We have evaluated the expression of the reporter beta-glucuronidase (GUS) gene driven by the cauliflower mosaic virus 35S (CaMV 35S) promoter in flowers and pollen from 14 independent transgenic strawberry lines. Of the 14 lines evaluated, 13 (92.8%) showed GUS activity--as estimated by the histochemical GUS assay--in some floral organs, with expression being most common in the flower stem, sepals, petals, ovary and stigma. Ten of these thirteen transgenic lines (77%) showed GUS activity in pollen, although the percentages of positive pollen per flower varied greatly among the different lines. A study of the GUS expression during pollen maturation showed that the (CaMV 35S) promoter showed low expression in pollen from flower buds before anthesis but was activated in mature pollen following anther dehiscence. The percentages of pollen grains that showed GUS activity ranged from 2.1% to 46.3%. These percentages were similar or even higher when mature pollen was stored dry at room temperature for 2 weeks. After 5 weeks of storage, the percentages of GUS-positive pollen decreased in two of the six lines analysed but remained at similar values in the other four lines. GUS activity was also measured in protein extracts of mature pollen by means of the fluorometric GUS assay, with the values obtained ranging from 3.8 micromol MU mg protein(-1) h(-1) to 0.26 micromol MU mg protein(-1) h(-1). Contrary to the generally held view that the CaMV 35S promoter is virtually silent in pollen, we conclude that it is highly expressed in transgenic strawberry pollen.

The effect of combining Agrobacterium tumefaciens infection and biolistic bombardment on the transformation of strawberry (Fragaria × ananassa Duch.) cv. Chandler, was evaluated. Bombarding leaf explants with uncoated gold particles followed by Agrobacterium infection did not improve transformation, and yielded similar percentages of shoot regeneration in the presence of kanamycin in bombarded and non-bombarded explants (7.2%). In a novel approximation, gold particles were coated with Agrobacterium cells and used to bombard leaf explants. Helium pressures of 4.5, 6.2 and 7.6 MPa and target distances of 3 and 9 cm were tested. An average of 96.2% of the explants showed β-glucuronidase (GUS) expression 15 d after bombardment, in comparison with 26.6% in explants bombarded with gold particles coated with the plasmid pGUSINT or 58.3% in non-bombarded Agrobacterium-infected explants. After 25 weeks of culture, the highest transformation frequency was obtained using a 6.2 MPa helium pressure and 3 cm target distance, yielding 69% kanamycin-resistant explants and a final transformation fre-quency of 20.7%. These values were 4.5 times higher for kanamycin-resistant explants (69% with biolistic vs 16% with Agrobacterium infection) and 2.9 times higher for transformation frequency (20.7 vs 7%,) compared with those obtained with standard Agrobacterium transformation procedures (Barceló et al. 1998, Plant Cell, Tiss. Org. Cult. 54, 29–36). More than 15 independent transgenic plants obtained by the Agrobacterium-coated particle system were acclimatized and confirmed as transgenics by GUS activity and PCR. Segregation analysis of kanamycin resistance has been performed in seven independent lines, three of which contained a single insertion of the T-DNA.