水稻台稉9號細胞培養及篩選耐白葉枯病菌細胞系之初探

Study on Cell Culture and Preliminary Screening for Bacterial Leaf Blight Tolerant Cell Lines of Oryza sativa var. japonica cv. Taiken 9

張雅嵐(Ya-Lan Chang)

指導教授 ： 鄭秋雄

國立屏東科技大學/農學院/植物醫學系所/碩士(2013年)

https://doi.org/10.6346/NPUST.2013.00001

摘要

本研究之目的為研發水稻癒傷組織誘導培養技術、懸浮細胞建立培養及初代培養技術與篩選抗白葉枯病細胞系。以台梗9號水稻成熟種子為培植體，探討2,4-D濃度、成熟種子儲藏時間及種子置床方式對癒傷組織誘導之影響，結果以收穫後儲藏（4 ℃）一年內之種子為培植體，並以胚朝上全埋之置床方式培養於含2~4 mg/l 2,4-D之CS-1培養基中之癒傷組織形成量最佳，其單位培植體癒傷組織鮮重約0.25 g，以此條件誘導之癒傷組織進行懸浮細胞建立培養之克鮮重癒傷組織懸浮細胞數最多。為研發懸浮細胞建立培養技術，探討建立培養天數、癒傷組織鮮重量及癒傷組誘導方式（種子置床方式）對懸浮細胞建立培養之影響，結果以垂直胚朝上全埋之種子置床方式誘導之癒傷組織鮮重3 g培養於含2 mg/l 2,4-D之CSW-1液體培養基中建立培養14天之懸浮細胞數最多，其克鮮重癒傷組織之懸浮細胞數約4.97~5.28 ×106 cells，以此條件建立之懸浮細胞做為初代培養及繼代培養之細胞材料可減緩細胞數下降速率。為建立初代培養技術，比較CS-1調整培養基與CSW-1調整培養基對懸浮細胞初代培養之影響，結果以建立培養14天之懸浮細胞培養於CSW-1調整培養基（新鮮培養液與使用過培養液為1：1）中培養3週之細胞增殖率最佳約123.3 %（1.23倍）。為開發適合水稻懸浮細胞初代培養之培養基，探討維他命B1含量、培養基（MS、N6、B5及CS-1）及碳水化合物對懸浮細胞初代培養之影響，結果以2~4×105 cells/ml之細胞數，培養於含2 mg/l 2,4-D及0.5 mg/l 維他命B1之CSW-1培養基（CS-1培養基添加30 g 蔗糖、3 g葡萄糖及0.3 g果糖）中培養2週之細胞增殖率約62~87 %為最佳。為篩選出抗水稻白葉枯病細胞系，以白葉枯病菌培養過濾濃縮液（濃縮至50 %）為篩選劑，處理水稻懸浮細胞48小時，結果細胞存活率約32.3 %。存活細胞（抗性細胞系）以CS-1培養基中培養四週後，繼以CS-1調整培養基培養3週，再以濃縮至50 %之培養過濾濃縮液處理48小時之細胞存活率約108.9 %，相較於非抗性細胞系之存活率27.7 %，顯示抗白葉枯病細胞系對白葉枯病菌培養過濾濃縮液具有抗性。

關鍵字

水稻台稉9號 ； 癒傷組織 ； 懸浮細胞培養 ； 建立培養 ； 初代培養 ； CSW-1培養基 ； 調整培養基 ； 水稻白葉枯病 ； 病菌培養過濾液 ； 抗水稻白葉枯病細胞系

並列摘要

The purposes of this study were to develop culture technique of callus induction, establishment culture and primary culture of suspension cell and to select the bacterial leaf blight-resistant cell lines in Oryza sativa var. japonica cv. Taiken 9. The mature seeds of O. sativa var. japonica cv. Taiken 9 served as the explant to evaluate the effects of 2,4-D concentration, seed storage time and place method of seed on callus induction. The results indicated that the seeds stored at 4℃ for less than one year as explants and embryo upward whole buried in CS-1 medium containing 2-4 mg/l 2,4-D were best in quantity of callus formation, which callus fresh weight per explant about 0.25 g. According to these culture conditions, the callus served as materials of establishment culture of suspension cell could obtain the largest number of suspension cells of the callus fresh weight per gram. In order to develop the techniques of establishment culture in suspension cell, effects of days of establishment culture, fresh weight of callus and methods (place method of seed) of callus induction on establishment culture of suspension cell were evaluated. 3 g callus that induced with vertical and embryo downward whole buried cultured in CSW-1 liquid medium containing 2 mg/l 2,4-D for 14 days was most effective to increase the number of suspension cells of callus fresh weight per gram, which about 4.97-5.28 ×106 cells. The suspension cells established with the culture conditions as the materials of primary culture and subculture could slow down the decreasing rate of cell number. In comparison of the effect of conditioned medium of CS-1 and CSW-1 on primary culture of suspension cell for establish the techniques of primary culture. The suspension cells, establish cultured for 14 days, cultured in CSW-1 conditioned medium (fresh/conditioned medium ratio 1：1) for 3 weeks provided the best result in proliferation rate of cell, which about 123.3 % (1.23-fold). In order to develop the medium for the primary culture of suspension cell, the effects of vitamin B1concentration, media (MS, N6, B5 and CS-1) carbohydrate on primary culture of suspension cell were evaluated. The best results indicated that 2-4×105 cells/ml cultured in CSW-1 medium (CS-1 supplemented 30 g/l sucrose, 3 g/l glucose and 0.3 g/l fructose) containing 2 mg/l 2,4-D and 0.5 mg/l vitamin B1 for 2 weeks in proliferation rate of cell, which about 62-87 %. In order to select the bacterial leaf blight-resistant cell lines, culture filtrate of Xanthomonas oryzae pv. oryzae (concentrated to 50 %) was prepared as the select agent, and then the suspension cells of rice were treated with the filtrate for 48 h. Result showed the survival rate of cells was about 32.3 %. The survival cells (bacterial leaf blight-resistant cell lines) were further cultured in CS-1 medium for 4 weeks and subcultured in CS-1 medium for 3 weeks and treated with culture filtrate (concentrated to 50 %) of X. oryzae pv. oryzae for 48 h, the survival rate of resistant cell was about 108.9 %. Compared with the non-resistant cell lines (survival rate was 27.7 %), the result indicated that the bacterial leaf blight-resistant cell lines had resistance to culture filtrate of X. oryzae pv. oryzae.

並列關鍵字

rice (Oryza sativa var. japonica cv. Taiken 9) ； callus ； suspension cell culture ； establishment culture ； primary culture ； CSW-1 medium ； conditioned medium ； bacterial leaf blight of rice ； culture filtrate of Xanthomonas oryzae pv. oryzae ； bacterial leaf blight-resistant cell lines

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