Xue-Feng Xu1,2, Hai-Yan Zhu1,2, Yin-Feng Ren1,2, Can Feng1,2, Zhi-Hao Ye1,2, Hui-Mei Cai1,2, Xiao-Chun Wan1,2, Chuan-Yi Peng3,4. 1. State Key Laboratory of Tea Plant Biology and Utilization, Anhui Agricultural University, Hefei, 230036, Anhui, People's Republic of China. 2. Key Laboratory of Food Nutrition and Safety, School of Tea and Food Science & Technology, Anhui Agricultural University, Hefei, 230036, Anhui, People's Republic of China. 3. State Key Laboratory of Tea Plant Biology and Utilization, Anhui Agricultural University, Hefei, 230036, Anhui, People's Republic of China. pcy0917@ahau.edu.cn. 4. Key Laboratory of Food Nutrition and Safety, School of Tea and Food Science & Technology, Anhui Agricultural University, Hefei, 230036, Anhui, People's Republic of China. pcy0917@ahau.edu.cn.
Abstract
BACKGROUND: Plant protoplasts constitute unique single-cell systems that can be subjected to genomic, proteomic, and metabolomic analysis. An effective and sustainable method for preparing protoplasts from tea plants has yet to be established. The protoplasts were osmotically isolated, and the isolation and purification procedures were optimized. Various potential factors affecting protoplast preparation, including enzymatic composition and type, enzymatic hydrolysis duration, mannitol concentration in the enzyme solution, and iodixanol concentration, were evaluated. RESULTS: The optimal conditions were 1.5% (w/v) cellulase and 0.4-0.6% (w/v) macerozyme in a solution containing 0.4 M mannitol, enzymatic hydrolysis over 10 h, and an iodixanol concentration of 65%. The highest protoplast yield was 3.27 × 106 protoplasts g-1 fresh weight. As determined through fluorescein diacetate staining, maximal cell viability was 92.94%. The isolated protoplasts were round and regularly shaped without agglomeration, and they were less than 20 μm in diameter. Differences in preparation, with regard to yield and viability in the tissues (roots, branches, and leaves), cultivars, and cultivation method, were also observed. CONCLUSIONS: In summary, we reported on a simple, efficient method for preparing protoplasts of whole-organ tissue from tea plant. The findings are expected to contribute to the rapid development of tea plant biology.
BACKGROUND: Plant protoplasts constitute unique single-cell systems that can be subjected to genomic, proteomic, and metabolomic analysis. An effective and sustainable method for preparing protoplasts from tea plants has yet to be established. The protoplasts were osmotically isolated, and the isolation and purification procedures were optimized. Various potential factors affecting protoplast preparation, including enzymatic composition and type, enzymatic hydrolysis duration, mannitol concentration in the enzyme solution, and iodixanol concentration, were evaluated. RESULTS: The optimal conditions were 1.5% (w/v) cellulase and 0.4-0.6% (w/v) macerozyme in a solution containing 0.4 M mannitol, enzymatic hydrolysis over 10 h, and an iodixanol concentration of 65%. The highest protoplast yield was 3.27 × 106 protoplasts g-1 fresh weight. As determined through fluorescein diacetate staining, maximal cell viability was 92.94%. The isolated protoplasts were round and regularly shaped without agglomeration, and they were less than 20 μm in diameter. Differences in preparation, with regard to yield and viability in the tissues (roots, branches, and leaves), cultivars, and cultivation method, were also observed. CONCLUSIONS: In summary, we reported on a simple, efficient method for preparing protoplasts of whole-organ tissue from tea plant. The findings are expected to contribute to the rapid development of tea plant biology.