The study focused on an economically-important ornamental outdoor shrub, Nandina domestica, with the aims to (i) optimize an effective in vitro conservation method, and (ii) develop a cryopreservation protocol for shoot tips by the PVS2 vitrification and droplet-vitrification techniques. For in vitro conservation of shoot cultures, the tested parameters were sucrose content in the storage medium (30, 45, 60 g/L) and storage temperature (4 °C or 8 °C). Cryopreservation was performed by applying the PVS2 vitrification solution, in 2-ml cryovials or in drops over aluminum foil strips, for 15, 30, 60 or 90 min at 0 °C, followed by the direct immersion in liquid nitrogen of shoot tips. Results show that N. domestica shoots can be conserved successfully for 6 months at both the temperatures tested, especially when 60 g/L sucrose is used in the storage medium. However, conservation at 4 °C showed to be more appropriate, as hyperhydricity was observed in post-conservation of shoots coming from storage at 8 °C. As for cryopreservation, a daily gradual increase of sucrose concentration (from 0.25 to 1.0 M) produced better protection to the samples that were stored in liquid nitrogen. Indeed, with this sucrose treatment method, a 30-min PVS2 incubation time was enough to produce, 60 days after thawing, the best recovery (47% and 50%) of shoot tips, cryopreserved with PVS2 vitrification and droplet-vitrification, respectively.
Cryopreservation of in vitro-grown apical meristems of Lilium by droplet-vitrification