, of 110 species (>80 genera) (Sakai and Engelmann, 2007) Such t

, of 110 species (>80 genera) (Sakai and Engelmann, 2007). Such techniques are now being applied on a large scale; for example, to protect the potato collection at the International Potato Center, Peru and the banana and plantain collection in the Laboratory of Tropical Crop Improvement, Catholic University of Leuven, Belgium. Some examples of the tree species cryopreserved

by vitrification methods are given in Table 3. Most of the species are of interest to commercial forestry or are valuable fruit trees. Vitrification of the intracellular constituents during cooling can be achieved by partial drying of the sample in air; for example, the embryos or embryonic GSK3 inhibitor axes of five species of citrus cryopreserved after desiccation to c. 12% MC (Malik et al., 2012). In this example, longevity of the partially-dried embryos at −20 °C was limited to a few months NLG919 clinical trial only, whilst the cryopreserved samples were reported to retain high levels of viability after 6–8 years. There remain species-specific and specimen-specific subtleties in successful embryo cryopreservation, as a result of differing physiological states, intraspecific genetic variation, morphological variation from the shoot to root poles, and large differences in chemical composition and visco-elastic properties. Necessary adjustments to the methods

relate to the pre-culture phase, exposure to loading solution prior to PVS treatment, and the unloading phase. Consequently, there has been a determination to devise and apply generic methods. Two such methods

are cathodic amelioration and vacuum-infiltration vitrification (VIV) cryopreservation. Innovations around in vitro storage technology are covered elsewhere in the literature ( FAO, 2013). Cathodic amelioration aims to counteract the reactive oxygen species produced during cryopreservative Fossariinae procedures; for example, both excision and dehydration of recalcitrant seed axes of Castanea sativa (sweet chestnut) are known to trigger the production of superoxide radicals ( Roach et al., 2008). Improved cryopreservation success (promoting shoot development) has been achieved through the immersion of axes of Strychnos gerrardii (coast monkey-orange) in cathodic water after cryopreservation ( Berjak et al., 2011). The strongly reducing, high pH cathodic water is produced by electrolysis of a solution containing calcium and magnesium chloride. Vacuum infiltration vitrification (VIV) cryopreservation seeks to increase the uniformity of PVS penetration into plant embryos that vary in permeability due to differences in tissue mass, morphology, hydrophobicity and visco-elasticity (Nadarajan and Pritchard, 2014). Such variability in tissue properties has previously demanded empirical determinations of PVS exposure times, to balance the benefits of tissue dehydration whilst avoiding excessive chemical toxicity.

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