**3.1.4 Cryoprotection**

494 Current Frontiers in Cryobiology

Treatment Water Content g/g dry weight % Survival

11.45 ± 0.50 100

0.58 ± 0.15 50 ± 1.8

0.56 ± 0.12 30 ± 3.6

0.14 ± 0..02 35 ± 10.6

6.09 ± 0.16 96.67 ± 0.8

0.79 ± 0.17 56.25 ± 1.14

0.37 ± 0.07 33.18 ± 0.58

0.16 ± 0.61 5 ± 2.4

10.51 ± 0.6 80 ± 2.08

0.42 ± 0.07 40 ± 0.11

0.29 ± 0.05 40 ± 0.11

0.08 ±±0.01 20 ± 0.09

7.42 ± 0.38 96.67 ±

0.31 ± 0.05 71 ± 0.09

0.11 ± 0.4 73 ± 0.10

dehydrated for 90 min 0.24 ± 0,05 70 ± 0.10

Table 1. Treating FP 002 with different sucrose concentrations during development of

Preculture conditions using nodal cuttings with single buds

0.058 M sucrose for 1 d 18.22 ± 0.96 100 ± 0 0.058 M sucrose for 3 d 22.37 ± 2.02 86.67 ± 4.7 0.058 M sucrose for 5 d 16.7 ± 0.65 100 ± 0 0.1 M sucrose for 1 d 12.93 ± 0.66 96.67 ± 0.86 0.1 M sucrose for 3 d 14.18 ± 0.03 100 0.1 M sucrose for 5 d 12.74 ± 0.4 100 0.3 M sucrose for 1 d 8.82 ± 0.4 100 0.3 M sucrose for 3 d 7.98 ± 0.73 96.67 ± 0.86 0.3 M sucrose for 5 d 6.17 ± 0.39 100 0.5 M sucrose for 1 d 4.13 ± 0.38 96.67 ± 0.86 0.5 M sucrose for 3 d 4.22 ± 0.41 86.67 ± 2.27 0.5 M sucrose for 5 d 3.37 ± 0.27 96.67 ±0.86

0.1 M sucrose dehydrated for 0 min

0.1 M sucrose dehydrated for 60 min

0.1 M sucrose dehydrated for 90 min

0.1 M sucrose dehydrated for 120 min

0.3 M sucrose dehydrated for 0 min

0.3 M sucrose dehydrated for 60 min

0.3 M sucrose dehydrated for 90 min

0.3 M sucrose dehydrated for 120 min

0.1 M mannitol for 3 d and dehydrated for 0 min

0.1 M mannitol for 3 d and dehydrated for 60 min

0.1 M mannitol for 3 d and dehydrated for 90 min

0.1 M mannitol for 3 d and dehydrated for 120 min

0.3 M mannitol for 3 d and dehydrated for 0 min

0.3 M mannitol for 3 d and dehydrated for 60 min

0.3 M mannitol for 3 d and

0.3 M mannitol for 3 d and dehydrated for 120 min

Explants treated with 0.1 and then 0.3 M sucrose or mannitol, on exposure to PVS2 indicated only about 20% survival (Table 3). This observation was contrary to report by Niino *et al*., (2000) that *S. rotundifolius* innala recorded high survival on treating with PVS2 and subsequently, 85% survival on exposure to liquid nitrogen. *Solenostemon rotundifolius* used in this study, were extremely sensitive to both the loading solution (0.4 M sucrose + 0.2 M glycerol, data not shown) and PVS2, which in other reported studies, led to successful cryopreservation of other crops including *S. rotundiflius* (Wang *et al*., 2003; 2001; Turner *et al*., 2001; Niino *et al*., 2000). On screening for appropriate vitrification (cryoprotection) solution, the following cryoprotection solutions listed in Table 3 were tested. It was indicative from results that DMSO and Ethylene Glycol at the concentration (15%) that they occur in PVS2 did not have any lethal effect on the explants. However, sucrose and Glycerol at the concentrations that they occur in PVS2 (0.4 M and 30% respectively) were found to be lethal to the tissues (Table 3). The use of PVS2 at half concentration and a combination of 2.5% Glycerol, 5% sucrose, 7.5% DMSO and 7.5% Ethylene glycol (coded PVSB) resulted in survival and growth of explants. The responses confirm indication that cryoprotectants at full-strength are toxic to plant cells (Rheinhoud *et al*., 1995). These treated explants, however did not survive on exposure to liquid nitrogen. Combining the cryoprotection treatment with dehydration (data not shown) as has been reported by other investigators as enhancing high cryosurvival (Wang *et al*., 2003; 2001; Turner *et al*., 2001), did not result in survival after cryopreservation in this study. Encapsulating explants prior to treatment with PVS2 also did not result in explant survival.

Ultrastructural studies indicated that tissues treated with ½PVS2 (Plate 3) and PVSB (Plate 4) for 15 min, which survived had well constituted cells, however, some tonoplasts were not

Cryopreserving Vegetatively Propagated Tropical Crops

S: sucrose, G: Glycerol, EG: Ethylene Glycol

FP 002

FP 003

FP 004

– The Case of *Dioscorea* Species and *Solenostemon rotundifolius* 497

distinct and some extent of autophagy was observed in ½ PVS2, treated tissues (Plate 3). Slight deposition of starch was observed in plastids of tissues treated with PVSB. Tissues, which did not survive after the 15min treatments (Plate 5a&b), showed highly plasmolysed

Increasing glycerol and sucrose concentration as well as long exposure duration led to reduced survival and regeneration of encapsulated nodal cuttings treated with the plant vitrification solutions as indicated in (Table 4). This observation is contrary to other crops where encapsulation vitrification has been highly recommended for successful cryopreservation (Charoensub *et al*., 1999; Wang *et al*, 2003) however confirms that cryoprotectants can be damaging to plant tissues and the extent of effect varies according to

3.23 M Glycerol + 0.4 M Sucrose + 0.017 Raffinose 33.64 ± 1.39 3.23 M Glycerol + 0.4 M Sucrose 22.71 ± 2.77 PVS2 + 0.017 M raffinose 8.28 ± 0.20 PVS2 20 ± 0 0.07 M S + 0.3 M G 15 min 100 0.07 M S + 0.3 M G+0.14 M S +0.6 M G, 15 min 100 0.14 M S + 0.6 M G, 15 min 100 0.64 M DMSO for 15 min 90±0 1.28 M DMSO for 15 min 100± 0 1.92 M DMSO for 15 min 100 ± 0 2.42 M EG + 0.017 M Raffinose 84.24±1.46 2.42 M EG 77.28±0.97 ½ PVS2 + 1% Raffinose 80.63±1.82 0.3 M G + 0.2 M S + 0.96 M DMSO + 2.42 M EG + 0.017 M Raffinose (PSVB) 69.17±2.26

**Treatment % Survival** 

cells and totally deranged cells, exposing the lethal effect of vitrification solution.

type and concentration of cryoprotectant as well as plant species (Berjak *et al*., 1996).

Table 3. Survival (± SD) of Frafra potato explants, pregrown (0.1 M mannitol) and precultured (0.3 M mannitol) prior to cryoprotection treatments. n=30 – 40, P<0.05

½PVS2 and PVSB were always supplemented with 0.017 M Raffinose Table 4. FP explants treated with Plant Vitrification Solution

Frafra potato Variety Treatment Water Content g/g dry wt % Survival

PVSB for 15 min 4.13 ± 0.16 55 ± 2.04 PVSB for 40 min 4.45 ± 0.25 55 ± 2.04 ½ PVS2 for 15 min 3.36 ± 0.06 38.75 ± 0.3 ½ PVS2 for 40 min 3.26 ± 0.17 0 PVS2 for 15 min 2.00 ± 0.10 20

PVSB for 15 min 2.66 ± 0.16 59.01 ± 1.5 ½ PVS2 for 15 min 2.58 ± 0.23 53.76 ± 2.3 Control 5.63 ± 0.31 100

PVSB for 15 min 2.91 ± 0.28 54.56 ± 1.4 ½ PVS2 for 15 min 2.7 ± 0.14 66.62 ± 3.47 Control 6.59 ± 0.62 100

Plate 3 & 4. Ultratructure of surviving Frafra potato after mannitol pregrowth and preculture treatments, followed by subjecting explants for 15 min to ½PVS2 (Plate 3) and PVSB (Plate 4). (**3**) Although cells were well organised, some extent of autophagy was concluded to have occurred, in terms of intravacuolar inclusions in the ½ PVS2-treated material. (**4**) Cells appeared exceptionally active but showed distinctly lobed nuclei (N). Other organelles that can be recognized are mitochondria (M), endoplasmic reticulum (ER), Golgi bodies (Gb), plastids (P), and vacuoles (V) with tonoplast (T) intact.

Plate 5a & b. Ultratructure of non-surviving Frafra potato following mannitol pregrowth and preculture treatments, then subjecting explants to ½PVS2 for 15 (Plate 5a) and 40 min (Plate 5b) Cells were highly plasmolysed (arrows) and damaged to the extent that organelles could not be easily recognized.

Plate 3 & 4. Ultratructure of surviving Frafra potato after mannitol pregrowth and preculture treatments, followed by subjecting explants for 15 min to ½PVS2 (Plate 3) and PVSB (Plate 4). (**3**) Although cells were well organised, some extent of autophagy was concluded to have occurred, in terms of intravacuolar inclusions in the ½ PVS2-treated material. (**4**) Cells appeared exceptionally active but showed distinctly lobed nuclei (N). Other organelles that can be recognized are mitochondria (M), endoplasmic reticulum (ER),

Plate 5a & b. Ultratructure of non-surviving Frafra potato following mannitol pregrowth and preculture treatments, then subjecting explants to ½PVS2 for 15 (Plate 5a) and 40 min (Plate 5b) Cells were highly plasmolysed (arrows) and damaged to the extent that organelles

could not be easily recognized.

Golgi bodies (Gb), plastids (P), and vacuoles (V) with tonoplast (T) intact.

distinct and some extent of autophagy was observed in ½ PVS2, treated tissues (Plate 3). Slight deposition of starch was observed in plastids of tissues treated with PVSB. Tissues, which did not survive after the 15min treatments (Plate 5a&b), showed highly plasmolysed cells and totally deranged cells, exposing the lethal effect of vitrification solution.

Increasing glycerol and sucrose concentration as well as long exposure duration led to reduced survival and regeneration of encapsulated nodal cuttings treated with the plant vitrification solutions as indicated in (Table 4). This observation is contrary to other crops where encapsulation vitrification has been highly recommended for successful cryopreservation (Charoensub *et al*., 1999; Wang *et al*, 2003) however confirms that cryoprotectants can be damaging to plant tissues and the extent of effect varies according to type and concentration of cryoprotectant as well as plant species (Berjak *et al*., 1996).


S: sucrose, G: Glycerol, EG: Ethylene Glycol

Table 3. Survival (± SD) of Frafra potato explants, pregrown (0.1 M mannitol) and precultured (0.3 M mannitol) prior to cryoprotection treatments. n=30 – 40, P<0.05


½PVS2 and PVSB were always supplemented with 0.017 M Raffinose

Table 4. FP explants treated with Plant Vitrification Solution

Cryopreserving Vegetatively Propagated Tropical Crops

content that might enhance cryosurvival.

**3.2** *Dioscorea rotundata*

growth of nodal explants.

– The Case of *Dioscorea* Species and *Solenostemon rotundifolius* 499

Table 5. Other attempts to acclimatize the new Frafra potato accession 99/053 to lower water

Comparatively, yam explants cultured on medium supplemented with 0.3 M sucrose for 3-5 d considerably reduced tissue water content from about 12.2 g g-1 dry mass to between 4.8 and 5.5 g g-1 dry mass before cryoprotection with modified PVS2 (MPVS2) or silica gel dehydration. Following cryoprotection with MPVS2 the Plate (6) below indicated the

Plate. 6. Growing cultures of yam explant subjected to pregrowth on 0.09 sucrose

supplemented medium for 5 weeks, precultured on medium containing 0.3 m sucrose for 3 or 5 d, treated with MPVS2 for varied duration and unloaded with rehydration solution containing 0.3 or 1 M sucrose and cultured on regeneration medium for six week

Six months in culture 8.1± 1.2 Shoot grown from tuber under sterile conditions (8 weeks) 19.5 ± 1.6 Vitrified shoot grown from tuber under sterile conditions (8 weeks) 24.7 ± 3.9 Normal shoots transferred to vented vessels (3 weeks) 8.2 ± 1.3 Vitrified shoots transferred to vented vessels (3 weeks) 12.7 ± 2.5 Normal shoots transferred to dry air-line (3 weeks) 7.2 ± 1.0 Normal shoots transferred to humid air-line (3 weeks) 18.4 ± 2.2 Cultures transferred to RITA vessels 13.0 ± 2.2

**Treatment Water content** 
