Silica and secondary metabolites as chemophenetic markers for characterization of bamboo species in relation to genetic and morphometric analysis.

Bamboo is a non-timber forest product and one of the most important grass plants of industrial and domestic use. It is widely distributed in tropical countries including India, China and Southeast Asian countries with wide genetic diversity. The diversity in the available genotypes becomes an important resource for the selection and improvement of the plants for ecological and commercial use. This study investigates eight commercially and ecologically important bamboo species of six genera (Bambusa, Dendrocalamus, Thyrsostachys, Vietnamosasa, Cephalostachyum and Indocalamus) from India, Thailand and Laos. These were evaluated for genetic differences by molecular makers, chemo-morphological variation and ability of silicon accumulation. The genetic cluster analyses of eight RAPD primers revealed genetic similarities in the ranges of 24-55%. The total silica content varied from 18.34 to 40.08 ppm in leaves of different bamboo species. Chemical analysis of the silicon content by ICP-OES and secondary metabolite profiling on TLC depicted the prominent distinction among the species. The PCA analysis of quantitative morphological data grouped the species in two major clusters and found to correlate with chemical pattern and genetic similarity to some extent. This is the first report that summarizes species-specific variability of leaf silica content, secondary metabolites, and quantitative morphological data towards delineation of genetic phylogeny of bamboo species.

Root transcripts associated with arsenic accumulation in hyperaccumulator Pteris vittata.

Hyperaccumulation of arsenic (As) by brake fern Pteris vittata has been described as an important genetic trait that provides an option for development of a sustainable phytoremediation process for As mitigation. Accumulation of very high concentration of arsenic in above-ground tissues may be the result of arsenic vacuole compartmentalization, but the mechanism(s) of arsenic uptake and transport by underground tissues are largely unknown. In this study, we made an attempt towards understanding the molecular mechanism of As hyperaccumulation in this plant. A time-dependent As accumulation study indicates an exponential accumulation of As from 7 to 30 days of arsenic exposure in fronds, and day 3-7 in roots. Root transcriptome analysis identified 554,973 transcripts. Further, subsets of 824 transcripts were differentially expressed between treated and control samples. Many of the genes of critical As-stress response, transcription factors and metal transporters, biosynthesis of chelating compounds involved in uptake and accumulation mechanisms were identified. The genes that were highly expressed such as cysteine-rich RLK, and ABC transporter G family member 26 needs further studies along with arsenite transmembrane transporter. The analysis of generated transcriptome dataset has provided valuable information and platform for further functional studies.

Characterization of UDP-glucosyltransferase from Indigofera tinctoria.

Indican is a secondary metabolite in Indigofera tinctoria; its synthesis from indoxyl and UDP-glucose is catalyzed by a UDP-glucosyltransferase (UGT). In this study, we partially purified UGT extracted from I. tinctoria leaves and analyzed the protein by peptide mass fingerprinting. We identified two fragments that were homologous to UGT after comparison with the transcriptomic data of I. tinctoria leaves. The fragments were named itUgt1 and itUgt2 and were amplified using rapid amplification of cDNA ends polymerase chain reaction to obtain full-length cDNAs. The resultant nucleotide sequences of itUgt1 and itUgt2 encoded peptides of 477 and 475 amino acids, respectively. The primary structure of itUGT1 was 89% identical to that of itUGT2 and contained an important plant secondary product glycosyltransferase (PSPG) box sequence and a UGT motif. The recombinant proteins expressed in Escherichia coli were found to possess high indican synthesis activity. Although the properties of the two proteins itUGT1 and itUGT2 were very similar, itUGT2 was more stable at high temperatures than itUGT1. Expression levels of itUGT mRNA and protein in plant tissues were examined by UGT assay, immunoblotting, and semi-quantitative reverse transcription polymerase chain reaction. So far, we presume that itUGT1, but not itUGT2, primarily catalyzes indican synthesis in I. tinctoria leaves.

Identification of arsenic resistant endophytic bacteria from Pteris vittata roots and characterization for arsenic remediation application.

Mitigation of arsenic (As) pollution is a topical environmental issue of high R&D priority. The present investigation was carried out to isolate As resistant endophytes from the roots of Indian ecotype Pteris vittata and characterize their As transformation and tolerance ability, plant growth promoting characteristics and their role to facilitate As uptake by the plant. A total of 8 root endophytes were isolated from plants grown in As amended soil (25 mg As kg(-1)). These isolates were studied for minimum inhibitory concentration (MIC), arsenite As(III) - arsenate As(V) transformation ability, plant growth promoting (PGP) characteristics through siderophore, indole acetic acid (IAA) production, phosphatase, ACC deaminase activity, and presence of arsenite oxidase (aox) and arsenite transporter (arsB) genes. On the basis of 16S rDNA sequence analysis, these isolates belong to Proteobacteria, Firmicutes and Bacteroidetes families under the genera Bacillus, Enterobacter, Stenotrophomonas and Rhizobium. All isolates were found As tolerant, of which one isolates showed highest tolerance up to 1000 mg L(-1) concentration in SLP medium. Five isolates were IAA positive with highest IAA production up to 60 mg/L and two isolates exhibited siderophore activity. Phosphatase activity was shown by only one isolate while ACC deaminase activity was absent in all the isolates. The As transformation study by silver nitrate test showed that only two strains had dual characteristics of As(III) oxidation and As (V) reduction, four strains exhibited either of the characteristics while other two didn't confirmed any of the two characteristics. Presence of aox gene was detected in two strains and arsB gene in six isolates. The strain with highest As tolerance also showed highest IAA production and occurrence of arsB gene. Present investigation may open up further scope of utilizing these endophytes for up gradation of phytoextraction process.

RNA-Seq analysis for indigo biosynthesis pathway genes in Indigofera tinctoria and Polygonum tinctorium.

Natural indigo is the most important blue dye for textile dyeing and valuable secondary metabolite biosynthesized in Indigofera tinctoria and Polygonum tinctorium plants. Present investigation is made to generation of gene resource for pathway enrichment and to understand possible gene expression involved in indigo biosynthesis. The data about raw reads and the transcriptome assembly project has been deposited at GenBank under the accessions SRA180766 and SRX692542 for I. tinctoria and P. tinctorium, respectively.

Molecular docking and ADME studies of natural compounds of Agarwood oil for topical anti-inflammatory activity.

Aquilaria agallocha Roxb. family, Thymelaeaceae, is an evergreen plant of South-East Asia, commonly described as aloe wood or agarwood. Traditionally, the bark, root and heartwood are used for their medicinal properties as a folk medicine for hundreds of years. Chemical analyses revealed that the bulk of the oil is constituted by agarospirol (12.5%), jinkoh-eremol (11.8%) and hinesol (8.9%) as major contributor. In the present work, a QSAR model for antiinflammatory activity of 10-epi-γ-Eudesmol, jinkoh-eremol, agarospirol and other compounds has been developed by multiple linear regression method. The r(2) and rCV(2) of a model were 0.89 and 0.81 respectively. In silico molecular docking study suggests that compound 10-epi-γ-Eudesmol, jinkoh-eremol and agarospirol are preferentially more active than other identified compounds with strong binding affinity to major anti-inflammatory and immunomodulatory receptors. The oil displayed a significant and dose dependent reduction of 12-O-tetradecanoylphorobol-13 acetate (TPA)- induced ear edema and MDA activity when compared with vehicle treated mice. Pro-inflammatory cytokines (IL-1ß, IL-6 and TNF-α) were also reduced significantly in a dose dependent manner in all the TPA treated groups as compared to control. The present study indicates that agarwood oil significantly reduced the skin thickness, ear weight, oxidative stress and pro-inflammatory cytokines production in TPA-induced mouse ear inflammation model and contributed towards validation of its traditional use to treat inflammation related ailments.

Impact of geographic range on genetic and chemical diversity of Indian valerian (Valeriana jatamansi) from northwestern Himalaya.

An effort was made to determine the impact of geographic range on genetic richness and chemical constituents of Valeriana jatamansi Jones, an herb indigenous to the northwestern Himalaya. The genetic structure of 16 accessions from two major divisions of Uttarakhand state (Kumaon and Garhwal) was analyzed by ISSR markers. Overall genetic diversity among the populations was 45 %, with a cumulative range of 35-92 % similarity for most of the high-altitude plants and a comparatively narrow range, 50-88 %, for the population below the altitude of 1,800 m. Likewise, a remarkable predictability was evident from the chemical constituents on an individual basis. In principal component analysis, most of the accessions fall into two major groups and are classified as chemotypes based on the percentage of similar chemical constituents; these are mostly correlated to altitude. Geographic distance seems to influence the genetic and chemical variability, indicating the genetic inbreeding within the population.

Molecular profiling for genetic variability in Capsicum species based on ISSR and RAPD markers.

The taxonomic identity of Capsicum species is found to be difficult as it displays variations at morpho-chemical characters. Twenty-two accessions of six Capsicum species, namely, C. annuum, C. baccatum, C. chinense, C. eximium, C. frutescens, and C. luteum were investigated for phenotypic diversity based on flower color and for genetic differences by molecular makers. The genetic cluster analyses of 27 RAPD and eight ISSR primers, respectively, revealed genetic similarities in the ranges of 23-88% and 11-96%. Principal component analysis of the pooled RAPD and ISSR data further supports the genetic similarity and groupings. Different species showed variations in relation to corolla shade of flower. C. annuum accessions formed a single cluster in the molecular analysis as maintaining their flower characteristic. C. chinense accession shared flower features with the accessions of C. frutescens and were found to be closer at genotypic level. C. luteum was found to be rather closer to C. baccatum complex, both phenotypically and genetically. The only accession of C. eximium presenting purple flowers falls apart from the groupings. The floral characteristics and the molecular markers are found to be useful toward the delineation of the species specificity in Capsicum collection and identification of genetic stock.

Genetic and chemical diversity of high mucilaginous plants of Sida complex by ISSR markers and chemical fingerprinting.

A method was developed based on multiple approaches wherein DNA and chemical analysis was carried out toward differentiation of important species of Sida complex that is being used for commercial preparation. Isolated DNA samples were successfully performed through PCR amplification using ISSR markers and degree of genetic diversity among the different species of Sida is compared with that of chemical diversity. For genetic fingerprint investigation, selected 10 ISSR primers generating reproducible banding patterns were used. Among the total of 63 amplicons, 62 were recorded as polymorphic, genetic similarity index deduced from ISSR profiles ranged from 12 to 51%. Based on similarity index, S. acuta and S. rhombifolia found to be most similar (51%). High number of species-specific bands played pivotal role to delineate species at genetic level. Investigation based on HPTLC fingerprints analysis revealed 23 bands representing to characteristic chemicals and similarity index ranged from 73 to 91%. Prominent distinguishable bands were observed only in S. acuta, while S. cordifolia and S. rhombifolia shared most bands making them difficult to identify on chemical fingerprint basis. This report summarizes the genotypic and chemotypic diversity and the use of profiles for authentication of species of Sida complex.

An efficient protocol for high-frequency direct multiple shoot regeneration from internodes of peppermint (Mentha x piperita).

A simple, repeatable and efficient protocol for direct multiple shoot regeneration from internodal explants has been defined in peppermint (Mentha x piperita var. Indus). In vitro regenerated shoots of peppermint were excised into 4 to 8 mm long internodes and cultured on Murashige and Skoog's medium supplemented with different cytokinins. In the hormonal assay, 3.0 mg L(-1) zeatin or 6-isopentenyl adenine independently supplemented to half strength MS medium exhibited multiple shoot regeneration, while thiaduzorn (0.1-3.0 mg L(-1)) showed no morphogenetic effect. A maximum of 85% in vitro cultured explants showed multiple shoot formation with an average of 7 shoots per explant on MS medium supplemented with zeatin. Multiple shoots were initiated within three weeks of cultivation. Internodes with regenerated multiple shoots were transferred to half- strength MS medium without supplementing with any plant growth hormone for shoot elongation and rhizogenesis. Rooted plants acclimatized and grew to maturity under glasshouse conditions. The plantlets developed were phenotypically identical to the parent plant and exhibited 96% survival.