Improvement Of Technologies For Agrobacterium-mediate Genetic Transformation Of Citrus

In this present research, to obtain the high-efficiency in citrus transformation system, the internode stem segments of seedling kumquat (Fortunella spp. Swingle), sucarri orange (C.sinensis (L.) Osbeck cv.Succari) and eureka lemon [Citrus, limon (L.) Burm.f. cv Eureka] were used as explants and various factors that might affect the regeneration and transformation efficiency were investigated. Based on the optimized genetic transformation technology system, the ’Gene-deletor’ and ’seedless fruit’ genes were successfully transferred into kumquat and sweet orange. The main results were as follows:1. Establishment of seedling kumquat efficient regeneration and transformation technology system.The regeneration results showed that the internode stem segments from35days completely dark cultured seedling, were level set on regeneration medium consisting of MS basal medium supplemented with3mg·L-16-BA for50days, then cut the adventitious buds from explants and transferred on rooting medium consisted of1/2basal medium supplemented with3mg·L-1IBA and1g·L-1AC, which can finally contribute to90%of adventitious bud regeneration rate and86%of the rooting rate.The genetic transformation results showed that internode stem segments from35days age of seedling which cultured for25days in dark and10days in light were immersed infected for30min by agrobacterium which was resuspended by the co-cultivation liquid medium consisting of MS basal medium supplemented with2mg·L-16-BA,1mg·L-1NAA,1mg·L-1KT and20mg·L-1AS when its OD600nm value reached0.3, then the infected internode stem segments were cultivated horizontally side-by-side in co-cultivation medium (CCM) for3days under dim light at25℃, finally, transferred it into the selection medium consisted of MS basal medium supplemented with3mg·L-16-BA,50mg·L-1Kan and400mg·L-1Cef,which can result in optimum GUS positive rate. Based on this efficient genetic transformation technology system, the’Gene-deletor’and’seedless fruit’ genes were successfully transferred into the kumquat with6.07%of the transformation efficiency and obtained23transgenic lines. 2. Optimization of seedling succari orange transformation technology systemThe results revealed that, when using internode stem segments from30days age of seedling which cultured20days in dark and10days in light as explants,20mg·L-1as AS concentration,5.7as co-cultivation medium pH value and19℃as co-cultivation temperature, the GUS positive rate could reached at29.4%. When resistant buds were cultured on stem elongation medium consisting of MS basal medium with0.2mg·L-16-BA,0.2mg·L-1IAA and0.2mg·L-1GA3,95%of the resistance buds appeared elongation and the elongation length reached at an average of2.3cm. When resistant buds were transferred on the rooting medium consisting of1/2MS basal medium with0.5mg·L-1NAA,0.1mg·L-1IBA and1g·L-1AC,80.2%of the resistance buds appeared rooting and the rooting length reached at an average of5.83cm. On the basis of this optimized transgenic technology system, the ’seedless fruit’ gene was successfully transferred into seedling sucarri orange and obtained7transgenic lines.3. Optimization of seedling lemon transformation technology systemThe results revealed that, when using internode stem segments from30days age of seedling which cultured20days in dark and10days in light as explants, after infection explants were transferred to co-cultivation medium (MS+3mg·L-16-BA+20mg·L-1AS, pH value5.7) co-cultured3d and transferred to selection medium (MS+0.5mg·L-16-BA+75mg·L-1Kan+500mg·L-1Cef, or MS+0.5mg·L-16-BA+10mg·L-1Basta+500mg·L-1Cef, pH value5.7). Based on the transgenic technology system, eYFP gene was successfully transferred into the cells of lemon and obtained5YFP-positive buds, YFP-positive rate was2.7%.