We investigated the effects of plant growth regulators [6-benzyladenine (BA), kinetin (Kin), 6-c, c-dimethylallylaminopurine (2iP), thidiazuron (TDZ) and a-naphthaleneacetic acid (NAA)], modified Murashige and Skoog (MS) medium containing 10 mM NH 4 ? and 5 mM NO 3 -and supplemented with 2iP, BA, Kin and NAA (MSM medium), and two elicitors [jasmonic acid (JA), and salicylic acid (SA)], on plant growth and accumulation of hypericins (hypericin and pseudohypericin) and hyperforin in shoot cultures of Hypericum hirsutum and H. maculatum. Our data suggested that culture of shoots on MS medium supplemented with BA (0.4 mg l -1 ) or Kin (0.4 mg l -1 ) enhanced production of hypericins in H. maculatum and hyperforin in H. hirsutum. Hypericins and hyperforin concentrations decreased in both species when TDZ (0.4 mg l -1 ) was added to the MS medium. Also, TDZ induced hyperhydric malformations and necrosis of regenerated shoots. Cultivation of H. maculatum on MSM medium resulted in approximately twofold increased production of hypericins compared to controls, and the growth of H. hirsutum shoots on the same medium led to a 6.16-fold increase in hyperforin production. Of the two elicitors, SA was more effective in stimulating the accumulation of hypericins. At 50 lM, SA enhanced the production of hypericin (7.98-fold) and pseudohypericin (13.58-fold) in H. hirsutum, and, at 200 lM, enhanced the production of hypericin (2.2-fold) and pseudohypericin (3.94-fold) in H. maculatum.

The preservation and reproduction of gene resources of rare and endemic rowan species were the main aims of this study. Rowan species represent important woody trees from the aspect of forest biodiversity. There are only several endemic rowan species in the Czech Republic which are of hybridogenous origin from the whitebeam (Sorbus aria) range. Most of them have been described only at the end of the 20 th century. For these species, the new procedures of vegetative reproduction were developed. In vitro cultures from dormant buds of 57 mature trees were established. The successful induction of organogenesis was achieved in a MS modified medium with 0.2 mg·l-1 of BAP, 0.1 mg·l-1 of IBA, 200 mg·l-1 of glutamine, 2 mg·l-1 of glycine, 200 mg·l-1 of casein hydrolysate, 30 mg·l-1 of sucrose, and 6 mg·l-1 of agar. The pH was adjusted to 5.8. NAA in the concentration of 13.5 mg·l-1 was efficient for the rooting of microcuttings. An efficient protocol for the reproduction of endemic rowan species by means of organogenesis induction in apical meristems of dormant buds is reported.

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Shoot tips of five tomato (Lycopersicon esculentum Mill.) genotypes were successfully cryopreserved by droplet vitrification. Recovered plants were studied for genetic stability by two different assays: amplified fragment length polymorphism (AFLP) using 4 primer combinations and sequencing of lycopene β-cyclase gene (LCY-B) from leaves. The highest shoot regeneration after cryopreservation was 70% (Pontica) following 24 h osmoprotection in 0.5 M sucrose and 30 min dehydration in plant vitrification solution 2 (PVS2). The data inferred from AFLP showed no genetic dissimilarities between in vitro regenerants derived from cryopreserved tissues compared with the noncryopreserved controls. Although a single nucleotide polymorphism, a G→T transversion, was identified in position 1139 in Capriciu and Darsirius, this was not due to the cryopreservation process itself, since it was detected in both cryopreserved and control samples. Thus, sequencing of LCY-B gene from leaves revealed no DNA damages after cryopreservation and subsequent in vitro regeneration. Our results indicate that cryostorage by droplet vitrification is an efficient and reliable technique to preserve the selected tomato genotypes and to regenerate true-to-type plants.

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Romanian tomato (Lycopersicon esculentum Mill.) cultivars have been cryopreserved by encapsulation-dehydration and successfully acclimatized to ex vitro growth conditions. Shoot tips were excised from in vitro grown plants then precultured for 24 h in various sucrose concentrations, dehydrated up to 6 h in laminar air flow prior to direct immersion in liquid nitrogen (−196 °C) for 24 h. Different parameters have been studied: the effects of osmoprotection and desiccation duration on the regrowth of cryopreserved shoot tips, the effects of various IBA concentrations on rooting and the ex vitro acclimatization of plants recovered from liquid nitrogen. The highest frequency of regrowth (72% cv. 'Pontica') was obtained when encapsulated explants were precultured in 0.5 M sucrose and the moisture content (fresh weight basis) of alginate beads was 23%. The highest rooting rates (58% to 77%) for all cultivars were observed for shoots grown on MS medium supplemented with 1.0 mg/l IBA. The rooted plants could be easily acclimatized ex vitro with up to 100% survival.

Romanian tomato (Lycopersicon esculentum Mill.) cultivars have been cryopreserved by encapsulation-dehydration and successfully acclimatized to ex vitro growth conditions. Shoot tips were excised from in vitro grown plants then precultured for 24 h in various sucrose concentrations, dehydrated up to 6 h in laminar air flow prior to direct immersion in liquid nitrogen   (−196°C) for 24 h. Different parameters have been studied: the effects of osmoprotection and desiccation duration on the regrowth of cryopreserved shoot tips, the effects of various IBA concentrations on rooting and the ex vitro cclimatization of plants recovered from liquid nitrogen. The highest frequency of regrowth (72% cv. ‘Pontica’) was obtained when encapsulated explants were precultured in 0.5 M sucrose and the moisture content (fresh weight basis) of alginate beads was 23%. The highest rooting rates (58% to 77%) for all cultivars were observed for shoots grown on MS medium supplemented with 1.0 mg/l IBA. The rooted plants could be easily acclimatized ex vitro with up to 100% survival.

Apple (Malus × domestica Borkh.) genotypes originating from different plant collections (field collection, in vitro plant collections undergoing or not undergoing cryopreservation) were screened and characterized by SSR markers. Shoot tips excised from plants grown in vitro were successfully cryopreserved by encapsulation-dehydration. The highest regrowth frequency (69%, cultivar Goldrush) of cryopreserved apices was achieved after 24 h of osmoprotection in 0.5 M sucrose, 3 h of desiccation, and 24% water content of alginate beads. No differences in morphological characteristics including shoot length and number and length of roots were observed between controls and plants recovered after cryopreservation. SSR markers were used for calculation of genetic similarities between plants from the field collection, in vitro-micropropagated plants, or plants regenerated after liquid nitrogen storage. The set of microsatellite markers showed a low level of polymorphism among the studied genotypes, which could be distinguished by a specific combination of alleles generated by CH03g07, CH05c02, CH05d11, and CH05e03 primers. The CH03g07, CH05c02, CH05d11, CH05e03, GD96, GD147, and GD162 SSR markers exhibited low levels of polymorphism, while CH04AE07, CH04g10, GD100, and GD142 were nonpolymorphic. The Dice coeffi cient confirmed the effectiveness of SSRs for distinguishing between plants from ex situ collections and preserved plants. No major differences between ex situ plants, micropropagated plants, and plants recovered after cryopreservation were observed.