Enhancement Of Somatic Embryogenesis And Genetic Transformation With Barnase And PaFT Genes In Cinnamomum Camphora L.

Camphor tree(Cinnamomum camphora L.),native to China,is an important ornamental and economical tree species,which is cultivated all around the world.However,the application of the species is seriously limited by its prolific seed production.So the present research was carried out for the purpose of breeding sterile camphor tree plantlets. By enhancing the protocols for somatic embryogenesis and genetic transformation, transgenic plantlets were finally obtained successfully with the stable integration of GFP gene in the genome of C.camphora.Based on the improved systems,genetic transformation with Barnase and PaFT genes was carried out,and Barnase transgenic plantlets were obtained.The main results were as follows:(1) Enhancement of somatic embryogenesis in C.camphora.Improvement of somatic embryo(SE) induction,embryogenic callus initiation and plant regeneration using immature zygotic embryos as explants were studied.The results showed that,high osmotic stress pretreatment of explants with a sucrose solution was the key factor affecting SE formation,which improved embryogenesis frequency from 16.29 to 93.2%, and average number of SEs per explant from 3 to 12.57.Activated charcoal(AC) addition and 10-day medium renewal improved the quality of SEs.Basal medium and sucrose concentration in induction medium did not influence embryogenesis significantly,while Murashige and Skoog(MS) medium with 30 g/L sucrose increased average number of SEs.Light conditions had no significant effect on the frequency of embryogenesis,but culturing under light yielded more SEs.Addition of various plant growth regulators (PGRs) had negative effects on embryogenesis,even though some showed improved efficiency,the changes were not significant.After cultured on MS medium in darkness for 2 months,the primary SEs were transferred to MS medium supplemented with 0.1 mg/L TDZ and 0.2 mg/L IBA for germination,among which more than 80%of the embryos converted to plantlets.When cultured on MS medium with 0.1 mg/L NAA,SEs proliferated rapidly via secondary somatic embryogenesis.In addition to the process of proliferation,secondary somatic embryos(SSEs) were also formed on the surface of matured or even germinated SEs.SSEs were found difficult to achieve germination, which needed a maturation treatment with 0.5 mg/L ABA before cultured on germination medium,and the regeneration efficiency decreased with the increasing generation of subculture.Embryogenic calluses were observed during the process of SE induction and proliferation.With these experiments,the quality of SEs was improved,meanwhile,the efficiencies of SE induction,embryogenic callus initiation and plant regeneration were enhanced significantly.(2) Secondary somatic embryogenesis and plant regeneration in C.camphora.The process of secondary somatic embryogenesis of embryogenic lines preserved for＞4 years were studied by histological observation,and factors affecting maturation of SSEs as well as culture conditions for germination were investigated.The results showed that, SEs were proliferated rapidly via secondary somatic embryogenesis.SSEs were produced on the surface of the cotyledons and radicular ends of maternal SEs,which departed from maternal tissues at cotyledonary stage.Histological observations of the various stages of secondary embryo development revealed four typical stages namely,globular, heart-shaped,torpedo and cotyledonary.Multiple SSEs at different stages of development could be found on a single maternal SE,showing that they were formed asynchronously. The process of secondary embryogenesis continued in a cyclic way,with each newly formed embryo producing a subsequent generation of secondary embryos.Treatments of the long-term cultured SSEs with TDZ,ABA,GA₃,glycerin or PEG6000 promoted embryos maturation and germination,among which 0.5 mg/L ABA showed the most significant effect on maturation with final germination frequency of more than 50%.MS medium containing 0.1 mg/L TDZ and 0.2 mg/L IBA was suitable for germination,on which the germination frequencies of SSEs from different embryogenic lines(L9,L14, L21 and L23) had no significant differences.(3) Genetic stability assessment of long-term cultured SSEs and the regenerants in C. camphora.Genetic stability of SSEs(L1,L9,L14 and L23) and regenerants(L9 and L23) subcultured for＞6 years were assessed by ISSR markers.The results showed that,15 primers out of 38 ISSR primers screened,were found to produce clear reproducible bands resulting in a total of 96,98,96,96,96 and 95 distinct bands,of which 93 were monomorphic across all of the SSEs and regenerants tested and 1,3,1,1,1,2 showed polymophisms in each 12 samples of SSEs(L1,L9,L14 and L23) and regenerants(L9 and L23)(1.04,3.06,1.04,1.04,1.04 and 2.11%polymorphism),respectively.The results indicated SSEs and the regenerants remained high genetic stability after long-term subculture.(4) Transgenic plantlets production by Agrobacterium-mediated transformation of embryogenic calluses using GFP as a scorable marker.Via resuspending bacteria pellets, shortening the desiccation time of embryogenic calluses after infection,improving culture conditions for induction of resistant embryos,and applying the enhanced somatic embryogenesis system,transgenic plantlets were obtained.The results showed that, bacteria pellets resuspension had no significant effect,while shortening desiccation time of calluses after infection increased frequency of resistant calluses significantly.NAA promoted the induction and proliferation of resistant calluses,but appeared negative to resistant embryo production.MS medium with antibiotics was suitable for induction of resistant embryos,on which AgNO₃ addition seemed to have a positive effect,but it made embryos become aging and should be used carefully.Culturing under light significantly increased resistant embryo production.Decreasing the concentration of kanamycin inhibited the browning of SEs during maturation treatment.Transgenic plantlets derived from regenerated resistant embryos were proliferated and confirmed by green fluorescence assay,PCR and Southern blotting analyses,which indicated the stable insertion of GFP gene into the genome of C.camphora.(5) Study of genetic transformation of C.camphora with Barnase and PaFT genes. Introductions of Barnase,PaFT and Barnase-PaFT genes into embryogenic calluses were studied,and second transformation of resistant calluses already introduced Barnase with PaFT was carried out.The results showed that,Barnase possibly had been integrated to the genome of transgenic plantlets verified by PCR analysis.PaFT stimulated resistant SE development from resistant calluses,and PCR analysis using PaFT primer confirmed the introduction of PaFT into the genome of resistant embryos.