Studies On Markers-Free Transformation Tomato With Isoflavone Synthase Gene

At present, most of the transgenic plants use the selectable marker genes such as antibiotic and herbicide resistanse to select transplants.Recent years,more and more people worry about the safety of transgenic product.Although there is no report shows that selectable marker genes can affect the human health and the environment security.Marker-free transgenic system is a rapidly developed method which can eliminate the safty concern of transgenic food.This paper studied on Cre/loxp recoganization system, and agrobacterium-mediated transformation of tobacco with Cre/loxp system which constructed can delete the marker genes, and obtained the co-transformation tobacco plant.Isoflavones are a group of secondary metabolites common to leguminous plants that play roles in plant defense and nodulation and have many benefits to human health including relief of menopausal symptoms,reduction of osteoporosis and lowering risk of certain hormone-related cancers. Isoflavone synthase(IFS),which is the key enzyme in isoflavone biosynthesis. This study optimizated the transgenetic regeneration system of tomato, Constructed the pBi121-ifs verctor, and Agrobacterium-mediated transformation of processing tomato as well as obtained the transgenetic plant. The fruit tree have certain difficulty and particularity in genetic transformation. To obtain highly effective genetic transformation method about strawberry and apple, the optimization of regeneration System of strawberry leaf and apple leaf were established.The study includes three major conclusions as followed:1. In this experiment ,DNA was extracted from co-transformation tobacco leaves of 22 plants and was detected by PCR.The results showed that gus gene had been deleted successfully from 10 plants and delete rate was 45.5%.2. Agrobacterium-mediated transformation of processing tomato: The pre-culture medium was MS+B5 supplied with IAA1.0 mg/L +6-BA2.0mg/L,The high efficient media for selection was MS+B5 supplied with IAA1.0 mg/L +6-BA 2.0 mg/L +Cef500 mg/L +Kan50mg/L,The high efficient media for root induction was MS+B5 supplied with IBA2.0 mg/L + Cef300 mg/L +Kan50mg/L, DNA and RNA was extracted from the 11 Genetic Transformation of Processing Tomatoes and detected by PCR and RT-PCR.the results showed that ifs gene had been successfully transfered into 4 plants and the transformation efficiency was 36.4%.3. Establishment of regeneration system of strawberry: The best effects of stalk culture in medium was l/2(MS+B5) supplied with NAA0.5mg/L +TDZ0.5 mg/L,The best effects of buds induction culture in medium was MS+B5 supplied with NAA0.3 mg/L+TDZ3.0 mg/L,Establishment of regeneration system of strawberr apple:The best effects of stalk culture in medium was l/2(MS+B5) supplied withNAA0.2mg/L +6-BA 1.0 mg/L,The best effects of buds induction culture in medium was MS+B5 supplied with NAA0.1mg/L+ZT1.0 mg/L.The research above provided the technology foundation for obtaining the transgene-safty fruits in the future.