Establishment Of High-efficiency Regeneration System For Koelreuteria Paniculata Laxm.in Vitro

As a native tree species,Koelreuteria paniculata Laxm.(family Sapindaceae)is widely distributed in China.It is an important ornamental tree species with important ecological restoration and economic medicinal value.However,K.paniculata mainly relies on seed reproduction.Cutting and grafting are also difficult to apply to K.paniculate.The lacks of effective methods for asexual reproduction makes it difficult for K.paniculata on breeding and large-scale reproduction.Therefore,a rapid and effective regeneration pathway through somatic embryogenesis was established for K.paniculate in this study,and propagation system of stem segnent and anther culture in vitro were also studied.This study not only provides methods for the breeding and preservation of elites,but also provides a basis for the subsequent genetic research and molecular breeding in K.paniculata.The main findings are as follows:1.In-vitro culture of zygotic embryos in K.paniculata:The immature seeds(embryos with complete structure)were used as initial materials,and the sterile seedlings were obtained through the sterilization of the explant and incubation on the germination medium.In this process,the germination rate decreased with the gradual maturity of seeds.2.Embryogenic callus induction in K.paniculata:The stem segments of aseptic seedlings were used as induction materials.The influence of medium types and plant growth regulators(PGRs)concentrations were tested for callus induction.The optimum medium type for callus induction was DKW medium,and the optimum concentration of PGRs was 0.5 mg L-1 6-BA+0.25 mg L-1 NAA+1.5 mg L-1 2,4-D.The highest callus induction rate was 80.25%.3.Somatic embryogenesis in K.paniculata:The different types and concentrations of PGRs were tested for somatic embryogenesis.In this process,compared with 2,4-D,the exogenous auxin NAA played an important role in the somatic embryogenesis.And the calluses cultured on medium supplemented by 0.1 mg L-1 NAA obtained the highest frequency of somatic embryogenesis(52%).4.Maturation and germination of somatic embryos in K.paniculata:Different culture conditions and concentrations of NAA were tested for primary somatic embryos maturation.The light was beneficial for somatic embryos maturation,and the optimum concentration of NAA was 0.2 mg L-1 NAA(92.81%).For secondary somatic embryos,different types and concentrations of PGRs were tested for maturation.The effects of PGRs were as follows:GA3>ABA>NAA.The highest maturation rate(83.33%)was obtained on DKW medium supplemented by 0.15 mg L-1 GA3.The mature somatic embryos were transferred to 1/2 DKW medium containing 0.1 mg L-1 IB A,20 g L-1 sucrose and 5.5 g L’1 agar to obtain the normal plant.5.Establishment of rapid propagation system in K.paniculata:The formulation of basic medium,concentrations and types of PGRs were tested for rhizogenesis of stem segments.The optimum basic medium was 1/4 DKW medium containing 1.0 mg L-1 IBA,20 g L-1 maltose and 5.5 g L-1 agar.The effects of PGRs were as follows:IAA>IBA>NAA.6.Preliminary study on anther culture in K.paniculata:The observation of flowers development showed that the flowers are cross-pollination,and the development process is not synchronous.Anther disinfection results showed that the concentration and treatment time of sodium hypochlorite solution significantly affected the rate of anther infection and injury.It is more suitable to disinfect the explants that the proportion of sodium hypochlorite solution was higher than 1/20 and the disinfection time was more than 10 min.The callus induction results showed that the induction medium had a significant effect on the anther callus induction,and only the anther from the IEM3 and IEM4 had obtained the calluses.