Adventitious shoot regeneration and Agrobacterium-mediated transformation of red raspberry

Regeneration and transformation protocols for 'Comet' red raspberry (Rubus ideaus L.) were optimized with the purpose of making the Agrobacterium-mediated gene transfer system efficient for this crop. Adventitious shoot regeneration was significantly improved when 10 mm diameter leaf discs were used and when subculturing occurred after 4 weeks. The medium consisted of Murashige-Skoog (MS) basal medium containing 1 mg 1(-1) N-phenyl-N-1,2,3-thiadiazol-5-ylurea (thidiazuron) and 0.5 mg 1(-1) indole-3-butainoic acid. Regeneration was not affected by the presence of cefotaxime sodium (500 mg 1(-1)) in the medium but was moderately reduced when explants were inoculated with Agrobacterium, and inhibited at kanamycin levels of 40 mg 1(-1) or more. Of the seven wild-type Agrobacterium strains tested, C58 was the most virulent at inducing tumors both in vivo and on leaf discs in vitro. A disarmed version of C58 (LBA4404), containing the binary vector pBI121, was used to transform 'comet' leaf discs. One transformed shoot was produced from 270 inoculated explants. Transformation was confirmed by the capacity of the shoot to grow in medium containing 30 mg 1(-1) kanamycin, by the presence of GUS activity, and by the detection of the GUS gene by Southern analysis.