| Mei flower(Prunus mume Sieb.et Zucc.)native to China has high ornamental value and long cultivation history.However,it is limited to its cold resistance that it is difficult to plant and apply for overwintering in most open areas of North China.The use of genetic engineering techniques can accelerate and accurately develop Mei genetic resources for cold resistance.But,few mature genetic transformation systems are currently reported.In this study,using the cotyledon of the P.mume ’Xue Mei’ ×P.mume’Fenpi Gongfen’ hybrid as material,based on the former progress of research group,the cotyledon size,storage temperature,6-BA and ZT concentration,activated carbon,gelling agent and sugar source were screened to optimize the conditions for regeneration of adventitious buds.The genetic transformation efficiency was improved from the optimization of pre-culture time,infection time,antibiotic concentration,and screening of hormone combinations in subculture medium.Using this genetic transformation system,via agrobacterium-mediation PmCBFs genes were transformed into mature embryo cotyledons,and,the positive rooting and grafted seedlings identified by PCR were obtained.In addition,the T2 Arabidopsis plants over-expressed PmMYBs genes,were used as materials for cold resistance genes identification,and three cold-tolerance genes were initially screened.The T3 generation over-expressed Arabidopsis plants of PmMYB3R-5 were tested for physiological indexes under cold stress.The main results were as follows:1.Optimization of regeneration system of Mei flower cotyledon.The effect of immature embryo cotyledon size on adventitious bud induction was analyzed,and the results showed that the adventitious bud induction rate was the highest(47.37%)when the cotyledon size was 5.5 mm;Comparing the effects of the regeneration of mature embryo cotyledons and elongation of adventitious bud,there was no significant difference between room and 4℃ low temperature;With the extend of storage time,the regeneration rate and elongation ratio decreased overall;Comparing the effects of adding different hormones and activated carbon on the adventitious bud induction of mature embryo cotyledons,the results showed that on the medium supplemented ZT 1.0mg/L+TDZ 1.0 mg/L+IBA 0.8 mg/L hormone combination,the regeneration rate is the highest,reaching 86.90%;Activated carbon had a greater effect on the adventitious bud induction of mature embryos cotyledons;When not added,adventitious buds are regenerated;After addition,there were only root induction,and at low concentration(0.25g/L),more roots are produced;The effects of agar+sucrose,gel+glucose on the regeneration of mature embryos cotyledons and elongation of adventitious buds were analyzed,and,the results showed that the regeneration rate of mature embryo cotyledon and adventitious bud elongation were better on agar+sucrose.2.Optimization of genetic transformation system of Mei flower mature embryo cotyledons.Based on the predecessors,the GUS transient expression rate of cotyledons under different pre-cultivation time(1d,2d,3d,4d)and Agrobacterium infection time(10,15,20,25,30,40,60min)were compared;The results showed that the highest expression rate(46.88%)was obtained at 3d pre-culture and 25min infection time;The optimal concentration combination of Cef+Tm was screened,and it was found that the best combination was Cef 200 mg/L+Tm 200 mg/L,which can effectively inhibit Agrobacterium contamination,meanwhile reducing the total concentration of antibiotics;The effect of different hormone combinations on the growth of resistant buds was analyzed,and the best hormone combination was 6-BA 1.Omg/L+IBA 0.1 mg/L;The application of test-tube seedling grafting technology improved the survival rate of resistant buds(88.46%),overcame the difficulty of resistant buds to take root and develop plantlets,and shorten the cultivation time.3.Transformation of PmCBFs gene with mature embryos cotyledons of P.mume.Via mediation of Agrobacterium carrying overexpression of PmCBFa,PmCBFb and PmCBFc gene vectors,after 40 days of selective cultivation,182 resistant buds were obtained from 4,723 infected explants;The DNA of 122 resistant buds was extracted and tested by PCR,36 of which were positive(15 PmCBFb plants and 21 PmCBFc ones);Two PmCBFb over-expressed plantlets have rooted and transplanted;PmCBFc over-expressed plantlets have been rooted and grafted.4.Cold resistance screening of over-expressed PmMYBs genes and analysis of cold resistance of PmMYB3R-5 gene.Seven T2 generation high-expression Arabidopsis lines were identified under cold stress treatment;The results show that compared with the wild type,the survival rate of the plants over-expressed Pm023786,Pm013277 and Pm015880 were significantly improved,and the plants over-expressed Pm005332,Pm011057,Pm014372 and Pm024326 had no significance;From the three over-expression Arabidopsis plants with improved survival rate,the over-expression plants of PmMYB3R-5(Pm023786)were selected to obtain the T3 lines;Morphological observations of T3 lines revealed that the number and length of rosette leaves increased when flowering;compared with the wild type,the relative electrolyte leakage and ROS accumulation of the over-expressed plants decreased under cold stress,while the antioxidant enzyme activity increased,indicating that the PmMYB3R-5 gene may increase the cold resistance of Arabidopsis thaliana by increasing antioxidant enzyme activity. |
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