Research On Tissue Culture And Protoplast Isolation Of Virus-free Technology Of Mulberry

Plant tissue culture is generally used for rapid propagation of plants, detoxification, breeding and germplasm conservation, secondary metabolite production and basic theoretical research. At present, have the use of different explants established mulberry variety of in vitro culture system.Protoplasts of each cell contain all the genetic information of the individual, in the appropriate culture conditions, and their parents with similar re-generate the individual all-round at the same time a large number of protoplasts were geneticaly homogeneous. In recent years, plant protoplast research more and more important, its focus from protoplast isolation, physiological and biochemical characteristics of protoplasts and protoplasts regenerated plants were transferred to the application of protoplast.In this study, 14 Nongsang winter buds as expLants to MS, B5, N6, WPM as the basic medium supplemented with different concentrations of cytokinins (6-BA), Kinetin (KT) and auxin (2, 4-D) and NAA (NAA) and so on medium supplemented with sucrose 3%, agar 0.8%, pH value of 5.8 to study the establishment of mulberry aseptic system, different media components and the hormone concentration on Mulberry Tissue Seedlings, selected the optimum start-up, differentiation, rooting medium. The results showed that: in vitro of mulberry using different disinfection combined the best disinfectant; basal medium with MS medium; add the 6-BA and 2,4-D, the tissue culture growth height maximum of 4.1cm, the most suitable for the growth of mulberry tissue culture; optimal medium for the startup MS +6- BA 1.5 mg / L + 2,4-D 0.1 mg / L; differentiation medium was MS + 6-BA 2.0 mg / L + 2 ,4-D 0.1 mg / L; rooting medium was MS + NAA 0.1 mg / L.By Nongsang 14 secondary meristem tip tissue culture test known: meristem tip cultured on mulberry, 39℃heat treatment 15 d, virus-free plants reached the maximum average height of 3.8 cm; tip size is 0.5 mm, average height of the largest virus-free plants for the 2.1 cm; light is beneficial to induce germination of mulberry shoot tip tissue culture, growth, differentiation; activated carbon will help improve training tip of the switch to green detoxification rate, differentiation rate, and the degree of tip growth high; virus-free seedlings of the best medium for MS + 6-BA 2.0 mg / L + NAA 0.2 mg/L; secondary meristem tip than a detoxification can improve the detoxification rate of 6.67%.On protoplast isolation test that mulberry: 24 h dark treatment could protoplast viability by 5%; low temperature can protoplasm 72h activity increased 6%; protoplast isolation using 1.00% cellulase (Cellulase "Onzuka" RS ) + 0.05% pectinase (Pectinase Y-23) + 0.20% isolation enzyme (Macerozyme R-10) of enzymatic hydrolysis was the best combination of enzyme solution, the protoplast yield was 8.3×106个/ mL; 26℃in the dark conditions, hydrolysis 12-14 h isolated protoplast yield increased by 5 times and the activity increased by 4 times.