| The Content Variation of Natural Product Induced by Different Factor(s) | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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| Species Name: Brassica juncea (var. RLC-1) | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| Factor Name: K2CrO4 Treatment; Na2SeO4 Treatment | [1] | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| Experiment Detail |
The seeds were surface sterilized and then soaked for two hours and sown in soil mixture having 3 parts of garden soil, 1 part of sand and 1 part of manure. The experiment was carried out in earthen pots of uniform size each containing 5 Kg of the soil mixture. Before sowing, the soil was amended with K2CrO4 for Cr treatments (0 µM/Kg), and Na2SeO4 for Se treatments (0 µM/Kg), both alone and in combinations. The concentration used for Cr was 50% inhibitory concentration (IC50), while for Se, the most stimulatory concentrations as observed from preliminary studies were used. The pots were kept in natural environmental conditions and were watered regularly. The experiment was conducted in triplicates. The harvesting of the plants was done after 30 days of sowing and stored at -20 ℃ . Some harvested plants were also dried by keeping them in hot air oven for 24 h at 80 ℃.
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| Factor Function |
Se application aided in improving plant growth, reducing the oxidative damage and strengthening the antioxidative defence system in plants raised in soils with binary combinations of Cr and Se. Photosynthesis, which is one of the vital physiological processes, was positively influenced with application of Se. It helped in minimising the toxicity of Cr and enhanced the contents of pigments. The efficiency of photosynthetic machinery was further strengthened by the increase of net photosynthetic rate, transpiration rate, stomatal conductance and intercellular CO2 concentration, and hence indicated its importance in combating stress. The study also highlighted the role of Se in enhancing the contents of secondary metabolites which play an important role in heavy metal chelation, complex formation and ROS scavenging, thereby reducing the chances of Cr to cause physiological damage.
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| Mechanism |
A significant modulation in gene expression was observed in B. juncea in response to Cr and Se. The gene responsible for H2O2 production is respiratory burst oxidase (RBO) which showed a significant upregulation in its expression by 3.63 folds in response to Cr treatment. Se at 2 µM/Kg in combination with 300 µM/Kg Cr caused decrease by 1.62 folds in the expression of RBO gene.An increase in expression was observed SOD, CAT and GST-1 by 2.75 folds, 2.82 folds and 2.03 folds respectively in response to Cr. However, Cr treatment resulted in a reduction of relative expression of POD and GR genes by 0.54 and 0.61 folds respectively in leaves of B. juncea plants. The combined treatment of Se and Cr aided in reducing Cr toxicity by increasing the expression of genes coding all these enzymes. Maximum increase in expression in case of CAT (4.68 folds), GR (2.08 folds) and GST-1 (2.98 folds) was observed at binary combination of 4 µM/Kg Se and 300 µM/Kg Cr. For SOD, 4.25 folds increase in gene expression was observed at 6 µM/Kg Se and 300 µM/Kg Cr. The expression of POD enhanced by 1.75 folds at the concentration of 2 µM/Kg Se and 300 µM/Kg Cr. The genes coding forcholrophyllase (CHLASE) chalcone synthase (CHS) and phenylalanine ammonialyase (PAL) showed enhanced expression of 2.47 folds, 1.79 folds and 2.07 folds respectively in the plants raised in Cr spiked soils. The co-application of Se and Cr helped in increasing the expression of CHS and PAL, while aided in reducing the expression of CHLASE. The concentration of 4 µM/Kg for Se proved to be most beneficial for enhancing the gene expression of PAL by 3.92 folds, while the same concentration caused a decline in the expression of CHALSE by 1.65 folds. However, for CHS expression, 6 µM/Kg Se caused an increase by 2.52 folds. Statistical analysis by one-way ANOVA and MLR supported the observations. The values of beta-regression coefficients for Se indicated the stress alleviating effects of Se for all the genes.
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| Factor | Part | Location | NP Content | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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0 µ/Kg K2CrO4 + 0 µ/Kg Na2SeO4 (Control)
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Fresh leaves | Ludhiana, Punjab, India. |
NP Content: 0.0259 ± 0.0005 mg/g fresh weight
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0 µ/Kg K2CrO4 + 2 µ/Kg Na2SeO4
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Fresh leaves | Ludhiana, Punjab, India. |
NP Content: 0.0275 ± 0.0003 mg/g fresh weight
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0 µ/Kg K2CrO4 + 4 µ/Kg Na2SeO4
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Fresh leaves | Ludhiana, Punjab, India. |
NP Content: 0.0294 ± 0.0007 mg/g fresh weight
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0 µ/Kg K2CrO4 + 6 µ/Kg Na2SeO4
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Fresh leaves | Ludhiana, Punjab, India. |
NP Content: 0.0256 ± 0.0005 mg/g fresh weight
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300 µ/Kg K2CrO4 + 0 µ/Kg Na2SeO4
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Fresh leaves | Ludhiana, Punjab, India. |
NP Content: 0.0217 ± 0.0005 mg/g fresh weight
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300 µ/Kg K2CrO4 + 2 µ/Kg Na2SeO4
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Fresh leaves | Ludhiana, Punjab, India. |
NP Content: 0.0242 ± 0.0005 mg/g fresh weight
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300 µ/Kg K2CrO4 + 4 µ/Kg Na2SeO4
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Fresh leaves | Ludhiana, Punjab, India. |
NP Content: 0.0255 ± 0.0007 mg/g fresh weight
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300 µ/Kg K2CrO4 + 6 µ/Kg Na2SeO4
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Fresh leaves | Ludhiana, Punjab, India. |
NP Content: 0.0244 ± 0.0012 mg/g fresh weight
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| Factor Name: CdCl2 Treatment; Earthworms Treatment | [2] | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| Experiment Detail |
The experiments were conducted under controlled conditions using plastic pots having lower diameter of 7.8 cm, upper diameter of 13.5 cm and 12 cm in height. The soil was collected from the top layer (0-20 cm) from the Botanical Garden of the university. Soil was air dried crushed and sieved through 2 mm filter autoclaved at 121 ℃ for 2 h. The soil was autoclaved to exclude soil pathogens and other microorganisms if any. The autoclaved soil was poured in pots and kept in the growth chamber. The pots were filled with 500 g uncontaminated soil and partially decayed compost (cow manure) (2:1) and was used as growing medium. The cow dung was added into the soil for better performance of earthworms. A subsample of the study soil before mixing with compost was analyzed for its physicochemical characteristics. The soil used for the experiment was sandy loam soil having pH 7.8 , EC (Electrical conductivity) (µS/cm) =184.25 , TDS (Total Dissolved Solids) (mg/kg) = 130 , N (Nitrogen) (mg/kg) = 103 , P (Phosphorus) (mg/kg) = 10.6 , K (Potassium) (mg/kg) = 0.343 , %OC = 0.894, Cd (mg/kg) = ND (not detected by AAS).The Cd treatment was given by using anhydrous CdCl2 (Minimum assay: 95.0%) procured from Hi-Media laboratories. The CdCl2 anhydrous was added to the soil to make different concentrations of Cd 0.50 mM, 0.75 mM, 1.00 mM, and 1.25 mM (i.e. 56 mg/Kg , 84 mg/Kg , 112 mg/Kg and 140 mg/Kg respectively). The various treatments given are as shown below:(1)C0 (Control): (Cadmium absence);(2)C1: (0.5 mM Cd);(3)C2: (0.75 mM Cd);(4)C3: (1.00 mM Cd);(5)C4: (1.25 mM Cd).Each Cd treatment was given in soils without as well as with earthworms (WTE = without, WE = with earthworms). Earthworms (3 earthworms per pot) were inoculated after seven days of Cd treatment and incubated for 7 d in soil with earthworms. The seeds after surface sterilization were sown in soil containing different concentration of Cd and earthworms in plastic pots. These pots were kept in seed germinator under controlled conditions i.e. 25 ℃ temperature and 16:8 h dark: light photoperiod (1700 lx) for 15 d. Seedlings were harvested after 15 d followed by washing with distilled water. The growth and biochemical analysis was done on these seedlings.
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| Factor Function |
Increased Cd uptake in plants in presence of earthworms enhances the total antioxidative capacity, metal chelating compounds and content of other antioxidants in plants grown under metal polluted soils. Earthworms can improve plant growth by improving nutrient availability to plants through their vermicasting activity. Their role in modifying soil pH and increasing metal phytoavailability made their use ideal in phytoremediation of polluted soils. Increased uptake and accumulation of Cd in plants activates the antioxidative system of plants takes place by addition of earthworms to soil.
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| Mechanism |
The gene expression for the key enzymes involved in organic acid metabolism was studied to understand the role of earthworms in organic acid metabolism in plants under Cd metal stress. It was observed that in comparison to control (C0) seedlings the expression of CS, SUCLG1, SDH and FH was enhanced 1.72, 1.58, 1.65 and 1.88 folds in seedlings given C4 treatment with 1.25 mM dose of Cd respectively . However, after supplementation of earthworms to Cd treated soils given C4 treatment resulted in further enhancement in expression of CS (2.53 fold), SUCLG1 (2.35 fold), SDH (2.13 fold) and FH (3.06 fold) .
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| Factor | Part | Location | NP Content | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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0.5 mM CdCl2 + without earthworms
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NA | Ludhiana, India. |
NP Content: 0.217 ± 0.006 mg/g
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0.75 mM CdCl2 + without earthworms
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NA | Ludhiana, India. |
NP Content: 0.321 ± 0.026 mg/g
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1.00 mM CdCl2 + without earthworms
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NA | Ludhiana, India. |
NP Content: 0.357 ± 0.014 mg/g
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1.25 mM CdCl2 + without earthworms
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NA | Ludhiana, India. |
NP Content: 0.408 ± 0.012 mg/g
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0 mM CdCl2 + with earthworms
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NA | Ludhiana, India. |
NP Content: 0.207 ± 0.006 mg/g
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0 mM CdCl2 + without earthworms
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NA | Ludhiana, India. |
NP Content: 0.197 ± 0.009 mg/g
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0.5 mM CdCl2 + with earthworms
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NA | Ludhiana, India. |
NP Content: 0.233 ± 0.006 mg/g
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0.75 mM CdCl2 + with earthworms
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NA | Ludhiana, India. |
NP Content: 0.353 ± 0.038 mg/g
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1.00 mM CdCl2 + with earthworms
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NA | Ludhiana, India. |
NP Content: 0.404 ± 0.016 mg/g
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1.25 mM CdCl2 + with earthworms
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NA | Ludhiana, India. |
NP Content: 0.480 ± 0.016 mg/g
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| Species Name: Crocus sativus L. (saffron) | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| Factor Name: AMF Inoculation; Harvest Time Variation | [3] | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| Experiment Detail |
AMF Inoculation in Pot : Saffron corms with horizontal diameters of 1.3 to 2.8 cm were sown in pots (4 L; 1 corm per pot) in the last ten days of August 2016. Pots were filled with sterile quartz sand (3 L per pot) on a layer of sterilized expanded clay (1 L per pot). Corms were treated with two inocula (MycAgro Lab, Breteniere, FR), one composed of a single fungus Rhizophagus intraradices (Ri) and one of R. intraradices and Funneliformis mosseae (Ri + Fm). Ten grams of each inoculum were placed under each corm in order to guarantee the contact between the inoculum and the roots and therefore to favor the symbiosis between AMF and roots. Saffron corms used as controls were not inoculated (AMF-). Corms were not treated against fungal pathogens. A randomized block design was used with a total of 48 pots displayed in two experimental plot units (24 pots per unit) and three treatments (8 pots per treatment). Cultivation lasted for one cycle (August 2016-April 2017) in a heated glasshouse of the Department of Agricultural Forest and Food Sciences (DISAFA) of the University of Torino (Italy, 45° 06′ 23.21″ N Lat, 7° 57′ 82.8″ E Long; 293 m a.s.l.), with an average temperature of 22 ℃ during the day and 16 ℃ in the night. Irrigation water (pH 7.4, EC 505 µS cm) was added weekly (250 mL per pot) with a drip system. The corms were fertilized by fertigation (VIGORFLOR, AL.FE. srl, MN, Italy) every two weeks starting from the emergence of the spate, in quantities of 1.5 g/L of water. No flowering occurred because of the small size of the corms.AMF Inoculation in Open Field : Saffron corms with horizontal diameters of 2.5 to 3.5 cm were planted in the last ten days of August 2016 in two Alpine experimental sites located in the municipality of Morgex (45° 45′ 35″ N; 7° 02′ 37.3″ E; 1000 m a.s.l.) and Saint Cristophe (45° 45′ 06″ N; 7° 20′ 37″ E; 700 m a.s.l.) in Italy and cultivation lasted for two cycles (2016-2017 and 2017-2018). Both sites were cultivated with saffron for at least the previous three years. Before starting the experiment both fields were milled. To assess the effects of AMF inocula on saffron cultivation and production, the same treatments used in the pot trial were applied (Ri, Ri + Fm or AMF-). A randomized block design was used, with three experimental plot units (blocks). Each plot unit consisted of 56 corms, planted in a 1.44 m2 area (39 corms m-2). Inter-row planting distance was of 7 cm, while between-row distance was 25 cm. Plots were separated from each other with at least 4 m distance. Before planting, 10 g of inoculum was placed under the corms to ensure contact between plant and the treatment. Irrigation was provided when needed and hand weeding control was conducted during cultivation, while no preplanting fertilization, tillage, or treatments against pathogens were applied. The two Alpine sites were characterized by semicontinental climate, with a long and cold winter . In general, both sites had a sandy-loam texture according to the USDA classification and similar chemical characteristics.
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| Factor Function |
The inoculum composed by R. intraradices and F. mosseae was particularly effective in increasing flower production and saffron yield, while R. intraradices alone increased the content of some bioactive compounds-picrocrocin, quercitrin, crocin II-as well as antioxidant activity.
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| Factor | Part | Location | NP Content | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Harvesting time: 2016-2017
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Powdered saffrons | Italy |
NP Content: 76 mg/100g dry weight
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Harvesting time: 2017-2018
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Powdered saffrons | Italy |
NP Content: 67 mg/100g dry weight
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Rhizophagus intraradices and Funneliformis mosseae inoculation
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Powdered saffrons | Italy |
NP Content: 71 mg/100g dry weight
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Rhizophagus intraradices inoculation
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Powdered saffrons | Italy |
NP Content: 70 mg/100g dry weight
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Non-AMF inoculation (Control)
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Powdered saffrons | Italy |
NP Content: 73 mg/100g dry weight
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