Abstract

BackgroundGeneration 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.ResultsIn 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.ConclusionWe 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.

Highlights

  • Generation of marker-free transgenic plants is very important to the regulatory permission and commercial release of transgenic crops

  • Our results indicate that the systems developed in this study were effective in removing selectable marker genes, in this particular case herbicide resistant B. napus plants

  • The highest frequency (81%) co-transformation was attained by using the mixed-strain method as compared with the “double T-DNA” vector system of pDB1300-3300 (65%) or pBID RT Enhanced (32%) in ­T0 plants

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Summary

Introduction

Generation of marker-free transgenic plants is very important to the regulatory permission and commercial release of transgenic crops. Transgenic plants with marker genes impair public acceptance due to the concerns of public health and increase the environmental risk of the introgression of marker genes into weedy relatives and non-transgenic crops [14]. These factors complicate the regulatory process for the commercialization of genetically modified plants [15, 16]. Modified B. napus varieties without marker genes have been commercially released in a small number of countries, including the United States, Canada, Australia and Chile far [17]

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