Agrobacterium cells as microprojectile coating:: a novel approach to enhance stable transformation rates in strawberry

The effect of combining Agrobacterium tumefaciens infection and biolistic bombardment on the transformation of strawberry (Fragaria x 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 beta -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 frequency 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 (Barcelo 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.