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 e xplants were investigated. An attachment of disarmed A. tumefaciens st rain to hop tissue was demonstrated by means of scanning electron micr oscope. The sensitive method of agroinfection by means of A. tumefacie ns harboring infectious vector containing dimeric cDNA of hop latent v iroid (HLVd) was used to assay T-DNA delivery into hop cells. Six week s after Agrobacterium application was found 70% HLVd infected hop plan ts, suggesting specific interaction of A. tumefaciens with hop cells. For hop transformation plant expression vector containing gene for kan amycin resistance (NptII) and beta-glucuronidase reporter gene (GUS) f used 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 transf ormed plants were of chimeric nature. The differences observed in the lengths of PCR products amplified from hop genomic DNA in comparison w ith 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 tran sformation, 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-potent iated amoxicillin) enabled us to increase significantly the yield of h op regenerants rooting on medium containing kanamycin.