Study On The Establishment Of In Vitro Regeneration System From P. Calleryana Dcne. And RolB Gene Transformation

P.calleryana Dcne.is the mostly common used rootstock in South China,with high adaptability and resistance,which is compatible with both Chinese pear and European pear Cotyledons,stem pieces and leaves of P.calleryana Dcne.were used as explant to investigate some influencing factors and rolB gene was introduced into cotyledons of it mediated by Agrobacterium tumefaciens.A practical method was adopted for issue culture and gene transformation of P.calleryana Dcne.The main results were as follow:1.Two-time sterilized method and the medium for Cotyledon culture was optimized and selected.The cotyledons could regenerate the most adventitious buds on MS+6-BA 4 mg/L +NAA 0.2 mg/L.The buds proliferated about 9.12 times in every subculture during one to four subculture on MS+6-BA 1.0 mg/L+NAA 0.1 mg/L.Shoots higher than one centimeter rooted regularly induced on 1/4 MS+IBA 3mg/Ltwenty days then transferred onto MS+sugar5.0 g/L.Most of the rooted shoots survived after planted.2.Internode and leaves of P.calleryana Dcne.could produced callus after induced.Half of leaves produced buds on NN69+TDZ2.5 mg/L+ NAA 0.1 mg/L while 27.5 percent of the internodes produced buds on NN69+6-BA2.5 mg/L+NAA 0.2 mg/L.3.Factors which influenced the percentage of gene transformation such as pre-culture time, co-culture time,time of dipping and intension of selection were studied The tentative transformation system of P.calleryana Dcne was developed as follows:first the cotyledons were pre-cultured in darkness four days,then dipped into agrobacterium suspension for ten minutes.After the treated cotyledons were co-cultured in darkness for another sixty hours,they were cultured on the selection medium until the resistance callus formed The callus were sub-cultured in light to obtain resistance buds.Those buds were propagated after three to four times of sub-culture.4.In the study,seven P.calleryana Dcne plants with resistance to Km were obtained.Four of them were detected positive to PCR.Continued detection were expected to be carried out.