Elimination of selectable marker genes via segregation of uncoupled T-DNAs in populations of doubled haploid barley

Abstract

Barley represents one of the economicallymost important and most widely distributed crops worldwide, and genetic engineering is expected to play a crucial role in its further improvement. Once transgenic plants are obtained, the selectable marker gene is not any more necessary or even unwanted. Here we present a fast and efficient method to produce selectable marker-free, homozygous transgenic lines. Primary co-transgenic barley plants (T0), containing both the selectable marker gene (hygromycinphosphotransferase, in this case coupled with a green fluorescent protein gene) and a ’gene of interest’ (s-glucuronidase, without selectable marker) were generated via Agrobacterium-mediated gene transfer to immature embryos using separate T-DNAs. The binary vectors were introduced into the Agrobacterium strains AGL1 and LBA4404. The efficiency of the method was optimised by comparing more than ten different protocols to co-transfer independent T-DNAs to immature embryos. Uncoupled TDNAs present at hemizygous state in the primary transformants are randomly and independently distributed during male meiosis to the pollen grains. Homozygous selectable marker-free transgenic regenerants of such segregating androgenetic cultures can be efficiently produced and identified among doubled haploid individuals of the resultant T1. The method presented is not covered by complicated intellectual property rights as is the case in some alternative approaches. Moreover, only comparatively small populations are needed to find selectable marker-free, true-breeding transgenic T1plants, and further segregation analyses are unnecessary.