Study On Genetic Transformation And Optimization Of Brassica Campestris L. Subsp. Chinensis Making Var. Parachinensis Tsenet Lee Mediated By Asrobacterium Tumefaciens

In this experiment, the seeds of Youqingsijiu Brassica campestrisL.Subsp. chinensis Makino var. parachinensis Tsenet Lee were used as raw material.The high frequency regeneration system of petiole-cotyledon explants fromYouqingsijiu Brassica campestris L.Subsp. chinensis Makino var. parachinensisTsenet Lee, genetic transformation system and optimization of Chinese cabbagemediated by Agrobacterium tumefaciens was systematically discussed. The(E)-β-farnesene gene was transferred into the Youqingsijiu Brassica campestrisL.Subsp. chinensis Makino var. parachinensis Tsenet Lee successfully and thetransgenic plant had been initially identified with PCR.Factors effecting shoot differentiation of explants from Youqingsijiu Brassicacampestris L.Subsp. chinensis Makino var. parachinensis Tsenet Lee regenerationsystem were studied respectively, which included such as the concentration of AgNO3,the suitable concentration of plant growth hormone, the ratio of hormones andexplants seedling age. A highly effective regeneration system of petiol-cotyledonexplants from Youqingsijiu Brassica campestris L.Subsp. chinensis Makino var.parachinensis Tsenet Lee was established as follows:In ultra-clean work bench, with gauze wrapped seeds, were soaked in70%ethanol for45seconds, washed for3times with sterilized water, surface-sterilized in0.1%mercuric chloride solution for8~10minutes and keeping stirring the gauzeduring that time, washed for4~5times with sterilized water, sterile filter papersucking the seeds surface residual moisture, then sowed on1/2MS basic medium inculture bottle, about100seeds per bottle and the bottle were put in plant tissueculture room, under the culture condition of light intensity1600~2000lx,(25±1)℃,16h light/8h dark photoperiod, after3~7days to get seedlings. The petiol-cotyledonexplants was excised near the growing poingt from5days seedlings, and culturedunder the light on modified half of MS medium:1/2NH4+MS+30g/L sugrose+8g/Lagar+4mg/L6-BA+1mg/LNAA+7.5mg/L AgNO3(pH=5.8).20days later, buds began to regenerate from explants, and the frequency of shoots regeneration was48.57%.One method of plant genetic transformation, Agrobacterium-mediated plantgenetic transformation was used to study the genetic transformation and optimizationof Youqingsijiu Brassica campestris L.Subsp. chinensis Makino var. parachinensisTsenet Lee. Some important factors affecting frequency of transformation includingselection agent Kanamycin, bacteriostatic agent Ampicillin, pre-culture time ofexplants, concentration of Agrobacterium tumefaciens, time of infection, co-culturetime, co-culture temperature, concentration of acetosyringone were researched andone genetic transformation system mediated by Agrobacterium tumefaciens, which issuitable for Youqingsijiu Brassica campestris L.Subsp. chinensis Makino var.parachinensis Tsenet Lee, was established as follows:Taking some Agrobacterium tumefaciens strains (pBJ40/LBA)which was savedat-80℃were isolated and cultured in solid LB medium containing three kind ofantibiotics(50mg/LStr,50mg/L Kan,50mg/L Rif),200rpm,28℃, for one night. Thentransferring some of the bacterial suspension to liquid LB medium containing with50mg/LStr,50mg/L Kan,50mg/L Rif,200rpm,28℃, cultured in the same conditions.About A600=0.4, centrifugating (4℃,4000rpm,10min) the suspension to remove thehigh salt concentration and antibiotics, collecting colony, resuspending the colony byliquid MS medium, to get the infection bacterial suspension. Then the explants withpetiole-cotyledon from5days seedlings were put into infection bacterial suspensiongently, soaked for10minutes. Infected explants were co-cultured2days andtransferred to differentiation medium containing5mg/L Kan,300mg/L Amp. About20days later, regeneration shoots begin to occur and the frequency of transformationis2.98%. With the established Agrobacterium-mediated genetic transformationsystem,16plants transformation of Youqingsijiu Brassica campestris L.Subsp.chinensis Makino var. parachinensis Tsenet Lee were got. Lastly, the transformationplants were identified via PCR and positive incidence was87.50%.In this paper, factors affecting the shoot differentiation and transformation ratioof explants from Youqingsijiu Brassica campestris L.Subsp. chinensis Makino var.parachinensis Tsenet Lee was discussed. Results of the discussion supply the advicefor raising the regeneration frequency, the conversion of EβF gene and lay thefoundation for genetic transformation of Youqingsijiu Brassica campestris L.Subsp.chinensis Makino var. parachinensis Tsenet Lee.