Studies On Efficient Regeneration And Gene Editing Systems In Cucumber (Cucumis Sativs L.)

Cucumber（Cucumis sativus L.）is an annual climbing herb in Cucurbitaceae family.It is also an extremely sigificant vegetable crop.It is widely planted in temperate and tropical regions.The sex of cucumber flower is one of the main limiting factors for its yield.The sex determination is the result of selective retardation of bisexual flower primordia in cucumber to parthenogenesis.Previous studies have shown that the floral nature of cucumber is mainly affected by endogenous ethylene levels.Ethylene usually has the function of inhibiting stamen development and promoting pistil development.Cucumber Cs ACS2 and Cs ACS11 encode 1-aminopropane-1-carboxylate synthase（ACS）and play key roles in cucumber flower sex regulation.In this study,both two are used as target genes when we developed regeneration,genetic transformation and gene editing systems in cucumber.The lower efficiency in genetic transformation is the bottleneck for cultivating high-yield cucumber varieties through genetic engineering.Traditional cucumber gene editing system has many problems,such as extremely low transformation efficiency and limited gene editing vector delivery methods.In this study,"Xintai Mici"cucumber was used as the plant material.On the one hand,an efficient regeneration system and a genetic transformation system of cucumber basing on tissue culture were established to achieve the genes editing of Cs ACS2 and Cs ACS11.On the other hand,pollen transfection mediated by nano-magnetic particles in cucumber was explored,which will provide an alternative strategy for cucumber genetic transformation.The main results are as follows:1.Cucumber regeneration system1)Cucumber cotyledonary nodes were used as explants.The optimal hormone combination for adventitious bud induction in cucumber is MS with 1 mg/L 6-BA+0.5 mg/L ABA+2 mg/L Ag NO₃+0.065 m M L-glusate.The regeneration frequency of cucumber was 100%,and the mean of regenerated shoots per explant was 9.5.2)With different concentrations of FPX hormone,the regeneration frequency of cucumber was 100%at 500μM FPX,and the average number of regenerated buds per explant was increased to 10.9.2.Agrobacterium-mediated genetic transformation1)0.5 mg/L hygromycin could be used for selection.2)After testing the inhibitory effect of Temetine at different concentrations,400 mg/L was selected as a bacteriostatic agent.3)Agrobacterium strain EHA105 was selected for transformation with better infection ability than other strains.4)The best conversion method is to evacuate the syringe for 10minutes during infection.5)The resistant bud rate can reach 1.3%when the selection medium supplemented with 15μM FPX.6)The best screening medium for this experiment is 1/2 MS with 1mg/L 6BA+0.5 mg/L ABA+2 mg/L Ag NO₃+0.065 m M L-glusate+0.5 mg/L Hygromycine+15μM+400 mg/L Timentin.7)Twenty-one lines with hygromycine resistance were obtained using agrobacterium-mediated transformation.Two candidate plants were confirmed by PCR using GFP primers.3.Genetic transformation system of pollen transfection mediated by nano magnetic particles1)Introduce nano-magnetic particles and carrier complex into pollen.50 nm nanometer magnetic transfection particles can the germination pores for expression.2)The optimal transfection time is 5 minutes.Using 1 volume of magnetic nanoparticles into pollen,the transformation efficiency is1.33%,and using 5 volumes of magnetic nanoparticles,the transformation efficiency is 1.44%.The difference between these two is insignificant.3)After transfecting,pollen grains were pollinated to the female flower of cucumber,and 453 seeds were finally obtained.Unfortunately,none of the planlets are transgenic lines which were verified by PCR for T1 generation plants.