Establishment And Optimization Of The Peanut Cotyledon Regeneration System And Transformation System

Peanut(Arachis hypogaea L.) is an important economic and oil crop.Disease,insect and pest grass are easily affecting the productivity and quality of peanuts.Peanut varieties Yuanza9102、Hua410and Hua416were used for the study onestablishment of peanut cotyledon system,induced bud clump of age,from screeningsuitable concentration of Km and explore suitable infection of bacterial concentrationand infection time and total culture time4aspects improve the Agrobacteriummediated genetic transformztion efficiency in peanut.This study is first to establish a system of efficient plant regeneration in tissueculture of peanut.Base on the regeneration system,Agrobacterium tumefaciensmediated transformation method Was innoduced into peanut.Through GUS stainingshowed the Agrobacterium plasmid into peanut,obtained transgenic plant.Results wereshowed as following:1.Suitable for induction of bud medium hormone ratio: setting the bud inductionmedium with different hormone concentration ratio, a total of15treatment.Cotyledonas explant in induction medium on the induction of multiple buds formation, about2weeks to observe the bud induction rate. The results showed that,(1)The frequenciesof adventitious shoot formation were different due to the explant types.the youngleaflets were obviously superiorto epicotyl,hypocotyls and cotyledon.(2) The resultshowed that the medium supplemented with1mg/L2,4-D and0.3mg/L TDZ wasuseful for adventitious shoot formation of Yuanza9102cotyledon,the highestpercentage of regeneration is70%.The calli with adventitious shoot were transferred onto the MS mediumsupplemented with6-BA(0,2,4mg/L) for stem elongation.The suitable concentrationof6-BA was considered as0mg/L.The frequency of plant regeneration in Hua410、Hua416and Yuanza9102reached to84%,80%,76%.Plantlets were transferred tomedium supplemented with1mg/L NAA for root formation.2.Research on selection antibiotic shows:The feltration medium supplementedwith150mg/L Km,can efficiently inhibit shoots regeneration from cotyledon inpeanut.The bacterial strain mediun supplemented with300mg/L Cef can efficientlyinhibit tested bacterial strains,LBA4404growing and its effect on frequency ofmultiple buds formation from cotyledon does not show strong difference. 3.Study on influencing factors of peanutg enetic transformation:(1) Difference ofbacterial concentration affected frequency of genetic transformation,suitable bacterialconcentration was OD600=0.5.(2) Suitable infection time was15min.(3)Co-cultivationfor3d is suitable for transformation.4.Glucnase gene mediated by Atumefaciens was introduced into peanutcultivar,yuanza9102.PCR analysis confirmed the integration of GUS gene into thegenome of transgenic plants.5.The tissue culture and transformation condition optimization of Agrobacteriummediated, put forward LBA4404Peanut Genetic transformation technique processes:the peanut varieties far miscellaneous9102ripe and plump seed.After surfacedisinfection,useing pre-culture for2d,The precultured explants were infected byAgrobacterium tumefaciens LBA4404(OD600=0.5) for15min. and then co-culturewith Agrobacterium for3d, explains were transferred to MS+l.0mg/L2,4-D+0.3mg/LTDZ+150mg/LKm+300mg/LCef to induce bur tuft.After2weeks,calli with budswere transferred to MS+150mg/LKm+300mg/L Cef to induce bur elongate.They weresubcultured for2weeks.When the shoots grew into3-4cm in length,they were cutfrom the calli and transferred into root fomlation medium MS+150mg/LKm+300mg/Lcef.