Research In Effective Improved On The Technique Of Bt Gene CryIAc Transforming Capsicum By Agrobacterium Tumefaciens

Pepper(Capsicum annuum) is a kind of major vegetables in agriculture.During the recent three five-year-plans,China regarded breeding of pepper which can resist disease as the important projectAs the major harming pepper pest,Helicoverpa assulta,a kind of papilio xuthus insect,whose larva feeds on flower,bud and fruit of pepper and can make lots of rot fruit to drop the yield.CryIA(c) gene,a kind of Bt genes expressing insecticidal crystal protein of Bacillus thuringiensis,codes 133kD crystal protein and just has a insecticidal scope of Papilio xuthus insect.So we improved the technique that the CryIA(c) gene carried in the T-DNAs of binary vectors plasmid pKUC which contain nptII gene and gusA gene,were used to transform the explants of pepper via Agrobacterium transformation.Through stringent selection of the infected explants on the medium added with Kanamycin,the transgenic pepper plants were regenerated from the Kanamycin resistant explants,then test the instantly express of GUS gene to ascertain the CryIA(c) gene whether has integrated into pepper's genome;test the exist of Bt protein to ascertain the CryIA(c) gene whether could express normally;then search for the condition of transform explants'rootage,establish the basement for breeding of pepper resistant to insects,has active ecological significance and economical significance.1.The improved on the technique of Bt gene CryIAc transformingcapsicum by AgrobacteriumWhen transforming capsicum by Agrobacterium tumefaciens,research and analysis five factors:transform material, concentration of Agrobacterium and infect time , ablution procedure , the addition of AgNO3, antibiotic selection concentration.Regarding the frequency of Km resistant callus explantsstandard,result shows: transform material affect the transform efficiency mostly, the concentration of Agrobacterium and infect time , ablution procedure determine on the explants' putridness rate and contamination rate.We have improved the technique of Bt gene CryIAc transforming capsicum by Agrobacterium tumefaciens.Establish a universal high efficient and stability pepper genetic transformation system.2.Genetic transformation explant detection and identificationThrough stringent selection of the infected explants on the medium added with Kanamycin,eliminated some explants which without resistance to Kanamycin.Then the expression of GUS gene of the plants,regenerated from the Kanamycin resistant explants,were detected by histochemical method.Blue was found in organs of transformed plants.Prove that the report gene gusA carried by plasmid pKUC has been transformed into the Km resistant callus explants.Precise detect the CrylAc gene expression condition in the transformed explants with Envirologix QuantiPlate Kit for CryIAb/CryIAc.it shows the gene CrylAc that integrated in capsicum genome could express efficiently.3.The preliminary explore to the condition of genetic transformation plants'rootageThe pepper is regarded as the plant that especial hard to transform.The high efficient genetic transformation system is not already establish,we ameliorate the transform material and collocate with right medium,induce the adventitious root from Cotyledon petiole successfully.Establish the basement for breeding of pepper resistant to insects for production.