**3.1.2 Preculture**

490 Current Frontiers in Cryobiology

cryovials. The explants to be cryopreserved were then exposed to cryogenic conditions for specified durations, rewarmed, vitrification solution removed, rehydrated, and incubated as

Water content of yam explants was determined following the same procedure as described

Yam explant survival was determined following the same procedure as described above of

Shoot tips which were pretreated with high sucrose concentrations; pretreated and cryoprotected with MPVS2; pretreated, cryoprotected, and vitrified, were cultured on growth medium for 5-7 d following which they were transferred to a 0.1% aqueous solution of 2,3,5-triphenyl tetrazolium chloride (TTZ) and incubated in the dark overnight. Control material was obtained from cultures under standard growth room conditions. Patchy red staining, located around the meristematic region as a result of respiratory activity in viable

Water contents of Frafra potato nodal cutting explants following pregrowth on medium supplemented with 0.058 M (2%) sucrose was extremely high (22.25+1.7 gH2O g-1 dry wt) to enhance successful cryopreservation. As explant size and geometry have a marked effect on the success of freezing hydrated material (Wesley-Smith *et al*., 1995), nodal cuttings (two buds per explant) to be used for cryopreservation experiments were split into two halves with one bud per explant, this adequately lowered water content of explants (8.78±1.07 gH2O/g dry wt). The water content of the single (well trimmed) bud is similar to that of explants excised from greenhouse established plant (10.16±0.98 g/g dry wt. data shown in Table 2). Since cultures grown on medium supplemented with 2% sucrose were extremely wet for cryopreservation, higher sucrose or mannitol concentration (0.1 M) was employed to

Pregrowth of explants on medium supplemented with 0.1 M mannitol lowered the water content of explants from 19.5 under control condition (medium supplemented with 0.058 M sucrose), to 10.4 gg-1, which did not affect survival (Fig. 1). The ultrastructure was as well constituted as that of the control explants (Plate 1a), with ongoing metabolism indicated by abundant cristate mitochondria (Plate 1b), Golgi bodies and profiles of endoplasmic recticulum (insert). Growth on 0.1 M sucrose supplemented medium, also lowered the water

cells was scored as the tissue having survived the various treatments.

enhance desiccation tolerance, which subsequently improves cryotolerance .

described above.

**2.2.6 Yam assessment** 

for Frafra potato above.

Frafra potato.

**2.2.6.2 Survival assessment** 

**2.2.6.1 Water content determination** 

**2.2.6.3 Tetrazolium test for viability** 

**3. Results and discussion** 

**3.1 Frafra potato 3.1.1 Pregowth** 

Culturing individual Frafra potato buds on 0.3 M sucrose for 3 d (Table 1), lowered water content from 11.4 g g-1 (after growth on 0.1 M sucrose medium) to 7.3 g g-1 and explant survival was at 100 %. This level of sucrose has been applied in other crops such as carrots (Dereuddre *et al*., 1991), wasabi (Mastumoto *et al*., 1998), and African violet (Shibili *et al*., 2004). Similarly, explant on medium supplemented with 0.3 M mannitol which were derived from 0.1 M mannitol supplemented medium, water contents reduced further from 10.47 to 7.42 gg-1 and survival was still at 100 % (Table 1). Mannitol and its isomer, sorbitol have been used for pre-treatment of plant tissues before cryopreservation (Wang *et al*., 2001) as well as in long term storage culture media as osmoticums (Ashun, 1996; Egnin et al, 1998). The growth of explants on regrowth medium following preculture varying sucrose media is shown in the Plate 2.

**Frafra potato Pregrowth Treatment** 

Fig. 1. Survival and water content of Frafra potato cultures on three pregrowth media + SD. Survival P >0.05, n=30, and WC P< 0.05, n=15-30

Cryopreserving Vegetatively Propagated Tropical Crops

at 3-weeks.

**3.1.3 Dehydration** 

of *Coffee canephora* (Hatanaka *et al*., 1994).

– The Case of *Dioscorea* Species and *Solenostemon rotundifolius* 493

Plate 2. Development of *Solenostemon rotundifolius* explants pregrown and precultured on medium supplemented with increasing sucrose concentrations 0.1 M, 0.3, 0.5 M, and 0.7 M for 1 d, 2 d, 3 d, 4 d, and 5 d and grown on growth medium in 90mm Petri plates. Data taken

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

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

Plate 1. Ultrastructure of *Solenotemon rotundifolius* explant meristematic cells. Legend are as follows: Ch, heterochromatin, V, vacuoles, T, tonoplast, P, plastids, M, mitochondrial, ER, endoplasmic reticulum, N, nucleus Gb, Golgi bodies, pl, plasma membrane.

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 profiles of endoplasmic reticulum.

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 reduce water content.

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 predisposed these explants negatively to subsequent steps.

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 intracellular deterioration, nuclear remains; and plasma membrane.

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 illustration of what was probably a non-surviving explant.

Plate 2. Development of *Solenostemon rotundifolius* explants pregrown and precultured on medium supplemented with increasing sucrose concentrations 0.1 M, 0.3, 0.5 M, and 0.7 M for 1 d, 2 d, 3 d, 4 d, and 5 d and grown on growth medium in 90mm Petri plates. Data taken at 3-weeks.
