The Transformation Of Grape Rootstock '5BB' With RolC Gene

An effective petiole adventitious bud regeneration system of grape rootstock'5BB'(Vitis berlandieri xV. riparia) in vitro was developed in this study.Agrobacterium-mediated and Sonication-assisted Agnobacterium-mediated transformation(SAAT) were adopted to introduce the roIC gene into '5BB'. In this research, factors thatcan increase the efficiency of grape plant regeneration and transformation were studiedsystematically, the major technique parameters of agrobacterium-mediated transformationwere optimized, and built the genetic transfrormation system of SAAT. The detailed resultswere listed as follows:1. Adventitious buds were induced from petiole of grape rootstock '5BB'. The effectsof diferent basic media, explant types, plant growth regulators and their concentrationcombinations on regeneration frequency of grape rootstock '5BB' were studied in vitro. Theresults indicated that the preferable basic medium for inducing adventitious bud frompetiole was MS basic medium; The better explant type was petiole which nodal position isthe second and the third; The felicitous growth regulator combination was BA 2.5mg/L andIBA 0.05mg/L; The highest regeneration frequency of petiole was 43.33ï¼….2. Transgenic plants were generated from petioles of grape rootstock '5BB' byagrobacterium-mediated transformation, and the percentages of GUS transient expression wereanalyzed to study the optimum parameters of this transformation system. The resultsshowed that 40 percentage of GUS transient expression could be realized under the followingcondition, namely explants being dipped into agrobacterium suspension for 4 minutes, twodays of co-cultivation and pre-culture on MS with Km 2.5mg/L, and Cef 250mg/L. Resistantbuds were obtained after maintainable culture on MS supplemented with BA2.5mg/L+IBA0.05mg/L+Cef250mg/L+Km2.5mg/L.3. The genetic transformation of SAAT was built: The tiptop percent (40ï¼…) GUStransient expression was obtained when the better explant type was stem, Sonication treatmenttime was 8s., Sonication treatment power was 100W and AS concentrateion was 75mg/L.4. The rolC gene was transformed into grape rootstock '5BB' for the first time. The new idioplasm of grape rootstock '5BB' with fastness of kanamycin was obtained. One linewith fastness of kanamycin had approved that roIC gene was conformitied into '5BB'genome by GUS histochemistry coloration and PCR.