Introduction
Refrigeration of working and active seed collections
Freeze-storage and cryopreservation
History of cryopreservation
Biological material | Procedure | Effectiveness | Remarks | References |
---|---|---|---|---|
S. lycopersicum ‘Ailsa Craig’ seeds | Seeds dried to 6–7% MC and sealed into aluminum foil strips → direct immersion in LN (180–1095 d) → rewarming at 40 °C (30 s) | 100% Explants viability | Difficult access to the paper | Grout (1980) |
S. betaceum ‘Amarillo’, ‘Común’, ‘Mora’, seeds | Seeds air dried to a level of 6.7–7.3% MC and directly immersed in LN (> 1 h) → rewarming in water at 37 °C (1 min) → recovery on semi-solid (0.45% agar) MS medium with 0.577 µM GA3 | 37% Germination ‘Amarillo’ 86% Germination ‘Mora’ 94% Germination ‘Común’ | Approximately 69–88% of non-germinated cryopreserved seeds remained viable according to the TTC test. Rapid desiccation and storage in LN probably induced dormancy in those seeds | Montoya et al. (2000) |
S. lycopersicum seeds | Seeds with 12% MC directly immersed in LN (7–28 days) | 53–57% Germination of both control and cryopreserved seeds. Recovered shoots were phenotypically similar with the control | The levels of cell wall-linked, free and total phenolics changed in seeds and recovered plantlets, depending on the duration of storage in LN | Zevallos et al. (2013, 2014) |
S. lycopersicum transgenic seeds | Seeds dried over silica gel and directly immersed in LN (> 1 h) → recovery in darkness | 74–100% Germination, depending on the line | No information about the rewarming procedure | Al-Abdallat et al. (2017) |
S. lycopersicum pollen | Dehydration to 6.8–9.3% MC by equilibration in air with 50–60% RH → storage in LN (22 months) → rewarming → rehydration → germination on semi-solid medium | 56.8–57.4% germination | The ability of pollen to set fruit and to produce seeds was not affected by cryostorage period | Karipidis et al. (2007) |
Vials with fresh pollen immersed in LN (1 min) → vials transferred to a freeze-dryer and dried with vacuum (30 min) → storage at − 20 °C (270 days) → rewarming in water at 35 °C (1 min) | Seed set after pollination with cryopreservation-recovered pollen perceived as “acceptable” | No precise data about pollen survival and fertility | Grout and Crisp (1995) | |
Fresh samples immersed directly in LN (1 year) | 25% Germination | Reduction in viability probably due to damage occurring during freezing and/or rewarming | Towill (1985) | |
Fresh samples wrapped in aluminum foil packets immersed directly in LN | 85% Germination | No attempt was made to set seed with this material | ||
S. lycopersicum ‘Ailsa Craig’ seedling meristem with a small piece of hypocotyl | Explants incubated in MS medium with 0.09 M sucrose cooled to 0 °C (20 min) → dehydration in MS medium with 15% DMSO (40 min) → cooling in LN vapor to − 120 °C → storage in LN (> 24 h) → rewarming at 40 °C (1.5 min) → explants rinsed 3 × with MS medium without DMSO → recovery on MS medium with 3 mg l−1 GA3 and 1 mg l−1 casein hydrolysate | 51% Recovery from cryopreserved young seedlings with 2 cm radicle 38% recovery from cryopreserved 7-day seedlings | Cooling rate changing continuously from 20 to 55 °C min−1 between 0 and − 120 °C required for optimal survival | Grout et al. (1978) |
S. lycopersicum ‘Capriciu’, ‘Darsirius’, ‘Kristin’, ‘Pontica’, ‘Siriana’ shoot tip, 2–3 mm in length, with 2-4 leaf primordia | Shoot tips osmoprotected in liquid MS medium with 0.5 M sucrose (24 h) → dehydration with PVS2 (20 min) → transfer to aluminum foils strips in PVS2 droplets → storage in LN (24 h) → rewarming in liquid MS medium with 0.09 M sucrose at room temperature → recovery on semi-solid (0.5% agar) MS medium | No information about explant survival and recovery which are very interesting, since rewarming was performed at room temperature | DNA of ‘Siriana’ suffered the weakest structural changes upon cryogenic storage. On the contrary, genomic DNA of ‘Pontica’ was the most responsive to cryopreservation process. Particularly, both C20-endoanti and C3′-endoanti conformations were detected. No structural changes of genomic DNAs from ‘Siriana’, ‘Darsirius’ and ‘Kristin’ were found upon cryopreservation | Muntean et al. (2015) |
Shoot tips embedded in 3% calcium alginate (30 min) → osmoprotection in liquid MS medium with 0.5 or 0.75 M sucrose → 3- or 4-h air desiccation (21-35% moisture content) → storage in LN (24 h) → rewarming in water at 38 °C (2 min) → recovery on semi-solid (0.55% agar) MS medium | 58% (‘Darsirius’)–72% (‘Pontica’) regrowth | Low MC of the alginate bead is critical to ensure the regrowth of cryopreserved explants Addition of plant growth regulators (cytokinins and/or auxins) into the recovery medium could improve the protocol efficiency | Coste et al. (2014) | |
Shoot tips osmoprotected in liquid MS medium with 0.5 M sucrose (24 h) → dehydration with PVS2 (30 min) → transfer to aluminum foil strips (0.5 × 2 cm) in PVS2 droplets (6 µl) → storage in LN (24 h) → rewarming in liquid MS medium with 0.09 M sucrose → recovery on semi-solid (0.5% agar) MS medium with 8.8 µM BA and 2.69 µM NAA | 60% (‘Kristin’)–70% (‘Pontica’) regrowth | Sequencing of lycopene β-cyclase gene from leaves (LCY-B; a carotenogenic gene which codifies an essential enzyme involved in the synthesis of α- and β-carotene by the cyclization of lycopene) revealed no DNA damages after cryopreservation and subsequent in vitro recovery Rapid rewarming in a water bath could improve the protocol efficiency | Coste et al. (2015) | |
Transgenic S. lycopersicum ‘Moneymaker’ shoot tip, 3 mm in length | Explant preculture on MS medium with 0.3 M sucrose (3 d, under dark) → encapsulation in 3% Na-alg with 0.3 M sucrose (30 min) → osmotic dehydration in liquid MS medium with 0.4–0.8 M sucrose (1–3 days) → desiccation (0–6 h) → storage in LN (> 1 h) → rewarming in water at 38 °C (3 min) → recovery on MS medium with 0.1 M sucrose (first 7 days under dark) | 0% survival and regrowth recorded in all lines subjected to storage in LN | High survival (100%) and regrowth (90%) rates of encapsulated-dehydrated but non-LN-stored explants | Al-Abdallat et al. (2017) |
Explant preculture on MS medium with 0.3 M sucrose (3 d, under dark at 4 °C) → explants placed on aluminum V-cryoplates and embedded in 3% Na-alg (15 min) → osmoprotection in LS (2 M glycerol + 1 M sucrose, 20 min) → dehydration with PVS2 (20 min) → storage in LN → rewarming in unloading solution (1.0 M sucrose) at room temperature → recovery on MS medium with 0.1 M sucrose (first 7 days under dark) | 35% (negative control)–70% (transgenic lines) survival of cryopreserved explants No regrowth of cryopreserved shoot tips | Cryopreserved shoot tips of all transgenic lines showed lower survival rates when compared with pretreated but non-LN-stored shoot tips. Regrowth rates of non-LN-stored explants were lower than observed survival percentages |