The elimination of a selectable marker gene in the doubled haploid progeny of co-transformed barley plants

Abstract

Following the production of transgenic plants, the selectable marker gene(s) used in the process are redundant, and their retention may be undesirable. They can be removed by exploiting segregation among the progeny of co-transformants carrying both the selectable marker gene and the effector transgene. Here we show that the doubled haploid technology widely used in conventional barley breeding programmes represents a useful means of fixing a transgene, while simultaneously removing the unwanted selectable marker gene. Primary barley co-transformants involving hpt::gfp (the selectable marker) and gus (a model transgene of interest) were produced via Agrobacterium-mediated gene transfer to immature embryos using two respective T-DNAs. These plants were then subjected to embryogenic pollen culture to separate independently integrated transgenes in doubled haploid progeny. A comparison between 14 combinations, involving two Agrobacterium strains carrying various plasmids, revealed that the highest rate of independent co-transformation was achieved when a single Agrobacterium clone carried two binary vectors. Using this principle along with Agrobacterium strain LBA4404, selectable marker-free, gus homozygous lines were eventually obtained from 1.5 per 100 immature embryos inoculated. Compared to the segregation of uncoupled T-DNAs in conventionally produced progeny, the incorporation of haploid technology improves the time and resource efficiency of producing true-breeding, selectable marker-free transgenic barley.Electronic supplementary materialThe online version of this article (doi:10.1007/s11103-012-9988-9) contains supplementary material, which is available to authorized users.

Efficient Generation of Marker-Free Transgenic Rice Plants Using an Improved Transposon-Mediated Transgene Reintegration Strategy

“…For the cotransformation, site-specific recombination, and other T-DNA-based strategies for marker gene removal (Ebinuma et al, 2001;Wang et al, 2011;Kapusi et al, 2013;Yau and Stewart, 2013), the complex transgene integration patterns such as multiple T-DNA loci, tandem-linked T-DNA copies, T-DNA truncations, and cotransferred non-T-DNA vector backbone sequence in transgenic plants (Kim et al, 2003;Rai et al, 2007) may complicate the molecular analysis of transgene integration and affect the stability of transgene expression. In this study, we validated that the maize Ac/Ds transposable element can be routinely used to generate marker-free transgenic plants in rice.…”

“…Several strategies have been developed to remove selectable markers from transgenic plants while retaining the gene of interest (GOI; Ebinuma et al, 2001;Cotsaftis et al, 2002;Wang et al, 2011;Kapusi et al, 2013;Yau and Stewart, 2013;Oliva et al, 2014). Transposition is an advantageous strategy for marker gene removal, because it allows intact transgene insertion with defined boundaries and requires only a few primary transformants (Cotsaftis et al, 2002).…”

Barley

“…Another strategy aims at androgenetic generation (haploid technology) of a segregating population of homozygous plants raised from pollen of primary transgenic barley plants produced via Agrobacterium infection of immature embryos. The results demonstrated that selectable marker-free homozygous transgenic plants can be effi ciently generated (Coronado et al 2005 ;Kapusi et al 2013 ). However, even though haploid technology represents an elegant solution and accelerates time and resource effi ciency of generating true-breeding, selectable markerfree transgenic barley, the major limitation is its strong genotype dependency.…”

Haploid and Doubled Haploid Techniques in Perennial Ryegrass (Lolium perenne L.) to Advance Research and Breeding

“…[13]. Microspores of tobacco, rapeseed, wheat, and barley are exploited in transformation and mutagenesis programs, in order to fix mutations and transgenes in a single step through subsequent DH induction [38][39][40][41][42]. For example in Brassicas, microspore mutation studies have enabled modifications of disease resistance, cold tolerance, and fatty acid composition [43].…”