No abstract

Conditions were developed for microclonal propagation, callus induction and proliferation, plant regeneration, and long-term maintenance of fast-growing normal root cultures of Gentiana species from the Ukrainian flora. The basic growth parameters were evaluated for cultured tissues. Extensive growth and considerable biomass yield was achieved in most cultures. The ability to form tissue and organ cultures depended on the original genotype, type of explant, growth regulator, and mineral composition of the nutrient medium. The efficiency of regeneration from cultured tissue declined with the duration of callus maintenance.

This chapter presents the results of cytogenetic and molecular genetic studies on tissue and organ cultures from some Gentiana species. Cytogenetic analysis of calli from seven gentians showed that all of them (except for G. punctata) consisted mainly of the cells with diploid and/or near-diploid chromosome sets with low anaphase aberration. Species specificity of cytogenetic structure was established for cell populations in vitro. An increase in the proportion of polyploid cells was found for G. acaulis callus after long-term culture. RAPD analysis of tissue cultures from six gentians showed that culture resulted in genetic changes, with the species differing in the level of genetic variation. RAPD and ISSR analyses of G. pneumonanthe and G. lutea genetic variation in tissue and organ cultures and regenerated plants revealed the most significant changes in isolated root cultures as well as in long-term cultured tissues. At the same time, direct and indirect regenerants had the least genetic difference compared to donor plant. The restriction maps of nuclear 18S-25S ribosomal DNA from G. acaulis, G. punctata, G. lutea, and G. asclepiadea were constructed based on the results of blot hybridization. The decrease of 18S-25S rDNA amount was found in cultured tissues. In contrast to other species, G. lutea showed intragenome heterogeneity of rRNA genes as well as qualitative rDNA changes in tissue culture, such as the appearance of repeats with altered length. A relationship has been suggested between the peculiarities of the structural organization of the 18S-25S rRNA gene cluster and its rearrangements. Rearrangements of 5S rDNA were not found in cultures of Gentiana L. species, except for the variation in nucleotide methylation pattern at restriction sites.