Hygromycin resistance is an effective selectable marker for biolistic transformation of black spruce (Picea mariana)

Using particle bombardment of mature somatic embryos followed by the induction of secondary embryogenesis in the presence of hygromycin, we produced over 90 Lines of transgenic embryonal masses expressing beta-glucuronidase from two genotypes of black spruce. Transformation efficiencies of up to 7% (1 transgenic line per 14 embryos bombarded) were achieved by extending the period of selection from 8 to 12 weeks. Proliferation of transformed embryonal masses in the presence of hygromycin had no effect on either embryogenicity or embryo maturation. Southern blot hybridization and PCR amplification confirmed the presence of the hygromycin phosphotransferase gene in genomic DNA. The expression of the beta-glucuronidase gene in the needles of regenerated seedlings support the potential for long-term transgene expression in spruce.