The RolB Gene Transformation Via Agrobacterium Tumefaciens Mediated In Pear Rootstock ’Pyrus Betulifolia Bge.’

Pyrus betulifolia Bge. is native to China, and has been widely using as pear rootstockfor thousands years. More than ninety percent of the pear trees in current North China aregrafted on this rootstock. Pyrus betulifolia is able to tolerate poor soil conditions likedrought and salt in some extent. But it has a poor rooting ability and the trees grafted onthis rootstock tend to have too much vigor. This study aimed at reducing the plant vigorand improving the rooting ability by transferring the rolB gene into Pyrus betulifolia viaAgrobacterium tumefaciens of strain EHA105mediated method. The adventitious budregeneration system and genetic transformation system have been established by studyingthe effect of some factors on the efficiency of regeneration and transformation, and thetransgenic lines were obtained.Results were as followed：1. Taking the undifferentiated cotyledon, hypocotyls and cotyledon after embryoculture as explants, analyzed the regeneration ability of the three kinds of explants. Theresults showed that among of the three kinds of explants, the regeneration effect of theundifferentiated Pyrus betulifolia cotyledon was the best, and the cotyledon after embryoculture took second place while the hypocotyls was worst.2. Taking cotyledon in Pyrus betulifolia as materials, the effects of the differentdevelopment stage explants, culture time in darkness, basic medium, kind andconcentration of plant growth regulator, the concentration of sugar on adventitious budregeneration from cotyledon were studied. The results showed that the best medium wasNN69, the rate of regeneration was68.9%in NN69supplemented with6-BA5.0mg/L+NAA0.05mg/L+30g/L sugar and the number of regenerated buds per explant reached4.80, without darkness culture.3. The optimal concentration of Kanamycin was20mg/L when the cotyledon wereused as the explants for the transformation mediated by EHA105. And the optimalconcentration of Km on the screening of transformed plants was50mg/L.4. The effects of concentration of Agrobacterium bacteria, infection time, co-culturetime and pre-culture time on transformation efficiency were determined. The suitable protocol for this gene transformation was that firstly to pre-culture the cotyledon indarkness for three days, then infect with the solution of Agrobacterium (OD600=0.7) for7min, and then co-culture on regeneration medium for3days. The resistant budsregenerated from the cotyledons were cultured on select medium supplemented with20mg/L Kan and500mg/L Cef. Of11.8%regenerated buds showed Kan resistant.5. Acquired resistance buds to successive transfer culture after three generations in thescreening culture medium were tested by PCR analysis, the results showed that in thedetection of plant has16positive plants, preliminary showed that exogenous gene Du Lisuccessfully integrated into the genome.