A Transgenic Insect Resistance And Herbicide Tolerance Cowpea Expressing Cry-Vip3 And CP4EPSPS

Cowpea(Vigna unguiculata[L.]Walp.)is an economically important legume crop for its abundant nourishment and affordable price.It is widely cultivated in Africa,South and Central America,East/Southeast Asia and Southern Europe.Approximately 9 million tons of dry cowpea grains are produced annually across the world on about 15 million ha(FAOSTAT 2021).In China,cowpea is one of the top ten vegetable crops and nearly 12 million tons of fresh pods are produced annually.However,during the growth stage,cowpea suffers from the stress of lepidopteran pests,among which legume pod borer(Maruca testulalis Geyer)could cause as high as 25-85%loss.To control the insect pests,spraying chemical pesticides is currently the leading method.However,large quantity of chemical pesticide applications increased the risk of breaching the limits of pesticide residues and causes food safety issue.In 2010,a"poisoned cowpea" with elevated level of pesticide residues was reported in Hainan Province,one of the main planting area of cowpea in China,and caused alarm to the consumers.Therefore,developing an environmentally friendly and sustainable strategy for insect control for cowpea is urgent.Transgenic crops had been proved as an environmentally friendly crop technology that has been adapted worldwide since first commercial event approved in 1996.The adoption of transgenic technology was most in corn,soybean and cotton.Nevertheless,there is one transgenic cowpea event was approved in Nigeria in 2019,which expressing a single Cry1Ab protein for lepidopteran pest control.In this study,we developed an efficient cowpea transformation system using glyphosate as the selection agent.Cowpea is sensitive to glyphosate.Early reports of cowpea transformation methods were mostly based on antibiotic selection,which is prone to considerable false positive transformants and introduces antibiotic selection marker into plant genome.Hence,we developed a transformation and selection system based on glyphosate selection.A Bt fusion gene cry-vip3(named as CAL)and a selection marker gene cp4epsps were introduced into cowpea simultaneously by Agrobacterium-mediated method.We tested different explants,tissue culture conditions and glyphosate concentrations to optimize cowpea transformation protocol.Total of 37 transformants were obtained with no false positive,and were bio assayed on beet armyworm(BAW)and tobacco cutworm(TCW).An event named CAP5 was selected for further study based on its express levels of transgenes and its agronomic performance.CAP5 has one copy of T-DNA integrated into the CAP5 genome,with no annotated functional gene was inserted.The CAP5 leaf,flower and pods were active against legume pod borer(LPB),BAW and TCW.Furthermore,glyphosate spraying test in greenhouse suggested that CAP5 could tolerant 4 times of the recommended glyphosate dose.The agronomic traits investigation revealed that there was no significant difference of pod length,pod weight,plant height and weight per 100-grain between CAP5 and its non-transgenic cowpea line.In conclusion,CAP5 is an elite cowpea transformant with strong insect resistance and herbicidal tolerance,and a good candidate for commercial release.