Study Of Agrobacterium-mediated Transformation Of Shanxinyang For Bt And Spider Insecticidal Peptide Gene

Poplar is a main forest tree species used as timber,windbreak and greening tree species, are widely distributed and extensively cultivated in Chinese provinces.Because it suffered lots of diseases and insect pests species,was harmed severely,always result in huge loss for forestry production and ecological construction.In recent times,it is appealed that ecology and environmental safety is strengthened day by day,large scale use of chemical pesticide will be controlled,the measures of biological control are weakness,and sustainable control of poplar harmful biology is the biggest problem in front of forest protection.In this research, Shanxinyang was used as receipt material,induced Bt and spider insecticidal peptide gene into the receipt material by Agrobacterium-mediated,this research is not only provided a theoretical basis for Shanxinyang genetic transformation and resistance breeding,but also provided propagation material for propagating pest resistant Shanxinyang clone and cultivating in the field.The different concentration of Cefotaxime sodium effected leaves formation and differentiation were tested,result showed:leaves from Shanxinyang have certain resistance of Cefotaxime sodium,600mg/L Cefotaxime sodium plus in selective medium have little effect on the growth of adventitious bud.Kanamycin 10mg/L can be used as the critical concentration in the leaves differentiation, higher than this concentration it will inhibit leaves differentiating;15mg/L is the best critical concentration in the rooting phase,over 20mg/L,it will restrain the growth of tissue culture seedling.In Shanxinyang transformation,pre-culture time for 2d was the best;the frequency of resistant buds was 23.3%for 2days,the frequency of resistant buds was 20%for 3 days,the frequency of resistant buds were both decreased when pre-culture time was less than 2 days or more than 4 days.If the bacterial concentration was too high would lead the recipient material being brown and dying soon,but if the bacterial concentration was too low would decreased the frequency of resistant buds,the optimal concentration is OD₆₀₀=0.1～0.2 for Shanxinyang genetic transformation.Infect 2～5min is propitious to Shanxinyang transformation.The frequency of resistant buds was increased from 2min to 5min with certain bacterial concentration.Co-culture time for 2-3d was appropriate,over 4d not only increased false positive buds, but also became the growth of explants weak,then etiolated leaves and buds.Optimized the genetic transformation system of Shanxinyang,pre-cultured 2～3d, OD₆₀₀=0.1～0.2,incubated 2～5min,co-cultured 2～3d and the co-cultivation medium plus acetosyringone 200μmol/L was the best combination.1570 leaves were transformed,86 Kan-resistant buds were obtained,after PCR analysis, 21 positive transformants were gained,accounted for 24.42%after antibiotic selection.PCR-Southern analysis showed that there are 21 expressions normal,the positive rate was 100%, conversion rate was 1.34%...