Studies On The Efficiency Factors Of Gene Transformation Via Pollen-Tube Pathway In Walnut

In this study, the seed embtyo of walnut (Juglans regia L.) was cultured to improve the seeding and proliferation rate. The growth of continued generation of Liaoning 5 and embryo of Qingxiang were treated with Hygromycin and Kanamycin in different concentration to research the impact of selection pressure screening, further improve the gene conversion system.Using the pollen tube pathway transformation, exogenous DNA was transferred into walnuts cultivar Zhonglin 1, Lvbo, Jinlong and Qingxiang. This article was intended to introduce the optimum introducting period, methods and concentration of DNA for the direct introduction of the exogenous DNA into plant genome and the factors that affect the effectiveness of the pollen-tube pathway, to further develop the use of genetic engineering and improve the status on the efficiency of regeneration and transformation.1. The stage of embryo germination: optimal germination mediun was DKW medium with 6-BA 0.5~1.0 mg/L+IBA 0.01 mg/L +sugar 55 mg/L for the embryo culture. The suitable proliferation mediun was DKW medium with 6-BA 1.0~1.5 mg/L+IBA 0.03 mg/L for adventitious bud.2. The effects of Hm and Km on subcuiture plantlets and embryo: 18 mg/L of hygromycin and 300 mg/L of Kanamycin of were the suitable screening concentration for the growth of subcultured explants of Liaoning 5; 20 mg/L of Hyg and 150 mg/L of Kan were added to as suitable screen pressure in embryo culture of Qingxiang.3. The suitable transformation time by dropping was 14~48 hours after pollination and the pollen tube extended to base part of the style. The optimum dripping time of introducing exogenous DNA into its ovule was approximately 24~48 hous after pollination, the pollen tube extended to embryo sac.4. Introduction methods showed different influence on setting rate of recepting plants, ovary injection method gave lower setting rate than stigma dropping. The Introduction method was stigma dropping after cut-style, which was helpful exogenous DNA to embryo sac and reduced the damage to guarantee the Rate of survival fruit and conversion rate.5. The best concentration of DNA was 200-500μg/mL, the rate of expression of GUS was gained with 13.9%-14.9% .6. The transgenic cotyledons were further tested by GUS analysis, partly showed GUS+. The transgenic fruits had been growing in culture added 15mg/L of Hm, the transgenic lines were further tested by PCR analysis. Four plants showed specific positive band as same as the plasmid control,the result indicated that the exogenous DNA has been integrated into the genome.