Molecular cloning and heterologous expression analysis of 1-Deoxy-D-Xylulose-5-Phosphate Synthase gene in Centella asiatica L.

Many genes involved in triterpenoid saponins in plants control isoprenoid flux and constitute the precursor pool, which is channeled into various downstream pathways leading to the synthesis of triterpenoid saponins in C. asiatica. Full-length 1-Deoxy-D-Xylulose-5-Phosphate-Synthase (CaDXS) gene was isolated for the study from the previously annotated Centella asiatica leaves transcriptomic data. The CaDXS gene sequence was submitted to the NCBI databases with GenBank accession number MZ997832. The full-length CaDXS gene contained a 2244 base pair open reading frame that encoded a 747 amino acid polypeptide. The predicted molecular weight (MW) and theoretical pI of DXS are 76.28 kDa and 6.86, respectively. Multiple amino acid sequence alignment of amino acids and phylogenetic studies suggest that CaDXS shares high similarities with DXS from other plants DXS belonging to different families. A phylogenetic tree was constructed using Molecular Evolutionary Genetic Analysis (MEGA) version 10.1.6. Structural analysis provided fundamental information about the three-dimensional features and physicochemical parameters of the CaDXS protein. Quantitative expression analysis showed that CaDXS transcripts were maximally expressed in leaf, followed by petiole, roots, and node tissues. CaDXS was cloned into the expression vector pET28a, expressed heterologously in DH5α bacteria, confirmed by sequencing, and subsequently characterized by protein expression and functional complementation. The study focused on understanding the protein structure, biological significance, regulatory mechanism, functional analysis, and gene characterization of the centellosides biosynthetic pathway gene DXS for the first time in the plant. It would provide new information about the metabolic pathway and its relative contribution to isoprenoid biosynthesis.

New Insights into Phylogenetic Relationship of Hydrocotyle (Araliaceae) Based on Plastid Genomes.

Hydrocotyle, belonging to the Hydrocotyloideae of Araliaceae, consists of 95 perennial and 35 annual species. Due to the lack of stable diagnostic morphological characteristics and high-resolution molecular markers, the phylogenetic relationships of Hydrocotyle need to be further investigated. In this study, we newly sequenced and assembled 13 whole plastid genomes of Hydrocotyle and performed comparative plastid genomic analyses with four previously published Hydrocotyle plastomes and phylogenomic analyses within Araliaceae. The plastid genomes of Hydrocotyle exhibited typical quadripartite structures with lengths from 152,659 bp to 153,669 bp, comprising a large single-copy (LSC) region (83,958-84,792 bp), a small single-copy (SSC) region (18,585-18,768 bp), and a pair of inverted repeats (IRs) (25,058-25,145 bp). Each plastome encoded 113 unique genes, containing 79 protein-coding genes, 30 tRNA genes, and four rRNA genes. Comparative analyses showed that the IR boundaries of Hydrocotyle plastomes were highly similar, and the coding and IR regions exhibited more conserved than non-coding and single-copy (SC) regions. A total of 2932 simple sequence repeats and 520 long sequence repeats were identified, with specificity in the number and distribution of repeat sequences. Six hypervariable regions were screened from the SC region, including four intergenic spacers (IGS) (ycf3-trnS, trnS-rps4, petA-psbJ, and ndhF-rpl32) and two coding genes (rpl16 and ycf1). Three protein-coding genes (atpE, rpl16, and ycf2) were subjected to positive selection only in a few species, implying that most protein-coding genes were relatively conserved during the plastid evolutionary process. Plastid phylogenomic analyses supported the treatment of Hydrocotyle from Apiaceae to Araliaceae, and topologies with a high resolution indicated that plastome data can be further used in the comprehensive phylogenetic research of Hydrocotyle. The diagnostic characteristics currently used in Hydrocotyle may not accurately reflect the phylogenetic relationships of this genus, and new taxonomic characteristics may need to be evaluated and selected in combination with more comprehensive molecular phylogenetic results.

Biotechnological and endophytic-mediated production of centellosides in Centella asiatica.

In vitro culture of a plant cell, tissue and organ is a marvellous, eco-friendly biotechnological strategy for the production of phytochemicals. With the emergence of recent biotechnological tools, genetic engineering is now widely practiced enhancing the quality and quantity of plant metabolites. Triterpenoid saponins especially asiaticoside and madecassoside of Centella asiatica (L.) Urb. are popularly known for their neuroprotective activity. It has become necessary to increase the production of asiaticoside and madecassoside because of their high pharmaceutical and industrial demand. Thus, the review aims to provide efficient biotechnological tools along with proper strategies. This review also included a comparative analysis of various carbon sources and biotic and abiotic elicitors. The vital roles of a variety of plant growth regulators and their combinations have also been evaluated at different in vitro growth stages of Centella asiatica. Selection of explants, direct and callus-mediated organogenesis, root organogenesis, somatic embryogenesis, synthetic seed production etc. are also highlighted in this study. In a nutshell, this review will present the research outcomes of different biotechnological interventions used to increase the yield of triterpenoid saponins in C. asiatica. KEY POINTS: • Critical and updated assessment on in vitro biotechnology in C. asiatica. • In vitro propagation of C. asiatica and elicitation of triterpenoid saponins production. • Methods for mass producing C. asiatica.

Molecular cloning and characterization of Triterpenoid Biosynthetic Pathway Gene HMGS in Centella asiatica (Linn.).

BACKGROUND: The flux of isoprenoids and the total accumulation of triterpenoid saponins known as centellosides in C. asiatica are controlled by the key genes of the Mevalonate pathway (MVA). These genes were reported to have positive regulation of the pathway in providing isoprenoid moieties. Though, some information is available on the pathway and secondary metabolites. However, most of the pathway steps are not characterized functionally. METHODOLOGY AND RESULTS: For the study, full-length pathway gene Hydroxymethyl glutaryl-CoA-synthase (CaHMGS; GenBank accession number: MZ997833), was isolated from previously annotated transcriptome data of Centella asiatica leaves. HMGS has been successfully cloned and heterologously expressed in bacteria E. coli strain DH5α. The cloned gene has been sequenced and further characterized through in silico studies by different bioinformatics tools. Also, the gene sequences have been submitted in NCBI. In silico studies of isolated gene sequence revealed the nature, characteristics of genes. The ORF of HMGS is 1449 bp encoding 482 amino acids. Predicted molecular weight (MW) of HMGS was 48.09 kDa and theoretical pI was 5.97. Blast results and Multiple sequence alignments of the gene showing the similarity with HMGS of other plants of their respective families. The Molecular Evolutionary Genetic Analysis (MEGA) version 10.1.6 was used to construct a phylogenetic tree. Differential tissue-specific expression of different plant parts was also checked. Tissue expression patterns unveiled that the highest expression level of the CaHMGS had been seen in the roots and lowest in the node of the plant. Functional complementation experiment of the CaHMGS in Saccharomyces cerevisiae wild strain YSC1021 and haploid strain YSC1021 which lack HMGS protein confirmed that the CaHMGS gene encodes functional CaHMGS that catalyzed the biosynthesis of mevalonate in yeast. CONCLUSIONS: The gene was reported, cloned and characterized first time in Centella asiatica. Understanding this biosynthetic pathway gene will further help in the improvement of plants for enhanced secondary metabolites production.

Transcriptome sequencing revealed the influence of blue light on the expression levels of light-stress response genes in Centella asiatica.

Centella asiatica is rich in medical and cosmetic properties. While physiological responses of C. asiatica to light have been widely reported, the knowledge of the effects of light on its gene expression is sparse. In this study, we used RNA sequencing (RNA-seq) to investigate the expression of the C. asiatica genes in response to monochromatic red and blue light. Most of the differentially expressed genes (DEGs) under blue light were up-regulated but those under red light were down-regulated. The DEGs encoded for CRY-DASH and UVR3 were among up-regulated genes that play significant roles in responses under blue light. The DEGs involved in the response to photosystem II photodamages and in the biosynthesis of photoprotective xanthophylls were also up-regulated. The expression of flavonoid biosynthetic DEGs under blue light was up-regulated but that under red light was down-regulated. Correspondingly, total flavonoid content under blue light was higher than that under red light. The ABI5, MYB4, and HYH transcription factors appeared as hub nodes in the protein-protein interaction network of the DEGs under blue light while ERF38 was a hub node among the DEGs under red light. In summary, stress-responsive genes were predominantly up-regulated under blue light to respond to stresses that could be induced under high energy light. The information obtained from this study can be useful to better understand the responses of C. asiatica to different light qualities.

Genetic diversity and structure revealed by genomic microsatellite markers in Centella asiatica (L.) Urb., a plant with medicinal potential.

BACKGROUND: Around the world, medicinal plants are utilised for various purposes. Centella asiatica is one of the important medicinal plants widely used in many medicinal systems. Nevertheless, analysis of the genetic diversity would pave the way for its most suitable utilisation. METHODS AND RESULTS: The present study analyses the genetic diversity and structure of eighty C. asiatica accessions collected from the southern states of India, using ten genomic microsatellite markers. The mean Nei's gene diversity (0.46) indicates considerable genetic diversity. Analysis of molecular variance (82.48%) exhibited significant genetic variance between samples within the population. The cluster analysis brought out the structure of the analysed populations as three subpopulations based on the genetic differentiation. CONCLUSIONS: The study showed significant intra-population variation, predominant inbreeding and population differentiation in C. asiatica. The findings will help better understanding of the genetic structure and gene pool of the plant.

De novo chromosome-level assembly of the Centella asiatica genome.

Centella asiatica is a herbaceous, perennial species indigenous to India and Southeast Asia. C. asiatica possesses several medicinal properties: anti-aging, anti-inflammatory, wound healing and memory enhancing. The lack of available genomics resources significantly impedes the improvement of C. asiatica varieties through molecular breeding. Here, we combined the 10× Genomics linked-read technology and the long-range HiC technique to obtain the genome assembly. The final assembly contained nine pseudomolecules, corresponding to the haploid chromosome number in C. asiatica. These nine chromosomes covered 402,536,584 bases or 93.6% of the 430-Mb assembly. Comparative genomics analyses based on single-copy orthologous genes showed that C. asiatica and the common ancestor of Coriandrum sativum (coriander) and Daucus carota (carrot) diverged about 48 million years ago. This assembly provides a valuable reference genome for future molecular studies, varietal development through marker-assisted breeding and comparative genomics studies in C. asiatica.

Expression of Escherichia coli Heat-Labile Enterotoxin B Subunit in Centella (Centella asiatica (L.) Urban) via Biolistic Transformation.

BACKGROUND: Heat-Labile enterotoxin B subunit (LTB) produced by Escherichia coli, a non-toxic protein subunit with potential biological properties, is a powerful mucosal and parenteral adjuvant which can induce a strong immune response against co-administered antigens. OBJECTIVE: In the present study, LTB protein, encoded by the optimized ltb (also known synthetic ltb, s-ltb) gene in centella plant (Centella asiatica) for use as an antigen, has been discussed. METHODS: The s-ltb gene was cloned into a plant expression vector, pMYO51, adjacent to the CaMV 35S promoter and was then introduced into centella plant by biolistic transformation. PCR amplification was conducted to determine the presence of s-ltb gene in the transgenic centella plant. The expression of s-ltb gene was analyzed by immunoblotting and quantified by ELISA. In vitro activity of LTB protein was determined by GM1-ELISA. RESULTS: PCR amplification has found seven transgenic centella individuals. However, only five of them produced LTB protein. ELISA analysis showed that the highest amount of LTB protein detected in transgenic centella leaves was about 0.8% of the total soluble protein. GM1-ELISA assay indicated that plant LTB protein bound specifically to GM1-ganglioside, suggesting that the LTB subunits formed active pentamers. CONCLUSION: The s-ltb gene that was successfully transformed into centella plants by the biolistic method has produced a relatively high amount of plant LTB protein in the pentameric quaternary structure that has GM1-ganglioside binding affinity, a receptor on the intestinal epithelial membrane.

Correlations between genetic, epigenetic and phenotypic variation of an introduced clonal herb.

Heritable epigenetic modifications may occur in response to environmental variation, further altering phenotypes through gene regulation, without genome sequence changes. However, epigenetic variation in wild plant populations and their correlations with genetic and phenotypic variation remain largely unknown, especially for clonal plants. We investigated genetic, epigenetic and phenotypic variation of ten populations of an introduced clonal herb Hydrocotyle vulgaris in China. Populations of H. vulgaris exhibited extremely low genetic diversity with one genotype exclusively dominant, but significantly higher epigenetic diversity. Both intra- and inter-population epigenetic variation were related to genetic variation. But there was no correlation between intra-/inter-population genetic variation and phenotypic variation. When genetic variation was controlled, intra-population epigenetic diversity was related to petiole length, specific leaf area, and leaf area variation, while inter-population epigenetic distance was correlated with leaf area differentiation. Our study provides empirical evidence that even though epigenetic variation is partly under genetic control, it could also independently play a role in shaping plant phenotypes, possibly serving as a pathway to accelerate evolution of clonal plant populations.

Constraints on the evolution of phenotypic plasticity in the clonal plant Hydrocotyle vulgaris.

The evolution of phenotypic plasticity of plant traits may be constrained by costs and limits. However, the precise constraints are still unclear for many traits under different ecological contexts. In a glasshouse experiment, we grew ramets of 12 genotypes of a clonal plant Hydrocotyle vulgaris under the control (full light and no flood), shade and flood conditions and tested the potential costs and limits of plasticity in 13 morphological and physiological traits in response to light availability and flood variation. In particular, we used multiple regression and correlation analyses to evaluate potential plasticity costs, developmental instability costs and developmental range limits of each trait. We detected significant costs of plasticity in specific petiole length and specific leaf area in response to shade under the full light condition and developmental range limits in specific internode length and intercellular CO2 concentration in response to light availability variation. However, we did not observe significant costs or limits of plasticity in any of the 13 traits in response to flood variation. Our results suggest that the evolution of phenotypic plasticity in plant traits can be constrained by costs and limits, but such constraints may be infrequent and differ under different environmental contexts.

A Novel Multifunctional C-23 Oxidase, CYP714E19, is Involved in Asiaticoside Biosynthesis.

Centella asiatica is widely used as a medicinal plant due to accumulation of the ursane-type triterpene saponins asiaticoside and madecassoside. The molecular structure of both compounds suggests that they are biosynthesized from α-amyrin via three hydroxylations, and the respective Cyt P450-dependent monooxygenases (P450 enzymes) oxidizing the C-28 and C-2α positions have been reported. However, a third enzyme hydroxylating C-23 remained elusive. We previously identified 40,064 unique sequences in the transcriptome of C. asiatica elicited by methyl jasmonate, and among them we have now found 149 unigenes encoding putative P450 enzymes. In this set, 23 full-length cDNAs were recognized, 13 of which belonged to P450 subfamilies previously implicated in secondary metabolism. Four of these genes were highly expressed in response to jasmonate treatment, especially in leaves, in accordance with the accumulation patterns of asiaticoside. The functions of these candidate genes were tested using heterologous expression in yeast cells. Gas chromatography-mass spectrometry (GC-MS) analysis revealed that yeast expressing only the oxidosqualene synthase CaDDS produced the asiaticoside precursor α-amyrin (along with its isomer ß-amyrin), while yeast co-expressing CaDDS and CYP716A83 also contained ursolic acid along with oleanolic acid. This P450 enzyme thus acts as a multifunctional triterpenoid C-28 oxidase converting amyrins into corresponding triterpenoid acids. Finally, yeast strains co-expressing CaDDS, CYP716A83 and CYP714E19 produced hederagenin and 23-hydroxyursolic acid, showing that CYP714E19 is a multifunctional triterpenoid oxidase catalyzing the C-23 hydroxylation of oleanolic acid and ursolic acid. Overall, our results demonstrate that CaDDS, CYP716A83 and CYP714E19 are C. asiatica enzymes catalyzing consecutive steps in asiaticoside biosynthesis.

Piriformospora indica cell wall extract as the best elicitor for asiaticoside production in Centella asiatica (L.) Urban, evidenced by morphological, physiological and molecular analyses.

Vascular plants synthesise a multitude of organic molecules or phytochemicals, referred to as "secondary metabolites". These molecules are involved in a variety of roles in the life span of plants, ranging from structural ones to protection. Centella asiatica (L.) Urban has probably been used since prehistoric times and has been reported to have been used for various medicinal and cosmetic purposes. The plant contains several active constituents, of which the most important is asiaticoside, a triterpenoid. Asiaticoside content in C. asiatica can be enhanced by the use of biotic elicitors like Piriformospora indica. P. indica has been used as a model to study the mechanisms and evolution of mutualistic symbiosis. P. indica is similar to Arbuscular Mycorrhizal (AM) fungi in terms of plant growth promotional effects. The autoclaved fraction from P. indica (PiCWE) was found to be the most active fraction in promoting the plant biomass and asiaticoside content. To date, there are no reports on the potential role of PiCWE in enhancement of asiaticoside over the control and P. indica colonized plants, which was evidenced by the differential expression of key genes involved and final asiaticoside content along with the determination of phytohormones. Moreover, differential expression of selected miRNAs in PiCWE - C. asiatica root interactions over the control and P. indica treated C. asiatica leaf samples was also scrutinized. The important consequence of induction with PiCWE was the significant enhancement of asiaticoside in the PiCWE induced plants in comparison with the asiaticoside content in control and P. indica-C. asiatica interaction. In addition, the role of miRNAs in C. asiatica - PiCWE would enable more in-depth studies for deciphering the molecular and physiological mechanisms of the association and regulation of PiCWE - C. asiatica interactions.

Characterization of the Asiatic Acid Glucosyltransferase, UGT73AH1, Involved in Asiaticoside Biosynthesis in Centella asiatica (L.) Urban.

Centella asiatica (L.) Urban contains two ursane-type triterpene saponins, asiaticoside and madecassoside, as major secondary metabolites. In order to select candidate genes encoding UDP-glucosyltransferases (UGTs) involved in asiaticoside biosynthesis, we performed transcriptomic analysis of leaves elicited by methyl jasmonate (MeJA). Among the unigenes, 120 isotigs and 13 singletons of unique sequences were annotated as UGTs, including 37 putative full-length cDNAs, and 15 of the putative UGT genes were named according to the UGT committee nomenclature protocols. One of them, UGT73AH1, was characterized by heterologous expression in Escherichia coli BL21 (DE3) cells. After induction with IPTG, a total protein extract was assayed with UDP-glucose and asiatic acid. UPLC-QTOF/MS analysis showed that UGT73AH1 catalyzes the glycosylation of asiatic acid to its monoglucoside. It remains unclear whether glycosylation occurs on the triterpene C-2α, C-3ß, C-23, or C-28 position. However, it is very likely that UGT73AH1 glucosylates the C-28 position, because only C-28 bears a glucose moiety in the final pathway product of asiatic acid, while C-2α, C-3ß, and C-23 remain un-conjugated.

Molecular cloning of an ester-forming triterpenoid: UDP-glucose 28-O-glucosyltransferase involved in saponin biosynthesis from the medicinal plant Centella asiatica.

Triterpene saponins include bioactive compounds with structures consisting of triterpene aglycones (sapogenins) and one or more sugar moieties linked through acetal or ester glycosidic linkages at one or more sites. Centella asiatica (L.) Urban is a medicinal plant that contains bioactive ursane-type saponins, such as madecassoside and asiaticoside. In this work, glucosylation of triterpenoids in C. asiatica was investigated starting with plant extracts. An enzyme capable of glucosylating asiatic and madecassic acids was partially purified. Proteomics methods and cDNA sequence data were employed as tools to obtain a full-length cDNA clone encoding a glucosyltransferase. The recombinant gene product, UGT73AD1, was functionally expressed in Escherichia coli and purified by immobilized metal-affinity chromatography. Purified recombinant UGT73AD1 was found to have a narrow specificity, glucosylating asiatic and madecassic acids at the C28 carboxyl. mRNA accumulated in all tissues tested (leaves, stems, roots and flowers), with highest expression in leaves. Thus, UGT73AD1 was identified as a triterpenoid carboxylic acid: UDP-glucose 28-O-glucosyltransferase that appears to be involved in saponin biosynthesis in C. asiatica.

From Plant Extract to a cDNA Encoding a Glucosyltransferase Candidate: Proteomics and Transcriptomics as Tools to Help Elucidate Saponin Biosynthesis in Centella asiatica.

Centella asiatica (L.) Urban (Apiaceae), a small annual plant that grows in India, Sri Lanka, Malaysia, and other parts of Asia, is well-known as a medicinal herb with a long history of therapeutic uses. The bioactive compounds present in C. asiatica leaves include ursane-type triterpene sapogenins and saponins-asiatic acid, madecassic acid, asiaticoside, and madecassoside. Various bioactivities have been shown for these compounds, although most of the steps in the biosynthesis of triterpene saponins, including glycosylation, remain uncharacterized at the molecular level. This chapter describes an approach that integrates partial enzyme purification, proteomics methods, and transcriptomics, with the aim of reducing the number of cDNA candidates encoding for a glucosyltransferase involved in saponin biosynthesis and facilitating the elucidation of the pathway in this medicinal plant.

Molecular cloning, characterization and expression analysis of 3-hydroxy-3-methylglutaryl coenzyme A reductase gene from Centella asiatica L.

3-Hydroxy-3-methylglutaryl-CoA reductases (HMGR) plays an important role in catalyzing the first committed step of isoprenoid biosynthesis in the mevelonic (MVA) pathway (catalyzes the conversion of HMG-CoA to MVA) in plants. The present manuscript reports the full length cDNA cloning of HMGR (CaHMGR, GenBank accession number: KJ939450.2) and its characterization from Centella asiatica. Sequence analysis indicated that the cDNA was of 1965 bp, which had an open reading frame of 1617 bp and encoded a protein containing 539 amino-acids with a mol wt of 57.9 kDa. A BLASTp search against non-redundant (nr) protein sequence showed that C. asiatica HMGR (CaHMGR) has 65-81% identity with HMGRs from different plant species and multi-alignment comparison analysis showed the presence of two motif each corresponding to HMG-CoA-binding and NADP(H)-binding. The Conserved Domain Database analysis predicted that CaHMGR belongs to Class I hydroxymethylglutaryl-coenzyme A (HMG-CoA) reductase. Three-dimensional modeling confirmed the novelty of CaHMGR with a spatial structure similar to Homo sapiens (PDB id: 1IDQ8_A). Tissue Expression analysis indicates that CaHMGR is ubiquitous albeit differentially expressed among different tissues analysed, Strong expression was recorded in the nodes and leaves and low in the roots. The present investigation confirmed that nodes are vital to terpenoid synthesis in C. asiatica. Thus, the cloning of full length CDS, characterization and structure-function analysis of HMGR gene in Centella facilitate to understand the HMGR's functions and regulatory mechanisms involved in mevalonate pathway in C. asiatica at genetic level.

E-microsatellite markers for Centella asiatica (Gotu Kola) genome: validation and cross-transferability in Apiaceae family for plant omics research and development.

Abstract Centella asiatica (Gotu Kola) is a plant that grows in tropical swampy regions of the world and has important medicinal and culinary use. It is often considered as part of Ayurvedic medicine, traditional African medicine, and traditional Chinese medicine. The unavailability of genomics resources is significantly impeding its genetic improvement. To date, no attempt has been made to develop Expressed Sequence Tags (ESTs) derived Simple Sequence Repeat (SSR) markers (eSSRs) from the Centella genome. Hence, the present study aimed to develop eSSRs and their further experimental validation and cross-transferability of these markers in different genera of the Apiaceae family to which Centella belongs. An in-house pipeline was developed for the entire analyses by combining bioinformatics tools and perl scripts. A total of 4443 C. asiatica EST sequences from dbEST were processed, which generated 2617 nonredundant high quality EST sequences consisting 441 contigs and 2176 singletons. Out of 1776.5 kb of examined sequences, 417 (15.9%) ESTs containing 686 SSRs were detected with a density of one SSR per 2.59 kb. The gene ontology study revealed 282 functional domains involved in various processes, components, and functions, out of which 64 ESTs were found to have both SSRs and functional domains. Out of 603 designed EST-SSR primers, 18 pairs of primers were selected for validation based on the optimum parameter value. Reproducible amplification was obtained for six primer pairs in C. asiatica that were further tested for cross-transferability in nine other important genera/species of the Apiaceae family. Cross-transferability of the EST-SSR markers among the species were examined and Centella javanica showed highest transferability (83.3%). The study revealed six highly polymorphic EST-SSR primers with an average PIC value of 0.95. In conclusion, these EST-SSR markers hold a big promise for the genomics analysis of Centella asiatica, to facilitate comparative map-based analyses across other related species within the Apiaceae family, and future marker-assisted breeding programs. To the best of our knowledge, this is the first report of development of EST-SSRs in Centella asiatica by in silico approaches, which offers a veritable potential in further use in plant omics research and development.

Morphological, chemical and molecular characterization of Centella asiatica germplasms for commercial cultivation in the Indo-Gangetic plains.

Centella asiatica germplasm collected from north, north-eastern and southern parts of India was compared for biomass and centellosides productivity under uniform agro-climatic conditions of the Indo-Gangetic plains at Lucknow. The highest biomass accumulation (411.9 g FW/m2 area) was recorded in accession A from north India, followed by 284.0, 135.7 and 29.2 g FW/m2 in accessions M, B and E from southern, eastern and north-eastern regions, respectively. Accession M possessed the highest asiaticoside content (52.1 mg/gDW) that was 1.58, 2.34 and 21.7 folds more than accessions A, B and E, respectively. The madecassoside level in leaves of accessions B and M was comparable (28.9 and 25.7 mg/gDW) and two folds more than accession A (13.9 mg/gDW). The madecassic and asiatic acid content in leaf tissue of all four accessions remained low in Lucknow. Amplified fragment length polymorphism (AFLP) analysis with 23 primers yielded 696 fragments, 563 of which were polymorphic. Accession M out-grouped with genetic dissimilarity indices of 83, 85 and 95% from accessions A, E and B, respectively. Commercial cultivation of accessions M and A through a four months growth cycle (June to September) in agro-climatic conditions of the Indo-Gangetic plains is suggested.

Field evaluation of in vitro-induced tetraploid and diploid Centella asiatica (L.) Urban.

Centella asiatica-a medicinal plant that produces high-value active triterpenoids-is in increasing demand by the pharmaceutical and cosmetic industries. The aim of this study was to field-test one induced tetraploid and three diploid C. asiatica lines for the selection of high-quality plants with high phytomass and triterpenoid content and to determine their optimal harvesting time. All tested C. asiatica were micropropagated using an established protocol. One-month-old plantlets were acclimatized for the field experiment. The plants were grown in a randomized complete block design (RCBD) with three replications, ten plantlets per replication, and the experimental bed site was 0.6 × 1.0 m. Growth parameters, phytomass and the amounts of four active triterpenoids were evaluated. All lines exhibited the highest growth, yields and triterpenoids at 4 months after cultivation. The tetraploid line showed significantly better characteristics, i.e., larger leaf area, leaf width, petiole length, and greater yields, than diploid lines. Dry weight per cultivated area (77.53 ± 3.07 g/m(2)) and total triterpenoids (15.38 ± 0.76 % dry weight) were increased significantly in tetraploid plants of C. asiatica. Furthermore, the harvesting time had an effect on the yield and triterpenoid content (P < 0.001). In all tetraploid and diploid lines, the yields and triterpenoid content per cultivated area reached their maximum at 4 months after planting. Our results demonstrated that polyploidy induction is a beneficial tool that can be used to improve the medicinal value of C. asiatica.

De novo sequencing and assembly of Centella asiatica leaf transcriptome for mapping of structural, functional and regulatory genes with special reference to secondary metabolism.

Centella asiatica (L.) Urban is an important medicinal plant and has been used since ancient times in traditional systems of medicine. C. asiatica mainly contains ursane skeleton based triterpenoid sapogenins and saponins predominantly in its leaves. This investigation employed Illumina next generation sequencing (NGS) strategy on a pool of three cDNAs from expanding leaf of C. asiatica and developed an assembled transcriptome sequence resource of the plant. The short transcript reads (STRs) generated and assembled into contigs and singletons, representing majority of the genes expressed in C. asiatica, were termed as 'tentative unique transcripts' (TUTs). The TUT dataset was analyzed with the objectives of (i) development of a transcriptome assembly of C. asiatica, and (ii) classification/characterization of the genes into categories like structural, functional, regulatory etc. based on their function. Overall, 68.49% of the 46,171,131 reads generated in the NGS process could be assembled into a total of 79,041 contigs. Gene ontology and functional annotation of sequences resulted into the identification of genes related to different sets of cellular functions including identification of genes related to primary and secondary metabolism. The wet lab validation of seventeen assembled gene sequences identified to be involved in secondary metabolic pathways and control of reactive oxygen species (ROS) was established by semi-quantitative and real time PCR (qRT-PCR). The validation also included sequencing/size matching of a set of semi-quantitative PCR amplicons with their in silico assembled contig/gene. This confirmed the appropriateness of assembling the reads and contigs. Thus, the present study constitutes the largest report to date on C. asiatica transcriptome based gene resource that may contribute substantially to the understanding of the basal biological functions and biochemical pathways of secondary metabolites as well as the transcriptional regulatory elements and genetic markers. This work sets the stage for multi-faceted future improvement of the plant, through discovery of new genes, marker-assisted breeding or genetic engineering, on this species as well as for other species of Apiaceae and triterpene producing medicinal plants.