Abstract
A suspension culture from potato spindle tuber viroid (PSTV)-infected cells of the wild type potato (Solanum demissum) has been established, which is a suitable model system for studying PSTV replicationin vivo. The conditions for rapid growth of these cells and for permanent extensive viroid biosynthesis within them are described. Biosynthesis of PSTV in the potato cells was demonstrated by32P-incorporation into nucleic acids and their subsequent electrophoretic analysis on polyacrylamide gels. Under optimum culture conditions the amount of32P-orthophosphate incorporation into PSTV reached 10% of that incorporated into the 2 M LiCl-soluble cellular RNA. (+)PSTV and its complementary form, i.e. (−)PSTV were identified after their electrophoretic separation on polyacrylamide and agarose gels by molecular hybridization. This analysis revealed the presence of six high molecular weight(−)PSTV species, which are possibly multimers of the unit length(+)PSTV molecule consisting of 359 nucleotides.
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Abbreviations
- 2,4-D:
-
2,4-dichlorophenoxy acetic acid
- 6BA:
-
6-benzyladenine
- NAA:
-
naphtalene acetic acid
- B5 medium:
-
medium of Gamborget al. (6)
- EDTA:
-
ethylenediaminetetraacetic acid
- LS medium:
-
medium of Linsmaier & Skoog (11)
- Tris:
-
Tris-hydroxymethylaminomethane
- SDS:
-
dodecylsulphate sodium salt
- SSC:
-
0.15 M NaCl/0.015 M Na-citrate
- PCV:
-
packed cell volume
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The infectious and sequenced PSTV is arbitrarily designated (+)PSTV
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Mühlbach, HP., Faustmann, O. & Sänger, H.L. Contitions for optimal growth of a PSTV-infected potato cell suspension and detection of viroid-complementary longer-than-unit-length RNA in these cells. Plant Mol Biol 2, 239–247 (1983). https://doi.org/10.1007/BF01578642
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DOI: https://doi.org/10.1007/BF01578642