The High-efficiency Generation Of Somatic Embryos And Optimization Of Genetic Transformation System Of Two Lily Varieties

A stable and efficient genetic transformation system is of great significance for Lilium gene function research,trait improvement and new variety breeding,and a good receptor system is the basis for the realization of Lilium genetic transformation.In this experiment,using Lilium oriental and Lilium longiflorum Thunb as materials,the high-efficiency generation of somatic embryogenesis of the two varieties Lilium were explored,and the genetic transformation system was optimized on the basis of the previous research of the research group.The overexpression study of BPM3(BTB/POZ-MATH3)gene was carried out in Lilium oriental ’Siberia’ used the optimized transformation system.The main findings are as follows:1.The somatic embryogenesis induction technology system of two different Lilium explants were established respectively.Taking scales as explants,the best somatic embryo induction medium of ’Siberia’ was MS+2.0 mg/L PIC+30 g/L sucrose+7 g/L agar,and the best somatic embryo induction medium for the scales of Lilium longiflorum Thunb was MS+1.8mg/L PIC+30 g/L sucrose+7 g/L agar,and the somatic embryo induction efficiency of the two varieties Lilium could reach 100%.Using leaves as explants,the most suitable medium for the induction of ’Siberia’ somatic embryo was MS+1.5 mg/L PIC+30 g/L sucrose+7 g/L agar,the induction rate was 91.11%.The most suitable medium for the induction of somatic embryo of Lilium longiflorum Thunb was MS+1.0 mg/L PIC+30 g/L sucrose+7 g/L agar,and the induction rate was 94.44%.In the process of subculture of somatic embryos,the culture temperature of 20°C was beneficial to maintain the embryonic state of spherical embryos and inhibit the occurrence of adventitious buds,which prolonged the preservation time of somatic embryos,and the subculture cycle of somatic embryos in ’Siberia’ could be extended to 33 days,the somatic embryos subculture cycle of Lilium longiflorum Thunb can reach 60 days.2.The genetic transformation systems of the two lily varieties were established and optimized respectively.Through the antibiotic susceptibility test,the suitable bacteriostatic antibiotic for somatic embryos of Lilium longiflorum Thunb was determined to be Hyg,and the sublethal concentration of Hyg was 30 mg/L,thus establishing the genetic transformation system of Lilium longiflorum Thunb based on somatic embryogenesis.Antibiotic screening determined that the suitable bacteriostatic antibiotic for somatic embryos of Lilium oriental’Siberia’ was Kan,and the sublethal concentration of Kan was 150 mg/L.And the somatic embryo of ’Siberia’ was used as the genetically transformed receptor material,and the receptor material were wounded by puncturing a hole with a sterile syringe needle before infection,and the co-cultivation temperature was lowered to 21°C.For filter paper co-culture,3 μM chloroxynil(CX)was used in the resuspension and co-culture medium to replace the original100 μM acetosyringone(AS),and added 3 μM CX in the screening medium,etc.Optimization of various transformation test details,optimized the genetic transformation system of Lilium oriental ’Siberia’ based on somatic embryogenesis.3.Genetic transformation of Lo BPM3 gene.Using ’Siberia’ somatic embryos as the transformation receptor material,the target gene Lo BPM3 was successfully transformed.After PCR identification,4 PCR positive lines and 17 positive plants were obtained.And through the expression level detection,3Lo BPM3 overexpression lines and 14 overexpression plants were obtained,the transformation efficiency of 30.43% was obtained.The higher the expression level of Lo BPM3 in the transgenic lines,the lower the expression of RAV(Related to ABI3/VP1)in the plant,and the faster the growth rate of the corresponding organ tissue.This study based on the somatic embryogenesis system,established the genetic transformation system of Lilium oriental’Siberia’ and Lilium longiflorum Thunb,and used ’Siberia’ somatic embryos as transformation recipient materials,comprehensively and systematically optimized the genetic transformation system of Lilium oriental ’Siberia’,and obtained Lo BPM3 gene overexpressing plants,it laid a foundation for the research on gene function and molecular breeding of Lilium oriental and Lilium longiflorum Thunb,and also provided materials for the functional research of the BTB/POZ-MATH3 domain in the growth and development of Lilium.