Abstract

A transformation system was established for red raspberry, blackberry and blackberry X raspberry hybrids, utilizing the binary vector system of A. tumefaciens. Leaf discs or internodal stem segments were inoculated with Agrobacterium strain LBA4404 containing the binary vectors PBI121.X, which has the -glucuronidase (GUS) marker gene, or Bin 19, which has the neomycin phosphotransferase II (NPTII) gene. Regenerants were produced on media containing MS salts and either 0.1 mg IBA and 2 mg benzyladenine/litre for leaf discs, or 0.2 mg benzyladenine and 0.2 mg 2,4-D/litre for stem segments. Kanamycin sulfate, which was used as a selective agent for the NPTII gene, inhibited organogenesis at 50 mg/litre and was therefore unsuitable for use as a selectable marker gene in Rubus. All regenerants were assayed utilizing the fluorogenic assay procedure to determine whether the GUS gene had been transferred into the material and could therefore cleave the substrate 4-methyl-umbelliferyl--&smallcap;D-glucuronide. Seven GUS-positive plantlets were obtained which confirmed that this marker gene had been transferred. A dot blot assay was carried out on GUS-positive plant material to establish whether the NPTII gene had also been transferred to the plant material.