Assessment of transgene flow in eggplant germplasm conserved at National Genebank

Abstract

Increased cultivation of genetically modified (GM) crops concerns curators for unintentional introgression of transgenes with germplasm conserved in genebanks. Availability of transgenic counterparts in certain crop species necessitates screening of germplasm for adventitious presence of transgenes towards proper conservation under short- or long-term. Primers for CaMV 35S promoter region, <i style="mso-bidi-font-style:normal">NptII</i> gene, and <i style="mso-bidi-font-style:normal">β</i>-fructofuranosidase (endogenous reference gene) were designed using MPprimer1.4 software. Uniplex- or multiplex-PCR protocol had produced amplicons of 156, 557 and 287 bp length, respectively for CaMV 35S promoter, <i style="mso-bidi-font-style:normal">NptII</i> gene and <i style="mso-bidi-font-style:normal">β</i>-fructofuranosidase primers. Presence of 287 bp fragment for endogenous reference gene and absence of other two fragments for specific transgene elements in conserved germplasm indicated the absence of transgenes in germplasm being conserved in National Genebank. Presence of <i style="mso-bidi-font-style:normal">β</i>-fructofuranosidase gene in most of the crop plants justifies its use for screening germplasm across species against transgene flow. It would be very much useful in screening germplasm towards conservation for which transgenic counterparts with CaMV 35S promoter and <i style="mso-bidi-font-style:normal">NptII</i> gene are available at present. Multiplex-PCR protocol was validated for the risk of transgene flow using eggplant germplasms, which are being conserved since 2008 at our National Genebank. Implementing validated screening protocol for transgene detection as one of the major preparatory measures before conserving germplasm in genebanks will help curators in checking unintentional transgene flow.