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.
Highlights
Barley is one of our major arable crop species, and may well have been the first species to have been domesticated
Biolistic transformation was the earliest platform employed for this purpose (Wan and Lemaux 1994), but this has been largely replaced by Agrobacterium-mediated gene transfer, which can be applied to either immature embryos (Tingay et al 1997), embryogenic pollen (Kumlehn et al 2006) or isolated
Of the 41 independent co-transgenic plants analysed by DNA gel blot, the gene of interest (GOI) gus was present as a single copy in 48.8 %, as two copies in 17.1 %, and as three or more copies in 34.1 %
Summary
Barley is one of our major arable crop species, and may well have been the first species to have been domesticated. The simple genetics displayed by barley has for many years encouraged its exploitation as a genetic model, and more recently this has been extended into the field of transgenesis. Selectable markers are desirable for the efficient recovery of transgenic regenerants, they often have no further purpose once a transgenic plant has been developed. The presence of a selectable marker prevents the use of the same gene for any successive round of transformation using another effector gene. Retaining selectable markers which encode resistances to antibiotics is considered in some quarters to be somehow risky, and so commercially grown transgenic plants are often required to be free of those markers
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