Comparison of three -mediated co-transformation methods for generating marker-free transgenic plants.

Background: Generation of marker-free transgenic plants is very important to the regulatory permission and commercial release of transgenic crops. Co-transformation methods that enable the removal of selectable marker genes have been extensively used because they are simple and clean. Few comparisons are currently available between different strain/plasmid co-transformation systems, and also data are related to variation in co-transformation frequencies caused by other details of the vector design.

Results: In this study, we constructed three vector systems for the co-transformation of allotetraploid () mediated by and compared these co-transformation methods. We tested a mixed-strain system, in which a single T-DNA is harbored in two plasmids, as well as two "double T-DNA" vector systems, in which two independent T-DNAs are harbored in one plasmid in a tandem orientation or in an inverted orientation. As confirmed by the use of PCR analysis, test strips, and Southern blot, the average co-transformation frequencies from these systems ranged from 24 to 81% in T plants, with the highest frequency of 81% for 1:1 treatment of the mixed-strain system. These vector systems are valuable for generating marker-free transgenic plants, and marker-free plants were successfully obtained in the T generation from 50 to 77% of T transgenic lines using these systems, with the highest frequency of 77% for "double T-DNA" vector systems of pBID RT Enhanced. We further found that marker-free plants were more frequently encountered in the progeny of transgenic lines which has only one or two marker gene copies in the T generation. Two types of herbicide resistant transgenic plants, with phosphinothricin resistance and with phosphinothricin and glyphosate resistance, were obtained.

Conclusion: We were successful in removing selectable marker genes in transgenic plants using all three co-transformation systems developed in this study. It was proved that if a appropriate mole ratio was designed for the specific length ratio of the twin T-DNAs for the mixed-strain method, high unlinked co-insertion frequency and overall success frequency could be achieved. Our study provides useful information for the construction of efficient co-transformation system for marker-free transgenic crop production and developed transgenic with various types of herbicide resistance.