Table 3.
Five practical cryopreservation methods for in vitro grown shoot tips of potato
| Procedure | DMSO droplet method in IPK, Germany | Droplet vitrification method in CIP, Peru | Droplet vitrification method in NAC, RDA, Rep. Korea | Encapsulation vitrification method in CAES, HRO, Japan | V cryo–plate method in NIAS, Japan |
|---|---|---|---|---|---|
| Culture | Culture on solid Murashige and Skoog medium (MS) with 20 g/l sucrose under 16 h photoperiod at 22°C for 3–4 weeks. | Culture on solid MS with 2 mg/l glycine, nicotinic acid, 0.5 mg/l pyridoxine, 0.1 mg/l thiamine, 25 g/l sucrose and 2.8 g/l phytagel at 22°C, 45 μmol/m2/s illumination and 16 h light. | Culture nodal segments on solid MS with 30 g/l sucrose, 2.2 g/l phytagel (Sigma Co.) at 24°C, under 16 h light, 100–140 μmol/m2/s illumination for 3–7 weeks. | Culture nodal segments on solid MS with 0.5 g/l casamino acid, 30 g/l sucrose, 2.5 g/l gellan gum at 23°C and 16 h photoperiod with light intensity of 50 μmol/m2/s every 2 weeks. | Culture on solid MS with 30 g/l sucrose, 0.3 g/l CaCl2 and 8 g/l agar at 20°C and 16 h photoperiod with light intensity of 104 μmol/m2/s every 3 months. |
| Preconditioning | Cold preculture under 8 h photoperiod at 21/8°C day/night temperature for 7 days. | Shoot multiply on the MS using apical cutting (~0.7 cm) for 3 weeks. If need, cold preculture on MS medium with 24 g/l sucrose at 6°C for 7 days. | Culture nodal segments on the MS for 7 days under standard condition or for 1 day under standard condition, then transfer at 4°C and 12 h photoperiod with light intensity of 20 μmol/m2/s and for 3 weeks. | Culture nodal segments with a lateral bud (about 5 mm) on the solid MS for about 2 weeks in the standard condition. | |
| Shoot tips | Explants of 2–3 mm in length and 0.5–1 mm width. | Explants of 1.8–2.5 mm length apical shoot tips. | Axillary shoot tips (1.0–2.0 mm in size) are isolated by dissection from the upper to middle part of the mother-plants. | Excision shoot tips with 2 to 3 leaflets (about 1 mm length) from axillary buds. | Excision shoot tips with 2 leaflets (about 1.5 mm length) from shoots. |
| Preculture Mounting shoot tips on cryo-plate/ Encapsulation | Incubate in liquid MS with 30 g/l sucrose, 0.5 mg/l zeatin riboside, 0.2 mg/l GA3 and 0.5 mg/l IAA (MSTo) overnight at 22°C. | Place specimens on potato meristem medium, which is MS with 0.04 mg/l kinetin, 0.1 mg/l gibberellic acid, 24 g/l sucrose and 2.8 g/l phytagel (MMP) and incubated at RT for about 1 h. | Isolated shoot tips were precultured in liquid MS with 0.3 M sucrose for 8 h under standard conditions. Shoot tips were further precultured in liquid MS with 0.7 M sucrose for 18 h under the same conditions. | Preculture on MS with 0.3 M sucrose, 1 mg/l GA3, 0.01 mg/l BA and 1 μg/l NAA for 16 hr at 23°C. Encapsulation of shoot tip (one shoot tip/ bead); mixture of shoot tips and calcium-free MS with 2% Na-alginate and 0.4 M sucrose are dropped into 0.1 M CaCl2 soution with 30 g/l sucrose for 30 min, at 25°C. | Preculture on MS with 0.3 M sucrose overnight (16 hr) at 25°C. Pour 2% Na-alginates solution in the wells of cryo-plate. Place the precultured shoot tips one by one in the wells. Then, drop over them 0.1 M CaCl2 soution and wait for 15 min at 25°C for complete polymerization. |
| Osmoprotection | Treatment with cryoprotectant solution (10%DMSO in MSTo at RT for 1–3 h. | Treatment with osmoprotective solution (LS) with 2 M glycerol and 0.4 M sucrose at RT for 15 min. | Dehydration in 10 ml PVS2 solution for 20 min with continuous shaking (60 rpm). | Treatment beads with MS solution with 0.6 M sucrose, 2 M glycerol and the same plant hormones in preculture medium on rotary shaker (60 rpm) at 25°C for 90 min. | Treatment with LS solution contains 2 M glycerol + 0.8 M sucrose by transfer the cryo-plates in a 25 ml reservoir filled with 20 ml LS at 25°C for 30 min. |
| Dehydration | PVS2 treatment on ice. Shoot tips were exposed for 50 min to 2 ml ice-cooled PVS2 in glass vials. | Dehydration with PVS2 at 0°C for 3 hr. The beads in PVS2 are shaken (20 rpm) in a water bath. | Dehydration with PVS2 by placing cryo-plates in a 25 ml filled with 20 ml PVS2 at 25°C for 30 min. | ||
| Transfer the explants | Transfer the explants into droplets of 2.5 μl cryoprotectant solution one by one on aluminium foils (5 × 25 × 0.03 mm). | The 3 min before the end of each PVS2 treatment, the specimen were transfered to a PVS2 drop (10–15 μl) on an aluminum foil strip (0.5 × 2 cm). | A few min before plunging in LN, seven drops (2.5 μl each) of PVS2 solution are placed on an aluminum foil strip (7 × 20 mm) using a dispenser. One shoot tip is put in each of the seven PVS2 drops. | ||
| Storage | Put the aluminium foils directly into cryotube filled with LN. Close the cryotube refilled with LN and stock into the Dewar with LN. | The strip holding the shoots is then rapidly plunged into a LN filled cryo-tube in a Petri dish on ice. Close the cryotube refilled with LN and stock into LN tanks. | The foil strip is immediately plunged in LN. After a few min, two foil strips is transferred in one 2 ml cryovial, which have been previously filled with LN and stored in a LN tank. | Cryotube containing beads and PVS2, is directly plunged in LN. | Transfer the cryo-plate in 2 ml cryotube, which is held on cryo-cane, and plunge it directly in LN. |
| Rewarming | Putting aluminium foils quickly in liquid MS with 30 g/l sucrose at room temperature (RT). | Dropping the strips quickly in liquid MS with 1.2 M sucrose at RT and incubate for 20 min. | Foil strips are taken out of the cryovials and immediately plunged in 6 ml 0.8 M sucrose solution at 40°C for 30 s and 6 ml of the solution are added. Shoot tips are further incubated in the unloading solution at RT for 30 min. | Cryotube is put into water bath at 38°C for 3 min. After removing the PVS2 solution, the beads are washed with 1 ml of 1.2 M sucrose solution for 10 min. | Cryo-plate is retrieved from the cryotube in LN and immersed in 2 ml cryotube containing 2 ml MS basal medium with 1 M sucrose, in which it is incubated for 15 min at RT. |
| Regeneration | Shoot tips are plated on solid MSTo (10 g/l agar) under a 16 h photoperiod at 22°C. | Post-cryo culture in the dark on MMP with progressively decreased sucrose levels (daily transfers from 0.3, to 0.2, to 0.1 M and maintained on 0.07 M). One week after warming, shoot tips were transferred from the filter paper to fresh MMP (0.07 M sucrose) and incubated at 22°C under standard condition. | Shoot tips are post-cultured on semi solid MS with 0.05 mg/l IAA, 0.3 mg/l zeatin, 0.05 mg/l GA3, 30 g/l sucrose and 1.8 mg/l phytagel at 24°C under low light intensity for 7 days and then transferred to standard culture conditions. | Beads with shoot tips are plated on the solid MS with 30 g/l sucrose and the same plant hormones in preculture medium for 1 day, then transferred on the solid MS with 30 g/l sucrose and 0.5 μg/l GA3 under standard condition. | Shoot tips are plated on the solid MS and cultured under standard condition. |
| Reference |
Kaczmarczyk et al. 2011 Keller et al. 2008 |
Panta et al. 2014 |
Kim et al. 2006 Yoon et al. 2006 |
Hirai and Sakai 1999 |
Yamamoto et al. 2013 Yamamoto et al. 2011b |