Vanilla Species Research Articles

A genome-wide assessment of the genetic diversity, evolution and relationships with allied species of the clonally propagated crop Vanilla planifolia Jacks. ex Andrews

The Vanilla genus is a complex taxonomic group characterized by a vegetative reproduction mode combined with intra- and inter-specific hybridizations, and polyploidy events. These factors strongly impact the diversification of the genus and complicate the delimitation of taxa. Among the hundred Vanilla species, Vanilla planifolia Jacks. ex Andrews and Vanilla × tahitensis J. W. Moore are the main cultivated aromatic species. We applied Genotyping-by-Sequencing to explore the genetic diversity of these two cultivated vanilla species, seven closely related species and nineteen interspecific hybrids. The inter- and intra-specific relationships of 133 vanilla accessions were examined based on 2004 filtered SNPs. Our results showed a strong genetic structuring between the nine species studied, with wild species showing much lower heterozygosity levels than cultivated ones. Moreover, using Bayesian clustering analyses, the kinship of several hybrids could be verified. We evidenced in particular that Vanilla sotoarenasii and Vanilla odorata C.Presl may be the parental species of V. x tahitensis. The analysis of 1129 SNPs for 84 V. planifolia accessions showed a clear genetic demarcation between the vegetatively propagated traditional vanilla cultivars compared to the accessions derived from sexual reproduction, and a higher genetic diversity and lower heterozygosity of the latter (Ho = 0.206) compared to the former (Ho = 0.362). Our data are consistent with a single-step domestication for V. planifolia in accordance with the recent history of its cultivation. It also opens avenues to breed new V. planifolia varieties adapted to biotic and abiotic constraints and to reduce mutational load induced by clonal propagation.

Trick or treat? Pollinator attraction in Vanilla pompona (Orchidaceae)

AbstractNatural pollination of Vanilla species remains poorly understood. Our research aimed at better understanding the pollinator attraction mechanism of the Neotropical species Vanilla pompona. Based on our results, we hypothesize that the identified pollinator Eulaema cingulata is attracted via a dual mechanism combining floral fragrance rewards and food deception.Abstract in Spanish is available with online material.

Evolutionary disruption in the pollination system of Vanilla (Orchidaceae).

Pollination of the pantropical Vanilla has been linked to melittophily and food deception. Here we investigated the role of flower traits on the reproduction of Neotropical Vanilla. We also studied the evolution of pollination systems in order to understand the origin of production of flower resources and the diversification of pollinators in this orchid genus. Our study was founded on data of adaptations in flower morphology, production of resources, scent release, pollinators and breeding systems of Vanilla and presenting new data on reproductive biology of V. palmarum. Data on reproductive biology of Vanilla were mapped onto a phylogeny to address our queries on the evolution of pollination systems in this genus. Vanilla palmarum shows a mixed mating system, with its facultative autogamous flowers being pollinated by hummingbirds. Its yellow flowers are scentless and produces nectar. Mapping of the pollination system onto trees resulted in one origin for bird pollination and at least two origins for autogamy in Vanilla. Nectar secretion has a single origin in the Neotropical thick-leafed lineage. Bird pollination of Vanilla is shown for the first time. The origin of ornithophily within a bee-pollinated clade is supported by flower morphology. Floral transitions to ornithophily have been favoured by the occupation of a distinct niche from that of the other thick-leafed Vanilla species. Despite its specialized pollination, V. palmarum is autogamous. A mixed mating system can promote reproductive assurance in the case of a decline in pollinator populations, or in areas where pollinator services are irregular or absent.

Quantification of the aromatic potential of ripe fruit of Vanilla planifolia (Orchidaceae) and several of its closely and distantly related species and hybrids

Vanilla flavor is derived from the cured fruits of two orchid species Vanilla planifolia and V. × tahitensis. About twenty other Vanilla species are also credited with scented fruits, but very little is known about their aromatic features. We investigated the aromatic compound content of the ripe fruits of 30 vanilla accessions obtained from eight species and three hybrids. Glucovanillin plus six phenolic and one anisic aglycones, extracted after enzymatic hydrolysis, were quantified by High-Performance Liquid Chromatography and Ultra-Violet detection. The analysis of the aromatic contents and the principal components showed clear-cut differences in the aromatic content of the species and varieties studied. The total content of aromatic compounds separated the three species most distantly related to V. planifolia, namely V. lindmaniana, V. crenulata and V. imperialis, from the other species notably by the absence of vanillin potential and a very low total aromatic potential (< 0.45% w/w dry matter). A clear gradient was observed between the different genotypes of V. planifolia, in particular for the total aromatic content (range 3.07–5.00% dry weight) and the vanillin potential (1.80–4.58%). Among all the analyzed samples, the greatest vanillin potential was observed in a V. planifolia x V. × tahitensis hybrid (7.46%). Anisyl alcohol was not detected in any of the 13 V. planifolia varieties but was detected in most of its close relatives such as V. sotoarenasii, V. bahiana, V. tahitensis, and V. pompona. Our data evidenced the very contrasting aromatic profiles of vanilla fruits, even among very close genotypes.

Drivers of population divergence and species differentiation in a recent group of indigenous orchids (Vanilla spp.) in Madagascar.

With over 25,000 species, orchids are among families with remarkable high rate of diversification. Since Darwin's time, major advances attributed the exceptional diversity of orchids to plant–pollinator interactions. However, unraveling the processes and factors that determine the phenotypic and genotypic variation of natural orchid populations remains a challenge. Here, we assessed genetic population structure and floral differentiation in recently diverged leafless Vanilla species in a world biodiversity hotspot, Madagascar, using seven microsatellite loci and 26 morphometric variables. Additionally, analyses were performed to test for the occurrence of any patterns of isolation by distance, isolation by environment, and isolation by adaptation and to detect possible physical barriers that might have caused genetic discontinuities between populations. Positive inbreeding coefficients detected in 22 populations were probably due to the presence of null alleles, geitonogamy and/or some admixture (sympatric species). In contrast, the only high‐altitude population showed an important rate of clonality leading to heterozygote excess. Genetic diversity was maximum in western populations, suggesting a postglacial colonization to the north and south. Clustering analyses identified seven genetic groups characterized by specific floral traits that matched five botanical descriptions in the literature. A contribution of montane refugia and river barriers on population differentiation was detected. We also detected combined effects of IBD/IBE and IBE/IBA on genetic differentiation and suggested this pattern is more likely determined by ecological isolation, although pollinator‐mediated divergent selection could not be ruled out for some of the species. Overall, this study provides further insights on speciation in orchids, a group for which Madagascar shows one of the world's highest level of endemism and confirms the importance of the peculiar biogeography of the island in shaping species differentiation.

NMR-based leaf metabolic profiling of V. planifolia and three endemic Vanilla species from the Peruvian Amazon

The fruit of Vanilla planifolia is broadly preferred by the agroindustry and gourmet markets due to its refined flavor and aroma. Peruvian Vanilla has been proposed as a possible source for genetic improvement of existing Vanilla cultivars, but, little has been done to facilitate comprehensive studies of these and other Vanilla. Here, a nuclear magnetic resonance (NMR) metabolomic platform was developed to profile for the first time the leaves – organ known to accumulate vanillin putative precursors – of V. planifolia and those of Peruvian V. pompona, V. palmarum, and V. ribeiroi, with the aim to determine metabolic differences among them. Analysis of the NMR spectra allowed the identification of thirty-six metabolites, twenty-five of which were quantified. One-way ANOVA and post-hoc Tukey test revealed that these metabolites changed significantly among species, whilst multivariate-analyses allowed the identification of malic and homocitric acids, together with two vanillin precursors, as relevant metabolic markers for species differentiation.

A reappraisal of Neotropical Vanilla. With a note on taxonomic inflation and the importance of alpha taxonomy in biological studies

Despite the long-standing cultural importance and botanical interest in Vanilla, many taxa belonging to the genus remain poorly understood. Vanilla species generally have broad geographical and ecological distributions. Most species are found in multiple countries, while local endemics are rare. Many names proposed in the eighteen and nineteenth centuries remain cryptic and unused despite having priority over more recently proposed names. Relatively few Vanilla species have been well-documented, both locally and across their entire distribution range, while a significant portion of novelties have been proposed on the basis of very few specimens that are compared only with local floras. After a careful inspection of the type materials, living plants, botanical illustrations, photographs and hundreds of additional herbarium specimens of Vanilla we tentatively recognize 62 species for the Neotropics. The taxonomy of Vanilla columbiana, V. hartii, V. inodora, V. karenchristianae, V. marowynensis, V. mexicana, V. odorata, V. phaeantha, V. planifolia, and V. pompona is revised. An updated typification, description, photographs, illustrations, list of studied specimens, distribution map, extent of occurrence and discussion is provided for each of the ten species. Taxonomic proposals include 28 new synonyms, 14 lectotypifications, and one neotypification. We stress on the importance of alpha-taxonomy for biological studies, emphasizing on the detrimental effects of taxonomic inflation and incorrect species determination on the inference of speciation rates, the understanding of biogeographical patterns, the correct estimation of ecological niches, seed dispersal studies, phylogenetic and genomic studies, and the assessments of conservation priorities, among others. Finally, the recently proposed genus Miguelia is placed under the synonymy of Vanilla.&#x0D; Key Words: Conservation, distribution, Miguelia, typification, Vanilla columbiana, V. hartii, V. inodora, V. karen-christianae, V. marowynensis, V. mexicana, V. odorata, V. phaeantha, V. planifolia, V. pompona

A phased Vanilla planifolia genome enables genetic improvement of flavour and production.

The global supply of vanilla extract is primarily sourced from the cured beans of the tropical orchid species Vanilla planifolia. Vanilla plants were collected from Mesoamerica, clonally propagated and globally distributed as part of the early spice trade. Today, the global food and beverage industry depends on descendants of these original plants that have not generally benefited from genetic improvement. As a result, vanilla growers and processors struggle to meet global demand for vanilla extract and are challenged by inefficient and unsustainable production practices. Here, we report a chromosome-scale, phased V. planifolia genome, which reveals sequence variants for genes that may impact the vanillin pathway and therefore influence bean quality. Resequencing of related vanilla species, including the minor commercial species Vanilla × tahitensis, identified genes that could impact productivity and post-harvest losses through pod dehiscence, flower anatomy and disease resistance. The vanilla genome reported in this study may enable accelerated breeding of vanilla to improve high-value traits.

The Leafless Vanilla Species-Complex from the South-West Indian Ocean Region: A Taxonomic Puzzle and a Model for Orchid Evolution and Conservation Research

The pantropical genus Vanilla is a member of the Orchidaceae family, one of the largest and most diverse families of angiosperms in the world. It originated in America and differentiated in America, Africa, and Asia. About 132 species of Vanilla have now been accepted since the discovery of the genus. Within the Vanilla genus, leaflessness has appeared at least three independent times, in Africa, Asia, and America as an adaptation to drought conditions. The South-West Indian Ocean (SWIO) region, a biodiversity hotspot for orchids, is home to seven leafless species. Chloroplast markers failed to differentiate species within this recent SWIO clade. Morphological traits used for description are very similar between species and do not provide reliable identification. Moreover, some of the species have overlapping flowering periods and geographic distribution in Madagascar, increasing the probability of finding some sympatric species and, therefore, hybrids. Leafless Vanilla species from the SWIO islands are thus an excellent model for understanding the evolution of orchids. For their conservation, and to confirm the identity of these leafless species, an integrated approach with classical taxonomy using a large number of samples, intense fieldwork on biology and ecology, and molecular studies using variable markers is necessary.

English

Diversity assessment of vanilla (Vanilla species) in Kenya is a key strategy for germplasm conservation and improvement. Production of vanilla crop in Kenya is limited due to inadequate knowledge on genetic diversity. This study was carried out to characterize 76 vanilla accessions from five counties of Kenya using 14 microsatellite DNA markers. POPGENE version 1.32 was used to compute variety factors. Amplicons ranged between 1 and 4. A total of 27 (96.43%) alleles were observed and their number ranged from 1.00 to 2.00 with a mean of 1.93. Effective allele values ranged from 1.00 to 1.99 with a mean of 1.63. Gene diversity ranged from 0 to 0.50 with a mean of 0.35, mean Shannon information index was 0.50 and Polymorphic information content values ranged from 0 to 0.38 with a mean of 0.35. Jaccard’s similarity coefficient ranged from 0.08 to 1.00 with an average of 0.54. Unrooted phylogenetic tree was constructed in DARwin 6.0.8 using Unweighted Pair Group Method with Arithmetic Mean, clustering the samples into 3 main clusters (A 99.6%, B 98.96% and C 100%) and 6 sub-clusters (A1, A2, B1, B2, B3 and C1). Vanilla accessions grown in Kenya have a broad genetic background but low genetic diversity. Results inform the need to introduce other vanilla species as sources of genetic variation for breeding. Key words: Genetic variation, DNA markers, breeding, Kenya.

Vanilla distribution modeling for conservation and sustainable cultivation in a joint land sparing/sharing concept

AbstractVanilla, an expensive but popular spice used in many industries, faces problems related to its supply. Some of these problems are due to the fact that vanilla cultivation is based on clonal material of a single species (Vanilla planifolia) and is dominated by just a few countries located outside the native growing areas of aromatic vanilla species, which is the neotropics. Despite the economic importance of this crop, relatively little attention has been paid to its wild relatives, in particular with respect to their biology, ecology, and potential use. We hypothesized that species distribution models (SDMs) can identify suitable areas for both the conservation and cultivation of vanilla crop wild relatives (CWRs), following a joint land sparing/land sharing (SPASHA) approach, thus offering alternative sourcing areas and production methods. This is the first study that explored the use of ensemble SDMs to provide applicable land use maps related to the conservation and sustainable cultivation of wild vanilla species in Costa Rica, contributing to a solution for the problems related to current vanilla production systems. We focused on four aromatic vanilla CWRs, native to Costa Rica, to make land use policy recommendations for this country, and more specifically for the biological corridor Osa and its surroundings within our study region Área de Conservación Osa (ACOSA). The resulting distribution maps, with a mean AUC of 0.89, reflected their current potential distribution (ranging from unsuitable to suitable) in Costa Rica. Combining them with recent land use and conservation area maps of our study region, we defined (1) areas for vanilla conservation and (2) areas for sustainable vanilla cultivation within agroforestry systems. These land use recommendations can now be integrated within the National Bio‐Corridor Program (PNCB) that aims at making biological corridors more productive by proposing alternative income generation for local communities living within these areas. Our approach can be applied to identify priority areas for implementing the SPASHA approach on other vanilla CWRs and in more regions across its native growing ranges, given the availability of land use maps and enough occurrence records to build accurate SDMs.

Development of species-specific molecular markers in Vanilla for seedling selection of hybrids.

Vanilla planifolia is the primary botanical source of vanilla extract used globally in various foods and beverages. V. planifolia has a global distribution based on a few foundational clones and therefore has limited genetic diversity. Many Vanilla species easily hybridize with V. planifolia and could be a source of valuable genetic traits like increased vanillin content, disease resistance, or early flowering. While breeding Vanilla hybrids may improve plant performance, basic molecular tools for this species are lacking. DNA-based molecular markers are the most efficient method to validate hybrid progeny, detect hybrids in commercial plantings, and identify unknown accessions. This study used publicly available sequence data to develop species-specific, qRT-PCR-based molecular markers for Vanilla. Over 580,000 assembled sequence fragments were filtered for species specificity and twenty-two targets were selected for qRT-PCR screening. Ten targets differentially amplified among V. planifolia, V. pompona, V. phaeantha, and V. palmarum with ΔCT values as high as 17.58 between species. The ten targets were used to validate the parentage of hybrid progeny from controlled crosses with most hybrid progeny showing amplification patterns similar to both parents. The ten targets were also used to screen sixteen Vanilla species for specificity, and supported species assignments for unknown accessions including the detection of putative hybrids. This is the first report using species-specific, qRT-PCR-based molecular markers in Vanilla. These markers are inexpensive, simple to develop, and can rapidly screen large populations. These methods will enable the further development of species-specific molecular markers when creating Vanilla interspecific hybrid populations.

C and H stable isotope ratio analysis using solid-phase microextraction and gas chromatography-isotope ratio mass spectrometry for vanillin authentication

It is possible to distinguish precious vanillin from Vanilla species (planifolia or tahitensis) from much less expensive synthetic and nature-identical vanillin on the basis of the stable isotope ratios of H and C (2H/1H, 13C/12C). Analysis is usually performed using GC-IRMS (Gas Chromatography - Isotope Ratio Mass Spectrometry) after solvent extraction of vanillin from the sample. Recently, head-space solid-phase microextraction (HS-SPME) has been proposed as an alternative for determining 13C/12C.The aim of this study was to develop a method to analyse 2H/1H in vanillin using SPME-GC-IRMS for the first time, by testing different operating conditions and comparing the results with those obtained after solvent extraction. The ultimate scope was to develop a quick, robust and effective method to measure 2H/1H and 13C/12C in vanillin to assess the authenticity of labelling.Almost 50 authentic samples from vanilla pods, nature-identical (ex) and synthetic vanillin and 4 commercial food products were taken into account. All the samples were subjected to HS-SPME-GC-IRMS analysis and most of them to GC-IRMS analysis after solvent extraction of vanillin.The SPME method developed for 2H/1H analysis guarantees the absence of isotopic fractionation, repeatability and reproducibility standard deviation of below 7‰ and savings in terms of time (from 30 to 5 min) and solvent.HS-SPME GC-IRMS analysis of δ2H and δ13C can be proposed as a rapid and robust method to discriminate different types of vanillin and assess the authenticity of natural vanillin, also contained in food matrices.

Detection of Vanilla Species by Employing Image Processing Approach

Image analysis approach is adopted for identifying two species of vanilla; a popular spice yielding orchid. Vanilla species namely Vanilla planifolia (cultivated species) and Vanilla andamanica (wild species) were compared using image analysis of their leaves which is otherwise done using plant taxonomical methods, mainly flower morphology. Geometric features of the leaves of the two species were computed and four features were identified for distinguishing these two species. Support Vector Machine classifier was employed to get accurate results. The algorithm was implemented in OpenCV-Java. This facilitates porting the application to an embedded platform which is in progress. The approach is helpful for identifying these two species of Vanilla before flowering.

Wild Vanilla planifolia and its relatives in the Mexican Yucatan Peninsula: Systematic analyses with ISSR and ITS

Background: Vanilla planifolia , a threatened species, is distributed naturally in semi-deciduous and evergreen rain forests of southeastern Mexico and parts of Central America. In the wild, it is difficult to diagnose from another sympatric Vanilla species, and individuals with reproductive structures are usually required.&#x0D; Questions: Can ISSR discriminate wild individuals of Vanilla planifolia from other sympatric species of the genus? Can phylogenetic analyses of ITS recover the monophyly of V. planifolia and help identify Vanilla species?&#x0D; Studied species: The vanilla ( V. planifolia ), the second-most important economically spice in the world.&#x0D; Study site and years of study: Plant material was collected during 2014 in the Mexican Yucatan Peninsula.&#x0D; Methods: We analyzed 88 wild individuals of several Vanilla species. Individuals of five species of Vanilla reported for the region were used as reference. 84 ISSR loci were analyzed using three clustering methods. A phylogenetic inference using ITS was performed.&#x0D; Results: ISSR markers clearly discriminate wild Vanilla planifolia , finding definite genetic structure within the species. The three clustering methods identified genetic relationships with congruent patterns. Five groups were found and they corresponded with the species studied. Phylogenetic inference of ITS sequences supported the monophyly of the Vanilla and the resulting cladograms were coherent with the clustering pattern observed in the ISSR studies.&#x0D; Conclusions: Both ISSR and ITS analyses are able to identify V. planifolia . Molecular data suggest the presence of V. pompona and a new species of Vanilla in the Yucatan Peninsula.

A new vanilla species from Costa Rica closely related to V. planifolia (Orchidaceae)

We describe a new vanilla species growing in sympatry with Vanilla planifolia Jacks. ex Andrews (Orchidaceae) in the province of Limón, Caribbean coast of Costa Rica. The morphology of the reproductive and vegetative organs observed on vines cultivated under shade-house, the nuclear (Internal Transcribed Spacer) and plastid (matK) nucleotide sequences, as well as the contents of aromatic compounds measured in ripe fruits, show that this species is close to but distinct from V. planifolia. The name V. sotoarenasii M.Pignal, Azofeifa-Bolaños &amp; Grisoni sp. nov. is proposed for this new Vanilla species endemic in Costa Rica. It is especially distinguished from V. planifolia by a reduction of about 30% of the size of the fruits and flowers, by a divergence of ITS sequences for at least two species-conserved nucleotides compared to seven other species of the V. planifolia group, and by the presence of anisic compounds and low content of phenolic compounds (including vanillin) in the fruits. These results confirmed the extension of the area of distribution of V. planifolia southward to Costa Rica, where a recent speciation process occurred. Because of its particular agronomic and aromatic properties, V. sotoarenasii sp. nov. could represent a valuable biological resource for the vanilla industry.

Colombian vanilla and its microbiota. III. Diversity and structure of the endophytic community

Endophytic fungal communities are well-recognised entities within plants worldwide. They hold species with potential in medicinal affairs, biological control of pests, industry, and more. Nevertheless, ecological data about structure and dynamics of endophytic communities are scarce. In this study we sampled root, stem and leaf tissues of Colombian vanilla species in order of both, identifying endosymbionts and characterising the community they belong to. An interesting array of endophytic species was found, including taxa that function as pathogens, saprotrophs, and dermatophytes in other plants/scenarios. Ecological parameters show a moderate diversity with a lognormal arrangement of species quite similar to communities of macroorganisms. Many species here reported belong to taxa broadly reported as sources of biologically active compounds, so they are good candidates for bioprospecting research.

An integrated strategy for the conservation and sustainable use of native Vanilla species in Colombia

The natural vanilla essence is obtained principally from the fruits of the species Vanilla planifolia, a member of the Vanilla aromatic clade, and native to the neo-tropics. Colombia is an important center of diversity for the genus with 22 Vanilla species reported, of which 18 belong to the aromatic clade. Colombian native Vanilla species comprise important genetic resources for the vanilla crop. Although there is no tradition of cultivation of vanilla in the country, these species have potential for establishment in sustainable agroforestry systems. Nonetheless, Vanilla, like many orchid species, is subject to both intrinsic and extrinsic conservation threats. This article outlines an integrated strategy for conservation, incorporating in situ, ex situ and circa situm measures to ensure the conservation of Vanilla species in Colombia, and to promote their sustainable use in community-based cultivation programs. This proposed strategy is also relevant for conservation managers in other countries with native Vanilla species.

Combining pollination ecology and fine-scale spatial genetic structure analysis to unravel the reproductive strategy of an insular threatened orchid

In Vanilla species (Orchidaceae), the influence of sexual and asexual mating systems on the spatial structuring of population genetic diversity is understudied. These elements are crucial in restoration program to limit inbreeding depression and to maintain the genetic diversity of natural populations. In the remnant fragments of tropical dry forest in Mayotte (Comoros Archipelago, Indian Ocean), the clarification of the reproductive strategies of the orphan leafless Vanilla humblotii Rchb. f. will provide a better understanding of its fine-scale spatial genetic structure. Approaches combining reproductive biology and fine-scale spatial genetic structure analyses using ten microsatellite markers in 49 individuals sampled in the only remaining large population of V. humblotii were employed to unravel the reproductive strategies of this species. The results showed that V. humblotii displays unscented flowers and is allogamous and pollinator-dependent although also self-compatible. A total absence of pollen movements and a low level of natural fruit set (~1%) are reported, although a wild bee (Allodape obscuripennis Strand, Xylocopinae) and a bird (Nectarinia coquerelli, Nectarinidae) visited the flowers. A high genotypic diversity (G/N=0.88) and a phalanx clonal growth are detected, and seed dispersal is higher than pollen dispersal. The phalanx distribution of the repeated genotypes (ramets arisen from the same genet) is responsible for significant autocorrelations detected at small distances. Limited inbreeding was detected although geitonogamy could have been enhanced by vegetative reproduction. This study highlights the need to perform interdisciplinary studies to unravel the reproductive strategy of clonal plant species with a deceptive pollination system.

Protocols for Biotechnological Interventions in Improvement of Vanilla (Vanilla planifolia Andrews.).

Vanilla (Vanilla planifolia Andrews (syn. V. fragrans Salisb.), a native of Central America, is the primary source of natural vanillin and plays a major role in the global economy. The gene pool of vanilla is threatened by deforestation and overcollection that has resulted in disappearance of natural habitats and wild species. Continuous vegetative propagation and lack of natural seed set and sufficient variations in the gene pool hamper crop improvement programs. In vitro techniques, one of the key tools of plant biotechnology, can be employed for overcoming specific problems, viz. production of disease-free clones, inducing somaclonal variations, developing hybrids, gene pool conservation, incorporating desired traits by distant hybridization, genetic engineering, etc. However, realization of these objectives necessitates standardization of protocols. This chapter describes the various protocols optimized for crop improvement in Vanilla species.