**2.2.5 Cryoprotection with modified plant vitrification solution 2(MPVS2)**

Explants that had been pregrown and precultured were exposed to 1 ml plant vitrification solution 2 (PVS2) as designed by Sakai and colleagues (Sakai et al., 1990), but modified as follows (MPVS2): basic MS medium, 30% glycerol, 15% ethylene glycol, 15% DMSO (v/v), 0.4 M sucrose (w/v), 0.1 M CaCl2.2H2O and 1% D-raffinose at pH 5.7±0.1. Inclusion of calcium chloride and raffinose has been found to be beneficial in promoting recovery after cryopreservation in other species (Mycock, 1999). Explants were treated for 0, 10, 20, 30 or 40 minutes in cryovials. The vitrification solution was decanted and the explants washed three times in 1 ml rehydration solution (as described above) for 30 minutes, then cultured on growth medium and incubated in the dark. Cultures were transferred to the alternating light/dark conditions once signs of growth and development were observed. Prior to being cooled to -70 or -196ºC, cryoprotected explants were suspended in fresh 0.5 ml MPVS2 in

Cryopreserving Vegetatively Propagated Tropical Crops

subsequent steps.

**3.1.2 Preculture** 

media is shown in the Plate 2.

% Survival Water content

Survival P >0.05, n=30, and WC P< 0.05, n=15-30

0

20

40

60

**% Survival**

80

100

120

– The Case of *Dioscorea* Species and *Solenostemon rotundifolius* 491

content of explants (11.4 gg-1) as shown in Fig. 1, this observation is in agreement with response of oil palm explant water content when treated with sucrose (Dumet *et al*., 1993) and while this did not reduce survival it resulted in severely distended organelles and evidence of tonoplast disruption and lobed nuclei (Plate 1d and e). Sucrose has been extensively used to treat plant tissues prior to cryopreservation (Panis *et al*., 2002; Grospietsch *et al*., 1999; Gonzalez-Benito & Perez, 1994; Santos & Stushnoff 2003), studies have however, not investigated the structural effect of sucrose on tissue. The damage revealed by ultrastructure (Plate 1f) could have predisposed explants negatively to

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

> 0.058 M suc 0.1 M suc 0.1 M mann **Frafra potato Pregrowth Treatment**

Fig. 1. Survival and water content of Frafra potato cultures on three pregrowth media + SD.

0

5

10

15

**Water content g g-1 dry wt.** 

20

25

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