Quantification of the tissue-culture induced variation in barley (Hordeum vulgare L.)

Abstract

Background: When plant tissue is passaged through in vitro culture, many regenerated plants appear to be no longer clonal copies of their donor genotype. Among the factors that affect this so-called tissue culture induced variation are explant genotype, explant tissue origin, medium composition, and the length of time in culture. Variation is understood to be generated via a combination of genetic and/or epigenetic changes. A lack of any phenotypic variation between regenerants does not necessarily imply a concomitant lack of genetic (or epigenetic) change, and it is therefore of interest to assay the outcomes of tissue culture at the genotypic level.

Results: A variant of methylation sensitive AFLP, based on the isoschizomeric combinations Acc65I/MseI and KpnI/MseI was applied to analyze, at both the sequence and methylation levels, the outcomes of regeneration from tissue culture in barley. Both sequence mutation and alteration in methylation pattern were detected. Two sets of regenerants from each of five DH donor lines were compared. One set was derived via androgenesis, and the other via somatic embryogenesis, developed from immature embryos. These comparisons delivered a quantitative assessment of the various types of somaclonal variation induced. The average level of variation was 6%, of which almost 1.7% could be accounted for by nucleotide mutation, and the remainder by changes in methylation state. The nucleotide mutation rates and the rate of epimutations were substantially similar between the andro- and embryo-derived sets of regenerants across all the donors.

Conclusion: We have developed an AFLP based approach that is capable of describing the qualitative and quantitative characteristics of the tissue culture-induced variation. We believe that this approach will find particular value in the study of patterns of inheritance of somaclonal variation, since non-heritable variation is of little interest for the improvement of plant species which are sexually propagated. Of significant biological interest is the conclusion that the mode of regeneration has no significant effect on the balance between sequence and methylation state change induced by the tissue culture process.

Figure 1

A schematic to interpret the outcomes on regenerant AFLP profiles of (epi)mutation events induced during tissue culture. The possible bases of AFLP profile changes induced during tissue culture are shown in terms of methylation of one or more sites and point mutation at the enzyme recognition site(s). All possible patterns were assigned a binary code. Top row: A single KpnI/Acc65I recognition site within the resolvable amplicon; lower two rows: a pair of KpnI/Acc65I sites within the resolvable amplicon. Grey circle – represents MseI site; pink circle – methylated site digested by KpnI but not Acc65I; green circle – non-methylated site digested by KpnI and Acc65I; red pentagon – former Acc65I/KpnI after mutation; red and black lines – donor and regenerant DNAs, respectively.