Research On The Glyphosate-resistant Corn

As one of three major grain crops in the world,Zea mays L.is an important food crop,fodder and industrial processing raw material.Weeds are main factor that result in the output reduction of maize,because they compete with maize in water,nutrient and sunlight,it also caused the occurrence of plant diseases and insect pests.Consequently,it has became meaningful to research how to efficiently and economically controlled weeds in field.With the development of biotechnology,employing the approach of gene engineering to achieve the Glyphosate resistant crop varieties have been viewed as a ideal method.Since 1998,the GA21(Glyphosate resistant crop varieties)was approved to be plant for commercial object,several transgenic maize varieties with excellent agronomic traits were emerged such as glyphosate-resistant,insect resistant,disease-resistant varieties.So far,transgenic maize has become the most widely planted GM(genetically modified)food crops in the world.In this study,we use two methods——ultrasonic assisted pollen mediated reforming process and ultrasonic assisted agrobacterium mediated seed germination process to transfer the target gene of CP4-EPSPS into maize inbred Chang 7-2.We choose the vector pM3301UbiSpCP4 that carry a strong constitutive promoter(corn ubiquitin promoter pZmUbi-1)to start express.Furthermore,in order to direct the CP4-EPSPS to the chloroplast,a chloroplastid transit peptide from Zea mays was added in the vector.These two new transgenic methods are very efficient and simple,have no dependence on genotype,because we employed pollen and germinating seed as receptor,and avoided the progress of tissue culture regeneration plant.So,the two methods provide efficient and simple operational process for the plant breeders.In addition,maize inbreds Chang 7-2 is an excellent inbred line have been widely used,which with good agronomic characters and it can be fast applied in production after transgene.Finally,the results are follows:Use the ultrasonic assisted pollen mediated method of genetically modified to processing 1552 stains maize,we obtained 943 maize seeds.There are 29 strains growing healthy in 648 transgenic trains of T1 generation after daubing 2%concentration glyphosate.We employ PCR to detect the target gene in the healthy plant and obtain positive bands in 16 strains.RT-PCR shows that exogenous gene transcribe normally in the positive strains.Furthermore,the test of PCR-Southern blot demonstrates that the next generation has inherited exogenous genes CP4-EPSPS stably.Planting the seeds of the 16 strains of T1 generation,selfing to obtained T2 generation,we screened five transgenic strains(including Anti-3,11,12,23 and 29)with good resistence to glyphosate through dauding 2%concentration glyphosate on the strains of T2.The transgenic plants were identified by PCR?Southern blot?ELISA test.We achieved three single copy transgenic strains with high resistence on glyphosate and high expression quantity on herbicide resistance protein,Anti-3,23,29.This study laid a foundation of researching on herbicide resistance transgenic corn and provides valuable research materials for the breeding work.