Potential off biolistic transformation of barley microspores based on viability and transient β-glucuronidase activity

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.