Cryopreservation of Thymus cariensis and T. vulgaris shoot tips: comparison of three vitrification-based methods.

Thymus is an important genus of the Lamiaceae family, comprising more than 400 perennial aromatic thyme species, which are used extensively for medicinal and culinary purposes. The present study focused on the development of cryopreservation procedures for Thymus vulgaris and T. cariensis, the latter being an endemic and endangered species of Turkey. For cryopreservation of T. vulgaris shoot tips, PVS2-based one-step freezing methods, i.e., PVS2 vitrification, encapsulation-vitrification and droplet-vitrification, were compared. Cold hardening and sucrose preculture were also optimized before the cryopreservation trials. For T. cariensis, a droplet-vitrification method was applied to cold-hardened shoot tips, and after sucrose preculture. In all the methods tested, PVS2 was applied for up to 120 min. The best T. vulgaris cryopreservation was achieved with a droplet-vitrification method, that involved 2-weeks cold hardening of shoot cultures, 48 h preculture of shoot tips on MS medium supplemented with 0.25 M sucrose, and a 90 min PVS2 treatment in droplets. After direct immersion in LN, thawing and plating, 80% of shoot-tips recovered. Post-thaw recovery was significantly lower when the same procedure was applied to T. cariensis shoot tips; however also here 90 min PVS2 treatment produced the highest survival (25 percent) and recovery (25 percent) levels.

Cryopreservation of redwood (Sequoia sempervirens) in vitro buds using vitrification-based techniques.

In this study, the efficiency of three vitrification-based cryopreservation techniques, i.e. vitrification, encapsulation-vitrification and droplet-vitrification were compared for cryopreserving Sequoia sempervirens apical and basal buds sampled from in vitro shoot cultures. The effect of cold-hardening of mother-plants and of bud culture medium and sucrose preculture was also investigated. Culture of apical and basal buds sampled from cold-hardened mother-plants on Quoirin and Lepoivre medium with activated charcoal had a positive effect on regrowth. Only droplet-vitrification ensured survival and regrowth after cryopreservation. After cryopreservation, regeneration of apical buds was possible for PVS2 exposure durations between 90 and 180 min but it remained low, with a maximum of 18 percent after 135 min treatment. With basal buds, regeneration after cryopreservation was possible over a larger range of PVS2 treatment durations, between 30 and 180 min. The highest regeneration percentage was slightly higher (22 percent) than that measured with apical buds, and was also achieved after 135 min PVS2 exposure.

Cryopreservation of Fraxinus excelsior L. embryogenic callus by one-step freezing and slow cooling techniques.

An efficient cryopreservation protocol for the safe storage of Fraxinus excelsior L. embryogenic callus cultures is reported. The cryopreservation methods tested included one-step freezing by means of (i) encapsulation-vitrification; or (ii) encapsulation-dehydration; and (iii) slow cooling using the Nalgene Freezing container, Mr Frosty, which produces a temperature decrease of about 1 masculineC min-1 when placed in a -70 degree C freezer. None of the one-step freezing techniques was effective for cryopreservation of encapsulated callus masses, irrespective of the cryoprotective treatment applied, i.e., treatment with the PVS2 vitrification solution or physical dehydration with silica gel before direct immersion in liquid nitrogen. On the contrary, when a slow cooling protocol was applied to embryogenic callus which had been pretreated for 60 min with a 210 g per liter (0.61 M) sucrose-7.5 percent DMSO cryoprotective solution, up to about 1.3 g per Petri dish of proliferating callus was observed 42 days after recovery from liquid nitrogen, and cultures were able to produce somatic embryos 8 weeks after transfer to semi-solid medium. TTC staining of callus cultures provided a fast evaluation of culture viability.

Cryopreservation of Pistacia spp. seeds by dehydration and one-step freezing.

Cryopreservation protocols by dehydration and one-step freezing were developed for seeds from three Pistacia species, i.e., P. vera, P. terebinthus and P. lentiscus, which were characterised by different initial germination percentages (100%, 17% and 81%, respectively). In P. vera, a maximum of 90% germination was obtained following 8 hours drying in silica gel (corresponding to 11.7% moisture content on a FW basis) and direct immersion in LN. In P. terebinthus and P. lentiscus, shorter periods of dehydration (1 hour and 15 min, respectively) were sufficient to reduce their moisture content to about 20%, which resulted in peak seed germination percentages from cryostorage of 16% and 47%, respectively. Following cryopreservation, the seeds germinated better on semi-solid MS medium, than on cotton wool wetted with dH(2)O or liquid MS medium. Finally, in P. vera and P. lentiscus, high and significant correlation coefficients were obtained between the TTC viability test and seed germinability after recovery from LN, provided that seeds which were considered positive in the test showed completely or partially red embryonic axes coupled to completely red cotyledons.