Research On Cell Suspension Culture Of Pinellia Ternata And Technology Of Artificial Seeds

Pinellia ternata (Thunb.) Berit is a perennial medical plant. Their tubers are used as a crude drug of traditional Chinese medicine. P. ternata degeneration caused by virus diseases usually results in a serious loss of both tuber yield and quality, which has been a main hinder in P. ternata production for long time.Virus-elimination through meristem culture has been widely used in the seedling and microtubers of P. ternata.and the development of biotechnology is important channels to resolve the production of artificial seeds and the improvement yield and quality. This thesis was mainly studied by different plant growth substances which induced callus, the synchronization of suspension cell and the differentiation of microtuber which was used as material for the production of artificial seeds ,and technology of artificial seeds for certain extent. This paper was provided theoretical and technical basis for production and application of artificial seeds. The main results were as follows.(1) Effects of different plant growth substances and the proportion on petiole callus'induction were compared by using the method of orthogonal design. Four plant growth substances were as follows 2,4-D,NAA, Picloram and KT. Statistic analysis showed that 2,4-D and Picloram were the most important factor on callus induction, followed by KT and NAA .The optimal medium to induce and subculture was MS + 2,4-D 0.5 mg·L^-1 + NAA 1.0 mg·L^-1 + Picloram 1.0 mg·L^-1 + KT 1.5 mg·L^-1.(2) The loosened calli of P. ternata were used as materials that has been subcultured in MS liquid medium added with 0.5 mg/L 6-BA, 1.0 mg·L^-12, 4-D and 300 mg·L^-1 CH,which were cultured from 3 to 4 generations. Then measured its growth period of suspended cells, finding out its exponential phase growth ranged from 15 to 21 days, which was the best subculture time. (3) It showed that the best suspension liquid medium for cell culture was MS + 2, 4-D 0.5 mg·L^-1 + 6-BA 0.5 mg·L^-1 through the experiments on different plant growth substances regulating.(4) Effects of low temperature on cell synchronization division was studied in suspension culture cell by the method of orthogonal experiment, the obtained experimental data was compared and analyzed through the analysis of range and analysis of variance. The results indicated that the cell division index was increased in various degrees after the low temperature treatment. When the temperature was set at 8℃for 24 hours and then recovered for 36 hours, the mitotic index was significantly increased to 11.3%.(5) The suspended cells in synchronization were used as materials by using the method of orthogonal design. Four plant growth substances were as follows 6-BA, KT, 2, 4-D and NAA. The optimal liquid medium to differentiate microtuber was MS + 6-BA0.5 mg·L^-1+ NAA0.5 mg·L^-1+ 2, 4-D 0.25 mg·L^-1 + sucrose 30 g·L^-1(6) The microtubers of P. ternata were used as materials for producing artificial seeds. .The influence of different concentration of chitosan, polyglycol,badistan and sodium benzoate on the germination ratio and sprout rate of artificial seeds were researched . The results demonstrated that one kind of artificial seed coat was 3% sodium alginate + 2% CaCl₂ + 0.05% CoCl₂ + 0.05% CuCl₂ + 0.1 mg·L^-1 GA₃ + 4 % chitosan + 2 % polyglycol glycol + 0.4 % sodium benzoate were the best, and its germination rate up to 85%. The conclusion was indicated that the other kind of artificial seed coat was 3% sodium alginate + 2% CaCl₂ + 0.05% CoCl₂ + 0.05% CuCl₂ + 0.1 mg·L^-1 GA₃ + 4 % chitosan + 4 % polyglycol + 0.2 % badistan,and its seedlings rate was as high as 95%.