biologia plantarum

International journal on Plant Life established by Bohumil Němec in 1959

Biologia plantarum 36:285, 1994

Instability of potato spindle tuber viroid (PSTVd) cDNA inAgrobacterium tumefaciens

J. Matoušek1, L. Trněná1, S. Rakouský1, D. Riesner2
1 Institute of Plant Molecular Biology, Academy of Sciences of the Czech Republic, České Budějovice, Czech Republic
2 Institut für Physikalische Biologie, Heinrich-Heine-Universität, Düsseldorf, FRG

Potato spindle tuber viroid (PSTVd, severe strain) was isolated from tobacco plants transformed with a dimeric infectious expression construct which was maintained for a long time inAgrobacterium tumefaciens. Afterin vitro hybridization of PSTVd to complete minus transcripts of PSTVd (lethal), the resulting heteroduplexes were analyzed by electrophoresis under native conditions. Electrophoretic analysis revealed an appearence of electrophoretically distinguishable heteroduplexes, suggesting an accumulation of mutated sequence variants of PSTVd had occurred in the plant transformants. TGGE analysis of PSTVd cDNA, re-cloned from the original expression vector pCB1413 to plasmid pUC18 confirmed the accumulation of mutations in the cDNA and the instability of this sequence inA. tumefaciens maintained at 4°C for 2.5 years. One of these point mutations was identified by sequencing the PSTVd cDNAs isolated from the individualE. coli colonies. This transition (GC→AT) was localized at the position of 81 in the PSTVd genome, causing the change of C to U in PSTVd plus RNA. Transformation of tobacco with the freshly prepared expression vector containing the dimeric sequence of PSTVd lethal KF440-2 lead to the propagation of PSTVd electrophoretically identical to that derived from the original sequence and maintained in the tomato by a conventional infection.

Received: February 2, 1993; Accepted: September 13, 1993; Published: June 1, 1994  Show citation

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Matoušek, J., Trněná, L., Rakouský, S., & Riesner, D. (1994). Instability of potato spindle tuber viroid (PSTVd) cDNA inAgrobacterium tumefaciens. Biologia plantarum, 36(2), 285
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