Endangered Medicinal Herb Research Articles

Molecular characterization and differential expression of an aromatic heptaketide producing type III plant polyketide synthase from Himalayan rhubarb

Rheum australe (Himalayan Rhubarb, Polygonaceae), an endangered medicinal and vegetable herb owes its age-old remedying properties to the bio-active phyto-constituents viz. anthraquinones, stilbenoids, chromones and dietary flavonoids. Polyketide pathway primarily involving the intricate Type III polyketide synthases (PKSs) contributes to the biosynthesis of these phyto-constituents. In the present study, we perform a homology-based approach to isolate an 1176 bp full-length cds sequence of the RaALS gene showing an equitable level of sequence similarity to related Type III PKSs at both nucleic acid and amino acid levels. In silico characterization revealed the presence of highly conserved amino acid residues found in nearly all Type III PKSs including the conserved active-site residues, signature motif and cyclization pocket residues with an exception of Ile256 and Gly258. Docking studies established major interactions between the starter acetyl-CoA and RaALS. Copy number analysis suggested slender evolution in Type III PKS in R. australe having a single copy of RaALS gene. qRT-PCR analyses revealed corroboration between the higher expression of RaALS in leaves followed by stem and root with that of the metabolite concentration. Expression studies further showed a direct increase of RaALS transcripts with the growing metabolite accretion in relation to altitude suggesting a probable involvement of specific Type III PKS in biosynthesis of the major phyto-constituents. Furthermore, abiotic stressors viz. methyl jasmonate, salicylic acid and UV light enhanced RaALS transcription hinting towards its role in defense mechanism in R. australe and highlighting the significance of RaALS as a prospective target for metabolic engineering.

Stress-Responsive cis-Regulatory Elements Underline Podophyllotoxin Biosynthesis and Better Performance of Sinopodophyllum hexandrum Under Water Deficit Conditions

Sinopodophyllum hexandrum is an endangered medicinal herb known for its bioactive lignan podophyllotoxin (PTOX), which is used for the preparation of anticancer drugs. In its natural habitat, S. hexandrum is exposed to a multitude of adversities, such as fluctuating temperatures, water deficit, and high UV radiations. Transcriptional regulation of genes, which is regulated by the condition-specific binding of transcriptional factors to precise motifs in the promoter region, underlines responses to an environmental cue. Therefore, analysis of promoter sequences could ascertain the spatio-temporal expression of genes and overall stress responses. Unavailability of genomic information does not permit such analysis in S. hexandrum, especially on regulation of PTOX pathway. Accordingly, this study describes isolation and in silico analysis of 5′-upstream regions of ShPLR (PINORESINOL-LARICIRESINOL REDUCTASE) and ShSLD (SECOISOLARICIRESINOL DEHYDROGENASE), the two key genes of the PTOX biosynthetic pathway. Data showed a range of motifs related to basal transcription, stress-responsive elements, such as those for drought, low temperature, and light, suggesting that the expression of these genes and resulting PTOX accumulation would be affected by, at least, these environmental cues. While the impact of temperature and light on PTOX accumulation is well studied, the effect of water deficit on the physiology of S. hexandrum and PTOX accumulation remains obscure. Given the presence of drought-responsive elements in the promoters of the key genes, the impact of water deficit on growth and development and PTOX accumulation was studied. The results showed decline in relative water content and net photosynthetic rate, and increase in relative electrolyte leakage with stress progression. Plants under stress exhibited a reduction in transpiration rate and chlorophyll content, with a gradual increase in osmoprotectant content. Besides, stressed plants showed an increase in the expression of genes involved in the phenylpropanoid pathway and PTOX biosynthesis, and an increase in PTOX accumulation. Upon re-watering, non-irrigated plants showed a significant improvement in biochemical and physiological parameters. Summarily, our results demonstrated the importance of osmoprotectants during water deficit and the revival capacity of the species from water deficit, wherein PTOX synthesis was also modulated. Moreover, isolated promoter sequences could be employed in genetic transformation to mediate the expression of stress-induced genes in other plant systems.

Assessment of Genetic Diversity of Rheum species (Endangered Medicinal Herb of Indian Himalayan Region) using Molecular Markers

The Rheum species are important medicinal plants that are facing extinction due to their unplanned development and overexploitation by pharmaceutical industries. DNA polymorphisms are not prone to environmental modifications, thus they are widely used for the identification and characterization of plants. The use of different molecular markers has enabled the researchers for the valuation of genetic variability and diversity in its natural zone of distribution. The conventional approach may take several years to yield this information. For the estimation of molecular and genetic variations in geographical zone of distribution, various molecular markers technique are available like RAPD (Randomly Amplified Polymorphic DNA), RFLP (Restriction fragment length polymorphism), ISSR (Inter-Simple Sequence Repeats), SSR and AFLP. The uses of different molecular markers for the study of genetic diversity have been discussed in the review.

De novo transcriptome analysis provides insights into formation of in vitro adventitious root from leaf explants of Arnebia euchroma

BackgroundAdventitious root formation is considered a major developmental step during the propagation of difficult to root plants, especially in horticultural crops. Recently, adventitious roots induced through plant tissue culture methods have also been used for production of phytochemicals such as flavonoids, anthocyanins and anthraquinones. It is rather well understood which horticultural species will easily form adventitious roots, but the factors affecting this process at molecular level or regulating the induction process in in vitro conditions are far less known. The present study was conducted to identify transcripts involved in in vitro induction and formation of adventitious roots using Arnebia euchroma leaves at different time points (intact leaf (control), 3 h, 12 h, 24 h, 3 d, 7 d, 10 d and 15 d). A. euchroma is an endangered medicinal Himalayan herb whose root contains red naphthoquinone pigments. These phytoconstituents are widely used as an herbal ingredient in Asian traditional medicine as well as natural colouring agent in food and cosmetics.ResultsA total of 137.93 to 293.76 million raw reads were generated and assembled to 54,587 transcripts with average length of 1512.27 bps and N50 of 2193 bps, respectively. In addition, 50,107 differentially expressed genes were identified and found to be involved in plant hormone signal transduction, cell wall modification and wound induced mitogen activated protein kinase signalling. The data exhibited dominance of auxin responsive (AUXIN RESPONSE FACTOR8, IAA13, GRETCHEN HAGEN3.1) and sucrose translocation (BETA-31 FRUCTOFURANOSIDASE and MONOSACCHARIDE-SENSING protein1) genes during induction phase. In the initiation phase, the expression of LATERAL ORGAN BOUNDARIES DOMAIN16, EXPANSIN-B15, ENDOGLUCANASE25 and LEUCINE-rich repeat EXTENSION-like proteins was increased. During the expression phase, the same transcripts, with exception of LATERAL ORGAN BOUNDARIES DOMAIN16 were identified. Overall, the transcriptomic analysis revealed a similar patterns of genes, however, their expression level varied in subsequent phases of in vitro adventitious root formation in A. euchroma.ConclusionThe results presented here will be helpful in understanding key regulators of in vitro adventitious root development in Arnebia species, which may be deployed in the future for phytochemical production at a commercial scale.

Polyphenol Rich Ajuga bracteosa Transgenic Regenerants Display Better Pharmacological Potential.

Ajuga bracteosa Wall. ex Benth. is an endangered medicinal herb traditionally used against different ailments. The present study aimed to create new insight into the fundamental mechanisms of genetic transformation and the biological activities of this plant. We transformed the A. bracteosa plant with rol genes of Agrobacterium rhizogenes and raised the regenerants from the hairy roots. These transgenic regenerants were screened for in vitro antioxidant activities, a range of in vivo assays, elemental analysis, polyphenol content, and different phytochemicals found through HPLC. Among 18 polyphenolic standards, kaempferol was most abundant in all transgenic lines. Furthermore, transgenic line 3 (ABRL3) showed maximum phenolics and flavonoids content among all tested plant extracts. ABRL3 also demonstrated the highest total antioxidant capacity (8.16 ± 1 μg AAE/mg), total reducing power, (6.60 ± 1.17 μg AAE/mg), DPPH activity (IC50 = 59.5 ± 0.8 μg/mL), hydroxyl ion scavenging (IC50 = 122.5 ± 0.90 μg/mL), and iron-chelating power (IC50 = 154.8 ± 2 μg/mL). Moreover, transformed plant extracts produced significant analgesic, anti-inflammatory, anticoagulant, and antidepressant activities in BALB/c mice models. In conclusion, transgenic regenerants of A. bracteosa pose better antioxidant and pharmacological properties under the effect of rol genes as compared to wild-type plants.

The first draft genome of Picrorhiza kurrooa, an endangered medicinal herb from Himalayas

Picrorhiza kurrooa is an endangered medicinal herb which is distributed across the Himalayan region at an altitude between 3000–5000 m above mean sea level. The medicinal properties of P. kurrooa are attributed to monoterpenoid picrosides present in leaf, rhizome and root of the plant. However, no genomic information is currently available for P. kurrooa, which limits our understanding about its molecular systems and associated responses. The present study brings the first assembled draft genome of P. kurrooa by using 227 Gb of raw data generated by Illumina and PacBio RS II sequencing platforms. The assembled genome has a size of n = ~ 1.7 Gb with 12,924 scaffolds. Four pronged assembly quality validations studies, including experimentally reported ESTs mapping and directed sequencing of the assembled contigs, confirmed high reliability of the assembly. About 76% of the genome is covered by complex repeats alone. Annotation revealed 24,798 protein coding and 9789 non-coding genes. Using the assembled genome, a total of 710 miRNAs were discovered, many of which were found responsible for molecular response against temperature changes. The miRNAs and targets were validated experimentally. The availability of draft genome sequence will aid in genetic improvement and conservation of P. kurrooa. Also, this study provided an efficient approach for assembling complex genomes while dealing with repeats when regular assemblers failed to progress due to repeats.

A SIMPLE AND EFFICIENT MICROPROPAGATION PROTOCOL FOR DEVELOPING PLANTLETS OF EXACUM BICOLOR ROXB. – AN ENDANGERED, ORNAMENTAL, AND ANTIDIABETIC HERB

Objective: Exacum bicolor Roxb. is an endangered medicinal herb due to overexploitation by humans and its inefficient vegetative reproduction. Here, we report an efficient and simple procedure for the regeneration of E. bicolor Roxb. using leaf as an explant.
 Methods: The optimal concentrations of the hormones needed for callus induction were determined by full factorial method using DOE (Design expert ver. 8.0). The hormones selected based on literature were kinetin, indole acetic acid, and 6-Benzylaminopurine (BAP). Multiple shoot regeneration was carried out in liquid and solid media with the optimal concentrations of the hormones obtained by DOE. Rooting was initiated using Murashige and Skoog media containing naphthalene acetic acid 0.5 mg/l, indole butyric acid (IBA) 1.0 mg/l, and gibberellic acid 3 0.5 mg/l along with 0.2% of activated charcoal.
 Results: Analysis of full factorial design run showed that BAP in combination with kinetin was effective for the growth of callus and multiple shoot regeneration was higher in liquid media (81.25%). The rate of rooting was observed to be 88.23% and the average number of roots was 0.26. Plantlets with budding apical region and well-established leaves and roots were observed in 30 days.
 Conclusion: The protocol reported here can be used for effective production of E. bicolor plants in a shorter duration compared to the conventional approach.

Optimisation of gentiopicroside production in Gentiana kurroo Royle from adventitious root cultures in a liquid culture system

Gentiana kurroo Royle is a critically endangered medicinal herb of the Indian Himalaya. It has bioactive seco-iridoid glycosides, notably gentiopicroside, in the rhizome and roots. In this study, we report on the in vitro production of gentiopicroside (GPD) from adventitious (AD) roots induced directly from the leaf, nodal, and suspension cell cultures of G. kurroo. Murashige and Skoog (MS) media fortified with Indole-3-butyric acid (IBA) 2 mg L−1 + Naphthaleneacetic acid (NAA) 0.5 mg L−1 and IBA 2 mg L−1 + Indole-3-acetic acid (IAA) 1 mg L−1 produced the maximum number of roots. Suspension cell cultures derived AD roots showed a 1.41-fold higher biomass production than adventitious roots induced from leaf and nodal explants. Among the various concentrations of MS media salts evaluated, half-strength MS suspension media favored the higher biomass and GPD accumulation. The maximum accumulation of GPD (2.58 mg g –l dry weight (DW)) and AD root biomass (18.96 g L−1 DW) was observed on the 48th day. Furthermore, GPD produced from AD root cultures were separated by using TLC, characterized using Nuclear Magnetic Resonance (NMR) and High-Resolution Liquid Chromatograph-Mass Spectrometer (HR LC-MS) and quantified with High-Performance Liquid Chromatography (HPLC).

In-vitro cytotoxicity in relation to chemotypic diversity in diploid and tetraploid populations of Gentiana kurroo Royle

Ethnopharmacological relevanceGentiana kurroo is a multipurpose critically endangered medicinal herb prescribed as medicine in Ayurveda in India and exhibits various pharmacological properties including anti-cancer activity. The species is rich repository of pharmacologically active secondary metabolites together with secoiridoidal glycosides. Aim of the studyThe study aimed to investigate the chemical diversity in different populations/cytotypes prevailing in G. kurroo to identify elite genetic stocks in terms of optimum accumulation/biosynthesis of desired metabolites and having higher in-vitro cytotoxicity potential in relation to chemotypic diversity. Material and methodsThe wild plants of the species were collected from different ranges of altitudes from the Kashmir Himalayas. For cytological evaluation, the standard meiotic analysis was performed. The standard LC-MS/MS technique was employed for phytochemical analysis based on different marker compounds viz. sweroside, swertiamarin, and gentiopicroside. Different tissues such as root-stock, aerial parts, and flowers were used for chemo-profiling. Further, the methanolic extracts of diploid and tetraploid cytotypes were assessed for cytotoxic activity by using MTT assay against four different human cancer cell lines. ResultsThe quantification of major bioactive compounds based on tissue- and location-specific comparison, as well as in-vitro cytotoxic potential among extant cytotypes, was evaluated. The comprehensive cytomorphological studies of the populations from NW Himalayas revealed the occurrence of different chromosomal races viz. n = 13, 26. The tetraploid cytotype was hitherto unreported. The tissue-specific chemo-profiling revealed relative dominance of different phytoconstituents in root-stock. There was a noticeable increase in the quantity of the analyzed compounds in relation to increasing ploidy status along the increasing altitudes. The MTT assay of methanolic extracts of diploid and tetraploid cytotypes displayed significant cytotoxicity potential in tetraploids. The root-stock extracts of tetraploids were highly active extracts with IC50 value ranges from 5.65 to 8.53 μg/mL against HCT-116 colon cancer. ConclusionThe chemical evaluation of major bioactive compounds in diverse cytotypes from different plant parts along different altitudes presented an appreciable variability in sweroside, swertiamarin, and gentiopicroside contents. Additionally, the concentrations of these phytoconstituents varied for cytotoxicity potential among different screened cytotypes. This quantitative difference of active bio-constituents was in correspondence with the growth inhibition percentage of different tested cancer cell lines. Thus, the present investigation strongly alludes towards a prognostic approach for the identification of elite cytotypes/chemotypes with significant pharmacological potential.

Comparative transcriptome analysis of Rheum australe, an endangered medicinal herb, growing in its natural habitat and those grown in controlled growth chambers

Rheum australe is an endangered medicinal herb of high altitude alpine region of Himalayas and is known to possess anti-cancerous properties. Unlike many herbs of the region, R. australe has broad leaves. The species thrives well under the environmental extremes in its niche habitat, therefore an understanding of transcriptome of R. australe to environmental cues was of significance. Since, temperature is one of the major environmental variables in the niche of R. australe, transcriptome was studied in the species growing in natural habitat and those grown in growth chambers maintained at 4 °C and 25 °C to understand genes associated with different temperatures. A total of 39,136 primarily assembled transcripts were obtained from 10,17,74,336 clean read, and 21,303 unigenes could match to public databases. An analysis of transcriptome by fragments per kilobase of transcript per million, followed by validation through qRT-PCR showed 22.4% up- and 22.5% down-regulated common differentially expressed genes in the species growing under natural habitat and at 4 °C as compared to those at 25 °C. These genes largely belonged to signaling pathway, transporters, secondary metabolites, phytohormones, and those associated with cellular protection, suggesting their importance in imparting adaptive advantage to R. australe in its niche.

Comparative transcriptome analysis infers bulb derived in vitro cultures as a promising source for sipeimine biosynthesis in Fritillaria cirrhosa D. Don (Liliaceae, syn. Fritillaria roylei Hook.) - High value Himalayan medicinal herb

Fritillaria cirrhosa D. Don (Liliaceae, syn. Fritillaria roylei Hook.) is a critically endangered medicinal herb of immense importance due to its pharmaceutical bioactive compound, especially sipeimine, used for the treatment of chronic respiratory disorders. However, the industrial demand for sipeimine solely depends on its endangered natural habitat. Therefore; there is an utmost need for its biodiversity conservation as well as for the sustainable utilization of phytochemicals. Plant cell culture and transcriptomics-based molecular bioprospection of key regulatory genes involved in sipeimine biosynthesis as such will play a crucial role in exploring the unexplored traits, that are in supply crisis or nearly in extinction stage. De novo comparative transcriptome sequencing of the bulb (in vivo), callus, and regenerated plantlets (in vitro) resulted in more than 150 million high-quality paired-end clean reads that assembled into final 31,428 transcripts. Functional annotation and unigenes classification with multiple public databases such as KEGG, Refseq, Uniprot, TAIR, GO, and COG, etc. along with chemical structures and functional biocatalytic activity analysis of different steroidal alkaloids facilitated the identification of 30 unigenes specific to sipeimine biosynthesis. Additionally, ABC transporters and TFs like bHLH, MYC, MYB, and WRKY suggests their possible role in metabolite translocation and regulation in vivo as well as in vitro tissues. Differential gene expression and quantitative analysis revealed that the MVA pathway probably the predominant route for 5C intermediate (IPP & DMAPP) biosynthesis. Further, the genes involved in the downstream biosynthesis pathway viz. SQLE, CAS1, SMT1, SMO1, SMO2, SC5DL, DHCR7, DHCR24, CYP710A, 3β-HSD, CYP90D2, and CYP374A6 shown similar expression pattern with RNA-Seq and qRT-PCR findings. The positive correlation between higher expression of proposed biosynthetic pathway genes and relatively higher accumulation of sipeimine in differentiated naturally grown bulb tissues (in vivo), undifferentiated cells (callus), and de-differentiated tissues i.e. regenerated plantlets (in vitro) has been evident from the present study. Comprehensive genomic resources created in F. cirrhosa will provide strong evidence of bulb derived in vitro culture as an alternative promising source for steroidal alkaloids biosynthesis and metabolite upscaling through genetic and metabolic engineering.

Micropropagation of Atropa acuminata Royle ex Lindl. (a critically endangered medicinal herb) through root callus and evaluation of genetic fidelity, enzymatic and non-enzymatic antioxidant activity of regenerants

Atropa acuminata Royle ex Lindl. (family Solanaceae) is a critically endangered therapeutic herb, known for its immense therapeutic potential against innumerable ailments. In view of protecting this species from brink of extinction, present work focuses upon development of an effectual micropropagation protocol via indirect organogenesis using root-derived callus. Best callusing was observed on Murashige and Skoog (MS) medium augmented with 10 µM Thidiazuron (TDZ), where 99% of root explants induced whitish-green and compact calluses within 45 days. Upon sub-culturing on same medium (10 µM TDZ), these callus pieces differentiated an average of 5.54 ± 0.65 shoots having 2.38 ± 0.37 cm of length in 71.5% of the explants, after 28 days. MS + 1 µM indole-3-butyric acid (IBA) resulted in optimum rooting response where an average of 8.08 ± 0.19 roots having an average length of 7.28 ± 0.11 cm were produced. Micropropagated plants with well-developed and thick roots were acclimatized under greenhouse conditions. Genetic fidelity of ex vitro acclimatized plants using start codon targeted (SCoT) and sequence-related amplified polymorphism (SRAP) markers, revealed 96% similarity between mother plant and micropropagated plants. These plants showed higher yield of phenols, flavonoids and exhibited better antioxidant (DPPH and phosphomolybdenum assay) activities in comparison with mother plant. Higher levels of photosynthetic pigments and antioxidant enzymes were also observed in these plants as compared to the mother plant. Besides, quantification of atropine and scopolamine using high-performance liquid chromatography (HPLC) in different plant parts of acclimatized plantlets revealed higher amount of both the alkaloids in leaves. The present protocol shall be gainfully employed for large-scale micropropagation, conservation and extraction of biotherapeutic molecules for commercial purposes.

Two light responsive WRKY genes exhibit positive and negative correlation with picroside content in Picrorhiza kurrooa Royle ex Benth, an endangered medicinal herb.

Picrorhiza kurrooa is an endangered herb known to produce the medicinally important picrosides through isoprenoid pathway. The present work showed the functionality of WRKY motifs (TGAC cis-acting elements) present in the promoters of regulatory genes 3-hydroxy-3-methylglutaryl coenzyme A reductase (Pkhmgr) and 1-deoxy-d-xylulose-5-phosphate synthase (Pkdxs) of the picrosides biosynthetic pathway by electrophoretic mobility shift assay. Also, the two WRKY genes, PkdWRKY and PksWRKY, were characterized and found to contain double and single characteristic WRKY domains, respectively along with a zinc-finger motif in each domain. Expression analysis revealed that PkdWRKY and PksWRKY exhibited a positive and negative correlation, respectively, with picrosides content under the environment of light and in different tissues. Functional evaluation in yeast showed DNA binding ability of both PksWRKY and PkdWRKY; however, only PkdWRKY exhibited transcriptional activation ability. Transient overexpression of PkdWRKY and PksWRKY in tobacco modulated the expression of selected native genes of tobacco involved in MVA and MEP pathway suggesting functionality of PkdWRKY and PksWRKY in planta. Collectively, data suggested that PkdWRKY and PksWRKY might be positive and negative regulators, respectively in the picrosides biosynthetic pathway.

Green synthesis of silver nanoparticles using Indian Belladonna extract and their potential antioxidant, anti-inflammatory, anticancer and larvicidal activities.

Atropa acuminata aqueous leaf extract biosynthesized silver nanoparticles showed strong antioxidant, anticancerous (HeLa cells) and anti-inflammatory activities. Besides, this bio syn-AgNP also proved effective against mosquito vectors causing malaria, dengue and filariasis. Present study highlights eco-friendly and sustainable approach for the synthesis of silver nanoparticles (AgNP) using aqueous leaf extract of A. acuminata, a critically endangered medicinal herb. The addition of 1mM silver nitrate to aqueous leaf extract resulted in the synthesis of AgNP when solution was heated at 60°C for 30min at pH 7. Absorption band at 428nm, as shown by UV-Vis spectroscopy confirmed the synthesis of AgNP. XRD patterns revealed the crystalline nature of AgNP and TEM analysis showed that most of the nanoparticles were spherical in shape. Zeta potential of AgNP was found to be -33.5mV which confirmed their high stability. FT-IR investigations confirmed the presence of different functional groups involved in the reduction and capping of AgNP. The synthesized AgNP showed effective DPPH (IC50-16.08µg/mL), H2O2 (IC50-25.40µg/mL), and superoxide (IC50-21.12µg/mL) radical scavenging activities. These plant-AgNP showed significant inhibition of albumin denaturation (IC50-12.98µg/mL) and antiproteinase activity (IC50-18.401µg/mL). Besides, biosynthesized AgNP were found to have strong inhibitory effect against a cervical cancer (HeLa) cell line (IC50-5.418µg/mL) as well as larvicidal activity against 3rd instar larvae of Anopheles stephensi (LC50-18.9ppm, LC90-40.18ppm), Aedes aegypti (LC50-12.395ppm, LC90-36.34ppm) and Culex quinquefasciatus (LC50-17.76ppm, LC90-30.82ppm) and were found to be non-toxic against normal cell line (HEK 293), and a non-target organism (Mesocyclops thermocyclopoides). This is the first report on the synthesis of AgNP using aqueous leaf extract of A. acuminata, validating their strong therapeutic potential.

Enhancement of production of pharmaceutically important anti-cancerous compound; cucurbitacin E via elicitation and precursor feeding of in vitro culture of Citrullus colocynthis (L.) Schard

Citrullus colocynthis (L.) Schard (Cucurbitaceae) is an endangered medicinal herb. It contains glycosides, cucurbitacins (colocynthin and colocynthetin), cucurbitacins A, B, C, D, E, I, J, K and L which are having medicinal values in various parts of the plant. Cucurbitacin E, a highly oxidated steroid consisting of a tetracyclic triterpene which is more potential triterpenoid for anti-cancer. In this study, we made an attempt to investigate the effect of elicitors with methyl jasmonate (MeJA), salicylic acid (SA), precursors like squalene and mevalonic acid as abiotic elicitors. Precursors were used for enhancement production of cucurbitacin E through cell suspension culture and in vitro plant cultures of Citrullus colocynthis. Based on HPLC results, MeJA is better elicitor than SA and in precursors squalene is better than mevalonic acid for cucurbitacin E production from Citrullus colocynthis cell suspension and in vitro plant cultures. Among all the concentration of elicitors MeJA, SA and precursors squalene, mevalonic acid, 75 µM concentrations have shown best results and MeJA showed highest peak area of 17.97 with 8.63 fold in cell suspension culture. Whereas, the effect of elicitor and precursor feeding studies in in vitro plant culture have produced high amount of cucurbitacin E at 100 µM concentration. Hence, the protocol established in the present study provides an opportunity for enhancement of biological active (anticancer) compound cucurbitacin E.

Conservation of Picrorrhiza kurroa with arbuscular mycorrhizal fungi: An endangered and highly economic medicinal herb of Himalaya

Arbuscular mycorrhizal (AM) fungi are prevalent from arctic to tropics in most agricultural and natural ecosystems, play an important role in the conservation of some valuable medicinal plants. Picrorrhiza kurroa Royle ex Benth is distinguished endangered medicinal herb, naturally distributed in alpine and sub-alpine region of Himalayas. In present investigation, spores density of AM fungi along root colonization were studied from the rhizospheric soils of P. kurroa collected from different locations of Himachal Pradesh. Highest spore number (60) was found in the soil samples collected from forest nursery Rajkunda while AM root space colonization was highest in the samples of forest nursery Devidhar. The biodiversity of AM fungi was studied; the number of AM species was also highest in these two forest nursery samples. Funneliformis mosseae and Acaulospora laevis were dominant species associated with P. kurroa. After mass multiplication of F. mosseae and A. laevis, bioinoculation experiment was performed on Picrorrhiza kurroa seedlings for the biomass production and nutrient uptake analysis. Seedlings were inoculated with single and in combined mycorrhizal treatments with different combinations of farmyard manure and vermicompost. All inoculated seedlings showed significant biomass production than control seedlings. Root length (8.7cm), fresh and dry biomass was observed to be maximum in the plants inoculated with F. mosseae and vermicompost @ 10t ha−1 while percent AM root colonization was highest (60.33) in the roots of plants inoculated with F. mosseae plus A. laevis with vermicompost @10t ha−1. The phosphorus content was more in seedlings inoculated with consortium of two native AM fungi followed by F. mosseae and A. laevis, separately. In overall, consortium of native AM fungi i.e. F. mosseae and A. laevis was found to be best for the conservation of the P. kurroa in the presence of vermicompost and FYM, respectively.

Metabolite and expression profiling of steroidal alkaloids in wild tissues compared to bulb derived in vitro cultures of Fritillaria roylei – High value critically endangered Himalayan medicinal herb

Bulbous Fritillaria roylei (known as Jangli lahsan) is an important critically endangered medicinal herb of Astavarga group and widely used in traditional medicinal system. Being a medicinal potential but endangered herb, biotechnological interventions are urgently needed for its restoration and production of bioactive compounds. In the present study, in vitro cultures were established from bulb scales as explant by using optimized sterilization protocol (79.67% survival). From different combinations and concentrations of plant growth regulator tested; high efficiency callus induction (88.89%) and in vitro plantlets regeneration (77.78%) response was obtained in Murashige and Skoog medium supplemented with 0.5 mg/L thidiazuron +2.0 mg/L picloram and 1.0 mg/L kinetin +0.5 mg/L naphthaleneacetic acid, respectively. For the quantification of steroidal alkaloids i.e. sipeimine and peimine; selective, sensitive, and rapid liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS) analytical method was developed and validated. Quantification of sipeimine and peimine was done for the first time in established in vitro cultures and naturally grown tissues of F. roylei i.e. bulbs, leaves, stem and flower bud. Among in vitro raised cultures, higher sipeimine content (1.98 μg/g) was detected in callus. Whereas, maximum sipeimine content (599.72 μg/g) was observed in floral bud compared to other wild tissues. Peimine content was detected in bulb (1.13 μg/g), leaf (0.78 μg/g) and stem (0.67 μg/g). Qualitative and quantitative phytochemical screening revealed potentiality of this important medicinal herb for commercial utilization in drug formulation to the industries. Maximum antioxidant activity (IC50) was exhibited in floral buds (0.11 mg/mL), while among in vitro raised cultures; regenerated plantlets (0.80 mg/mL) shown higher scavenging activity than callus (2.71 mg/mL). Higher total phenol content (42.86 μg gallic acid/mg) was observed in bulb whereas, flavonoids content is higher in leaves (68.36 μg quercetin/mg) followed by callus (58.22 μg quercetin /mg). Putative steroidal alkaloids gene expression studies using RT-qPCR showed positive correlation with metabolite content. For the first time, present investigation concluded that floral buds, leaves and stem could also be used as an alternative potential source for steroidal alkaloids and callus culture as sustainable approach for metabolite production to meet industrial standards and demands.

Genetic Structure and Diversity among Species of Salacia: An Endangered Medicinal Herb of Western Ghats, India

Salacia is one of the medicinally valuable genus, distributed throughout tropical areas which include India, Sri Lanka, Southern China and other Southern Asian Countries. The genus Salacia is represented by 21 species in India, among them eight species are recorded from the state of Karnataka in the Southern part of India. Despite its pharmaceutical importance, very little information exists about the genetic diversity of Salacia at molecular level. Hence the present study was carried out to evaluate the genetic among six species of Salacia namely S. chinensis, S. malabarica, S. oblonga, S. macrosperma, S. reticulata and S. gambleana with the help of ISSR marker analysis. Dendrogram and genetic distance were generated adopting Unweighted Paired Group Method with Arithmetic mean (UPGMA) in the NTSYS-pc software. Basic genetic parameters were calculated by analysing the genetic data with Pop gene 1.32 and GenAlEx 6.2 software. The overall polymorphism across the ten primers screened revealed 26 % polymorphism. A 60% polymorphism was scored for the primer UBC 841, whereas, no polymorphism was observed for primer UBC 840 and ISSR 6. The average observed heterozygosity was more than expected heterozygosity. Observed heterozygosity (Ho) ranged from 0.15 (UBC 841) to 0.38 (ISSR 6) with an average of 0.25, whereas expected heterozygosity (He) ranged from 0.10 (UBC 843) to 0.35 (ISSR 6) with an average of 0.23 for Salacia species. The higher heterozygosity pointed towards increased genetic diversity amongst the species. ISSR marker analysis showed high level of inter and intra population genetic differentiation.

Complete plastid genome sequence of the rare and endangered medicinal herb Psammosilene tunicoides, endemic to China

Psammosilene tunicoides W.C. Wu et C.Y. Wu, a monotypic species and endemic to Southwest China, is a rare and endangered traditional medicinal herb with satisfactory effects on multifold pathology. In order to provide crucial data for protection, we reported and analyzed the complete plastid genome of P. tunicoides as the foundation of germplasm conservation. The complete plastid genome is a typical quadripartite circular molecule of 153,957 bp in length, including a large single copy (LSC) region of 83,972 bp and a small single copy (SSC) region of 17,495 bp separated by two inverted repeat (IR) regions of 26,245 bp. In total of 111 unique genes were predicted, including 78 protein-coding genes (PCGs), 29 transfer RNA genes, and four ribosomal RNA genes. The overall GC content of P. tunicoides is 36.5%. The phylogenetic analysis revealed that P. tunicoides and Dianthus caryophyllus formed an independent clade with a 100% bootstrap support.

Impact of light-emitting diodes (LEDs) on the growth and morphogenesis of encapsulated shoot buds of Curculigo orchioides Gaertn., an endangered medicinal herb

The present study describes the impact of light-emitting diodes (LEDs) on synseed germination and subsequent synseed-derived seedling growth of Curculigo orchioides, an endangered medicinal plant under in vitro conditions. Shoot buds regenerated on Murashige and Skoog’s medium (MS) containing 4 mg/l 6-benzylaminopurine (BAP), and 3% sucrose were utilized for encapsulation. Encapsulation was achieved by suspending the excised shoot buds into sodium–alginate gel matrix and dropping them into 100 mM CaCl2 solution. Synseeds thus produced were incubated under different light conditions. Among the various light treatments, irradiation of synseeds with blue LEDs (BL) strongly influences the growth of the synseed-derived seedlings. A significant increase in length, fresh and dry biomass of the synseed-derived seedlings was observed under BL as compared to light treatments of conventional fluorescence lamps (FL) and a combination of blue and red LEDs with equal proportion (BRL, 1:1). The trifoliate leaf area and weighted density of leaves were measured from the digital images of synseed-derived seedlings using MATLAB® 2013a Image Processing Toolbox. Image analysis of the synseed-derived seedlings also revealed the improved growth in terms of increase in trifoliate leaf area and weighted density under BL. Exposure to red LEDs (RL) resulted in poor synseed germination and severely retarded the growth of the synseed-derived seedlings. The leaves of synseed-derived seedlings grown under BL had higher chlorophyll a, carotenoid, total phenols, and flavonoid contents than that of FL and BRL treatments. The synseed-derived seedlings developed under BL also displayed enhanced levels of antioxidant activity. Ex vitro performance of the synseed-derived seedlings raised under BL was found to be better with improved plant canopy as expressed in terms of increase in trifoliate leaf area and weighted density than other light treatments. The present study for the first time demonstrates the potential of BL irradiation on encapsulation technology with the ability of direct conversion of synseeds to synseed-converted seedlings leading to the vigorous growth of the plantlets which may make the encapsulation strategy ideal for supply of healthy plant propagules for transfer to field conditions and germplasm conservation.