Cryopreserving Vegetatively Propagated Tropical Crops – The Case of Dioscorea Species and Solenostemon rotundifolius

Marian D. Quain1, Patricia Berjak2, Elizabeth Acheampong3 and Marceline Egnin4 1Council for Scientific and Industrial Research Crops Research Institute 2School of Biological and Conservation Sciences, University of KwaZulu-Natal, Westville Campus, Durban 3Tissue Culture Laboratory, Department of Botany, University of Ghana, Legon, Accra 4Plant Biotechnology and Genomics Research Laboratory, Tuskegee University 1,3Ghana 2South Africa 4USA


Introduction
Root and tuber crops in the Sub-Saharan African region play a major role in daily diet, accounting for over 50% of the total staple.Dioscorea spp.and Solenostemon rotundifolius are among the tuber staples in West Africa.Solenostemon rotundifolius (Poir) J.K. Morton is an edible starchy tuber crop known to have originated in tropical Africa (Schippers, 2000).It occurs in western, central, eastern and southern Africa.In Ghana, it is popular in the northern part of the country and its common name is Frafra potato (Tetteh and Guo, 1993).In South Africa, it occurs mainly in coastal KwaZulu-Natal, eastern Mpumalanga and northwestern Cape and it is commonly known as Zulu round potato (Schippers, 2000).It is used to combat famine as it has high protein content, and has medicinal and social values (Kay, 1973).It flowers profusely, yet has rare seed production and is therefore propagated vegetatively by means of vine cuttings and tuber sprouts.Storage of the tuber in hot climates is a problem.In Ghana, it is stored in dry places or left on the ground under trees where conditions are cool.The tuber is stored buried in the ground to maintain the good quality for about two months.Otherwise the tuber sprouts within a shorter period.However, in South Africa, the tuber stores well through the winter months (Schippers 2000).
The germplasm is endangered because although field and in vitro gene banks are being used for conservation, these serve short to medium term purposes, and are expensive.Efforts to conserve the germplasm in the longer-term under slow growth in vitro are hampered by the relatively rapid growth of the cultures.Cryostorage which is recognised as the very safe cost effective option for the long-term conservation of genetic resources, especially vegetatively propagated species and crops with recalcitrant seeds (Engelmann & Engels., 2002) therefore provide a viable alternative to the long-term storage, and ensure recovery of stable germplasm (Gonzalez-Arnao et al., 1999).
Dioscorea species, colloquially known as yams, of family Dioscoreaceae are perennial monocotyledonous climbers with underground tubers which, in some species are edible and serve as major staples in sub-Saharan Africa.Propagation is routinely vegetative, using either the tubers or vine cuttings.Farmers ensure the production of true-to-type crops by using clonal planting material, because of the social and staple importance attached to yams in sub-Saharan Africa.Hence the conservation of clonal germplasm of yam is extremely important.Dioscorea spp.has about 700 species within the family, nine of which are medicinal plants that accumulate steroid saponins in their rhizomes.Six species of Dioscorea D .bulbifera, D. cayenensis, D. dumentorum, D. prahensilis, D. alata and D. rotundata contain mealy starch with a good level of vitamin C and other nutritive substances, which serve as major staples in sub-Saharan Africa.Dioscorea rotundata is native of West Africa, where it plays important role in the socio cultural life of the people.Dioscorea alata is the most widespread worldwide and is most cultivated in Southeast Asia, the Caribbean and West Africa.Dioscorea rotundata is now utilised in other parts of the world, and it has become a foreign exchange earner particularly in Ghana.
In vitro slow growth tissue culture methods have been used in conserving the germplasm (Ashun 1996;Ng & Daniel 2000;Ng & Ng 1991).Although this method usefully complements the traditional form of conservation, it serves only short-to medium-term storage purposes.Thus cryopreservation, which imposes a stasis on metabolic and deteriorative processes, is a worthwhile option to be explored.
Explant treatment to attain low water content which is critical for cryopreservation has in some protocols been by exposing tissues to stress, which enhance desiccation and cold tolerance (Withers 1985;Jitsuyama et al., 2002).Such stress has been induced by abscisic acid, sugars, mannitol and sorbitol (Mastumoto et al., 1998;Jitsuyama et al., 2002;Veisseire et al, 1993;Panis et al., 2002;Walter et al., 2002).The use of cryoprotectants, which exert osmotic stress and lead to loss of free water from tissues and vitrification when frozen has also been induced by using reagents such as sucrose, glycerol, DMSO, ethylene glycol, proline and many others (Engelmann et al., 1994;Harding & Benson, 1994;Matinez-Montero et al, 1998;Plessis et al., 1993;Nishizawa et al., 1993).Desiccation of tissues on activated silica gel (Hatanaka et al., 1994;Cho et al., 2002), in laminar air flow cabinets (Gonzalez-Benito & Pezez, 1994; Thammasiri, 1999) and flash driers (Berjak et al., 1999;Pammenter et al., 1991;Wesley-Smith et al., 1992;Walter et al., 2002;Potts & Lumkin 1997) have all been used to appreciably reduce water content to enhance cryotolerance.Although these treatments have all been reported to be successful in enhancing cryopreservation of some tissues, there are differences in response to known protocols which have been mainly attributed to specie and variety specificity (Gonzalez-Benito et al., 2002;Martinez-Montero et al., 1998;Panis et al., 2002;Gonzalez-Arnao et al., 1999).
The prevention of the formation of lethal ice crystals when tissue is exposed to sub-zero temperatures is essential for successful cryopreservation, of vegetatively propagated germplasm.This chapter looks at the various attempts made to cryopreserve germplasm of Solenostemon rotundifolious and possible underlying mechanism that might have led to failure of tissues to respond to all methods utilized.Tissue survival, water contents and ultrastructure are used as parameters for analyzing response to various treatments.Also, response of yam in vitro-grown explants (shoot tips and axillary buds) to various desiccation Cryopreserving Vegetatively Propagated Tropical Crops -The Case of Dioscorea Species and Solenostemon rotundifolius 487 procedures and their ability to survive after exposure to cryogenic temperatures is investigated here, with the ultimate aim of developing a simple protocol for long-term conservation of the germplasm of Dioscorea species via cryopreservation.Parameters that need critical investigation are discussed.

Source of explant
In vitro cultures of Solenostemon rotundifolius accession number UWR 002 was obtained from the in vitro gene bank that had been maintained under slow growth conditions at 18 o C. In vitro cultures were multiplied on Murashige and Skoog (MS) medium (Murashige and Skoog, 1962) supplemented with 2% sucrose and 0.7% agar.Subculturing was carried out at four-weeks intervals.Cultures were maintained under a 16 h photoperiod (40 µM /m 2 /s 1 ) at 25 o C +1 o C.

Conditioning donor plant material in culture (pregrowth)
Nodal cuttings were cultured on MS medium supplemented with either 0.058 M (2%) or 0.1 M sucrose or 0.1 M mannitol and 0.8% agar.Cultures were incubated for two to three weeks after which uniformly developed plantlets were used for various experiments.

Conditioning excised explants in culture (preculture)
Solenostemon rotundifolius nodal cuttings consisting two buds (having lateral buds which are microscopically globular and covered by leaf primodia as described by Niino et al., 2000) were obtained from pregrown cultures, positioned on sterile nylon mesh cut side down and placed on fresh pregrowth media overnight.Explants were then transferred on mesh to media with higher sucrose concentrations (0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 and 1.0 M) sucrose for one to seven days.Media supplemented with 0.1 and 0.3 M mannitol were also used as pregrowth treatment.Incubation was under a 16 h photoperiod (40µM/m 2 /s -1 ), at 25°C+1°C.

Silica gel dehydration
Explants were placed on oven-sterilised aluminium foil and dehydrated over approximately 35 g activated silica gel in covered 90 mm glass Petri dishes for 30 min to 16 h under sterile conditions.

Rehydration
Explants were rehydrated following cryoprotection treatment, silica gel dehydration and cooling.This was carried out in cryovials containing liquid MS medium supplemented with 0.1 M sucrose, 1mM MgCl 2 .6H 2 O and 1 µM CaCl 2 .2H 2 O for 30 minutes.Re-hydrated buds were cultured on growth medium, incubated under continuous dark conditions till signs of growth and development were observed (at least one week) before they were transferred to a dual photoperiod.

Cryoprotection
Liquid medium (MS) supplemented with 0.2, 0.4 M, 0.07 M, 0.14 M, sucrose, 2.0 M, 0.64 M, 1.28 M, 3.23 M glycerol, 2.42 M ethylene glycol, 0.017 M raffinose, 0.64 M, 1.28 M and 1.92 M DMSO, in varied combinations, plant vitrification solution II (PVS2), (Sakai et al., 1990) and half strength PVS2 were used to cryoprotect nodal cutting explants for 5, to 40 minutes.Explants used were obtained from shoots grown on 0.1 M mannitol for 2-3 weeks.Excised explants were cultured on 0.3 M mannitol for 72 hours, and cryoprotected in cryovials, using 1 ml cryoprotectant solution (the 1 ml cryoprotectant was decanted and replace with 0.5 ml during cryoprotection).To enhance explant cryoprotection, dehydration over activated silica gel for 60 minutes either before or after cryoprotection was also investigated.Following cryoprotection, the cryoprotectant solution was decanted and explants were washed three times with rehydration solution (described above).However, explants subjected to cooling, (LN or freezing to -70°C) were immediately on retrieval, rewarmed in water bath at 40 o C for two -three minutes.Following rewarming, tissues were then allowed to stay for 30 min in the rehydration solution before blotting dry, and cultured on growth medium.Incubation was in continuous dark conditions till signs of growth and development were observed.Developing cultures were transferred to 16 h photoperiod.

Water content determination
Individually weighed explants were oven-dried at 80°C for 48 hours to determine dry mass.Water content was determined individually for 5-10 explants, and expressed on a dry mass (g H 2 O g -1 dry mass) basis.

Survival
Explant survival was assessed weekly for three weeks after culturing.Generally, 8-10 explants were used per treatment and experiments were replicated three times.Surviving explants were those which showed shoots with buds, leaves, and root development.

Transmission electron microscopy
A standard glutaraldehyde-osmium fixation method was used, followed by dehydration through an acetone series embedding in a low viscosity epoxy resin (Spurr, 1969).Sections of the meristematic regions of axillary buds and shoot tips were collected on 200 mesh hexagonal copper 3.05 mm grids.Sections were post-stained with uranyl acetate and lead citrate, washed with distilled water, and viewed and photographed with a JEOL 100-S transmission electron microscope.

Source of explant
In vitro cultures of Dioscorea rotundata ("Pona"), accession number PS 98 013 were obtained from the in vitro gene bank of the Department of Botany, University of Ghana, Legon, where the plants were maintained under long-term slow growth conditions at 18ºC.Cultures were multiplied and sub-cultured at six-week intervals on Murashige and Skoog (MS) medium (Murashige and Skoog 1962) with 2.5 μM kinetin, 20 mg l-1 L-cysteine, 2% (0.056 M) sucrose, 0.7% agar, and maintained under a 16 h photoperiod (40 μ mol m-2 s-1) at 25±1°C.

Conditioning excised explants in culture (preculture)
Yam shoot tips (~1 -2 mm) were excised from cultures grown on MS pregrowth medium [as above, except containing 3% (0.09 M) sucrose instead of 2% sucrose] for five weeks, placed on sterilised nylon mesh, which was then positioned, explant cut side down, on fresh medium, overnight.Explants were then transferred on the mesh to semi-solid medium with higher sucrose concentrations (0.3, 0.5, 0.7 and 1.0 M, the control material continuing to be exposed to 0.09 M sucrose) in 90 mm Petri dishes for one, three, five or seven days, each followed by transfer either to growth-enhancing medium (MS complete salts with vitamins, 3% sucrose, 5 μM kinetin, 20 mg l-1 L-cysteine, 0.8% agar, 1% filter-sterilised casein hydrolysate at pH 5.7 ± 0.1), or further conditioned for cryopreservation.

Silica gel dehydration and cooling of explants
Yam explants dehydrated using the same methodology as described above for Frafra potato.Dehydrated explants were placed in cryovials, which were plunged into, and maintained in, liquid nitrogen for one hour, or cooled at 1ºC min-1 in a Nalgene cryo freezing container (Mr Frosty™), to -70°C, and maintained for at least four hours at this temperature.Rewarming was effected immediately on retrieval from the cryogen, in a water bath at 40°C for two to three minutes for all treatments.

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 CaCl 2 .2H 2 O 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 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.

Water content determination
Water content of yam explants was determined following the same procedure as described for Frafra potato above.

Survival assessment
Yam explant survival was determined following the same procedure as described above of Frafra potato.

S h o o t t i p s w h i c h w e r e p r e t r e a t e d w i t h h i gh 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 cells was scored as the tissue having survived the various treatments.

Pregowth
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 gH 2 O 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±1.07gH 2 O/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 enhance desiccation tolerance, which subsequently improves cryotolerance .
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 www.intechopen.comCryopreserving Vegetatively Propagated Tropical Crops -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 subsequent steps.

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

Cryoprotection
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 i n n a l a r e c o r d e d h i g h s u r v i v a l o n t r e a t i n g w i t h P V S 2 a n d 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 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).
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.

Cryopreservation
Although explants treated with mannitol and dehydrated over activated silica gel for 90 minutes had water content 0.24 gg -1 and survival was 70 % (Table 1), when exposed to liquid nitrogen, survival was nil.Ultrastructure indicated extensively degraded cells with withdrawn and broken plasmalemma, cytoplasm and nucleoplasm were all damaged (Plate not shown).Explants from all dehydration treatments did not survive on exposure to liquid nitrogen as well as ultra-cold liquid nitrogen (slash), although, it has been reported that rapid cooling enhance cryosurvival (Wesley-Smith et al., 1992).Having dehydrated explant to water content of 0.11gg-1, it is obvious from ultrastructure (not shown) that the prolonged stress exerted decreased explant ability to withstand freezing since there is a level below which dehydration stress is increasingly apparent (Wesley-Smith et al., 1992).Unlike the loss of viability in S. rotundifolius, explant at higher water contents have been reported to survive on exposure to liquid nitrogen (Berjak et al., 1995;Kioko et al., 1998 and2000) During cryopreservation all metabolic processes cease, it is possible that mannitol treated explants were too active metabolically judging from the high number of mitochondria occurring in the cytoplasm (Plate 1b).Hence bringing the systems to a halt caused a breakdown in all the plant metabolic systems causing cytoplasm to lose its viability since following dehydration, only few organelles could be observed in cytoplasm (Plate 1f).It is also possible that with the occurrence of high number of small vacuoles in mannitol treated explant (Plate 1c) which is a characteristic whereby, large vacuoles volumes are reduced by redistributing them into smaller vesicles on exposure to mannitol (Gnanapragasam & Vasil, 1992), being an advantage for survival since water contents are relatively low (Reinhoud et al., 1995).However, it is probably that, the water present in the vacuoles did not have high viscosity, which would prevent the formation of ice crystals during cooling and thawing hence causing degeneration of plant cell integrity.
Sucrose treated explant, ultrastructure indicated cytoplasmic breakdown at all stages of treatment.Although the plant cell were not in a high metabolic state prior to exposure to liquid nitrogen, cellular degeneration had already set in and may have had a major role to play, leading to loss of viability on exposure to liquid nitrogen.
The above and all associated factors need to be investigated further.These will help optimise plant cell structure prior to cryopreservation.Based on the ultrastructural studies carried out, it is obvious that the use of mannitol for pregrowth and preculture treatment, the plant tissues develop capability to tolerate other stress (desiccation).S. rotundifolius tissues besides yielding high explant survival, results in stable ultrastructure for further plant growth and development.However the treatment does not necessarily result in survival on exposure to cryopreservation.The use of higher concentration of mannitol may enhance cryotolerance.Other critical factors that have to be investigated include maturation of explant supported by constitution of ultrastructure and related water content which play crucial rôle in cryopreservation (Chandal et al., 1994;Berjak et al., 1993).However, the extremely high water content (18.7 -9.64 g/g dry wt) of plant even in the greenhouse (graph not shown), may still make it difficult to cryopreserved tissues of local accessions of S. rotundifolius.Several attempts were made to adequately harden Frafra potato (Table 5) prior to subjecting explants to various treatments and then cooling however, none of them resulted in explants survival after cooling.

Conclusions
Undoubtedly, cryopreservation has high potential for the long-term storage of vegetative explants.It is, however, vital to achieve appropriate tissue water content and the quality of the initial material.This study has shown that vitrification-based cryopreservation protocol is useful for yam explants.However, Frafra potato is extremely sensitive to the vitrification based protocol while explants of the latter easily become hyperhydric and are impossible to dehydrate sufficiently for cryopreservation The findings are relevant for cryopreservation of a range of yam germplasm (Quain et al., 2009) and also provide a sound basis for further attempts to cryopreserve Frafra potato genetic resources.The technique represents developed simple, cost-effective and potentially reliable methodology that does not require sophisticated equipment.Such procedures should be adapted for germplasm conservation of other species, using limited resources in laboratories in sub-Saharan Africa.
Findings in this present study suggest that in order to achieve an optimal recovery of cryopreserved explants, the donor plants should be adequately conditioned and the recovery media enriched while testing the different cryogenic procedures.Although encapsulation vitrification and encapsulation dehydration procedures have been used in cryopreservation procedure, for the yams used in this study, the use of non-encapsulated explant proved to be a better option.The encapsulation of explants after cryopreservation in the production of e.g.synthetic seeds (Naidoo, 2006;Perán et al., 2006), is, however, worth exploring.
The key to successful cryoprotection has been suggested as inducing tolerance to vitrification solutions and the ability of explants to tolerate dehydration treatment by cryoprotectants has been hypothesised by several researchers as the determining factor for successful cryosurvival (Langis & Steponkus, 1990;Reinhoud et al., 1995).It is still not conclusive whether having been hardened, explants being used in procedures would survive cryopreservation.However, Frafra potato explants were not amenable to conditioning by any of these pretreatments.The requirements for successful cryopreservation differ for different species.There is the possibility that the optimum developmental stage of the explant for successful cryopreservation varies from species to species.Therefore there is the need to ascertain and test many parameters on the basis of each species.However, culture conditions especially those that will obviate hyperhydricity, are of paramount importance, as presently indicated for Frafra potato.
It can be concluded from the experiments that:  Successful cryopreservation of Dioscorea rotundata is possible using a simple vitrification protocol.


The procedure incorporates pregrowth of the donor plant on 0.09 M sucrose-supplemented medium for five weeks, -preculture on 0.3 M sucrose supplemented medium for 5 d -MPVS2 solution for 40 min, -Rapid cooling in liquid nitrogen or slow cooling to -70ºC.


For the first time successful cryopreservation of Dioscorea rotundata accession 'Pona' which is an elite variety in Ghana has been achieved.


The technique represents developed simple, cost-effective and potentially reliable methodology that does not require sophisticated equipment.


Procedures can be adapted for germplasm conservation of other species, using limited resources in laboratories in sub-Saharan Africa.


To achieve an optimal recovery of cryopreserved explants the donor plants should be adequately conditioned.


Frafra potato is extremely sensitive to the vitrification based protocol.
Frafra potato explants easily becomes hyperhydric, and are impossible to dehydrate sufficiently for cryopreservation, this provide a sound basis for further attempts to cryopreserve Frafra potato genetic resources.These observations therefore make available information for further investigation towards development of cryopreservation protocol.
Ultrastructural studies indicated that cells had deposits of starch in plastids following sucrose treatments.Survival for D. rotundata shoot tips treated with MPVS2 vitrification solution, and cooled to -70ºC, was 16% for 15 min treatment and 44% for 40 min.Explant rehydration was in 1.0M sucrose supplemented solution.After the 40 min MPVS2 treatment the TTZ test indicated 88% viability retention of explants cooled to -70ºC, and 44% at -196ºC.Plantlet development was obtained for -70ºC-cooled shoot tips, whereas only callus development occurred from tissues exposed to liquid nitrogen.Explant regeneration was not obtained with silica gel dehydration techniques.It was concluded that vitrificationsolution based cryopreservation presently offers the best option for conservation of this Dioscorea species.