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ThesisItem Restricted Effect of priming with Moringa oleifera leaf extract on sorghum under salt stress(Punjab Agricultural University, Ludhiana, 2022) Anand Kumar; Oberoi, Harpreet KaurIn the present study, seeds of fourteen sweet sorghum genotypes were treated with different NaCl concentrations (0, 50, 100, 150, 200 mM). All germination traits decreased with increased salinity. Based on germination traits and dendogram obtained at 100 mM NaCl, PHULE VASUNDHARA and SPV 2074 were selected as tolerant and susceptible genotypes, respectively. Seeds of selected genotypes were primed with 10% Moringa oleifera leaf extract and grown in Hoagland‟s solution with and without 100 mM NaCl concentration. NaClinduced the activities of SOD (in roots), POX and PPO and reduced the activities of NR, NiR and GOGAT, however, significant improvement in the activities were observed with MLE in shoots and roots of two contrasting genotypes at 10 DAG. Salt stress resulted in increased Na+/K+ ratio, H2O2, MDA, sugars, phenols and decreased total chlorophyll. Gene expression of antioxidative enzymes was upregulated under salt stress in shoots and roots of both primed genotypes. Salt stress resulted higher PPO, GOGAT, NiR and NR gene expressions in shoots of both contrasting genotypes with priming conditions. Expression of HAK gene was higher in primed SPV 2074 shoot and root without salt stress. In both tissues of tolerant and susceptible genotypes, expression of HKT-6 was higher with and without priming under both control and salt stress conditions. Therefore, the present investigation suggests that moringa seed priming impact the growth, enzymatic activities as well as gene expression under salt stress in sweet sorghum.ThesisItem Embargo Evaluation of Momordica spp. for bioactive components and antidiabetic potential(Punjab Agricultural University, 2022) Singla, Diksha; Sangha, Manjeet KaurThe present study evaluated antidiabetic potential of bioactive components from immature fruits (60 genotypes) of Momordica spp. in streptozotocin-induced diabetic Wistar rats. The fruit and its parts; epicarp, mesocarp, endocarp, and seeds were evaluated for biochemicals. PAUBG-407 possessed higher phenolics, fiber components, carotenoids, saponins, macro and microminerals; PAUBG-195 (alkaloids); PAUBG-130 (ascorbic acid and tocopherols); PAUBG-218 (proteins) and Punjab-15 (most bioactives). Whole fruit was rich in many bioactives. Charantin, soluble dietary fiber and momordicin-II were purified from whole fruits of PAUBG-407 while vicine and momordicin-I were purified from whole fruit of PAUBG- 195 and PAUBG-89, respectively and characterized on NMR and FT-IR. Their antidiabetic potential was evaluated in 7-8 week old rats, orally administered bioactives @100mg/kg b.w, daily for 4 weeks. Nine groups were made; normal (), untreated diabetic (II), diabetic + (metformin (I), charantin (IV), SDF (V), vicine (VI), momordicin-I (VII), momordicin-II (V), and diabetic+ all bioactives mixed in equal ratio (IX)). Their effect was assessed weekly and at 4 weeks (some parameters). Fasting and random blood glucose; kidney and Iiver CAT, SOD, GPx, GST, GSH. and TBARS: KFT (urea, creatinine, uric acid, Na', K and Ca); LFT (bilirubin. direct and indirect bilirubin, SGOT, SGPT, ALP, total protein, albumin and globulin): plasma insulin, Hb and HbAl c; serum lipid profile (TC, TG, VLDL, DL and LDL), hepatic enzymes- hexokinase, glucose-6-phosphatase and fructose-6- phosphatase; histological aberrations observed in liver, kidney, pancreas and brain improved SIgnificantly (p<0.0001) in diabetic rats treated with all bioactives but highly with momordicin-II and bjoactive mixture. PAUBG-407 may be used in improvement programmes generating antidiabetic karela. Future plans may envisage in testing antidiabetic potential of f targeted bioactives or use of targeted advanced line in diabetic human subjects.ThesisItem Restricted Biochemical basis of defense response in pigeonpea against maruca vitrata (fabricius) infestation(Punjab Agricultural University, 2022) Sukhmanpreet Kaur; Grewal, Satvir KaurThe present investigation was carried out to identify the biochemical basis of Maruca vitrata (pod borer) infestation in pigeonpea. Two pigeonpea genotypes, AL 1747 (moderately resistant) and MN 1 (susceptible) were compared for methylglyoxal detoxification, proline metabolizing enzymes along with free radical scavenging activities in leaves, flowers and pods under control and infested conditions. Lower accumulation of methylglyoxal (MG) in AL 1747 genotype was due to higher activities of enzymes of glyoxylase (Glyoxylase I, Glyoxylase II, Glyoxylase III) and non-glyoxylase (methylglyoxal reductase, MGR) pathway that converts MG into lactate. Higher activity of lactate dehydrogenase (LDH) in AL 1747 indicates the utilization of end product of MG detoxification pathway for energy production. Higher glutathione content in AL 1747 genotype might be responsible for efficient working of MG detoxification pathway under insect infestation. Higher activity of γGCS in AL 1747 genotype maintains the glutathione pool, necessary for the proper functioning of glyoxylase pathway to carry out the detoxification of methylglyoxal, thereby enhancing resistance. Higher activities of GST and GPX in AL 1747 genotype might be responsible for detoxification of toxic products that accumulates upon insect infestation. However, decreased activities of glyoxylase enzymes and MGR in MN 1 resulted in accumulation of MG which limits plant growth and development. The reciprocal regulation of proline dehydrogenase (ProDH) with proline biosynthesizing enzymes such as ornithine aminotransferase (OAT), glutamate dehydrogenase (GDH), Δ1-pyrroline-5-carboxylate synthetase (P5CS) in AL 1747 suggests that proline accumulation was initiated as part of induced defense response against M. vitrata infestation. Higher free radical scavenging activities such as 2,2-diphenyl-1-picryl hydrazyl (DPPH), superoxide anion, hydroxyl ion, nitric oxide, Ferric reducing antioxidant power (FRAP) might be responsible for lowering ROS accumulation upon insect infestation. Hence, the resistance of AL 1747 to M. vitrata might be due to higher activities of enzymes of glyoxylase and non-glyoxylase system; proline metabolizing enzymes as well as free radical scavenging activities thereby mitigating insect infestation induced oxidative stress.ThesisItem Restricted Exploring Garlic Biochemical Markers Against Root Knot Nematodes(Punjab Agricultural University, 2022) Shakya, Sandeep; Chawla, NeenaThe aim of the present study was to explore garlic biochemical markers and to evaluate garlic extract against root knot nematode of selected genotypes. The study included dry matter, total sugars, reducing sugars, starch, pyruvic acid, allicin and crude fibre in garlic of thirty genotypes. On the basis of allicin per cent, PG-114, PG-119 and PG-128 were selected as the best genotypes for evaluation of nematicidal properties of garlic extract keeping allicin as the main phytochemical. The garlic extract was made from three selected genotypes using different solvents (methanol and distilled water) of different concentrations (S1, S2, S3, S4 and S5). Out of these, PG-119 garlic extract prepared in methanol showed the best results in maximum egg hatching inhibition per cent over untreated control (84.01%, 83.63% and 82.57%) and juvenile mortality per cent (92.35%, 94.47% and 95.67%) after 24h, 48h and 96h respectively at S5 concentration (5.11 ug/ml) as compared to other concentrations applied for in-vitro observations. On the basis of the best performance of PG-119 under an in-vitro trial, it was selected for an in-vivo trial in which a single dose, as well as a double dose of S4 concentration (17.74 ug/ml), showed maximum reduction in root gall index (3.124 and 2.111), final soil population (381.791 and 284.323) and a number of egg mass of root-knot nematode (21.333 and 11.000) as compared to the untreated control. Also, maximum enhancement in plant growth such as plant height and plant weight were observed with the same concentration. Overall, the nematicidal properties of the garlic extract exhibited concentration and time-dependent behaviour.ThesisItem Restricted Malt quality and thermostability studies in mutants, hulled, hull-less and wild genotypes of barley (Hordeum vulgare L.)(Punjab Agricultural University, 2023) Heena Rani; Bhardwaj, Rachana DDespite various attempts to develop malt hydrolytic enzymes, the catalytic activity and thermostability of these enzymes are still far from sufficiency for their use in the malting process. The present study explored diverse barley germplasm to identify the genotypes with high activity and thermostability of enzymes alongwith the proteins responsible for high malt yield. Based on enzymatic assays, we selected BL1714T, DWRB137T and BL2105T with highly activity and thermostability of α and β amylases and limit dextrinase, which are key diastatic power enzymes. The barley proteome of these genotypes was analyzed before and after kilning using label-free quantitative global proteomic approach against the control genotypes; BL1718Ctrl, DWRUB64Ctrl and BL2083Ctrl, respectively. Among various isoforms of hydrolytic enzymes Bamy 1 and Amy1_2 were more abundant in thermostable genotypes indicating their role in controlling the activity of these enzymes at high temperatures. Gene ontology enrichment analysis indicated disulfide reductase, exopeptidase, serine-type peptidase (serpin), cell wall hydrolase and oxidoreductase activities as most common enriched terms in thermostable genotypes. These proteins work to release the bound form of hydrolytic enzymes, resulting in their increased overall activity. These proteins could be employed as biochemical indicators for identifying barley lines with high activity and thermostability. Using GC-MS/MS, we identified maltose, glucose, talose, sucrose, fructose, turanose and other minor sugars in green and kilned malt. Talose and turanose were identified among major sugars for the first time during malting. Further, malt quality analysis suggested that βglucanase and malt β-glucan can be used as additional traits to produce malt with superior quality.ThesisItem Embargo Biochemical and molecular shifts in Solanum melongena alien introgression lines following JASSID, Amrasca bigutulla bigutulla (Ishida) infestation(Punjab Agricultural University, 2023) Handa, Sysha; Rimaljeet KaurBrinjal jassid, Amrasca biguttula biguttula (Ishida) is emerging as devastating pest which significantly affects its yield and production. Varietal resistance could be an imperative and effective strategy to combat pest feeding. Vegetable specialist at PAU, Ludhiana developed S. melongena – S. insanum derived lines which displayed excellent variability to resistance against jassid infestation. Key resistance determinants at morphological, biochemical and transcriptional levels have not been explored so far. Therefore, we planned our research with the objective of determining jassid responsive defensive traits which encompasses phenotypic assessment, morphological characterization, biochemical alterations and gene expression profiling in S. melongena – S. insanum against jassid feeding. Phenotypic assessment of introgression lines (ILs) on the basis of jassid population per plant and pest percent intensity categorized genotypes into highly resistant, moderately resistant, moderately susceptible and highly susceptible genotypes. An increase in affected leaf area was determined with increase in the incidence of A. bigutulla bigutulla infestation. Genotypes with high trichome density and a short trichome length were resistant to pest attack. The increased expression of APX transcripts and ascorbate peroxidise (APX) activity along with a decrease in jassid count in resistant genotypes determined the pivotal role of APX in S. melongena genotypes against jassid attack. This candidate would be used in deployment of S. insanum resistance during brinjal breeding programs. On the contrary, susceptible genotypes showed higher expression of catalase (CAT) activity and CAT transcripts during jassid feeding. APX primarily decomposed H2O2 in resistant lines, whereas CAT primarily did so in susceptible genotypes. During high jassid infestation, H2O2 concentration rapidly increased in test genotypes. In comparison to resistant genotypes, H2O2 buildup was higher in susceptible genotypes. Increased total sugar and sucrose content in susceptible genotypes following jassid infestation suggests their contribution to the pest susceptibility. The genotypes with high jassid infestation displayed high oxidative stress (H2O2 and MDA), indicating that an increase in liposygenase (LOX) activity might cause high production of hydroperoxides, which in turn led to an increase in CAT, polyphenol oxidase (PPO), and phenolic accumulation. The association between JPP and H2O2, LOX, CAT, PPO, MDA, and total phenols was significant and positive. Thus, our findings suggest that susceptible genotypes experienced more oxidative stress than resistant genotypes.ThesisItem Restricted Effect of sucrose as signaling molecule on carbohydrate metabolism and antioxidant capacity in wheat (Triticum aestivum L.) under high temperature stress(Punjab Agricultural University, 2023) Tagde, Sanjana Sanjay; Bavita AsthirIn the present study, the effect of sucrose was studied on the antioxidant defense system and carbohydrate metabolism of two wheat (Triticum aestivum L.) genotypes (Unnat PBW 343 and C 306) under high temperature (HT) stress. The analysis was carried out on the seedling raised in the laboratory (25oC and 32oC) in the petri plates on germinating paper for six days. High temperature significantly decreased the activity of antioxidant enzymes such as ascorbate peroxidase (APX), guaiacol peroxidase (GPX), catalase (CAT), superoxide dismutase (SOD) and glutathione reductase (GR) and non-enzymatic antioxidants, viz., ascorbate, glutathione reduced and glutathione oxidized content. Apparently, the activities of acid and neutral invertase, sucrose synthase (cleavage, synthesis), α- and β-amylase, and the glyoxalase enzymes (Gly I, Gly II, and Gly III) increased on the supply of exogenous sucrose in the wheat seedlings. High temperature significantly reduced shoot/root lengths, fresh weight and dry weight of both the genotypes, whereas sucrose mitigated this effect by increasing these attributes. As a defense reaction, exogenous sucrose application upregulated levels of antioxidants involved in the ascorbate-glutathione cycle in germinating seedlings under heat stress condition. Sucrose mediated amelioration of heat stress seems to be associated with the production of reactive oxygen species like H2O2, accumulation of proline which improved overall thermotolerance and increased membrane stability with the decline in the malondialdehyde content. Carbohydrate metabolism in terms of total sugars and reducing sugars increased under both control and stress condition. High temperature accentuated sucrolytic enzymes viz. invertases and sucrose synthase. Likewise, a significant increase in the glyoxalase (Gly I, Gly II and Gly III) activity along with methyl glyoxal (MG) content was recorded. Higher build up of sugars and starch particularly in the shoot of Unnat PBW 343 indicates that sugars play a very important role in heat stress tolerance as it reduces oxidative stress by decreasing the content of H2O2 and increasing antioxidants. Growth related parameters were significantly improved with sucrose under HT stress. Hence, the interplay of several biochemical parameters including osmolyte may be involved in improving seed germination under stress conditions. Thus, sucrose could be used as an effective signaling molecule for improving seedling growth under stress condition.ThesisItem Restricted GPC- B1 gene mediated shifts in nitrogen metabolism in wheat under organic and conventional farming practices(Punjab Agricultural University, 2023) Manjot Kaur; Bhatia, SurekhaComparison of protein content in Gpc-B1 gene mediated wheat lines in relation to activities of nitrogen metabolism enzymes was investigated under organic and conventional systems. Different high protein wheat lines PBW 821, BWL1663, BWL 7495, BWL 7502, BWL 7504, BWL 7509, BWL 7511, BWL 7548, PBW 725 and PBW 826 were grown under both organic and conventional conditions. All wheat lines were analysed for morpho-physiological parameters. Flag leaf and developing grains were analysed for protein content and activities of nitrogen metabolism enzymes at different developmental stages. In different wheat lines, anthesis stage and maturity came after 90-94 and 134-139 days after sowing under organic conditions whereas these took 58-61 and 105-107 days after sowing under conventional conditions. Tillers per meter row length, spikelet per spikes, grains per spike and thousand grain weight in all wheat lines were found significantly less under organic conditions than under conventional conditions. However, the spikes per plant and harvest index and root: shoot biomass were found significantly higher under organic conditions than under conventional conditions. In all wheat lines, total chlorophyll content in flag leaf was increased from tillering to anthesis and then declines at post anthesis stage under both farming conditions and showed non-significant difference between both farming conditions. A gradual increase in protein content and activities of enzymes nitrate reductase (NR) and glutamate dehydrogenase (GDH) were found in flag leaf from tillering to 7day post anthesis (DPA) and in developing grains from 7-35 DPA in all wheat lines under both farming conditions. Whereas activities of enzymes glutamine synthetase (GS) and glutamine oxoglutarate aminotransferase (GOGAT) declined gradually. However, at all stages protein content, NR and GDH activities were higher under conventional conditions. Whereas, GOGAT activity was higher under organic conditions. The protein content, total soluble sugars and free amino acids was found significantly higher in harvested grains of wheat lines under conventional than under organic conditions. The wheat line BWL 7509 was analysed for amino acid profiling. Aspartic acid has highest values followed by asparagine and glutamine in wheat line under both farming conditions. In conclusion, protein content in wheat lines grown under conventional condition were higher than organic conditions and this was directly related to the nitrate reductase and glutamate dehydrogenase.ThesisItem Embargo CRISPR/CAS9 ribonucleoprotein complex mediated editing of TaASN2 to develop low acrylamide wheat(Punjab Agricultural University, Ludhiana, 2022) Prabhjot Kaur; Suneja, YadhuProcessed food products of wheat have been found to possess acrylamide, a potent neurotoxin and a potential carcinogenic compound. It is formed non-enzymatically when free reducingsugars and free asparagine present in wheat flour react at high temperature cooking via Maillard reaction. Asparagine synthetase 2 (TaASN2) has been identified to be responsible for free asparagine accumulation in grain, the concentration of which is directly related to its acrylamide forming potential. In the present study, CRISPR/Cas9 based genome editing approach was used to generate TaASN2 specific knock-outs to develop low acrylamide genetic stocks of wheat. BWL 5429, a high yielding, disease resistant and a high grain protein wheat line known for its superior chapatti quality was used as the target genotype. To achieve DNA-free editing, vectorless direct delivery approach that uses pre-assembled ribonucleoproteins was employed. Target specific gRNAs were designed using CRISPOR and Wheat CRISPR online available softwares. D-genome specific gRNA [having MM (MorMateos) = 83, CFD (Cutting Frequency Determination) = 81, MIT Specificty Score = 63)] was designed from exon 4 of TaASN2, which showed cleavage of the amplified TaASN2 gene segment upon in vitro cleavage, so was chosen for particle gene gun mediated genetict ransformation. Ready-to-use Cas9 was procured commercially. Pre-assembled RNP complex was used to genetically transform three explants- ‗mature embryo‘, ‗mature embryo with endosperm intact‘ and ‗immature embryo‘. 323 TaASN2-specific and 185 TaPDS-specific T1 spikes were harvested from T0 regenerated embryo/seeds transplanted in pots. ∼1500 spikes of TaASN2 specific knock-outs were harvested in the following crop season. DNA extractedfrom T0 and T1 putative genome edited plants was subjected to amplification of exon 4 of TaASN2 specific to D-genome copy. Upon restriction digestion of exon 4 specific amplicon with HaeIII, the appearance of an intact 267 bp band in a few plants enabled the detection of positive genome edited events. These positive identified TaASN2 knockouts may be further confirmed via amplicon sequencing.
