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Comparative Study
. 2018 Nov 26;13(11):e0207763.
doi: 10.1371/journal.pone.0207763. eCollection 2018.

Product Authenticity Versus globalisation-The Tulsi Case

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Free PMC article
Comparative Study

Product Authenticity Versus globalisation-The Tulsi Case

Gabriele Jürges et al. PLoS One. .
Free PMC article

Abstract

Using the Indian medicinal plant Tulsi (Holy Basil) as a case study, we have tested to what extent the discrepancy between vernacular and scientific nomenclature can be resolved, whether the presumed chemical diversity underlying the medicinal use of Tulsi has a genetic component, and whether it is possible to detect this genetic component using genetic barcoding markers. Based on four plastidic markers, we can define several haplotypes within Ocimum that are consistent across these markers. Haplotype II is congruent with O. tenuiflorum, while haplotype I extends over several members of the genus and cannot be resolved into genetically separate subclades. The vernacular subdivision of Tulsi into three types (Rama, Krishna, Vana) can only be partially linked with genetic differences-whereby Rama and Krishna Tulsi can be assigned to O. tenuiflorum, while Vana Tulsi belongs to haplotype I. This genetic difference is mirrored by differences in the profiles of secondary compounds. While developmental state and light quality modulate the amplitude to which the chemical profile is expressed, the profile itself seems to be linked with genetic differences. We finally develop an authentication assay that makes use of a characteristic single nucleotide polymorphism in one of the barcoding markers, establishing a differential restriction pattern that can be used to discriminate Vana Tulsi.

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Representative specimens of Ocimum accessions used in the current study in the flowering stage.
The upper row shows the inflorescence, the lower row shows close-ups of individual flowers with details of morphology. Size bar 10 mm.
Fig 2
Fig 2. Molecular relationships inferred from the RubisCo large subunit (rbcL) marker.
A Evolutionary relationship of 65 sequences for the rbcL marker from Ocimum, along with one sequence from Nepeta cataria and Hanceola sinensis, respectively, used as outgroups inferred using the Neighbour-Joining algorithm. Bootstrap values are derived from 1000 replicates. O bas O. basilicum, O americ O. americanum, O x afric O. x africanum, O kilim O. kilimandscharicum, O tenuifl O. tenuiflorum, O campech O. campechianum, O fil O. filamentosum, O grat O. gratissimum. GenBank accession numbers are shown with each accession, circles represent sequences that had been isolated in the course of the current study, red circle represents the sequence for ‘Vana Tulsi’ that clusters outside of O. tenuiflorum. Blue arrows indicate two accessions that have been reported as ‘Krishna Tulsi’ (both as O. tenuiflorum), green arrows indicate two accessions that have been reported as ‘Vana Tulsi’. Roman numbers represent four main haplotypes. B Signatures of the four main haplotypes defined by the rbcL marker, numbers indicate the nucleotide position in the alignment (S1 Appendix). Support is defined as the number of sequences deposited in GenBank that show this signature. Note that the sequence of ‘Vana Tulsi’ falls into cluster I comprising O. basilicum, O. americanum, O. x africanum, and O. kilimandscharicum.
Fig 3
Fig 3. Molecular relationships inferred from the informative positions in a composite alignment of rbcL, matK, trnF-L intergenic spacer, and the trnH-psbA intergenic spacer based on the 79 informative positions in this composite alignment.
The inset shows the flower morphology of the different clades. The colour code for upper (circles) and lower (triangles) lips is green long, yellow intermediate, red short. Haplotypes that deviate from the consensus for the respective clade (b basilicum/americanum/x africanum/kilimandscharicum clade, g gratissimum clade, t tenuiflorum clade) are indicated by numbers).
Fig 4
Fig 4. Chemical profiling of different Ocimum accessions raised side by side in the greenhouse under white light over six months.
Flavonoids (A) and essential oils (B) were probed by HT-TLC along with reference standards (in case of flavonoids, rutin and hyperoside, in case of essential oils methyl eugenol and ursolic acid). Arrows indicate type-specific bands, green arrow indicates a prominent band in the flavonoid band that is characteristic for accessions belonging to haplotype I in Fig 3, while red arrow indicates a flavonoid band that is characteristic for accessions belonging to haplotype II and IV accessions in Fig 3.
Fig 5
Fig 5. Effect of light quality on chemical profiles and glandular scale number in Ocimum tenuiflorum, ‘Rama Tulsi’, ‘Krishna Tulsi’, and ‘Vana Tulsi’ accessions raised side by side in the greenhouse under equal fluence rates (108 μM.sec-1.m-2 ± 30 μM.sec-1.m-2) of either white light (WL), white light supplemented with red and blue light (655 nm, 447 nm, RB), long-wavelength UV (365 nm, UVA) or short-wavelength UV (310 nm, UVB) over six months.
Flavonoids (A) and essential oils (B) were probed by HT-TLC along with reference standards (in case of flavonoids, rutin and hyperoside, in case of essential oils methyl eugenol and ursolic acid). Profiles for one accession are shown side by side, indicated by colour symbols (RB, red-blue squares, WL white squares, UVB dark-violet squares, UVA light-violet squares). C mean densities of glandular scales on the upper, adaxial, and the lower, abaxial, side of fully expanded leaves given in relative units. Data represent mean values and standard errors from two independent experimental series comprising each three individual plants per accession and ten leaves per plant for each genotype.
Fig 6
Fig 6. Discrimination of haplotype I and haplotypes II-IV using restriction length polymorphism of the trnH-psbA IGS marker.
A Sequence polymorphism between the haplotypes leading to a differential Hinf I recognition site in case of haplotypes II-IV. B Prediction of the fragment size obtained by Hinf I restriction. C Predicted banding pattern for haplotype I and haplotypes II-IV. D restriction patterns obtained after Hinf I digestion of trnH-psbA IGS amplicons in different accessions of Ocimum. Sample 1 and 2 are two commercial samples declaring to contain ‘Rama’, ‘Krishna’, and ‘Vana’ Tulsi.

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References

    1. Page JE, Balza F, Nishida T, Towers GHN. Biologically active diterpenes from Aspilia mossambicensis, a chimpanzee medicinal plant. Phytochemistry. 1992; 10: 3437–3439. - PubMed
    1. Berlin B, Breedlove DE, Raven PH. Folk Taxonomies and Biological Classification. Science. 1966. 254: 273–275 - PubMed
    1. European Commission. Regulation (EC) No 258/97 of the European Parliament and of the Council of 27 January 1997 concerning novel foods and novel food ingredients. Official Journal of the European Communities. 1997. L43(14/02/97) http://ec.europa.eu/food/safety/novel_food/catalogue_en
    1. Ayerza R Jr., Coates W. Chia Rediscovering a Forgotten Crop of the Aztecs 2005. The University of Arizona Press
    1. Horn T, Häser A. Bamboo tea: reduction of taxonomic complexity and application of DNA diagnostics based on rbcL and matK sequence data. Peer Journal. 2016. 4: 2781. - PMC - PubMed

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Grant support

This work was not specifically funded except a Ph.D. fellowship to Daniela Rios Rodriguez from the German Academic Exchange Service (91643072). This funding organisation had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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