research on tulsi plant

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The Clinical Efficacy and Safety of Tulsi in Humans: A Systematic Review of the Literature

Affiliation.

1 School of Health and Biomedical Sciences, RMIT University, Melbourne, VIC, Australia.
PMID: 28400848
PMCID: PMC5376420
DOI: 10.1155/2017/9217567

Tulsi, also known as holy basil, is indigenous to the Indian continent and highly revered for its medicinal uses within the Ayurvedic and Siddha medical systems. Many in vitro, animal and human studies attest to tulsi having multiple therapeutic actions including adaptogenic, antimicrobial, anti-inflammatory, cardioprotective, and immunomodulatory effects, yet to date there are no systematic reviews of human research on tulsi's clinical efficacy and safety. We conducted a comprehensive literature review of human studies that reported on a clinical outcome after ingestion of tulsi. We searched for studies published in books, theses, conference proceedings, and electronic databases including Cochrane Library, Google Scholar, Embase, Medline, PubMed, Science Direct, and Indian Medical databases. A total of 24 studies were identified that reported therapeutic effects on metabolic disorders, cardiovascular disease, immunity, and neurocognition. All studies reported favourable clinical outcomes with no studies reporting any significant adverse events. The reviewed studies reinforce traditional uses and suggest tulsi is an effective treatment for lifestyle-related chronic diseases including diabetes, metabolic syndrome, and psychological stress. Further studies are required to explore mechanisms of action, clarify the dosage and dose form, and determine the populations most likely to benefit from tulsi's therapeutic effects.

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Selection and micropropagation of an elite melatonin rich tulsi ( ocimum sanctum l.) germplasm line.

1. Introduction

2. materials and methods, 2.1. plant materials and germplasm line development, 2.2. determination of antioxidant potential using dpph bioassay, 2.3. culture establishment and propagation, 2.4. shoot multiplication, 2.5. rooting and acclimatization in the greenhouse, 2.6. folin-ciocalteu phenolic assay, 2.7. detection and quantification of neurotransmitters, 2.8. statistical analysis, 4. discussion, supplementary materials, author contributions, data availability statement, acknowledgments, conflicts of interest.

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Click here to enlarge figure

Compound	Transition	Cone Voltage	Collision Voltage
Serotonin	177 > 160	45	10
Serotonin	177 > 115	45	27
Melatonin	233 > 159	30	23
Melatonin	233 > 174	30	15

Source	Melatonin (ng/g)	Serotonin (ng/g)
Wildtype	42.8 ± 4.95 b	1684.7 ± 82.04 a
Vrinda	66.4 ± 16.5 a	1305.1 ± 139.43 b

Source/Plant Parts	Leaf		Root
Source/Plant Parts	MEL (ng/g)	SER (ng/g)	MEL (ng/g)	SER (ng/g)
Field	327.17 a	685.71 a	365.05 a	605.26 a
Greenhouse	341.83 a	497.95 a	378.64 a	501.79 a

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Shukla, M.R.; Kibler, A.; Turi, C.E.; Erland, L.A.E.; Sullivan, J.A.; Murch, S.J.; Saxena, P.K. Selection and Micropropagation of an Elite Melatonin Rich Tulsi ( Ocimum sanctum L.) Germplasm Line. Agronomy 2021 , 11 , 207. https://doi.org/10.3390/agronomy11020207

Shukla MR, Kibler A, Turi CE, Erland LAE, Sullivan JA, Murch SJ, Saxena PK. Selection and Micropropagation of an Elite Melatonin Rich Tulsi ( Ocimum sanctum L.) Germplasm Line. Agronomy . 2021; 11(2):207. https://doi.org/10.3390/agronomy11020207

Shukla, Mukund R., Annaliese Kibler, Christina E. Turi, Lauren A. E. Erland, J. Alan Sullivan, Susan J. Murch, and Praveen K. Saxena. 2021. "Selection and Micropropagation of an Elite Melatonin Rich Tulsi ( Ocimum sanctum L.) Germplasm Line" Agronomy 11, no. 2: 207. https://doi.org/10.3390/agronomy11020207

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Research article
Open access
Published: 28 August 2015

Genome sequencing of herb Tulsi ( Ocimum tenuiflorum ) unravels key genes behind its strong medicinal properties

Atul K. Upadhyay 1 ,
Anita R. Chacko 1 ,
A. Gandhimathi 1 ,
Pritha Ghosh 1 ,
K. Harini 1 ,
Agnel P. Joseph 1 ,
Adwait G. Joshi 1 , 3 ,
Snehal D. Karpe 1 ,
Swati Kaushik 1 ,
Nagesh Kuravadi 2 ,
Chandana S Lingu 2 ,
J. Mahita 1 ,
Ramya Malarini 2 ,
Sony Malhotra 1 ,
Manoharan Malini 1 ,
Oommen K. Mathew 1 , 4 ,
Eshita Mutt 1 ,
Mahantesha Naika 1 ,
Sathyanarayanan Nitish 1 ,
Shaik Naseer Pasha 1 , 3 ,
Upadhyayula S. Raghavender 1 ,
Anantharamanan Rajamani 2 ,
S Shilpa 2 ,
Prashant N. Shingate 1 , 3 ,
Heikham Russiachand Singh 2 ,
Anshul Sukhwal 1 , 4 ,
Margaret S. Sunitha 1 ,
Manojkumar Sumathi 2 ,
S. Ramaswamy 2 ,
Malali Gowda 2 &
Ramanathan Sowdhamini 1

BMC Plant Biology volume 15 , Article number: 212 ( 2015 ) Cite this article

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Krishna Tulsi, a member of Lamiaceae family, is a herb well known for its spiritual, religious and medicinal importance in India. The common name of this plant is ‘Tulsi’ (or ‘Tulasi’ or ‘Thulasi’) and is considered sacred by Hindus. We present the draft genome of Ocimum tenuiflurum L (subtype Krishna Tulsi) in this report. The paired-end and mate-pair sequence libraries were generated for the whole genome sequenced with the Illumina Hiseq 1000, resulting in an assembled genome of 374 Mb, with a genome coverage of 61 % (612 Mb estimated genome size). We have also studied transcriptomes (RNA-Seq) of two subtypes of O. tenuiflorum, Krishna and Rama Tulsi and report the relative expression of genes in both the varieties.

The pathways leading to the production of medicinally-important specialized metabolites have been studied in detail, in relation to similar pathways in Arabidopsis thaliana and other plants. Expression levels of anthocyanin biosynthesis-related genes in leaf samples of Krishna Tulsi were observed to be relatively high, explaining the purple colouration of Krishna Tulsi leaves. The expression of six important genes identified from genome data were validated by performing q-RT-PCR in different tissues of five different species, which shows the high extent of urosolic acid-producing genes in young leaves of the Rama subtype . In addition, the presence of eugenol and ursolic acid, implied as potential drugs in the cure of many diseases including cancer was confirmed using mass spectrometry.

Conclusions

The availability of the whole genome of O.tenuiflorum and our sequence analysis suggests that small amino acid changes at the functional sites of genes involved in metabolite synthesis pathways confer special medicinal properties to this herb.

Plants of the genus Ocimum belong to the family Lamiaceae (Order Lamiales) and are widely distributed in the tropical, sub-tropical and warm temperate regions of the world [ 1 ]. These plants are known to produce essential oils comprising of a number of aromatic compounds and Tulsi is rightly known as the “Queen of Herbs” for this reason. In India, these plants are mostly grown at homes for worship and as offerings in temples. Among plants with medicinal value, those belonging to the genus Ocimum are very important aromatic herbs or shrubs.

The genus Ocimum is highly variable and possesses wide genetic diversity at intra and inter-species levels. Nine species of Ocimum viz., O. teniuflorum L., O. basilicum L., O. gratissimum L., O. kilimandscharicum, O. micranthum L. , O. campechianum L., O. americanum L., O. minimum L., and O. citriodorum L., are found in India, three of which ( O. americanum L., O. minimum L., and O. citriodorum L.) are exotic [ 2 ]. It is difficult to distinguish all these species on the basis of leaf morphology alone (Fig. 1 ). The metabolites (essential oils) of genus Ocimum have been reported to possess antioxidant and antifungal properties and to cure many diseases including bronchitis in Ayurveda, an Indian system of medicine [ 3 ]. Plants produce specialized metabolites as part of their defense mechanisms and these metabolites have significant medicinal properties that cure several human diseases. They can be isolated from various parts of the plant, including leaves, flowers, roots, bark, seeds and stem [ 4 ]. Pharmacological screening and the systematic study of the chemical constituents of plant metabolites provide a basis for developing new drugs. Some of the important metabolites reported from Ocimum species include linalool, linalyl, geraniol, citral, camphor, eugenol, methyleugenol, methyl chavicol, methyl cinnamate, thymol, safrol, taxol, urosolic acid etc. [ 4 ]. These metabolites are of immense value in the pharmaceutical, perfume and cosmetic industries. Metabolites derived from Ocimum species have been found to contain many medicinally relevant properties including anti-cancer, antioxidant, antifungal and anti-inflammatory virtues, and are also recommended for the treatment of malaria, bronchitis, diarrhea, dysentery, etc. [ 5 ]. Essential oils produced as specialized metabolites found in leaves, seeds, flowers and roots of Ocimum species are used in pharmaceutics and many systems of traditional Indian medicine [ 3 , 4 ]. Genome and transcriptome sequencing of medicinal plants serve as a robust tool for gene discovery and downstream biochemical pathway discovery of medicinally important metabolites [ 6 ]. Recently, an abundance of transcripts for biosynthesis of terpenoids in O. sanctum and of phenylpropanoids in O. basilicum [ 7 ] was reported during an attempt to compare transcriptomes of the two species of Ocimum . Despite its important role in traditional Indian medicine and its impressive arsenal of bioactive compounds, our understanding of Krishna Tulsi biology is limited. In this paper, we present the draft genome sequence of the non-model plant O. tenuiflorum (subtype Krishna), along with transcriptomes of two subtypes, Krishna and Rama Tulsi from leaf samples. We have identified a large set of genes involved in the production of specialized metabolites of medicinal interest such as apigenin, luteolin, rosmarinic acid pathway, eugenol, and ursolic acid.

Plant and leaf morphology of five Ocimum species prevalent in India viz., O. tenuiflorum subtype Krishna, O. tenuiflorum subtype Rama, O. gratissimum , O. sacharicum , O. kilmand. Leaf morphologies are quite different for the five species

Genome sequencing and assembly of the non-model plant O. tenuiflorum subtype Krishna

The paired-end (PE; 2x100-bp) and mate-paired (MP; 2x50-bp) DNA libraries were generated for Krishna Tulsi subtype using Illumina protocols. In total we obtained 373 million reads of PE and 166 million reads of MP data for Krishna Tulsi. Low quality (LQ) sequence reads were trimmed (Additional file 1 : Figure S1 and Additional file 2 : Figure S2) and reads with quality scores of less than Q30 were removed. The good quality reads were used for de-novo genome assembly. Median insert size of PE data was 335 (with a median absolute deviation of 21), whereas median insert size of MP data was 2473 (with a median absolute deviation of 704). K-mer 43 was opted as the best assembly from the statistical analysis of different k-mers. We obtained a maximum scaffold length of 184.7 Kb (Table 1 ) with an N50 length of 27.1 Kb. This assembly gives rise to a total of 78,224 scaffolds including equal to or more than 100 bp. The current draft assembly of Krishna Tulsi genome is 374.8 Mb in length. The genomic content of Krishna Tulsi is 0.72 pg/2C which is equivalent to 704.6 Mb [ 8 ], but the estimated genome size by k-mer method is 612 Mb and 61 % of the estimated genome size was assembled. The genome size reported in the literature [ 8 ], may be of a different cultivar. This lower genome coverage may be due to limited sequencing data (only two libraries were used in sequencing) or due to a high percentage of repeats (42.9 %). In terms of depth of sequencing, we sequenced 59× of the genome with paired-end (100 bp) and mate-pair (50 bp) libraries (since one lane can produce approximately 30Gb of data, even assuming that reads cover the entire 612 Mb of the estimated genome size). Ocimum species are characterized by the different basic chromosome numbers x = 8, 10, 12, or 16 [ 9 , 10 ]. In case of O. tenuiflorum individuals with 2n = 32, 2n = 36, and 2n = 76 have been recorded and the chromosome number of O. tenuiflorum is observed to be 2n = 36 [ 8 ].

A comparative analysis of the assemblies generated using PE data alone and with both PE and MP data show that the size and quality of the genome assembled using PE data alone improved substantially with the inclusion of MP data (Additional file 3 : Figures S3 and Additional file 4 : Figure S4, Additional file 5 : Table S1 and Additional file 6 : Table S2).

Validation of de novo genome assembly, annotation and repeat content of Ocimum tenuiflorum subtype Krishna genome

The de novo genome assembly was validated by mapping raw reads to the assembled genome. On an average, 74 % of reads were mapped back to the assembled genome. Almost 83.3 % of the RNA-seq reads were mapped to the assembled genome. The completeness of de novo genome assembly and annotations were also checked with two other approaches i.e., by using CEGMA (Core Eukaryotic Genes Mapping approach) [ 11 ] and DEG (Database of Essential Genes) [ 12 ] (please see Methods for details). First, we searched for essential eukaryotic genes in the O. tenuiflorum assembly. This resulted in the mapping of 85.1 % of complete core proteins (CEGMA) and more than 95 % including partial genes against our genome assembly (Additional file 7 : Table S3). Secondly, we searched for the predicted genes from the final assembly of essential genes recorded in the DEG database. We observed that about 89 % of essential genes were included within the assembly. These genes were also validated using Pfam domain annotation and were of comparable domain lengths as the classical members of that family (Additional file 8 : Table S4). Phylogenetic trees for highly conserved essential genes like glyceraldehyde 3-phosphate dehydrogenase (Additional file 9 : Figure S5), cytochrome P450 (Additional file 10 : Figure S6) and actin (Additional file 11 : Figure S7) from Krishna Tulsi and their respective homologues were analyzed and compared with other plant species. Krishna Tulsi genes were found to cluster with genes belonging to related species namely, Solanum lycopersicum, Cucumis sativus and even with distantly related Arabidopsis thaliana, indicating that highly conserved genes, essential to plant growth and functioning, have been detected in O. tenuiflorum assemblies. These trends further support the quality of the genome assembly.

Regarding the repeat content of the genome, we identified 78224 repeat regions, with a GC content of 36.1 %, adding to 160889218 bp (160 Mb), which constituted 42.9 % of assembled genome which is 374806882 bp (374 Mb) long (Additional file 12 : Table S5). Long terminal repeats (LTRs) are found in large numbers in plant genomes (Schmidt T , 1999) and a similar trend is also found in the type of repeats identified in the Tulsi genome.

Genome annotation

We identified 36768 putative gene models in the initial genome draft (version 1.2) of O. tenuiflorum genome. At least one gene was observed in each of the 10012 scaffolds, with an average of three to four genes per scaffold. During the process of refined gene prediction, 16384 gene models were observed to have expression evidence (RNA-Seq data from leaves of Tulsi (Krishna and Rama)). A total of 19384 gene models have been identified by ab initio means (without any RNA or protein evidence) (Table 2 ).

All the gene predictions, with or without RNA/protein evidences, were screened based on length (>100 bp). In case of sequential overlaps between different gene models, the gene models which are of longer length and with RNA or protein evidence for a given region of scaffold were preferred over the ones without any evidence.

There are 31,020 genes with at least one homologue in NRDB and 24,607 genes which contain at least one Pfam domain. In total, 3929 unique Pfam domains were identified for all the predicted genes in Tulsi (please see URL: http://caps.ncbs.res.in/Ote for the full list of predicted genes). A majority of domains identified were protein kinases or LRR-containing domains (Additional file 13 : Figure S8). Further comparison of Pfam results, with assembled plant genomes of similar size, reveals that the number of predicted gene models is in overall agreement in numbers as well as gene boundaries.

Orthology of Tulsi genes

The orthology relationships were deduced between Krishna Tulsi ( O. tenuiflorum ; Ote) and four other species viz. Arabidopsis thaliana (Ath), Mimulus guttatus (Mgu), Solanum lycopersicum (Sly) and Oryza sativa (Osa) (please see Methods for details). We observe 8370 clusters which contain a total of 89922 gene products from the five plant species (Fig. 2a ). M. guttatus and O. tenuiflorum share the same order (Lamiales), but belong to different families (Phrymaceae and Lamiaceae, respectively), which was evident from the presence of the highest number of common gene families (11707) between them. This was followed by Solanum lycopersicum (11022), Arabidopsis thaliana (10206) and Oryza sativa (9154) as expected from taxonomic hierarchy (Fig. 2a ). We found 17584 genes to be orthologous to any of the above four species. Considering all the 36768 Ote genes, 1282 groups contained only Ote Krishna Tulsi genes (3302). We obtained 16 Ote genes which lack traceable orthology to 22 other plant species and homology relationships (list of these genes is available on the database). Few of these unique Ote genes are transposons.

Distribution and clustering of orthologous genes of Tulsi genome to other related plant genomes. a . Distribution of gene families among five plant genomes. Ocimum tenuiflorum (Ote - green), Arabidopsis thaliana (Ath – black rectangle), Oryza sativa (Osa – red), Solanum lycopersicum (Sly – blue) and Mimulus guttatus (Mgu – black circle). The numbers in the Venn diagram represent shared and unique gene families across these 5 species obtained by OrthoMCL. b . Horizontal stacked bar plot of all the genes in 23 different genomes. This figure shows ortholog group distribution in all 23 plant species including Tulsi. Each row represents a plant species - Physcomitrella patens (Ppa), Selaginella moellendorffii (Smo) , Oryza sativa (Osa) , Setaria italic (Sit) , Zea mays (Zma) , Sorghum bicolor (Sbi) , Aquilegia caerulea (Aca) , Ocimum tenuiflorum (Ote) , Mimulus guttatus (Mgu) , Solanum lycopersicum (Sly) , Solanum tuberosum (Stu) , Vitis vinifera (Vvi) , Eucalyptus grandis (Egr) , Citrus sinensis (Csi) , Theobroma cacao (Tca) , Carica papaya (Cpa) , Brassica rapa (Bra) , Arabidopsis thaliana (Ath) , Fragaria vesca (Fve) , Prunus persica (Ppe) , Glycine max (Gma) , Medicago truncatula (Mtr) , Populus trichocarpa (Ptr). The bar graph represents ortholog protein groups for that species subdivided into 22 categories depending on the degree of sharing with the other 22 plant species e.g., category 2 represents the number of orthologous groups that have representatives from the species of interest and from one more species out of the 23 species selected for the study

In order to inspect in detail the distribution of the orthologous relationship of Ocimum genes across different species and taxonomic levels, 22 fully-sequenced plant genomes (Additional file 14 : Table S6) were considered. The orthologous groups from all 23 species were organized according to the clustering. Three hundred and thirty four clusters of genes are present across all the 23 species chosen for the study. Common genes across all species, comprising of their respective orthologous group, are plotted as a horizontal stacked bar plot (Fig. 2b ). The pattern of sharing orthologous groups is quite unique to primitive plant genomes (like lycophyte and bryophyte) and monocots. However, the pattern observed in the Tulsi genome is quite similar to that of M. guttatus (Mgu). Interestingly, this pattern is somewhat different for two members of Solanacea, which have more genes shared only in two out of 23 genomes, perhaps due to other features such as polyploidy.

Genes involved in synthesis of specialized metabolites of medicinal value: comparative analysis between O. tenuiflorum (Ote, Krishna Tulsi) and other plant genomes

Next, we performed a restricted analysis of the genes involved in the metabolite production in Ote and the genomes of a few plant species that are either closely-related ( S. lycopersicum , V. vinifera) or well-characterised ( M. truncatula , and A. thaliana ). We observed 121 (72.45 %), 130 (77.84 %), 106 (63.47 %) and 94 (56.28 %) scaffolds and contigs from the selected four representative genomes associated with 167 metabolite-related scaffolds and contigs in Ote Krishna Tulsi (Fig. 3 ) respectively . In terms of the number of orthologous genes from this selected plant genome associated with metabolite genes of Ote, we observed a similar trend of association as 601, 620, 570 and 556 genes in S. lycopersicum , V. vinifera, M. truncatula , and A. thaliana respectively. These numbers agree with the taxonomic phylogeny and hierarchy, suggesting that the evolution of genes involved in metabolic pathways is not a cause of recent expansions or sudden drifts.

When compared against 11,389 scaffolds (greater than 10Kb in size) from Ote, 10032, 9997, 8648 and 8277 scaffolds were found to be associated with the four reference plant genomes (Additional file 15 : Figure S9, Additional file 16 : Figure S10 and Additional file 17 : Figure S11 for three genomes and Additional file 18 : Table S7 for four genomes). Further, most of the metabolite-related scaffolds in Ote Krishna Tulsi were associated with chromosomes 1, 6, 8, and 10 of tomato (Fig. 4 ). In particular, gene products that are likely associated in luteolin synthesis pathway are observed to cluster in scaffolds, which are similar to nucleotide stretches in Chromosomes 3, 5, 6, 8 and 10 of the tomato genome (Fig. 4 ).

Transcriptome de novo assembly of Krishna and Rama Tulsi mature leaf samples

De novo transcriptome assembly was performed for the mature leaf samples of subtype Krishna Tulsi. The best assembly resulted in 109291 contigs with N50 of 893 bp and longest sequence of 12.1 Kb. All these contigs added up to 49.5 Mb with a GC content of 42.9 %. Scaffolding of these contigs resulted in 89878 scaffolds with N50 of 1597 bp and longest sequence of 12.7 Kb. All these scaffolds added up to 56.3 Mb with a GC content of 42.9 % (Table 3 ). Similarly, assembly was performed for the subtype Rama Tulsi and combined reads (Krishna and Rama Tulsi) as well (Table 3 ).

Differential expression of transcripts

The differentially expressed genes found in the transcriptomes of both the Tulsi subtypes were analysed. We observe a substantial number of genes up-regulated and down-regulated in Krishna Tulsi, compared to Rama Tulsi. Some of the highly expressed genes were also confirmed by q-RT-PCR technique in different tissue samples i.e., stems, leaves and flowers and also in five species viz. O. tenuiflorum subtype Krishna and Rama, O. gratissimum , O. basilicum , and O. kilmand.

For a comparison, we generated a heat map of the top 50 differentially more abundant genes in Krishna Tulsi samples (Fig. 5a ). Similarly, top 50 differentially more abundant genes in Rama with respect to Krishna sample were also plotted (Fig. 5b ). Gamma-cadinene synthase is one of the top 50 differentially expressed transcripts with RPKM values of 577.0 and 31.7 in Krishna and Rama Tulsi samples, respectively (please see below for details). Other highly expressed transcripts in Krishna Tulsi sample are Heat shock cognate protein 80, Cellulose synthase A catalyic subunit 6 (UDP-forming), Fructose-biphosphate aldolase (chloroplatic), Phototropin-2, and Rubisco activase 1 (chloroplatic). The chalcone synthase or naringenin-chalcone synthase (CHS) is one of the enzymes important for coloration of plant parts, which is observed to be highly expressed. Abundance values of all the transcripts, along with their functional annotations by NCBI BLAST results and their corresponding Krishna Tulsi genomic scaffold, show several genes involved in the synthesis of specialized metabolites implicated to be of medicinal value (Additional file 19 : Table S8).

Transcript expression of Tulsi Krishna and Rama subtypes are expressed as RPKM values. Highly significant differentially abundant RNA scaffolds/transcripts were defined to have RPKM of atleast 5 in both and the fold-change difference between two subtypes should be atleast 8 times. Only the transcripts, for which the 95 % lower-confidence-bound of more abundant subtype and 95 % upper-confidence-bound of less abundant subtype, and had at least 8 times difference, were retained. Of these differentially abundant transcripts, top-50 in Krishna and Rama subtype were plotted in the form of heat-map. a . Differentially more abundant transcripts in Krishna. b . Differentially more abundant transcripts in Rama. (please look in Additional file 24 : Text B and C for transcript IDs for a. and b)

Dark purple coloration of the leaves and stem of subtype Krishna Tulsi is one of its characteristic phenotypes, which distinguishes it from other subtypes and species of genus Ocimum. Chalcone synthase (CHS) is an enzyme belonging to a family of polyketide synthases which catalyzes the initial step for flavonoid biosynthesis. Flavonoids are important plant specific metabolites that perform various functions such as pigmentation, antifungal defense etc . Reviewed protein sequence for CHS from UniProt (Universal Protein resource) database [ 13 ] was employed to search against annotated protein sequences of Krishna Tulsi genome and six transcripts were obtained as possible hits. The best hit could be identified with 95 % query coverage and 99 % sequence identity. The extent of abundance of this hit (protein sequence) was checked in the leaf transcriptome of both the Tulsi subtypes viz. Krishna and Rama. Abundance (in terms of RPKM) of the six transcripts was, on an average, two times more in case of Krishna as compared to Rama (please see Fig. 5 ), and may be involved in the coloration phenotype of Krishna subtype plants [ 14 ]. For further confirmation of expression of these transcripts, q-RT-PCR was performed. As expected, anthocyanin producing gene was observed to be more abundant in Krishna young leaf samples and mature leaf samples (used as control) (Fig. 6a and b ). In contrast, the chlorophyll binding protein was more abundant in Krishna mature leaf samples. In addition, we also examined the presence of gamma-cadeninene synthase gene which is responsible for aroma [ 15 ]. This gene was found to be more abundant in Rama root sample and young leaf samples of O. Saccharum , but not observed in higher quantities in O. kilmund .

Expression quantification of selected genes by q-RT-PCR method. a . Fold changes of genes involved in color production, obtained through q-RT PCR. Blue colour horizontal bar is for chlorophyll a-b binding protein, red to denote Gamma-cadenine synthase and green to denote Anthocyanin. Mature leaf of Krishna subtype was used as control. It can be seen that, genes responsible for color production such as Chlorophyll a-b binding protein and gene in anthacyanin pathway are down-regulated as compared to mature Krishna leaf, which corresponds to phenotypic characteristics. b . Fold changes of genes involved in ursolic acid biosynthetic pathway, as obtained through qRT-PCR for 5 different Tulsi subtypes. Blue colour horizontal bar is for squalene epoxidase, red to denote alpha-amyrin synthase and green to denote Cytochrome P450 monooxygenase. Mature leaf of Krishna subtype was used as control. Mature leaf of Rama subtype has high expression of genes while expression in Ocimum kilmund is low. Expression of these genes are uniformly high in small, developing plants. Samples are as follows: 1) O. tenuiflorum (Rama) - Sampling Leaf. 2) O. tenuiflorum (Rama) - Sampling Root. 3) O. tenuiflorum (Rama) - Mature Leaf. 4) O. tenuiflorum (Krishna) - Sampling Leaf. 5) O. tenuiflorum (Krishna) - Sampling Root. 6) O. gratissimum - Sampling Leaf. 7) O. gratissimum - Sampling Root. 8) O. gratissimum - Mature Leaf. 9) O. sacharicum - Sampling Leaf. 10) O. sacharicum - Sampling Root. 11) O. sacharicum - Mature Leaf. 12) O. kilmund - Sampling Leaf. 13) O. kilmund - Sampling Root. 14) O. kilmund - Mature Leaf

Specialized metabolites detection and validation

Nearly 30 specialized metabolites (Fig. 7a ) are reported form the genus Ocimum which are found to have medicinal values or properties [ 4 ]. Amongst these, 14 metabolites belonging to five basic groups were found to have complete pathway information in PlantCyc database ( http://www.plantcyc.org/ ) [ 16 ] (Additional file 20 : Figure S12). Hence, genes involved in these pathways were chosen for further analysis and searched against the assembled genome of O. tenuiflorum . Figure 7b highlights the distribution of the genes identified in various classes of metabolites of disease relevance (i.e., these metabolites are well-known as drugs in the cure of human diseases).

A total of 458 genes were identified in Ote genome, which are either homologous or directly code for enzymes involved in the synthesis of specialized metabolites (Fig. 8 ) (details of gene IDs of these proteins are provided in Table 4 and Additional file 21 : Table S9). Twenty eight O. tenuiflorum gene products were annotated as putative terpene synthases using BLAST sequence searches with E-value of 10 −4 and query coverage filter of >75 % (Additional file 22 : Table S10).

Phylogeny of terpene synthases of representative sequences of six classes from the plant kingdom along with putative Tulsi terpene synthases genes: The tree is color coded as tpsa:red, tbsb:blue, tpsc:yellow, tpsd: green, tpse: blue and tpsf:purple

Among these specialized metabolites, we focused on ursolic acid, belonging to sesquiterpenes, since it is known to have anti-inflammatory, anti-microbial, anti-tumour and anti-cancer properties. The synthesis of ursolic acid from squalene is a three-step process starting from squalene (Fig. 9 ). α-Amyrin is formed by concerted cyclization of squalene epoxide, while ursolic acid is eventually synthesized by the catalytic activity of multifunctional cytochrome P450. The enzymes involved are, therefore, squalene epoxidase, alpha-amyrin synthase and alpha-amyrin 2, 8 monoxygenase. Sequence search algorithms were employed to search for the three enzymes of this pathway in the Tulsi genome, starting from protein sequences for each of these enzymes from PlantCyc database as queries. The search for squalene epoxidase in Tulsi, using the sequence of this enzyme in Oryza sativa japonica (LOC_Os02g04710.2) as a query, gave rise to a hit (C3776143), with 50 % sequence identity covering 80 % of the query length (Additional file 23 : Figure S13). Using Amyrin synthase LUP2 from A. thaliana (Q8RWT0) and 13 other well-accepted alpha/beta amyrin synthases as a query, four hits were identified in the Tulsi genome (scaffold16333, scaffold20801, scaffold12312 and maker-C3776143). In classical amyrin synthases, a QW structural motif repeats six times in the entire sequence [ 17 , 18 ], while there are two functional motifs, viz ., a well conserved SDTAE [ 19 ] motif which is believed to form the catalytic pocket and the MWCYCR [ 20 ] motif that is shown to play a crucial role in catalysis. These motifs are observed in the four hits in Tulsi genome (Additional file 24 : Text D). Further, a phylogenetic tree was constructed using 16 query sequences and these four hits (Fig. 10 ). One of the Tulsi hits, (scaffold 16333_mrnal) clusters with a well-characterized alpha amyrin synthase from C. roseus (H2ER439) suggesting that this particular scaffold might indeed retain an alpha amyrin synthase.

The synthesis of ursolic acid from squalene is a three-step process starting from squalene. A: Squalene epoxidase, B: α-amyrin synthase, C1: α-amyrin 28-monooxygenase [Multifunctional], C2: Uvaol dehydrogenase [Multifunctional] and C3: Ursolic aldehyde 28-monooxygenase. Squalene epoxidase and alpha amyrin synthase, along with alpha amyrin 28 mono-oxygenase, uvol dehydrogenase and ursolic aldehyde 28 mono-oxygenase, play important role in synthesis of ursolic acid. These three genes have been chosen for quantification of gene expression by q-RT PCR method in different tissues and species

Phylogenetic tree of sixteen amyrin query sequences and four putative amyrins from Tulsi. Tulsi hits are marked in blue clour, red ones are alpha amyrin synthase, greens are beta amyrin synthase and cyan ones are proteins from other class of amyrin. The presence of motifs and position in the phylogeny indicate that the hits obtained in O. tenuiflorum genome are likely to be alpha-amyrin synthases

Interestingly, many genes involved in the synthesis of specialized metabolites of relevance in the treatment of diseases are also more abundant, as observed in the assembled transcriptome (Additional file 21 : Table S9). Similarly, genes involved in the synthesis of 16 other specialized metabolites (Additional file 25 : Table S11), are also equally interesting. However, this requires detailed understanding of the mechanism of synthesis and enzymes involved in the pathways. We analysed RNA-Seq data of two leaf samples in order to compare the genes related to important metabolite pathways and the peculiar phenotype of O. tenuiflorum subtype Krishna with subtype Rama Tulsi. There were 104 transcripts, whose fold change in expression was observed to be eight times more in Krishna Tulsi than in Rama Tulsi. Likewise, there were 229 transcripts whose fold change expression was eight times more in Rama Tulsi as compared to Krishna Tulsi. These are available for download at- (caps.ncbs.res.in/download/tdat_data/Supplementary_tables/Supplementary Table 8.txt).

In the case of the multifunctional Cytochome P450 (which catalyses the last three steps in the synthesis of urosolic acid, Fig. 9 ), a predicted gene from scaffold2032 was obtained as a hit, when a reviewed UniProt entry F1T282 from V. vinifera was considered as query and searched in the Tulsi genome assembly using BLAST. This hit retains 61 % sequence identity and the alignment covers 90 % of the length of the query (alignments are shown in Additional file 23 : Figure S13). This scaffold contains a total of three predicted genes viz., Ote100020320011, Ote100020320001 (similar to UHRF1-binding protein) and Ote100020320031 (gene of interest).

From the available transcriptome assembly, these genes, identified as involved in the synthesis of urosolic acid, were analysed for their levels of expression. The RPKM values were also high for these three genes (please see Additional file 21 : Table S9). To further validate the levels of expression of these genes, q-RT-PCR was performed using sequence-specific primers. The presence of these three enzymes is generally high in all the mature leaf samples and highest in Rama subtype (using Krishna subtype as control). Alpha-amyrin synthase is more abundant in mature leaf samples of O. gratissimum and O. sacharicum species. However, interestingly, the three enzymes are found to be more abundant in the young leaf samples of Rama subtype; in contrast, atleast one of the three genes is less in the Krishna leaf sample and in all root samples. The expression of the three genes implicated in urosolic acid synthesis is uniformly low in samples of O. kilmund.

Next, to correlate gene expression and to quantify the presence of ursolic acid and eugenol, chemical profiling was performed using LC-Mass spectrometry from different tissues and samples. Eugenol and ursolic acid were observed in the highest quantities in mature leaf sample of Rama subtype and in relatively low quantities in O. kilmund . The amount of eugenol in the leaf sample of O. tenuiflorum subtype Rama (2235 ng/mg) is considerably high followed by O. kilmund (1472 ng/mg), O. sacharicum (651 ng/mg) and lowest in O. gratissimum (73 ng/mg). In all stem samples, the amount of eugenol is consistently low with the highest in O. tenuiflorum subtype Rama (24 ng/mg), O. tenuiflorum subtype Krishna (17 ng/mg), O. kilmund (15 ng/mg) and below limits of quantification in O. gratissimum and O. sacharicum . The presence of oleanolic acid is also severely reduced in stem samples of Rama subtype (2869 ng/mg) and in Krishna subtype (1088 ng/mg) in comparison to the mature leaf samples (7556 ng/mg for Rama and 4630 ng/mg for Krishna). The presence of urosolic acid is 50 % less in stem samples of Rama subtype (2883 ng/mg) when compared to the mature leaf samples (4597), whereas it is much lower in the stem samples of other species as compared to the leaf sample. The amount of ursolic acid in the stem samples of Krishna subtype (746 ng/mg) is 4.6 times less than that of the mature leaf samples (3471 ng/mg) (please see Table 5 ).

O. tenuiflorum subtype Krishna Tulsi is one of the non-model plants of great medicinal value, for which there has been no genomic information available till date. We have performed genome sequencing of O. tenuiflorum subtype Krishna of the paired-end (PE; 2x100-bp) and mate-paired (MP; 2x50-bp) DNA libraries by Illumina Hiseq 1000. The best de novo assembly was obtained at k-mer 43 by SOAPdenovo2, an eukaryotic de novo genome assembler. Repeats were identified and masked, and gene prediction and annotation was carried out using the MAKER annotation pipeline by using genomic, transcriptomics and EST data. The nearest species whose genome has been sequenced is the monkey flower ( M. guttatus ), which shares its order Lamiales with O. tenuiflorum (Ote) but falls in a different family (Phrymaceae). Orthology search of Ote Krishna Tulsi genes in four genomes viz. A. thaliana (Ath), M. guttatus (Mgu), S. lycopersicum (Sly) and O. sativa (Osa) also confirmed the close relationship between Krishna Tulsi and M. guttatus (Mgu), in terms of the number of common gene families i.e., 578 out of 2488 total genes. When we considered all the 36,768 predicted genes from the Krishna Tulsi genome, we found that 1282 ortholog groups have Ocimum- only genes. These 1282 groups contain 13,306 Ocimum genes and hence they are referred to as paralogs by OrthoMCL. Of the remaining Ote genes, 17,584 genes were found to be orthologous to any of the other four species studied in this case. We performed an analysis of the genes involved in the metabolite production in Ote and the genomes of a few other related plant species. Based on the direct evidence or homology a total of 458 genes were identified in Ote genome, which are involved in coding of enzymes implied in the synthesis of specialized metabolites. Comparative analysis of transciptomes of O. tenuiflorum subtype Krishna and Rama was performed to detect potential differentially-regulated genes and their involvement in metabolite synthesis. On comparing both the transcriptomes, differentially expressed genes were observed with a substantial number of genes more abundant and others less abundant in either subtypes. Gamma-cadinene synthase is more abundant in Krishna sample (RPKM value 577.047) as compared to Rama sample (RPKM value 31.73). To confirm some of the more abundant genes along with Gamma-cadinene synthase, we performed q-RT-PCR in different tissue samples i.e., stem and leaves and also in five species viz. O. tenuiflorum subtype Krishna and Rama, O. gratissimum , O. basilicum , and O. kilmand. Expression of Gamma-cadinene synthase is found more in Krishna samples as compared to Rama by q-RT-PCR also. Likewise, Chalcone synthase (CHS) is an anthocyanin-producing gene, which is observed to be more abundant in young leaf samples of Krishna and mature leaf samples in transcriptome data. Subsequently, this has been confirmed by q-RT-PCR and from mass spectrometry readings of ursolic acid and eugenol from different tissue samples and from different species.

We present a draft genome of O. tenuiflorum Krishna Tulsi subtype Krishna Tulsi. The habitat of genus Ocimum is tropical climate and it is wide spread over Asia, Africa, Central and South America. High RNA-seq expression values of the genes responsible for the purple coloration of the plant parts in Krishna subtype, as compared to Rama subtype, were observed. We also identified a fFew unique genes (16) of Ote, which lack any traceable orthology and homology relationships from all the 22 species used in this study.

Krishna Tulsi is described in the Vedas and Puranas (ancient scriptures of Hindus) and has a long history of cultivation, of roughly 3000 years, and is therefore assumed to be of Indian origin [ 21 ]. In literature, it is also referred to as the “Queen of Herbs”. Major genes involved in the synthesis of medicinally important specialized metabolites in the plant could be unraveled despite limited data on sequencing and coverage [ 22 ]. Expressions of these genes were confirmed by complementing with RNA-seq data and q-RT-PCR method. We also investigated one of the important metabolic pathways involving the production of ursolic acid in detail, by mass-spectrometry and q-RT-PCR methods. Synthesis of specialized metabolites or their precursors appear to begin in the young leaves of Tulsi. Subsequently, the mature leaves retain the medicinally relevant metabolites. O. tenuiflorum Rama subtype retains the high abundance of key medicinally relevant metabolites like eugenol and ursolic acid, as observed in the transcriptome, metabolite quantifications and q-RT-PCR expression values consistent with its high medicinal values. Our main emphasis was to unravel the important metabolite genes by using genomic and transcriptomic data despite limited sequencing information.

Isolation of genomic DNA from O. tenuiflorum subtype Krishna Tulsi

Young leaves of Tulsi subtype Krishna and Rama were used for genomic DNA isolation. About one gram of leaves were crushed using liquid Nitrogen and DNA extraction buffer (200 mM TrisHCL [pH-8.0], 200 mM NaCl, 25 mM EDTA and 1 % PVP) was added [ 23 ]. The ground material along with 1/10th volume of 20 % SDS solution was incubated at 65 °C for 30 min. The tubes were centrifuged at 14,000 RPM for 10 min at room temperature to remove the debris. The supernatant was transferred into a fresh tube and treated with equal volume of phenol: chloroform: isoamyl alcohol (25:24:1) and mixed gently for 5 min. The mixture was centrifuged at 12,000 RPM for 10 min to separate the phases. The aqueous phase from the centrifuged tube was transferred to a fresh tube and DNA was precipitated with 1/5th volume of 2 M NaCl and 2 volumes of ice-cold ethanol. The DNA was pelleted by centrifugation at 12,000 RPM for 10 min. Precipitated DNA pellet was taken as a starting material for purification using the Sigma Genelute plant DNA isolation kit (G2N70, Sigma). The DNA was run on a 1 % agarose gel to assess the quality. The A260/280 ratio and quantity were determined using the nanodrop.

Genome sequencing, assembly and annotation

Genome sequencing was performed by using Illumina HiSeq 1000 technology in the Next Generation Genomics Facility at Centre for Cellular and Molecular Platforms (C-CAMP). Genomic DNA paired-end and gel free mate-pair library preparation was performed for Krishna Tulsi using TruSeq DNA sample preparation kit (FC-121-2001) and Nextera mate-pair sample preparation kit (FC-132-1001) from Illumina ( www.illumina.com ). FASTX-Toolkit [ 24 ] and FastQC tools [ 25 ] were used for pre-processing of raw reads and for quality check of the reads. Genome assembly from reads of PE and MP together was done by using SOAPdenovo2, a de novo draft genome assembler [ 26 ]. Preliminary assemblies were performed based on k-mers from 21 to 63 with an interval of two. Gene prediction and annotation was carried out using the MAKER annotation pipeline [ 27 ] with predicted gene models using AUGUSTUS [ 28 ] and A. thaliana genes as reference for initial prediction. The gene models were refined using homology searches against all protein sequences from Viridaeplantae kingdom.

Validation of genome assembly and annotations

To validate genome assembly, we have mapped raw reads on to the de novo assembled genome by using REAPR (SMALT) [ 29 ], SAMtools [ 30 ] and Picard tools ( http://broadinstitute.github.io/picard/ ). Maximum and minimum insert size of 500 bp and 0 bp respectively were selected for mapping. We report an alignment pairing with best score, using standard Smith-Waterman scores. The threshold minimum score used was calculated by the formula to be: < Minimum score > = < world length > + step size – 1. Here the word length of 13 is used with a step size of 6. Estimation of the genome size of the Tulsi genome was done using the k-mer distribution analysis by Jellyfish [ 31 ]. Essential genes implicated in the regulation, assembly and functioning of plant cells, have been identified in the Krishna Tulsi assembled genome using a two-way approach. Firstly, using CEGMA which was derived from the KOG database [ 32 ] (for eukaryotic genomes) and core proteins in any eukaryotic genome (including ones in draft stages), essential genes were annotated. Secondly, a subset of A. thaliana genes were extracted from a well-characterized Database of Essential Genes (DEG) and compared against Krishna Tulsi assemblies. Validation of the extracted genes was performed by Pfam domain annotation approaches. Putative essential genes from the Krishna Tulsi dataset were further searched using BLASTP [ 33 ] against the NCBI (NR) database and closely-related homologues were aligned and phylogenetic tree constructed.

Repeat identification

Repeat elements in the assembled genome were identified using RepeatScout (version 1.0.5) [ 34 ] and RepeatMasker (version 4.0.3) [ 35 ]. The library of ab initio repeats generated by RepeatScout was classified into known repeat classes using the RepeatClassifier module of RepeatScout (Additional file 12 : Table S5). The RepBase library of RepeatMasker and the non-redundant library of ab-initio classified repeats were then used to mask the repeat elements in the assembled genome. The repeat-masked genome assembly was then used for genome annotation.

The repeat-masked assembled genome of Krishna Tulsi was processed through the MAKER annotation pipeline [ 27 ]. AUGUSTUS [ 28 ] was used for gene prediction, trained on A. thaliana gene models. RNA-seq data obtained from leaf samples was used as EST evidence to refine the gene models. Initial gene models of protein sequences belonging to Viridaeplantae kingdom, obtained from the NCBI database, were used as protein evidence for refining gene prediction. Both EST and protein evidence were prepared using EXONERATE [ 36 ] and used for gene prediction refinement through AUGUSTUS. All the protein sequences of these gene models were subjected to validation based on identification of homologues through BLASTP search against NRDB at E-value cutoff of 10 −3 . Pfam release 27 was consulted for all domain predictions with an E-value cutoff of 10 −5 using HMMER3 package [ 37 ].

Orthology detection

All the predicted gene models from Krishna Tulsi were used with OrthoMCL tool [ 38 ] to identify clusters between selected species of A. thaliana (Ath), O. sativa (Osa), S. lycopersicum (Sly), M. guttatus (Mgu). In order to inspect distribution of the orthologous relationship of Ocimum genes across different species and taxonomic levels, ProteinOrtho tool [ 39 ] was implemented on Krishna Tulsi (Ote) gene models along with 22 different species: Aquilegia caerulea (Aca), Glycine max (Gma), Setaria italic (Sit), Mimulus guttatus (Mgu), Solanum lycopersicum (Sly), Arabidopsis thaliana (Ath), Medicago truncatula (Mtr), Selaginella moellendorffii (Smo), Brassica rapa (Bra), Oryza sativa (Osa), Solanum tuberosum (Stu), Carica papaya (Cpa), Physcomitrella patens (Ppa), Theobroma cacao (Tca), Camellia sinensis (Csi), Prunus persica (Ppe), Vitis vinifera (Vvi), Eucalyptus grandis (Egr), Populus trichocarpa (Ptr), Zea mays (Zma), Fragaria vesca (Fve), Sorghum bicolor (Sbi). All the complete proteome sets were obtained from Phytozome resource [ 40 ]. Phylogenetic tree reconstruction was carried out using ‘RbcS’ (Rubisco small subunit) coding sequences from all 23 species. CLUSTALW [ 41 ] and Phylip package [ 42 ] were employed for multiple sequence alignment (MSA) and subsequent clustering using Neighbor Joining (NJ) method, respectively. Distant homology relationships were verified through PSI-BLAST [ 33 ] at different set of E-value cutoffs. Gene products for which we were unable to establish any homology or orthology relationships, but consisted of a Pfam domain, were referred to as unique genes specific to Ote.

Comparative analysis between Krishna Tulsi and other plant genomes

The most recent version of whole genome sequences of S. lycopersicum, V. vinefera, M. tranculata and A. thaliana were downloaded from NCBI ( ftp://ftp.ncbi.nlm.nih.gov/genomes/ ). BLAT [ 43 ] was employed for sequence searches using S. lycopersicum, V. vinefera, M. tranculata and A. thaliana genomes against two sets of Tulsi genome data: one containing 11389 scaffolds (which are greater than 10000 bp) and another containing 167 scaffolds and contigs with metabolite-related genes (identified earlier on the Krishna Tulsi genome). The figures were prepared using in-house software written for this purpose.

Isolation of RNA from Tulsi subtypes, Krishna and Rama, and RNA-seq library preparation

RNA isolation was carried out with 100 mg of the leaf tissue (Rama and Krishna) using the Sigma Spectrum Plant Total RNA Kit (STRN50, Sigma). DNA contamination was removed by DNAse treatment using DNA-free™ kit (AM1906, Ambion). The DNase free RNA quality was determined using the Agilent Bioanalyzer. The RNA Integrity Number (RIN) values of all the samples were greater than 6. The A260/280 ratio and the quantity were determined using the nanodrop. RNA-seq library preparation was done with 1 μg of total RNA following the TruSeq RNA sample preparation from Illumina (RS-122-2001).

Transcriptome sequencing and assembly

We assembled all the mRNA reads having HQ scores of all the bases more than 20, of Krishna and Rama subtype separately and also by combining the reads from both of these subtypes by using SOAPdenovo-trans [ 26 ] at different K-mers starting from 19 to 63 at an interval of two. An insert size of 350 was used for the assembly of transcriptomes. RNA-seq reads were mapped to the assembled genome by Tophat2 [ 44 ], which uses Bowtie2 [ 45 ] as a mapping tool. We used a minimum and maximum intron length of 50 and 500000 bp respectively. Maximum multi hits (parameter that dictates the number of alignments to the reference for a given read) was assigned as 20 and transcriptome max hits (maximum number of mappings allowed for a read, when aligned to the transcriptome) of 60 was used.

Transcript differential expression comparison

To quantify expression in terms of reads per kilo base per million (RPKM), non-redundant combined assembled transcript sequences (at 90 % sequence similarity by CD-hit EST [ 46 ]) were taken as reference. This non-redundant transcriptome was used as the reference transcriptome to calculate differential expression of transcripts in both the samples [ 6 , 47 ]. The reads of RNA-seq experiments from Krishna and Rama subtypes were mapped back on to the reference transcriptome by using SeqMap (version – 1.0.12) [ 48 ] and RPKM values were determined by using rSeq: RNA-seq analyzer (version 0.1.1) [ 49 ].

The dataset obtained after gene prediction on the assembled genome was employed to search for enzymes involved in secondary metabolite production. There are 14 metabolites (flavonoids (2), phenylpropanoids (4), terpenes (2), sesquiterpenes (5) and sterols (1)), which are reported to be present in Ocimum and have known pathway information in PlantCyc ( http://www.plantcyc.org/ ) [ 16 ]. Reviewed entries from the UniProt database and all the known sequences of the enzymes from other species possessing these enzymes were used as queries to search in the full dataset of scaffolds and contigs, using PSI-BLAST at E-value of 10 −5 and three iterations. The protein hits obtained in our dataset were further subjected to validation using a query coverage filter of 75 %.

In order to study the expression of genes involved in the synthesis of specialized metabolite (s), the assembled transcriptome of both Ocimum species were searched, employing the reviewed entry corresponding to each enzyme in the UniProt database. These searches were performed using TBLASTN at an E-value of 10 −3 , and the best hit in our dataset was selected based on the least E-value. If the reviewed entry for any of the enzyme was not present, unreviewed entries from PlantCyc database were employed.

Quantification of eugenol and ursolic acid using UHPLC-MS/SRM method

A Vantage TSQ triple stage quadrupole mass spectrometer (Thermo Fisher Scientific, San Jose, CA, USA) equipped with a heated electro spray ionization (HESI) source was used for the analysis of eugenol and an APCI probe was used for the ursolic acid analysis. The mass spectrometer was interfaced with an Agilent 1290 infinity UHPLC system (Agilent Technologies India Pvt. Ltd., India) equipped with a column oven (set at 40 °C), auto sampler and a thermo-controller (set at 4 °C). The needle was washed from outside with acetonitrile (0.1 % formic acid) before every injection to avoid any potential carry-over problems. Separations were performed using a shim-pack XR-ODSIII column (2 × 150 mm, 2 μm). For Eugenol: Mobile phase A was water (10 mM Ammonium acetate) containing 0.1 % formic acid, and mobile phase B was acetonitrile containing 0.1 % formic acid. For Ursolic acid: Mobile phase A was water (10 mM Ammonium acetate), and mobile phase B was acetonitrile: methanol (3:1). Injections of 10 μL were performed using flow through a needle

Eugenol was quantified after derivatizing with pyridine sulfonyl chloride using estrone-d4 as an internal standard. Methanol was used to extract eugenol from fresh leaves (2 mg/mL) and dried stem powder (20 mg/ml). Briefly 10 μL of extract and 10 μL of internal standard (from 2.5 μg/mL) were added into 200 μL of buffer [acetone: NaHCO3 (1:1)]. To this 10 μL of pyridine sulfonyl chloride (10 mg/mL) was added and incubated at 60 °C for 15 min. After incubation the derivative was extracted with 800 μL of MTBE and the organic layer was dried and reconstituted in 50 μL of methanol followed by 10 μL injection for the analysis. A gradient (0–2 mins:30 %B, 2–5 mins:30–90 %B, 5–7 mins:90–100 %B, 7–10 mins:100 %B, 10–10.1 mins:100–30 %B, 10.1–15 mins:30) was then initiated at a flow rate of 200 μL/min. Operating conditions were as follows: spray voltage, 3000 V; ion transfer capillary temperature, 270 °C; source temperature 100 °C; sheath gas 20, auxiliary gas 5 (arbitrary units); collision gas, argon; S-lens voltage was optimized for individual metabolites; scan time of 50 millisec/transition; and ion polarity positive. A standard curve was constructed from 0.078 to 5ngon column to quantify eugenol. The SRM transition used for the analysis of eugenol is (306.1 → 79) and for estrone-d4 (416.3 → 274.1).

Ursolic Acid:

Ursolic acid was quantified using estrone-d4 as an internal standard. A brief extraction was done from 2 mg/mL of dry powder using 1 mL of methanol (sonication-3 min, centrifugation −5 min). The extract was further diluted to 0.2 mg/mL in methanol. From this extract 10 μL was added along with 10 μL of internal standard (0.1 ug/mL) to 30 μL of methanol and 10 μL was injected for the analysis. A gradient (0–2 mins:20 %B, 2–8 mins:20–100 %B, 8–14.5 mins:100 %B, 14.5–14.6 mins:100–20 %B, 14.6–20 mins:20 %B) was then initiated at a flow rate of 200 μL/min. Operating conditions were as follows: Discharge current 4 μA; ion transfer capillary temperature, 270 °C; source temperature 300 °C; sheath gas 20, auxiliary gas 5 (arbitrary units); collision gas, argon; S-lens voltage was optimized for individual metabolites; scan time of 50 millisec/transition; and ion polarity positive. A standard curve was constructed from 0.034 to 2.5 ng on column to quantify ursolic acid. The same standard curve was used for the analysis of oleanolic acid. The SRM transition used for the analysis of both ursolic and oleanolic acid is (439.4 → 119) and for estrone-d4 (275.3 → 257.1).

Availability of supporting data section

Information on the genes identified in Tulsi, along with the scaffold numbers, are provided in http://caps.ncbs.res.in/Ote .

BioProject : PRJNA251328

SRA id : SRP051184

Accession number of O. tenuiflorum : JQCZ00000000

Also please see DOI for supporting data: https://mynotebook.labarchives.com/share/National%2520Centre%2520for%2520Biological%2520Sciences/MTkuNXw2MjMwNC8xNS9UcmVlTm9kZS80MjAwNTk4MTM5fDQ5LjU =

Data available from the Dryad Digital Repository: http://dx.doi.org/10.5061/dryad.6f1r2

Abbreviations

Mate paired

Core eukaryotic genes mapping approach

Database of essential genes

Long terminal repeats

Ocimum tenuiflorum

Arabidopsis thaliana

Mimulus guttatus

Solanum lycopersicum

Oryza sativa (Osa)

Simple sequence repeats

chalcone synthase

Rubisco small subunit

Multiple sequence alignment

Neighbor joining

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Acknowledgements

We thank Ms. R. Savithri and Dr. Anna Spudich for useful discussions. We thank NCBS and InStem for infrastructure and other facilities. Sequencing was done at C-CAMP (BT/PR3481/INF/22/140/2011). We thank Mr. Kannan for metabolite confirmation done at C-CAMP. We acknowledge the financial support of NCBS scholarship to AU and RS, Glue Grant (BT/PR15352/MED/15/70/2011) funded by the Department of Biotechnology, India, to AC., Centre of Excellence Grant (BT/01/COE/09/01) funded by the Department of Biotechnology, India to AG, OKM, AS, SN, SNP and MS., University Grants Commission Scholarship, India to PG and Kothari fellowship to MM, Bridge postdoctoral fellows, NCBS and Instem India to KH, APJ, MN, PS and MSS., Indo-Japan Grant (BT/IC/ Japan(BI)/01/2010) funded by the Department of Biotechnology, India to KH., Council of Scientific and Industrial Research, India to AGJ, SDK, JM, AS and USR, Department of Biotechnology Scholarship, India to SK, EM, PS and SM, Extramural Grant (37/1606/13/EMR-II) funded by Council of Scientific and Industrial Research, India to SN., InStem to SR, Department of Biotechnology, Government of India (Ramalingaswami Fellowship Grant; BT/HRD/35/02/2006) to MG.

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Correspondence to Malali Gowda or Ramanathan Sowdhamini .

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The authors declare that they have no competing interests.

Authors’ contributions

Conceived and designed experiments: RS, SR, MG. Data generation: NP, CS, RM, SS, MS, AR, SN, MN. Data analysis and presentation: AKU, AS, MS, KH, EM, APJ, AGJ, OKM, PNS, USR, SM, SK, AG, NK, HRS, ARC, SDK, JM, PG, MM, MSS, HRS, SN, SNP. Writing of the manuscript: AKU, AS, MS, KH, EM, APJ, AGJ, OKM, PNS, USR, SM, SK, AG, NK, HRS, ARC, SDK, JM, PG, MSS, HRS, SN, SNP. Provided resources and tools and critical reviewed manuscript: RS, MG. All authors read and approved the final manuscript.

Additional files

Additional file 1: figure s1..

Per base sequence quality of R1 reads of PE sequences used in final genome assembly.

Additional file 2: Figure S2.

Per base sequence quality of R2 reads of PE sequences used in final genome assembly.

Additional file 3: Figure S3.

Distribution of assembled scaffolds according to their length.

Additional file 4: Figure S4.

Distribution of scaffold length difference between paired end and paired with mate pair end assembly.

Additional file 5: Table S1.

Scaffold lengths distribution in the MP + PE and PE assemblies.

Additional file 6: Table S2.

Statistics of scaffold length comparison from assemblies of PE and MP + PE together.

Additional file 7: Table S3.

Completeness of assembly and presence of essential genes by CEGMA results for O. tenuiflorum at two levels; (a) only in PE assembly (b) in PE + PM assembly.

Additional file 8: Table S4.

Presence of essential genes in O.tenuiflorum (Tulsi) at three levels; a) in only paired end assembly (ab-initio gene prediction), b) in paired end and mate-pair assembly’s Level 2 [evidence from RNAseq, EST and known tulsi genes], c) in paired end and mate-paired assembly’s Level 1 (gene prediction).

Additional file 9: Figure S5.

Phylogenetic trees of essential gene, cytochrome P450 from O.tenuiflorum and their respective homologues.

Additional file 10: Figure S6.

NJ tree for glyceraldehydes phosphate dehydrogenase protein in O. tenuiflorum (Tulsi, marked in red) and its nearest homologues.

Additional file 11: Figure S7.

Phylogenetic trees of essential genes, actin from O.tenuiflorum and their respective homologues.

Additional file 12: Table S5.

Repeat elements identified in Tulsi genome assembly and classified in different groups of repeats.

Additional file 13: Figure S8.

Pie chart of distribution of protein domains (Pfam) of all the predicted genes in O. tenuiflorum subtype Krishna genome.

Additional file 14: Table S6.

List of species used for phylogeny analysis along with Ocimum to depict taxonomical distribution of this species in plant kingdom.

Additional file 15: Figure S9.

Circular representation of O. tenuiflorum metabolite-related genes mapped onto Vitis vinefera plant genome. Color indicate blue = < 2 genes, green =2 genes, yellowgreen = > 2 genes, red = Metabolite related genes. Connecting line between scaffolds and chromosome represents postion of the scaffold in genome. Red color of connecting line represents presence of metabolite related genes.

Additional file 16: Figure S10.

Circular representation of O. tenuiflorum metabolite-related genes mapped onto Medicago tranculata plant genome. Color indicate blue = < 2 genes, green =2 genes, yellowgreen = > 2 genes, red = Metabolite-related genes. Connecting line between scaffolds and chromosome represents postion of the scaffold in genome. Red color of connecting line represents presence of metabolite related genes.

Additional file 17: Figure S11.

Circular representation of O. tenuiflorum metabolite-related genes mapped onto Arabidopsis thaliana plant genome. Color indicate blue = < 2 genes, green =2 genes, yellowgreen = > 2 genes, red = Metabolite-related genes. Connecting line between scaffolds and chromosome represents postion of the scaffold in genome. Red color of connecting line represents presence of metabolite-related genes.

Additional file 18: Table S7.

Associations of O. tenuiflorum a) scaffolds related to metabolite-related genes, b) longer length scaffolds (greater than 10Kb in size) to four different plant genomes.

Additional file 19: Table S8.

All the transcripts with their expression and validation at different level such as genome hit and blast hit results against non-redundant database of NCBI.

Additional file 20: Figure S12.

Pathways of all the 14 important medicinal metabolites of the Tulsi genome which were studied in detail.

Additional file 21: Table S9.

Gene IDs involved in specialized metabolite production for each of the metabolites with known pathways.

Additional file 22: Table S10.

Sequences of putative terpene synthases in O. tenuiflorum genome.

Additional file 23: Figure S13.

a. Sequence alignment of metabolite protein predicted from Scaffold 14352 from Ocimum and O65402 protein sequence from Arabidopsis. b. Sequence alignment of protein sequence predicted in scaffold16333 from Ocimum genome and Q8RWT0 protein sequence from Arabidopsis. c. Sequence alignment of protein sequence predicted in scaffold2032 from Ocimum genome and F1T282 protein sequence from Vitis proteome.

Additional file 24:

Text A. List of scaffolds as marked from START to END in Fig. 4 . Text B. List of IDs of transcripts more abundant in Krishna as compared to Rama subtype (from top to bottom) marked in Fig. 5a . Text C. List of IDs of transcripts more abundant in Rama as compared to Krishna subtype (from top to bottom) as marked in Fig. 5b . Text D. Multiple sequence alignment of amyrin synthases with hits in Tulsi genome.

Additional file 25: Table S11.

Metabolites with unknown pathways with their disease implications. There are 15 medicinally relevant metabolites in Ocimum sp. with unknown pathways.

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Upadhyay, A.K., Chacko, A.R., Gandhimathi, A. et al. Genome sequencing of herb Tulsi ( Ocimum tenuiflorum ) unravels key genes behind its strong medicinal properties. BMC Plant Biol 15 , 212 (2015). https://doi.org/10.1186/s12870-015-0562-x

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Published : 28 August 2015

DOI : https://doi.org/10.1186/s12870-015-0562-x

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Plant Cell and Tissue Culture: Propagation, Improvement, and Conservation of Medicinal Plants

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Maryam Khezri 2 ,
Rasool Asghari-Zakaria 2 &
Nasser Zare 2

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Plant tissue culture, a revolutionary technique in plant biology, is a highly effective method for multiplying, biodiversity conservation, and genetic manipulation and enhancement of plants. Over the decades, technological breakthroughs and a deeper understanding of plant physiology have transformed this technique from curiosity to a widely used method with wide-ranging applications. Micropropagation usually involves four key stages: initiation (explant culture under an aseptic environment), propagation, rooting, and acclimatization. The optimal growth and development of plant tissues at each stage are substantially influenced by the application and proper balance of phytohormones, growth regulators, and culture media composition . Advanced genetic technologies based on plant tissue culture are used in addressing global food security and are helpful in the sustainable production of crops. As technology advances, incorporating plant tissue culture with other cutting-edge approaches promises to unlock plants’ full potential for humanity’s benefit. This review examines the key principles, recent advances, and diverse applications of plant tissue culture in medicinal plants.

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In 2024, fusion technology will finally make the transition from basic research to commercial application. The reason for that will be the construction and completion of the first commercial fusion demonstrators. These cutting-edge facilities are smaller than fusion power plants. For instance, a laser-based fusion demonstrator might use five to ten laser beams, while a commercial power plant can use several hundred. However, they have a crucial role—to prove that fusion technology works on a small scale, paving the way for the construction of larger fusion-power plants. In 2024, they will do just this, starting to build devices that will finally achieve the elusive goal of energy gain– in other words, outputting more energy than the quantity needed to kickstart the fusion process. Hitting this milestone is a critical step in addressing the steeply increasing global energy demand, as fusion energy has the potential to provide an abundant, carbon-free source of power.

In 2022, researchers at the National Ignition Facility (NIF) in California became the first to demonstrate experimentally that a fusion process could indeed produce a net energy gain. This experiment used high-power lasers to deposit energy in a small fuel target—a millimeter-sized capsule containing frozen deuterium and tritium—creating the conditions for fusion to occur. The lasers delivered 2.05 megajoules of energy to the target, resulting in a fusion energy production of 3.1 megajoules. This was a scientific experiment—unlike fusion demonstrators, the NIF is not designed to operate continuously like a power plant. However, as a result of this scientific breakthrough, nuclear fusion has attracted considerable research, political, and investor attention in recent months.

National fusion strategies have been developed in the US, UK, Japan, Germany, and other countries to advance research and testing of the technology. Currently, the US and the UK are leading the race: The US Department of Energy funds fusion research with an annual budget of about $1.4 billion and encourages private enterprises to accelerate commercialization. The UK similarly fosters public-private partnership by raising a fusion cluster with universities and companies combining their expertise. High-profile investors recognize the opportunity of fusion technology, with over $5 billion of private capital flowing into fusion companies in the last two years.

The initiatives are bearing fruit: Several fusion companies worldwide, including Commonwealth Fusion Systems, Helion Energy, and General Fusion have announced plans to begin constructing facilities in 2024 to demonstrate their technological approach. According to the latest report by the Fusion Industry Association , over half of all fusion companies believe that fusion energy will be delivered to the public power grid during the 2030s. In May 2023, Microsoft signed a power purchase agreement with Helion Energy, to secure a supply of fusion-generated electricity by 2028. In August 2023, Marvel Fusion (a fusion energy firm I cofounded) announced a partnership with Colorado State University worth $150 million, the largest public-private partnership to date, with the aim of building the only laser facility tailored to a commercial laser-based fusion technology and the most powerful short-pulse laser system in the world. With these advances and commitments in place, 2024 is set to show that fusion is no longer a distant dream but an achievable future of clean and sustainable energy.

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New research illuminates the ecological importance of gray wolves in the American West

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A study published in the journal BioScience sheds light on the importance of gray wolves in the western United States. Led by William Ripple, a scientist at Oregon State University and the Conservation Biology Institute, the research delves into the implications of large predator absence on plant and animal communities, and ecosystem functions. It calls attention to "shifting baselines" wherein increasingly degraded conditions are viewed as reflecting the historical state of a system.

"By the 1930s, wolves were largely absent from the American West, including its national parks . Most published ecological research from this region occurred after the extirpation of wolves," explains Ripple. "This situation underscores the potential impact of shifting baselines on our understanding of plant community succession, animal community dynamics, and ecosystem functions ."

Age structure data for deciduous trees reveal substantial ecological impacts of elk and other ungulates following the removal of gray wolves from Yellowstone, Olympic, and Wind Cave National Parks. This has led to declines in long-term tree recruitment, influencing plant communities and ecological processes.

The study highlights the necessity of characterizing historical context and reference conditions when exploring areas where large predators, like wolves, are either absent, functionally extinct, or persist in reduced densities. The authors note that such areas likely occur in many regions of the world as a result of the widespread loss of large predators.

Where applicable, the authors recommend that researchers include a discussion of how the presence or absence of large predators may have influenced their results and conclusions in future ecological studies in national parks.

"In addition to the loss or displacement of large predators, there may be other potential anthropogenic legacies within national parks that should be considered, including fire suppression, invasion by exotic plants and animals, and overgrazing by livestock," adds Dr. Robert Beschta, co-author of the study and emeritus professor at Oregon State University.

To address the effects of predator loss and other potential legacy factors, the study suggests that researchers investigate park archives to exploit historical data and information. National park archives can provide valuable insights into the history of predators and their prey, enabling scientists to discern among competing explanations for shifting ecological baselines.

"Studying altered ecosystems without recognizing how or why the system has changed over time since the absence of a large predator could have serious implications for wildlife management, biodiversity conservation , and ecosystem restoration," emphasizes Ripple.

The research underscores the importance of integrating historical context into ecological studies to provide a more comprehensive understanding of ecosystem dynamics. By acknowledging the historical presence of large predators and other anthropogenic legacies, as well as their potential ecosystem effects, researchers can contribute to more effective conservation and management strategies in national parks and beyond.

Recently, a coalition comprising nearly twelve conservation organizations initiated legal action against the U.S. Fish and Wildlife Service and the U.S. Department of the Interior. Their aim is to reinstate safeguards for gray wolves in Montana and Idaho, contending that the states' forceful hunting strategies endanger these wolf populations.

The research has implications for the long-term conservation of wolves and other large predators , including current gray wolf management and litigation in the West.

"We hope our study will be of use to both conservation organizations and government agencies in identifying ecosystem management goals," added Ripple.

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J Indian Soc Periodontol
v.20(2); Mar-Apr 2016

Antimicrobial efficacy of Tulsi leaf ( Ocimum sanctum ) extract on periodontal pathogens: An in vitro study

Sajjanshetty mallikarjun.

Department of Public Health Dentistry, Manipal College of Dental Sciences, Manipal University, Manipal, Karnataka, India

Ashwini Rao

Gururaghavendran rajesh, ramya shenoy, background:.

Periodontitis is an infection of the periodontal complex with severe forms of disease associated with specific bacteria colonizing the subgingival area. Widespread use of drugs has resulted in the emergence of side effects, uncommon infections, and resistance. Plant medicine like Tulsi has been used in many clinical conditions, and it appears to be a suitable alternative to manage conditions affecting the oral cavity. Hence, the objective was to assess the in vitro antimicrobial activity of Tulsi leaves extract ( Ocimum sanctum ) on periodontal pathogens with doxycycline as standard, as doxycycline has been used as an adjunct to nonsurgical therapy in periodontitis patients.

Materials and Methods:

Ethanolic extract of Tulsi was prepared by cold extraction method. Extract was diluted with an inert solvent, dimethyl formamide, to obtain five different concentrations (0.5%, 1%, 2%, 5%, and 10%). Doxycycline was used as a positive control and dimethyl formamide, as a negative control. The extract and controls were subjected to the microbiological investigation against Aggregatibacter actinomycetemcomitans , Prevotella intermedia , and Porphyromonas gingivalis . Agar well diffusion method was employed to determine the concentration at which Tulsi gave an inhibition zone, similar to doxycycline. Data were analyzed using one-way analysis of variance and Tukey post-hoc test was used for inter- and intra-group comparisons.

At 5% and 10% concentrations, Tulsi extracts demonstrated antimicrobial activity against A. actinomycetemcomitans , similar to doxycycline with similar inhibition zones ( P > 0.05). P. gingivalis and P. intermedia , however, exhibited resistance to Tulsi extract that showed significantly smaller inhibition zones ( P < 0.05).

Conclusions:

Tulsi demonstrated effective antimicrobial property against A. actinomycetemcomitans , suggesting its possible use as an effective and affordable “adjunct” along with the standard care in the management of periodontal conditions. However, further research assessing the toxicity, durability, and other assessments followed by clinical trials is necessary to explore the potential of Tulsi in combating oral conditions.

INTRODUCTION

Oral infections, particularly periodontitis and other periodontal conditions have been long known to be associated with aerobic and anaerobic pathogens. Oral cavity houses more than 500 different types of bacteria that are reportedly associated with different oral diseases.[ 1 ] Aggregatibacter actinomycetemcomitans has been recognized as one of the most common micro-organism, responsible for the initiation of periodontitis and is commonly associated with active periodontal lesions.[ 2 ] Levels of Porphyromonas gingivalis , Prevotell intermedia , and other anaerobic bacteria have been commonly seen to increase in chronic periodontitis.[ 3 ] While P. intermedia is more commonly associated with the advancement of necrotizing ulcerative gingivitis,[ 4 ] P. gingivalis is predominantly associated with the development of localized aggressive periodontitis.[ 5 , 6 , 7 ]

The emergence of drug resistance in human and animal pathogenic bacteria, as well as undesirable side effects of certain antibiotics, has triggered immense interest in the search for new antimicrobial alternatives of plant origin. The most important advantage claimed for therapeutic use of medicinal plants in various ailments is their safety besides being economical, effective, and easy availability.[ 8 ]

Medicinal plants are used widely by traditional medical practitioners in their day-to-day practice for curing various diseases. Phytomedicine (use of medicinal herbs for treatment), for oral disorders such as dental caries and periodontal disease, has also been well practiced in traditional medicine of Indian, Egyptian, Greek, and Chinese civilizations.[ 9 , 10 ] Different parts of Ocimum sanctum Linn (known as Tulsi), a small herb seen throughout India, have been used for various medicinal purposes. Tulsi has long been recognized as possessing antioxidant properties,[ 11 ] as a COX2 inhibitor,[ 12 ] and to provide protection from radiation poisoning[ 13 ] and cataracts.[ 14 ] Studies have also demonstrated anti-hyperlipidemia and cardioprotective effects of Tulsi in rats fed on a high-fat diet,[ 15 ] and it is also known to promote immune system function.[ 16 ] A decoction prepared from Tulsi plant is hepatoprotective, immunomodulatory, analgesic, antipyretic, and is used as a diaphoretic in malarial fever.[ 17 ] Tulsi has also been used as an important pot herb in folklore practices for a number of ailments and diseases.[ 18 ] This scenario is ever applicable to Tulsi for a wide range of conditions ranging from relatively minor illnesses such as cold or a cough to various severe conditions. O. sanctum (Tulsi) has been studied on a major scale and has shown a plethora of biological and pharmacological activities benefiting humans since ages.

However, literature pertaining to the antimicrobial activity of Tulsi on periodontal bacteria has not been reported earlier. Therefore, the present study was conceptualized as the initial step to comprehensively report the antimicrobial potential of Tulsi by assessing the inhibition zones with agar well diffusion method against three pathogens viz., A. actinomycetemcomitans , P. gingivalis and P. intermedia , which are commonly associated with periodontitis. The findings were then compared with those obtained with doxycycline, a drug used commonly as an adjunct to nonsurgical therapy for treatment of periodontitis, as a standard.[ 19 ]

MATERIALS AND METHODS

The study employed an in vitro experimental design. Ethical clearance to conduct the study was obtained from the Institutional Ethics Committee of Manipal College of Dental Sciences, Mangalore (Ref No: MCODS/198/2014).

Tulsi leaves were obtained from courtyards and local market in Mangalore city. Specimens were identified by a botanist and a pharmacognosist for their authenticity. Leaves were separated from the stem, washed in clear water and dried until they were adequately dry to be ground (dried for 7 days). Dried leaves were powdered separately in an electric grinder until a homogenous powder was obtained. Ethanolic extract was prepared from the powder obtained using “cold extraction method.”[ 20 ] A total of 250 g of finely powdered Tulsi was macerated with 100% ethanol for 3 days. The alcoholic decoction was subjected to filtration with Whatman #1 filter paper to obtain a clear filtrate. The filtrate thus obtained was reduced at a low temperature of <60°C to obtain a solid residue of Tulsi extract [ Figure 1 ]. From the 250 g of Tulsi powder dissolved in 1 L of ethanol, approximately 18 g of solid residue (extract) was obtained.

An external file that holds a picture, illustration, etc.
Object name is JISP-20-145-g001.jpg

Tulsi ( Ocimum sanctum ) extract preparation; (a) Ocimum sanctum plant; (b) leaves separated and dried; (c) leaves ground to powder; (d) extract obtained

One gram of this extract was dissolved in 10 ml of dimethyl formamide to obtain a 10% concentration of extract. Similarly, concentrations of 0.5%, 1%, 2%, and 5% of Tulsi extract were obtained by diluting with appropriate amounts of solvent. These extracts were collected in sterile containers and transported for microbiological assays. In this study, doxycycline, one of the drugs commonly employed in the treatment of aggressive periodontitis, was used as a positive control and dimethyl formamide, the solvent used in the extract preparation, was used as a negative control.

Microbiological assay

Agar well diffusion method was used to determine the antimicrobial activity of Tulsi leaves extract in vitro . Blood agar was used to culture different micro-organisms examined in this study. Colonies of microorganisms were transferred to the agar plates using a swab, and their turbidity was visually adjusted with the broth to equal that of a 0.5 McFarland turbidity standard that had been vortexed. Within 15 min of adjusting the inoculum to a McFarland 0.5 turbidity standard, a sterile cotton swab was dipped into the inoculum and rotated against the wall of the tube above the liquid to remove excess inoculum. The entire surface of agar plate was then swabbed 3 times with the cotton swab, transferring the inoculum, while the plates were rotated by approximately 60° between streaks to ensure even distribution. The overall procedure of inoculum preparation and inoculation of culture media remained the same for all three bacteria. Each bacterium was inoculated on five agar plates for five respective concentrations (0.5%, 1%, 2%, 5%, and 10%) of the Tulsi extract. Therefore, a total of 15 plates were inoculated to test all the three bacteria.

The inoculated plate was allowed to stand for at least 3 min but no longer than 15 min, before making wells for different compounds to be tested. A hollow tube of 5 mm diameter was heated and pressed above the inoculated agar plates. It was removed immediately by making a well in the plate; likewise, three wells on each plate were made, one each for positive control, negative control and Tulsi extract [ Figure 2 ]. Each well received 5 µl of respective compound assigned for it. Plates were incubated at 37°C in an incubator within 15 min of compound application. Incubation was done for 48 h for both aerobic ( A. actinomycetemcomitans ) and anaerobic ( P. gingivalis and P. intermedia ) bacteria. For anaerobic organisms, plates were incubated in the McIntosh and Filde's anaerobic jar, while aerobic micro-organism was cultured in the incubator at 37°C for 48 h.

An external file that holds a picture, illustration, etc.
Object name is JISP-20-145-g002.jpg

Wells made in blood agar for Tulsi, doxycycline, and dimethylformamide

After the incubation period, plates were read only if the lawn of growth was confluent or nearly confluent. The diameter of inhibition zone was measured to the nearest whole millimeter by using a Vernier Calliper. The microbiological procedure was repeated 4 times for each bacterium, and corresponding four values of zones of inhibition for each concentration of Tulsi extract along with doxycycline and dimethyl formamide were obtained for each of the three bacteria. The values so obtained were compared within the group (same concentration of extract) and with different groups (different concentrations of extract) and also with the positive control (doxycycline) for different bacteria [ Figure 3 ].

An external file that holds a picture, illustration, etc.
Object name is JISP-20-145-g003.jpg

ones of inhibition produced by Tulsi at different concentrations. (a) Against Porphyromonas gingivalis ; (b) 5% Tulsi against Aggregatibacter actinomycetemcomitans ; (c) 10% Tulsi against Aggregatibacter actinomycetemcomitans ; (d) against Prevotella intermedia

Statistical analysis was done using the software Statistical Package for Social Sciences (SPSS, version 16.0; SPSS Inc., Chicago, IL, USA). The disc diffusion values of different concentrations of Tulsi leaves extract, positive and negative controls against all the bacteria were entered in the SPSS software for statistical analysis. Descriptive statistics was retrieved, and data were analyzed using one-way analysis of variance (ANOVA), and Tukey post-hoc test was used for comparison within the group and with different groups. Statistical significance level was established at P < 0.05.

Zones of inhibition displayed by Tulsi extract (at different concentrations), positive and negative controls against A. actinomycetemcomitans , P. gingivalis and P. intermedia are compiled in Table 1 . The least zones of inhibition were displayed by the negative control and doxycycline exhibited the widest zones of inhibition against all the three bacteria. Tulsi leaves' extract showed increasing zones of inhibition with increasing concentration against all the three bacteria. Mean zone of inhibition for each concentration and each bacterium was calculated for analysis [ Figure 4 ]. All the concentrations of Tulsi and dimethyl formamide showed minimal inhibitory zones against P. gingivalis and P. intermedia . Against A. actinomycetemcomitans , Tulsi leaves extract at a concentration of 0.5%, 1%, and 2% and dimethyl formamide showed a minimal zone of inhibition. However, Tulsi displayed wide inhibition zones at 5% and 10%. All the bacteria tested showed susceptibility to doxycycline explained by the wide inhibition zones displayed [ Table 1 ].

Zones of inhibition (in mm)

An external file that holds a picture, illustration, etc.
Object name is JISP-20-145-g004.jpg

Mean zones of inhibition by Tulsi, doxycycline and dimethylformamide against Aggregatibacter actinomycetemcomitans , Porphyromonas gingivalis and Prevotella intermedi

One-way ANOVA revealed an increase in the mean zones of inhibition against all the three bacteria with increasing concentration of Tulsi, which was statistically significant ( P < 0.001). Comparison with doxycycline revealed insignificant inhibition zones by all the concentrations of Tulsi against P. gingivalis and P. intermedia . Similar results were obtained on a comparison of inhibition zones exhibited by Tulsi, at 0.5%, 1%, and 2% concentrations and doxycycline against A. actinomycetemcomitans . However, at 5% and 10%, Tulsi showed wider zones of inhibition against A. actinomycetemcomitans , which were similar to doxycycline in comparison [ Table 2 ].

Antimicrobial efficacy of Tulsi leaf extracts (ANOVA and post-hoc -Tukey)

An external file that holds a picture, illustration, etc.
Object name is JISP-20-145-g006.jpg

Post-hoc tests revealed a significant difference in the antimicrobial efficacy of doxycycline and Tulsi against P. gingivalis and P. intermedia , with mean zone of inhibition obtained by doxycycline clearly exceeding the mean zones of inhibition obtained by different concentrations of Tulsi. The only exception in this phenomenon was that of Tulsi at a concentration of 5% and 10%, both of which showed antimicrobial efficacy against A. actinomycetemcomitans , similar to that of doxycycline [ Table 2 ].

With due consideration to available evidence pertaining to side effects and emergence of uncommon infections with the usage of synthetic antimicrobial agents,[ 21 , 22 ] including tetracyclines,[ 23 ] particularly doxycycline[ 24 , 25 ] which is used most commonly for management of aggressive periodontitis; this study was conducted in the quest of identifying Tulsi as a possible alternative or an “adjunct” in the treatment of aggressive periodontitis. Several plant products such as Tulsi, neem, lemon, and others have been tested for their antimicrobial properties in the past[ 26 , 27 , 28 , 29 , 30 ] with considerable success. Resistance to currently used chemotherapeutics is the major factor that necessitates the search for alternative safe, efficacious, and cost-effective treatment options, particularly in developing countries.[ 31 ]

In this study, we attempted to obtain information on the antimicrobial efficacy of Tulsi, particularly against three periodontal pathogens namely A. actinomycetemcomitans , P. intermedia and P. gingivalis ; as these microbes are more commonly associated with initiation and progression of various periodontal diseases, especially aggressive periodontitis.[ 2 , 32 , 33 , 34 , 35 , 36 , 37 , 38 ] Results in this in vitro experiment showed that Tulsi at a concentration of 5% and 10% can effectively inhibit the growth of A. actinomycetemcomitans , comparable to that of doxycycline. Different mechanisms of action of Tulsi have been proposed by many authors earlier. Vishwabhan et al .[ 39 ] proposed the antimicrobial activity of Tulsi by virtue of its essential oil content. Various other studies also project the idea that Tulsi yields various essential oils which are responsible for the medicinal uses including antimicrobial, antioxidant, antifungal, and anti-inflammatory activities that can probably explain its activity against the microbes discussed so far.[ 40 , 41 , 42 ] It has also been postulated that Tulsi has an immunomodulatory effect and acts by increasing the levels of interferon, interleukin-4 and T helper cells that can strengthen host response to infections.[ 16 ] Singhal et al .[ 41 ] proposed that the antibacterial activity of Tulsi leaf extract could be attributed to its ability to reduce silver ions to silver nanoparticles that possess antibacterial properties against both Gram-negative and Gram-positive bacteria.

Although many previous studies like those conducted by Agarwal et al .,[ 20 , 26 ] Rathod[ 43 ] Shah et al .[ 44 ] and Prasannabalaji[ 45 ] have all shown the antimicrobial properties of Tulsi against different organisms, and there also is evidence claiming plant products being used as effective therapy against periodontitis.[ 46 ] The present study is one of the first to assess the antimicrobial efficacy of Tulsi leaves extract against periodontal pathogens, in particular associated with aggressive periodontitis. Comparisons with previous studies are not justified here due to variation in the organisms tested against Tulsi for its antimicrobial effect. Since there were scarce literature available that could depict the efficacy of Tulsi against periodontal microbes specifically, the present study encourages researchers to carry out further studies assessing toxicity, durability and other assessments followed by clinical trials to provide an insight into the activity of Tulsi against periodontal pathogens on a transient as well as longitudinal basis to establish clear implications of Tulsi in periodontal disease management. With the basic limitations of the study design, generalizability is a possibility with further accumulation of evidence in this regard. However, within the limitations of the present study it could be concluded that Tulsi, as an adjunct, “if” found effective and safe on further research would be considered as a potential “adjunct” along with the standard care in the management of periodontitis to overcome the side effects of synthetic drugs, especially in this era of ever advancing clinical dentistry.

Financial support and sponsorship

Conflicts of interest.

There are no conflicts of interest

Acknowledgement

Dr. Ashok Shenoy, head of the department, Department of Pharmacology, Kasturba Medical College, Mangalore, Manipal University, Karnataka, for aiding in on the Tulsi leaves extract preparation. Dr. Kishore G Bhat, head of the department, Department of Molecular Biology and Immunology, Maratha Mandal's Nathajirao G Halgekar Institute of Dental Sciences and Research Centre, Belgaum, Karnataka, for the support provided in conducting microbiological assays.

Fields Of Discovery: A hunger to research carnivorous plants

Story by Zachary Hodges
Photos by Katherine Jacobson
June 17, 2024

A web banner reading "Fields of Discovery."

T hree years ago, high school senior Jonathan Lu attended a Zoom meeting for prospective students with Purdue University’s Department of Botany and Plant Pathology . At the meeting, Lu chatted feverishly with botany professor Scott McAdam about their shared passion for plants. While the meeting eventually ended, their shared pursuit of knowledge had only begun.   

Once Lu started as a botany student at Purdue, Lu enrolled in Botany 120: Principles of Plant Biology 1 with McAdam. Lu was drawn in by McAdam’s approach to the course. “I liked his way of thinking about the world.”  Not thinking that McAdam would remember their interaction over Zoom, Lu ended up asking McAdam if he researched carnivorous plants. To Lu’s surprise, McAdam told Lu that he recalled their conversation about Venus flytraps. “Before I knew it, I was brought into the lab. He just said, ’Make yourself at home.’” 

Today, Lu works in McAdam’s lab researching the anatomy, morphology and desiccation tolerance of carnivorous plants. Generally, the team is examining how carnivorous plants like Venus flytraps withstand severe droughts. 

Another facet of Lu's research is in the anatomy of Nepenthes pitcher plants. “These plants are tropical pitcher plants where bugs fall in their traps. Inside the traps, there are these little moon-shaped lunate structures. These structures were once believed to be stomata, which are structures that allow plants to breathe. Upon looking closely at the structures, we found that that’s not true. We’re just finding out now that these structures are unique. Now, I'm trying to discover how they develop.” 

Lu’s passion for carnivorous plant research began early. When he was four years old, he got his first Venus flytrap. “It grew, and it grew, and then I killed it,” Lu recalls. “And then, we got a new one, and I killed that. And then, we got another one. It was a cycle.” 

Finally, Lu was able to keep a Venus flytrap alive for one year. Soon, he started a collection of carnivorous plants at home in a small dish. Over time, that collection grew and expanded into kiddie pools and grow racks.

Jonathan Lu examines a plant in the Lilly Greenhouses.

Lu explains that carnivorous plants are a niche topic in a secluded field. “In my eyes, these are understudied plants,” he explains. 

The lab examines the new ways these plants approach stimuli. For example, they might look at what structures a carnivorous plant uses to kill bugs. To complement that research, they could find a non-carnivorous plant with the same structures. This would allow them to examine the similarities and differences between the plant structures to learn more about how plants function.  

“The work is very foundational,” says Lu. 

Lu expects to graduate in Spring 2026. Post graduation, he plans to go into academia and potentially work towards higher degrees. Ultimately, he wants to delve into the mysteries of the function and evolution of carnivorous plants and why they are so rare. 

Like his beloved carnivorous plants, Lu has a hunger. He seeks to understand these under-valued plants. It takes care and resilience to study a field that many overlook, but Lu is used to the process of tending to his work. From kiddie pools to collegiate labs, he’s been doing it all his life.

Jonathan Lu sits among the plants he is researching this summer.

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COMMENTS

Tulsi
Modern day scientific research into tulsi demonstrates the many psychological and physiological benefits from consuming tulsi and provides a testament to the wisdom inherent in Hinduism and Ayurveda, which celebrates tulsi as a plant that can be worshipped, ingested, made into tea and used for medicinal and spiritual purposes within daily life.
A Review Paper on Tulsi Plant (Ocimum sanctum L.)
Tulsi (Ocimum sanctum L.) in Hindi or Tulasi in Sanskrit (holy basil in English) is an exceptionally adored cul inary. and rest orative frag rant herb from the family Lamiaceae that is indigenous ...
The Clinical Efficacy and Safety of Tulsi in Humans: A Systematic
The study durations ranged from 2 to 13 weeks and tulsi dosage and frequency varied from 300 mg to 3000 mg given as 1-3 times per day as tulsi leaf aqueous extract; 300 mg-1000 mg once or twice per day as tulsi leaf ethanolic extract; 6 g to 14 g per day as the tulsi whole plant aqueous extract; and 10 g fresh tulsi leaf aqueous extract ...
Tulsi
Of all the herbs used within Ayurveda, tulsi (Ocimum sanctum Linn) is preeminent, and scientific research is now confirming its beneficial effects. There is mounting evidence that tulsi can address physical, chemical, metabolic and psychological stress through a unique combination of pharmacological actions. Tulsi has been found to protect ...
The Cultural and Commercial Value of Tulsi (Ocimum tenuiflorum L
Tulsi is part of various traditional medical systems including: Ayurveda, Siddha, and Unani. Ayurvedic scriptures refer to Tulsi as one of the main pillars of herbal medicine, first mentioned in the Rig Veda around 1500 BC . Tulsi is one of the most sacred plants on the Indian subcontinent, as it represents a Hindu goddess, Virinda Tulsi.
(PDF) A review medicinal and traditional uses on Tulsi plant (Ocimum
The research suggests that tulsi is a potential therapy option for way of life and is stronger than conventionally utilised methods. ... The Tulsi plant has a variety of jobs. Tulsi leaves are widely
The Clinical Efficacy and Safety of Tulsi in Humans: A ...
Many in vitro, animal and human studies attest to tulsi having multiple therapeutic actions including adaptogenic, antimicrobial, anti-inflammatory, cardioprotective, and immunomodulatory effects, yet to date there are no systematic reviews of human research on tulsi's clinical efficacy and safety. We conducted a comprehensive literature review ...
Tulsi
The aromatic perennial plant (Ocimum sanctum L.) belongs to the Lamiaceae family (Labiatae). Ocimum sanctum [] is known as "Holy Basil" in English and "Tulsi" or "Tulasi" in Hindi and is regarded as a sacred plant in Hindu culture.Tulsi is a popular plant in India, especially in religious gardens and residential locations Tulsi is naturally grown.
Ocimum tenuiflorum
Ocimum tenuiflorum, commonly known as holy basil or tulsi, is an aromatic perennial plant in the family Lamiaceae. It is native to tropical and subtropical regions of Australia, Malesia, Asia, and the western Pacific. It is widely cultivated throughout the Southeast Asian tropics. This plant has escaped from cultivation and has naturalized in many tropical regions of the Americas.
Tulsi (Ocimum sanctum)
Tulsi (Ocimum sanctum) is an omnipresent, multipurpose plant and regarded as holy plant in Hindu religion finds place in front of every Hindu household. This herb is a member of Lamiaceae family ...
Plants
Tulsi (Holy basil, Ocimum tenuiflorum L., Lamiaceae), native to Asia, has become globalised as the cultural, cosmetic, and medicinal uses of the herb have been popularised. DNA barcoding, a molecular technique used to identify species based on short regions of DNA, can discriminate between different species and identify contaminants and adulterants. This study aimed to explore the values ...
PDF A Review Paper on Tulsi Plant (Ocimum sanctum L.)
JETIR2201574 Journal of Emerging Technologies and Innovative Research (JETIR) www.jetir.org g515 A Review Paper on Tulsi Plant (Ocimum sanctum L.) Dr. Sudhir Kumar Mishra, Principal, S.S. College, Jehanabad, ABSTRACT Tulsi (Ocimum sanctum L.), holly basil, is indigenous to the Indian mainland and profoundly respected for its restorative uses ...
PDF Multiple health benefits of Tulsi plants
regard tulsi (Ocimum sanctum Linn), and scientific research has confirmed its benefits. With its unique combination of pharmacological actions, tulsi has demonstrated its ability to address physical, chemical, ... Tulsi plant's roots, stem, fruit, and leaves have several phytochemical constituents that impart different
PDF Tulsi-botanical variants, uses, constituents, and mode of working
Tulsi is one among the plants that has potential to do so. The traditional sacred plant of India known for its medicinal and other uses is more relevant today than ever before. ... Research results affirmed that the plant's unique composition of the pharmacological constituents can lower metabolic and psychological stress often resulting in ...
Selection and Micropropagation of an Elite Melatonin Rich Tulsi ...
Tulsi (Ocimum sanctum L.) is a sacred plant of medicinal and spiritual significance in many cultures. Medicinal properties of Tulsi are ascribed to its phytochemicals with antioxidant capabilities. The current study was undertaken to screen a large seed population of Tulsi to select germplasm lines with high antioxidant potential and to standardize protocols for micropropagation and biomass ...
An Update on the Therapeutic Anticancer Potential of
The tulsi plant is a bushy shrub or small tree (Figure 1 A) native to the tropics and subtropics. It smells and tastes completely different from anything else. ... The possible health benefits of tulsi leaves have been the subject of research . A recent study by Enegide et al. reviewed the Ocimum species and its ethnomedicinal uses in 2021 .
Genome sequencing of herb Tulsi
Krishna Tulsi, a member of Lamiaceae family, is a herb well known for its spiritual, religious and medicinal importance in India. The common name of this plant is 'Tulsi' (or 'Tulasi' or 'Thulasi') and is considered sacred by Hindus. We present the draft genome of Ocimum tenuiflurum L (subtype Krishna Tulsi) in this report. The paired-end and mate-pair sequence libraries were ...
Plant Cell and Tissue Culture: Propagation, Improvement, and ...
Plant tissue culture, a revolutionary technique in plant biology, is a highly effective method for multiplying, biodiversity conservation, and genetic manipulation and enhancement of plants. ... the identification and production of mutants are essential to the molecular research progress on model plant species. 11.4 Germplasm Conservation. More ...
(PDF) Review Article Traditional Indian Herbal Plants Tulsi and Its
The Rama Tulsi is the effective remedy for the severe acute Respiratory Syndrome. Juice of its leaves gives relief in cold, fever, bronchitis and cough. Tulsi oil is also used as the ear drop ...
Interaction with insects accelerates plant evolution
Oct. 26, 2020 — A research group has discovered that insects have a decisive influence on the biodiversity and flowering phases of plants. If there is a lack of insects where the plants are ...
More than 800 coal plants worldwide could be profitably decommissioned
More than 800 coal-fired power plants in emerging countries could be decommissioned and profitably replaced by cleaner solar energy starting from the end of the decade, research on Monday showed.
Fusion Sparks an Energy Revolution
These cutting-edge facilities are smaller than fusion power plants. For instance, a laser-based fusion demonstrator might use five to ten laser beams, while a commercial power plant can use ...
Blessing in disguise: Mycoviruses enhance fungicide effectiveness
Aug. 16, 2019 — Plants can tell the time, and this affects their responses to certain herbicides used in agriculture according to new research. The study found that plant circadian rhythms ...
Non-native plants and animals expanding ranges 100-times faster than
Interaction with insects accelerates plant evolution, research finds. 11 hours ago. Look to women for sustainable livestock farming bordering the Amazon rainforest, says study. Jun 18, 2024.
PDF A review on: Indian traditional shrub Tulsi (ocimum sanctum): The
Tulsi plant are present. 1. Tulsi plants with green leaves known as Ram Tulsi 2. Tulsi plants with purple leaves known as Krishna Tulsi. Tulsi medicinal property as per ayurveda Tulsi has anti-inflammatory properties as it undermined vata. So its external application on swollen area helps to reduce swelling and pain.
PDF The Cultural and Commercial Value of Tulsi (Ocimum tenuiflorum L
whole plant can be used, seeds, roots, leaves, ﬂowers and even the stem [6]. Fresh or dried Tulsi leaves can be consumed, infused in teas, or used in cooking for systemic effects. Bathing in Tulsi infused water has been recommended for topical skin conditions such as eczema. A variety of commercial Tulsi products are also available including ...
New research illuminates the ecological importance of gray wolves in
Non-native plants and animals expanding ranges 100-times faster than native species, finds new research 16 hours ago How glacier algae are challenging the way we think about evolution
Antimicrobial efficacy of Tulsi leaf (Ocimum sanctum) extract on
Plant medicine like Tulsi has been used in many clinical conditions, and it appears to be a suitable alternative to manage conditions affecting the oral cavity. Hence, the objective was to assess the in vitro antimicrobial activity of Tulsi leaves extract ( Ocimum sanctum ) on periodontal pathogens with doxycycline as standard, as doxycycline ...
(PDF) TULSI: An Effective Medicinal Plant
The plant is rich in tannins, phenols, ald ehydes, fats and saponin. It is also rich in many. essential oil components like eugenol, ecarvacrol, etc. Propagation and Growth. Tulsi plant is ...
Fields Of Discovery: A hunger to research carnivorous plants
Generally, the team is examining how carnivorous plants like Venus flytraps withstand severe droughts. Another facet of Lu's research is in the anatomy of Nepenthes pitcher plants. "These plants are tropical pitcher plants where bugs fall in their traps. Inside the traps, there are these little moon-shaped lunate structures.

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The Clinical Efficacy and Safety of Tulsi in Humans: A Systematic Review of the Literature

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Genome sequencing of herb Tulsi ( Ocimum tenuiflorum ) unravels key genes behind its strong medicinal properties

Conclusions

Genome sequencing and assembly of the non-model plant O. tenuiflorum subtype Krishna

Validation of de novo genome assembly, annotation and repeat content of Ocimum tenuiflorum subtype Krishna genome

Genome annotation

Orthology of Tulsi genes

Genes involved in synthesis of specialized metabolites of medicinal value: comparative analysis between O. tenuiflorum (Ote, Krishna Tulsi) and other plant genomes

Transcriptome de novo assembly of Krishna and Rama Tulsi mature leaf samples

Differential expression of transcripts

Specialized metabolites detection and validation

Isolation of genomic DNA from O. tenuiflorum subtype Krishna Tulsi

Genome sequencing, assembly and annotation

Validation of genome assembly and annotations

Repeat identification

Orthology detection

Comparative analysis between Krishna Tulsi and other plant genomes

Isolation of RNA from Tulsi subtypes, Krishna and Rama, and RNA-seq library preparation

Transcriptome sequencing and assembly

Transcript differential expression comparison

Quantification of eugenol and ursolic acid using UHPLC-MS/SRM method

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Plant Cell and Tissue Culture: Propagation, Improvement, and Conservation of Medicinal Plants

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