Post-cryopreservation treatments are equally as important as the pre-cooling steps to obtain satisfying (at least 40%) explant recovery after cryostorage. Rewarming is performed either at room temperature (as in the case of droplet-vitrification technique) or quickly in a 35–40 °C water bath to avoid ice recrystallization (aggregation of smaller crystals into bigger ones).

PVS are highly effective when performing dehydration or binding to water, preventing it from crystallizing; however, some components of these solutions are toxic to the cell (e.g., DMSO). Consequently, it is necessary to completely remove the PVS solution from the biological material and to secure the cells against uncontrolled rehydration after LN storage [111]. Therefore, after rewarming, the explants are washed with a concentrated sucrose solution, usually liquid MS salts with 1.2 M sucrose for 2 × 1.5 min, the so-called Sakai’s unloading solution (RS) [28]. Maintaining the temperature of the cells at 0 °C during the de- and rehydration processes increased cell survival in wheat [49].

The selection of an appropriate recovery medium plays a critical role in plant regrowth by controlling totipotency, growth, and development of cells and tissues. It is recommended to perform the recovery culture of non-encapsulated explants on a medium with increased osmotic potential and with an addition of appropriate plant growth regulators (PGRs), such as gibberellic acid (GA) during the first few days of recovery [112]. Recent studies demonstrated a higher efficiency of meta-topolin (mT), a non-conventional cytokinin, than N6-benzyladenine (BA) when used during the recovery phase in the LN-derived lateral buds of hazelnut (Corylus avellana L.) [113]. Conventional cytokinins, such as BA, are reported to cause certain morpho-physiological, anatomical, and biochemical disorders [114,115]. Meta-topolin varies from BA in the conversion of its major metabolic product O-glucoside, which translocates quickly to different parts of the explants [116]. By such means, mT alleviates in vitro-influenced disorders such as leaf senescence, hyperhydric shoots, and shoot apex and leaf necrosis [115,117,118].

Physical conditions in the growth room should also be considered. Light quantity and quality (i.e., its intensity, photoperiod, and spectral composition) affect morphogenetic responses of in vitro plants. Modification of light spectra both before LN storage and during recovery after cryopreservation improves survival and recovery. Yoon et al. [45] reported that several potato cultivars grown under high light intensity (140 nmol m−2 s−1) prior to cryopreservation resulted in significantly higher post-cryopreservation recovery. Edesi et al. [119] showed that blue light promoted growth potential, photomorphogenesis, and subsequent survival after cryopreservation of potato clones, while Mølmann et al. [120] showed that the red light produced strong inhibition of sprout elongation even at low irradiances 10–100 nmol m−2 s−1 in the same species. Nonetheless, the effect of modified light conditions on cryopreservation efficiency is still not well studied.

Successful recovery appears to be dependent upon the presence of antioxidant protection from reactive oxygen species (ROS), often occurring after cryostorage [121]. The addition of vitamin additives into the culture medium such as ascorbic acid and tocopherol were shown to reduce oxidative damage. Moreover, the use of non-vitamin antioxidants (antiradicals) such as lipoic acid, glutathione, glycine betaine, and polyvinylpyrrolidone is beneficial. These natural compounds showed interesting results for viability in Rubus spp. shoot tips, which increased by 25% after LN storage [122].

The use of NPs in recent years has received attention since they are proving benefits to improve in vitro plant properties and regeneration rate, depending on their type, size, shape, and concentration [123,124]. It is presumed that the addition of NPs into the recovery medium could result in pores formation in the roots stimulating a greater uptake of water and, consequently, in significant plant growth. This phenomenon appeared in the case of manganese nanoparticles (MnNPs), which produced a differential change in the morphology and physiology of deadly nightshade (Atropa belladonna L.) with lower doses at 50 mg·L−1 [125]. Silver nanoparticles (AgNPs) were applied to rice (Oryza sativa L., cv. Swarna) and described as phytostimulants in some active compounds (i.e., chlorophyll and carotenoids) without negative impacts on the plant [126]. On the other hand, the NP excess could produce phytotoxicity, oxidative stress damage, and genotoxic effects [125,127]. For example, growth inhibition, especially in the root zone, was evident in wheat (Triticum aestivum L.) using copper nanoparticles (CuNPs) [128]. Overall, nanoparticles have become an attractive alternative to improve agrobiotechnological production; even so, there are still limitations that must be studied, analyzed, and controlled to successfully apply NPs during the pre- and post-LN storage steps.

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