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In vitro storage and safe international exchange of yam (Dioscorea spp.) Germplasm Bernard Malaurie
**IRD (Institut
de Recherche pour le Developpement.) formerly ORSTOM. Keywords: Active and base in vitro genebanks', Chemotherapy, Cryopreservation, Disease-free techniques, Indexation techniques, Slow growth condition culture, Virus eradication, Yam viruses.
Yam edible tubers feed million of peoples in the intertropical area, where they represent 12% of human feeding. However, as a vegetatively propagated crop, yam is seriously affected by an accumulation of pathogens. Establishing in vitro germplasm collection is a process that clean the plants from all diseases but viruses. It give a good control on the preservation of the yam genetic resources and facilitate international exchanges of healthy plant material. Two kinds of in vitro germplasm preservation were considered : slow growth condition culture for mid-term preservation, and direct cryopreservation in liquid nitrogen for long-term preservation. Virus eradication was approached by meristem culture and chemo and thermotherapy. Production of virus-free plants was controlled by enzyme linked immunosorbent assay. Mid-term conservation of yam germplasm is used routinely, and from these conditions a direct acclimatization is possible. On the cryopreservation aspect, experiments are under way to applied the optimized protocol to genotypes more representative of the diversity, to insure a routinely use. More work can be conducted now on virus eradication, based on knowledge accumulated on potyvirus diversity, on several immunological and molecular tests available for yam indexing and on new virus eradication techniques. Introduction Yams are plants characterized by their production of tubers, aerial tubers, or rhizomes. They take on a very important economic interest for human being by their steroidal compounds, for medicinal species, and by their source of carbohydrates for the edible species and relatives. Millions of people in Africa, the Caribbean and the south Pacific are concerned by this important food crop. Yams belongs to Dioscorea genus which correspond to more than 600 species (Coursey 1967), distributed for most of them in the intertropical humid area. For the edible yams and relatives, two groups are considered, such as, the domesticated species, and the wild species. Eleven species are cultivated among the forty to fifty domesticated species which are occasionally used (Martin & Degras 1978) (Table 1), but only 6 represent an important part of feeding with a world annual production of over 28 millions tons (D. alata, D. cayenensis-D. rotundata complex, D. bulbifera, D. dumetorum, D. esculenta, D. trifida). Table 1 . Main edible species of yam
Sources: Malaurie et al. (1998d)
From the Table 1, we
observed that main edible species of yams are, for some of them,
native of a continent, and cultivated in the same continent, or/and
cultivated in an other. This observation imply very strong links
to exchange problems. In Table 2, 43 countries
have been observed with Dioscorea germplasm (FAO
1996, IBPGR 1986). These countries are supposed
to be concerned by an international exchange of yam germplasm. Some
of them, have, in our knowledge, already developed in vitro
germplasm collection (Malaurie et al. 1998a).
Table 2 . Countries 1 and geographic zones where yam collections have been observed
(Sources : IBPGR 1986, FAO
1996, Malaurie et al 1998a) In vitro conservation These three levels in vitro genebanks (short, medium and long term conservation) have been previously introduced in vitro from tuber or seed. These introduction have to be linked to an obligatory phytosanitary control from mother plants and from in vitro material after introduction. Medium term conservation, which correspond to in vitro culture in slow growth conditions, could be obtained by using the different behaviour of the plant against physiological, mineral, biochemical, chemical, or physical state (Malaurie et al. 1998a, d).Medium-term conservation
This in vitro germplasm collection of yam is maintained in test tubes, at IRD (Montpellier, France), with a total of 6 test tubes by accession, with two different places of storage for the replicates ; the minimal growth conditions allow to maintain most of the accessions up to 2 years. Technical constraints in the collection management lead to subculture the accessions every 6-8 months (Malaurie et al. 1998c). Table 3 . Listing of different species of yam maintained in an in vitro collection, under slow growth culture condition (GeneTrop, GAP unit, IRD, Montpellier, France)
Long-term conservation Long term conservation correspond to cryopreservation in liquid nitrogen, at -196 °C. Different techniques have been set up for plant cryopreservation in liquid nitrogen. On the one hand, so-called conventional techniques, using two steps of slow freezing, with the addition of cryo-protector (Sakai 1984), and on the other hand, new techniques, characterized by a very rapid freezing, about 1000°C/ min, by direct immersion in liquid nitrogen (Table 4) (Dereuddre et al. 1991; Uragami 1993). The aim of these techniques is to try to control water flow and ice formation, and tend to a vitrificated state, avoiding crystal formation during thawing, and to protect the cell from thermic shocks.Table 4 . Long term conservation : cryopreservation in liquid nitrogen, -196°C
Sources : Uragami (1993), Malaurie et al. (1998a). Most of the results about cryopreservation have been obtained from conventional techniques on suspension cells of medicinal yam, D. deltoidea being the most used (Popov & Volkova 1994). More recent works have been done on rapid cryopreservation of callus (Chulafich et al. 1994), by direct immersion in liquid nitrogen, of two other medicinal yams (D. balcanica, D. caucasica). Since 1996, new results have been obtained using encapsulation/dehydration of shoot apices (Mandal et al. 1996; Malaurie & Trouslot 1996; Malaurie et al. 1998b,f, Mandal 1998), or later on, using encapsulation/vitrification (Mandal 1998), or vitrification alone (Mandal 1998; Ng & Ng 1998, Kyesmu & Takagi 1998).
Figure 1. 1) microprogation of shoots; 2) ánd 3) apex excision; 4) encapsulation of apical shoot-tips in alginate beads; 5) sucrose pretreatment; 6) dehydration over silica gel in airtight boxes; 7) apical shoot-tips in sterile cryotubes; 8) rapid immersion of the cryovials into liquid nitrogen. Indexation and Disease-free germplasm production Indexation Table 5. Viruses of yam: group and type viruses, yam species affected and reference works
1 YMMV:
is it a new potyvirus or a strain of the YMV? Indexation for virus detection were systematically developed on the introduced clones, during the, establishment of the yam in vitro germplasm collection, in the biotechnology laboratory of ORSTOM, afiterwards IIRSDA Adiopodoumé research station, near Abidjan, Côte d'lvoire (Malaurie et al. 1993; Malaurie et al. 1988) Later on, one other indexation on new genotypes of the yam in vitro germplasm collection allowed to show that detection of viruses serologically links to PVX and to PVY, in differents yam species, was possible, even with the same frequencies than with YMV (Urbino et al. 1998). Virus eradication techniques The use of in vitro techniques allows to, be free from fungus, bacteria, and other pest. Only viruses could be present on the plant and have to be eradicated. Different techniques exist and are already applied on yam. There are meristem culture, thermotherapy and/ or chemotherapy. They could be use alone or associated (Table 6). Table 6 . Yam disease eradication techniques
Success in meristem culture depends on the size and location of the explant excised, and on the growth regulator ratio. 'Meristem culture', on Table 6, concern works using meristem-tips (0.2-0.5 mm long) as well as shoot-tips (0.6-2.5 mm long). Works about production of virus-free in vitro plants of yam through yam meristem culture alone are very rare (Saleil et al. 1990, Malaurie, unpublished results), and data are not sufficient for the production of virus-free plants, routinely. Production of virus-free in vitro plants of yam. has been attempted through thermotherapy, chemotherapy associated or not, firom mother in vivo plants, nodal cuttings or apices (Balagne 1985; Mantell 1993; Mantell et al. 1980; Salazar & Fernandez 1988). None of them described clearly the percentage rate of virus-free plants obtained through these techniques. Meanwhile, the production of plantlets free from virus is described by Mantell Mantell(1993) on D. alata cv. Kinabayo, after the action of antiviral agents on nodal microcuttings infected by a potyvirus.Other available techniques could be electrotherapy used on potato, or apex micrografting, used on Lemon tree or vine, routinely. If different works have already been done on yam sanitation, only a few of them conducted to an eradication of virus with more or less importance. Safe international exchange Exchange and distribution of plant material could be done by two ways: 1) with non aseptic plant material (tubers, aerial tubers, seeds, nodal cuttings from the vine), 2) with plant material in aseptic conditions (micro-nodal cuttings, microtubers, aerial microtubers, apices, zygotic or somatic embryos, callus and cells suspension).Exchange in non-aseptic conditions was used in the past, but required severe quarantine measures. Since 1989, with the FAO/IBPGR technical guidelines for the safe movement of yam germplasm, recommendation has been given to use in vitro condition for exchange and distribution. For that, safe movement of yam germplasm could be done easily by three ways: 1) micro-nodal cuttings, 2) micro-tubers, 3) or encapsulated apices (Malaurie et al. 1998e).Safe movement of yam germplasm by micro-nodal cuttings is the most common way and has been frequently used (Malaurie et al. 1998a). In Table 7, the use of laboratories with in vitro and quarantine facilities allowed the indexation, in vitro introduction and micropropagation for a safe diffusion of various genotypes from different geographical origin. Table 7. Enrichment of the genetic diversity of a country by transfer and introduction of in vitro yam genotypes from different geographic origins*
*All plant material from the sending countries were, at first, tubers sent to laboratories with quarantine and in vitro culture facilities (1988-89: Orstom & Iirsda, Adiopodourné, Côte d'lvoire; 1992-95: Orstom, LRGAPT, Montpellier) for their in vitro introduction and micropropagation, preliminary to all safe intemational exchange. Tuber potentiality shown by a great number of in vitro yams (aerial and basal micro-tubers) could be also used for a safe transfer of yam germplasm. They could increase the percentage of success during their acclimatation in field (Malaurie et al. 1993; Mantell 1993; Ng & Mantell 1997). These tubers developed in vitro are dormant at maturity and they still keep their dormancy from 2 to 5 months, as tubers developed in vivo, for most of the species, the other showing longer dormancy period from 6 to 12 months (Ng & Ng 1997).Recently a new method, experimented over three yam species (D. alata, D. opposita, D. rotundata), has been proposed by Hasan & Takagi (1995). They use encapsulation technique, with the embeddment of nodal cuttings in alginate beads, for a concept of a material transfer. In front of the increasingly movement of yam germplasm in the next few years, linked to the increasing demand of plant material for feeding people, we need, for an integrated international safe movement of yam germplasm, to develop a chain of facilities with quarantine, indexation, sanitation and in vitro conservation (Malaurie et al. 1998e) Concluding remarks Yam in vitro germplasm conservation and its safe intemational exchange need to develop further investigations and respect the recommendations which have been done already (FAO/IBPGR 1989, Hanson 1986, Ashmore 1997). Yam in vitro culture contribute to the safeguard of the biodiversity of the genus Dioscorea. At the present time, we are already able to manage routinely yam in vitro genebanks in slow growth culture, and provide germplasm for intemational exchange. The use of new techniques in addition to the existent ones, for pathogen eradication, and for the obtention of resistant plants to some viruses, should guarantee to yam a state of virus-free plant and allow international exchanges, and in long term, distribution to the farmer of cultivar free from virus. Further research have to be done on cryopreservation which should allow a transfer of technology. For an efficient distribution - transfer - utilisation of yam germplasm, we should develop a chain of facilities with - quarantine - indexation -sanitation - in vitro conservation - fully and cost efficient, linked in a unique location or distributed over several locations. But, we never forget that, for a better security of germplasm conservation, different methods of conservation have to be combined -in situ - Field Genebanks - , ex situ - Seed Genebanks, in vitro Genebanks and, to prevent material loss, duplication of yam germplasm has to be considered (Hanson 1986). 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Cryopreservation of in vitro yarn (Dioscorca sp.) apices by the encapsulation-dehydration technique. Eucarpia, Meeting on Tropical Plants, Marsh 11-15 1996, France, Abstract, p 252. Malaurie, B., Trouslot, M.-F. and Berthaud, J. (1998)a. Conservation et échange de germoplasme chez les ignames (Dioscorea spp.).'L'igname: Plante Séculaire et Culture d'Avenir'. eds. J. Berthaud, N. Bricas, J.-L. Marchand, Montpellier 3-6 Juin 1997, In : Acte du séminaire international Cirad-lnra-Orstom-Coraf, France. 135-161 Malaurie, B., Trouslot, M.-F., Engelmann, F. and Chabrillange, N. (1998)b. Effect of pretreatment conditions on the cryopreservation of in vitro-cultured yam (Dioscorea alata 'Brazo Fuerte' and. D. bulbifera 'Nouméa Imboro') shoot apices by encapsulation-dehydration. Cryo-Letters 19 : 15-26 Malaurie, B., Trouslot, M.-F., Berthaud, J., Chabrillange, N., Récalt, C. and Dussert, S. (1998)c. 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In VIlth Symposium of the International Society for Tropical Root Crops, Gosier (Guadeloupe), 1-6 July 1985, INRA Ed., Paris, p. 439-445 Saleil, V., Degras, L. and Jonard, R. (1990). Obtention de plantes indemnes du virus de la mosaïque de l'igname (YMV) par culture in vitro des apex chez l'igname americaine Dioscorea trifida L. Agronomie, 10: 605-615 Shirako, Y. and Ehara, Y. (1986). Rapid diagnosis of chinese yam necrotic mosaic virus infection by electro-blot immunoassay. Ann. Phytopath. Soe. Japan, 52: 453-459 Uragami, A. (1993). Cryopreservation of cultured cells and organs of vegetables. In Reprint from JICA GRP REF no. 6, 111-135. Urbino, C., Bousalem, M., Pinel, A., Fargette, D. and Dubem, J. (1998). Les Virus de I'igname: aspects épidémiologiques et variabilité.'L''ignarne: Plante Séculaire et Culture d'Avenir'. eds J. Berthaud, N. Bricas, J.L. Marchand, Montpellier 3-6 Juin 1997, In: Acte du séminaire international Cirad-lnra-Orstom-Coraf, France. 205-211
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