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

• Main Authors: Chang, Ya-Lan, 張雅嵐
• Other Authors: Cheng, Chiu-Chsiung
• Format: Others
• Language: zh-TW
• Published: 2013
• Online Access: http://ndltd.ncl.edu.tw/handle/08663234527476784521

碩士 === 國立屏東科技大學 === 植物醫學系所 === 101 === 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.