Use of the GUS gene as a selectable marker for Agrobacterium-mediated transformation of Rubus

A transformation system was established for red raspberry, blackberry and blackberry x raspberry hybrids, utilizing the binary vector system of Agrobacterium 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 (NPT II) gene. Regenerants were produced on media containing MS salts, 20 gl^-1 sucrose, 7 gl^-1 agar, 100 mgl^-1 inositol, 0.5 mgl^-1 nicotinic acid, 0.5 mgl^-1 pyridoxine-HCl, 0.1 mgl^-1 thiamine, and either 0.1 mgl^-1 IBA and 2 mgl^-1 BAP for leaf discs, or 0.2 mgl^-1 BAP and 0.2 mgl^-1 2,4-D for stem segments. Kanamycin sulphate, which was used as a selective agent for the NPT II gene, inhibited organogenesis at 50 mgl^-1 and was therefore unsuitable for use as a selectable marker gene in Rubus. All regenerants were assayed utilizing the fluorogenic assay procedure to determine if the GUS gene had been transferred into the material and could therefore cleave the substrate 4-methyl-umbelliferyl--D-glucuronide. Seven GUS-positive plantlets were obtained which confirmed that this marker gene had been transferred into Rubus. A dot blot assay was carried out on GUS-positive plant material to establish if the NPT II gene had also been transferred to the plant material.