在PCR-Southern產生訊號，選擇其中5棵進行Southern blot分析時只有2棵具有gfp基因之訊號。進行台灣原生百合花色基因轉殖，總共獲得34棵篩選植株，經由PCR-Southern分析，共有10棵產生dfr基因雜合訊號，5棵具有f3h基因雜合訊號，1棵具有chi基因雜合訊號；分別進行Southern blot分析時，5棵篩選植株僅只有3棵具有dfr基因雜合訊號，6棵篩選植株僅只有1棵具有f3h基因雜合訊號。 (3) 觀察轉殖植株不同部位之GFP螢光表現：經篩選之玉米轉殖植株之種子及葉片均可觀察出具有GFP之螢光表現。台灣原生百合花粉轉殖時，其花粉管具有螢光表現，且篩選植株之種子、葉片及花瓣均有GFP之螢光表現。 (4) 轉殖植株之成份分析：台灣原生百合轉殖dfr基因之篩選植株，其鱗莖外觀產生黃色與紅色之變化，因此利用薄層色層分析(TLC)及高效液相層析(HPLC)進行山奈酚(kaempferol)、楊梅黃素(myricitin)及檞皮素(quericitin)之成份分析：在TLC分析方面，發現共有4株疑似具有楊梅黃素成份，但在HPLC分析時所有樣品均無類黃酮累積。

Summary The main objectives of the present study were: (1) to standardize a protocol for transfer of foreign genes into maize (Zea mays. L.) and Lilium spp. by using a mixture of Agrobacterium tumefaciens strain EHA105 harboring a standard binary vector pCAMBIA1302 and pollen. (2) to investigate the factors like concentration of Agrobacterium and acetosyringon (AS), and duration of co-culture for efficiency of transformation (3) to confirm the transformation events by detection of GFP in transgenic plants by fluorescence microscope; and other molecular techniques like PCR, PCR-Southern and Southern. (4) to analyze transgenic plants for flavonoids by TLC and HPLC. The results obtained during the course of the study are as follows: (1) Protocols of genetic transformation in maize (Zea mays. L.), Lilium longiflorum and Lilium formosanum with Agrobacterium tumefaciens mediated T-DNA delivery system were standardized. In maize, Agrobacterium at 0.45×108 CFU/ml, acetosyringon (AS) at 1 mM, 6 hours co-cultivation of pollen and Agrobacterium were found optimum for transformation. The mixture of Agrobacterium and pollen was used for pollination. After 45-60 days of pollination, seeds obtained were kept at selection pressure of hygromycin at 20 mg/L. Using optimum conditions, it was observed that each pollination treatment in maize resulted into an average of 0.66 plant surviving selection pressure. In Lilium longiflorum, Agrobacterium concentration at 9×108 CFU/ml and AS concentration at 1 mM and 0 hours co-cultivation with pollen, resulted in an average of 2.67 plants surviving selection pressure per pollination treatment. While in case of Lilium formosanum, Agrobacterium concentration at 0.45×108 CFU/ml and AS concentration at 1 mM and 24 hours co-cultivation with pollen, resulted in an average 2.33 plants surviving selection pressure per pollination treatment. (2) Molecular analysis to confirm transformation: In maize, 23 hygromycin -resistant plants were obtained after one month at selection pressure. PCR analysis showed that out of 13 transgenic lines examined, only 9 contained hptⅡ and gfp genes. The GFP gene was identified by PCR-Southern. Out of 3 PCR positive plants, Southern blot showed only one plant contained hptⅡ and gfp gene in maize. In Lilium formosanum, 30 hygromycin -resistant plants were obtained after two months at selection pressure. PCR analysis showed that out of 13 transgenic lines examined, only 10 contained gfp gene. The GFP gene was identified by PCR-Southern. Out of 5 PCR positive plants, Southern blot showed only two plants contained of gfp gene in Lilium formosanum. In another set of experiment, Lilium formosanum was transformed with Agrobacterium strains containing 3 independent flower color genes (dfr, f3h, chi). 34 hygromycin-resistant plants were obtained after two months at selection pressure under optimum conditions. Ten out of 34transgenic lines confirmed by PCR and PCR-Southern were found to contain dfr gene. Southern blot analysis of 5 PCR positive plants showed that 3 plants had dfr gene fragment. While, 5 out of 34 plants were PCR-Southern positive for f3h gene and one was found to contain f3h gene fragment by Southern blot analysis. One out of 34 hygromycin-resistant plants, only one showed PCR-Southern positive for chi gene. (3) Detections of GFP in transgenic plant by fluorescence microscope: GFP expression was observed in seed and leaf of transgenic maize and pollen tube, seed, leaf and petal of Lilium formosanum transgenic plant. (4) The flavonoids contents in the transgenic Lilium formosanum bulb and root were determined by high performance liquid chromatography (HPLC) and thin layer chromatography (TLC), Four samples showed myricetin like compounds as detected by TLC, but HPLC analysis did not show presence of such compounds.