Establishment Of Regeneration System And The Transformation With Ipt Gene Of Zanthoxylum Piperitum DC Var. Inerme Makino

In this study,an efficient plant regeneration protocol in vitro and transformation with ipt gene mediated via Agrobacterium tumefaciens method of Zanthoxylum piperitum DC var.inerme Makino were achieved firstly.The establishment of efficient regeneration system and Agrobacterium tumefaciens-mediat -ed transformation of Zanthoxylum piperitum DC var.inerme Makino will make substantial contribution as an essential and useful means to further fundamental study of breeding by the application of genetic engineering technology.1.Establishment of high efficient regeneration system of Zanthoxylum piperitum DC var. inerme MakinoIn this study,the explants(leave petioles and stems) were cultured in vitro on WPM medium supplanted with N-6benzyladenine(6-BA),α-naphthaleneacetic acid(NAA) and indole-3-butyric acid (IBA),and established the plant regeneration system of Zanthoxylum piperitum DC vat.inerme Makino. The results showed that the most suitable culture medium for callus induction and shoots reproduction was WPM+6-BA0.5 mg·L^-1+NAA0.2 mg·L^-1.Induction of rooting was achieved by transferring the shoots to the half strength macro-element WPM medium.The plantlets were transplanted to soil after acclimatization.2.Establishment the Gentetie transformation System of Zanthoxylum piperitum DC var. inerme Makino mediated via Agrobacterium tumefaciensThe genetic transformation system of Zanthoxylum piperitum DC vat.inerme Makino was optimized by using the cotyledon and hypocotyl as the receptors mediated with Agrobacterium tumefaciens.The content of kanamycin(Kan) selection pressures were determined as 30 mg·L^-1 for shoot regeneration of petioles and stems.To monitor the induction rate of Kanamycin-resistant callus,the optimal parameters (such as pre-culture time,bacterium concentration,infection time,co-culture time.) for A.tumefaciens-mediated transformation of Z.piperitum DC van inernme Makino were examined.The results showed that 3 days' pre-culture,10 min Agrobacterium(OD₆₀₀ about 0.5-0.7) infection with 100μmol·L^-1 acetosyringone(AS),and 3 days' co-culture would lead to an increase in transformation efficiency.3 Detection of transgenic Z.piperitum DC var.inerme Makino plantsThe binary expression element with ipt gene is transformed to Z piperitum DC var.inerme Makino via Agrobacterium mediated method.The results showed that many transformants showed blue when they were dyed and many also showed positive with PCR checking.All of these results prove that aimed gene has been transformed into Z piperitum DC var.inerme Makino.The expression of ipt gene has effectively promoted the adventive bud induction.Z.piperitum DC vat.inerme Makino with ipt gene could produce a great many branches and could easily be propagated,but rhizogenesis of transformants was restrained.