Regeneration of protoplasts to permit expression of genetic variants resulting from precocious events

Heterozygous, homozygous and hetero-allelic situations were created in the case of Nicotiana tabacum L. and Petunia hybrida Hort. with marker genes regulating chloroplast differentiation or anthocyanin production in order to test the somatic stability of the genome, both in normal conditions and after mesophyll protoplast regeneration into plants. In the case of tobacco it was observed that the frequency of variance in double heterozygous genotypes was approximately 400 times greater than in the palisade parenchyma. A set of different genotypes at the same locus showed that heterozygosity was the most important factor leading to the observed frequency of variation. A genotype analysis of the regenerated variant plants showed that the discontinuous changes were always due to changes in genotype involving the marker genes. The problem of a preferential time of occurrence for the genotype events leading to variation was tackled by subcloning protoplast-derived colonies. The variant sectors involved two or three quadrants; none of the subcloned colonies was shown to be homogeneously variant. This can only be explained by a genetic event occurring, at the earliest, in the differentiated leaf cell and, at the latest, before the second division of the microcolony. These two observations - high frequency of variant plants and early appearance - argue in favour of the practical value of variability between the protoplast-derived clones. The process was tentatively verified by means of another system; an interline F1 hybrid of Petunia carrying seven marker genes showed a variant frequency that is most probably far higher after protoplast regeneration than in normal plant tissue. It appears, however, that there are very likely genome regions more favoured in somatic variation. (author)