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the Endangered Genetic Resources of Vanilla. Michel Grisoni and Marion Moles. Pascale Besse and Séverine Bory. UMR PVBMT, CIRAD / UR. CIRAD, Pôle de ...

Towards an International Plant Collection to Maintain and Characterize the Endangered Genetic Resources of Vanilla Michel Grisoni and Marion Moles UMR PVBMT, CIRAD / UR CIRAD, Pôle de Protection des Plantes Saint Pierre, La Réunion France

Pascale Besse and Séverine Bory UMR PVBMT, CIRAD / UR Université de la Réunion Saint Denis, La Réunion France

Marie-France Duval CIRAD UPR Multiplication Végétative Montpellier France

Rémi Kahane CIRAD / Global Horticultural Initiative Secretariat at AVRDC Shanhua Taiwan ROC

Keywords: aromatic crops, orchid virus, patrimonial resources, Vanilla planifolia Abstract The primary gene pool of Vanilla planifolia Jackson, the main source for commercial vanilla, is heavily threatened in its area of origin (Central America) by habitat fragmentation and destruction. In addition, ex situ conserved material is often endangered by viruses such as Cymbidium mosaic virus that easily spread by contact in plant collections. Therefore, there is an urgent need to protect the genetic resources that could be a supply of desirable traits for vanilla production. These resources should include the local varieties cultivated around the centre of origin and in the areas of dispersion, as well as the vanilla relatives estimated to 110 species distributed worldwide. In this respect, a germplasm collection of vanilla species and varieties has been set up by CIRAD in Reunion Island since 2003. It is structured according to three key rules: 1) a strict phytosanitary control of the planting material that is certified free of the viruses known to infect vanilla, 2) a clear specification of the origin and property rights attached to the material in fulfilment with international conventions on biodiversity, and 3) an accurate characterization of the accessions using molecular, morphological and aromatic markers. This collection presently gathers 250 accessions tentatively classified into 22 species. Most of the material belongs to the V. planifolia species and originates from the South-Western Indian Ocean area. The tropical and contrasted climatic conditions of Reunion Island should enable blooming and fruiting of most accessions, thus facilitating taxonomic identification and agronomic evaluation. Preliminary results and perspectives regarding the management and characterization of this collection are presented and the standpoint of networking with similar initiatives in favour of vanilla preservation is advocated. INTRODUCTION Vanilla is a universally appreciated flavour used in an increasing diversity of agroindustrial products such as baked goods, ice cream, soda, snack food and body care (Rownell, 2005). The vanilla flavour is exclusively produced from the fruits of aromatic orchids of the genus Vanilla (Vanilloideae, Orchidaceae). It differs from synthetic and biosynthetic vanillin by its natural origin and curing process as well as by its aromatic complexity (Klimes and Lamparsky, 1976; Pérez-Silva et al., 2006). Vanilla extract remains therefore highly demanded by agro-food industry despite its high and fluctuating price. However, the market now tends to request higher quality standards, more diverse and specific aromatic profiles and lower production costs. These goals could typically be reached through the genetic improvement of the cultivated plant material. However, little attention has been paid so far to the taxonomy and genetic diversity of the Vanilla genus, and due to its easy vegetative propagation through cuttings, only a few accessions are

Proc. XXVII IHC-S1 Plant Gen. Resources Ed.-in-Chief: K.E. Hummer Acta Hort. 760, ISHS 2007


cultivated all around the world. There is therefore a need to characterize, maintain and improve the genetic resources. Vanilla planifolia Jackson (syn. V. fragrans Salisb.) is the most cultivated species and accounts for more than 95% of the commercial vanilla. This species is believed to originate from the humid forests of southern Mexico and has been spread all around the world from the beginning of the 16th century. More than one hundred relative vanilla species have been described in the world, but most of the aromatic species originates from tropical America (Portères, 1954). Following the general trend of worldwide biodiversity erosion resulting from the degradation of biotopes, recent surveys indicated that vanilla genetic resources were particularly endangered. In Mexico, for instance, which represents the primary gene pool for vanilla, a very limited number of in situ genuine wild vanilla subsists due to deforestation and over-collection (Lubinsky, 2003; Soto Arenas, 2006). In Madagascar, where a vast ex situ collection had been established in the mid 1900’s, it seems that only a very few accessions are still available as a consequence of poor management, virus infection and repeated climatic disasters (Grisoni et al., 1997; Leclercq-Le Quillec and Nany, 2000). In this context where in situ and ex situ gene pools are endangered, specific measures are to be taken to preserve the genetic resources. This paper briefly depicts the scheme that is being developed by the International Cooperation Centre in Agronomic Research for Development (CIRAD) in Reunion Island (France) in order to preserve the biological diversity of vanilla species. MATERIALS AND METHODS Origin of the Material The collection was initiated in 2003, starting from a local stock of 30 accessions that had been maintained by tissue culture for several decades. It was incremented by prospecting French territories (Réunion, Guadeloupe, Martinique, and Guyane) and thanks to contributions from many entities around the world, including botanical gardens, research institutes, vanilla growers, private companies or orchid hobbyists. Legal Aspects of Material Transfers In fulfilment with international regulations and policies, the origin of all the accessions entering the collection was recorded and a Material Transfer Agreement (MTA) was established with each provider. The MTA specified in particular that the accessions remained provider’s property, were not transferable to third party without explicit consent of the provider and that any valorisation of results originating from the material should rely on the principle of equitable benefice sharing with the provider. According to application of European regulation on plant pathogens, importations of vanilla cuttings into La Réunion were subjected to prior establishment of an Official Letter of Authorisation (LOA). Disease Management The vanilla cuttings were introduced into the quarantine facility of the Pôle de Protection des Plantes at Saint Pierre (La Réunion, France). They were grown in individual pots for at least six month in order to check for the absence of pests, diseases symptoms and viruses. As a vegetatively propagated crop, vanilla may be threatened by the accumulation of viruses. Cymbidium mosaic virus (CymMV, Potexvirus) is the most frequently found virus in vanilla plantations worldwide (Grisoni et al., 2004; Leclercq Le Quillec et al., 2001), followed by the potyviruses for which seven species at least have been identified (Grisoni et al., 2006). Cucumber mosaic virus (CMV, Cucumovirus), Odontoglosum ringspot virus (ORSV, Tobamovirus) and an uncharacterized Rhabdovirus have been also reported in few locations. 84

The accessions were tested for viruses using standard Elisa protocols with commercially available antisera (Table 1). Plants that tested free of viruses in at least three successive Elisa tests were transferred to the plant collection. Virus-infected plants were destroyed or transferred to in vitro culture in order to proceed with virus elimination through meristem-tip culture. Maintenance of the Material in Collection Healthy vanilla plants were maintained under an insect-proof shade house (60% shading) located at Saint-Pierre, La Réunion (55°30’E – 23°20’S, 80 m above sea level). Three cuttings per accession were grown on wooden support in separate 0.8 m² trays supplied with a 50/50 mixture of urban compost and coco fibre (Biogrow, Montescot-FR). Plants were sprayed regularly to prevent root diseases (Fusarium and Phytophthora) and scale infestation (Conchaspis angraeci). The virological status of the vines was checked annually by visual inspections and bulked Elisa tests. Tissue Culture In vitro cultures were established from surface sterilized axillary buds placed on Murashige and Skoog (MS) medium. Axenic shoots were then propagated on MS medium complemented with 0.5 % benzylaminopurin (BAP). In vitro plants were grown in a climatic chamber at the following conditions: 12 hours day at 26°C, 100-120 µmol s-1m-2 photosynthetic radiation, and 12 hours night at 24°C. For each accession, a total of twelve individuals were conserved in vitro. Genetic and Taxonomic Characterization The value of the biological collections depends of the quantity and quality of the information attached to the accessions. In particular the material should be taxonomically identified and its biological traits as much as possible described. Many of the vanilla accessions incorporated in the collection were poorly documented. They were therefore analysed using RAPD (Besse et al., 2004) and AFLP (Duval et al., 2006) as well as morphology of the reproductive and vegetative apparatus, when available. Herbarium specimens from the French National Museum of Natural History (MNHN) were used as reference for both the morphological and the molecular identification. RESULTS AND DISCUSSION Provisory Inventory of the Collection By July 15th, 2006, the vanilla collection gathered 498 accessions belonging to 36 species. It is divided into 250 accessions (13 species) available as DNA extract only and 248 accessions (23 species) available also as living plants. Among these, 147 have already been indexed and are maintained virus-free in an insect-proof shade house (Fig. 1). The reminder (201 accessions) is in the indexing or sanitation process. The majority (46 %) of the plants in the living collection were collected in the Indian Ocean area, followed by America (28%) and Europe (18%) (Table 2). All the plant accessions are being progressively transferred in vitro for tissue culture. At present, only short-term and medium-term (low nutrient content and low temperature) in vitro conservation are considered. These modes of active conservation enable rapid amplification or distribution of the accessions and direct acclimatization of the plants. Conversely, they require frequent transplantation of the accessions (two to three times a year). Characterization of the Accessions RAPD and AFLP molecular markers allowed clear cut differentiation of American vs non American vanilla species and distinction between close species such as V. planifolia, V. tahitensis, V. bahiana and V. pompona (Fig. 2). It thus enabled to authenticate or to correct species identification for most accessions as well as confirm or 85

assume the hybrid status for a few of them. The genetic resources maintained in the collection are listed in Table 3. Each section and subsection of the Vanilla genus (Portères, 1954) is represented but the Foliosiae-Lamellosae is majoritary with in particular V. planifolia (303 accessions), V. pompona (31), V. tahitensis (19) and V. bahiana (16). Forty-seven accessions were not taxonomically identified so far because we did not get flower yet and no reference AFLP profile was available. Twenty-one accessions are hybrids having V. planifolia as one of the parents. Additional tools are currently been developed to identify the vanilla relatives SSR, CAPS and assess genetic diversity within and between taxa. The expected data should help in elucidating the phylogeny of the vanilla and give some insight to the diversity and structuration of the genus as well as to the history of its dissemination (Duval et al., 2006). Indexing and Phytosanitary Status By June 2006, 208 accessions introduced as cuttings in quarantine had been tested by Elisa. Virus identification was further confirmed for all infected samples tested by sequencing of RT-PCR amplicons. CymMV was the most frequently found virus (41 accessions), far ahead from potyviruses (1 accession) and ORSV (1 accession). There was an obvious relationship between the origin of the material and the rate of infection by CymMV (Table 4). All the specimens collected in the wild were virus free, while the specimens originating from botanical gardens were infected for more than 40% of them. A similar pattern between wild and cultivated has been reported for ornamental orchids and likely results from the high rate of mechanical transmission of CymMV (Wisler et al., 1979). It is possible that other unrecorded virus infects some accessions even in the absence of symptoms. To improve phytosanitary security, this should be checked using broad range detection methods such as electronic microscopy or PCR using degenerated primers. Goals and Prospects for Sustainable Conservation of Vanilla Diversity Our preliminary data provided some insights on vanilla biodiversity and enabled to initiate a conservation program for this biodiversity. However additional research is needed to improve our comprehension of intra- and inter-specific diversity of the genus and to adjust accordingly our conservation strategies. This research should primarily focus on the development of new molecular markers of genetic and epigenetic polymorphisms, the adaptation of biotechnologies for plant sanitation and cryoconservation and the assessment of the aromatic and biological traits of the vanilla accessions. Interestingly, meanwhile this project was being implemented in Reunion Island; similar initiatives were set up in other regions. As far as we know, three projects are presently maintaining a significant number of accessions i) in the Pacific region, the Etablissement Vanille de Tahiti and the Secretariat of the Pacific Community, handle a collection of about 250 accessions covering 10 ecotypes of V. tahitensis from French Polynesia and a few V. planifolia and hybrids (Guarino, 2004), ii) in India, the Indian Institute of Spices Research maintains 300 genotypes including indigenous and exotic vanilla species as well as seedling progenies (Minoo et al., 2006), iii) in Mexico, about 200 accessions (20 species) are preserved, covering most of the biodiversity found in Mesoamerica (P. Lubinski, pers. com.). These working collections aim not only at preserving the vanilla genetic resources, but also at characterizing them genetically and phenotypically. Furthermore, attention has been paid to insure optimal phytosanitary status to the accessions and develop technologies for long- or medium-term storage of the material. Therefore, there is a convergence of objectives which should strengthen the collaboration between the distinct regional projects. By so, we could achieve the global inventory of the genetic 86

resources of vanilla, identify the area that requires further survey in order to cover as completely as possible the whole genetic diversity, identify the sources of desirable traits in primary and secondary gene pools, clarify the taxonomy and phylogeny of the genus, define legal and technical procedures that facilitate and secure material exchanges, and define sound criteria for the establishment of core collections that combine restricted size and large diversity. Such a cooperative networking would thus enable a better understanding and securing of vanilla biodiversity, and improve the quality of the planting material for the vanilla industry. ACKNOWLEDGEMENTS Authors express their thanks to the many contributors who supplied the collection with vanilla material, to Katia Jade and Jean-Bernard Dijoux for technical work at CIRAD Réunion and to Marc Pignal (MNHN, Paris) for very helpful assistance in specimen recollection and species identification. Literature Cited Besse, P., Da Silva, D., Bory, S., Grisoni, M., Le Bellec, F. and Duval, M.-F. 2004. RAPD genetic diversity in cultivated vanilla: Vanilla planifolia, and relationships with V. tahitensis and V. pompona. Plant science 167, 379-385. Duval, M.-F., Bory, S., Andrzejewski, S., Grisoni, M., Besse, P., Causse, S., Charon, C., Dron, M., Odoux, E. and Wong, M. 2006. Diversité génétique des vanilliers dans leurs zones de dispersion secondaire. Les Actes du BRG 6, 181-196. Grisoni, M., Côme, B. and Nany, F. 1997. Projet de relance de la vanilliculture dans la région du Sava: Compte rendu de mission à Madagascar du 05 au 18 mai 1997, CIRAD / FOFIFA, Saint Pierre, 11 p. Grisoni, M., Davidson, F., Hyrondelle, C., Farreyrol, K., Caruana, M.L. and Pearson, M. 2004. Nature, incidence and symptomatology of viruses infecting Vanilla tahitensis in French Polynesia. Plant disease 88, 119-124. Grisoni, M., Moles, M., Farreyrol, K., Rassaby, L., Davis, R.I. and Pearson, M.N. 2006. Identification of the potyviruses infecting vanilla by direct sequencing of a short RTPCR amplicon. Plant Pathology 55, 523-529. Guarino, L. 2004. Collection of plant genetic resources collections in the Pacific island countries and territories, Secretariat of the Pacific Community, Suva, Fiji Islands, 144 p. Klimes, L. and Lamparsky, D. 1976. Vanilla volatiles - a comprehensive analysis. International Flavours and Food Additives 7, 272-291. Leclercq Le Quillec, F., Riviere, C. and Lagorce, A. 2001. Spread of Cymbidium mosaic potexvirus and potyviruses in vanilla plants grown in shade houses in Reunion Island. Fruits (Paris) 56, 249-260. Leclercq-Le Quillec, F. and Nany, F. 2000. Compte rendu de mission à Madagascar 18 fev. - 15 mars 2000, CIRAD/FED/FOFIFA, 11 p. Lubinsky, P. 2003. Conservation of wild vanilla. Poster presented at the First International Congress on the future of the Vanilla business. Princeton, NJ, USA Minoo, D., Nirmal Babu, K. and Peter, K.V. 2006. Conservation of Vanilla species, in vitro. Scientia Horticulturae., 110, 175-180. Pérez-Silva, A., Odoux, E., Brat, P., Ribeyre, F., Rodriguez-Jimenes, G., Robles-Olvera, V., García-Alvarado, M.A. and Günata, Z. 2006. GC–MS and GC–olfactometry analysis of aroma compounds in a representative organic aroma extract from cured vanilla (Vanilla planifolia G. Jackson) beans. Food Chemistry 99, 728-735. Portères, R. 1954. Le genre Vanilla et ses espèces. In G. Bouriquet (Ed.): Le vanillier et la vanille dans le monde, Editions Paul Lechevalier, Paris 599 p. Rownell, B. 2005. Crucial raw materials : the state of vanilla challenges, pricing, trends and applications. Perfumer and flavorist 30, 36-39. Soto Arenas, M.A. 2006. Vainilla: los retos de un cultivo basado en una especie amenazada con una historia de vida compleja. Poster presented at the Congreso 87

Internacional de Productores de Vainilla, May 26-28, 2006, Papantla, Veracruz, Mexico. Wisler, G.C., Zettler, F.W. and Sheehan, T.J. 1979. Relative incidence of Cymbidium mosaic and Odontoglossum ringspot viruses in several genera of wild and cultivated orchids. Proceedings of the Florida State Horticultural Society 92, 339-340.


Table 1. Antibodies used for virus detection in enzyme linked immunosorbent assay (Elisa). Virus Cymbidium mosaic virus Potyvirus group Cucumber mosaic virus Odontoglossum ringspot virus

Elisa format Indirect Double Antibody Sandwich Plate trapped Antigen Plate trapped Antigen Plate trapped Antigen

Provider Agdia (Elkhart-IN, US) Agdia (Elkhart-IN, US) Bioreba (Marnes la Coquette, FR) DSMZ (Braunschweig, DE)

Table 2. Geographic origin of the material in collection.

V. planifolia V. pompona V. bahiana V. tahitensis Other species TOTAL


Indian Ocean 76 15 0 3 20 114







19 6 16 0 28 69

10 4 0 1 30 45

12 1 0 1 0 14

2 0 0 0 2 4

1 0 0 0 1 2

120 26 16 5 81 248

Table 3. List of the vanilla genetic resources maintained in the collection. Section

Subsection Lamellosae

Foliosae Membranaceae Papillosae Not determined Aphyllae


Not determined

Not determined

Vanilla species africana, albida, bahiana, bicolor , chamissonis, crenulata, cucullata, ensifolia, hartii, leprieurii, odorata, planifolia, polylepis, pompona, ramosa, siamensis, tahitensis, zanzibarica inodora, ovata (cf.) , palmarum annamica, françoisii, grandifolia, imperialis grandiflora (cf.), mexicana aphylla, claviculata, decaryana, dilloniana, humblotii, madagascariensis, phalaenopsis, roscheri, wightiana Hybrid and unidentified species

Table 4. Incidence of Cymbidium mosaic virus in vanilla cuttings according to the origin of the material. Origin of the material Botanical collection Production plot Wild vanilla Total

Tested 108 90 10 208

Number of accessions Healthy 67 89 10 166

CymMV 41 1 0 42



Fig. 1. View of the insect-proof shade house harbouring the vanilla collection at SaintPierre (La Réunion, France).

Fig. 2. Differentiation of vanilla species based on the phylogenetic tree derived from 337 AFLP markers and 90 accessions of the collection.