Phenolics and flavonoids from Polygonum posumbu and comparision of flavonoid compounds content in different tissues (leaves, stems and roots).

The growing global need for antioxidative phenolics and flavonoids for maintenance of human health resulted into search of new sustainable unexplored medicinal plants used by the traditional healers for various ailments. Many synthetic based products of phenolics and flavonoids have been used, however the demand of eco-friendly, natural herbal based products are increasing. As a result, the current study aims to explore traditional potential of Polygonum posumbu related to its phenolics and flavonoids. Optimization of extraction parameters were employed which includes: solvent selection (water, ethanol, methanol, acetone and ethyl acetate), ethanol composition (40-100%), solvent to sample ratio (30-70 ml/g), temperature (50-80 °C) and time (1-5 h). Under optimal conditions, total phenolics (TPC), total flavonoids (TFC), the extract yield (EY) and antioxidant activities of leaves extract were 162.79 ± 2.28 mg GAE/g, 56.57 ± 6.22 mg QE/g 27.96 ± 0.91%, and 27.34 ± 0.98 µg/ml respectively. Seven flavonoids were quantified in different tissues with significant (p ≤ 0.05) differences found in flavonoids contents in different parts of the plant. Highest concentration of flavonoids was observed in stems: (-)-epicatechin-53.19 ± 1.13 mg/g, myricetin-15.90 ± 0.13 mg/g, quercetin-50.66 ± 0.08 mg/g, luteolin-43.10 ± 0.47 mg/g, apigenin-16.73 ± 0.43 mg/g. Leaves and roots had the highest amount of genistein (05.06 ± 0.01 mg/g) and kaempferol (11.13 ± 0.06 mg/g) respectively. From the study it had been found that Polygonum posumbu possess a very good amount of phenolics and flavonoids and this study details first ever investigation on this plant in terms of phenolics and flavonoids. Therefore, this study enhanced the importance of this bioresource in functional food or nutraceutical industries.

Metabolic engineering of plant secondary metabolites: prospects and its technological challenges.

Plants produce a wide range of secondary metabolites that play vital roles for their primary functions such as growth, defence, adaptations or reproduction. Some of the plant secondary metabolites are beneficial to mankind as nutraceuticals and pharmaceuticals. Metabolic pathways and their regulatory mechanism are crucial for targeting metabolite engineering. The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-mediated system has been widely applied in genome editing with high accuracy, efficiency, and multiplex targeting ability. Besides its vast application in genetic improvement, the technique also facilitates a comprehensive profiling approach to functional genomics related to gene discovery involved in various plant secondary metabolic pathways. Despite these wide applications, several challenges limit CRISPR/Cas system applicability in genome editing in plants. This review highlights updated applications of CRISPR/Cas system-mediated metabolic engineering of plants and its challenges.

Phytochemical Composition and Antimicrobial Activity of Essential Oil from the Leaves of Artemisia vulgaris L.

Artemisia vulgaris is an enormously useful aromatic plant known for its insecticidal, antifungal, parasiticidal, and medicinal values. The main aim of this study is to investigate phytochemical contents and the potential antimicrobial activities of Artemisia vulgaris essential oil (AVEO) from the fresh leaves of A. vulgaris grown in Manipur. The AVEO isolated by hydro-distillation from A. vulgaris were analyzed by gas chromatography/mass spectrometry and solid-phase microextraction-GC/MS to describe their volatile chemical profile. There were 47 components identified in the AVEO by GC/MS, amounting to 97.66% of the total composition, while 97.35% were identified by SPME-GC/MS. The prominent compounds present in AVEO analyzed by direct injection and SPME methods are found to be eucalyptol (29.91% and 43.70%), sabinene (8.44% and 8.86%), endo-Borneol (8.24% and 4.76%), 2,7-Dimethyl-2,6-octadien-4-ol (6.76% and 4.24%), and 10-epi-γ-Eudesmol (6.50% and 3.09%). The consolidated component in the leaf volatiles comes to the terms of monoterpenes. The AVEO exhibits antimicrobial activities against fungal pathogens such as Sclerotium oryzae (ITCC 4107) and Fusarium oxysporum (MTCC 9913) and bacterial cultures such as Bacillus cereus (ATCC 13061) and Staphylococcus aureus (ATCC 25923). The percent inhibition of AVEO against the S. oryzae and F. oxysporum was found up to 50.3% and 33.13%, respectively. The MIC and MBC of the essential oil tested for B. cereus and S. aureus were found to be (0.3%, 0.63%) and (0.63%, 2.5%), respectively. Finally, the results revealed that the AVEO characterized by the hydro-distillation and SPME extraction yielded the same chemical profile and showed potent antimicrobial activities. Further research into A. vulgaris's antibacterial properties can be performed in order to use it as a source for natural antimicrobial medications.

Reactive oxygen species turnover, phenolics metabolism, and some key gene expressions modulate postharvest physiological deterioration in cassava tubers.

Rapid postharvest physiological deterioration (PPD) in cassava (Manihot esculenta Crantz) tuber is a significant concern during storage. The freshly harvested tubers start spoiling within 24 to 72 h. Accumulation of H2O2 is one of the earliest biochemical events that occurred during PPD, which was detected using the 3,3 diaminobenzidine (DAB) in two contrast cassava genotypes, MNP Local A (29-57 µg g-1) and Sree Prakash (64-141 µg g-1). Accumulating the fluorescence hydroxycoumarin compounds emitted by the cassava tubers observed under an ultraviolet (UV) lamp showed significant variations at 0, 3, 6, 9, 12, and 15 days of storage. The total phenolics and carotenoids significantly and negatively correlated with PPD progression; however, the anthocyanin and flavonoids positively correlated with the PPD-anchored ROS accumulation. The primary compound, Phthalic acid, di(2-propylpentyl) ester, was identified in both the cassava tubers, Sree Prakash (57.21 and 35.21%), and MNP Local A (75.58 and 60.21%) at 0, and 72 h of PPD, respectively. The expression of PPD-associated genes APX-2, APX-3, PAL, and AP was higher at 6-12 days of PPD, which signified the synthesis of ROS turnover and phenylpropanoid biosynthesis. A significant, strong, and positive correlation was established between the secondary metabolites and PPD signaling gene expression, which was inversely correlated with hydroxycoumarin and H2O2 accumulation. MNP Local A tubers exhibited longer storage life of 15 days with a low PPD score, higher metabolites synthesis, and gene expression. The PPD-resistant lines may be used to augment cassava breeding strategies for large-scale commercial and industrial use.

Internal Transcribed Spacer (ITS) Region of Nuclear Ribosomal DNA as a Suitable DNA Barcode for Identification of Zanthoxylum armatum DC. from Manipur.

Zanthoxylum armatum DC. is a plant with many medicinal values which is extensively used in traditional system of medicine for curing various diseases and ailments, including cancer. The aim of the present study is to identify Zanthoxylum armatum collected from different parts of Manipur, India, at molecular level. Molecular markers like internal transcribed spacer (ITS) region and other DNA barcoding genes such as matK, rbcL, psbA-trnH and trnL-trnF were targeted to find out the most suitable DNA barcode for identifying this species. Sequences obtained using the five primer pairs-ITS An5 and ITS An4, matK-413f-1 and matK-1227r-1, rbcL-1F and rbcL-724R, psbA-F and trnH-R and trnL-F and trnF-R were submitted to GenBank, NCBI. Amongst the five DNA barcoding targets, one nuclear and four chloroplast genes were successfully amplified by PCR (100%) and sequencing (100%) in all the eight plant samples. Sequence similarity of total ITS region (620 bp) when compared to the reference sequence were found to be between 98.55 and 99.68%. In our study, ITS sequence in combination with DNA barcoding sequences of rbcL, trnH-psbA and trnL-trnF was very successful in identification of Z. armatum and differentiate other species clearly in the phylogeny analysis. Our work shows ITS region to be the most suitable DNA barcode which formed a monophyletic group of the species in the phylogenetic tree analysis. The sequences of the barcoding genes of Z. armatum DC. obtained from this study adds to the already available resources which will be helpful in the future research endeavours.

Molecular detection and pathogenicity of a nucleopolyhedrovirus isolated from looper caterpillar (Hyposidra talaca), a tea pest.

Hyposidra talaca is a major defoliating pest of tea plants in north-eastern part of India. In this study, we look for variations (if any) in the attacking virus. Viral samples were collected from different regions of the northern part of West Bengal in India and were analyzed by PCR technique to study the variations across the region. The partial segment of the HytaNPV polyhedrin gene was cloned and sequenced. A 527 bp nucleotide sequence containing highly conserved region from polyhedrin gene of HytaNPV was observed. The blast homology search for studied polyhedrin gene showed 98% sequence identity with the sequence of previous reported NPV of H. talaca, H. infixaria and Buzura suppressaria. Pathogenicity study against second instar H. talaca indicated that the LC50 values ranged from 4.61 × 105 to 7.57 × 105 polyhedral occlusion bodies per ml (POBs/ml) and the LT50 values ranged from 4.2 to 6.66 days. Sequencing result reveals that the same HytaNPV strain dominates across this area and the pathogenicity indicates its potential as an alternative to chemical insecticides to control H. talaca.

Enhanced resistance to blister blight in transgenic tea (Camellia sinensis [L.] O. Kuntze) by overexpression of class I chitinase gene from potato (Solanum tuberosum).

Tea is the second most consumed beverage in the world. A crop loss of up to 43 % has been reported due to blister blight disease of tea caused by a fungus, Exobasidium vexans. Thus, it directly affects the tea industry qualitatively and quantitatively. Solanum tuberosum class I chitinase gene (AF153195) is a plant pathogenesis-related gene. It was introduced into tea genome via Agrobacterium-mediated transformation with hygromycin phosphotransferase (hpt) gene conferring hygromycin resistance as plant selectable marker. A total of 41 hygromycin resistant plantlets were obtained, and PCR analysis established 12 plantlets confirming about the stable integration of transgene in the plant genome. Real-time PCR detected transgene expression in four transgenic plantlets (T28, C57, C9, and T31). Resistance to biotrophic fungal pathogen, E. vexans, was tested by detached leaf infection assay of greenhouse acclimated plantlets. An inhibitory activity against the fungal pathogen was evident from the detached leaves from the transformants compared with the control. Fungal lesion formed on control plantlet whereas the transgenic plantlets showed resistance to inoculated fungal pathogen by the formation of hypersensitivity reaction area. This result suggests that constitutive expression of the potato class I chitinase gene can be exploited to improve resistance to fungal pathogen, E. vexans, in economical perennial plantation crop like tea.

Biochemical evaluation of triploid progenies of diploid × tetraploid breeding populations of Camellia for genotypes rich in catechin and caffeine.

To verify the quality of triploid varieties of Camellia tea species at the secondary metabolite level, we tested caffeine and catechin profiles of 97 F(1) segregating progenies in two breeding populations with a common tetraploid parent and diploid parents of two geographic and varietal origins. Catechin and caffeine levels of the triploid progenies were quantified and compared against their diploid parent. Some of the progenies showed better performance than their diploid parent. Most of the progenies of the diploid C. sinensis × tetraploid cross showed heterosis for caffeine and EGCG. Progenies of the C. assamica subsp. lasiocalyx × tetraploid cross showed heterosis for +C, EC, EGC, and TC. The genomic contributions of the diploid parent seem to be the main factor in the variation between the two populations. Our studies showed quantitative enhancement of some of the quality-related parameters in tea, providing a platform to refocus on this classical breeding approach for developing quality cultivars in tea.

Molecular analysis of drought tolerance in tea by cDNA-AFLP based transcript profiling.

A cDNA-AFLP approach was used to identify transcript and/or genes specifically expressed in response to drought in tea. Drought was artificially induced and whole genome transcript profiling was done at three different stages-6 days before wilting, 3 days before wilting and at wilting stage of both tolerant and susceptible cultivars, and genetic differences was thus visualized as polymorphisms in the transcriptome. The cDNA-AFLP technique allowed genes and transcripts to be identified in the tolerant genotype (TV-23) whose expression is responsive to drought stress. The cluster analysis revealed two types of clustering-type I separated the tolerant and susceptible cultivar, whereas type II separated the time point of sample and this may be grouped as early and late responsive transcripts. 108 transcript derived fragments were identified as differentially expressed in tolerant genotypes of which 89 sequences could be obtained. Fifty-nine of them showed homology in the public databases. Functional ontology showed genes related to carbohydrate metabolism, response to stress, protein modification process and translation. Cluster I includes five fragments and cluster II includes 25 fragments. Other genes strongly expressed in response to drought in tolerant genotype would help us in identifying and determining the genetic basis of mechanisms involved in conferring drought tolerance in tea.

Identification of drought tolerant progenies in tea by gene expression analysis.

Understanding the genes that govern tea plant (Camellia sinensis) architecture and response to drought stress is urgently needed to enhance breeding in tea with improved water use efficiency. Field drought is a slow mechanism and the plants go through an adaptive process in contrast to the drastic changes of rapid dehydration in case of controlled experiments. We identified a set of drought responsive genes under controlled condition using SSH, and validated the identified genes and their pattern of expression under field drought condition. The study was at three stages of water deficit stress viz., before wilting, wilting and recovery, which revealed a set of genes with higher expression at before wilting stage including dehydrin, abscissic acid ripening protein, glutathione peroxidase, cinnamoyl CoA reductase, calmodulin binding protein. The higher expression of these genes was related with increase tolerance character of DT/TS-463 before wilting, these five tolerant progenies could withstand drought stress and thus are candidates for breeding. We observed that physiological parameter like water use efficiency formed a close group with genes such as calmodulin related, DRM3, hexose transporter, hydrogen peroxide induced protein, ACC oxidase, lipase, ethylene responsive transcription factor and diaminopimelate decarboxylase, during wilting point. Our data provides valuable information for the gene components and the dynamics of gene expression in second and third leaf against drought stress in tea, which could be regarded as candidate targets potentially associated with drought tolerance. We propose that the identified five tolerant progenies on the basis of their drought tolerance can thus be utilised for future breeding programmes.

Understanding Darjeeling tea flavour on a molecular basis.

Darjeeling teas are the highest grown teas in the world and preferred for its flavour, aroma and quality. Apart from the genetic makeup of the plant, earlier reports suggest that insect infestation, particularly jassids and thrips triggers the aroma and flavour formation in Darjeeling tea. The present work encompasses the identification of the genes/transcriptomes responsible for the typical flavour of Darjeeling tea, besides understanding the role of jassids and thrips in particular, in producing the best cup character and quality. The quantitative real time PCR analysis was based on a suppression subtractive hybridisation forward library of B157 (tea clone infested with thrips), providing us transcripts related to aroma and flavour formation. We observed the expression of genes like leucine zipper, ntd, nced, geraniol synthase, raffinose synthase, trehalose synthase, amylase, farnesyl transferase, catalase, methyl transferase, linalool synthase, peroxidases, elicitor responsive proteins, linamarase, nerolidol linalool synthase 2, 12-oxophytodienoate reductase, glucosidase, MYB transcription factor, and alcohol dehydrogenase, highly regulated due to insect infestation, manufacturing stresses and mechanical injury. The first report on gene expression dynamics in thrips infested Darjeeling tea leaves can be extrapolated with increase in volatiles which is responsible for enhancing the quality of Darjeeling tea, specially the flavour and aroma of the infusion. We hope to model these responses in order to understand the molecular changes that occur during Darjeeling tea flavour formation.

New record of nucleopolyhedroviruses in tea looper caterpillars in India.

Tea production in North-East India hit a record loss due to the widespread severe outbreak of a mixed brood of three species of looper caterpillar pests of geometrid moths (Lepidoptera) in 2008-2010. In addition to Buzura suppressaria, two newly recorded geometrids, viz., Hyposidra infixaria and Hyposidra talaca have caused widespread severe damage in recent years. In the present study we report the nucleopolyhedroviruses (NPV) isolated from the tea looper caterpillar from North-East India. We identified and characterized the NPV by cloning and sequencing a partial segment of polyhedrin gene of virus infected larvae of B. suppressaria, H. talaca and H. infixaria. A comparison of deduced amino acids of polyhedrin gene among H. talaca, H. infixaria and B. suppressaria showed that same strain was found to infect all the three loopers in India, which show high sequence identity with B. suppressaria Chinese isolates. Based on the polyhedrin sequence homology, it is predicted that a variant of B. suppressaria Chinese isolate of NPV found to infect H. talaca, H. infixaria and B. suppressaria in India.

Transgenic mimicry of pathogen attack stimulates growth and secondary metabolite accumulation.

Plant secondary metabolites, including pharmaceuticals, flavorings and aromas, are often produced in response to stress. We used chemical inducers of the pathogen defense response (jasmonic acid, salicylate, killed fungi, oligosaccharides and the fungal elicitor protein, cryptogein) to increase metabolite and biomass production in transformed root cultures of the medicinal plant, Withania somnifera, and the weed, Convolvulus sepium. In an effort to genetically mimic the observed effects of cryptogein, we employed Agrobacterium rhizogenes to insert a synthetic gene encoding cryptogein into the roots of C. sepium, W. somnifera and Tylophora tanakae. This genetic transformation was associated with stimulation in both secondary metabolite production and growth in the first two species, and in growth in the third. In whole plants of Convolvulus arvensis and Arabidopsis thaliana, transformation with the cryptogein gene led, respectively, to increases in the calystegines and certain flavonoids. A similar transgenic mimicry of pathogen attack was previously employed to stimulate resistance to the pathogen and abiotic stress. In the present study of biochemical phenotype, we show that transgenic mimicry is correlated with increased secondary metabolite production in transformed root cultures and whole plants. We propose that natural transformation with genes encoding the production of microbial elicitors could influence interactions between plants and other organisms.