Seven new species of Rinorea (Violaceae) from the Neotropics.

Over the course of revising the genus Rinorea (Violaceae) from Colombia, field observations and herbarium studies revealed seven new species. Several of the new species described here belong to species complexes that required examination of herbarium material from across the Neotropics. Each of the new species described here have oppositely arranged leaves and belong to Rinoreasect.Pubiflorae, a section restricted to the Neotropics. Two new species are segregated from the R.ovalifolia species complex: Rinoreachiribiquetensis from Chiribiquete National Park in the Colombian Amazon and Rinoreastevensii from the Orinoco River near the border of Colombia and Venezuela. Two new species are segregated from the Rinoreahirsuta species complex: Rinoreagaleanoae-bernalii and Rinoreacogolloi, both from the eastern slopes of the Andean Central Cordillera along the mid-Magdalena River Valley in Colombia. From the widely distributed R.pubiflora species complex, we segregated one new species, Rinoreacallejasii, from southeast Panama and the Chocó in Colombia. In addition to these five new taxa segregated from widely distributed species complexes, we discovered two previously unknown species with affinities to other Neotropical Rinorea. Rinoreaaymardii is described from the Alto Orinoco-Casiquiare Biosphere Reserve in Venezuela and most closely resembles R.melanodonta from Colombia. Rinoreabetancurii is segregated from R.macrocarpa and occurs in the Amazonian Regions of Colombia, Brazil, Peru and Venezuela. In this study, we provide descriptions, illustrations and distribution maps of the new species and make preliminary assessments of the risk of extinction using the IUCN Red List Categories and Criteria. We also furnish an identification key to the species of Rinoreasect.Pubiflorae in Colombia.

A complete chloroplast genome sequence of Viola albida Palibin 1899 (Violaceae), a member of VIOLA ALBIDA complex.

The VIOLA ALBIDA complex is a complicated group with taxonomic problems having continuous leaf variations and composed of taxa related to the following names: Viola albida, V. albida var. takahashii, and V. chaerophylloides. As a first step to understanding the genomic nature of this complex, this study identified the whole chloroplast genome of V. albida. The genome is 157,692 bp in length (36.3% of GC content) and contains four subregions: a large single copy region of 86,220 bp, a small single copy region of 17,248 bp, and a pair of inverted regions of 27,112 bp each. An annotation of the gene identifies 111 unique genes, including 77 protein-coding genes, four rRNA genes, and 30 tRNA genes. The phylogenetic analysis of this genome with selected cp genomes from Viola identifies the close relationship between V. albida and V. ulleungdoensis. It is noteworthy that V. chaerophylloides, traditionally recognized as a member of the VIOLA ALBIDA complex, is genetically distant from V. albida and forms a sister group of all other members of the subsection Patellares. Our genome report is expected to serve as a basis for understanding the identity of the VIOLA ALBIDA complex.

A Revised Phylogenetic Classification for Viola (Violaceae).

The genus Viola (Violaceae) is among the 40-50 largest genera among angiosperms, yet its taxonomy has not been revised for nearly a century. In the most recent revision, by Wilhelm Becker in 1925, the then-known 400 species were distributed among 14 sections and numerous unranked groups. Here, we provide an updated, comprehensive classification of the genus, based on data from phylogeny, morphology, chromosome counts, and ploidy, and based on modern principles of monophyly. The revision is presented as an annotated global checklist of accepted species of Viola, an updated multigene phylogenetic network and an ITS phylogeny with denser taxon sampling, a brief summary of the taxonomic changes from Becker's classification and their justification, a morphological binary key to the accepted subgenera, sections and subsections, and an account of each infrageneric subdivision with justifications for delimitation and rank including a description, a list of apomorphies, molecular phylogenies where possible or relevant, a distribution map, and a list of included species. We distribute the 664 species accepted by us into 2 subgenera, 31 sections, and 20 subsections. We erect one new subgenus of Viola (subg. Neoandinium, a replacement name for the illegitimate subg. Andinium), six new sections (sect. Abyssinium, sect. Himalayum, sect. Melvio, sect. Nematocaulon, sect. Spathulidium, sect. Xanthidium), and seven new subsections (subsect. Australasiaticae, subsect. Bulbosae, subsect. Clausenianae, subsect. Cleistogamae, subsect. Dispares, subsect. Formosanae, subsect. Pseudorupestres). Evolution within the genus is discussed in light of biogeography, the fossil record, morphology, and particular traits. Viola is among very few temperate and widespread genera that originated in South America. The biggest identified knowledge gaps for Viola concern the South American taxa, for which basic knowledge from phylogeny, chromosome counts, and fossil data is virtually absent. Viola has also never been subject to comprehensive anatomical study. Studies into seed anatomy and morphology are required to understand the fossil record of the genus.

A chloroplast genome sequence of Viola arcuata distributed in Korea.

Recently, the chloroplast genome of Viola verecunda from a sample collected in Japan has been published. Although the name is often recognized as a taxonomic synonym of Viola arcuata, the genetic identity of the two species has never been compared intensively. We report the complete chloroplast genome sequence of V. arcuata, of which sample was collected from Seoul, Korea. The cp genome of V. arcuata (OM301625) has 157,870 bp in length and is composed of four regions: 86,366 bp of a large single-copy (LSC) region, 17,298 bp of a small single-copy (SSC) region, and 27,103 bp of a pair of inverted repeats (IRs). The complete genome contains 130 genes, including 84 protein-coding genes, eight rRNA genes, and 37 tRNA genes. When comparing chloroplast genomes between V. verecunda, and V. arcuata, 34 different loci were recognized: 12 SNPs and 22 indels. In the coding regions, there were two amino acid insertions (ndhI) caused by one base deletion, three synonymous substitutions (ndhF, ccsA, and ndhI), and six nonsynonymous substitutions (matK, rpoC2, ndhF, ycf1, and two rpl2s on each IR region). In non-coding regions, variants of 19 polyN sites, one microsatellite, two insertions, and two SNPs were recognized. Phylogenetic analysis confirms a sister or nearly identical relationship between two genomes. This study will provide the genetic basis for solving a taxonomic problem between V. arcuata and V. verecunda.

Anchietins A-E: 30-norfriedelane-type triterpenes from Anchietea pyrifolia.

Five undescribed norfriedelane triterpenoids, anchietins A-E, along with three known norfriedelane triterpenoids, 21ß-hydroxycaloncobalactone, and welwitschiilactones B and C, were isolated from the vine of Anchietia pyrifolia. The compounds were characterized by spectroscopic and crystallographic methods, including 2D NMR spectroscopy and single crystal X-ray crystallography. The isolated compounds were evaluated for the cytotoxic activity against HeLa and HL60 cells and for the inhibitory activity against lipopolysaccharide-induced NO production. Further, inhibitory effects on the inducible nitric oxide synthase mRNA expression levels were also assessed.

Megastigmane sesquiterpenoids from whole plants of Viola kunawurensis.

Investigation on the chemical constituents of Viola kunawurensis resulted in the isolation of seven undescribed megastigmane sesquiterpenoids including four bicyclic megastigmane glucosides, kunawuronoside A-D, two megastigmane glucosides, kunawuronoside E-F, and a megastigmane, kunawurone A, together with ten known megastigmane sesquiterpenoids. Their structures were established by comprehensive 1D, 2D-NMR and HRESIMS analyses, and their absolute configurations were determined by comparing their calculated ECD data with the experimental ones. Evaluations of the anti-inflammatory activity revealed that kunawuronoside A-D and compounds 14-15 inhibited COX-2 expression with inhibition rates ranging from 36.7% to 58.5%, while the NO production induced by lipopolysaccharide (LPS) was suppressed by the kunawuronoside A-D in a dose-dependent manner in RAW264.7 macrophage cell line.

Rapid evolution of selfing syndrome traits in Viola arvensis revealed by resurrection ecology.

PREMISE: As part of global change, climate warming and pollinator decline are expected to affect plant phenology and plant-pollinator interactions. This paper aims at characterizing rapid evolution of life history traits and floral traits over two decades in the wild pansy (Viola arvensis), a common weed in agrosystems. METHODS: We used a resurrection ecology approach with genotypes sampled in 1991 and 2012 from a population in Burgundy (France). The species has a mixed mating system (hereafter: mixed selfer) and presents a floral polymorphism. To correct for maternal effects, we measured plant traits in the second generation in a common garden (after a refreshing generation) to characterize plant evolution during the two decades. In addition, historical population selfing rates in 1991 and 2012 were inferred from microsatellites markers through heterozygote deficiency and identity disequilibrium. RESULTS: Phenotypic data revealed a significant advance in flowering date, reduced flower sizes and a higher propensity of plants to set seed by autonomous selfing. Moreover, we detected a change in color morph frequency with an increase of the pale morph frequency. In accordance with phenotypic data, the neutral genetic data revealed an increase in historical selfing rates from 0.68 in 1991 to 0.86 in 2012. CONCLUSIONS: Taken together, such data suggest that the wild pansy, a mixed selfer, is evolving a selfing syndrome that may be the consequence of reduced pollinator activity in agrosystems.

The complete plastid genome sequence of Viola selkirkii Pursh ex Goldie (Violaceae).

Viola selkirkii, belonging to the genus Viola, has heart-shaped leaves and pale purple flowers, and it is widely distributed in the Northern Hemisphere, including Europe, North America, and Asia. In this study, the plastid genome of V. selkirkii was sequenced and phylogenetic analysis was performed on 11 Viola plastid genomes. The length of the plastid genome length of V. selkirkii was 156,774 bp, and it was identified as having a typical quadripartite structure with a large single-copy region (85,930 bp), a small single-copy region (17,982 bp), and two inverted repeat regions (26,431 bp each). A phylogenetic analysis was conducted with 77 protein-coding genes from the complete plastid genomes of 11 Viola and nine Salicaceae species; the complete plastid genome of Erythroxylum novogranatense was used as an outgroup. Viola formed a monophyletic clade, and V. selkirkii was closely related to V. ulleungdoensis. These results contribute to the clear identification of the phylogenetic position of V. selkirkii in Viola.

Climate change is associated with increased allocation to potential outcrossing in a common mixed mating species.

PREMISE: Although the balance between cross- and self-fertilization is driven by the environment, no long-term study has documented whether anthropogenic climate change is affecting reproductive strategy allocation in species with mixed mating systems. Here, we test whether the common blue violet (Viola sororia; Violaceae) has altered relative allocation to the production of potentially outcrossing flowers as the climate has changed throughout the 20th century. METHODS: Using herbarium records spanning from 1875 to 2015 from the central United States, we quantified production of obligately selfing cleistogamous (CL) flowers and potentially outcrossing chasmogamous (CH) flowers by V. sororia, coupled these records with historic temperature and precipitation data, and tested whether changes to the proportion of CL flowers correlate with temporal climate trends. RESULTS: We find that V. sororia progressively produced lower proportions of CL flowers across the past century and in environments with lower mean annual temperature and higher total annual precipitation. We also find that both CL and CH flower phenology has advanced across this time period. CONCLUSIONS: Our results suggest that V. sororia has responded to lower temperatures and greater water availability by shifting reproductive strategy allocation away from selfing and toward potential outcrossing. This provides the first long-term study of how climate change may affect relative allocation to potential outcrossing in species with mixed mating systems. By revealing that CL flowering is associated with low water availability and high temperature, our results suggest the production of obligately selfing flowers is favored in water limited environments.

Effects of temperature on seed dormancy and germination of the coastal dune plant Viola grayi: germination phenology and responses to winter warming.

PREMISE: In temperate sand dunes, rising air temperature from climate change could not only further elevate surface soil temperatures during summers, but also drastically change the range of soil temperatures in other seasons. Winter warming may shift the timing of seed germination of dune species that require cold stratification for dormancy release. METHODS: We assessed the effects of temperature on dormancy and germination of Viola grayi seeds and evaluated whether winter warming could affect its germination phenology by conducting germination experiments and analyzing soil temperature data in cold and warm winters. RESULTS: Viola grayi seeds were dormant when dispersed in spring. One-month moist-chilling treatment (4°C) effectively released dormancy, while short, intermittent lower temperatures (alternating 20°/5°C) did not. Continuous higher temperatures induced secondary dormancy in nondormant seeds. During a cold, snowy winter, the surface soil temperatures of the sand dune remained at 0°-2°C for approximately 1 month owing to the accumulated snow, while the period of such stable low soil temperatures was much shorter during a warm, less-snowy winter, and the highest soil temperature reached 20°-25°C. These results suggest that dispersed seeds germinate in the following spring after winter chilling, but they may remain dormant after warm winters. CONCLUSIONS: With winter warming, seed dormancy of V. grayi seeds could be prolonged and the associated germination delayed. Assessing the minimum requirements for dormancy release and the potential to form persistent soil seed banks is important for judging the necessity and urgency of conservation efforts for temperate dune species.

Comparative analysis of cyclotide-producing plant cell suspensions presents opportunities for cyclotide plant molecular farming.

Cyclotides are a class of ribosomally-synthesized plant peptides that function in plants as a defense against insects and fungal pathogens. Their unique structure comprises a cyclized peptide backbone threaded by three disulfide bonds, that imparts structural stability, a desirable quality for peptide-based therapeutics or insecticides. Producing these peptides synthetically is challenging due to the amount of chemical waste produced and inefficiency of folding certain cyclotides. Thus, it is desirable to develop a means to access cyclotide biosynthesis in their native hosts, cultured in defined conditions, at both laboratory and commercial scale. Here we developed suspension cell cultures from two species previously unexplored for cyclotide production in suspension cells, Clitoria ternatea L., Hybanthus enneaspermus F. Muell., as well as with Oldenlandia affinis (Roem. & Schult.) DC., a species reported previously to accumulate cyclotides in cell suspensions. We assessed the growth rate, cyclotide production and gene expression for the various species. We found that while many cyclotides had reduced expression in Oldenlandia affinis suspension cells when compared to plant organs, those in Clitoria ternatea and Hybanthus enneaspermus maintained or increased expression levels. The cyclotides that continued to be expressed in suspension cultures shared similar sequence and biophysical properties as a group, regardless of phylogenetic origin of the host. Of particular interest was the discovery of inducibility by NaCl of cyclotide expression in O. affinis, cycloviolacin O2 expression in O. affinis, and the scale up of cycloviolacin O2 production in H. enneaspermus. Together the results presented here highlight the utility of plant cell suspensions as modalities to produce macrocyclic peptides.

The complete chloroplast genome sequence of Viola verecunda (Violaceae).

Viola verecunda is a perennial plant native to the mountainous areas of Northeast Asia. Here the complete chloroplast genome of V. verecunda and its phylogenetic relationships to other species within the genus Viola are reported. The complete chloroplast genome of V. verecunda is 157,843 bp in length and circular in structure with four regions: a large single-copy region (86,345 bp), a small single-copy region (17,292 bp), and a pair of inverted repeat regions (27,103 bp each). The chloroplast genome contains 111 unique genes comprising 77 protein-coding, 30 unique tRNA, and 4 unique rRNA genes. Based on the protein-coding gene sequences from eight Viola chloroplast genomes, with Balanops balansae designated as the outgroup, maximum likelihood tree analysis indicates that V. verecunda is more closely related to V. raddeana than to other Viola species. The complete chloroplast genome of V. verecunda contributes to a better understanding of the phylogenetic relationships among Viola species.

Selective forces on the maintenance of outcrossing in an almost exclusively cleistogamous violet species.

PREMISE: Cleistogamous species constitute interesting study systems to resolve the longstanding question of how outcrossing is maintained given that seed production is ensured through selfing. In this work, we investigate the selective forces that allow the persistence of producing self-pollinated cleistogamous (CL) and chasmogamous (CH) flowers in Viola jaubertiana Marès & Vigin. METHODS: We monitored three populations at different elevation for two years, and studied the flowering phenology and the relative contribution of each flower morph to parental fitness. We tested whether allocation to CH and CL flowers differed across populations and if it covaried with herbivory and water stress conditions. We also performed hand-pollination and bagging experiments in CH flowers to estimate inbreeding depression and heterosis. RESULTS: The CH flowers open in winter under unfavorable conditions for pollination, show high pollen limitation and no-delayed selfing, and thus produce a low amount of seeds. Conversely, CL flowers appear in early spring, are physiologically cheaper to produce (i.e., dry weight is 3.4 times lower than that of CH flowers), and yield approximately 100 times more seeds than CH flowers. The CH flowers were favored under water stress and low herbivory. Crosses between populations showed up to 25% greater fitness than those within populations. CONCLUSIONS: Despite the great pollen limitation in CH flowers, we suggest that the interaction among different environmental determinants and heterosis are probably sufficient forces to maintain chasmogamy in this long-lived species, reducing deleterious fixed mutations in the selfed lines.

Tropical vibes from Sri Lanka - cyclotides from Viola betonicifolia by transcriptome and mass spectrometry analysis.

Cyclotides are an extremely stable class of peptides, ubiquitously distributed in Violaceae. The aim of the present study was to investigate the presence of cyclotides in Sri Lankan Violaceae plants, using combined tools of transcriptomics and mass spectrometry. New cyclotides were discovered for the first time in the wild flora of Sri Lanka, within Viola betonicifolia, a plant used in traditional medicine as an antimicrobial. Plant extracts prepared in small scale from Viola betonicifolia were first subjected to LC-MS analysis. Subsequent transcriptome de novo sequencing of Viola betonicifolia uncovered 25 new (vibe 1-25) and three known (varv A/kalata S, viba 17, viba 11) peptide sequences from Möbius and bracelet cyclotide subfamilies as well as hybrid cyclotides. Among the transcripts, putative linear acyclotide sequences (vibe 4, vibe 10, vibe 11 and vibe 22) that lack a conserved asparagine or aspartic acid vital for cyclisation were also present. Four asparagine endopeptidases (AEPs), VbAEP1-4 were found within the Viola betonicifolia transcriptome, including a peptide asparaginyl ligase (PAL), potentially involved in cyclotide backbone cyclisation, showing >93% sequence homology to Viola yedoensis peptide asparaginyl ligases, VyPALs. In addition, we identified two protein disulfide isomerases (PDIs), VbPDI1-2, likely involved in cyclotide oxidative folding, having high sequence homology (>74%) with previously reported Rubiaceae and Violaceae PDIs. The current study highlights the ubiquity of cyclotides in Violaceae as well as the utility of transcriptomic analysis for cyclotides and their putative processing enzyme discovery. The high variability of cyclotide sequences in terms of loop sizes and residues in V. betonicifolia showcase the cyclotide structure as an adaptable scaffold as well as their importance as a combinatorial library, implicated in plant defense.

The complete chloroplast genome of Viola philippica (Violaceae).

Viola philippica, as a traditional Chinese medicine, has great value in treating various diseases. Here, we report the chloroplast genome of V. philippica and its phylogenetic feature. The complete chloroplast genome is 156,744 bp in length, assembled from 22,346,570 reads, and its GC contents ratio is 36.26%. Its long single-copy (LSC) region is 85,892 bp. The small single-copy (SSC) region covers 18,006 bp and inverted repeat (IR) is 26,423 bp. It encodes 77 genes, including 43 protein genes, 4 rRNA genes, and 30 tRNA genes. Moreover, according to the phylogenetic analysis for a total of 12 chloroplast sequences, V. philippica demonstrated close relationship within genus Viola.

Comparison of glucosinolate diversity in the crucifer tribe Cardamineae and the remaining order Brassicales highlights repetitive evolutionary loss and gain of biosynthetic steps.

We review glucosinolate (GSL) diversity and analyze phylogeny in the crucifer tribe Cardamineae as well as selected species from Brassicaceae (tribe Brassiceae) and Resedaceae. Some GSLs occur widely, while there is a scattered distribution of many less common GSLs, tentatively sorted into three classes: ancient, intermediate and more recently evolved. The number of conclusively identified GSLs in the tribe (53 GSLs) constitute 60% of all GSLs known with certainty from any plant (89 GSLs) and apparently unique GSLs in the tribe constitute 10 of those GSLs conclusively identified (19%). Intraspecific, qualitative GSL polymorphism is known from at least four species in the tribe. The most ancient GSL biosynthesis in Brassicales probably involved biosynthesis from Phe, Val, Leu, Ile and possibly Trp, and hydroxylation at the ß-position. From a broad comparison of families in Brassicales and tribes in Brassicaceae, we estimate that a common ancestor of the tribe Cardamineae and the family Brassicaceae exhibited GSL biosynthesis from Phe, Val, Ile, Leu, possibly Tyr, Trp and homoPhe (ancient GSLs), as well as homologs of Met and possibly homoIle (intermediate age GSLs). From the comparison of phylogeny and GSL diversity, we also suggest that hydroxylation and subsequent methylation of indole GSLs and usual modifications of Met-derived GSLs (formation of sulfinyls, sulfonyls and alkenyls) occur due to conserved biochemical mechanisms and was present in a common ancestor of the family. Apparent loss of homologs of Met as biosynthetic precursors was deduced in the entire genus Barbarea and was frequent in Cardamine (e.g. C. pratensis, C. diphylla, C. concatenata, possibly C. amara). The loss was often associated with appearance of significant levels of unique or rare GSLs as well as recapitulation of ancient types of GSLs. Biosynthetic traits interpreted as de novo evolution included hydroxylation at rare positions, acylation at the thioglucose and use of dihomoIle and possibly homoIle as biosynthetic precursors. Biochemical aspects of the deduced evolution are discussed and testable hypotheses proposed. Biosyntheses from Val, Leu, Ile, Phe, Trp, homoPhe and homologs of Met are increasingly well understood, while GSL biosynthesis from mono- and dihomoIle is poorly understood. Overall, interpretation of known diversity suggests that evolution of GSL biosynthesis often seems to recapitulate ancient biosynthesis. In contrast, unprecedented GSL biosynthetic innovation seems to be rare.

The complete chloroplast genome sequence of Viola prionantha (Violaceae).

Viola Prionantha belongs to the family Violaceae. It has been widely used for a traditional Chinese herb with antibacterial activity and is grown as an early spring ornamental species in China. In this study, we determined the complete chloroplast genome sequence of V. prionantha which forms a circular structure. The whole chloroplast genome was 156,501 bp in length, consisting of a pair of inverted repeats (IR) of 26,404 bp, a large single-copy (LSC) region of 85,689 bp, and a small single-copy (SSC) region of 18,004 bp. We annotated 131 genes, including 84 coding sequences, 8 rRNA sequences, 37 tRNA sequences and 2 pesudogenes. Among the annotated genes, 17 genes contained one or two introns. Furthermore, a phylogenetic analysis revealed that V. prionantha and V. seoulensis clustered together as sisters to other Violaceae species.

The complete chloroplast genome sequence of Viola prionantha (Violaceae).

Viola prionantha belongs to the family Violaceae. It has been widely used for a traditional Chinese herb in China. We determined the complete chloroplast genome sequence of V. prionantha. The whole chloroplast genome was 156,501 bp in length, consisting of a pair of inverted repeats (IR) of 26,404 bp, each, a large single-copy (LSC) region of 85,689 bp, and a small single-copy (SSC) region of 18,004 bp. We annotated 131 genes, including 84 coding sequences, 8 rRNA sequences, 37 tRNA sequences, and 2 pesudogenes. Among the annotated genes Phylogenetic analysis revealed that V. prionantha and Viola seoulensis clustered together as sisters.

The mechanism of neuroprotective effect of Viola odorata against serum/glucose deprivation-induced PC12 cell death.

OBJECTIVE: Oxidative stress is associated with the pathogenesis of brain ischemia and other neurodegenerative disorders. Previous researches have shown the antioxidant activity of Viola odorata L. In this project, we studied neuro-protective and reactive oxygen species (ROS) scavenging activities of methanol (MeOH) extract and other fractions isolated from V. odorata in PC12 cell line in serum/glucose deprivation (SGD) condition. MATERIALS AND METHODS: The PC12 neuronal cells were pretreated for 6 hr with MeOH extract and fractions of V. odorata (1 to 25 µg/ml) followed by 24 hr incubation under SGD condition. Cell viability was measured by Alamar Blue® assay. The level of ROS was calculated using DCFH-DA. Also, Bax/Bcl-2 protein ratio was analyzed by western blot assay. RESULTS: SGD condition significantly decreased cells viability (p<0.001). Pretreatment with EtOAc (12.5 and 25 µg/ml), BuOH (12, 25, 50 µg/ml) and CH2Cl2 (1.5 µg/ml) fractions of V. odorata reduced SGD-induced cytotoxicity. MeOH extract could not increase the viability significantly. All four semi polar fractions (EtOAc, BuOH, CH2Cl2 and MeOH) decreased SGD-induced ROS production and changed Bax/Bcl-2 ratio. CONCLUSION: V. odorata showed promising effects against SGD condition; further mechanistic and clinical studies are warranted before application of V. odorata as a neuro-protective agent.

Production of bioactive cyclotides in somatic embryos of Viola odorata.

Viola odorata L. (Violaceae), an Indian medicinal plant, contains a plethora of cyclotides, which are a class of cyclic peptides derived from plants, possessing several applications. Somatic embryo culture of V. odorata was developed, via indirect somatic embryogenesis, to serve as an alternative to natural plant biomass for sustainable and continuous production of its bioactive ingredients, such as cyclotides. Among the various combinations of phytohormones tested, Murashige and Skoog medium supplemented with 1 mg/l thidiazuron gave rise to the maximum frequency of induction (86.7%) and a high number of somatic embryos (3) from an embryogenic callus. Identification and characterization of cyclotides in the somatic embryos were carried out using a Fourier transform mass spectrometer coupled with liquid chromatography (LC-FTMS). Among the cyclotides identified in the study, few were found to be exclusively present in the somatic embryo culture. Furthermore, the relative abundance of the cyclotides was higher in somatic embryo extract than in the natural plant extract. The biological activities (cytotoxic, haemolytic and antimicrobial) of the somatic embryos and the parent plant were compared. Unlike the natural plants, the somatic embryo extracts demonstrated specificity i.e. they were found to be potent against cancerous cells but not against non-cancerous cell line or red blood cells. In contrast to the plant extract, the somatic embryos extracts were found to be potent against Escherichia coli and Staphylococcus aureus. These results suggest that somatic embryos of V. odorata (rich in cyclotides) can be used as an alternative to plant biomass for its therapeutic applications and germplasm conservation.