A Study On Adventitious Bud Regeneration And FmWIND1 Gene Cloning And Expression In Callus Of Fraxinus Mandshurica

In this study,the hypocotyls,roots,cotyledons and stem segments of Fraxinus mandshurica seedlings were used to induce callus and adventitious buds,optimize the factors affecting in vitro regeneration and establish callus regeneration technology.The contents of intro hormones,the expression of related genes and the changes of apparent modification in callus regeneration were analyzed in this paper.Furthermore,the relationship among them was analyzed at the same time.The mechanism of adventitious bud formation was analyzed.Gene cloning,sequence analysis and expression pattern of the key regulatory gene FmWIND1 were conducted in regeneration process.The main results are as follows:1.Factors affecting formation of callus and differentiation of adventitious bud.The callus induction rate and callus status of different 6-BA and TDZ hormone combinations among hypocotyl,root,cotyledon and tissue cultural seedlings.High concentrations of TDZ were beneficial to callus induction and low concentrations of 6-BA lead to the loose state callus formation.The TDZ played a crucial role in the formation of adventitious buds.The callus induction rates order of different explants was stem cotyledon(100%)>segment(98.54%)>hypocotyl(92.56%)>root(50.71%).Callus from stem segments and hypocotyls plantlets had the ability to regenerate,and the differentiation rates were 33.33%and 10.52%.The callus formed in the wound part of stem segment was more favorable to the formation and had more differentiation of adventitious buds.Compared with different basic media,MSB5 was more benefit to the differentiation of adventitious buds from stem segments plantlets cultured in vitro.2.Establishment of an efficient callus adventitious bud regeneration system and callus regeneration technology.The results showed that the suitable medium for callus induction and adventitious bud differentiation were C12 medium(MSB5+30g/L sucrose+7g/L Agar+5mg/L 6-BA+8mg/L TDZ+2mg/L glycine+0.lmg/L IBA+5%coconut water).The induction rates of callus and adventitious bud were 99.15%and 33.33%,respectively.The addition of 2.4mg/L DNA demethylation reagent 5-azacytidine(5-aza)and 0.15mg/L histone deacetylase inhibitor trichostatin A(TSA)could increase the rates of adventitious bud induction,which could reach 50.79%and 51.11%.3.Callus pathway regeneration technique in the stem segment.The optimum medium for inducing callus and adventitious buds was MSB5+30g/L sucrose+7g/L agar+5mg/L 6-BA+8mg/L TDZ+2mg/L glycine+O.lmg/L IBA+5%coconut water+0.15mg/L TSA,and the induction rate of adventitious buds was 51.11%after 30 day-culture.Transferring to the secondary proliferation medium(WPM+20g/L sucrose+7g/L agar+8mg/L 6-BA),the multiplication coefficient after a secondary 30 day-culture was 4.28.The epigenetic root rate of 3～5cm long strong adventitious branches into rooting medium(WPM+20g/L sucrose+7g/L agar+1.4mg/L IBA+0.7mg/L NAA)after 20 day-culture was 80.54%.After acclimatization,the seedlings were transplanted to the mixed matrix containing peat soil and vermiculite(peat soil:vermiculite=3:1),and the survival rate reached about 90%.4.Analysis of hormone contents and related gene expression changes during the differentiation of adventitious buds in callus.IAA,ZR and GA3 involved in callus formation during the callus formation in the stem segment of the tissue culture seedlings in Fraxinus mandshurica,and ZR was involved in the formation of adventitious buds.FmWIND1,FmYUC1,Fm WOX5,FmREV and FmPLT3 involved in the formation of adventitious buds.5.Preliminary exploration in the relationship between epigenetic modification and regeneration-related gene expression in callus regeneration process.The level of DNA methylation decreased in the formation stage of callus in the stem segment of the Fraxinus mandshurica,and the level of DNA methylation recovered again in the formation stage of adventitious buds.The activity of HDAC enzyme decreased with the increase of TSA concentration in the formation stage of indeterminate buds.6.The key gene FmWIND1 for callus formation and bud regeneration was cloned and bioinformatics analysis was carried out.The length of the FmWIND1 gene was 990bp.The FmWIND1 protein sequence encoded 329 amino acids,with 6.72 of isoelectric point,which was an unstable hydrophilic protein,and the sub-cells were located in the nucleus.Besides,there was one AP2 conserved domain in the FmWIND1 protein.The evolutionary tree of FmWINDl protein was constructed,and FmWIND1 was found to be closely related to WEND1 protein from European olive and sesame.Through Quantitative Real-time PCR(qRT-PCR)analysis,the expression characters of FmWIND1 gene in different tissues were:root>stem>flower>leaf,the rapid response rose in 12h after being stimulated by wound,and the expression increased again after the buds were regenerated.This work laid the foundation for micropropagation in tissue culture and the estab-lishment of genetic transformation teclinology of micropropagation,and enriched the re-generation theory.