Plant Biotechnology
EJB Electronic Journal of Biotechnology ISSN: 0717-3458
1998 by Universidad Catlica de Valparaso -- Chile  
BIP REVIEW ARTICLE

In vitro storage and safe international exchange of yam (Dioscorea spp.) Germplasm

Bernard Malaurie
GeneTrop - Unité de Génétique et d'Amélioration des Plantes - Centre ORSTOM** - 911 avenue Agropolis - BP. 5045 -F. 34032. Montpellier Cedex 1, France.
Tel: Bureau (33)-- 4 67 41 62 44.Standard (33)-- 4 67 41 61 00
Fax: (33)-- 4 67 54 78 00

E-mail : Bernard.Malaurie@mpl.ird.fr

http://www.mpl.ird.fr

**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.

BIP Article

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

Species

Zone of origin

Zone of culture

     

Enantiophyllum Section

   

D. alata L.

South East Asia

Inter-tropical humid

D. cayenensis Lamk.

D. rotundata Poir.

complex

West Africa

West & Central Africa, and Caribbean

D. nummularia Lamk.

Indonesia, Oceania

Indonesia, Oceania and

Micronesia

D. opposita Thunb.

D. japonica Thunb.

complex

Temperate area from:

China, Corea, Taiwan

Japan

Temperate area from:

China, Corea, Taiwan

Japan

D. transversa Br.

South Pacific

South Pacific

Lasiophyton Section

   

D. dumetorum (Kunth) Pax.

West Africa

West Africa

D. hispida Dennst.

India, South-China, New Guinea

India, South-China, New Guinea

D. pentaphylla L.

Himalaya and Oceania

Himalaya and Oceania

Combilium Section

   

D. esculenta (Lour.) Burk.

South East Asia

Inter-tropical humid

Opsophyton Section

   

D. bulbifera L.

South East Asia and Africa

Inter-tropical humid

Macrogynodium Section

   

D. trifida L.

Guyana, Amazonian basin

Caribbean

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

Europe

West Indies

America

Pacific

Asia

Africa

France 2

Barbados *

Brazil 2

Cook Islands

Bengladesh

Bénin

United Kingdom 2

Cuba 2

Colombia

Fiji

India

Burkina Faso

 

Guadeloupe 2

Costa Rica

Niue Islands

Indonesia

Cameroun

 

Jamaïca 2

Guatemala

New Caledonia 2

Japan 2

Ivory Coast 2

 

Saint-Dominique

Mexico

Papua NewGuinea

Malaisia

Ghana 2

 

Trinidad- Tobago

Panama

Salomon Islands

Nepal

Nigeria 2

   

USA

Tonga

Philippines 2

South Africa

     

Vanuatu

Sri Lanka

Togo

     

Western Samoa 2

Thailand

Uganda

       

Viet Nam

 

(Sources : IBPGR 1986, FAO 1996, Malaurie et al 1998a)

* in vitro maintenance for production purpose
1 This country listing is not exhaustive, and take into account only sources in our possession
2 Countries with in vitro collection (according to sources in our possession)

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

At IRD, we choose to maintain the in vitro yam collection in a medium with low mineral nutrient and a low sucrose concentration. We succeeded in the introduction and maintenance of 14 species of yam (Malaurie et al. 1993). Since this time, this collection is continuously enriched by new genotypes and comprises 21 species (Table 3).

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)

Species

Number of accessions

 

D. abyssinica Hochst. Ex Kunth

10

D. alata L.

91

D. bulbifera L.

8

D. cayenensis Lamk.

D. rotundata Poir.

complex

89

D. burkilliana J. Miège

11

D. dumetorum (Kunth) Pax. Edible type
  Wild type

3

2

D. esculenta (Lour.) Burk.

10

D. hirtiflora Benth.

1

D. mangenotiana J. Miège

15

D. minutiflora Engl.

2

D. opposita Thunb.

D. japonica Thunb.

complex

1

D. praehensilis Benth.

18

D. preussii Pax

3

D. sagittifolia

6

D. sansibarensis Pax

1

D. schimperana Hochst. Ex Kunth

1

D. smilacifolia De Wild

2

D. togoensis Knuth

8

D. transversa Br.

1

D. trifida L.

4

Interspecific Hybrids: D. cayenensis-D. rotundata complex cv. 'Krengle' X D. praehensilis

14

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 1000C/ 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, -196C

Steps

Conventional techniques

 

New techniques

   
   

Air-drying

Vitrification

Encapsulation

/ Vitrification

Encapsulation

/ Dehydration

           

Encapsulation

     

+

+

           

Sucrose pretreatment

+/-

+

(+ABA)

   

+

           

Cryoprotector

+

 

++++

++++

 
           

Desiccation

 

+

   

+

           

Slow-freezing

+

0C to -40C

(0.3 to 1C/min)

       
           

Rapid-freezing

+

-40C to -196C

(200C/min)

+

+25C to -196C

(720C/min)

+

+25C to -196C

(400 to 1100C/min)

+

+25C to -196C

(400 to 1100C/min)

+

+25C to -196C

(720C/min)

           

Thawing

500C/min

120C/min

120C/min

120C/min

120C/min

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).

 

Click here to enlarge

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
In vitro germplasm conservation present different advantages such as: 1) to be free from genetic erosion, 2) to have the possibility for the establishment of core collection with long term genebanks, 3) to be free from fungus and bacteria, 4) to be not expensive, when in vitro facilities are already present, 5) easy and convenient for international distribution. But International exchanges need more for safe international exchange. We need to know the plant material on genetic level, and over all on the phytosanitary level. On the phytosanitary level, various viruses have been described on edible and medicinal yams on their production area. Different works, depending virus and virus group, are reported (Table 5). Indexing techniques allow to highlight a certain number of viruses on yam: Poty, potex, badna and cucumo-viruses, where yam mosaic virus (YMV) provokes the most important loss.


Table 5. Viruses of yam: group and type viruses, yam species affected and reference works

Virus Group

Virus

Authors

Cucumovirus

CMV

Migliori 1977; Fauquet & Thouvenel 1987

'Carlavirus' cf.

ChYNMV

(Chinese yam necrotic mosaic virus)

Shirako & Ehara 1986

Badnavirus

DBV

(Dioscorea bacilliform badnavirus)

Mantell & Haque 1978

 

DaBV

(D. alata bacilliform virus)

Degras 1986

 

DbBV

(D. bulbifera bacilliform virus)

Degras 1986

Potexvirus

DLV

(Dioscorea latent virus)

Phillips & Brunt 1988

 

PVX

(Potato virus X)

Urbino et al. 1998

Potyvirus

YMV

(yam mosaic virus)

Aleman 1996; Goudou-Urbino et al. 1996; Dallot et al. 1998

 

YMMV 1

(yam mild mosaic virus)

Mumford & Seal 1997

 

D. trifida virus 2

Migliori 1977

 

DGBMV 2

(Dioscorea green banding mosaic virus)

Porth & Nienhaus 1983

 

DaRMV 3

(D. alata ring mottle virus)

Porth & Nienhaus 1983

 

DaV 4

(D. alata virus)

Reckhaus & Nienhaus 1981

 

D. dumetorum potyvirus

Mumford & Seal 1997

 

D. esculenta potyvirus

Mumford & Seal 1997

 

DGBV (Dioscorea greenbanding potyvirus)

Phillips et al. 1986

 

PVY

(Potato virus Y)

Urbino et al. 1998

1 YMMV: is it a new potyvirus or a strain of the YMV?
2 D. trifida virus and DGBMV have been shown as YMV strains (Porth et al.,1987)
3 DaRMV should be a 'yam strain' of the beet mosaic potyvirus transmissible on N. benthamiana (Porth et al.,1987)
4 DaV is serologically links toYMV but differ by is non-transmissibility (Porth et al.,1987)

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

Eradication techniques

Species

Authors

 

 

 

meristem culture

D. cayenensis-rotundata,

D. japonica, D. opposita,

D. praehensilis,

D. rotundata, D. trifida, Dioscorea spp.

Cortes Monllor et al 1982; Kobayashi 1991; Malaurie et al 1988a, 1995a,b; Matsubaru & Ishira 1988; Mikami 1984; Saleil et al 1990;

Thermotherapy in vivo + meristems culture

 

D. alata

Mantell et al 1980

Nodal microcutting or apices + Thermotherapy

D. alata, D. trifida

Balagne 1985; Salazar & Fernanadez 1988

Nodal microcutting + Chemotherapy

D. alata

Mantell 1993

Nodal microcutting + Thermotherapy &/or Chemotherapy

D. praehensilis

Malaurie, unpublished results

Meristem culture + Thermotherapy &/or Chemotherapy

D. cayenensis-rotundata,

D. praehensilis

Malaurie, unpublished results



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*

Species

Sending countries

Receiving countries

D. alata

Côte d'Ivoire

Brazil

Nouvelle Calédonie

West Indies

Nouvelle Calédonie

Côte d'Ivoire

D. bulbifera

Nouvelle Calédonie

Côte d'Ivoire

D. cayenensis-D. rotundata complex

Côte d'Ivoire

Brazil

Côte d'Ivoire

Nouvelle Calédonie

*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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