Studies On Origin Of Secondary Somatic Embryogenesis And Detection Of Somaclonal Variation Of Regenerated Plants Of Hevea Brasiliensis

The innovation of the rubber tree planting material is an important driving force for the development of the natural rubber industry.With fast-growing, high-yield, high resistance characteristics, Hevea brasiliensis self-rooting juvenile-type clone is a new generation of rubber tree planting material. It has promising application and will be the future main planting material. The establishment of Hevea brasiliensis secondary somatic embryogenesis technological system laid a solid technical foundation for the production and promotion of the rubber tree planting material in our country and the world.To study the origin of Hevea brasiliensis somatic embryogenesis and the stability of regenerated plants genetic,the material of Hevea brasiliensis Reyan7-33-97clone secondary embryos and regenerated plants were used in this study,which of the origin of secondary germ cell observation, the chromosome number of10multiplication generations, the content determination of DNA and the EST-SSRs variation analysis research were finished. The experiment results provided an onslaught of useful information for rubber new planting material(Hevea brasiliensis self-rooting juvenile-type clone) production and application.The dominate results were listed as followings:1. Paraffin section technology was used to observe the development of secondary embryos, the results showed that Hevea brasiliensis secondary embryos derived from single cells and mainly developed from scutellum epidermal cells.2. Studying the relationship between embryogenesis and culture time found that a large number of callus induction started from the12th day and on the20th day reached the highest number of embryogenesis. After1month differentiation of somatic embryo, cotyledinous embryo began to mass produce. Simultaneously the total number of globular embryogenesis, heart-shape embryogenesis and the torpedo embryogenesis are close to the number of cotyledons embryogenesis in the differentiation stage of somatic embryo, which indicated that the maturity of somatic embryo can be completed under the culture conditions.3. Normal chromosome tabletting method and FCM were applied to detect the chromosome number of10multiplication passages and the change of ploidy. The results showed that the chromosome number was36from1to10generation and the content of DNA was the same as the reference, which indicated that somatic embryo strains chromosome mutation of those10multiplication passages did not happen. 4.12primers were applied to analysis molecular fingerprinting of somatic embryo plants of10multiplication passages. A total of3060loci were detected, including35mutation sites and the total loci aberration rate was1.14%. A total of22mutation strains were detected in the90regenerated plants generated from1to10passages. The mutation strain rate was24.4%and the frequency of single plant mutation loci above3.0%accounted for11.1%of the total testing plants. The mutation rate was increased with increasing secondary embryos cycle-index and peaked at2.61%in passage3. The biggest drop occurred between passage4and6with0.98%and0.65%mutation rate respectively. Both of the mutation rates dropped to0.65%in passage9and10with the beginning dropped from7th passage. The DNA loci mutation rate was2.94%-8.82%in the detected22mutation strains.The strains with DNA loci mutation rate above3.0%accounted for45.5%of the total mutation strains,12strains below3.0%(accounted for54.5%),7strains between3.0%and6.0%(accounted for31.8%) and only3strains above6.0%(accounted for13.6%). The average genetic distance (GD) among passages was0.0119, illustrating small genetic differences between passages. The average genetic line (GI) was0.9881, illustrating the high similarity coefficient among passages. Clustering result between individual showed that the height of90regenerated plants was similar to Reyan7-33-97Clones budding seedlings. The average genetic coefficient between reference and regenerated plants was0.9891,75.6%(68/90) of regenerated plants and Reyan7-33-97Clones budding seedlings reached1.5. The function of the12EST-SSRs loci gene was analyzed and the primers S98and S159genes with highest mutation rate were related to the resistance, which indicated that the stability of stress-resistant gene determined mutation rates.