Study On Peach Anther Culture And Genetic Transformation Of Plum

Peach belong to family of Rosaceae(Rosaceae)and is one of important economic crop in China.Owing to limitation of the traditional breeding to the development of peach industry in China,it is necessary to develop a high-efficiency method for peach breeding.With the rapid development of modern biotechnology,genetic transformation has been used frequently to improve the work of fruit tree breeding.However,the technology of regeneration and genetic transformation of peach are not high-efficiency and stable,which limits significantly improvement of peach germplasm by genetic transformation.In this study,in order to establish efficient regeneration system of peach,the anthers and petals from three peach cultivars,including ‘qiumihong’,‘huangshuimi’ and ‘dajiubao’ were selected to establish sterile system,and then the best inducible callus culture medium were studied and screened.In addition,the genetic transformation of plum was also researched in this study owing to the same genus plum and peach belong to.Hypocotyl slices and cotyledons of ‘Cui Hong’ and and ‘Hei Baoshi’ plum were used as explants,and the expression and dyeing level of GUS gene by different strains and different concentrations were explored.Both of these experiments will lay the foundation for the subsequent verification of peach gene function and the innovation of germplasm resources through genetic transformation.The main contributions of this work are as follows:1.The culture of peach anther and induction of callus(1)The best tissue is the anther during mononuclear phase from small flower bud of peach.During this time,the anther color is yellow with red dots.(2)Submerging explants in 75% alcohol + 0.1% Na Cl O for 5 min is the best way to disinfect the flower organs.(3)The medium suitable for anther and petal callus induction is medium 1 containing NN69,60 g·L-1 Suc,3 g·L-1 plant Gel,2.0 mg·L-1 6-BA and 1.0 mg·L-1 2,4-D and medium 2 containing NN69,60 g·L-1 Suc,3 g·L-1 plant Gel,1.0 mg·L-1 6-BA and 0.5 mg·L-1 2,4-D,respectively.Comparing callus induction rate of different varieties,the results suggest that the callus formation rate of ‘Qiumihong’ is relatively high.For instance,the formation rate of the anther callus is as high as 22.08% in the medium 1.And,the formation rate of its petal callus is up to 100% under the condition of 2.(4)Three kinds of callus statuses were identified based on the morphology and histology.embryogenic calli with Yellow compact and green compact were identified and light yellow loose were non-embryogenic.2.The study on the genetic transformation system of plum(1)Genetic transformation of ‘Cui Hong’ PlumUnder the condition of the EHA105 strain with the 0.5 concentration,hypocotyl sections from ‘Cui Hong’ Plum were used as explants for genetic transformation.The GUS transient expression rate of ‘Cui Hong’ Plum hypocotyls is 73.33%;the GUS transient expression rate of the ‘Cui Hong’ Plum cotyledon up to 100%.Among them,the GUS histochemical dyeing with deep blue state is 62.5%(2)The effect of different medium on the browning of hypocotyl slices of ‘Cui Hong’ PlumThe effect of PVP and the different ratio between TDZ and IBA on anti-browning of the hypocotyl slices of ‘Cui Hong’ Plum were explored during the process of genetic transformation.The study shows that the medium suitable for hypocotyl regeneration is 3/4MS medium with IBA 0.05mg·L-1+TDZ 3.0mg·L-1+Tim 300mg·L-1+Kan 80mg·L-1 + PVP 1.0 g·L-1.(3)The genetic transformation of ‘HeiBaoshi’ plum hypocotyl slicesUnder the condition of the GV3101 strain with the 0.5 concentration of bacteria(OD600),hypocotyl sections from ‘Hei Baoshi’ plum were used as explants for genetic transformation.The GUS transient expression rate of the ‘Hei Baoshi’ plum hypocotyls is 60% while the GUS transient expression rate of the ‘Hei Baoshi’ plum cotyledon up to 100%.Among them,the GUS histochemical dyeing with deep blue state is 79.24%.