Establishment And Optimization Of Peanut Tissue Culture And Genetic Transformation System

China is a major peanut producer in the world,ranking first in terms of unit yield,total output,and export volume.Since the 1990s,the peanut industry has developed rapidly.With the in-depth identification of peanut germplasm resources and the acceleration of breeding work,the variety and quantity of cultivated peanut varieties are increasing day by day.Parental varieties that combine high yield and quality breeding objectives are increasingly similar in blood relationship.The incompatibility between cultivated and wild peanut resources has limited conventional breeding and narrowed the genetic basis between varieties,The number of similar varieties increases and the convergence becomes stronger.The development of molecular biology and the application of plant transgenic technology can accurately and directionally transfer beneficial target genes into crops to improve their resistance and quality,providing an effective means for breeding excellent new peanut varieties.In this study,epicotyl of four peanut varieties（pearl bean type:9326,JNH 7;multigrain type:JNH 8,Northeast King）suitable for popular cultivation in Northeast China were used as explants,and four test treatments were set up（A1:1%sodium hypochlorite,disinfection treatment for 7 min,A2:1%sodium hypochlorite,disinfection treatment for 15 min,A3:10%sodium hypochlorite,disinfection treatment for 7 min,A4:10%sodium hypochlorite,disinfection treatment for 5 min）.To investigate the effect of different sodium hypochlorite concentrations and times on the disinfection of epicotyl and to conduct a study on the regeneration technique of peanut somatic cell embryogenesis plants,as well as to analyze the effect of genotype and hormone ratios in peanut tissue culture.Using the epicotyl and callus of mature peanut as receptors,agrobacterium tumefaciens was used to carry out gene transformation in peanut,and the main factors affecting the transformation rate were discussed in order to establish an efficient and stable peanut gene transformation system.The test results are as follows:1.Four different gradients of disinfection were set up with epicotyl of JNH 7.A1had 97.33%germination rate,23.33%infection rate and 2.67%mortality rate.A2 had94.67%germination rate,12.22%infection rate and 5.33%mortality rate.A3 had78.89%germination rate,5.33%infection rate and 18.89%mortality rate.A4 had96.89%germination rate,5.33%infection rate and 3.11%mortality rate.The germination rate of A4 was 96.89%,the infection rate was 5.33%,and the mortality rate was 3.11%.The experimental results showed that A4 maintained the same germination rate with 97.33%germination rate but the lowest staining rate and mortality rate of 5.33%and 3.11%,respectively,compared to the other treatments.Therefore,it can be inferred that A4:10%sodium hypochlorite disinfection for 5 min was the most effective.2.Determination of the optimum hormone ratios for the induction media of different genotypes of peanut:Hormone ratio:The optimal medium for callus induction in JNH 7 was determined to be MS-B5+Pic 3 mg/L+Gln 3 mg/L,with an embryogenic callus induction rate of 68.66%;The optimal medium for callus induction of 9326 was MS-B5+Pic 3 mg/L+Gln 3 mg/L,with an embryogenic callus induction rate of 65.33%;The optimal medium for callus induction of JNH 8 was MS-B5+Pic 3 mg/L+Gln 2 mg/L,with an embryogenic callus induction rate of66.67%;The best medium for callus induction of Northeast King was MS-B5+Pic 3mg/L+Gln 2 mg/L,with an embryogenic callus induction rate of 66.67%.3.Hormone ratios for different genotypes of peanut somatic cell embryo induction medium for seedling formation were determined:The optimal medium for bud induction of JNH 7 was determined to be MS-B5+NAA 0.1 mg/L+6-BA 1 mg/L,with the highest seedling formation rate of 48.00%;The optimal medium for bud induction of 9326 was MS-B5+NAA 0.1 mg/L+6-BA 0.4 mg/L,with the highest seedling rate of 47.00%;The optimal medium for bud induction of JNH 8 was MS-B5+NAA 0.1 mg/L+6-BA 3 mg/L,with the highest seedling rate of 48.67%;The best medium for inducing Northeast King bud is MS-B5+NAA 0.1 mg/L+6-BA 3mg/L,with the highest seedling rate of 50.00%.4.Optimization of Agrobacterium mediated genetic transformation conditions:Infestation was carried out at the concentration of OD₆₀₀:0.6-0.8 in the bacterial solution.The results showed that the optimal infection time for callus tissue and epicotyl of multi grain type（JNH 8,Northeast King）was 15 min,and the optimal co culture time was 3 d.The optimal infection time for callus of pearl bean type（9326）is12 min,co culture time is 2 d,explant infection time is 12 min,and co culture time is3 d.The optimal infection time for callus and explants of pearl bean type（JNH 7）is15 min,and the optimal co culture time is 3 d.