Study On Genetic Transformation Of Pokeweed Antiviral Protein Into Gerbera Jamesonii Bolus

Pokeweed antiviral protein(PAP) is one kind of broad-spectrum resistance protein which can resist various kinds of virus.For example:Cucumber mosaic virus(CMV),Tobacco mosaic virus(TMV), Tobacco necrosis virus (TNV),Alfalfa mosaic virus(ALMV),Southern bean mosaic virus (SBMV),Potato virus X, (PVX),Potato virus Y(PVY),Cauli flower mosaic virus(CaMV),African cassava mosaic virus (ACMV),Turnip mosaic virus (TuMV). Plant can obtain the broad-spectrum resistance by integrating PAP into it's own genome through Agrobacterium-mediated transformation in order to increase plant productivity and quality.Gerbera jamesonii Bolus which is one kind of most popular cut flower in the world could be infected by virus easily, especially while the amount of China's cut flowers increase dramaticly and the cultivated area expand rapidly. When the infection occurs the leaf of Gerbera jamesonii Bolus would be abnormal and the colour of leaf would be faded or even die. The traditional prevent method to resist virus is to spray pesticide,while it can increase the virus's resistance and tolerance to drug..Also widely use of peticide can induce the environmental pulltion.So it is significant to get anti-virus Gerbera jamesonii Bolus through transgenic plant technology.The research established efficient stable systems of regeneration and genetic Agrobacterium-mediated transformation for Gerbera jamesonii Bolus.Through the systems PAP has been integrated into Gerbera jamesonii Bolus.After inoculating Tobacco mosaic virus(TMV)on transgenic Gerbera jamesonii Bolus,the activities of SOD andCAT have been tested.The results showed that transgenic Gerbera jamesonii Bolus could express resistance to virus.The main results are as follows:1. An efficient system of regeneration has been established through no vaccine's leaf and petiole base as explant,using different concentrations of hormone to induce callus, adventitious buds,roots.The results eventually showed we should using petiole base as explant, MS+BA9.0mg/L+NAAO.1mg/L as callus induction medium, MS+BA0.8mg/L+NAAO.lmg/L as adventitious buds induction medium, 1/2MS+NAA0.5 mg/L as roots induction medium.2. An efficient system of genetic Agrobacterium-mediated transformation for Gerbera jamesonii Bolus has been established.The results showed that highest transformation frequency was obtained when the OD600 of Agrobacterium was about 0.3, the Agrobacterium infect 8 min,36 hours co-culture, and 2 days of detention screening. MS+BA0.8 mg/L+NAA 0.1mg/L+100umol/L AS as co-culture medium, MS+BA0.8mg/L+NAA0.1mg/L+Cef500mg/L as delayed medium. MS+BA0.8mg/ L+NAA0.1mg/L+Hyg13 mg/L+Cef500mg/L as bud screening medium.1/2MS+NAA0.5 mg/L+Hyg8mg/L+Cef500mg/L as root screening medium.3. Through this system we finally transferd pokeweed antivral protein gene into Gerbera jamesonii Bolus.Results of PCR proved that the transgene were integrated into 10 Gerbera jamesonii Bolus plants. After inoculating Tobacco mosaic virus(TMV)on transgenic Gerbera jamesonii Bolus,through regular observations of plant morphology there were 4 plants expressing apparent resistance to TMV.We also confirmed the gene has beenn integrated into plant by testing transgenic and un-transgenic plants'activities of SOD and CAT.