| The establishment of plant protoplast regeneration system have important significance on somatic hybridization, genetic transformation and theoretical studies. Lilium regale wilson is a very importmant breeding material because of its strong disease resistance,and it can provide good parental characters for somatic hybridization. We first identified the embryogeny of two kinds of calli induced from cultured bulb of Lilium regale Wilson, adjusted the callus state, subsequently isolated, cultured the protoplast and optimized the system.We identified the embryogeny of two kinds of calli with the result that the yellow callus of Lilium regale Wilson was embryogenic callu, the white one was non-embryogenic callus. Its characteristic showed that the yellow callus was tight, glossy and firm, and there were many spherical protuberances on the callus surface which were easily peeled off; its cell ultrastructure showed that this kind of callus had thin cell wall, narrow intercellular space, big nucleus, thick cytoplasm, small and few vacuoles, and large number of organelles. The characteristic of white callus showed that it was loose, soft, dim and had no spherical protuberances; its cell ultrastructure showed that this kind of callus had thick cell wall, broad intercellular space, small nucleus, and big vacuole which could fill the entire cell.We used the non-embryogenic calli induced from cultured bulb as the material to study the effects of CoCl2 and sucrose on the calli state. The results showed that CoCl2 and sucrose with high concentration (0.05μmol/L,90g/L) were beneficial to the transformation of non-embryogenic callus to embryogenic callus. Different concentration of CoCl2 and sucrose had extremely significant influences on the growing status, proliferation coefficient, and proliferation volume of white callus. CoCl2 and sucrose with low concentration (0.01umol/L,30g/L) were beneficial to the growing status, proliferation coefficient, and proliferation volume of white callus. With increasing of CoCl2 and sucrose concentration, the growing status, proliferation coefficient, and proliferation volume of white callus were decreased.Viable protoplasts were isolated from cell suspension or calli of Lilium regale Wilson by enzymatic digestion. We studied the effects of different cellulases and manntitols concentration, digestion time and manners on the protoplasts yield. The results showed that mannitol concentrations and the digestion time had extremely significant influences on the yield of protoplasts. The optimum manntitol concentration and digestion time for protoplast yield were 140g/L and 6 hours respectively. The protoplast yield was up to 7.17×105 per gram. and the vitality was 70.6%. The cellulase concentrations and digestion manners had not significant influences on the yields of protoplasts.We use the isolated protoplast as material to study the effects of mesh size of screen, the revolution speeds and centrifugation time on the protoplasts purity. The results showed that the protoplast diameter that is 30-80μm and we choosed the screen of 180 mesh size. The protoplasts purity and yield was highest under 300 r/min and 4 or 5 minutes.We cultured the protoplast via liquid shallow culture method, and studied the effects of different kind and concentration of suger on cell division. We drawed that the glucose was superior to the sucrose, and the optimum glucose concentration for cell division was 60 g/L. In this condition, the protoplasts divided first and the first division frequency was highest. The protoplasts became large and oval in shape after 2 days of culture. They completed the first division after 5 days culture, and the cell mass composed of 8～16 cells formed in 10 days of culture. The first division frequency was about 5%, and the cell colony frequency was about 2.5%. |
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