IRKUTSK, USSR

Siberian Inst. Plant Physiol. & Biochem.

KRASNODAR, USSR

Krasnodar Research Inst. Agriculture

Possible free radical mechanism of appearance of somaclonal variations in maize and other plant species --Yu. M. Konstantinov and A. S. Mashnenkov
 
 
Somaclonal variations which have been shown recently in many plant species appear in vitro mainly in callus, cell suspension and isolated protoplast cultures (P. J. Larkin, W. R. Scowcroft, Theor. Appl. Genet. 60:197-214, 1981). These somatic variations can result from cytological changes, point mutations and other less well-defined alterations to the nuclear and cytoplasmic (mitochondrial and chloroplast) genomes. A knowledge of the factors inducing somaclonal variations in plant cell culture is very important since to solve particular biotechnological problems, it is necessary for the cell population to be of high genetic homogeneity or on the contrary it is possible for the culture to be genetically unstable which serves in the latter case as a non-traditional source of economically valuable genotypes.

It is well known that oxygen radicals are generated in plant cells in vivo and in vitro as by-products of normal oxidative metabolism. Being highly reactive, these free radicals can attack cellular DNA directly (J. Imlay, S. Lin, Science 240:1302-1309, 1988). Alternatively, it has been reported that in complex biological systems oxygen radicals can cause DNA damage indirectly by initiating lipid peroxidation (LP) (A. Hruczkewycz, Biochem. Biophys. Res. Commun. 153:191-197, 1988). It is generally agreed that malondialdehyde produced as a peroxidation by-product is a potent cross-linking agent which can inactivate critical biomolecules including enzymes, nucleic acids, lipids (A. Tappel, Fed. Proc. 32:1870-1874, 1972). Besides, oxygen dependent mutagenesis in Escherichia coli lacking superoxide dismutase has been reported (S. Farr et al., PNAS 83:8268-8272, 1986).

Oxidative stress of isolated plant cell and tissue cultures seems to be responsible for the appearance of somaclonal variations. The main reasons for the oxidative stress are obviously the following: 1) altered oxygen regime in plant cells in vitro as compared to in vivo; 2) the disturbance in vitro of the normal antioxidant status of plant cells specific for in vivo conditions and provided with the system of antioxidant defence of the whole organism; 3) the presence in the cell cultivation media in vitro of strong pro-oxidants capable of initiating the LP reactions.

The oxidative stress of plant cell cultures results in a sharp increase of free radical lipid peroxidation of membranes and the resulting products can serve as powerful mutagenic factors and cause various mutations and modifications in the genetic system of plant cells including genomes of nuclei, mitochondria and chloroplasts.

The primary, secondary and final products of LP (free radicals including oxygen radicals, such components as conjugated diene, malondialdehyde and Schiff's bases) can be directly responsible for the plant genome instability during the oxidative stress. It should be emphasized that the LP initiation in certain membrane compartments of plant cells can cause spreading of the peroxidation to other organelles due to distant effect of LP products. When intracellular concentrations of natural antioxidants (a-tocopherol, thiol-containing compounds etc.) are reduced, LP products can induce structural rearrangements of DNA causing single- and double-strand breaks of polynucleotide chains and possibly other structural changes.

At the same time, LP products can greatly affect genetic processes by disturbing normal functions of enzyme systems providing different stages of realization and reproduction of genetic information (transcription, translation, replication) including the systems of repair and recombination of DNA. The enhanced level of transposition of mobile genetic elements can also indicate cell genome instability during LP reactions induced by the oxidative stress in culture systems.

It is well known that the level of DNA methylation determined by the activity of the enzyme system of methylases plays an important role in the regulation of gene expression. In this connection, certain changes in gene functioning in somaclonal variants appear to be determined by disturbances in the normal function of cell methylases affected by LP products.

In addition, a significant amount of resulting chromosomal and other mutations in cell cultures seems to be eliminated at the stage of plant regeneration and at the stage of gametogenesis in asexually and sexually propagated species, respectively.

The analysis of the composition of the most widely used plant tissue culture media argues in favour of the advanced free radical mechanism of the appearance of somaclonal variations. Thus, the Murashige and Skoog medium (T. Murashige, F. Skoog, Physiol. Plant. 15:473-497, 1962) and other plant tissue culture media (E. Linsmaier, F. Skoog, Physiol. Plant. 18:100-127, 1965; J. Nagy, P. Maliga, Z. Pflanzenphysiol. 78:453-455, 1976) contain Fe-EDTA complex (ca. 0.1 mM) with a strong pro-oxidant effect. Consequently, metabolic conditions of plant cells cultivated in artificial plant cell media (increased O2 concentration, the presence of pro-oxidants and reducing equivalents) contribute to the initiation and proceeding of enzymic (NADH- or NADPH-dependent) and nonenzymic (ascorbate-dependent) lipid peroxidation.

Now the experiments to check the validity of the advanced hypothesis are being carried out using cell cultures of maize and other cereals.

In conclusion, significant changes in the oxygen regime of plant tissue and cells during their in vitro cultivation can obviously result in increase of the level of free radical oxidation of membrane lipids, whose products affect the genetic system of the cell and induce the formation of somaclonal variations.


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