Study On M.sativa And A,thaliana Transformation Of GR79Mt Gene Meidated By A.tumefaciens

Glyphosate,commercially called Roundup,is a broad-spectrum and highly-efficient herbicide that isused widely in agriculture production.Medicago sativa is a perennial legume forage,famous for it’s richnutritions and good palatability,and is widely cultivated in China.A large number of weed not onlyincreased the cost of inputs, but also affect the yield and quality of forage.In this study,we transformedglyphosate-resistant gene GR79Mt into arabidopsis and alfalfa, in order to obtain the plants withglyphosate resistance.We concluded the following results:1．The regeneration system of Medicago sativa L.Zhongmu No.6was built.Type of explantsaffects callus induction rate, hypocotyls was better than cotyledons.SH medium containing2mg/L2,4-Dand0.2mg/L KT was the best induction medium,the embryo can be obtained on differentiation mediumcontaining only0.2mg/L KT. Casein hydrolyzate(1g/L) played an important role in the production andmaturation of embryoid. Embryoids or plantlets induced rooting on1/2MS medium without growthregulators.2．According to the the known sequence of glyphosate-resistent gene GR79Mt,added a chloroplasttransit peptide gene(CTP2) at its5’ end.The genes were promoted by the enhanced CaMV35s promoterand stoped by E9gene’s terminator from pea.All genetic elements were synthesized after adding PvuⅠrestriction enzyme site at both ends of the sequences.The plant expression vector pCAMBIA2300wasdigested with PvuⅠ,then ligated with the synthetic sequence.The reconstructed vector named pCA-GM.3．Transformation of Arabidopsis thaliana inflorescences were mediated by Agrobacteriumtumefaciens.The steriled seeds were screened on MS medium containing60mg/L glyphosate.When thetransgenic plants growed up, extracted its genomic DNA,the PCR,RT-PCR detection showed that thetarget genes were expressed at the transcriptional level.The GR79Mt gene was integrated into thegenome of Arabidopsis thaliana.4．Double boundary binary vector pCA-GM was transformed into alfalfa calli mediated by A.tumefaciens.Callus induction medium containing2mg/L2,4-D,0.2mg/L KT and60mg/L glyphosate.Inthe period of embryoids induction,KT concentration was adjusted to0.4mg/L, the glyphosateconcentration was increased to80mg/L. Root was induced in1/2MS medium without growthregulators.Finally,12regeneration plants were got,two of them were positive through PCR testing.Preliminary indication the target gene had been integrated into the alfalfa genome.