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Subject: Biochemistry × End date: 2022 × Thesis Type: M.Sc ×
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    ThesisItemOpen Access
    Physiological and biochemical changes in response to iron deficiency in pearl millet
    (CCSHAU, Hisar, 2022-08) Ritu; Dr. Vinod Kumar
    Iron (Fe) is the most essential micronutrient required for plant growth and development, being required as a redox active metal involved in various physiological and metabolic processes. About 1/3rd of world soil is deficient in Fe affecting plant growth and development leading to poor yield as well as nutritional quality. It is difficult to manage Fe deficiency to plants through agronomic practices. However, recently developed Fe biofortified crop varieties with higher Fe uptake potential provides an attractive alternate for evaluation for their performance under prevailing Fe deficiency in soils. In this study, Biofortified pearl millet hybrid HHB-299 developed by CCSHAU, Hisar was used along with popular non-biofortified hybrid HHB-67(I) for evaluating these contrasting Fe containing genotypes for their growth performance under varied Fe supply (100, 50, 10 and 0 µM Fe in nutrient solution, respectively) under controlled conditions. Effect of Fe deficiency as compared to Fe sufficiency was studied at 5-leaf and panicle initiation stages of pearl millet genotypes with analysis of various morpho-physiological (root length, shoot length, chlorophyll content and leaf area) and biochemical parameters (SOD and catalase activity, total antioxidant activity, and content of MDA, H2O2, proline, phytosiderophores and Fe). It was observed that with increase in Fe deficiency under treatments T1 to T3, shoot length was decreased in the range of 0.92- 38.9% and 9.5-41.7% at 5-leaf and panicle initiation stage, respectively, for genotype HHB-67(I), as compared to control. In comparison, significantly, less decrease in shoot length was observed for biofortified genotype HHB-299. Similarly, increased rate of chlorosis was observed in both the genotypes, with increased Fe deficiency. Overall analysis of data for antioxidant activity revealed its values in the range of 18-76% at 5-leaf stage and 32.3- 56.1% at panicle initiation stage, respectively, for both the genotypes. With increase in Fe deficiency, the phytosiderophores content was noticed an increase in the range of 55.5-69.2% and 53.3-68.1% at 5-leaf and panicle initiation stages of genotype HHB-67(I), respectively. In the genotype HHB-299, it was increased up to 81.7% and 75% at 5-leaf and panicle initiation stage under increased Fe deficiency treatments. The Fe content in shoots and roots was reportedly decreased under Fe deficient conditions, for both the stages and genotypes. Based on the outcome of study, it is concluded that the contrasting pearl millet genotypes have varied biochemical and morpho-physiological responses towards Fe deficiency. Preliminary findings in this study suggested HHB-299 as better performer under given Fe deficiency treatments over HHB-67(I) owing to its less affected biochemical and morphological responses as observed. The study might have implications in development, evaluation and promotion of biofortified or Fe deficiency tolerant crops for cultivation in soils with prevailing Fe deficiency.
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    ThesisItemOpen Access
    Immobilization of xylanase from Bacillus pumilus over aluminum oxide beads
    (CCSHAU, Hisar, 2021-09) Tanwar, Ekta; Nagar, Sushil
    In present investigation the xylanase from Bacillus pumilus SV-85S was covalently immobilized over aluminum oxide beads. The immobilization yield and efficiency were 48.05 and 83.13%, respectively. Some surface modifications were observed through SEM analysis on xylanase bound aluminum oxide beads. The bands at 3440 and 1635 cm-1 were observed through FTIR analysis over aluminum oxide beads, glutaraldehyde treated beads and immobilized xylanase. Optimum pH and temperature for free and immobilized enzyme were 7.0 and 55ᵒC, respectively. Increment of Km and Vmax values was observed in immobilized enzyme by around 28.96 and 34.61 % respectively. The pH, thermal and storage stability were improved after immobilization. The Immobilized xylanase retained 65 % of its initial activity even after 5 cycles of reusability. The increase in Half-life (t1/2) and D- value whereas decrease in kd value after immobilization, indicates that thermal stability of xylanase was enhanced. Thermodynamic parameters (ΔH, ΔG and ΔS) were enhanced after immobilization of xylanase. The decrease in activation energy in case of immobilized xylanase indicates that catalytic efficiency was enhanced. Hence, the present investigation concludes that immobilization of enzyme is important due to its considerable reusability and stability, and advantageous for industrial application.
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    ThesisItemOpen Access
    Comparative studies of free and immobilized Aspergillus phytase
    (CCSHAU, Hisar, 2021-09) Mallesh, Gampa; Nagar, Sushil
    In this investigation the phytase from Aspergillus oryzae SBS50 was immobilized over activated aluminum oxide beads. The immobilization yield and efficiency were 47.4 and 71.2 %, respectively. Morphology and functional groups of activated beads and immobilized phytase were determined through SEM and FTIR analysis. The immobilized phytase retained 51.5 % of its initial activity even after 5 cycles of reusability. The optimum pH and temperature were shifted from 5.5 to 4.0 and 50 to 60 C after immobilization. The Km value was 2.61 and 3.09 mM, whereas as Vmax value was 32.25 and 43.29 IU/ml for free and immobilized phytase, respectively. The pH stability, thermal stability, storage stability and thermodynamic parameters (H, G, and S) were enhanced after immobilization The increase in Half-life (t1/2) and D-value whereas decrease in Kd value after immobilization indicates that thermal stability of phytase was enhanced. Thus, the investigation concludes that immobilization of phytase causes in improvement of many properties, so it is highly beneficial to immobilize the phytase.
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    ThesisItemOpen Access
    Effect of brassinolide on morpho-physiological and biochemical parameters under drought stress in Indian mustard
    (CCSHAU, Hisar, 2020-02) Naveen; Kumari, Nisha
    The present experiment was conducted in Oilseed Research Area during Rabi 2018-19 with two Indian mustard varieties viz. drought-tolerant (RH-725) and drought-sensitive (RH-749) to find the efficiency of brassinolide in two concentrations of 10 ppm and 20 ppm sprayed at 42 and 52 days after sowing. The experiment was laid out in RBD design with three replications. Results revealed that: The activities of antioxidative enzyme (SOD, CAT and POX) increase in both the varieties but this increase was more pronounced in RH-725 with 20 ppm concentration of brassinolide spray. The concentration of non-enzymatic antioxidants (carotenoid, ascorbic acid and proline) also increase significantly in RH-725 as compared to RH-749 at 20 ppm spray of brassinolide. The oxidative stress indicators (hydrogen peroxide, malondialdehyde and electrolyte leakage) decrease to a significant level under brassinolide spray in tolerant variety at 50 % flowering. The exogenous application of brassinolide improved the physiological framework in the leaves of RH-725 at 50 % flowering. The yield and yield attributing characters such as plant height (cm), number of primary branches/plant, main shoot length (cm), number of siliquae on main shoot, number of seeds/siliqua, siliqua length (cm), seed yield/plant (g) were significantly increased among the treatments as well as varieties. Due to spraying of brassinolide the percentage of increase in seed yield ranged from 11.84 % in RH-725 and 5.77 % in RH-749 at 10 ppm concentration over control. In view of the present findings, the higher concentration of non-enzymatic antioxidants, higher activity of antioxidative enzymes and improved physiological parameters and maximum yield was found in RH-725 i.e. drought-tolerant at 20 ppm concentration of brassinolide spray.
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    ThesisItemOpen Access
    Biochemical studies on fatty acid esterase isolated from pearl millet and wheat flours
    (CCSHAU, Hisar, 2020) Nisha; Chugh, L. K
    The present investigation was conducted on pearl millet cv. HC 20 and wheat cv. WH 1105. Pearl millet fatty acid esterase (PM-FAE) and wheat fatty acid esterase (WH-FAE) were partially purified from by ammonium sulphate fractionation (30-60 % saturation) followed by dialysis by 3.03 and 7.75 folds with specific activity of 15.35 and 22.5 units/mg protein and recovery of 57and 56 % of the activity, respectively. PM-FAE and WH-FAE, both exhibited optimum pH of 8.0. Optimum temperature for PM-FAE (40oC) was lower than WH-FAE (45oC). WH-FAE was comparatively more stable at high temperature (70OC). Km value of PM-FAE (0.129 μM p-NPB) was lower than that of WH-FAE (0.28 μM p-NPB). Km values of PM-FAE for p-NPB and NPO were about 50 % lower compared to that of WH-FAE. Affinity of PM-FAE and WH-FAE for NPD was approximately equal. Ascorbic acid and palmitoyl ascorbate inhibited both FAEs in a concentration dependent manner. After 5 days of storage, reduction of 73 % in total buildup of fat acidity (FA), 84% reduction in rate of increase in FA, 55 % inhibition of in vitro activity of FAE, 52 % inhibition in situ activity of FAE and 50 % inhibition of activity of lipoxygenase (LOX) in 0.58 % ascorbic acid-fortified flour compared to unfortified flour were observed. Inhibitory effect of ascorbic acid fortification on in vitro activity of FAE, in situ activity of FAE and LOX increased with fortified concentration of ascorbic acid whereas rate of increase in FA was almost unaffected. Inhibition of LOX (62-69 %) in 0.84 % ascorbic acid-fortified flour was comparable to that (68-74 %) of blanched grain flour (BGF) throughout storage period. It was concluded that comparatively higher activity of PM-FAE in flour, lower Km value for its substrates and lower optimum temperature might be responsible for faster in situ hydrolysis of lipids and hence poor shelf life of pearl millet flour than wheat flour. Indirect fortification of flour by ascorbic acid treatment of pearl millet grains was effective in negatively modulating activities of FAE and LOX in flour for arresting not only in situ hydrolysis but also enzymatic oxidation of lipids.
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    ThesisItemOpen Access
    In vitro antioxidant and antimicrobial activities of mustard extracts
    (CCSHAU, Hisar, 2021-09) Jyoti; Nisha Kumari
    Mustard is the third most important oilseed crop after oil palm and soybean. Although mustard is widely used as edible oil and condiment, the residue left after oil extraction is popularly used as livestock and poultry feed with excellent protein quality. However, the information regarding antioxidative and antimicrobial potential of mustard seed meal is still scanty. Therefore, the objective of the present study was to extract the polyphenols from brown and yellow mustard seed meal with different solvents i.e 80% methanol, 80% ethanol, 80% acetone and 80% isopropanol. Each extract was analysed for antioxidant potential using DPPH and ABTS scavenging activity, ferric ion reducing antioxidant power (FRAP) assay, total antioxidant activity, iron chelating activity and β-Carotene content. The 80% acetone extracted maximum polyphenols followed by 80% ethanol, 80% methanol and 80% isopropanol. Brown mustard exhibited a higher amount of polyphenols as compared to yellow mustard. Yellow mustard exhibited higher antioxidant potential in terms of DPPH and ABTS scavenging activity, ferric ion reducing antioxidant power, iron chelating activity and β-Carotene content. Correlation analysis indicated a negative association between total phenolic content and DPPH, ABTS and FRAP. Antimicrobial potential was evaluated against two gram-negative (Escherichia coli and Xanthomonas campesteris), two gram positive (Bacillus cereus and Lactobacillus plantarum) bacteria and three fungi (Fusarium oxysporum, Macrophomina phaseolina and Candida albicans). Yellow mustard exhibited higher antibacterial activity while brown mustard had higher antifungal activity. Both the mustard seed meal extracts were ineffective against probiotic bacteria Lactobacillus plantarum. Gallic acid, caffeic acid, ferulic acid and sinapic acid were identified in mustard seed meal by HPLC analysis.
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    ThesisItemOpen Access
    Effect of phytase supplementation on micronutrients dialyzability in wheat bran
    (CCSHAU, Hisar, 2021-09) Kiran; Vinod Kumar
    Bread wheat, a staple cereal, have considerably low amount of iron (Fe) and zinc (Zn) bioavailability mainly due to the presence of phytic acid (PA; myo-inositol-1,2,3,4,5,6-hexakisphosphate), a phosphorus storage form in seeds, as a potent chelator of divalent cations. Wheat bran (WB) is a residue of the rolled milled wheat grain and it is rich source of dietary fibres, minerals, inorganic phosphates, vitamins, and tannins. The major portion of PA is accumulated in aleurone layer and globoid crystals of wheat. The PA chelates of Fe and Zn is a major source of concern for monogastric animals as lack of suitable digestible enzyme make this complex insoluble and decreases bioavailability of these micronutrients. Supplementation of exogenous phytase for dephytinization and value addition of cereals based foods is considered as a promising approach. In present study, experimental five wheat varieties were taken and their bran samples were collected for determining their chemical and biochemical properties (including content of PA, tannin, total dietary fibre, protein, inorganic phosphate, Fe and Zn) affecting the micronutrient bioavailability by either way. Significant variation was noticed in dietary fibre, crude protein and micronutrient content. Exogenous phytases from two different sources (fungal phytase from Aspergillus orizae strain SBS50 and wheat phytase from Sigma-Aldrich) were used to treat the wheat bran samples and determine their effects on above parameters. Phytase untreated samples were taken for comparative analysis of results obtained. For phytase treatments, 0.2 U/g phytase enzyme was used for both enzyme and results revealed that the PA content and tannin content was reduced by phytase treatment in the range of 37.2-39.5 % with wheat phytase and 7.90-33.33 % with Aspergillus phytase, respectively. Wheat phytase treatment was found more effective for PA, whereas, the Aspergillus phytase was found more effective in increasing the inorganic P content in treated bran samples. The inorganic P content in phytase treated bran sample was increased in the range of 16.30-29.62 % over untreated with maximum content was obtained in bran of WH 711 &WH 1021. Protein content after phytase treatment was increased for crude protein, whereas, soluble protein content was reduced to different extent. It was revealed that under given experimental conditions, phytase treatment resulted in decreased micronutrient dialyzability to different extent, with 7.11-45% reduction with wheat phytase treatment. Overall, wheat phytase treatment was found as comparatively better over Aspergillus phytase under given experimental conditions and bran of WH 1021 was found superior in studied quality attributes under given treatments. It is proposed to conduct the similar study with higher dose of phytase enzyme and in vitro digestion similar to gastric conditions before dialysis could give a greater and better insight into this area. The decreased dialyzability might be associated to experimental conditions and lower phytase dose. However, the results of the study showed that adding phytase to wheat bran improved its nutritional value for food applications.
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    ThesisItemOpen Access
    Effect of exogenous melatonin on cotton seed germination and seedling under saline condition
    (CCSHAU, Hisar, 2021-09) Jakhar, Sonika; Mandhania, Shiwani
    Cotton is an important fiber crop grown throughout the world and contributes significantly to global economy. Salinity is most destructive abiotic stress particularly at seed germination and seedling stage which considerably affects cotton productivity. Melatonin is regarded as a plant growth-promoting molecule and helps in mitigating several different abiotic stresses including salinity. Therefore, the present investigation was carried to study the effect of melatonin on seed germination and biochemical constituents in cotton under salinity stress. Results showed that salinity causes negative effects on cotton seed germination and decreases seed vigor. It also enhanced relative stress injury (in terms of electrolyte leakage), H2O2 and MDA content of seedlings by increasing membrane permeability to ions and solutes. Exogenous application of melatonin at the concentration of 20 μM has profound effect on seed germination, seedling vigor traits and antioxidant defense system in cotton under salinity soil. This indicates that seed priming with 20 μM melatonin mitigates the salinity effect and significantly improves cotton seed germination and vigor index traits. Melatonin might improve cotton germination under saline stress by mitigating the negative effect of oxidative stresses through elevating antioxidant defense system. This study provides an important information to improve cotton productivity in salt affected soil.
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    ThesisItemOpen Access
    Effect of phosphorus and potassium application on quality of sorghum (Sorghum bicolor (L.) Moench)
    (CCSHAU, 2019) Seema; Singal, H.R.
    Sorghum (Sorghum bicolor (L.) Moench) locally known as Jawar or Chari is an important kharif season crop which is grown for both fodder and grains. Fertilizers constitute an integral part of improved crop production technology. Application of optimum quantity of phosphorus and potassium is important in improving quality and yield of forage sorghum. The present investigation was carried out to study the effect of phosphorus and potassium application on quality parameters. Sorghum variety HJ 541 was grown in field and different doses of phosphorus (15, 30. 45, 60 and 90 kg P2O5/ha) and potassium (20, 30, 40, 50 and 60 kg K2O/ha) were given. Plant/leaf samples were collected at 30 DAS, 50 DAS and 90 DAS. Biochemical parameters namely HCN, IVDMD, crude protein, nitrogen content, phosphorus content, potassium content, iron content, NDF, ADF, total soluble solids, total soluble sugars, reducing and non- reducing sugars, morpho- physiological parameters namely fresh weight, dry weight, stem girth, internode length and number of leaves and cell wall components namely cellulose, hemicelluloses, lignin and pectin were studied. It was observed that HCN content was less than permissible value (200 μg/g at fresh weight) with the application of phosphorus and potassium. IVDMD, crude protein, nitrogen content, phosphorus content, potassium content, iron content, total soluble solids, total soluble sugars, reducing and non- reducing sugars increased with the application of phosphorus and potassium. Maximum fresh and dry weight, stem girth internode length and number of leaves increased with the application of phosphorus and potassium. NDF, ADF, cellulose, hemicelluloses, lignin and pectin decreased with the application of phosphorus and potassium. The results obtained revealed that the quality of forage sorghum improved with the application of phosphorus and potassium.