Establishment Of Somatic Embryogenesis Regeneration System And Preliminary Study On Genetic Transformation In Litchi Chinesis ‘Heiye’

Litchi（Litchi chinesis Sonn.）belonging to Sapindaceae family is a south subtropical famous green fruit trees in south China.Litchi is generally modified by conventional methods.With the progress of biotechnology,it has become a trend using modern breeding methods such as cell engineering and gene engineering to improve litchi germplasm.Anther and immature zygotic embryos at different periods of Heiye were used as experimental materials to induce callus,then the callus were used to establish in vitro regeneration system by somatic embryogenesis pathway.Efficient transgenic system of Heiye litchi using embryogenic callus was optimized by the GUS gene transient expression.The important results were described as follows:（1）Using Heiye anther as explant,the best medium for callus induction and subculture were MS medium containing 1.0 mg l^-1 2,4-D,0.5 mg l^-1 TDZ,30 g l^-1sucrose,7.0 g l^-1 agar and MS medium containing 1.0 mg l^-1 2,4-D,0.5 mg l^-1 6-BA,30g l^-1 sucrose,7.0 g l^-1 agar,respectively.The results also showed that the quality of the induced callus by the anther was poor and the callus was watery and white.（2）The immature zygotic embryos at 25 days after anthesis of Heiye were more suitable for callus induction.The results showed that the best medium for callus induction and subculture were both MS medium containing 2.0 mg l^-1 2,4-D,30 g l^-1sucrose,7.0 g l^-1 agar.There are two kinds of callus in this research.Non-morphogenic callus was whitish and watery with weak proliferation ability,while embryogenic callus were light yellow and has strong proliferation ability.It is composed of uniform cells with obvious cell wall.（3）The optimal medium for induce somatic embryos induction was MS medium containing 0.5 g l^-1 AC,30 g l^-1 sucrose,7.0 g l^-1 agar.The highest induction rate reached to 100%,and most somatic embryos were normal,the normal somatic embryo rate as high as 79.11%.All the normal somatic embryos were potential to germinate into regeneration plants.The somatic embryo medium was 1/2MS added with 1.0 g l^-1AC,20 g l^-1 sucrose,8.0 g l^-1 agar.Somatic embryos could root and germinate,the germination rate was 0.6%.The optimal regeneration medium was MS medium containing 1.0 g l^-1 AC,1.0 mg l^-1 ZT+30 g l^-1 sucrose and 7.0 g l^-1 agar,and the regeneration rate was 0.9%.（4）Embryonic callus consist of big,irregular cells with loose cell structure and visible cell walls.Somatic embryo exhibited various morphologies,for example globular,heart,torpedo and cotyledonary embryos.They contained embryonic cells that circle,uniform size and compact.（5）The factors affecting gene transfer efficiency in callus of Heiye through agrobacterium tumefaciens were studied using GUS gene.The results showed that the optimum microbial suspension was OD₆₀₀=0.8.Infection for 2 hours will get the best transformation effect.The genetic transformation rate was up to 85.28%.