Vanilla

Michel Roux-Cuvelier and Michel Grisoni

Importance of Plant Genetic Resources

Plant genetic resources are a major strategic challenge for all activities linked to agriculture and the agro-food industry, especially in the present context of climate change. Globally, since the 1950s, population increase and the development of intensive agriculture have contributed to a reduction in the diversity of plant species. Today, the protection of these resources is of vital importance in achieving sustainable food security for the populations.

From the 1970s, a number of initiatives were developed to safeguard the diversity of cultivated species. During 1971, the initiation of the CGIAR (Consultative Group on International Agricultural Research) provided an initial response to the problem of the loss of genetic diversity for the major agricultural species. At present, 11 of the 15 CGIAR centers are responsible for maintaining international gene banks for the preservation and dissemination of the plant genetic resources that provide the basis of world food security (http://www.cgiar.org/index.html). More recently, initiatives aimed at securing collections of genetic resources have been launched. These include the creation of the OECD’s BRCs (Biological Resource Centres) or of the Global Crop Diversity Trust (http://www.croptrust.org/main/), that is behind operational projects such as the Svalbard Global Seed Vault that currently conserves almost 660 different genera and 3300 species from all continents (http://www.croptrust.org/ main/arctic.php?itemid=211).

While the major agricultural species are covered by recognized conservation systems, few large-scale initiatives have been taken to preserve underutilized and orphan species. However, to meet the challenges of the future, the study and protection of agricultural biodiversity must not be based solely on a limited number of species, but must be envisaged from a broad, open perspective, presenting each species as being interdependent on the others and a representative of its own specific evolutionary process.

Today, a conservation program cannot be implemented without first defining the objectives for the use of resources and for acquiring knowledge of intra- or inter-species genetic diversity. Once the objectives have been established, questions should be asked about the representativeness of genetic diversity (what should be preserved and in what quantities?) and the cost of setting up and maintaining these collections.

In situ collections consist of preserving species in their natural or human- modified ecosystems, in other words, the place in which they developed their distinctive characteristics. These collections mainly concern wild species and are often represented by the national or regional parks that protect the ecosystems as a whole. While this type of conservation represents the ideal model in that it maintains the selection pressure of its environment, its effectiveness in terms of conservation often depends on the degree to which local populations are involved in the management of the region and its resources. Moreover, it provides no protection against climate or biological risks (pathogens, plant pests, or invasive species).

Ex situ collections consist of preserving genetic resources outside of their natural habitat. This conservation method freezes the resources at the genetic level. The degree of protection of resources and their independence in relation to the natural environment is highly variable depending on the type of conservation. Whole plants may be kept in the form of field collections, arboreta, in greenhouses, or in tissue culture. Fragments of plants (meristems, embryos, tissues), which can be used to regenerate a whole plant, may be preserved at very low temperature in liquid nitrogen. Seeds are commonly held in cold storage, under controlled humidity conditions. Conserving pollen at low temperature means that a stock of haploid material that can be maintained. By conserving as much diversity as possible at lower cost, DNA banks can be used for intra- and interspecies genetic studies. Within ex situ collections, a distinction can be made between safeguarding and active or working collections, which give rise to specific activities that involve the resources stored, such as genetic diversity studies and plant breeding research.

These ex situ conservation methods are, nevertheless, reliant on human activity and on the smooth functioning of the conservation facilities and structures.

Finally, herbaria (see Chapter 4)—the first method for conserving plant species (sixteenth century)—and spirit collections are essential, especially for conducting taxonomic studies.

Although the economic cost of maintaining collections is linked to the number of accessions conserved, the issue of the number of individuals that represent diversity and ensure the security of the accession is crucial. The principle of the “core collection” may provide a response. Noirot et al. (1996) developed a method for creating a core collection based on quantitative data. This method (Principal Component Scoring) aims to include the maximum diversity from the base collection in a sample of minimum size, while avoiding redundancies.

For plant genetic resources, each conservation system has its advantages and disadvantages and often only a combination of these different systems will make it possible to optimize and to secure the conservation of resources.

In addition to the quantitative aspect (the number of species and accessions conserved), the quality of data and research programs linked to resources is now a key element in justifying the material and human investments. The value and accuracy of these data also contribute to the development of resources.

The Case of Vanilla

The genus Vanilla, which belongs to the Orchid family, includes 90–100 species depending on the author (Bory et al., 2008c). Most of these species are wild; only two of them, V. planifolia and V. tahitensis, are grown to produce commercial vanilla, with V. planifolia providing 95% of the world production. The taxonomy of vanilla is very old (Portères, 1954; Rolfe, 1896), incomplete, and still imprecise (see Chapter 1). It must therefore be thoroughly revised, especially in light of the findings of recent molecular genetics and cytogenetics research (Bory et al., 2008a, 2008b; Cameron, 2003, 2009; Schluter et al., 2007; Soto Arenas, 2003; Verma et al., 2009). For example, several recent research studies on the origin of V. tahitensis suggest that this species is the result of cross-breeding between V. planifolia and V. odorata (Besse et al., 2004; Bory et al., 2008d; Lubinsky et al., 2008b).

The species of the genus Vanilla are mainly found in natural habitats in tropical and subtropical regions of the American, African, and Asian continents. Most are threatened by the destruction of their original habitats, which is accentuated by climate change. The species V. planifolia is particularly endangered, as the primary gene pool in its region of origin (southern Mexico) is subject to considerable pressure linked to deforestation and the overexploitation of natural resources (Soto Arenas, 1999). In secondary diversification zones such as the islands of the Indian Ocean, and especially Réunion—the point of entry for the species into this region in the nineteenth century—there is considerable intraspecies homogeneity, indicating that cultivation may rely on a very restricted genetic base, which probably developed from one single individual through vegetative reproduction. Molecular studies have confirmed the very low level of genetic diversity in the vanilla plants cultivated throughout the world (V. planifolia) (Bory et al., 2008d; Lubinsky et al., 2008a; Minoo et al., 2008a). The vegetative reproduction process, which is predominant for the species of vanilla grown, does not make it possible to maintain and extend the gene pool. Nevertheless, an interesting phenotypic diversity is observed, which may be explained by the accumulation of somatic mutations, by the possibility of natural seed germination in the case of sexual reproduction, but also by the variable ploidy level that can be found in cultivated vanilla species (see Chapter 2). However, due to varietal homogeneity, vanilla cultivation is particularly vulnerable to environmental hazards, such as climate change and the emergence of plant pests.

The secondary gene pool of vanilla includes some 100 species that have diversified in America, Africa, and Asia. These species have individual properties that may be of particular interest for the genetic improvement of cultivated vanilla, such as autofertility, resistance to disease (fusariosis, viruses), the capacity to bear a large amount of fruit, a lower dependence on the photoperiod for the induction of flowering, a higher vanillin content, the presence of other aromatic or medicinal metabolites, and resistance to drought. The establishment of gene banks, in the form of collections, is thus extremely urgent for vanilla in order to safeguard the endangered endemic and patrimonial genetic resources (Grisoni et al., 2007; Lubinsky et al., 2008a; Pandey et al., 2008; Soto Arenas, 2006). Their interesting characteristics could thus be used in genetic crop improvement programs, as has begun in India (Minoo et al., 2006, 2008b; Muthuramalingam et al., 2004).

Overview of the Conservation of Vanilla Genetic Resources

The most effective means of protecting the diversity of vanilla species ought to be in situ protection in their areas of origin. However, the natural areas where the primary gene pools are found are very often subject to strong demographic pressure that endangers the different species. This type of conservation can therefore only be envisaged if it is associated with a global conservation strategy at the level of a territory (Soto Arenas, 1999). In Mexico, the success and experience of the CONABIO (Comisión Nacional para el Conocimiento y Uso de la Biodiversidad) may enable the implementation of this conservation method. In South Africa, the iSimangaliso reserve focuses particularly on conserving V. rosheri, which is second in the list of rare and endangered endemic species (Combrink and Kyle, 2006).

However, the diversity of vanilla, including more than 100 species distributed over three continents and living in varied biotopes, makes it difficult to set up in situ conservation systems. The establishment of ex situ collections therefore appears to be a necessary strategy for protecting vanilla genetic resources.

Initiatives of varying degrees of importance have been taken in the past in certain vanilla-producing countries where the genetic resources are found (Puerto Rico, Madagascar, Costa Rica, Mexico). Thus, from the 1940s onward on the Mayaguez station in Puerto Rico, research was conducted to characterize and improve vanilla plants (Childers et al., 1959). At about the same time in Madagascar, the Ambohitsara vanilla station near Antalaha began to collect and hybridize a wide range of vanilla plants (Dequaire, 1976). However, this collection was decimated by repeated cyclones, a lack of maintenance, and the propagation of viruses (Grisoni, 2009). In the late 1970s, the CATIE (Centro Agronómico Tropical de Investigación y Enseñanza) in Costa Rica collected and maintained about 32 vanilla accessions from Central America. A part of this collection was safeguarded by passage in vitro (Jarret and Fernandez, 1984). In Mexico, alongside the CONABIO program, a collection of clones representing the diversity of the country was established, but unfortunately could not be maintained (M.A. Soto Arenas, pers. comm.).

Today, the most important collections are found in France—Réunion (CIRAD), French Polynesia (EVT), and Cherbourg (council/MNHN), in India (ICRI), and in the United States (Table 3.1). Several botanical gardens and research institutions also have varying quantities of vanilla specimens in their greenhouses (Les Serres d’Auteuil, Jardin du Luxembourg and Jardin Botanique de Nancy in France, the Royal Botanic Gardens, Kew and the Copenhagen Botanical Garden in Europe, or

Data kindly provided by the curators of the collections.

Rutgers University, the New York Botanical Garden, and the Rio de Janeiro Botanical Garden in Brazil, the Secretariat of the Pacific Community in Fiji, to name but a few). Private collections belonging to orchid lovers are another source of sometimes rare specimens. In total, around 50% of global diversity (in terms of the number of species) is thus conserved in these collections, essentially in the form of whole plants, in vivo or sometimes in vitro.

However, in most cases, the material is poorly identified in terms of taxonomy and the conservation methods used do not guarantee complete security for the resources. In vivo collections are not shielded from plant health risks (introduction of parasitic fungi or viruses and vectors of viral disease in greenhouses) or climate risks (storms, cyclones). Viral indexing has shown that 40% of the vanilla plants conserved in botanical gardens are infected by the Cymbidium mosaic virus (Grisoni et al., 2007). In vitro culture techniques for vanilla plants have been mastered (see Chapter 5) and ensure protection of plant material from contamination. However, in vitro conservation of collections requires regular maintenance operations that imply a large number of qualified workers. Furthermore, the succession of subcultures means that somaclonal variants may appear and the original genetic resources may be lost. For these reasons, methods to secure the collections must be developed as a matter of urgency. Cryopreservation could be an option for the future. In India, cryopreservation of pollen has been successfully conducted as a part of interspecies hybridization research. This technique solves the problem of the synchronization of flowering in different species. Likewise, a protocol for the cryopreservation of the apex of vanilla plants has been standardized for the storage of genetic resources (see Chapter 5). Protocols for the cryopreservation of meristems are being studied, particularly in Mexico (Gonzalez-Arnao et al., 2009) and France.

In Reunion Island the collection of vanilla genetic resources is the central element of the VATEL Biological Resource Centre, which was accredited in 2009. This recognition implies resource management that follows a quality process similar to the ISO 9001 international standard, and requires compliance with procedures on conservation technologies, the introduction of biological material, and the dissemination of genetic resources.

Rules on the Transfer of Vanilla Genetic Resources

Similar to the rest of the Orchid family, the genus Vanilla is protected by the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES), also known as the Washington Convention (http://www.cites.org/index. html). The aim of this international convention, signed by 175 countries, is to ensure that international trade in specimens of wild animals and plants does not endanger the survival of the species to which they belong. Indeed, although we can consider that cultivated vanilla is not endangered, almost all the species grow in natural forests located in high-risk zones. As the Orchid family is listed in Appendix 2 of the CITES convention, which includes over 28,000 plant species, vanilla is subject to the application of strict rules on the transfer of and trade in plant material between States (Figure 3.1). This protection concerns all parts of plants and all by-products, with a few exceptions, especially concerning fruits and their parts and products of cultivated vanilla.

However, point 6 of Article VII of the convention (“Exemptions and Other Special Provisions Relating to Trade”) facilitates exchanges of noncommercial plant material for scientific purposes.

Each member country of CITES must implement a legislation to guarantee compliance with the convention at the national level. Countries have the right to adopt more binding legislation.

The second level that regulates access to plant genetic resources in general and to vanilla in particular is the Convention on Biological Diversity (CBD), or Rio Convention (http://www.cbd.int/). At the level of the plant kingdom, the CBD covers all species that are not included in the International Treaty on Plant Genetic Resources for Food and Agriculture (http://www.planttreaty.org/index_en.htm). The CBD came into force on December 29, 1993. It has 191 parties (member countries) and its three main objectives are: to conserve biological diversity, to use biological diversity in a sustainable fashion and to share the benefits of biological diversity fairly and equitably.

Compliance with the rules for exchanging plant genetic resources between countries, set out in the Convention, translates in practical terms into the drafting and signing of an MTA (Material Transfer Agreement) by parties. This document defines, of a common accord, the rights and obligations of the parties concerned by the transfer, especially with regard to the issue of the use of resources and the sharing of benefits and advantages arising from this use. It now seems vital to use an MTA in any exchange of plant material in order to prevent disputes between suppliers and users of genetic resources.

Contrary to the CITES convention, there are no exemptions from the rules of the CBD for the use of plant genetic resources for scientific purposes.

The genetic resources held by a country prior to December 29, 1993 are not subject to the rules of the Convention.

Over and above the CBD and the rules on transfers, any exchange of plant material between countries must comply with the obligations of the International Plant Protection Convention (http://www.ippc.int/IPP/En/default.jsp), especially concerning the phytosanitary certificate that is required for any transfer of plant material. An initial guide was drawn up by the IBPGR, now Biodiversity International (Pearson et al., 1991), but the knowledge acquired over the last 15 years regarding vanilla viruses (see Chapter 7) calls for this guide to be updated.

Conclusion and Prospects

Similar to numerous other plant species, vanilla genetic resources are threatened with extinction or genetic erosion in many areas of origin and diversification. Any in situ initiatives for conserving species must therefore be encouraged, but the creation of ex situ collections is essential for protecting the diversity. This conservation method means resources are more secure, thanks to in vitro or cryopreservation mechanisms, and also makes it easier to promote resources and to acquire scientific data. Indeed, despite many research studies that have been made possible particularly by the widespread use of molecular biology tools, knowledge of vanilla genetics, in terms of the taxonomy of the genus and the properties of different species, is still incomplete. This knowledge gap is even more striking when it comes to usage and customs linked to the vanilla that are grown by local communities in regions where the genetic resources are found.

The creation of a global network of in situ and ex situ collections of genetic resources, based on branches on the three continents (America, Africa, and Asia) that hold most of these resources, could result in considerable progress in terms of the conservation and the scientific and economic improvement of vanilla. The development of increasingly effective genomics, associated with biotechnology techniques, means plant breeding programs can be set up. Exploiting the specific characteristics of wild species, such as resistance to drought in aphyllous vanilla, could provide a means of diversifying vanilla production areas and anticipating future climate change. The creation and development of vanilla plants that are more disease resistant, more productive, and richer in vanillin and other aromatic compounds could improve the living conditions of small-scale growers in vanilla production areas.

To ensure that these research studies and conservation network initiatives are more effective, plant material exchanges between collections and research programs should be facilitated, especially by relaxing the rules of the CBD in line with the existing exemption in the CITES convention.

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