Exploring DNA barcode for accurate identification of threatened Aconitum L. species from Western Himalaya.

BACKGROUND: Aconitum species, belonging to Ranunculaceae, have high medicinal importance but due to their overexploitation come under IUCN (International Union for Conservation of Nature) red list. The precise identification of the Aconitum species is equally important because they are used in herbal formulations. The present study aimed to develop an efficient DNA barcode system for the authentic identification of Aconitum species. METHODS AND RESULTS: A set of 92 barcode gene sequences (including 12 developed during the present study and 80 retrieved from NCBI) of 5 Aconitum species (A. heterophyllum, A. vialoceum, A. japonicum, A. napellus, and A. stapfianum) were analyzed using three methods (tree-based, distance-based, and similarity-based) for species discrimination. The PWG-distance method was found most effective for species discrimination. The discrimination rate of PWG- distance ranged from 33.3% (rbcL + trnH-psbA) to 100% (ITS, rbcL + ITS, ITS + trnH-psbA and rbcL + ITS + trnH-psbA). Among DNA barcodes and their combinations, the ITS marker had the highest degree of species discrimination (NJ-40%, PWG-100% and BLAST-40%), followed by trnH-psbA (NJ-20%, PWG-60% and BLAST-20%). ITS also had higher barcoding gap as compared to other individual barcodes and their combinations. Further, we also analyzed six Aconitum species (A. balfourii, A. ferox, A. heterophyllum, A. rotundifolium, A. soongaricum and A. violaceum) existing in Western Himalaya. These species were distinguished clearly through tree-based method using the ITS barcode gene with 100% species resolution. CONCLUSION: ITS showed the best species discrimination power and was used to develop species-specific barcodes for Aconitum species. DNA barcodes developed during the present study can be used to identify Aconitum species.

Mutagens in commercial food processing and its microbial transformation.

Mutagens are chemical molecules that have the ability to damage DNA. Mutagens can enter into our body upon consumption of improperly cooked or processed food products such as high temperature or prolonged cooking duration. Mutagens are found in the food products can be classified into N-nitroso derivatives, polycyclic aromatic hydrocarbons, and heterocyclic aromatic amines. Food products with high fat and protein content are more prone to mutagenic formation. Microorganisms were found to be a potent weapon in the fight against various mutagens through biotransformation. Therefore, searching for the microorganisms which have the ability to transform mutagens and the development of techniques for the identification as well as detection of mutagens in food products is much needed. In the future, methods for the identification and detection of these mutagens as well as the identification of new and more potent microorganisms which can transform mutagens into non-mutagens are much needed.

Neural networks and genetic algorithm as robust optimization tools for modeling the microbial production of poly-ß-hydroxybutyrate (PHB) from Brewers' spent grain.

This research work has been carried out to establish the combinatorial impact of various fermentation medium constituents, used for poly-ß hydroxybutyrate (PHB) biosynthesis. Model development was performed with an optimized medium composition that enhanced the biosynthesis of PHB from the biowaste material Brewers' spent grain (BSG). The latter was used as a carbon substrate in submerged fermentation with Bacillus sphaericus NCIM 2478. Three independent variables: BSG, yeast extract (YE), and salt solution concentration (SS) and one dependent variable (amount of PHB produced) were assigned. A total of 35 microbial fermentation trials were conducted by which a nonlinear mathematical relationship was established in terms of neural network model between independent and dependent variables. The resulting artificial neural networks (ANNs) model for this process was further optimized using a global genetic algorithm optimization technique, which predicted the maximum production of PHB (916.31 mg/L) at a concentration of BSG (50.12 g/L), concentration of YE (0.22 g/L), and concentration of SS (24.06%, v/v). The experimental value of the quantity of PHB (concentration ∼916 mg/L) was found to be very close to the value predicted by the ANN-GA model approach.

Nitric oxide secures reproductive efficiency in heat-stressed lentil (Lens culinaris Medik.) plants by enhancing the photosynthetic ability to improve yield traits.

Rising temperatures, globally and locally, would be detrimental for cool- and summer-season food legumes, such as lentil (Lens culinaris Medik.). Lentil is highly sensitive to supra-optimal temperatures (> 30 °C), particularly during reproductive growth, resulting in flower and pod losses. Thus, suitable strategies are needed to introduce heat tolerance in this legume. Here, we evaluated the efficacy of nitric oxide (NO)-applied as foliar treatment of 1 mM sodium nitroprusside (SNP), twice (one day before final exposure to high temperature, and again five days later)-on heat-stressed (32/20 °C) lentil genotypes, differing in heat sensitivity. As a result of heat stress, endogenous NO increased significantly in heat-tolerant genotypes (46-62% in leaves and 66-68% in anthers, relative to the respective controls), while it decreased in heat-sensitive (HS) genotypes (27-30% in leaves and 28-33% in anthers, relative to the respective controls). Foliar supplementation with SNP markedly increased endogenous NO in leaves and anthers of both the control and heat-treated plants. Heat stress significantly accelerated phenology, damaged membranes, chlorophyll, chlorophyll fluorescence, cellular viability, and decreased leaf water status, carbon fixing and assimilating ability, less so in plants treated with SNP. Heat stress plus SNP significantly improved carbon fixation (as RuBisCo activity) and assimilation ability, (as sucrose concentration (in leaves and anthers), sucrose synthase and vacuolar acid invertase activity, reducing sugars), as well as osmolyte accumulation (proline and glycine betaine) in leaves and anthers. Moreover, SNP-treated plants had significantly less oxidative damage-measured as malondialdehyde and hydrogen peroxide concentrations-in leaves and anthers, relative to the respective control. Reproductive function-assessed as pollen grain germination and viability, stigma receptivity, and ovular viability-decreased markedly in plants exposed to heat stress alone, more so in HS genotypes, but increased significantly with SNP treatment as a consequence of improved leaf and anther function, to significantly increase the pod and seed numbers in heat-stressed lentil plants, relative to heat-stress alone.

Heat Priming of Lentil (Lens culinaris Medik.) Seeds and Foliar Treatment with γ-Aminobutyric Acid (GABA), Confers Protection to Reproductive Function and Yield Traits under High-Temperature Stress Environments.

Gradually increasing temperatures at global and local scales are causing heat stress for cool and summer-season food legumes, such as lentil (Lens culinaris Medik.), which is highly susceptible to heat stress, especially during its reproductive stages of development. Hence, suitable strategies are needed to develop heat tolerance in this legume. In the present study, we tested the effectiveness of heat priming (HPr; 6 h at 35 °C) the lentil seeds and a foliar treatment of γ-aminobutyric acid (GABA; 1 mM; applied twice at different times), singly or in combination (HPr+GABA), under heat stress (32/20 °C) in two heat-tolerant (HT; IG2507, IG3263) and two heat-sensitive (HS; IG2821, IG2849) genotypes to mitigate heat stress. The three treatments significantly reduced heat injury to leaves and flowers, particularly when applied in combination, including leaf damage assessed as membrane injury, cellular oxidizing ability, leaf water status, and stomatal conductance. The combined HPr+GABA treatment significantly improved the photosynthetic function, measured as photosynthetic efficiency, chlorophyll concentration, and sucrose synthesis; and significantly reduced the oxidative damage, which was associated with a marked up-regulation in the activities of enzymatic antioxidants. The combined treatment also facilitated the synthesis of osmolytes, such as proline and glycine betaine, by upregulating the expression of their biosynthesizing enzymes (pyrroline-5-carboxylate synthase; betaine aldehyde dehydrogenase) under heat stress. The HPr+GABA treatment caused a considerable enhancement in endogenous levels of GABA in leaves, more so in the two heat-sensitive genotypes. The reproductive function, measured as germination and viability of pollen grains, receptivity of stigma, and viability of ovules, was significantly improved with combined treatment, resulting in enhanced pod number (21-23% in HT and 35-38% in HS genotypes, compared to heat stress alone) and seed yield per plant (22-24% in HT and 37-40% in HS genotypes, in comparison to heat stress alone). The combined treatment (HPr+GABA) was more effective and pronounced in heat-sensitive than heat-tolerant genotypes for all the traits tested. This study offers a potential solution for tackling and protecting heat stress injury in lentil plants.

Insomnia and its associated factors among older people of selected ward of Banepa municipality, Nepal.

Aims: The aim of this study was to calculate the prevalence of insomnia and identify the factors associated with it among older people of Banepa Municipality, Province No.3 of Nepal. Design: A quantitative descriptive cross-sectional study was done; data were collected for three months from September-November 2018. Methods: One hundred and fourteen older people were recruited. The data were collected through a structured interview questionnaire that included socio-demographic characteristics and Nepalese version of standard Pittsburgh Insomnia Rating Scale (20-item version). Information on general health conditions, alcohol intake and personal habits was also collected. Results: The mean age of the respondents (N = 114) was 76.04 (SD 7.81), years and 51.8% were females. The prevalence of insomnia was 71.1% in the older population. Significant associations were found between insomnia and advanced age (p = .002), illiteracy (p < .001), not working (p < .001), financially dependent on others (p < .001), presence of comorbid disease (p < .001) and taking regular medicine at present (p < .001).

Influence of drought and heat stress, applied independently or in combination during seed development, on qualitative and quantitative aspects of seeds of lentil (Lens culinaris Medikus) genotypes, differing in drought sensitivity.

Terminal droughts, along with high temperatures, are becoming more frequent to strongly influence the seed development in cool-season pulses like lentil. In the present study, the lentil plants growing outdoors under natural environment were subjected to following treatments at the time of seed filling till maturity: (a) 28/23 °C day/night temperature as controls; (b) drought stressed, plants maintained at 50% field capacity, under the same growth conditions as in a; (c) heat stressed, 33/28 °C day/night temperature, under the same growth conditions as in a; and (d) drought + heat stressed, plants at 50% field capacity, 33/28 °C day/night temperature, under the same growth conditions as in (a). Both heat and drought resulted in marked reduction in the rate and duration of seed filling to decrease the final seed size; drought resulted in more damage than heat stress; combined stresses accentuated the damage to seed starch, storage proteins and their fractions, minerals, and several amino acids. Comparison of a drought-tolerant and a drought-sensitive genotype indicated the former type showed significantly less damage to various components of seeds, under drought as well as heat stress suggesting a cross tolerance, which was linked to its (drought tolerant) better capacity to retain more water in leaves and hence more photo-assimilation ability, compared with drought-sensitive genotype.

Drought or/and Heat-Stress Effects on Seed Filling in Food Crops: Impacts on Functional Biochemistry, Seed Yields, and Nutritional Quality.

Drought (water deficits) and heat (high temperatures) stress are the prime abiotic constraints, under the current and climate change scenario in future. Any further increase in the occurrence, and extremity of these stresses, either individually or in combination, would severely reduce the crop productivity and food security, globally. Although, they obstruct productivity at all crop growth stages, the extent of damage at reproductive phase of crop growth, mainly the seed filling phase, is critical and causes considerable yield losses. Drought and heat stress substantially affect the seed yields by reducing seed size and number, eventually affecting the commercial trait '100 seed weight' and seed quality. Seed filling is influenced by various metabolic processes occurring in the leaves, especially production and translocation of photoassimilates, importing precursors for biosynthesis of seed reserves, minerals and other functional constituents. These processes are highly sensitive to drought and heat, due to involvement of array of diverse enzymes and transporters, located in the leaves and seeds. We highlight here the findings in various food crops showing how their seed composition is drastically impacted at various cellular levels due to drought and heat stresses, applied separately, or in combination. The combined stresses are extremely detrimental for seed yield and its quality, and thus need more attention. Understanding the precise target sites regulating seed filling events in leaves and seeds, and how they are affected by abiotic stresses, is imperative to enhance the seed quality. It is vital to know the physiological, biochemical and genetic mechanisms, which govern the various seed filling events under stress environments, to devise strategies to improve stress tolerance. Converging modern advances in physiology, biochemistry and biotechnology, especially the "omics" technologies might provide a strong impetus to research on this aspect. Such application, along with effective agronomic management system would pave the way in developing crop genotypes/varieties with improved productivity under drought and/or heat stresses.

Impact of heat stress during seed filling on seed quality and seed yield in lentil (Lens culinaris Medikus) genotypes.

BACKGROUND: Lentil, a cool-season food legume, is highly sensitive to high temperatures, which drastically reduce biomass and seed yield. The effects of heat stress on qualitative and quantitative aspects of seeds are not yet known. RESULTS: In this study, we assessed the effects of high temperatures on quantitative and qualitative aspects of seeds in a heat-tolerant (HT; FLIP2009) and heat-sensitive (HS; IG4242) genotypes in a controlled environment. Initially, the plants were raised in a natural, outdoor environment (22/10 °C mean day/night temperature, 1350 µmol m-2 s-1 light intensity, 60-65% relative humidity) from November to mid-February until 50% flowering (114-115 days after sowing). After that, one set of plants was maintained in a controlled environment (28/23 °C, as mean day and night temperature, 500 µmol m-2 s-1 light intensity, 60-65% relative humidity;control) and one set was exposed to heat stress (33/28 °C, as mean day and night temperature, 500 µmol m-2 s-1 light intensity, 60-65% relative humidity), where they remained until maturity. Compared to control, heat stress reduced the seed growth rate by 30-44% and the seed-filling duration by 5.5-8.1 days, which ultimately reduced the seed yield by 38-58% and individual seed weights by 20-39%. Heat stress significantly damaged cell membranes and reduced chlorophyll concentration and fluorescence, and the photosynthetic rate, which was associated with a significant reduction in relative leaf water content. The proximate analysis of seed reserves showed that heat stress reduced starch (25-43%), protein (26-41%) and fat (39-57%) content, and increased total sugars (36-68%), relative to the controls. Heat stress also inhibited the accumulation of storage proteins including albumins, globulins, prolamins and glutelins (22-42%). Most of the amino acids decreased significantly under heat stress in comparison to control, whereas some, such as proline, followed by glycine, alanine, isoleucine, leucine and lysine, increased. Heat stress reduced Ca (13-28%), Fe (17-52%), P (10-54%), K (12.4-28.3%) and Zn (36-59%) content in seeds, compared to the controls. CONCLUSIONS: High temperatures during seed filling are detrimental for seed yield and quality components in lentil genotypes, with severe impacts on heat-sensitive genotypes. © 2018 Society of Chemical Industry.

Effects of Drought, Heat and Their Interaction on the Growth, Yield and Photosynthetic Function of Lentil (Lens culinaris Medikus) Genotypes Varying in Heat and Drought Sensitivity.

Rising temperatures and drought stress limit the growth and production potential of lentil (Lens culinaris Medikus), particularly during reproductive growth and seed filling. The present study aimed to (i) investigate the individual and combined effects of heat and drought stress during seed filling, (ii) determine the response of lentil genotypes with contrasting heat and drought sensitivity, and (iii) assess any cross tolerance in contrasting genotypes. For this purpose, eight lentil genotypes (two drought-tolerant, two drought-sensitive, two heat-tolerant, two heat-sensitive) were either sown at the normal time (second week of November 2014), when the temperatures at the time of seed filling were below 30/20°C (day/night), or sown late (second week of February 2015) to impose heat stress (temperatures > 30/20°C (day/night) during reproducive growth and seed filling. Half of the pots in each sowing environment were fully watered throughout (100% field capacity) while the others had water withheld (50% of field capacity) from the start of seed filling to maturity. Both heat and drought, individually or in combination, damaged cell membranes, photosynthetic traits and water relations; the effects were more severe with the combined stress. RuBisCo and stomatal conductance increased with heat stress but decreased with drought and the combined stress. Leaf and seed sucrose decreased with each stress in conjunction with its biosynthetic enzyme, while its (sucrose) hydrolysis increased under heat and drought stress, but was inhibited due to combination of stresses. Starch increased under heat stress in leaves but decreased in seeds, but drastically declined in seeds under drought alone or in combination with heat stress. At the same time, starch hydrolysis in leaves and seeds increased resulting in an accumulation of reducing sugars. Heat stress inhibited yield traits (seed number and seed weight per plant) more than drought stress, while drought stress reduced individual seed weights more than heat stress. The combined stress severely inhibited yield traits with less effect on the drought- and heat-tolerant genotypes. Drought stress inhibited the biochemical processes of seed filling more than heat stress, and the combined stress had a highly detrimental effect. A partial cross tolerance was noticed in drought and heat-tolerant lentil genotypes against the two stresses.

Food Legumes and Rising Temperatures: Effects, Adaptive Functional Mechanisms Specific to Reproductive Growth Stage and Strategies to Improve Heat Tolerance.

Ambient temperatures are predicted to rise in the future owing to several reasons associated with global climate changes. These temperature increases can result in heat stress- a severe threat to crop production in most countries. Legumes are well-known for their impact on agricultural sustainability as well as their nutritional and health benefits. Heat stress imposes challenges for legume crops and has deleterious effects on the morphology, physiology, and reproductive growth of plants. High-temperature stress at the time of the reproductive stage is becoming a severe limitation for production of grain legumes as their cultivation expands to warmer environments and temperature variability increases due to climate change. The reproductive period is vital in the life cycle of all plants and is susceptible to high-temperature stress as various metabolic processes are adversely impacted during this phase, which reduces crop yield. Food legumes exposed to high-temperature stress during reproduction show flower abortion, pollen and ovule infertility, impaired fertilization, and reduced seed filling, leading to smaller seeds and poor yields. Through various breeding techniques, heat tolerance in major legumes can be enhanced to improve performance in the field. Omics approaches unravel different mechanisms underlying thermotolerance, which is imperative to understand the processes of molecular responses toward high-temperature stress.

Identification of High-Temperature Tolerant Lentil (Lens culinaris Medik.) Genotypes through Leaf and Pollen Traits.

Rising temperatures are proving detrimental for various agricultural crops. Cool-season legumes such as lentil (Lens culunaris Medik.) are sensitive to even small increases in temperature during the reproductive stage, hence the need to explore the available germplasm for heat tolerance as well as its underlying mechanisms. In the present study, a set of 38 core lentil accessions were screened for heat stress tolerance by sowing 2 months later (first week of January; max/min temperature >32/20°C during the reproductive stage) than the recommended date of sowing (first week of November; max/min temperature <32/20°C during the reproductive stage). Screening revealed some promising heat-tolerant genotypes (IG2507, IG3263, IG3297, IG3312, IG3327, IG3546, IG3330, IG3745, IG4258, and FLIP2009) which can be used in a breeding program. Five heat-tolerant (HT) genotypes (IG2507, IG3263, IG3745, IG4258, and FLIP2009) and five heat-sensitive (HS) genotypes (IG2821, IG2849, IG4242, IG3973, IG3964) were selected from the screened germplasm and subjected to further analysis by growing them the following year under similar conditions to probe the mechanisms associated with heat tolerance. Comparative studies on reproductive function revealed significantly higher pollen germination, pollen viability, stigmatic function, ovular viability, pollen tube growth through the style, and pod set in HT genotypes under heat stress. Nodulation was remarkably higher (1.8-22-fold) in HT genotypes. Moreover, HT genotypes produced more sucrose in their leaves (65-73%) and anthers (35-78%) that HS genotypes, which was associated with superior reproductive function and nodulation. Exogenous supplementation of sucrose to in vitro-grown pollen grains, collected from heat-stressed plants, enhanced their germination ability. Assessment of the leaves of HT genotypes suggested significantly less damage to membranes (1.3-1.4-fold), photosynthetic function (1.14-1.17-fold) and cellular oxidizing ability (1.1-1.5-fold) than HS genotypes, which was linked to higher relative leaf water content (RLWC) and stomatal conductance (gS). Consequently, HT genotypes had less oxidative damage (measured as malondialdehyde and hydrogen peroxide concentration), coupled with a higher expression of antioxidants, especially those of the ascorbate-glutathione pathway. Controlled environment studies on contrasting genotypes further supported the impact of heat stress and differentiated the response of HT and HS genotypes to varying temperatures. Our studies indicated that temperatures >35/25°C were highly detrimental for growth and yield in lentil. While HT genotypes tolerated temperatures up to 40/30°C by producing fewer pods, the HS genotypes failed to do so even at 38/28°C. The findings attributed heat tolerance to superior pollen function and higher expression of leaf antioxidants.

Use of Plackett-Burman design for rapid screening of nitrogen and carbon sources for the production of lipase in solid state fermentation by Yarrowia lipolytica from mustard oil cake (Brassica napus)

Mustard oil cake (Brassica napus), the residue obtained after extraction of mustard oil from mustard oil seeds, was investigated for the production of lipase under solid state fermentation (SSF) using the marine yeast Yarrowia lipolytica NCIM 3589. Process parameters such as incubation time, biomass concentration, initial moisture content, carbon source concentration and nitrogen source concentration of the medium were optimized. Screening of ten nitrogen and five carbon sources has been accomplished with the help of Plackett-Burman design. The highest lipase activity of 57.89 units per gram of dry fermented substrate (U/gds) was observed with the substrate of mustard oil cake in four days of fermentation.

Use of Plackett-Burman design for rapid screening of nitrogen and carbon sources for the production of lipase in solid state fermentation by Yarrowia lipolytica from mustard oil cake (Brassica napus).

Mustard oil cake (Brassica napus), the residue obtained after extraction of mustard oil from mustard oil seeds, was investigated for the production of lipase under solid state fermentation (SSF) using the marine yeast Yarrowia lipolytica NCIM 3589. Process parameters such as incubation time, biomass concentration, initial moisture content, carbon source concentration and nitrogen source concentration of the medium were optimized. Screening of ten nitrogen and five carbon sources has been accomplished with the help of Plackett-Burman design. The highest lipase activity of 57.89 units per gram of dry fermented substrate (U/gds) was observed with the substrate of mustard oil cake in four days of fermentation.