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

Ramie (Boehmeria nivea L. Gaud.), special product of China, is a herbaceousperennid which belongs to urticaceae family, boehmeria genus. Ramie is one of theimportant textile materials and has good textile nature. In addition, the roots, stems andleafs can be utilized extensively. But there are three big problems in ramie breedingprocedure, one is the period of traditional breeding is too long, the other is the lack oframie cultivated breed, another is the difficulty in distant hybridization. Biologyengineering provides new pathway for variety modification through converting excellentgenes to original biology which has no these excellent characteristics. In order to transferexcellent genes to receptor plant to create excellent varieties, this subject was carried outthrough studying the main elements in protoplast culturing and thry to establishing anefficient system to get regenerated plant. The main research content and results are asfollowings:1. Protoplast isolation and purificationThe centration of protoplasts isolation and purification penetrate pressure regulator(mannitol) on cotyledon of ramie was different with different genotype, '5041-3' neededmannitol 11ï¼…and WZ,XYL,HPD,YD,'1690' needed mannitol 9ï¼….This subject has also carried on dark pretreatment and the growth hormonepretreatment to ramie cotyledon. The result showed that after the two kinds ofprethreaments, the material were enzymolized more full, the protoplast output andviability were also increased greatly, but the inclusion of protoplasts was reduced. At thesametime, we found that if the dark treatment time shorter than 24 h the effect was notobvious, 36 h was the most optimum, and if longer than 72 h the yield and viability ofprotoplast would drop sharply.The best combination of enzymes for cotyledons protoplast isolation was usingCellulase R-10(3.0ï¼…)+pectinase(0.5ï¼…) treat cotyledons 9h or using CellulaseR-10(1.0ï¼…)+HemicellulaseR-10(2.0ï¼…)+MacerozymeR-10(1.0ï¼…)treat cotyledons 12 h. 2. Protoplast cultureProtoplasts cultured on grose-liquid double layer and alginate embedding growed betterthan cultured on Liquid thin culture, and were not easy to be polluted, more better for cellwall regeneration and cell division.Cotyledon protoplasts second divided in KM₈P medium supplemented withNAA(0.5mg/L),NAA(0.5mg/L)+BA(1.0mg/L),2,4-D(0.5mg/L)+KT(0.5mg/L),2,4-D(0.5mg/L)+KT(1.0mg/L), but continuous division has not been observed.3. Study of Ramie somatic embryogenesisVarious calli were produced on cotyledon explants. The variety and the developmentstage of explants affect the potential of morphogenesis. Characters of ramie callus weremultiplicity, among all of them, light yellow or green-yellow, friable embryogenic calliare easy to developed into embryos via somatic embryogenesis.The results indicated thatCPA and 2, 4-D were good for callus induction and proliferation, but the concentrationmust be lower than 1 mg/L. Gin, Asn, CH were crucial for ramie callus induction andproliferation, however, those callus proliferation too fast were difficult to produceembryogenic calli.Genotype is an important factor that influences somatic embryogenesis. Various calliwere produced when explants of cultivars were cultured on the same induction medium.The potentials of cultivars were different, under the same condition. WZ has the bestsomatic embryos potential and the fast multiplication. But calli of HPD were easy toturning to brown and die.4. Study on ramie organogenesisCotyledons were used to study direct organogenesis of Ramie, and direct regenerationshoots were obtained. The results indicated that the combination of hormone TDZ andNAA,BA and NAA could ensure the frequency of shoot regeneration at 60％～80ï¼….'5041-3' and WZ have the best ability of shoots regeneration in all of the used genotypes.