Transgene expression in regenerating cotyledons and embryogenic cultures of Scots pine

Regenerating cotyledons and embryogenic cultures of Scots pine (Pinus sylvestris L.) were used as targets for gene delivery via particle bombardment. Both target tissues differed in their response to selective agents, regulative sequences of transferred genes, and conditions optimal for particle bombardment. Of the gene constructs tested, the pBI410 including CaMV 35S promoter, AMV-translation enhancer and gusA : npt reporter and selectable marker gene fusion was the most suitable for cotyledon transformation, resulting on average in 3.7 (±0.1 SE) β-glucuronidase (GUS) expression units per cotyledon. In embryogenic cell masses the pB1221.1 gene construct including the gusA reporter gene driven by the 35S promoter gave the highest transient expression, 63 (±15 SE) GUS expression units of g−1 fresh weight of embryogenic cell mass. Kanamycin and geneticin concentrations suitable for selection of cotyledons were 10–15 mg dm−3 and 0.5–1.0 mg dm−3, respectively. Kanamycin (10 mg dm−3) and phosphinotricin (1 mg dm−3) as selectors caused a significant decline in growth, but geneticin did not significantly affect the growth of the embryogenic cultures during the 8 week cultivation period. The production of transgenic plantlets seems to be more dependent on the regeneration and multiplication efficiency of organogenesis and somatic embryogenesis of Scots pine than on gene delivery into regenerating tissues.