Blue Light Mediates Chloroplast Avoidance and Enhances Photoprotection of Vanilla Orchid.

Vanilla orchid, which is well-known for its flavor and fragrance, is cultivated in tropical and subtropical regions. This shade-loving plant is very sensitive to high irradiance. In this study, we show that vanilla chloroplasts started to have avoidance movement when blue light (BL) was higher than 20 µmol m-2s-1 and significant avoidance movement was observed under BL irradiation at 100 µmol m-2s-1 (BL100). The light response curve indicated that when vanilla was exposed to 1000 µmol m-2s-1, the electron transport rate (ETR) and photochemical quenching of fluorescence (qP) were significantly reduced to a negligible amount. We found that if a vanilla orchid was irradiated with BL100 for 12 days, it acquired BL-acclimation. Chloroplasts moved to the side of cells in order to reduce light-harvesting antenna size, and chloroplast photodamage was eliminated. Therefore, BL-acclimation enhanced vanilla orchid growth and tolerance to moderate (500 µmol m-2s-1) and high light (1000 µmol m-2s-1) stress conditions. It was found that under high irradiation, BL-acclimatized vanilla maintained higher ETR and qP capacity than the control without BL-acclimation. BL-acclimation induced antioxidant enzyme activities, reduced ROS accumulation, and accumulated more carbohydrates. Moreover, BL-acclimatized orchids upregulated photosystem-II-associated marker genes (D1 and PetC), Rubisco and PEPC transcripts and sustained expression levels thereof, and also maximized the photosynthesis rate. Consequently, BL-acclimatized orchids had higher biomass. In short, this study found that acclimating vanilla orchid with BL before transplantation to the field might eliminate photoinhibition and enhance vanilla growth and production.

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.

Efecto de la interacción luz-agua sobre la fotosíntesis de la Vanilla planifolia (Orchidaceae) / Light-water interaction effect on photosynthesis of the Vanilla planifolia (Orchidaceae)

Introducción: La vainilla (Vanilla planifolia Andrews) es una planta CAM de gran importancia económica en el mercado mundial de saborizantes y aromatizantes. Por ser una planta hemiepífita que inicialmente crece en el sotobosque sombreado y posteriormente asciende hasta el dosel, se aclimata a diferentes condiciones de radiación y humedad. La posibilidad de extender su cultivo a zonas secas donde sea menor la incidencia de enfermedades, dependerá de su respuesta a períodos prolongados de sequía. Objetivo: Evaluar los efectos de la interacción entre la radiación y la humedad sobre los parámetros de la fotosíntesis y funcionamiento del CAM de plantas de vainilla en sus fases iniciales de desarrollo vegetativo, y determinar si las condiciones de radiación en las que crece la planta pueden ayudar a moderar los efectos negativos de la sequía. Métodos: Se evaluaron plantas de vainilla que crecieron con riego durante 18 meses en casetas con iluminación relativa (IR) de 8, 17, 31 y 67 %; posteriormente se suspendió el riego y se fueron deshidratando durante 94 días hasta alcanzar un contenido de humedad crítico (contenido hídrico relativo, CHR ≤ 50 %), y finalmente se reinició el riego durante 22 días; se evaluó el efecto de estos procesos sobre la asimilación de CO2, la acidez titulable y la eficiencia cuántica del Fotosistema II (Fv/Fm). Resultados: Bajo condiciones intermedias de IR (17 % y 31 %), la asimilación de CO2 y laacumulación nocturna de ácidos orgánicos presentaron la menor afectación por el déficit hídrico, así como la mejor recuperación luego de la rehidratación. El CHR se vio más afectado por la disponibilidad de agua en el tejido vegetal, mientras que Fv/Fm lo fue por el ambiente de radiación. Los efectos combinados de estrés hídrico y alta radiación ocasionaron daños irreversibles en la fotosíntesis para el tratamiento de IR de 67 %. Conclusiones: En las plantas de vainilla, el impacto negativo de la sequía sobre los parámetros de la fotosíntesis fue mayor en alta radiación; sin embargo, en condiciones de baja radiación también aumentó la susceptibilidad de las plantas a la sequía, en comparación con los ambientes de radiación intermedia, en los cuales la densidad de flujo de fotones media fue de 340 μmol m-2 seg-1 (17 % de IR) y 620 μmol m-2 seg-1 (31 % de IR). Estos resultados sugieren el potencial de cultivar vainilla en zonas con sequía estacional bajo sistemas productivos de baja tecnificación, manteniendo estas condiciones de radiación.

Introduction: Vanilla planifolia Andrews is a CAM plant of economic importance in the global market of flavorings and essences. Being a hemiepiphyte which initially grows in the shady understory and later climbs to the canopy, it acclimates to different conditions of radiation and humidity. The possibility of extending this crop to dry areas where the incidence of diseases is lower will depend on the response to prolonged periods of drought. Objective: To evaluate the effects of the interaction of radiation and humidity on the parameters of photosynthesis and operation of CAM of vanilla plants, and to determine if the radiation environment could help to moderate the negative effects of drought. Methods: We evaluated well-watered vanilla plants grown for 18 months in relative illumination (RI) of 8, 17, 31 and 67 %, which underwent dehydration for 94 days until reaching critical water content (relative water content, RWC < 50 %) and then rehydration for 22 days; variables evaluated were: CO2 assimilation, titratable acidity, and quantum efficiency of Photosystem II (Fv / Fm). Results: Under intermediate conditions of radiation (17 % and 31 % RI), CO2 assimilation and nocturnal accumulation of organic acids were less affected by water deficit and also showed the best recovery after rehydration. The RWC was most affected by the water available in plant tissue, while Fv / Fm was affected by radiation. The combined effects of water stress and high radiation caused irreversible damage to photosynthesis for the 67 % RI treatment. Conclusions: In vanilla plants, the negative impact of drought on photosynthetic parameters was greater in high radiation; however, in low radiation conditions the susceptibility of plants to drought also increased, as compared to intermediate radiation environments, which were under a mean photon flux density of 340 µmol m-2 s-1 (17 % IR) and 620 µmol m-2 s-1 (31 % IR). These results suggest the potential for growing vanilla in areas with seasonal drought under low-tech production systems, maintaining these radiation conditions.

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.

Different continuous cropping spans significantly affect microbial community membership and structure in a vanilla-grown soil as revealed by deep pyrosequencing.

In the present study, soil bacterial and fungal communities across vanilla continuous cropping time-series fields were assessed through deep pyrosequencing of 16S ribosomal RNA (rRNA) genes and internal transcribed spacer (ITS) regions. The results demonstrated that the long-term monoculture of vanilla significantly altered soil microbial communities. Soil fungal diversity index increased with consecutive cropping years, whereas soil bacterial diversity was relatively stable. Bray-Curtis dissimilarity cluster and UniFrac-weighted principal coordinate analysis (PCoA) revealed that monoculture time was the major determinant for fungal community structure, but not for bacterial community structure. The relative abundances (RAs) of the Firmicutes, Actinobacteria, Bacteroidetes, and Basidiomycota phyla were depleted along the years of vanilla monoculture. Pearson correlations at the phyla level demonstrated that Actinobacteria, Armatimonadetes, Bacteroidetes, Verrucomicrobia, and Firmicutes had significant negative correlations with vanilla disease index (DI), while no significant correlation for fungal phyla was observed. In addition, the amount of the pathogen Fusarium oxysporum accumulated with increasing years and was significantly positively correlated with vanilla DI. By contrast, the abundance of beneficial bacteria, including Bradyrhizobium and Bacillus, significantly decreased over time. In sum, soil weakness and vanilla stem wilt disease after long-term continuous cropping can be attributed to the alteration of the soil microbial community membership and structure, i.e., the reduction of the beneficial microbes and the accumulation of the fungal pathogen.

A deep transcriptomic analysis of pod development in the vanilla orchid (Vanilla planifolia).

BACKGROUND: Pods of the vanilla orchid (Vanilla planifolia) accumulate large amounts of the flavor compound vanillin (3-methoxy, 4-hydroxy-benzaldehyde) as a glucoside during the later stages of their development. At earlier stages, the developing seeds within the pod synthesize a novel lignin polymer, catechyl (C) lignin, in their coats. Genomic resources for determining the biosynthetic routes to these compounds and other flavor components in V. planifolia are currently limited. RESULTS: Using next-generation sequencing technologies, we have generated very large gene sequence datasets from vanilla pods at different times of development, and representing different tissue types, including the seeds, hairs, placental and mesocarp tissues. This developmental series was chosen as being the most informative for interrogation of pathways of vanillin and C-lignin biosynthesis in the pod and seed, respectively. The combined 454/Illumina RNA-seq platforms provide both deep sequence coverage and high quality de novo transcriptome assembly for this non-model crop species. CONCLUSIONS: The annotated sequence data provide a foundation for understanding multiple aspects of the biochemistry and development of the vanilla bean, as exemplified by the identification of candidate genes involved in lignin biosynthesis. Our transcriptome data indicate that C-lignin formation in the seed coat involves coordinate expression of monolignol biosynthetic genes with the exception of those encoding the caffeoyl coenzyme A 3-O-methyltransferase for conversion of caffeoyl to feruloyl moieties. This database provides a general resource for further studies on this important flavor species.

Expression profiles of key phenylpropanoid genes during Vanilla planifolia pod development reveal a positive correlation between PAL gene expression and vanillin biosynthesis.

In Vanilla planifolia pods, development of flavor precursors is dependent on the phenylpropanoid pathway. The distinctive vanilla aroma is produced by numerous phenolic compounds of which vanillin is the most important. Because of the economic importance of vanilla, vanillin biosynthetic pathways have been extensively studied but agreement has not yet been reached on the processes leading to its accumulation. In order to explore the transcriptional control exerted on these pathways, five key phenylpropanoid genes expressed during pod development were identified and their mRNA accumulation profiles were evaluated during pod development and maturation using quantitative real-time PCR. As a prerequisite for expression analysis using qRT-PCR, five potential reference genes were tested, and two genes encoding Actin and EF1 were shown to be the most stable reference genes for accurate normalization during pod development. For the first time, genes encoding a phenylalanine ammonia-lyase (VpPAL1) and a cinnamate 4-hydroxylase (VpC4H1) were identified in vanilla pods and studied during maturation. Among phenylpropanoid genes, differential regulation was observed from 3 to 8 months after pollination. VpPAL1 was gradually up-regulated, reaching the maximum expression level at maturity. In contrast, genes encoding 4HBS, C4H, OMT2 and OMT3 did not show significant increase in expression levels after the fourth month post-pollination. Expression profiling of these key phenylpropanoid genes is also discussed in light of accumulation patterns for key phenolic compounds. Interestingly, VpPAL1 gene expression was shown to be positively correlated to maturation and vanillin accumulation.

Combined inoculation of Pseudomonas fluorescens and Trichoderma harzianum for enhancing plant growth of vanilla (Vanilla planifolia).

This study was conducted to evaluate the plant growth promoting efficiency of combined inoculation of rhizobacteria on Vanilla plants. Based on the in vitro performance of indigenous Trichoderma spp. and Pseudomonas spp., four effective antagonists were selected and screened under greenhouse experiment for their growth enhancement potential. The maximum percentage of growth enhancement were observed in the combination of Trichoderma harzianum with Pseudomonas fluorescens treatment followed by Pseudomonas fluorescens, Trichoderma harzianum, Pseudomonas putida and Trichoderma virens, respectively in decreasing order. Combined inoculation of Trichoderma harzianum and Pseudomonas fluorescens registered the maximum length of vine (82.88 cm), highest number of leaves (26.67/plant), recorded the highest fresh weight of shoots (61.54 g plant(-1)), fresh weight of roots (4.46 g plant(-1)) and dry weight of shoot (4.56 g plant(-1)) where as the highest dry weight of roots (2.0806 g plant(-1)) were achieved with treatments of Pseudomonas fluorescens. Among the inoculated strains, combined inoculation of Trichoderma harzianum and Pseudomonas fluorescens recorded the maximum nitrogen uptake (61.28 mg plant(-1)) followed by the combined inoculation of Trichoderma harzianum (std) and Pseudomonas fluorescens (std) (55.03 mg plant(-1)) and the highest phosphorus uptake (38.80 mg plant(-1)) was recorded in dual inoculation of Trichoderma harzianum and Pseudomonas fluorescens.

Metabolome of Vanilla planifolia (Orchidaceae) and related species under Cymbidium mosaic virus (CymMV) infection.

The genus Vanilla which belongs to the Orchidaceae family comprises more than 110 species of which two are commercially cultivated (Vanilla planifolia and Vanilla xtahitensis). The cured pods of these species are the source of natural vanilla flavor. In intensive cultivation systems the vines are threatened by viruses such as Cymbidium mosaic virus (CymMV). In order to investigate the effect of CymMV on the growth and metabolome of vanilla plants, four accessions grown in intensive cultivation systems under shadehouse, CR01 (V. planifolia), CR17 (V. xtahitensis), CR03 (V. planifolia × V. xtahitensis) and CR18 (Vanilla pompona), were challenged with an isolate of CymMV. CymMV infected plants of CR01, CR03 and CR17 had a reduced growth compared to healthy plants, while there was no significant difference in the growth of CR18 vines. Interestingly, CR18 had qualitatively more phenolic compounds in leaves and a virus titre that diminished over time. No differences in the metabolomic profiles of the shadehouse samples obtained by nuclear magnetic resonance (NMR) were observed between the virus infected vs. healthy plants. However, using in- vitro V. planifolia plants, the metabolomic profiles were affected by virus infection. Under these controlled conditions the levels of amino acids and sugars present in the leaves were increased in CymMV infected plants, compared to uninfected ones, whereas the levels of phenolic compounds and malic acid were decreased. The metabolism, growth and viral status of V. pompona accession CR18 contrasted from that of the other species suggesting the existence of partial resistance to CymMV in the vanilla germplasm.

Shoot differentiation from protocorm callus cultures of Vanilla planifolia (Orchidaceae): proteomic and metabolic responses at early stage.

BACKGROUND: Vanilla planifolia is an important Orchid commercially cultivated for the production of natural vanilla flavour. Vanilla plants are conventionally propagated by stem cuttings and thus causing injury to the mother plants. Regeneration and in vitro mass multiplication are proposed as an alternative to minimize damage to mother plants. Because mass production of V. planifolia through indirect shoot differentiation from callus culture is rare and may be a successful use of in vitro techniques for producing somaclonal variants, we have established a novel protocol for the regeneration of vanilla plants and investigated the initial biochemical and molecular mechanisms that trigger shoot organogenesis from embryogenic/organogenic callus. RESULTS: For embryogenic callus induction, seeds obtained from 7-month-old green pods of V. planifolia were inoculated on MS basal medium (BM) containing TDZ (0.5 mg l(-1)). Germination of unorganized mass callus such as protocorm -like structure (PLS) arising from each seed has been observed. The primary embryogenic calli have been formed after transferring on BM containing IAA (0.5 mg l(-1)) and TDZ (0.5 mg l(-1)). These calli were maintained by subculturing on BM containing IAA (0.5 mg l(-1)) and TDZ (0.3 mg l(-1)) during 6 months and formed embryogenic/organogenic calli. Histological analysis showed that shoot organogenesis was induced between 15 and 20 days after embryogenic/organogenic calli were transferred onto MS basal medium with NAA (0.5 mg l(-1)). By associating proteomics and metabolomics analyses, the biochemical and molecular markers responsible for shoot induction have been studied in 15-day-old calli at the stage where no differentiating part was visible on organogenic calli. Two-dimensional electrophoresis followed by matrix-assisted laser desorption ionization time-of-flight-tandem mass spectrometry (MALDI-TOF-TOF-MS) analysis revealed that 15 protein spots are significantly expressed (P < 0.05) at earlier stages of shoot differentiation. The majority of these proteins are involved in amino acid-protein metabolism and photosynthetic activity. In accordance with proteomic analysis, metabolic profiling using 1D and 2D NMR techniques showed the importance of numerous compounds related with sugar mobilization and nitrogen metabolism. NMR analysis techniques also allowed the identification of some secondary metabolites such as phenolic compounds whose accumulation was enhanced during shoot differentiation. CONCLUSION: The subculture of embryogenic/organogenic calli onto shoot differentiation medium triggers the stimulation of cell metabolism principally at three levels namely (i) initiation of photosynthesis, glycolysis and phenolic compounds synthesis; (ii) amino acid-protein synthesis, and protein stabilization; (iii) sugar degradation. These biochemical mechanisms associated with the initiation of shoot formation during protocorm-like body (PLB) organogenesis could be coordinated by the removal of TDZ in callus maintenance medium. These results might contribute to elucidate the complex mechanism that leads to vanilla callus differentiation and subsequent shoot formation into PLB organogenesis. Moreover, our results highlight an early intermediate metabolic event in vanillin biosynthetic pathway with respect to secondary metabolism. Indeed, for the first time in vanilla tissue culture, phenolic compounds such as glucoside A and glucoside B were identified. The degradation of these compounds in specialized tissue (i.e. young green beans) probably contributes to the biosynthesis of glucovanillin, the parent compound of vanillin.

Biological variation of Vanilla planifolia leaf metabolome.

The metabolomic analysis of Vanilla planifolia leaves collected at different developmental stages was carried out using (1)H-nuclear magnetic resonance (NMR) spectroscopy and multivariate data analysis in order to evaluate their variation. Ontogenic changes of the metabolome were considered since leaves of different ages were collected at two different times of the day and in two different seasons. Principal component analysis (PCA) and partial least square modeling discriminate analysis (PLS-DA) of (1)H NMR data provided a clear separation according to leaf age, time of the day and season of collection. Young leaves were found to have higher levels of glucose, bis[4-(beta-D-glucopyranosyloxy)-benzyl]-2-isopropyltartrate (glucoside A) and bis[4-(beta-D-glucopyranosyloxy)-benzyl]-2-(2-butyl)-tartrate (glucoside B), whereas older leaves had more sucrose, acetic acid, homocitric acid and malic acid. Results obtained from PLS-DA analysis showed that leaves collected in March 2008 had higher levels of glucosides A and B as compared to those collected in August 2007. However, the relative standard deviation (RSD) exhibited by the individual values of glucosides A and B showed that those compounds vary more according to their developmental stage (50%) than to the time of day or the season in which they were collected (19%). Although morphological variations of the V. planifolia accessions were observed, no clear separation of the accessions was determined from the analysis of the NMR spectra. The results obtained in this study, show that this method based on the use of (1)H NMR spectroscopy in combination with multivariate analysis has a great potential for further applications in the study of vanilla leaf metabolome.

Metabolic changes in different developmental stages of Vanilla planifolia pods.

The metabolomic analysis of developing Vanilla planifolia green pods (between 3 and 8 months after pollination) was carried out by nuclear magnetic resonance (NMR) spectroscopy and multivariate data analysis. Multivariate data analysis of the (1)H NMR spectra, such as principal component analysis (PCA) and partial least-squares-discriminant analysis (PLS-DA), showed a trend of separation of those samples based on the metabolites present in the methanol/water (1:1) extract. Older pods had a higher content of glucovanillin, vanillin, p-hydroxybenzaldehyde glucoside, p-hydroxybenzaldehyde, and sucrose, while younger pods had more bis[4-(beta-D-glucopyranosyloxy)-benzyl]-2-isopropyltartrate (glucoside A), bis[4-(beta-D-glucopyranosyloxy)-benzyl]-2-(2-butyl)tartrate (glucoside B), glucose, malic acid, and homocitric acid. A liquid chromatography-mass spectrometry (LC-MS) analysis targeted at phenolic compound content was also performed on the developing pods and confirmed the NMR results. Ratios of aglycones/glucosides were estimated and thus allowed for detection of more minor metabolites in the green vanilla pods. Quantification of compounds based on both LC-MS and NMR analyses showed that free vanillin can reach 24% of the total vanillin content after 8 months of development in the vanilla green pods.

Micropropagation and in vitro conservation of vanilla (Vanilla planifolia Andrews).

Vanilla (Vanilla planifolia Andrews (syn. V. fragrans Salisb.), a source of natural vanillin, plays a major positive role in the economy of several countries. A native to the Central America, its primary gene pool is threatened by deforestation and over collection that has resulted in disappearance of natural habitats and wild species. Therefore, multiplication and conservation of vanilla diversity is of paramount importance because of its narrow genetic base. It plays an important role in the production of disease free planting material for commercial cultivation. Simple protocols for micropropagation, in vitro conservation and synthetic seed production are described in this chapter which could further be applied to other related vanilla species as well.

Mycorrhizal fungi of Vanilla: diversity, specificity and effects on seed germination and plant growth.

Mycorrhizal fungi are essential for the germination of orchid seeds. However, the specificity of orchids for their mycorrhizal fungi and the effects of the fungi on orchid growth are controversial. Mycorrhizal fungi have been studied in some temperate and tropical, epiphytic orchids, but the symbionts of tropical, terrestrial orchids are still unknown. Here we study diversity, specificity and function of mycorrhizal fungi in Vanilla, a pantropical genus that is both terrestrial and epiphytic. Mycorrhizal roots were collected from four Vanilla species in Puerto Rico, Costa Rica and Cuba. Cultured and uncultured mycorrhizal fungi were identified by sequencing the internal transcribed spacer region of nuclear rDNA (nrITS) and part of the mitochondrial ribosomal large subunit (mtLSU), and by counting number of nuclei in hyphae. Vanilla spp. were associated with a wide range of mycorrhizal fungi: Ceratobasidium, Thanatephorus and Tulasnella. Related fungi were found in different species of Vanilla, although at different relative frequencies. Ceratobasidium was more common in roots in soil and Tulasnella was more common in roots on tree bark, but several clades of fungi included strains from both substrates. Relative frequencies of genera of mycorrhizal fungi differed significantly between cultured fungi and those detected by direct amplification. Ceratobasidium and Tulasnella were tested for effects on seed germination of Vanilla and effects on growth of Vanilla and Dendrobium plants. We found significant differences among fungi in effects on seed germination and plant growth. Effects of mycorrhizal fungi on Vanilla and Dendrobium were similar: a clade of Ceratobasidium had a consistently positive effect on plant growth and seed germination. This clade has potential use in germination and propagation of orchids. Results confirmed that a single orchid species can be associated with several mycorrhizal fungi with different functional consequences for the plant.

Genetic fidelity of long-term micropropagated shoot cultures of vanilla (Vanilla planifolia Andrews) as assessed by molecular markers.

Occurrence of genetic variants during micropropagation is occasionally encountered when the cultures are maintained in vitro for long period. Therefore, the micropropagated multiple shoots of Vanilla planifolia Andrews developed from axillary bud explants established 10 years ago were used to determine somaclonal variation using random amplified polymorphic DNA (RAPD) and intersimple sequence repeats markers (ISSR). One thousand micro-plants were established in soil of which 95 plantlets (consisting of four phenotypes) along with the mother plant were subjected to genetic analyses using RAPD and ISSR markers. Out of the 45 RAPD and 20 ISSR primers screened, 30 RAPD and 7 ISSR primers showed 317 clear, distinct and reproducible band classes resulting in a total of 30 115 bands. However, no difference was observed in banding patterns of any of the samples for a particular primer, indicating the absence of variation among the micropropagated plants. Our results allow us to conclude that the micropropagation protocol that we have used for in vitro proliferation of vanilla plantlets for the last 10 years might be applicable for the production of clonal plants over a considerable period of time.