Effect Of Different Culture Condition On Genetic Variation In Gala Apple

This work was mainly studied germinating of Gala on germ-free condition, callus induction, subculture, and monitor of variation in molecular level.The main results indicated:1. The sterilizing methods of the buds sprouted from the cutting shoots cultured in water in a greenhouse were investigated. The results showed that the optimal sterilizing method of buds was treating with 0.1% HgCl₂ plus Tween-20 (2 drops) for 7 min; the conditions of 4 g · L^-1 PVP, upper dark treatment for 10 days and changing medium every three day were beneficial to lightening browning; the most suitable medium for buds induction was MS supplemented with BA 2.0 mg· L^-1 and NAA 0.1 mg · L^-1,for multiplication was MS supplemented with BA 0.5 mg · L^-1 and NAA 0.1 mg · L^-1 the suited subculture time was 28-30 days.2. The optimal medium on the callus induction from Gala leaves of single bud system was MS containing 30 g · L^-1 sucrose,2,4-D 2.0 mg · L^-1 NAA 0.5 mg · L^-1 and BA 2.0 mg · L^-1, and the rate of callus induction reached 93.7%; 2,4-D was most important of all the hormonal regulants; It would take 14-21 days in darkness that the callusogenesis of the explants was switched on, then the callus amplification and its further differentiation must be in light;the eligible time of callus subculture was every 15-30 day.3. During the course of SSR-PCR amplification, orthogonal experiment of the factors of the system such as concentration of DNA,Mg²⁺, Taq DNA polymerase,dNTP and primer was designed.Then we established steady SSR-PCR system.Amplification reaction volumes were 25uL,each containinglO x Buffer 2.5 μ L,MgCl₂(25mM)4μL, dNTP(2.5 mM)4μ L, primer(10 μ M)1μ L, DNA35ng and Taq DNA polymerase 1U.One primer named CHO2D12 in twenty-six showed most polymorphism alleyes.Some variation of callus in different conditions was detected by using SSR-PCR technique. The influence of different substance in turn was sucrose, 2, 4-D,NAA,BA, the main level that callus variation rate was low of which was respective sucrose 30 g ·L^-1, 2,4-D0.05 mg · L^-1,NAA 0.5 mg ·L^-1 and BA 2.0 mg · L^-1; and the main level that callus variation rate was high of which was respective sucrose 10 g · L^-1,2,4 -D 2.0 mg · L^-1,NAA 0.1mg · L^-1 and BA 4.0 mg · L^-1;the callus variation in molecular level obviously increased along with the culture time.