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PRO LO NG ED CULTIVATIO N OF SOMATIC P LA N T C E LL
GENOMEVARIABILITY IN RAUWOLFIASERPENTINABENTH. CELLS
DURINGTHELONG-TERMCULTURING
E.V.Spiridonova, V.T.Solovyan, V .A.Kunakh
Institute ofMolecular Biology and Genetics NAS Ukraine, 252143 Kyiv, Zabolotnogo str.,
150 ,
Through the use of the DNA restriction assay, dot-blot hybridization of
a number o f cloned sequences and genes with DNA from several Rauwolfia
species (R.serpentina, R.verticillata, R.caffra, R.canescens, R.vomitoria)
and R.serpentina callus tissue passaged since 1964 there was revealed the
essential cultured cell genome rearrangement. The restriction fragment length
polymorphism (RFLP) involved was largely due to the variations in fractions
o f d iffe re n t sequences that were manifested in the am p lifica tion ,
deamplification, deletions and other alterations. Examination of the intact
plants from the different Rauwolfia species demonstrated that the varying
sequences in the cultured R.serpentina cells v/ere characterized by the
interspecies RFLP. In some cases the variations brought out in the cultured
cells were found to occur in their related intact counterparts.
By the using o f Mspl and Hpall restriction endonuclease isoshisomeres
it was shown that in cultured cells there take place modifications in
methylation o f the C*CGG sequence. This contributes a great deal to RFLP
as suggested by the blot-hybridization using 32P-labeled genes of ribosomal
DNA (pea), Rubisco (pea) and SAR o f Adhl-gene (maize). These modifi­
cations resulted to a certain extent in the interspecies variations as well.
Thus, during the long-term culturing there proceed the genome changes
arising from both the rearrangements o f certain sequences and DNA
modification via cytosine bases methylation. One may speculate the
existence o f parallelism between the genome evolution in culture and
interspecies variability found among the related Rauwolfia species. It is
hypothe-sizedthe canalized nature of the genome changes in cultured cell
populations.
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