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In the genus Hypericum, cryoconservation offers a strategy for maintenance of remarkable biodiversity, emerging from large inter- and intra-specific variability in morphological and phytochemical characteristics. Long-term cryostorage thus represents a proper tool for preservation of genetic resources of endangered and threatened Hypericum species or new somaclonal variants with unique properties. Many representatives of the genus are known as producers of pharmacologically important polyketides, namely naphthodianthrones and phloroglucinols. As a part of numerous in vitro collections, the nearly cosmopolitan Hypericum perforatum – Saint John’s wort – has become a suitable model system for application of biotechnological approaches providing an attractive alternative to the traditional methods for secondary metabolite production. The necessary requirements for efficient cryopreservation include a high survival rate along with an unchanged biochemical profile of plants regenerated from cryopreserved cells. Understanding of the processes which are critical for recovery of H. perforatum cells after the cryogenic treatment enables establishment of cryopreservation protocols applicable to a broad number of Hypericum species. Among them, several endemic taxa attract a particular attention due to their unique characteristics or yet unrevealed spectrum of bioactive compounds. In this review, recent advances in the conventional two-step and vitrification-based cryopreservation techniques are presented in relation to the recovery rate and biosynthetic capacity of Hypericum spp. The pre-cryogenic treatments which were identified to be crucial for successful post-cryogenic recovery are discussed. Being a part of genetic predisposition, the freezing tolerance as a necessary precondition for successful post-cryogenic recovery is pointed out. Additionally, a beneficial influence of cold stress on modulating naphthodianthrone biosynthesis is outlined.

The discovery of paclitaxel, a diterpene pseudalkaloid with antineoplastic properties has started a new medicinal use of yew. Although the ability of plant cell cultures to produce paclitaxel is well established, the manipulation of nutritional and environmental conditions, as well as the selection and cloning of new cell lines with appropriate genetic, biochemical and physiological properties is a crucial point for the future biotechnological production of taxanes.

An altered ploidy level was observed in plants regenerated by adventitious shoot formation from seedlings of Hypcricurn perforotlim L. (2n = 4 x = 32). Among the somaclones of the R, generation, the presence of diploids (2n = 2 x =16), triploids (2 n = 3 x = 24). tetraploids (2n = 4 x = 32) and mixoploids was detected. Cytogenetic analyses of the R , and R2 progenies showed that the chromosomal instahility of the Ro somadones wils transferred onto the next generations. While almost all the seed progeny of diploids (100%~ in R1 and 94% in R2) and more than 60% of tetraploids (61% in R1 and 73% in R2) retained their chromosome number, cytogenetic diversity was observed in the progeny of triploids, mixoploids and some tetraploids. Somaclones and their offspiing were analyzed for hypericin content. Statistical evaluation showed a comelation between hypericin content and ploidy during a two-year cultivation of Ro somaclones and in their R I and R2 progenies.

The time-course of expression of dbat and dbtnbt genes involved in the later steps of paclitaxel biosynthesis and the intracellular taxane accumulation were investigated through a 64-day subculture interval of VI/M1 and VI/M2 Taxus baccata callus cultures. HPLC proved traces of baccatin III and an intracellular content of paclitaxel up to 90 μg/g DW. The steadystate of the respective gene transcripts was measured by quantitative real-time RT-PCR. The expression profile of dbat and dbtnbt genes was slightly different and varied within the subculture. The highest level of dbat expression was detected 24 h after inoculation followed by a decrease in both cultures. In contrast with dbat no substantially high expression of the dbtnbt gene after inoculation was observed. The impact of the conditions during inoculation on gene expression is discussed. Although the increase in transcriptional activity of both genes positively correlated with callus growth, the intracellular accumulation of paclitaxel varied during subculture with the maximum in the stationary (VI/M1) or at the end of the linear (VI/M2) phase. The increase of the steady-state mRNA level of the dbtnbt gene was followed by paclitaxel accumulation with a delay of approx. 28 (VI/M1) and 14 days (VI/M2).

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