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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 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.ThesisItem Embargo Assessing genetic variability for asparagine synthetase expression in developing wheat grains(Punjab Agricultural University, Ludhiana, 2022) Ankit Singh; Suneja, YadhuWheat serves as a rich source of energy for large part of world population which consumes it daily. In addition to calories,a large proportion of dietary protein is also obtained from wheat based products. However, recent studies have identified grain asparagine concentration to influence the acrylamide formationduring high temperature processing (a neurotoxic and potentially carcinogenic compound).Accordingly, the present study was aimed at exploring genetic variation in wheat landraces (of Indian, Iranian and Mexican origin) and cultivars for asparagine synthetase activity and free asparagine accumulation in developing caryopsis at different stages of grain development. Initially, a larger set of wheat landraces and cultivars was evaluated for phenotypic characters and wheat yield related parameters. Plant height,peduncle length and spike length ranged between 57.50cm to 114.70cm, 18.83 cm to 49.57cm and 4.50cm to 11.57cm respectively. Average value of five spike weight, number of grain per spike and 100-grain weight was recorded as 7.12 g, 32 and 3.11g respectively. A representative set of ten wheat landraces and cultivars was thereafter short listed to explore variability in asparagine biosynthesis at 7 DPA, 14 DPA, 21DPA, 28DPA and 35 DPA. With respect to asparagine synthetase expression, highest specific activity was found at 21 DPA that varied from 4.23 μmol of β-aspartyl hydroxamate formed min-1 g -1 protein (Mex 1116) to 28.67 μmol of β-aspartyl hydroxamate formed min-1 g -1 protein (BWL5429). An incremental increase in free asparagine accumulation was observed during grain filling period i.e. from an average value of 35.57 μmol g -1 FW (7 DPA) to 75.69 μmolg -1 FW (35 DPA). With respect to free amino acid, the concentration increased from 7 DPA (1.99 mg g-1 FW) to 21 DPA (3.68 mg g-1 FW) and decreased thereafter (2.74 mg g-1 FW) at 35 DPA. As the grain filling progressed toward physiological maturity, highest total soluble protein concentration was found in CPAN-3004 (14.61mg g-1 FW). The information generated from the present work may be supplemented with expression studies and quantification of acrylamide from the processed food products of identified wheat genotypes.ThesisItem Embargo Quality Evaluation of Indigenous Pigmented Paddy Cultivars of North East India(Punjab Agricultural University, Ludhiana, 2022) Lalthlamuana; Arashdeep SinghPigmented rice varieties Fazu and Kc from Mizoram and Khurkhul Chakhao, Phouren Amubi, Chakhao Poireiton and Chakhao Amubi from Manipur were analysed to determine the quality of indigenous pigmented paddy cultivars of North-East India. Results showed that the length of pigmented grain ranged between 6.0 to 6.9mm with L/B ratio ranges between 2.27 to 3.09, which is in range of long grains. Thousand kernel weight, bulk density, and angle of repose of pigmented grains ranged between 20.21 to 23.15g, 0.81 to 0.87 g/ml and 14.02 to 27.39 Ɵ, respectively. Among all the cultivars Kc showed highest head rice yield, while highest milling recovery was observed in Chakhao Amubi. Khurkhul Chakhao was found to have highest fibre and amylose content while Chakhao Amubi had highest protein, fat and ash content. Flour from all the pigmented rice cultivar exhibits excellent functional properties, among which Fazu variety have highest water and oil absorption capacities (266.46 & 242.10%, respectively), while Chakhao Amubi showed highest swelling power (3.95g/g) and gel consistency (47.24mm). The Chakhao Amubi rice variety had the highest DPPH antioxidant activity (51.31 %), while lowest was found out to in Fazu variety from Mizoram (32.29 %). Total phenolic ranged between 411.46 to 618.24 mg GAE/100g and total flavonoid ranged between 34.47 to 44.23 mg quercetin/100g. The amounts of anthocyanin in the rice cultivars range from 11.37 mg/L (KC) to 16.94 mg/L (KC). FTIR spectra of the pigmented rice cultivars confirms the presence of phenolic compounds and anthocyanins in the pigmented rice samples. All rice cultivars exhibit minimum cooking time in the range of 20.01 to 28.01 min. The hardness of the cooked rice samples ranged from 21.69 to 91.37 N. The antioxidant activity of the pigmented rice cultivers was found to increase while the total phenolic, total flavonoid, and anthocyanin content decrease after cooking. All the pigmented rice samples showed their potential for cookies making. Gluten free cookies prepared from pigmented rice grain flour showed spread ratio in the range of 9.05 to 10.70. Among all the samples, cookies prepared from Kc rice cultivar had highest overall acceptability scores (7.91). The texture hardness of the cookies ranges between 15.73 to 57.12 N, which significant level of antioxidant activity, phenolic, flavonoid and anthocyanin content. Cooking and baking of rice was found to have a significant impact on the bioactive compounds and antioxidant properties of the samples.ThesisItem Embargo Biochemical evaluation of bitter gourd genotypes in response to yellow mosaic virus disease(Punjab Agricultural University, Ludhiana, 2022) Navneet Kaur; Sangha, Manjeet KaurThe study evaluated biochemical changes in resistant and susceptible bitter gourd plants; 4 cultivars and 11 advanced lines inoculated with viruliferous whiteflies for about 2 weeks for study of yellow mosaic virus disease (YMVD) caused by yellow mosaic virus (YMV). The disease severity was scored at 15, 30, 45, and 60 days of inoculation (DOI) and grouped the plants into highly resistant (Jhaar Karela, Punjab-15, and Punjab Kareli-1), resistant (PAUBG-407, PAUBG-200, and PAUBG-1514), moderately resistant (PAUBG-204), highly susceptible (Punjab-14, PAUBG-301, PAUBG-229, and PAUBG-341), susceptible (PAUBG128) and moderately susceptible (PAUBG-394, PAUBG-226, and PAUBG-353) groups. Further, PCR diagnosis indicated the presence of the virus in all samples. The infection decreased mean chlorophyll (a, b, total), carotenoids, and Fv/Fm ratio more in susceptible genotypes than resistant genotypes over healthy controls. The susceptible genotypes registered higher total carbohydrates and total soluble sugars but the contents of total soluble and crude proteins decreased with increasing DOI. The infection increased phenols, hydrogen peroxide, catalase (CAT), superoxide dismutase (SOD), ascorbate peroxidase (APX), phenyl ammonia-lyase (PAL), polyphenol oxidase (PPO), and peroxidase (POD) activity more in infected resistant genotypes than in susceptible genotypes. Data was significant (5%) w.r.t genotypes, stages, and treatments. A greater increase in biochemicals in resistant samples may give better protection and the resistant genotypes may aid in crop improvement programs against YMVD.ThesisItem Open Access 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.
