**3.1.3 Dehydration**

492 Current Frontiers in Cryobiology

Plate 1. Ultrastructure of *Solenotemon rotundifolius* explant meristematic cells. Legend are as follows: Ch, heterochromatin, V, vacuoles, T, tonoplast, P, plastids, M, mitochondrial, ER,

Plate 1a Control explants cultured on 2% sucrose supplemented medium. Cells show oval nuclei, normally-distributed heterochromatin, vacuoles each with a well-defined tonoplast, small, relatively dense plastids, circular mitochondrial profiles with dense matrices, and

Plates 1b (insert), & c. Explants pregrown on 0.1 M mannitol for three weeks cells (1b). Ongoing metabolic activity indicated by abundance of mitochondria, Golgi bodies and profiles of endoplasmic reticulum (insert), many plasmodesmata are visible. A group of relatively small vacuoles is shown (1c), which appeared typical of mannitol treatment to

Plate 1d After sucrose (0.1 M) pregrowth for three weeks, there was evidence of tonoplast disruption, lobed nuclei with possibility of vacuole fission or fusion. Plate 1(e) shows somewhat distorted plastids and a potentially autolysing cell (lower left) where vacuolar dissolution (tonoplast disruption) appears to have occurred. Such events would have

Plate 1f. Explants pregrown on 0.1 M sucrose, precultured on 0.3 M sucrose for 3 d and then dehydrated over activated silica gel for 120 min. Water content was 0.16 g g–1 while survival of the sample was only 2.5%. Most specimens presented this appearance of advances

Plate 1g. Explants pregrown on 0.1 M mannitol and then preculture on 0.3 M mannitol for 3 d, dehydrated over activated silica gel for 120 min, during which water content was lowered to 0.11 g g-1. This was accompanied by 38.1% survival. Nuclear and cytoplasmic derangement had occurred although some cells had few intact organelles, shown in this

endoplasmic reticulum, N, nucleus Gb, Golgi bodies, pl, plasma membrane.

predisposed these explants negatively to subsequent steps.

illustration of what was probably a non-surviving explant.

intracellular deterioration, nuclear remains; and plasma membrane.

profiles of endoplasmic reticulum.

reduce water content.

Dehydrating explants over silica gel, considerably lowered water content (Table 1). In experiments by other investigators, this technique has been successfully used to dehydrate and cryopreserve citrus axes, (Santos & Stushnoff, 2003) and encapsulated somatic embryos of *Coffee canephora* (Hatanaka *et al*., 1994).

Mannitol treated (0.3 M) explants of Frafra potato used in this particular experiment, when dehydrated over activated silica gel, the lowest water content (0.11 g g-1) was recorded for 120 min dehydration and survival was 73% (Table 1). However, sucrose treated (0.3 M) explants indicated lowest water content of 0.16 g g-1 which although, is higher than in mannitol (stated above), survival was as low as 5% (Table 1). It is possible that damage suffered by tissues as revealed by ultrastructure (Plate 1d&e) during sucrose treatment predisposed them to further damage on dehydration. It was evident here that mannitol treated tissue are more desiccation tolerant than sucrose treated tissues although other

Cryopreserving Vegetatively Propagated Tropical Crops

Grospietsch *et al*., 1999; Santos & Stushnoff, 2003).

months

**3.1.4 Cryoprotection** 

not result in explant survival.

reconstituted for normal plant growth and development to occur.

– The Case of *Dioscorea* Species and *Solenostemon rotundifolius* 495

reports have successfully used sucrose to induce dehydration tolerance (Dumet *et al*., 1993;

Sucrose treated tissues had totally been deranged after 120 minutes (Plate 1f) of dehydration compared with mannitol tissues (Plate 1g) which has some intact nuclei and few organelles present. These must be responsible for the survival recorded (Table 1). It is possible the presence of the intact nuclei and organelles in the mannitol treated cells could be

> **Crop accession Water content FP UER 001** 18 + 5.14 **FP UER 002** 10 + 0.9 **FP UWR 003** 9.6 + 2.7 **FP UER 004** 14.6 + 2.1 **99/053** 8.4 + 1.1 **99/1033** 11.2 + 0.6 **99/016** 11.2 + 1.6 **99/022** 11.35 + 0.6

Table 2. Water content of screenhouse of Frafra potato established in greenhouse for three

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

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


Table 1. Treating FP 002 with different sucrose concentrations during development of Preculture conditions using nodal cuttings with single buds

reports have successfully used sucrose to induce dehydration tolerance (Dumet *et al*., 1993; Grospietsch *et al*., 1999; Santos & Stushnoff, 2003).

Sucrose treated tissues had totally been deranged after 120 minutes (Plate 1f) of dehydration compared with mannitol tissues (Plate 1g) which has some intact nuclei and few organelles present. These must be responsible for the survival recorded (Table 1). It is possible the presence of the intact nuclei and organelles in the mannitol treated cells could be reconstituted for normal plant growth and development to occur.


Table 2. Water content of screenhouse of Frafra potato established in greenhouse for three months
