Viroid infection of hop (Humulus lupulus L) mediated by agrobacterium tumefaciens and conditions for hop transformation

Different steps of interaction of Agrobacterium tumefaciens with hop explants were investigated. An attachment of disarmed A. tumefaciens strain to hop tissue was demonstrated by means of scanning electron microscope. The sensitive method of agroinfection by means of A. tumefaciens harboring infectious vector containing dimeric cDNA of hop latent viroid (HLVd) was used to assay T-DNA delivery into hop cells. Six weeks after Agrobacterium application was found 70% HLVd infected hop plants, suggesting specific interaction of A. tumefaciens with hop cells. For hop transformation plant expression vector containing gene for kanamycin resistance (NptII) and beta-glucuronidase reporter gene (GUS) fused with viroid antisense RNA was used. The GUS- specific insert was amplified by PCR from hop genomic DNA and analyzed by electrophoresis and molecular hybridization. Our results suggested that some of transformed plants were of chimeric nature. The differences observed in the lengths of PCR products amplified from hop genomic DNA in comparison with control (plasmid) DNA extracted from A. tumefaciens suggested some T-DNA instability or/and variability in T-DNA integration. New tissue culture conditions were proposed for A. tumefaciens-mediated hop transformation, with included regeneration on medium containing MS, 40 g/l glucose, vitamins, 2.03 mg/l IBA, 2.25 mg/l BAP, 0.35 mg/l GA3; 25 mg/l kanamycin and 500 mg/l augmentin. A replacement of ticarpen, widely used as an antimicrobial antibiotic, by augmentin (clavulanate-potentiated amoxicillin) enabled us to increase significantly the yield of hop regenerants rooting on medium containing kanamycin.