Optimization Of Leaves Regeneration And Genetic Transformation System Of Several Blueberry Varieties

Blueberry is a fruit of great economic and health value.With the increasing demand of market diversification,it is urgent to improve the yield and quality of blueberry fruits through biotechnology breeding.Compared with the traditional method of cross breeding,agrobacterium-mediated plant genetic transformation in biotechnology has been successfully used in a variety of fruit trees,but there are still some problems such as low efficiency and difficulty in the transformation in blueberries.In this study,highbush blueberry varieties‘Northland',‘Millennia',‘Sierra',‘Legacy'and‘Bluesouth'were used as materials,through different hormone combination and concentration test,optimization of blueberry leaves adventitious bud regeneration system and establish the blueberry leaf callus regeneration system;by analyzing the effects of factors such as the infection concentration and time of agrobacterium tumeba,the number of days of co-culture,and the concentration of bacteriostasis agent,the genetic transformation system of blueberries was optimized to provide technical support for the subsequent research on the gene function and breeding of blueberries.The main findings are as follows:?1?The regeneration adventitious bud system of leaves of 4 blueberry varieties‘Northland',‘Millennia',‘Sierra'and‘Legacy'was optimized.WPM+4.0 mg/LZT+30 g/L sucrose+6.5 g/L Agar was selected as the best medium for inducing adventitious bud of 4 blueberry varieties.The rate of adventitious bud induction was 60%in‘Northland',81.67%in‘Millennia',83.33%in‘Sierra'and26.67%in‘Legacy'.?2?The leaf callus of the two cultivars of‘Northland'and‘Legacy'were induced successfully.The optimal leave-induced callus medium obtained was WPM+1.0 mg/L 2,4-D+0.4 mg/L 6-BA+30 g/L sucrose+6.5 g Agar.The effect of dark culture was the best,and the induction rate of leaf callus could reach 100%.?3?The leaf callus of the two cultivars of‘Northland'and‘Legacy'were successfully subcultured.The optimal callus subculture medium was WPM+1.5 mg/L 2,4-D+0.4 mg/L 6-BA+30 g/L sucrose+6.5 g Agar.The effect of dark cultivation was better under light conditions.?4?The concentration of kanamycin,the infection concentration and the infection time of agrobacterium and concentration of bacteriostatic agent in blueberry genetic transformation were optimized.The kanamycin concentration of leaves of‘Northland'and‘Millennia'was 15 mg/L and 20mg/L,respectively.The OD₆₀₀value of agrobacterium was 0.9 in the leaves of‘Northland'and 0.3-0.4in the leaves of‘Millennia'.The infection time was 60min and the co-culture time was 6 d.WPM+300mg/L Cef liquid medium and WPM+4.0 mg/L ZT liquid medium were used with sterilization.Using WPM+4.0 mg/L ZT+300 mg/L Cef+Km as the selective medium,blueberry leaves had the highest survival rate and could directly regenerate Km-resistant shoots.The induction period was 50-60 d.When the OD₆₀₀value of agrobacterium was 0.3,the Km-resistant shoots induction rate of‘Millennia'was 2%,and when the OD₆₀₀value was 0.4,the Km-resistant shoots induction rate of‘Millennia'was2.04%.When the OD₆₀₀value was 0.9,the Km-resistant shoots induction rate of‘Northland'was 2.5%.