Engineering Non-transgenic Gynoecious Cucumber Using An Improved Transformation Protocol And Optimized CRISPR/CAS9 System

Gynoecism permits higher,more concentrated yields.Additionally,the use of a gynoecious parent eliminates the need for hand emasculation.Using CRISPR/Cas9-mediated gene editing to generate gynoecious cucumber not only avoid the time-consuming and laborious process of crossing,but also the risk of the introduction of undesirable traits.However,Cucumber is well known intractable to transformation,which makes it a daunting task to apply these gene editing tools.In this study,successful transformation was obtained by applying vacuum infiltration with a syringe to promote Agrobacterium infection of the cells from which transgenic shoots regenerate.In addition,the use of a CO donor,hemin,reduces chlorosis and accelerates rooting of regenerated shoots.Thus,we established a simplified and more effective transformation protocol for cucumber and melon.We further optimized the CRISPR/Cas9 system by identifying the best CsU6 promoter.Inserting a GFP tag facilitates selection for both transformants and transgene-free mutants among the progeny.To validate our protocol for transformation and genome editing,we targeted three genes CsVFBl,CsML08 and CsGAD1 for mutation.The transformation efficiency approached around 1‰.Editing at the desired sites was detected in all TO plants indicating a higher mutation efficiency than previously reported.CmWIP1 acts as an inhibitor of carpel development,CmWIP1 mutant confers a gynoecious phenotype in melon.Modification of the CmWIPlortholog in cucumber,CsWIP1,may accelerate the development of gynoecious inbred.Knocking out of CsWIP1 were performed,transgene-free Cswipl homozygous mutants were obtained by using GFP to facilitates selection.Compared to monoecious wild-type plants,Cswipl is gynoecious,bearing seven times more female flowers than WT.Fruits produced from female flowers of Cswipl were indistinguishable from fruit of the WT.This made Cswipl great potential use in heterosis breeding.In summary,we established a simplified and more effective transformation protocol for cucumber and melon.By optimize the vector system,a higher gene editing efficiency in cucumber was obtained than previously reported.Development of gynoecious cucumber by editing of CsWIP1 approach provide a stable and fast strategy to convert monoecious lines to gynoecious lines.