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ThesisItem Open Access Physiological and biochemical changes in response to iron deficiency in pearl millet(CCSHAU, Hisar, 2022-08) Ritu; Dr. Vinod KumarIron (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.ThesisItem Open Access Biochemical and protein profiling in response to application of herbal nano-formulation(s) on shelf life of guava (Psidium guajava L.)(CCSHAU Hisar, 2022-09) Anju Rani; Tokas, JayantiThe guava (Psidium guajava L) is a resilient, easily adaptable and evergreen tree of the Myrtaceae family. Guavas are nutrient rich fruit having high content of vitamin A, vitamin C, antioxidants and minerals but the post-harvest losses stand at 20-40% of the produce. Herbal NFs were used to prevent post-harvest losses. Initially, different herbal extracts of giloy (leaves and stem) and ajwain seeds were prepared using different solvents and extraction procedure. Ajwain methanol (E2), ajwain methanol-acetone (7:3) (E3), giloy methanol (E16) and giloy acetone (E17) leaves extracts were selected for NFs synthesis. Three NFs viz. sodium alginate: CaCl2 (T1), sodium alginate: CaCl2: ajwain extract (T2), and sodium alginate: CaCl2: giloy extract (T3) were selected for further treatment. Physiological, quality, minerals, cell wall and ripening related parameters were analyzed every alternate day upto 10 DAT during storage at 25±2 °C and 32±2 °C. The results revealed that physiological loss in weight increased and fruit firmness decreased during storage. The quality parameters viz., total sugars, reducing sugars total soluble solids and ascorbate increased initially and then decreased at both the storage temperatures. The percent increase in total soluble sugars, reducing sugars and TSS on 10th day was maximum in control (34.8 and 36.6%; 70.7 and 76.3%; 31.8 and 34.8%) followed by T1 (29.3 and 30.0%; 64.6 and 66.2%; 15.7 and 25.1%), T3 (17.3 and 23.1%;42.5 and 49.4%; 5.1 and 9.5%), and T2 (14.9 and 20.7%; 36.4 and 44.6%; 3.7 and 8.9%) NFs at 25±2 °C and 32±2 °C. Whereas, Titratable acidity, crude proteins and antioxidant activity reduced during storage. Minerals content decreased during ripening in all the treatments and minimum decrease was recorded in T2 NFs treated fruits. Activity of cell wall degrading enzymes cellulase and polygalaturonase increased as the storage period increased whereas pectin methyl esterase activity increased initially and then decreased. The maximum percent increase of cellulase and PG was observed in control (272.6 and 365.3%; 285.3 and 325.3%) fruits followed by T1 (225.6 and 343.6%; 271.0 and 306.5%), T3 (211.2 and 318.7%; 224.1 and 272.2%) and T2 (201.3 and 292.9%; 215.2 and 266.5%) NFs treated fruits at 25±2 °C and 32±2 °C. Ethylene peak was observed on 4th and 6th day of treatment at 25±2 °C and 32±2 °C and similar trend was observed for ACC oxidase activity. The results revealed that Sodium alginate: CaCl2: Ajwain (T2) NF was most effective in controlling post-harvest losses and increased shelf life by upto 10-12 days. Lesser quality degradation took place at 25±2 °C as compared to 32±2 °C. The, control and T2 NF treated fruits were further analyzed for proteomics study using 2D-Electrophoresis and LC-MS/MS. Maximum proteins concentrated at higher pH and more in the range of 20-120 kD in 2D gel. Analysis of LC-MS/MS results using MASCOT software revealed more than 100 differentially expressed proteins (DEPs), out of which 35 proteins were up-regulated and 20 proteins were down-regulated having fold change ≥1.5 and ≤0.12, respectively, which were further analysis using GO software. Proteins were classified on the basis of molecular, biological and cellular functions. Protein profiling elucidated the expression of some novel proteins which might play a role in delayed ripening.ThesisItem Open Access Development of herbal nano-formulations for enhancing shelf life of guava (Psidium guajava L.) fruit(Chaudhary Charan Singh Haryana Agricultural University hisar, 2022-09) Gupta, Pernika; Tokas, JayantiGuava (Psidium guajava L.), member of the family Myrtaceae is an important fruit of tropical and subtropical area of world. It is also known as ―apple of the tropics‖. Nutritionally, guava fruit is a rich source of ascorbic acid but the post-harvest losses stand at 20-40% of the produce. Herbal extract based edible nano-formualtions were used to prevent post-harvest losses. Initially, different herbal extracts of ashwagandha and menthe leaves were prepared using different solvents. Three nanoformulations viz. Alg-Lys (T1), Alg-Lys:menthe extract (methanol) (T2 ), and Alg- Lys:ashwagandha extract (methanol) (T3) were selected for further treatments. Physiological, quality, cell wall components & degrading enzymes, antioxidative and ripening related parameters were analyzed every 3 day upto 12 DAT during storage at turning and mature green stage of Hisar Safeda and Hisar Surkha. The results revealed that physiological loss in weight increased and fruit firmness decreased during storage. The quality parameters viz., total soluble sugars and total soluble solids; antioxidative enzymes viz., superoxide dismutase, catalase, ascorbate peroxidase activity and ascorbate content increased initially and then decreased in both Hisar Safeda and Hisar Surkha. Overall decrease in total soluble solids was delayed in T3 treated guava fruits which was significantly higher than in control fruits in Hisar Safeda and Hisar Surkha. Whereas, titratable acidity, crude protein content, glutathione reductase and peroxidase activity reduced during storage. Activity of cell wall degrading enzymes cellulase and polygalaturonase increased as the storage period increased whereas pectin methyl esterase activity increased initially and then decreased. The amount of ethylene evolution and activity of ACC Oxidase in two cultivars of guava fruits increased initially and during further ripening process, the activity reduced. The results revealed that Alg-Lys:ashwagandha (T3) nanoformulation was most effective in controlling post-harvest losses and increased shelf life of Hisar Safeda upto 5 days and Hisar Surkha upto 3 days. The, control and T3 nanoformulation treated fruits were further analyzed 2D-Electrophoresis and LC-MS/MS and it was observed that maximum proteins had concentrated at higher pH and more in the range of 20-120 kD. Analysis of LC-MS/MS results using MASCOT software revealed more than 60 differentially expressed proteins (DEPs), which were involved in energy and carbohydrate metabolism, stress response, defense, biosynthesis, hydrolase, secondary metabolism, amino acid and nucleic acid, protein synthesis and biogenesis of cellular components having fold change ≥1.5 which were considered for further analysis. Using GO classification, major pathways involved in molecular, biological and cellular functions in Hisar Safeda fruit ripening were classified. Proteins were classified on the basis of molecular, biological and cellular functions. Protein profiling elucidated the expression of six novel proteins which might play a role in delayed ripening.
