Copper and Silver Nanoparticle Seed Priming and Foliar Spray Modulate Plant Growth and Thrips Infestation in Capsicum spp.

Nanoparticles (NPs) have the potential to improve plant health and secondary metabolite production. In the present study, three different NPs, i.e., Ag, Cu, and Cu-Ag NPs were produced in the range from 25 to 86 nm, with zeta potentials ranging from -28.8 to -38.5 mV. The synthesized NPs were used for seed priming and foliar spray on three varieties of Capsicum annuum. L, i.e., Arka Sweta (AS), Arka Meghana (AM), and Arka Harita (AH) plants grown under greenhouse conditions. Seed priming at various concentrations of NPs (1, 10, 20 ppm) enhanced the seed germination (96%), seedling vigor index (2494-3112.66), seedling length (6-49%), and biomass (46%) of 45 days old Arka Meghana seedlings. Additionally, all plant tissues accumulated significantly higher amounts of chlorophyll (51-142%), carotenoids (23-94.2%), total phenolic content (73%), and total flavonoid content (57%), compared with the control (p ≤ 0.05). The foliar spray of NPs (20-100 ppm) has a protective effect on the chili plants against thrips infestation (30-76%). The foliar spray enhanced chlorophyll (15-62%), carotenoids (15-50%), total phenolic content (20-62%), total flavonoid content (64-99%), reducing sugars (15-97%), total antioxidant activity (15-142%), ferric reducing antioxidant power assay (15-109%), DPPH (129-54 mg mL-1), and capsaicinoids (capsaicin and dihydrocapsaicin) (82-128%). This study illustrates that Ag, Cu, and Cu-Ag NPs suppress thrips infestation and proliferation with enhanced plant growth and biochemical activity, which is inversely proportional to the NP size. Chemical NPs play a crucial role in the economic significance of chili plants, offering a promising avenue for developing pesticides to effectively combat thrips infestation. This advancement holds great potential in enhancing the overall agronomic productivity of the chili crops.

Chitosan nanoparticles modulate plant growth, and yield, as well as thrips infestation in Capsicum spp.

The objective of this study was to investigate the physiological and biochemical effects of Chitosan nanoparticles on Capsicum annuum plants. The particle size, polydispersity index, composition, and structure of the synthesized chitosan-based nanoparticles (Chitosan (CS), Chitosan-Silver (CSAg), and Chitosan-Copper (CSCu) NPs) were determined by analyzing the zeta potential, FTIR, TEM, and XRD. The seedlings showed improved physiological and biochemical characteristics when 1, 10, and 20 ppm concentrations of nanoparticles (CS, CSAg, and CSCu) were used for 24-h seed priming. The application of nanoparticles in different concentrations (0, 20, 40, 60, 80, and 100 ppm) on the leaves of Capsicum spp. plants resulted in improved physiological traits and protection against thrips by 70-85 %. Furthermore, it enhanced the content of chlorophyll (20-75 %), carotenoids (20-30 %), total phenolics (20-45 %), total flavonoids (40-125 %), reducing sugars (15-40 %), total antioxidant activity (10-82 %), FRAP (10-100 %), DPPH (76-83 mg mL-1) activity, and total capsaicinoids (125-142 %). Therefore, the use of chitosan-based nanoparticles could be considered an environmentally friendly approach to enhance secondary metabolite production, disease resistance, and growth in Capsicum spp. plants for sustainable production.

Phytoconstituents, GC-MS Characterization of Omega Fatty Acids, and Antioxidant Potential of Less-Known Plant Rivina humilis L.

Rivina humilis L. (Petiveriaceae), commonly known as the pigeon berry, accumulates betalains in berries. The present study was focused on identifying the phytoconstituents, mineral content, fatty acid composition, phenolics, flavonoids, antinutritional factors, and antioxidant activities of different plant parts (leaf, stem, root, and seeds), which are otherwise not well explored. Phytoconstituent analysis revealed seeds as a potential source of carbohydrates (50.15 g/100 g), proteins (10.96 g/100 g), and fats (11.25 g/100 g). Roots showed the highest fat (17.66 g/100 g) and dietary fiber (81.49 g/100 g). Leaves and roots contain more iron (29.59 and 29.39 mg/100 g), whereas seed has high zinc content (12.09 mg/100 g). Leaf oil showed 47.83 g/100 g of omega-3-fatty acid, confirmed by GC-MS analysis. Seed oil showed 22.23 g/100, 44.48 g/100, and 24.04 g/100 g of palmitic, oleic, and linoleic acids, respectively. The leaf extract has the highest TPC (597.55 mg/100 g), followed by the seed (421.68 mg/100 g). The leaf's 80% ethanolic extract had high TFC (2442.19 mg/100 g), followed by 70% methanolic extract (1566.25 mg/100 g). The antinutritional profile indicated significant phytic acid and oxalates in the leaf (9.3 g/100 and 2.07 g/100 g) and stem (6.9 and 1.58 g/100 g) and low tannin content (<0.5 g/100 g). The leaf's 80% ethanolic extract exhibited double the TAA than 70% methanolic extract (1.52 g/100 g). The leaf with an 80% ethanolic extract had the lowest DPPH and ABTS radical scavenging EC50 (2.22 and 0.37 mg/mL). The leaf with an 80% ethanolic extract (479.73 mg/100 g) and seed (391.14 mg/100 g) had the highest FRAP activity. Our study proves that different parts of R. humilis had a good content of phytoconstituents, bioactives, and antioxidant activities. Hence, R. humilis leaves and seeds are a novel source of omega fatty acids and minerals reported for the first time and have potential applications in the food and pharmaceutical industries.

Genome-wide identification of MATE, functional analysis and molecular dynamics of DcMATE21 involved in anthocyanin accumulation in Daucus carota.

Anthocyanins are a subclass of flavonoids that are synthesized in the endoplasmic reticulum and then transported to the vacuole in plants. Multidrug and toxic compound extrusion transporters (MATE) is a family of membrane transporters that transport ions and secondary metabolites, such as anthocyanins, in plants. Although various studies on MATE transporters have been carried out on different plant species, this is the first comprehensive report to mine the Daucus carota genome to identify the MATE gene family. Our study identified 45 DcMATEs through genome-wide analysis and detected five segmental and six tandem duplications from the genome. The chromosome distribution, phylogenetic analysis, and cis-regulatory elements revealed the structural diversity and numerous functions associated with the DcMATEs. In addition, we analyzed RNA-seq data obtained from the European Nucleotide Archive to screen for the expression of DcMATEs involved in anthocyanin biosynthesis. Among the identified DcMATEs, DcMATE21 correlated with anthocyanin content in the different D. carota varieties. In addition, the expression of DcMATE21 and anthocyanin biosynthesis genes was correlated under abscisic acid, methyl jasmonate, sodium nitroprusside, salicylic acid, and phenylalanine treatments, which were substantiated by anthocyanin accumulation in the in vitro cultures. Further molecular membrane dynamics of DcMATE21 with anthocyanin (cyanidin-3-glucoside) identified the binding pocket, showing extensive H-bond interactions with 10 crucial amino acids present in the transmembrane helix of 7, 8, and 10 of DcMATE21. The current investigation, using RNA-seq, in vitro cultures, and molecular dynamics studies revealed the involvement of DcMATE21 in anthocyanin accumulation in vitro cultures of D. carota.

Nanomaterials in agriculture for plant health and food safety: a comprehensive review on the current state of agro-nanoscience.

In the modern epoch, nanotechnology took forward the agriculture and food industry with new tools that promise to increase food production sustainably. It also anticipated that it would become a driving economic force shortly. Nanotechnology has the potential to reduce agricultural inputs, enrich the soil by absorbing nutrients, manage plant diseases, and detect diseases. The aim of the present review is to cover the potential aspects of nanoscience and its trend-setting appliances in modern agriculture and food production. This review focuses on the impact of various nanomaterials on plant health to improve agricultural production and its cooperative approach to food production. Nanotechnology has great potential compared to conventional approaches. The appealing path of nanotrends in the farming sector raises hopes and illuminates the route of innovative technologies to overcome various diseases in plants with an enhanced yield to meet the growing global population's need for food security.

miR-7-5p Antagomir Protects Against Inflammation-Mediated Apoptosis and Lung Injury via Targeting Raf-1 In Vitro and In Vivo.

Exacerbated inflammation and apoptosis are considered upstream events associated with acute lung injury (ALI). microRNAs are critical regulators of genes responsible for inflammation and apoptosis and are considered potential therapeutic targets for ameliorating ALI. This study was undertaken to uncover the role of miR-7-5p in LPS-induced lung injury. A LPS-induced inflammation model was established using BEAS-2B cells and C57BL/6 mice. Bioinformatics analysis and the luciferase reporter assay confirmed that Raf-1 is a target of miR-7-5p and that its expression was inversely correlated with expression of proinflammatory markers and miR-7-5p, whereas miR-7-5p inhibition in vitro led to subsequent restoration of Raf-1 expression and prevention of apoptosis. Intranasal (i.n.) administration of antagomir using the C57BL/6 mouse model further confirmed that miR-7-5p inhibition suppresses LPS-induced inflammation and apoptosis via modulating the miR-7-5p/Raf-1 axis. Our findings indicate that blocking miR-7-5p expression by antagomir protects mice from LPS-induced lung injury by suppressing inflammation and activation of mitochondria-mediated survival signalling. In conclusion, our findings demonstrate a previously unknown pathophysiological role of miR-7-5p in the progression of ALI, and targeted i.n. administration of miR-7-5p antagomir could aid in the development of potential therapeutic strategies against lung injury.

Genome-wide identification, characterization of Serotonin N-acetyltransferase and deciphering its importance under development, biotic and abiotic stress in soybean.

Serotonin N-acetyltransferase (SNAT) is the penultimate enzyme involved in plant melatonin biosynthesis. Identifying its expression under development and stress will reveal the regulatory role in the soybean. To identify and characterize SNAT, we employed genome-wide analysis, gene structure, cis-acting elements, expression, and enzyme activity. We identified seven putative genes by genome-wide analysis and found chloroplast signal peptides in three GmSNATs. To elucidate GmSNATs role, expression datasets of more than a hundred samples related to circadian rhythm, developmental stages, and stress conditions were analysed. Notably, the expression of GmSNAT1 did not show significant expression during biotic and abiotic stress. The GmSNAT1 sequence showed 67.8 and 72.2 % similarities with OsSNAT and AtSNAT, respectively. The Km and Vmax of the purified recombinant GmSNAT1 were 657 µM and 3780 pmol/min/mg, respectively. To further understand the GmSNAT1 role, we supplemented different concentrations of serotonin and melatonin to in-vitro cultures and seed priming. These studies revealed that the GmSNAT1 expression was significantly up-regulated at higher concentrations of serotonin and down-regulated at higher melatonin concentrations. We speculate that a high concentration of melatonin during abiotic, biotic stress, and in-vitro cultures are responsible for regulating GmSNAT1 expression, which may regulate them at the enzyme level during stress in soybean.

Distinct GmASMTs are involved in regulating transcription factors and signalling cross-talk across embryo development, biotic, and abiotic stress in soybean.

N-Acetylserotonin O-methyltransferase (ASMT) is the final enzyme involved in melatonin biosynthesis. Identifying the expression of ASMT will reveal the regulatory role in the development and stress conditions in soybean. To identify and characterize ASMT in soybean (GmASMT), we employed genome-wide analysis, gene structure, cis-acting elements, gene expression, co-expression network analysis, and enzyme assay. We found seven pairs of segmental and tandem duplication pairs among the 44 identified GmASMTs by genome-wide analysis. Notably, co-expression network analysis reported that distinct GmASMTs are involved in various stress response. For example, GmASMT3, GmASMT44, GmASMT17, and GmASMT7 are involved in embryo development, heat, drought, aphid, and soybean cyst nematode infections, respectively. These distinct networks of GmASMTs were associated with transcription factors (NAC, MYB, WRKY, and ERF), stress signalling, isoflavone and secondary metabolites, calcium, and calmodulin proteins involved in stress regulation. Further, GmASMTs demonstrated auxin-like activities by regulating the genes involved in auxin transporter (WAT1 and NRT1/PTR) and auxin-responsive protein during developmental and biotic stress. The current study identified the key regulatory role of GmASMTs during development and stress. Hence GmASMT could be the primary target in genetic engineering for crop improvement under changing environmental conditions.

Salt Stress-Induced Anthocyanin Biosynthesis Genes and MATE Transporter Involved in Anthocyanin Accumulation in Daucus carota Cell Culture.

Anthocyanins biosynthesis is a well-studied biosynthesis pathway in Daucus carota. However, the scale-up production at the bioreactor level and transporter involved in accumulation is poorly understood. To increase anthocyanin content and elucidate the molecular mechanism involved in accumulation, we examined D. carota cell culture in flask and bioreactor for 18 days under salt stress (20.0 mM NH4NO3/37.6 mM KNO3) at 3 day intervals. The expression of anthocyanin biosynthesis and putative MATE (multidrug and toxic compound extrusion) transporter expression was analyzed by qRT-PCR. It was observed that there was a significant enhancement of anthocyanin in the bioreactor compared to the control culture. A correlation was observed between the expression of MATE and the anthocyanin biosynthesis genes (CHS, C4H, LDOX, and UFGT) on the 9th day in a bioreactor, where maximum anthocyanin accumulation and expression was detected. We hypothesize the involvement of MATE in transporting anthocyanin to tonoplast in D. carota culture under salt stress.

Physicochemical Composition and Characterization of Bioactive Compounds of Mulberry (Morus indica L.) Fruit During Ontogeny.

Mulberry fruit is well recognized as one of the richest sources of bioactive compounds. We investigated the physicochemical composition and characterized the bioactive compounds during different ripening stages of mulberry (Morus indica) fruit and evaluated their anti-quorum sensing activity on Chromobacterium violaceum. The proximate components such as carbohydrates, proteins and lipids were found to be high in the ripe fruit compared to unripe and mid-ripe fruit. The ripe fruit contained higher content of total phenolics and flavonoids (336.05 and 282.55 mg/100 g fresh weight (FW), respectively). Epicatechin and resveratrol were the major polyphenols detected in the fruit with the range 5.13-19.46 and 4.07-14.45 mg/100 g FW, respectively. Chlorogenic acid and myricetin were predominant in the unripe and mid-ripe fruit (7.14 and 1.84 mg/100 g FW, respectively). The fruit was found to be an excellent source of anti-diabetic compound 1-deoxynojirimycin. The highest content of 1-deoxynojirimycin was present in the mid-ripe fruit, with a content of 2.91 mg/100 g FW. Furthermore, fruit extracts exhibited anti-quorum sensing activity against Chromobacterium violaceum by effectively inhibiting violacein production. Ripe fruit extracts showed the highest activity of 76.30% at 1 mg/mL and thus, could be used as a potent anti-quorum sensing agent. The results could be promising in the selection of appropriate developmental stages for M. indica fruit commercial exploitation in the food formulations rich in potential health components.

Exogenous Serotonin and Melatonin Regulate Dietary Isoflavones Profoundly through Ethylene Biosynthesis in Soybean [Glycine max (L.) Merr.].

Serotonin and melatonin are important signaling and stress mitigating molecules. However, their role and molecular mechanism in the accumulation of isoflavones are not clearly defined. To elucidate their functions, serotonin and melatonin were applied to in vitro cultures of soybean at different concentrations and analyzed to assess the accumulation of isoflavone content followed by transcript levels of biosynthesis genes at different time intervals. Increased total phenolics, total flavonoids, and different forms of isoflavone content were observed in the treatments. Expression levels of critical genes in isoflavone, ethylene, jasmonic acid, abscisic acid, and melatonin biosynthesis and related transcription factor were quantified. A correlation was observed between the expression of ethylene biosynthesis genes (S-adenosylmethionine synthase and 1-aminocyclopropane-1-carboxylate oxidase) and isoflavone biosynthesis genes (chalcone synthase, chalcone reductase, and isoflavone synthase). We hypothesize that, under serotonin and melatonin treatments, ethylene biosynthesis may play a role in the increase/decrease in isoflavone content in soybean culture.

Nanoliposomal encapsulation mediated enhancement of betalain stability: Characterisation, storage stability and antioxidant activity of Basella rubra L. fruits for its applications in vegan gummy candies.

Betalains are violet-red, natural food grade pigments with health benefits; however, their stability limits its use in industrial food processing. This can be overcome by placing the betalains in lecithin nanoliposomes (NLs), which causes a 76% improvement of betalain colour and stability. Extended sonication time (8 min) lowered the zeta potential (-47.5 to -40.8), and particle size (74.23 to 55.35 nm). Zeta potential, particle size, and polydispersity index of Betalain NLs (BNLs) didn't change significantly during storage (40 days). Degradation in the colour of BNLs was observed only at 121 °C (20 min) while the native juice degraded at 100 °C (20 min). BNLs were incorporated in gummy candies (GuCa) to improve its colour stability. The betalain retention, colour, texture, antioxidant activity, and shelf-life of the GuCa during storage (5 °C, 28 days) demonstrated the efficacy of BNLs to be explored as a natural colourant for the food industry.

Influence of photoperiod on growth, bioactive compounds and antioxidant activity in callus cultures of Basella rubra L.

Basella rubra L. is an important green leafy vegetable vine and is known for its health benefits in traditional medicine. Light is a basic physical factor essential to the development and bioactive secondary metabolite production in in vitro callus cultures. The present study researched the impact of different photoperiods on biomass, bioactive compounds, and antioxidant activity in callus cultures of B. rubra. The in vitro seedling based cotyledonary leaf explants responded differently, when cultured on Murashige and Skoog (MS) medium with varying concentrations and combination of auxins and cytokinins. The best callus proliferation was found in MS medium with 0.1 mg.L-1 1-naphthaleneacetic acid (NAA) and 6 mg.L-1 6-benzylaminopurine (BAP), with greenish callus inception by about 2 weeks. The growth curve recorded for 6 weeks of culturing revealed that the photoperiod effect was found to be pivotal for acquiring biomass. At the fifth week, the continuous light supported maximum biomass (12.42 g) production followed by the 16:8 h photoperiod (9.02 g) and continuous darkness (4.28 g). The 80% ethanol extract of 1-week-old callus that grows under the 16:8 h photoperiod showed the highest total phenolic content (TPC) (74 mg.100 g-1 fresh weight, FW) when compared to all other extracts at different stages. The ferric reducing antioxidant power assay showed the highest (336.23 mg.100 g-1 FW) activity in methanol extractions of first-week callus cultures maintained in the continuous light condition. HPLC-UV identification and quantification of individual phenolics and flavonoids, such as gallic, trans-cinnamic, quercetin, protocatechuic and rutin, were highest in the callus cultures. The outcome of this study is significant to this plant, as B. rubra is familiar for its important health constituents with high-value bioactives and applications in the pharma and nutraceutical industries.

Evaluation of Various Drying Methods on Bioactives, Ascorbic Acid and Antioxidant Potentials of Talinum triangulare L., foliage.

The aim of this study was to assess the efficiency of different drying methods (room, sun, oven, microwave, cross-flow, infra-red, dehumidifier, and freeze-drying) on maximum retention of the nutritional and bioactive compounds profiling of Talinum triangulare, which is a less-known perishable leafy-vegetable. The evaluation of various drying methods is to learn the best appropriate strategy for a post-harvest drying method for retaining all the potential benefits with minor loss. Microwave and freeze-dried samples held the maximum ascorbic acid quantified by HPLC with 1.36 and 1.11 g/100 g DW, respectively. The main carotenoid compounds identified were violaxanthin, lutein, zeaxanthin, ß-carotene isomers, trans-ß-carotene, and cis-ß-carotenes. Gallic, protocatechuic, catechin, para-coumaric, ferulic, rutin, trans-cinnamic, and quercetin are the significant phenolics and flavonoids identified and quantified by liquid chromatography. The efficiency of different solvents on bioactive extractions uncovered that the methanol and 80% aqueous ethanol were good for retention of total phenolics, total flavonoids, and antioxidant compounds, which was affirmed through phosphomolybdate, DPPH, and FRAP assays. Dried T. triangulare foliage could be productively utilized as a promising raw material for food and pharma businesses because of its rich bioactive composition.

Chemical composition, nutraceuticals characterization, NMR confirmation of squalene and antioxidant activities of Basella rubra L. seed oil.

Basella rubra (Malabar spinach) is a commonly consumed green leafy vegetable in southern parts of India. The chemical composition, nutraceuticals characterization, squalene Nuclear Magnetic Resonance (NMR), in vitro antioxidant activities and cytotoxicity of B. rubra seed oil (33.08%) was investigated. Gas chromatography-mass spectrometry (GC/MS) analysis revealed the presence of palmitic (27.21 µmol%), oleic (33.83 µmol%) and linoleic acid (26.02 µmol%) with a total of 64.38 µmol% unsaturated fatty acids respectively. HPLC nutraceutical characterization showed a major constituent of gallic acid (11.23 mg%), γ-tocopherols (17.74 mg%), cycloartenylferulate (1.7 mg%), and squalene (1 g%). Squalene was further recovered (98%), purified (99.9%), and confirmed through 1H and 13C NMR. The in vitro antioxidant activities recorded by using 2,2-diphenyl-1-picrylhydrazyl (EC50 = 6 mg mL-1), ferric reducing antioxidant power (361.85 mM of Trolox Eq./100 g) and 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulphonic acid (EC50 = 56.19 mg mL-1) scavenging activity. In vitro cytotoxicity assessed on 3T3-L1 showed good cell survival without any toxicity (upto 400 µg mL-1). B. rubra seed oil has proven nutraceuticals and antioxidant potentials with least toxicity which can be recommended for functional foods applications.

In vitro shoot multiplication of Rumex vesicarius L., and quantification of ascorbic acid and major phenolics from its leaf derived callus.

An optimised method for enhanced in vitro shoot multiplication of Rumex vesicarius (Polygonaceae)-a branched succulent herb-was achieved. The in vitro seed pre-treatment with 4% urea was able to show 95% seed germination on MS medium within 2 weeks of culturing. In vitro shoot bud induction from shoot tip explants was best in the presence of 2 mg L-1 kinetin on Murashige and Skoog medium (MS medium), wherein 8.3 shoots/explants were observed. Shoot elongation was found to be high (3.75 ± 0.5 cm) in 2 mg L-1 BA comprising medium. The transfer of micro-shoots on to MS medium comprising 1.5 mg L-1 IBA and 1% activated charcoal (w/v) supported efficiently in vitro rooting (2.5-4.0 cm) in 3-4 weeks. Upon hardening, 70% rooted plants survived under greenhouse conditions. Though friable callus was produced on MS medium-containing 2 mg L-1 BA followed by 3 mg L-1 BA, no organogenesis was noticed. The ascorbic acid content of 78.62 ± 0.25 mg 100 g-1 FW was recorded in callus cultures grown on medium supplemented with 2 mg L-1 BA, and it is 1.74-fold more compared to normal ex vitro leaves of the same age. In vitro raised plant leaf showed 1.98-fold more ascorbic acid (89.42 ± 0.18 mg 100 g-1 FW) to that of ex vitro leaves. The total phenolic content was found to be 60 mg in callus as compared to 610 mg (per 100 g GAE FW) of ex vitro leaves. The major phenolic compounds quantified were synergic, chlorogenic, ferulic, and generic acids, respectively. This optimised protocol will facilitate to pursue scale-up studies for in vitro ascorbic acid production and also to further investigate the kinetics of biosynthetic pathway genes involved.

Mucilaginous polysaccharides from vegetative parts of Bixa orellana L.: Their characterization and antioxidant potential.

The aim of this study was to extricate adhesive mucilaginous polysaccharides (MPS) from vegetative parts of Bixa orellana and furthermore to explore proximate analysis and antioxidant potentials. The ethanol precipitated mucilage concentrates of leaf holds 57.7% ± 3.2% moisture, 31.7 ± 1.61 g/100 g aggregate starch content, 23.6 ± 0.07 g/100 g reducing sugar content, 14.5 ± 2.0 g/100 g pentose content, 9.37 ± 0.56 g/100 g uronic acid substance, 10.6 ± 0.98 g/100 g total phenolic content, and 36.9 ± 0.52 mg/100 g total protein content. In contrasted with twigs, the leaves MPS demonstrated two folds increment in phenolic content. The sugar composition characteristics of MPS by gas liquid chromatography demonstrated a noteworthy content of xylose (163.5 ± 6.6 mg/100 g), mannose (17.2 ± 0.6 mg/100 g), and galactose (13.7 ± 0.4 mg/100 g) in leaf. The MPS extract of leaves exhibited a dose-dependent antioxidant activity and free radical scavenging ability. The data obtained in this study open a new avenue to further investigate the broad applications of these annatto dye yielding plant polysaccharides. PRACTICAL APPLICATIONS: The mucilaginous polysaccharides (MPS) from plant sources are being used for different applications in food, pharma, and cosmetic enterprises. Nowadays there is a developing enthusiasm for MPS for their dietary and medicinal advantages. Particularly dietary polysaccharides are known to evoke immunomodulatory and anti-inflammatory properties. Here, we present the leaf and twig parts of Bixa orellana as rich sources of MPS, the MPS characterization and also antioxidant potential, which could substantiate B. orellana MPS as functional ingredients and furthermore in nourishment industry for expanding dairy foods consistency.

Combining bioinformatics and conventional PCR optimization strategy for one-time design of high-specificity primers for WRKY gene family using unigene database.

Gene families, like the conserved transcription factor families, evolve through gene duplications and share moderate similarity between member genes. Lack of genomic data makes it difficult to design high-specificity primers to the target genes. Furthermore, many primers under-perform in highly sensitive assays like quantitative PCR due to issues of thermodynamic nature, thereby increasing the cost and time for analysis. A methodology involving intra-species and inter-generic bioinformatic sequence comparison combined with thermodynamic estimation of primer performance was used for one-time design of gene specific primers for different WRKYs, Mitogen Activated Protein-kinases and N-methyltransferases of Coffea canephora without the aid of genome sequence resources. Out of a total 37 primer sets including 31 pairs of primers for WRKY from 34 mined WRKY Unigenes/ESTs and six pairs for genes coding for MAP kinases and NBS-LRR proteins, 32 sets exhibited high specificity of amplification upon genome analysis as well as in the high-resolution melt analysis. Furthermore, PCR optimization strategies-both in silico and experimental-indicated a superior performance of the primer sets for different applications like quantitative PCR and rapid amplification of cDNA ends. Only one set of primer resulted in mis-priming upon confirmation by DNA sequencing of the cloned amplicons. The intra-species differences and inter-generic similarities ensure high specificity of primers in all cases studied. The procedure allowed design of primers for the use in different downstream applications with high performance, specificity, yield and ease-of-use.

Primer design and amplification efficiencies are crucial for reliability of quantitative PCR studies of caffeine biosynthetic N-methyltransferases in coffee.

Primers having suboptimal amplification efficiencies were shown to falsely represent fold change expression of the N-methyltransferases gene family involved in caffeine biosynthesis in Coffea canephora. To study this phenomenon, the role of stability of the internal reference gene, as well as the amplification efficiency correction of the primers was investigated. GAPDH and Ubiquitin exhibited a good stability for studying the ontogeny of endosperm tissue, as well as the leaf transcriptome during stress from salicylic acid, methyl jasmonate, PEG-mediated drought and sudden exposure to light. Ubiquitin manifested low variation in Cq under all these stress regimes and in endosperm ontogeny with 30.1-30.9 in the best dataset and 28.8-30.9 in the most deviating dataset. It was observed that problems arising due to improper amplification efficiency of the target or reference genes or both could lead to misinterpretation of gene expression levels. Quantitative RT-PCR performed at a sub-optimal efficiency of GAPDH reference gene at 1.68 led to the faulty interpretation of 2.007 folds upregulation by the 2-ΔΔCt method and 1.705 folds upregulation by Efficiency method for the first NMT (Xanthosine methyltransferase), which actually is repressed during dark acclimatization of coffee plants. Efficiency correction improved the reliability of the expression data and also indicated a downregulation of this gene by 0.485 folds and 0.474 folds using 2-ΔΔCt and E method, respectively, in concordance to earlier reports. Hence, efficiency correction of the primers having suboptimal efficiencies is an absolute prerequisite for the accurate calculation of fold change using quantitative RT-PCR.

Differential expression of anthocyanin biosynthesis genes in Daucus carota callus culture in response to ammonium and potassium nitrate ratio in the culture medium.

Anthocyanins are major water-soluble and dynamic colouring plant pigment present in plant tissues with the high antioxidant properties. The role of ammonium and potassium nitrate in the culture medium on anthocyanin augmentation is probed thoroughly, but the mechanism of its biosynthesis continues to be unclear. Hence, the present study was undertaken to optimise nitrate ratio in the culture medium for anthocyanin augmentation and examination of its biosynthesis pathway in callus culture of Daucus carota. MS basal medium fortified with various ratio of NH4NO3:KNO3 was employed to find their impact on biomass, anthocyanin augmentation and the expression profile of anthocyanin biosynthesis genes in the callus culture. The data indicated that the highest anthocyanin content (9.30 ± 0.25 mg/100 g FW) was seen in callus grown on the medium supplemented with 20.0 mM NH4NO3:37.6 mM KNO3 and the least was seen in the medium which contained 40.0 mM NH4NO3:18.8 mM KNO3 (2.74 ± 0.27 mg/100 g FW). This indicates an optimal concentration of NH4NO3:KNO3 ratio is essential to produce a higher amount of anthocyanin in in vitro culture. Meanwhile, anthocyanin biosynthesis genes were differentially expressed as confirmed by qRT-PCR in the time interval of 5, 10, 15, 20 and 25 days. The transcript levels of nine anthocyanin biosynthesis genes were increased in the response of varying NH4NO3:KNO3 ratio in the medium. The transcript level of early genes PAL, 4CL, CHS and CHI increased by 19.5, 21.0, 16.2 and 9.98-fold, respectively, compared with control. In addition, late biosynthesis genes LDOX and UFGT resulted in the transcript level of 11.3 and 13.6-fold, respectively.