Biolistic transformation of haploid isolated microspores of barley (Hordeum vulgare L.)

Abstract

Transgenic barley plants were produced by the direct delivery of plasmid DNA into isolated microspores of barley cv. Igri using high velocity microprojectiles. The plasmid pAHC25 contained the uidA and bar genes, each under the control of a maize Ubi1 promoter. Bombarded microspores were cultured and selected on solid medium containing varying concentrations (2–5 mg/L) of the Basta herbicide active agent bialaphos. The effectiveness of selection with bialaphos depended on its interaction with the medium component glutamine. Six transgenic plants (R0) were obtained, and the presence of the uidA and bar genes and their integration into nuclear DNA in transformed R0 plants were confirmed by PCR and Southern blot analysis. Phosphinothricin acetyltransferase activity was observed in all six R0 transgenic plants, whereas none showed β-glucuronidase (GUS) activity in histochemical GUS assays. Two of the six R0 plants were haploid and sterile; one of them was trisomic and partially sterile; the remainder were diploid, but one of them was also sterile. Inheritance of the transgenes in progeny of three seed-producing transgenic plants was investigated. Southern blot analysis of genomic DNA from R1 plants showed that the introduced bar and uidA genes were hemizygous and stably cotransmitted to the R1 progeny derived from self-pollination. Analysis of Basta resistance and the integration of the bar gene by PCR analysis in R1 plants indicated that the bar gene was being inherited and expressed as a single dominant trait. Fluorescent in situ hybridization was performed on chromosomes of the trisomic plant to confirm the presence of transgenes in the genome.Key words: barley, microspore, biolistic transformation, bialaphos, haploid, FISH.