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This study was carried out on Kober 5BB (Vitis Berlandieri × V. riparia) grape rootstock shoot tips during the preparatory steps preceding the direct immersion in liquid nitrogen, in order to overcome until now unsuccessful cryopreservation with this species. The exposure of shoot tips to 0.3-0.4 M sucrose leads to a high cell solute concentration. The treatment with plant vitrification solution (PVS2) alone, i.e., not followed by storage in liquid nitrogen, markedly affected shoot tip survival. After a 30 min exposure, regrowth percentage of shoot tips decreased from 94 % (control) to 57 %, and dropped to 15 % when the treatment was prolonged up to 60 min. After a 90 min exposure, no regrowth occurred. In addition, plantlets regenerated from shoot tips which underwent 60 min or more exposure to PVS2 showed signs of malformation. Microscope observations of shoot tips treated with 0.3 or 0.4 M sucrose and 30 min PVS2 showed the presence of cells starting to plasmolyze, localized in the area surrounding the apical meristem. A limited presence of starch grains in meristem and bract cells was also noted. However, the most conspicuous consequence of prolonged PVS2 treatment was convex plasmolysis. The phenomenon was dependent on the time of PVS2 exposure. Indeed, after a 30 min treatment, plasmolysis was minimal or absent, but it increased with longer exposure to PVS2 at 4 °C.

A morphological and anatomical study of regenerants obtained from mandarin anther culture was carried out. Beside morphologically normal somatic embryos, abnormal structures were originated in the course of somatic embryogenesis. Anatomical anomalies can be found at several growth stages, causing the formation of slender stems, stubby structures, non-functional leaves. When too long, some structures are subject to shedding, with the formation of various abscission zones. Most of them are subject to degeneration, although many are capable of further, localized, morphogenesis. A thorough knowledge of morphology and anatomy of normal and abnormal regenerants could make possible to select and subculture the lines considered most suitable for conversion into plantlets.

Shoot tips from in vitro stock plants of red chicory ‘Rosso di Chioggia’ line were cryopreserved by one-step vitrification. After two days of cold-hardening on hormone-free MS medium and loading for 30 min in a mixture of 2 M glycerol and 0.4 M sucrose at 25°C, shoot tips were dehydrated with PVS2 vitrification solution at 0°C for 60 min and plunged directly into liquid nitrogen. The post-thaw survival of shoot tips was achieved 79% when was cultured on recovery medium containing 0.5 mM BA. Observed regrowth, after six weeks of culture in the same medium composition, was 100%. Rooted cryopreserved microshoots showed good quality when transferred to the greenhouse. Preliminary results proved that the genetic fidelity of the cryopreserved line was maintained. The same vitrification protocol was then applied to three other red chicory lines, ‘Rosso di Treviso precoce’, ‘Rosso di Treviso tardivo’ and ‘Castelfranco’. A simple and effective protocol for the cryopreservation of red chicory shoot tips has been successfully developed as a result of this study.

The application of three different vitrification-based freezing strategies for the cryostorage of white poplar (Populus alba L.) and hybrid aspen (P. tremula L. × P. tremuloides Michx.) have been assessed. The PVS2 vitrification protocol was successfully applied to two white poplar in vitro clones stored for more than 6 months in slow-growth conditions (4 °C, in darkness) and showing clear signs of explant etiolation and decay. After 60 min of PVS2 treatment, P. alba L. (cv. Villafranca) explants isolated from axillary buds demonstrated significantly better potential for post-freeze regrowth (64%) compared to those obtained from apical buds (17%). Similarly, a high level of survival (78%) of the frozen hybrid aspen shoot tips was recorded following the application of the same technique. Using the encapsulation-vitrification procedure, no toxic effects of the PVS2 treatment were noticed after 120 min exposure, however none of the cryopreserved (poplar and aspen) explants survived after 3 weeks. In contrast, the droplet-vitrification technique appeared to be very efficient in the cryopreservation of white poplar shoot tips, which increases the opportunities for wider application of this method in other woody species.;