ISSR and somaclonal variation: a new molecular technique for an important in vitro phenomenon
Financial support: Xavier Leroy is supported by a grant of the Brittany Regional Council
Keywords : Brassica, Cell proliferation, DNA-damage, ISSR, Somaclonal variation.
The phenomenon known as somaclonal variation has recently provided a source for development of variant plant lines. In theory, the regeneration of tissue explants into a mature plant should result in the production of clones of the parent plant, i.e., plants which are identical in genotype and phenotype to the plant from which the explant was obtained. In practice, this is in fact the result observed in the vast majority of cases. However, it has long been recognised that occasional abnormalities occur, resulting in variant plants which are generally discussed as 'artefacts' of the tissue culture process. In more recent years, it has become apparent that, rather than being an unexplainable aberration in an otherwise uniform regeneration process, the appearance of variants in tissue culture may be a routine occurrence for certain types of plants and/or specific explant sources. Even more, recently, it has been recognised that the existence of this phenomenon potentially provides a source of useful variation which can form the basis of developing agriculturally useful variant plant lines.
The types of variation which are frequently observed may differ from species to species, and it is often difficult to determine the genetic nature of the observed variation. One of the more frequent types of variation is a difference in chromosome number, i.e., aneuploidy, polyploidy, or mixoploidy. Chromosome changes are known to occur in high frequency in the early stages of callus or liquid cell cultures, and therefore the occurrence of such abnormalities is not particularly surprising. It is possible, however, to select an appropriate culture medium which will enhance chromosome stability in the explant used.
Variations in chromosome number are not only types which have been observed. Deletions and translocations have also been reported. Somaclonal variation has been shown to yield regenerated plants with altered growth habits, disease and pest resistance, fruit colour or size, among others. Although original reports of somaclonal variation were limited to plants which were normally asexually propagated, it is now known that these techniques can be successfully applied to sexually propagated plants as well. It is easy to see, given the right type of stability inherited variation, that such a tool can be extremely useful in establishing improved cultivars in a wide range of different plants, with the advantage of avoiding the numerous crossings usually required to identify and establish a useful hybrid line by traditional breeding methods. Although it appears to be possible, to some extent, to induce variation by manipulation of the culture medium, it is not possible to predict what type of somaclones, if any, may be obtained by the development of a variant of a particular type, or whether the somaclones produced will have value. In other words, the scientist is limited by what randomly occurring variation might appear in his culture; the possibility always exists, therefore, that no agriculturally useful variants will appear.
We have used inter-(simple sequence repeat) PCR (inter-SSR PCR) to simultaneously sample numerous diverse regions of the genome, specially those regions present between inverted abundant repetitive elements. Inter-SSR PCR uses a single primer based on repetitive DNA sequences. This technique produces amplification of those DNA sequences, typically less than 3 kb in size, which are present between relatively close inverted primer-binding repeat sequences. Exploiting abundant repetitive sequences such as CAA or GATA repeats generate approximately 30-40 PCR products. This process allows us to readily measure the occurrence of genomic events in variant regions, by comparing variant DNA to normal DNA from the same plant. As we have described, an estimate of the relative degree of genomic instability is readily obtained for each callus. We present the rigorous sequencing of an altered segment revealed by inter-SSR PCR, which manifests genomic damage. Inter-SSR PCR band alterations need not to be limited to insertions or deletions between the primer binding sites, nor to the deletion of a single primer binding site itself. Such processes indeed alter or eliminate bands, but larger-scale processes such as deletions of entire chromosomes or large fragments thereof could in one step eliminate more than one band.
Figures 1-5 Illustrations of genomic-instability forms and patterns, other than the pattern represented by microsatellite instability, detected by the ISSR method:
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