A Study On Culture Technology Of Protoplast In Ramie (Boehmeria Nivea L.Guad.)

Ramie(Boehmeria nivea L. Gaud.), special product of China and named "China grass" is a herbaceous perennid which belongs to urticaceae family ,boehmeria genus . Ramie is one of the important textile materials and has good textile nature and excellent dress-making performance. In addition, the roots, stems and leafs can be utilized extensively. Profound researches have been done to ramie material distribution, introducing culture, pedigree selection breeding, hybrid breeding, hybrid vigor utility, male sterility, inducing breeding, asexual propagation and plant tissue culture. But two big problems can hardly be solved in ramie breeding procedure, one is too long breeding period due to traditional breeding methods and the other one is fibre quality reduction due to impurity varieties. Biology engineering provides new pathway for variety modification through converting excellent genes to original biology which has no these excellent characteristics. In order to convert good genes to receptor plant and cell fusion and then create excellent varieties, this subject was carried out through studying the main elements in protoplast culturing and establishing a set of efficient system to get regenerated plant. The main research content and results are as following:1. Effect on different explants for protoplast isolationFor ramie seeds, calli were induced by adding hormones of 3.0mg/L high concentration. But the character of these calli are too bad and can not be used for further protoplast isolation. Different hormone combinations indicated very remarkable difference to callus induction ratio of ramie cotyledons and hypocotyls. KT0.5mg/L and 2,4-D0.5mg/L showed good effect on callus induction and 2,4-D0.5mg/L and KT 1.0 mg/L made calli grow stabilized. Yellow loosed calli are easy to form embryogenesis cell mass, which can be observed little cell with plenty of cytoplasm in microscope.2. Effect on callus induction of genotypeHypocotyls of different genotype such as Huazhu No.4, Xiangzhu No.2, Qianzhu No.l, Ruiyang Xiyelu, Enshi Qingma, 1690 can all produce calli and indicate very remarkable difference in induction ratio and calli characters. Huazhu No.4 was most suitable to MSB medium with adding 2, 4-D0.5mg/L KT l.Omg/L, GA3 0.2mg/L3. Subculture for suspension linesIn order to keep activity, suspension lines must be subcultured in every 7 days. The best materials for protoplast isolation is those which be cultured for 4-6 days in every subculture period.4. Protoplast isolationThe combination of enzymes for cotyledons protoplast isolation was Cellulase R-10 3.0%+Macerozyme 0.5% and Cellulase R-10 5.0%+Pectinase 1.0% was suitable to cell suspension lines. The protoplast yield was 1067ml and viability 88.6%~90.6%. 5. Protoplast cultureCotyledon protoplasts divided in KM8P medium supplemented with 2, 4-D 0.5 mg/L+NAA0.2 mg /L+ 6-BA0.2 mg/L+glucose0.7mol/L, but continuous division has not been observed. Difference was also very remarkable for cotyledon genotypes.