Immature pollen-derived doubled haploid formation in barley cv. Golden Promise as a tool for transgene recombination

Abstract

Barley transformation mediated by Agrobacterium tumefaciens is routinely performed in a number of laboratories. However, elimination of selectable marker genes and formation of plants homozygous for the transgene via conventional segregation is laborious and time-consuming. Here we suggest a concept that includes the production of primary transgenic plants via infection of immature embryos with A. tumefaciens followed by androgenetic generation of a segregating population of entirely homozygous plants. Selectable marker-free, truebreeding plants carrying a single-opy transgene integrant may thus be efficiently and rapidly obtained. However, amenability to Agrobacterium-mediated transformation as well as androgenetic potential is genotype-dependent. Efficient genetic transformation by infection of immature embryos is so far confined to the spring type cultivar ‘Golden Promise’ which, however, turned out to be recalcitrant in pollen embryogenesis. To facilitate androgenetic generation of homozygous segregants from primary transformants, we have established a method for embryogenic pollen culture in cv. Golden Promise that includes conventional cold-treatment and subsequent preculture of immature pollen under starvation conditions prior to transfer to complete nutrient medium. Further we show that conditioning of the pollen culture medium by co-culture of immature wheat pistils as well as addition of pistil-preconditioned medium considerably support androgenetic development. Employment of the established method using immature pollen of primary transgenic plants demonstrates that selectable marker-free, true-breeding transgenic progeny can be rapidly obtained pursuing the concept proposed. The protocol presented will be useful in functional genomics as well as in molecular breeding approaches.