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.
ABSTRACT
Microspores could be an excellent target for plant transformation, owing to their haploid nature, the availability of a large population of fairly synchronous single cells, and their potential to regenerate into plants through embryogenesis. Therefore, the potential for microspore transformation by biolistic procedures was examined cytologically, based on the viability and β-glucuronidase (GUS) activity of bombarded microspores. The microspores were bombarded with gold particles coated with the plasmid pAHC25. On average, 10.7% of the total number of microspores bombarded contained particles. Of these, 4.7, 1.2, and 4.7% received one, two, and three or more particles, respectively. Of the microspores receiving particles, ca. 7% had one or more particles in the nucleus. Viability of bombarded microspores was followed for 7 days in culture. Over this period, the frequency of viable microspores with particles was significantly reduced from 1.56% at day 1, to 0.72% at day 3, and finally to 0.05% at day 7, with this last group having only a single particle. While microspores that received multiple particles did not survive after 1 week in culture, initially they could be scored as positive for transient GUS activity. Microspores with particles delivered directly into the nucleus (vs. other cell compartments) showed enhanced uidA transient expression and these microspores were most likely the source of integration of the introduced DNA into the recipient genome. The potential for the recovery of transgenic barley plants following biolistic bombardment is discussed.
