No abstract

Hairy root-regenerated clones of Hypericum perforatum L. grown in vitro similarly to those successfully adapted to ex vitro conditions showed phenotype features typical for plants transformed with Agrobacterium rhizogenes T-DNA. These included reduced apical dominance, increased branching, dwarfing and reduced fertility. Transgenic clones differed in ability to develop root system as a necessary condition for transfer to the soil. One of the profiling characters, capability of hypericin biosynthesis was altered as well. Dark glands as the sites of hypericin accumulation and/or synthesis exhibited significantly higher densities on both, leaves and petals of transgenic clones comparing to controls. In the genome of transgenic clones, rolABC genes were detected. Both clones harboured similar copy number of individual rol genes. However, copy numbers descended from rolA to rolC gene in both clones.

Flow cytometry seed screen of mature seeds originating from several in vitro regenerated Hypericum perforatum L. somaclones and their seed progenies were used to screen the ways of reproduction of 4 subsequent generations of several somaclonal families and to search for the relation between the ploidy and prevalent mode of reproduction. The prevalent reproduction pathway of diploid plants was sexual reproduction. Seed samples of plants with higher ploidy levels showed an extensive variation in the mode of reproduction: BII and BIII hybrid formation and/or aposporous pseudogamy including parthenogenetic development of a reduced embryo sac.

The content of hypericins in in vitro regenerated Hypericum perforatum plants (R (0)) and four generations of their seed progeny (R (1)-R (4)) was compared. The mean content of hypericins in field-grown plants over the period 1992-2002 gradually increased under selection, and in the R (4) generation it was almost seven-times higher than that in the R (0) somaclones. Significant difference between hypericin content in diploids and tetraploids was detected in R (0), R (1) and R (3) generations. Hypericin content in four diploid and tetraploid lineages originated from a single somaclone was genotype dependent. To eliminate the influence of environmental conditions during different growing seasons, we used seeds of selected R (0)-R (3) plants to derive R'(1) to R'(4) generations cultivated during the same years. In this case no statistically significant difference in hypericin content was found between the R'(1)-R'(4) generations. Apomictically and sexually derived plants were distinguished by PCR using variable numbers of tandem repeats (VNTR) primers. The content of hypericins in apomictically derived progenies was compared.