Morphological characters, AFLP markers and flow cytometry were used to investigate the morphological and genetic variability and differentiation of Viola reichenbachiana and V. riviniana in non-metallicolous (NM) and metallicolous (M) populations. The aims were to clarify the taxonomic status of plants occurring in ore-bearing areas, to determine any relationship in V. reichenbachiana and V. riviniana from sites not polluted with heavy metals, and to examine the genetic variability and differentiation of M and NM populations of both species. Multivariate analyses based on morphological characters showed significant differences between V. reichenbachiana and V. riviniana from non-polluted sites, high levels of intra- and inter-population variability, and the occurrence of inter-specific hybrids. Plants from M populations showed hybrid characters but also fell within the range of V. riviniana or V. reichenbachiana. There were no significant differences in relative genome size between plants from polluted areas and V. riviniana from NM populations. Bayesian analysis of population genetic structure based on AFLP markers distinguished two main groups: V. reichenbachiana and V. riviniana together with the M populations. That analysis also revealed the occurrence of populations of inter-specific hybrids from non-polluted areas. Further Bayesian analysis of V. riviniana including NM and M populations separated all the studied M populations from NM populations. We conclude that plants forming the M populations are well adapted to a metal-polluted environment, and could be considered as stabilised introgressive forms resulting from unidirectional (asymmetric) introgression toward V. riviniana.

Genetic variation of five species belonging to the section Hartmania -Chara baltica, C. intermedia, C. hispida, C. polyacantha and C. rudis -was investigated using Amplified Fragment Length Polymorphism (AFLP) techniques. Chara intermedia can be distinguished from the others by the presence or absence of numerous morphological characters. However, probably because of the high variability of the morphological features and common occurrence of apparently transitional forms, more than 80 specimens of the five species were collected and analysed. The specimens were collected from a total of 24 sites, including freshwater localities and sites in the Baltic Sea, initially classified into the five species based on morphological features, and then analysed using AFLP; as an outgroup we used C. globularis. The specimens formed two groups, the first group comprising C. intermedia and C. baltica, the second C. hispida, C. rudis and C. polyacantha. No consistent genetic differences were found between species within each of the two groups. Transitional forms of C. hispida/C. rudis were placed in a clade between individuals of C. hispida and C. rudis. Local populations of some charophytes differ morphologically from one another, possibly as a result of local adaptation.

Hypericum sinaicum L. is an endangered Egyptian medicinal plant of high importance due to the presence of naphthodianthrones (hypericins), which have photodynamic properties and pharmaceutical potential. We sought to assess H. sinaicum ability to develop hairy roots that could be cultured in contained conditions in vitro and used as a source for hypericin production. We used four A. rhizogenes strains differing in their plasmids and chromosomal backgrounds to inoculate excised H. sinaicum root, stem and leaf explants to induce hairy root development. Additionally, inoculum was applied to shoots held in Rockwool cubes supporting their stand after removal of the root system. All explant types were susceptible to A. rhizogenes although stem explants responded more frequently (over 90%) than other explant types. The A4 and A4T A. rhizogenes strains were highly, and equally effective in hairy root induction on 66-72% of explants while the LBA1334 strain was the most effective in transformation of shoots. Sonication applied to explants during inoculation enhanced the frequency of hairy root development, the most effective was 60 s treatment doubling the percentage of explants with hairy roots. However, shoot transformation was the most effective approach as shoots developed hairy roots within 10 days after inoculation. Molecular analyses confirmed that the established hairy root cultures in vitro were indeed obtained due to a horizontal gene transfer from bacteria. These cultures grew fast and the hypericin content in hairy roots was about two fold higher than in H. sinaicum plants as determined by HPLC.

Chara tenuispina a. br. is an extremely rare species of the genus Chara L., which inhabits freshwater and shaded, shallow water environments on calcareous peat bogs, as compared to C. globularis ThUill., which is a widespread species. To re-examine the taxonomic system proposed by Wood & imahori (1965), who treated C. tenuispina as a variety of C. globularis, we studied the morphology of both species, as well as their phylogenetic relationships, based on three cpDNA gene sequences (atpB, matK, rbcL). In general, the species do not differ significantly in their general appearance, but certain details, such as prolonged stipulodes in upper rows below the branchlet, or the extremely long spine cells in C. tenuispina, may be noted as distinguishing characteristics. In addition, the results of sequence analysis demonstrated that these species are phylogenetically separated, forming distinct clades. This supports the taxonomic interpretation that C. tenuispina is a distinct species rather than variety of C. globularis.