Production of glucosinolates by tissue culture of Brassica oleracea var. italica

十字花科植物廣受大家喜愛，其所含有的二次代謝產物－硫代葡萄糖苷，已被許多文獻證實具有許多生理活性，如抑制腫瘤形成、抗氧化與提高人體中解毒酵素的活性等。本研究以十字花科中之青花菜為材料，希望以植物組織培養的方式來生產硫代葡萄糖苷。實驗分三階段進行。第一階段乃尋找適合青花菜形成癒傷組織的培養基。在嘗試不同濃度與種類的植物荷爾蒙後，發現以MS培養基添加1 mg/ L 2,4-D與0.5 mg/ L的BA有最好的誘導效果，成功誘導的癒傷組織高達80%。第二階段進行搖瓶培養，發現在七天後，glucobrassicin會增加。第三階段則以發酵槽放大培養，結果顯示硫代葡萄糖苷在癒傷組織內含量較多，僅有少量硫代葡萄糖苷會釋放到培養液中。其中又以氣舉式發酵槽通入5 %二氧化碳下，其癒傷組織中的硫代葡萄糖苷含量最多。而利用發酵槽培養癒傷組織，其水萃液(1 g/ 30 mL H2O)抗氧化能力又以氣舉式發酵槽不通入二氧化碳最好，其也含有較高的酚類物質。在癒傷組織中硫代葡萄糖苷之HPLC分析圖譜中，在滯留時間為27分鐘有一明顯波峰，經由質譜儀分析後，推測其結構可能為4-Methoxyglucobrassicin。 Brassicaceae family is popular vegetable around the world, and it contains secondary metabolites-glucosinolates, which are potent source of protective chemicals against cancer. Glucosinolates can induce the phase II enzyme system thus inhibiting the growth of tumor. The purpose of our efforts was to produce glucosinolates by plant tissue culture, and we chose broccoli, one of the important vegetables in Brassicaceae family, as material. The experiment was divided into three stages. Firstly, we tried to find out an optimal medium to induce broccoli to form callus. After investigating different kinds and concentrations of plant hormones, we found that addition of 1 g/ mL 2,4-D and 0.5 g/mL BA in the MS medium could obtain the best result. The percentage of health callus was above 80. In the second stage, shaker flask was used to cultivate broccoli callus in suspension culture. It was found that the glucobrassicin increased after 7 days of cultivation. Finally, fermentor was used to cultivate broccoli callus. We found that glucosinolates were mostly in the callus, and only a little released to the medium. The content of glucosinolates in callus cultivated by air lift fermentor with 5 % CO2 was the highest. However, the callus with the highest antioxidant activity was obtained in air lift fermentor without CO2, and it also had the highest phenolics content. The HPLC profile revealed that the major glucosinolates appeared at retention time 27 min, and MS analysis indicated that the compound is 4-Methoxyglucobrassicin