|
Electronic Journal of Biotechnology
Universidad Católica de Valparaíso
ISSN: 0717-3458
Vol. 1, Num. 3, 1998, pp. 103-117
|
Electronic Journal of Biotechnology, Vol.
1, No. 3, December, 1998
REVIEW ARTICLE
Medium-term and long-term in
vitro conservation and safe international exchange of yam (Dioscorea
spp.) germplasm
Bernard Malaurie*1, Marie-France
Trouslot2, Julien Berthaud3,
Mustapha Bousalem4, Agnès Pinel5
and Jean Dubern6
1GeneTrop Unité de Génétique
et dAmélioration 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.orstom.fr
2GeneTrop Unité
de Génétique et dAmélioration des Plantes - Centre ORSTOM**
911 avenue Agropolis - BP. 5045 -F. 34032. Montpellier Cedex 1, France.
3GeneTrop Unité de Génétique
et dAmélioration des Plantes - Centre ORSTOM** 911 avenue Agropolis
- BP. 5045 -F. 34032. Montpellier Cedex 1, France.
4Laboratoire de Phytovirologie des
Régions Chaudes (LPRC) CIRAD-ORSTOM** avenue du val de Montferrand
- BP. 5035 -F. 34032 - Montpellier Cedex 1, France.
5Laboratoire de Phytovirologie des
Régions Chaudes (LPRC) CIRAD-ORSTOM** avenue du val de Montferrand
- BP. 5035 -F. 34032 - Montpellier Cedex 1, France.
6Laboratoire de Phytovirologie des
Régions Chaudes (LPRC) CIRAD-ORSTOM** avenue du val de Montferrand
- BP. 5035 -F. 34032 - Montpellier Cedex 1, France. E-mail : dubern@melusine.mpl.orstom.fr
http://www.mpl.orstom.fr
*Corresponding author
**ORSTOM is now called IRD, Institut de Recherche pour le Developpement.
Code Number: ej98015
Abstract
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 cleans the plants from all diseases but viruses. It gives a good control
on the preservation of the yam genetic resources and facilitates international
exchanges of healthy plant material.
Two kinds of in vitro germplasm preservation
were considered : slow growth condition culture for mid-term preservation,
and cryopreservation using the encapsulation/dehydration technique for long-term
preservation. Virus eradication was approached by meristem culture and chemo
and thermotherapy. Production of virus-free plants was controlled by ELISA.
We succeeded in the introduction and maintenance
of 20 yam species, under slow growth conditions. Cryopreservation was applied
successfully on two edible yam species, Dioscorea. alata L and
D. bulbifera L. Virus-free plants were obtained by meristem culture in
D. cayenensis-D. rotundata complex and D. praehensilis. Indexation
allowed the detection of different virus (poty-, potex-, badna- and cucumovirus),
where the most important potyvirus was YMV.
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 apply the optimized
protocol to genotypes which are 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 tests available
for yam indexing (ELISA, rt/PCR, monoclonal antibodies) and on new sanitation
techniques.
Keywords: Active and base in vitro
genebanks, Chemotherapy, Cryopreservation, Disease-free techniques, Indexation
techniques, Slow growth condition culture, Virus eradication, Yam viruses.
Article
Yam belongs to
Dioscorea genus which has more than 600 species (Coursey,
1967) most of them distributed in the intertropical humid area. We will
distinguish two types of yam: 1) medicinal yams, 2) edible yams and relatives.
Medicinal yams concern about fifty species caracterized by their sapogenin content,
which are steroidal components. For the edible yams and relatives, we will observe
two groups: 1) domesticated species, 2) and wild species.
For domesticated species we consider that
forty to fifty species are occasionally used (Martin and Degras,
1978). From these only eleven are cultivated (Table 1).
From the 11 cultivated, 6 represent an important part of feeding (D. alata,
D. cayenensis-D. rotundata complex, D. bulbifera, D. dumetorum, D. esculenta,
D. trifida), 3 are scattered in all the intertropical humid area (D.
alata, D. bulbifera, D. esculenta), and yam belonging to the D. cayenensis-D.
rotundata complex take place most of them in West Africa and some in the
Caribbean area. The other yams are cultivated in their origin area (Degras,
1986).
Table 1 .
Main edible species of yam
Species 1
|
Zone of origin
|
Zone of culture
|
|
|
|
Enantiophyllum Section
|
|
|
D. alata L.
|
South East Asia
|
Inter-tropical humid
|
D. cayenensis Lamk.
D. rotundata Poir.
complex 2
|
West Africa
|
West and Central Africa, and Caribbean
|
D. nummularia Lamk.
|
Indonesia, Oceania
|
Indonesia, Oceania and
Micronesia
|
D. opposita Thunb.
D. japonica Thunb.
complex 3
|
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. (1998a)
1 Species have been regrouped in Section
by Knuth (1924), completed by Burkill
(1960)
2 Grouping together species of D. cayenensis and
D. rotundata in a Complex has been proposed by Ayensu
and Coursey (1972), Martin and Rhodes (1978),
Miège (1982)
3 Grouping together species of D. opposita and D.
japonica in a Complex has been proposed by Tanaka
(1977).
Main edible species of yams are: 1) native
of a continent, 2) and cultivated in the same continent, 3) or/and cultivated
in an other. This observation implies very strong links to exchange problems.
In Table 2, 36 countries have been observed by IBPGR
in 1986 with Dioscorea germplasm. 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.
Table 2 .
Countries¹ 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
|
Colombia
|
Fiji
|
India
|
Burkina Faso
|
|
|
|
|
|
|
|
Guadeloupe 2
|
Costa Rica
|
Niue Islands
|
Indonesia
|
Cameroun
|
|
|
|
|
|
|
|
Jamaïque
|
Guatemala
|
Nouvelle Calédonie 2
|
Japan 2
|
Côte dIvoire 2
|
|
|
|
|
|
|
|
Saint-Domingue
|
Mexico
|
Papua NewGuinea
|
Malaisia
|
Ghana
|
|
|
|
|
|
|
|
Trinidad
y Tobago
|
Panama
|
Salomon Islands
|
Nepal
|
Nigeria 2
|
|
|
|
|
|
|
|
|
USA
|
Tonga
|
Philippina
|
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)
Different genebank preservation levels exist.
A first group concerns non aseptic germplasm conservation with in field genebank
and seed genebank, where important disadvantages and heavy constraint of quarantine
measures explain the choice of in vitro germplasm conservation (Hanson,
1986; Malaurie et al., 1998a) (Table
3).
Table 3. Non
aseptic Germplasm Conservation
|
Non aseptic Genebanks
|
|
In field Genebanks
|
Seed Genebanks
|
Disadvantages
|
|
|
Genetic erosion
|
+++
|
|
Expensive
|
+++
|
|
Hard to manage
|
+++
|
|
Do not bread true
|
|
+++
|
Tuber shape
|
|
+++
|
Dormancy
|
|
+++
|
In vitro
conservation
Three levels of in vitro genebank
preservation levels could be considered: 1) short term conservation: this conservation
under normal growth conditions is suitable for temporary storage of germplasm
collections, and for international distribution, 2) medium term conservation,
which could be considered as an active in vitro genebank, 3) long term
conservation, considered also as a base in vitro genebank.
These in vitro genebanks have been
previously introduced in vitro from tuber or seed. These introductions
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 under slow growth conditions, could
be obtained by several ways: 1) physiological stage of the explant, 2) addition
of osmotic agents and growth moderators, 3) low storage temperature, 4) low
mineral or sucrose concentrations, 5) low oxygen pressure, 6) encapsulation
in alginate (Charrier et al., 1991; Withers,
1991; Engelmann, 1991; Malaurie et al.,
1998a).
Medium-term conservation
At ORSTOM**, 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 20 species (Table
4).
For yam, this simple solution of slow growth
is used routinely and from these culture conditions a direct acclimatization
is possible. This mode of conservation allows an international distribution
of the material and corresponds to an active genebank (Malaurie
et al. 1993,1998c, Malaurie and Trouslot
1995c).
This in vitro germplasm collection
of yam is maintained in test tubes, at ORSTOM** (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 4 .
Listing of different species of yam maintained in an in vitro collection,
under slow growth culture condition *
(GeneTrop, GAP unit, ORSTOM**, Montpellier,
France)
Species
|
Number of accessions
|
D. abyssinica Hochst.
Ex Kunth
|
6
|
D. alata L.
|
91
|
D. bulbifera L.
|
8
|
D. cayenensis Lamk.
D. rotundata Poir.
complex
|
63 (+ 17)
|
D. burkilliana J. Miège
|
11
|
D. dumetorum (Kunth) Pax.
|
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.
|
17
|
D. preussii Pax
|
1
|
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.
|
2 (+ 1)
|
Interspecific Hybrids: D.
cayenensis-D. rotundata complex cv. Krengle X D. praehensilis
|
14
|
so-called Igname de Pilimpikou
|
(+ 9)
|
* (+ ): Accessions recently
introduced
Different species maintained in the in
vitro collection, such as D. cayenensis-D. rotundata complex are
going to be enriched by cultivars from Burkina Faso for a sanitation program,
and from Benin for a genetic program linkeds to virologic aspect. Others species
supposed to be links to D. cayenensis-D. rotundata complex, such as D.
mangenotiana, D. praehensilis, D. minutiflora, or D. abyssinica,
D. praehensilis, are going to be enriched by further introduction.
Orstom** virologists are interested by D.
trifida because of its strong sensibility to virus, which provoked in Guadeloupe,
French West-Indies, its quite disappearance. Serological and molecular works
are developed to explain this virus sensibility.
Long-term conservation
Long term conservation correspond to cryopreservation
in liquid nitrogen, at 196 °C. Plant cryobiology, which begun in 1971 by Latta
works on carrot cell suspension, benefited from results on animals cell by Polge
et al in 1949. Since these dates, different techniques have been set up:
1) on one hand, the so-called conventional techniques, using two steps of slow
freezing, with the addition of cryo-protector (Sakai, 1984),
2) and on the other hand, new techniques, characterized by a very rapid freezing,
about 1000°C/ min, by direct immersion in liquid nitrogen (Table
5) (Dereuddre et al., 1990, 1991; Tannoury
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 5 .
Long term conservation : cryopreservation in liquid nitrogen, -196°C
Steps
|
Conventional techniques
|
|
New techniques
|
|
|
|
Air-drying
|
Vitrification
|
Encapsulation
/ Dehydration
|
Encapsulation
|
|
|
|
+
|
Sucrose pretreatment
|
+/-
|
+
(+ABA)
|
|
+
|
Cryoprotector
|
+
|
|
++++
|
|
Desiccation
|
|
+
|
|
+
|
Slow-freezing
|
+
0°C to -40°C
(0.3 to 1°C/min)
|
|
|
|
Rapid-freezing
|
+
-40°C to -196°C
(200°C/min)
|
+
+25°C to -196°C
(720°C/min)
|
+
+25°C to -196°C
(400 to 1100°C/min)
|
+
+25°C to -196°C
(720°C/min)
|
Thawing
|
500°C/min
|
120°C/min
|
120°C/min
|
120°C/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 ( Butenko et al.,
1984; Popov and Fedorovskii, 1992; Popov
and 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
by two different research teams, using encapsulation/dehydration of shoot apices.
On the one hand, Mandal et al. (1996) compared the
survival capacities of apices after the osmotic and thermic stress of the technique
of four species of yam - three edible (D. alata, D. bulbifera, D. wallichii),
and one medicinal (D. floribunda). Four of them have survived after immersion
in liquid nitrogen, with 26 to 71%, depending on the species. Meanwhile, only
two of them (D. alata, D. wallichii) allowed the recovery into
shoots after immersion in liquid nitrogen, with 21 and 37%, respectively.
On the other hand, ORSTOM** ability in different
aspects of the long term conservation on tropical plants (Engelmann,
1991), and on encapsulation/dehydration technique applied on coffee, cassava,
oil palm...etc, permitted to apply the process on apical shoot-tips of in
vitro plantlets of yam (Malaurie and Trouslot 1996). Malaurie
et al. (1998b) obtained survival rates over 50% for the two species (D.
alata, D. bulbifera), and recovery to rooted leafy shoots after immersion
in liquid nitrogen of at least 60% for D. bulbifera and 20% for D.
alata, three months culture after thawing.
Comparatively to previous works on cryopreservation
using encapsulation/dehydration technique, Malaurie et al. (1998b)
have used higher sucrose concentration (0.9, 1.0 and 1.1M), a wider range of
dehydration duration, up to 23h and a new and more accurate method for measuring
of dry weight.
The new and more accurate method for measuring
of dry weight used in our experiments consisted of desiccating alginate beads
for 30d in airtight boxes containing dry silica gel, to avoid mass loss due
to caramelization of sugar when drying at a temperature higher than 100°C. We
obtained a strong linear correlation between dry mass (DW30) and
sucrose molarity for sucrose-pretreated alginate beads. During the whole experiment,
we used DW30 values estimated by linear regression (Table
6).
Table 6. Dry mass
and water content of sucrose-pretreated alginate beads, determined
after 30d of drying with silica gel in airtight boxes at room temperature (1)
Sucrose concentration
|
DW30 (% FW) estimated
by linear regression (2)
|
Water content before dehydration
(g.g-1 DW)
|
0.75M
|
28.8
|
2.47
|
0.9M
|
33.3
|
2.00
|
1.0M
|
36.3
|
1.76
|
1.1M
|
39.3
|
1.54
|
(1)Source: Malaurie et al.
(1998b)
(2)From mean values over 13 to 15 replicates for each of the
four sucrose concentrations
(y= 6.4319 + 29.872x; N= 4; r= 0.999). Similar results were
obtained from replicate data
(y= 6.4177 + 29.883x; N= 55; r= 0.960). Data not shown.
For
the best sucrose pretreatments depending species, Figure
1(source: Malaurie et al., 1998b) shows that D. bulbifera
still has high survival with high sucrose concentration and after long duration
dehydration (up to 23h). For the two species, the water content of encapsulated
apices had to be decreased down to 0.15g H2O g-1 DW in order to obtain high
survival after freezing. The percentage of water loss was of 67, 62, 58 and
55% FW (± 1%) for 0.75, 0.9, 1 and 1.1M sucrose pretreatments, respectively.
Our results demonstrated that, in most cases, survival increased when dehydration
was extended to a defined threshold, around 0.13-0.15g. H2O g-1 DW, which was
obtained after desiccation periods from 10 to 18h. It seemed that, with this
soft dessication process, we could rub out differences in residual water-free,
which certainly exist between apices from a same plot.
Indexation and
disease-free germplasm production
Indexation
In vitro germplasm conservation presents
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 fungis 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 7). 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 7. Viruses
of yam: group and type viruses, yam species affected and reference works
Virus Group
|
Virus
|
Yam species affected
|
Geographic spreading
|
Disease importance
|
Authors
|
Cucumovirus
|
CMV
|
D. alata,
D. cayenensis-D. rotundata
complex, D. trifida
|
Caribbean and West-Africa
|
-
|
Migliori, 1977;
Fauquet and Thouvenel, 1987
|
|
|
|
|
|
|
Carlavirus cf.
|
ChYNMV
(Chinese yam necrotic mosaic virus)
|
D. batatas
|
Japan
|
-
|
Fukomoto and Tochihara,
1978; Shirako and Ehara, 1986
|
|
|
|
|
|
|
Badnavirus
|
DBV
(Dioscorea bacilliform
badnavirus)
|
D. alata
|
Barbade
|
+/-
|
Mantell and Haque,
1978
|
|
DaBV
(D. alata bacilliform virus)
|
|
|
-
|
Degras, 1986
|
|
DbBV
(D. bulbifera bacilliform
virus)
|
|
|
-
|
Degras, 1986
|
|
|
|
|
|
|
Potexvirus
|
DLV
(Dioscorea latent virus)
|
D. floribunda
D. composita
|
Puerto Rico
|
-
|
Hearon et al., 1978;
Phillips and Brunt 1988; Watterworth
et al., 1974
|
|
PVX
(Potato virus X)
|
|
In vitro collection
|
-
|
Urbino et al.,1998
|
|
|
|
|
|
|
Potyvirus
|
YMV
(yam mosaic virus)
|
D. alata, D. cayenensis-D.
rotundata complex,
|
All the intertropical area
|
+++
|
Thouvenel and Fauquet,
1979;Goudou-Urbino,1995;Goudou-Urbino
et al. 1996 a,b)
|
|
YMMV 1
(yam mild mosaic virus)
|
D. alata, D. cayenensis-D.
rotundata complex,
|
West-Africa
|
+++
|
Mumford and Seal,
1997
|
|
D. trifida virus 2
|
D. trifida
|
Guadeloupe
|
+++
|
Migliori, 1977
|
|
DGBMV 2
(Dioscorea green banding
mosaic virus)
|
|
Togo
|
++
|
Porth and Nienhaus,
1983
|
|
DaRMV 3
(D. alata ring mottle virus)
|
D. alata
|
Togo
|
++
|
Porth and Nienhaus,
1983
|
|
|
|
|
|
|
|
DaV 4
(D. alata virus)
|
D. alata
D. rotundata
|
Togo
|
+
|
Reckhaus and Nienhaus,
1981
|
|
D. dumetorum potyvirus
|
|
South-Pacific
|
-
|
Mumford and Seal,
1997
|
|
D. esculenta potyvirus
|
|
South-Pacific
|
-
|
Mumford and Seal,
1997
|
|
DGBV (Dioscorea greenbanding
potyvirus)
|
D. composita
D. floribunda
|
Puerto Rico
|
-
|
Hearon et al., 1978;
Phillips et al.,
1986
|
|
PVY
(Potato virus Y)
|
|
In vitro collection
|
|
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)
During the establishment of the yam in
vitro germplasm collection, in the biotechnology laboratory of ORSTOM**
(Malaurie et al., 1993), afterwards IIRSDA - Adiopodoumé
research station, near Abidjan, Ivory Coast - clones were systematically indexed
by ELISA directed to YMV, when introduced in vitro (Malaurie
and Thouvenel, 1988; Malaurie et al., 1988a,b;
Charrier and Hamon, 1991).
Later on, one indexation was carried out
by a virologist team on the duplicate of the yam in vitro germplasm collection
enriched by introduction of new genotypes. 92 samples, belonging to several
yam species, were used for the indexation : D. alata, D. bulbifera, D. cayenensis-
rotundata complex, D. dumetorum, D. esculenta, D. mangenotiana, D. praehensilis,
D. shimperiana, D. togoensis, D. trifida. These samples were originating
from various geographic areas: Africa, Caribbean, South America and Asia. Four
viruses were fetched by ELISA technique: PVX (potato virus X, potexvirus) PVY
(potato virus Y, potyvirus), CMV (cucumber mosaic cucumovirus), and YMV (Urbino
et al. 1998). Results are presented in Table 8.
Table 8. Indexation
of the ORSTOM** in vitro yam collection
ELISA results
|
PVX
|
PVY
|
CMV
|
YMV
|
% Positives
|
5,5
|
6,3
|
2,1
|
7,7
|
% Negatives
|
94,5
|
93,7
|
97,9
|
92,3
|
Source: Urbino et al. (1998)
This study allowed to show
that detection of viruses serologically linked to PVX and to PVY, in different
yam species, was possible, even with the same frequencies than with YMV. Further
works have to be done for a precise caracterization of these virus isolates,
and check their respective importance in natural environement. Experiments using
more sophisticated techniques for virus diagnostic (IC/rt/PCR) are developed
(Bousalem, 1995). Yam on molecular caracterization and molecular
diversity on potyvirus of the yam mosaic virus (YMV) have been developed on
the ILTAB/ORSTOM**-TSRI laboratories (Aleman, 1996; Aleman
et al 1996a,b) and from the LPRC laboratory (Bousalem,
1995; Urbino et al., 1998).
Virus eradication
techniques
The use of in vitro techniques allows
to be free from fungis, bacteria, and other pests. 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
(36°C during 1 to 2 weeks on in vivo or in vitro plants, use of
chemicals such as vidarabine, ribavirin and 2-thiouracil). They could be used
alone or associated (Table 9).
Table 9 . Yam
disease eradication techniques
|
Eradication techniques
|
Species
|
Authors
|
Type of use*
|
Virus eradication
|
(A)
|
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,b, 1992, 1995a,b;
Malaurie and Thouvenel, 1988;
Matsubaru and Ishira, 1988; Mikami ,1984; Saleil
et al., 1990;
|
E
|
+ / -
|
|
|
|
|
|
|
(B)
|
Thermotherapy in vivo + meristems
culture
|
D. alata
|
Mantell et al 1980
|
E
|
+
|
(C)
|
Nodal microcutting or apices + Thermotherapy
|
D. alata, D. trifida
|
Balagne 1985; Salazar
and Fernanadez, 1988
|
E, R (+ / -)
|
+
|
|
|
|
|
|
|
(D)
|
Nodal microcutting + Chemotherapy
|
D. alata
|
Mantell ,1993
|
E, R (+ / -)
|
+
|
(E)
|
Nodal microcutting + Thermotherapy
&/or Chemotherapy
|
D. praehensilis
|
Malaurie, unpublished results
|
E
|
+ / -
|
(F)
|
Meristem culture + Thermotherapy
&/or Chemotherapy
|
D. cayenensis-rotundata,
D. praehensilis
|
Malaurie, unpublished results
|
E
|
+ / -
|
* E: experimental use; R: routine use
Success in meristem culture depends on the
size and location of the explant excised, and on the growth regulator ratio.
Meristem culture, on Table 9, concerns works using meristem-tips
(0.2-0.5 mm long) as well as shoot-tips (0.6-2.5 mm long). Experiments on viability
and in vitro morphological development of meristem-tips of two sizes,
small (0.3-0.5 mm) and large (0.6-0.8 mm), have shown that it was better
to use large meristem size to increase the shoot elongation percentage. The
use of axillary or apical meristems did not induce difference and should allow
an important yield in micropropagating such material from excised meristem-tips.
Eleven months after meristem excision, production of plantlets was observed
with a rate of 82% and 39% from the survivors, for a clone of D. cayenensis-D.
rotundata complex and D. praehensilis genotype, respectively (Malaurie
et al., 1995a,b).
Meristem cultures have been done on 8 clones
of 5 Dioscorea species belonging to the in vitro germplasm collection.
Morphological development has been observed and data were recorded 60 days after
meristem inoculation. In our case, the production of rooted leafy shoots, 60
days after meristem inoculation, occurred in five clones out of eight, with
percentage shoot leaf production of 5 to 26 %, depending on the clone. Six months
later, the excised meristems of all clones developed into rooted leafy shoots,
where D. bulbifera, and D. dumetorum was not, to our knowledge,
mentioned in the literature (Table 10).
Table 10. Genotypic
effect on morphogenetic orientation
2 months after meristem excision of Dioscorea
spp *
|
Total meristems observed
|
Necrosis 1)
%
|
|
Organogenesis 2)
%
|
|
Regeneration 3)
%
|
|
D. alata
|
67
|
27
|
|
55
|
|
18
|
|
D. bulbifera
|
252
|
46
|
|
37
|
|
18
|
|
D. bulbifera
|
146
|
52
|
|
48
|
|
0
|
|
D. cayenensis-D. rotundata complex
|
23
|
65
|
|
35
|
|
0
|
|
D. cayenensis-D. rotundata complex
|
81
|
24
|
|
51
|
|
26
|
|
D. cayenensis-D. rotundata complex
|
81
|
26
|
|
54
|
|
20
|
|
D. dumetorum
|
24
|
83
|
|
17
|
|
0
|
|
D. praehensilis
|
117
|
41
|
|
54
|
|
5
|
|
1) Necrosis. 2) Organogenesis: callusing,
rooting, swelling were added together, 3) Regeneration: meristem development
into rooted leafy shoots and axillary bud development or bud neoformation.
*(Malaurie, unpublished results)
Works about production of virus-free in
vitro plants of yam through yam meristem culture alone are very rare. Saleil
et al. (1990) on D. trifida obtained YMV-free plants, after ELISA
indexation with a 27% rate through the total indexed plants. Nevertheless, other
unpublished results on 2 genotypes YMV-infected of 2 species (D. cayenensis-
D. rotundata complex, D. praehensilis) showed that meristem culture
allowed the production of virus-free plants with 76% and 17% plants indexed,
respectively (Malaurie, unpublished results).
Production of virus-free in vitro
plants of yam has been attempted through thermotherapy, chemotherapy associated
or not, from in vivo mother plants, nodal cuttings or apices (Balagne,
1985; Mantell, 1993; Mantell et al., 1980;
Salazar and Fernandez, 1988). None of them described clearly
the percentage of virus-free plants obtained through these techniques. Meanwhile,
the production of plantlets free from virus is described by Mantell
(1993) on D. alata cv. Kinabayo, after the action of antiviral agents
(vidarabin, ribavirin) on nodal microcuttings infected by a potyvirus. The production
of virus-free plants have been obtained 210 days after in vitro inoculation,
after 3 subcultures of 60, 120 and 30 days on a liquid/solid biphasic cuture
system with 10-5 M of antiviral agent.
Other available techniques could be electrotherapy
used on potato, with 60 to 100% success as compared to 25-40% with thermotherapy
(Lozoya-Saldaña et al., 1996; Bernal et
al., 1998), 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 conditions 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.
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 11, 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 11. Enrichment of the genetic diversity
of a country by transfer and introduction
of in vitro yam genotypes from
different geographic origins*
Species
|
Number of accession
|
Sending countries
|
Receiving countries
|
D. alata
|
6
|
Côte dIvoire
|
Nouvelle Calédonie
|
D. alata
|
5
|
West Indies
|
Nouvelle Calédonie
|
D. alata
|
1
|
Brazil
|
Nouvelle Calédonie
|
D. alata
|
5
|
West Indies
|
Côte dIvoire
|
D. alata
|
3
|
Nouvelle Calédonie
|
Côte dIvoire
|
D. alata
|
3
|
Brazil
|
Côte dIvoire
|
D. bulbifera
|
1
|
Nouvelle Calédonie
|
Côte dIvoire
|
D. cayenensis-D. rotundata complex
|
4
|
Côte dIvoire
|
Nouvelle Calédonie
|
D. cayenensis-D. rotundata complex
|
1
|
Brazil
|
Côte dIvoire
|
* 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, Adiopodoumé, Côte
dIvoire; 1992-95: Orstom**, LRGAPT, Montpellier) for their in vitro
introduction and micropropagation, preliminary to all safe international 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 (John et al.,1993; Malaurie
et al., 1993; Mantell, 1993; Ng, 1988;
Ng and 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.
Recently a new method, experimented over
three yam species (D. alata, D. opposita, D. rotundata),
has been proposed by Hasan and Takagi (1995). They use encapsulation
technique, with the embeddment of nodal cuttings in alginate beads, for a concept
of a material transfer. This process allow to maintain in the dark for at least
2 weeks. These 2 weeks in the dark allow to envisage a safe and easy international
exchange of genetic resources.
Concluding remarks
This paper tries to describe different studies
done and to be done on yam in vitro germplasm conservation and its safe
international exchange. Yam in vitro culture contributes to the safeguard
of the biodiversity of the genus Dioscorea. An application of the results
obtained on cryopreservation to more species should allow a transfer of technology.
The use of new techniques, in a one hand, for pathogen eradication (electrotherapy,
micrografting), in addition to the existent ones, and in the other hand, for
the obtention of plants resistant to somes viruses (transformation), 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.
To conclude we can say that we are already
able to manage routinely yam in vitro genebanks in slow growth culture,
to index for more viruses, and to produce some virus-free in vitro plantlets.
For an efficient distribution - transfer
- utilisation of yam germplasm, we should develop: 1) virus-free germplasm,
2) restricted size collection, with large diversity, so-called core-collections.
For that, in vitro conservation under slow growth condition and cryopreservation,
have to be applied routinely to more genotypes; virus-indexing has to be done
with more precise techniques (rt/PCR); therapy has to be done with several combined
techniques to become genotype independant.
But, we should not forget, as previously
said by Hanson (1986), 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).
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