Marker-free transgenic corn plant production through co-bombardment

The use of particle gun for the production of marker-free plants is scant in published literature. Perhaps this is a reflection of the widely held notion that the events generated through bombardment tend to have multiple copies of transgenes, usually integrated at a single locus, features which precludes segregating away the selectable marker gene. However, our previous studies have shown that single-copy integrants are obtained at a high frequency if limited quantity of DNA is used for bombardment. Also, the concatemerized insertion of transgenes has been demonstrated to be greatly reduced if "cassette DNA" is employed in place of whole plasmid DNA for bombardment. Based on the above findings, in the present study the feasibility of co-bombardment was evaluated for the production of marker-free plants in corn, employing a combination of limited quantity DNA and cassette DNA approaches for bombardment. Transgenic events were generated after co-bombardment of a selectable marker cassette containing the nptII gene (2.5 ng per shot) and a GUS gene cassette (15 ng per shot). Among these events single-copy integrants for nptII gene occurred at an average frequency of 68% within which the co-expression frequency of GUS and nptII genes ranged from 41% to 80%. Marker-free corn plants could be identified from the progeny of 28 out of the 103 R0 co-expressing events screened. The results demonstrate that by using cassette DNA and low quantities of DNA for bombardment, marker-free plants are produced at efficiencies comparable to that of Agrobacterium-based co-transformation methods.