Developments of Rice Cell Suspension Culture and a Novel Strategy for Screening New Resistant Lines to Rice Blight Disease Caused by Xanthomonas oryzae pv. oryzae

• Main Authors: RESTU RIZKYTA KUSUMA, 古書瑪
• Other Authors: Dr. Chiu-Chsiung Cheng
• Format: Others
• Published: 2013
• Online Access: http://ndltd.ncl.edu.tw/handle/91510835965043461466

碩士 === 國立屏東科技大學 === 植物醫學系所 === 102 === Rice is the staple food for more than half of the world population. Bacterial blight (BB) disease is one of the serious problems in rice production, especially in Asia. Development of cell culture system will help to solve some problems. As an alternative of cell suspension technologies, cell suspension culture has produced somaclonal variants with higher potential to screen novel lines for crop improvement. In this study, we developed a rice cell suspension system in primary culture and further screen resistant cell lines of Oryza sativa var. japonica cv. Taiken 9 to bacterial blight disease caused by Xanthomonas oryza pv.oryzae (Xoo). The culture cells obtained from the callus, cultured on CS-1 medium containing 3 % sucrose and 2 mg/L 2,4-D for 4 weeks, were offered as the materials and CS-1 medium as the basal medium for establishment culture of cell suspension for 2 weeks, and the effects of carbohydrate source, plant growth regulator, nitrogen, vitamin and conditioned medium on cell proliferation were evaluated. The results indicated that rice cell suspension in primary culture can be improved by modifying the composition of medium, such as carbohydrate source, plant growth regulator, nitrogen, vitamin and conditioned medium. The proliferation cell was signifcantly increased 1-fold in 3 weeks of primary culture in CS-1 conditioned medium (fresh/spent medium ratio 1:1) containing 3 % sucrose, 0.5 % glucose, 0.05 % fructose and 2 mg/l 2,4-D. This medium was further used to screen the bacterial leaf blight-resistant cell lines through applying culture filtrate of Xoo. In this strategy, we attend to screen a novel line which could produce high amounts of reactive oxygen species (ROS) while the variant could recognize pathogen-associated molecular patterns (PAMPs) from Xoo. In the begining, 1 % of Xoo filtrates were applied as environmental pressure in cell suspension culture. Then, the survival cells were further separated into 24-wells microplate in modified CS-1 medium for 1 week. After 10 % of Xoo filtrate was applied in cell suspension for 1 hour, the ROS production in cells was evaluated. Results showed 33 % cell lines were strong ROS-producing, two cell lines were selected and cultured for second round screening. The ratio of strong ROS-producing cell lines was increased up to 67 % in the third round screening. We observed that most the cells in selected cell lines were high to medium ROS-producing. The strong ROS-producing cell lines in third round screening can be further cultured for plant regeneration. The rice cell lines with high ROS production may have potential of resistant cell lines against Xoo. In this study, we developed a rice cell suspension system in primary culture and further establish a new screening strategy to obtain novel disease resistant cell lines against bacterial blight pathogen.