An efficient method for the production of marker-free transgenic plants of peanut (Arachis hypogaea L.)

Abstract

Recombinant genes conferring resistance to antibiotics or herbicides are widely used as selectable markers in plant transformation for selecting the primary transgenic events. However, these become redundant once the transgenic plants have been developed and identified. Although, there is no evidence that the selectable marker genes are unsafe for consumers and the environment, it would be desirable if the marker genes can be eliminated from the final transgenic events. The availability of efficient transformation methods can enable the possibility of developing transgenic events that are devoid of the marker gene/s upfront. Taking advantage of the high and consistent transformation potential of peanut, we report a technique for developing its transgenics without the use of any selectable marker gene. Marker-free binary vectors harboring either the phytoene synthase gene from maize (Zmpsy1) or the chitinase gene from rice (Rchit) were constructed and used for Agrobacterium tumefaciens-mediated transformation of peanut. The putative transgenic events growing in vitro were initially identified by PCR and further confirmed for gene integration and expression by dot blots assays, Southern blots, and RT-PCR where they showed a transformation frequency of over 75%. This system is simple, efficient, rapid, and does not require the complex segregation steps and analysis for selection of the transgenic events. This approach for generation of marker-free transgenic plants minimizes the risk of introducing unwanted genetic changes, allows stacking of multiple genes and can be applicable to other plant species that have high shoot regeneration efficiencies.