| Brazilian rubber tree is the main source of natural rubber,and natural rubber is an important strategic naterial in China.For a long time,China’s natural rubber dependence on imports has exceeded 80%,which has become a bottleneck affecting the healthy development of the rubber industry.The establishment of efficient and stable rubber tree tissue culture system and genetic transformation system,the use of transgenic technology to carry out functional gene research and genetic transformation of Brazilian rubber trees is an effective way to speed up the improvement of rubber tree varieties and cultivate high-yield and high-resistance rubber tree varieties.Plant suspension cell line is a good material for plant genetic transformation because of its fast proliferation.good dispersibility and homogeneity,as well as the advantages of regeneration through somatic embryogenesis and avoidance of chimera formation.However,the establishment and maintenance of plant embryogenic suspension system depend on variety characteristics,culture conditions and culture methods.In this study,the somatic embryogenic callus induced by explants was used as the initial tissue material for the anthers of the rubber tree anti-wind high product Reyan 7-33-97,which was widely planted in the gum-growing area of China.A stable rubber tree embryogenic suspension cell line was established and regeneration plants of embryogenic suspension cells were induced.On this basis,the rubber tree embryogenic suspension cell mass is used as the receptor for genetic transformation.Through screening the transformation conditions of embryogenic suspension cells of rubber tree somatic embryos mediated by Agrobacterium tumefaciens,the genetic transformation system was established.Using this genetic transformation system to transform the rubber tree pathogen model to stimulate the immune PTI signaling pathway gene HbLYKl gene.The transformed callus and transformed enbryoid body of the transgenic rubber HbLYK1 gene were obtained by hygromycin resistance screening and molecular detection,and the next step was effective germination and seedling culture.Meanwhile,transformed buds of HbLYK3,a homologous gene of transgenic rubber HbLYK1 were used as scions to graft on the rootstocks of buds and seedlings,and the surviving transformed rubber HbLYK3 plants were obtained.lt laid a foundation for establishing an efficient and stable genetic transformation system of embryogenic suspension cells in rubber trees and improving the seedling formation and transplanting technology system of transgenic regenerated plants.The main results are as follows:1.The immature anther somatic embryos of Hevea brasiliensis Reyan 7-33-97 were used as explants and inoculated into embryogenic callus induction medium.The callus was cultured on improved MS+2,4-D 2 mg.L-1+KT 0.5 mg.L-1 for 40-50 days to obtain crisp embryogenic callus with bright yellow color,uniform growth and loose structure.It was a good initial callus material for establishing embryogenic suspension cell lines of Hevea brasiliensis.2.The crisp embryogenic callus was inoculated into liquid proliferation medium(MS+2,4-D 1 mg.L-1+KT 1.5 mg.L-1+NAA 0.5 mg.L-1+5%coconut juice)for suspension culture,8 d subculture once;During the suspension culture,the cultures were screened for callus granules through 20 mesh,100 mesh and 170 mesh stainless steel mesh screens.After the suspension culture was screened for 1-2 months,a yellow dense embryogenic suspension cell line with good dispersibility,small granules and rapid proliferation could be obtained;The suitable initial callus inoculation amount was 1.2 g.50 mL-1,and the appropriate subculture ratio of suspension cells was 1:3.3.The embryogenic suspension cell mass was cultured on solid differentiation medium(modified MS+KT 0.5 mg/L-1+0.2 mg/L-1 IAA+0.5 mg/L-1 GA3),The incidence of somatic microembryos was 68.7%;On modified MS medium without hormone added,The induction rate of somatic embryo maturation was 53.08%,and the germination rate of somatic embryo was 24.7%;Plants regenerated from somatic embryos were transplanted through seedling refining,and the survival rate was over 90%;Grafting of buds and rootstocks for shoots that cannot be rooted,and survival rate of grafted shoots with top buds as scions is over 95%,The survival rate of grafted seedlings with axillary buds as scions was about 50%,The frequency of plant regeneration of embryogenic suspension cell lines was effectively increased by supplementing regenerated plants with bud seedling rootstock grafting.4.Subsequent maintenance of embryonic suspension cell line and crispy embryogenic callus in a "liquid-solid-liquid" alternate culture method is an effective way to purify and rejuvenate embryogenic cell mass.Subculture for one and a half years in this way、the embryogenic suspension cell line still maintains good embryogenicity and high plant regeneration frequency.5.Using somatic embryonic suspension cells as genetic transformation receptor and GUS gene as reporter gene,the genetic transformation system of somatic embryonic suspension cells of rubber tree was established.The concentration of hygromycin was 30 mg/L-1,the concentration of carboxybenzylpenicillin was 300 mg/L-1,the concentration of OD600 was 0.8,the infection time was 8 min,Acetyl syringone（AS）100 and the co-culture temperature was 22℃.It was suitable to co-culture in solid medium for 3 days,and the transformation rate of resistant callus was 15%.6.The PTI innate immune pathway gene HbLYK1 was transformed by the somatic embryogenic suspension cell genetic transformation system,and the transformant callus and transformant cell embryoid bodies of the rubber HbLYK1 were obtained,They were detected by PCR and Western blot.It was confirmed that the exogenous gene rubber HbLYK1 had been successfully integrated into rubber genomic DNA.7.The transformed buds of the HbLYK1 homologous gene HbLYK3 gene were used as sciones,and the transformed bud sprouts were grafted,and regeneration plants of transgenic rubber HbLYK3 gene with successful grafting survival.In conclusion,a stable somatic embryogenic cell suspension system and its genetic transformation system were preliminarily established in this study,Improve the seedling system of rubber tree transgenic plants,which can be further optimized to establish a genetic transformation system for efficient and stable somatic embryogenic suspension cell lines of rubber trees.To provide effective technical methods for the future research of functional genes of rubber trees,purposeful genetic improvement and creation of new rubber tree germplasm;Transgenic plant materials transformed with HbLYKs gene,a PTI signaling pathway gene stimulated by transforming the pathogenic pattern of rubber trees,also laid a foundation for the creation of new rubber tree germplasm resources with disease resistance. |
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