RIBOZYME-MEDIATED HIGH-RESISTANCE AGAINST POTATO SPINDLE TUBER VIROIDIN TRANSGENIC POTATOES

A hammerhead ribozyme [R(-)] targeting the minus strand RNA of potato spindle tuber viroid (PSTVd) and a mutated nonfunctional ribozyme [mR( -)] were designed, cloned, and transcribed. As predicted, both monomer and dimer transcripts of the active R(-) ribozyme gene could cleave t he PSTVd minus strand dimer RNA into three fragments of 77, 338, and 3 59 bases in vitro at 25 and 50 degrees C. The tandem dimer genes of R( -) and mR(-) were subcloned separately into the plant expression vecto r pROK2. Transgenic potato plants (cultivar Desiree) were generated by Agrobacterium tumefaciens-mediated transformation. Twenty-three of 34 independent transgenic plant lines expressing the active ribozyme R(- ) resulted in having high levels of resistance to PSTVd, being free of PSTVd accumulation after challenge inoculation with PSTVd, but the re maining lines showed weaker levels of resistance to PSTVd with low lev els of PSTVd accumulation, In contrast, 59 of 60 independent transgeni c lines expressing the mutated ribozyme mR(-) were susceptible to PSTV d inoculation and had levels of PSTVd accumulation similar to that of the control plants transformed with the empty vector. The resistance a gainst PSTVd replication was stably inherited to the vegetative progen ies.