薄葉嘉賜木癒合組織誘導及細胞懸浮培養

Abstract

薄葉嘉賜木為嘉賜木屬植物中少數分布於臺灣者，其枝葉中含clerodane 型的雙萜類產物，此類二次代謝物對於多種癌症皆具抑制功效，然過去的化學合成與萃取在經濟與技術層面皆面臨瓶頸。本研究以薄葉嘉賜木枝葉為原料進行細胞懸浮系統之建立。實驗結果以採集自南投竹山較為幼嫩的枝葉為試材，經農藥億力及鏈黴素溶液中浸泡 1 小時；水龍頭下流水處理 1 小時；濃度 70%的酒精浸泡 30 秒；浸於次氯酸鈣溶液中，在超 音波震盪器中表面消毒 12 分鐘後；移於無菌操作台以無菌水清洗；放入農藥億力溶液中，於不照光之 shaker 搖晃浸泡 21 小時；再以無菌水清洗，可將莖段與葉片之發霉率降至 30%以下。癒合組織誘導以在不照光的條件下，加入 2ppm 2,4-D、0.2ppm BA、CH(0.1g/L)之 MS 培養基效果最佳；加入 2ppm 2,4-D、0.1ppm BA、CH(0.1g/L)之 MS 培養基因誘導出之癒合組織較鬆軟，更適於懸浮培養。而繼代培養亦以上述加入 2ppm 2,4-D、0.2ppm BA 之生長效果最佳，每月約增加 1.55 克； 培養基中僅含 2,4-D 者會快速褐化，遠不及兩種生長調節劑共同使用。細胞懸浮培養以莖段誘導出的癒合組織為材料，在分別加入 2、4、6 ppm 2,4-D 及 0.1、0.2、0.3 ppm BA 之 MS 培養液能有細胞較為分散之效果，當中又以加入 4 ppm 2,4-D 及0.2 ppm BA 生長效果最佳，約能在 7 天後開始大量增殖，25-30 天後約增加 11 毫升體積。然嘗試多種培養基組合皆無法使細胞於懸浮液中完全散開，其生長效果 有待改進。無菌播種由 2、3、4 月以同株樹木結果所得之種子為材，於加入活性碳之 MS 培養基中培養，予以消毒後無發霉問題但發芽率低，其中 3 月發芽率為 2月之兩倍，其發芽率不佳之原因推測可能為種子發育階段不了解所致；另無菌苗組織亦可成功誘導出癒合組織。

The genus Casearia rarely distributes in Taiwan, Casearia membranacea Hance is one of them. Previously research shows the species contain a series of clerodane diterpenoids which is anti-tumor in leaves and twigs. However, it is difficult to extract the diterpenoids through the traditional way. This study aims to establish the cell suspension culture system of the Casearia membranacea Hance. The results showed that using the young leaves and twigs of C. membranacea from Chushan, Nantou as material, sterilized with Ca(ClO)2 for 12 mins, benomyl for 21 hours and washed thoroughly with sterilized distilled water, the contamination rate could be lower than 70 %. Callus induction succeeded in MS medium with 2.0 mg/l 2,4-D, 0.2 mg/l BA and CH (0.1g/L) without light, and the more friable callus was induced on MS medium with 4.0 mg/l 2,4-D, 0.2 mg/l BA, which is more suitable for cell suspension culture. The subculture showed the best reproduction rate with the MS medium contained 2.0 mg/l 2,4-D, 0.2 mg/l BA and CH(0.1g/L), which increased 1.5g per month. Besides, callus increased more with the medium contained two growth regulators interaction. The cell suspension culture system took stem callus as material showed the better distribution effect with mediums contain 2、4、6 ppm 2,4-D and 0.1、0.2、0.3 ppm BA, reached the best production rate with MS medium contained 4.0 mg/l 2,4-D, 0.2 mg/l BA. Our results revealed that the cells aggregate tightly on the MS medium with all kinds of growth regulators combination we tried so far, the growth rate remains a problem that should be solved. Culturing aseptic seedlings of C. membranacea used seeds collected during February to April, with proper sterilization, the contamination rate could be nearly zero but the germination rate was lower than 10%. The germination rate of the seeds picked in March is double than that in February. The lower germination rate may be caused by lacking knowledge about the physiology of the seeds. Callus could also be inducted by tissues from aseptic seedlings.