Genetic Transformation And Resistance Evaluation Of Glyphosate Resistance Gene In Brassica Napus L

Rapeseed is one of the important oil crops in China.The quality and yield are seriously affected by the Monocotyledonous and dicotyledonous weeds sometimes.Among chemical weed control agents,the factors that herbicide types,application dosage and spraying time have more restrictions to the application of traditional selective herbicide in weeding.As non-selective herbicide,glyphosate is favored for its high efficacy,low cost low environmental resistance,flexible application requirement and safety,and is world’s most commonly used herbicide.However,glyphosate rarely applied directly in rapeseed field due to its toxic to plants.Therefore,glyphosate resistant rapeseed can greatly decrease the cost of production and provide a more effective weed management strategy.In this research,we transformed a novel glyphosate-resistant gene I.variabilis-EPSPS^*into the inbreed line―Y127‖,and obtained glyphosate-resistant germplasm for applying in commercial production of herbicide resistant breeding.The main resultss are as follows:1.In I.variabilis-EPSPS^*transformation experiment,30 seedlings were obtained after 50mg/L glyphosate selection in regeneration tissue cultured medium,and PCR assay demonstrated that 8 seedlings were I.variabilis-EPSPS^*positive（the positive rate was 26.6%）.Only 1 indepentdent trangentic plant named as E₁ was obtained after spraying 800X diluted solution of 41%glyphosate isopropylamine salt（Roundup）.2.The seed germination experiment showed that the I.variabilis-EPSPS^*E1T₁transgenic plants from the E₁ grew normally in the medium with different concentrations of glyphosate.In contrast,the growth of non-transgenic plants had been severely inhibited in the medium added with glyphosate.The measurements of root and hypocotyl of seedlings derived from the medium of different concentrations of glyphosate showed the plants with Bna C04EPSPS modified（RG1 and RE1）and I.variabilis-EPSPS^*transgenic T₁plants from E₁were glyphosate tolerant.3.In different concentration of 41%glyphosate isopropylamine salt（Roundup）treatment,the injury level of transgenic and non-transgenic plants will enhanced with the increase of glyphosate concentration.The shikimic acid assay of transgenic plants grown in the greenhouse showed that the content of shikimic acid in the plant increased in the primary period,and then slowly decreased after spraying of roundup.However,the shikimic acid content in non-transgenic plant“Y127”gradually increased and finally reached at a stable level.Agronomic traits assay results indicated that no significant differences between the I.variabilis-EPSPS^*transgenic and non-transgenic plants in silique length,seed number per silique and 1000 seed weight etc.4.QRT-PCR analysis showed that the expression level of I.variabilis-EPSPS^*gene would increase after roundup treatment and would not change with different glyphosate concentration.Southern blot indicated that E₁T₁has two I.variabilis-EPSPS^*cassettes integrated in plant genome.5.Foliar glyphosate application in the field showed that after spraying600×dilution of 41%glyphosate isopropylamine salt（roundup）,E₁T₁ plants grew normally,while“Y127”plants appeared chlorotic youngest leaves plus severe meristem defects.Field investigation showed that the yield of E₁T₁ transgenic plants were rarely affected by glyphosate.Analysis of T₂ generation plants indicated that glyphosate resistance conferred by I.variabilis-EPSPS^*could be stably inherited,and the the semi-lethal dosage(I₅₀)of glyphosate for E₁T₁ was 321.50mg/L.