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

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.

In this study, we constructed three vector systems for the co-transformation of allotetraploid Brassica napus (B. napus) mediated by Agrobacterium tumefaciens 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 T₀ 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 B. napus plants, and marker-free plants were successfully obtained in the T₁ generation from 50 to 77% of T₀ 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 B. napus plants were more frequently encountered in the progeny of transgenic lines which has only one or two marker gene copies in the T₀ generation. Two types of herbicide resistant transgenic B. napus plants, Bar ⁺ with phosphinothricin resistance and Bar ⁺ EPSPS ⁺ GOX ⁺ with phosphinothricin and glyphosate resistance, were obtained.

We were successful in removing selectable marker genes in transgenic B. napus 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 B. napus with various types of herbicide resistance.