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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.ThesisItem Open Access Effect of fall army worm (Spodoptera frugiperda) infestation on ascorbate, glutathione and polyamine metabolism of maize(Punjab Agricultural University, Ludhiana, 2022) Randhawa, Jagdeep Singh; Kamaljit KaurThe investigation was carried out to find out the effect of Fall Armyworm infestation on ascorbate, glutathione and polyamine metabolism of maize and teosinte. Seeds of CML72, LM24, teosinte and BC (LM24 ×teosinte) were sown in the experimental fields in randomized block design (RBD) with three replicates. Fall armyworm larvae were released on the whorl of 15 days old plants at the rate of 15 larvae per plant. During infestation, specific activity of monodehydroascorbate reductase increased in all the genotypes at all the growth stages examined. Maximum specific activity of dehydroascorbate reductase was observed in CML 72 and teosinte leaves at 14 and 21 day after infestation (DAI). During infestation, teosinte leaves had shown the maximum glutathione reductase (GR) specific activity at 21 DAI stage that was comparable with that of BC (LM24×teosinte) leaves. Ascorbate content increased in all the genotype at 14 and 21 DAI. Glutathione content was highest in the infested teosinte leaves which could be responsible for its enhanced stress tolerance behaviour. H2O2 content increased in the leaves of all the genotypes at 14 and 21 DAI stages. The maximum glutathione-s-transferase and polyamine oxidase (PAO) activities in the infested leaves of teosinte and BC (LM24×teosinte) showed that the corresponding genes might be dominantly expressed in these genotypes. During infested conditions, BC (LM 24× teosinte) remained unaffected with respect to diamine oxidase (DAO) specific activity. Ornithine decarboxylase (ODC) specific activity increased in all the genotypes at 14 DAI, with BC (LM 24× teosinte) showing the maximum values. Arginine decarboxylase (ADC) specific activity increased at all the growth stages in LM 24 and BC (LM 24× teosinte) plants. Teosinte leaves showed unaltered ADC specific activity at all the growth stages examined. The increased putrescine content of infested teosinte leaves might be due to its high PAO activity while that of LM 24 plants could be due to increased ODC activity. The spermidine content of CML 72 also became comparable with LM 24 at 14 DAI. During infestation, spermine content increased in all the genotypes. Maximum spermine content was observed in LM 24 and BC (LM24×teosinte) at 21 DAI.ThesisItem Open Access Phytochemicals, antioxidant activity and grain quality in pigmented and non-pigmented rice(Punjab Agricultural University, 2023) Gosangi Avinash; Sharma, NeerjaPigmented rice are rich source of bioactive compounds especially polyphenolic components, tocopherols, (TOC), tocotrienols (T3) and γ- oryzanol, which impart positive effects on human health. The present study, was therefore, planned to study the variation in phytochemical composition, antioxidant activity and grain quality of a set of rice genotypes, comprising 9 pigmented (PR, 3 black and 6 red ) and 7 non-pigmented (NP) rices. The results revealed that PR rices (black followed by red) had higher ash, crude protein, crude fat and crude fibre content compared to NP rices in the bran, whole grain and milled rice fractions. Total dietary fibre content was highest in black rice genotypes. Insoluble dietary fibres were ~10 - folds higher than soluble dietary fibres in all the three colour groups. NP rices were observed to have the best milling, cooking and physicochemical quality parameters. Black rice genotypes displayed the highest total phenolic content (TPC) and the contribution of bound phenolics to TPC in all 16 genotypes under study was higher than free phenolics. The in vitro antioxidant activity of black rice group was highest followed by red and NP group. The total flavonoid and anthocyanin content was highest in black rice group while proanthocyanidins were highest in red rice group in both bran and whole grain fraction. In all the three colour groups, bound phenolic acid content was higher than free phenolic acids. The main bound phenolic acids were t-ferulic acid, p-coumaric acid, and 2,5 - hydroxybenzoic acid (2,5-DHA), while in the free form, p- hydroxybenzoic acid and syringic acid were the major phenolic acids in both the grain fractions of all three colour groups. 2, 5 - DHA was quantified for the first time in bran and whole grain fractions of NP rices. The total γ- oryzanol content in bran was highest in black rice followed by red and NP group. 24 methylene cycloartenyl ferulate and cycloartenyl ferulate were the major contributors to total γ- oryzanol content in all 16 genotypes. Total vitamin E content was highest in black rices followed by red and NP rices. In bran, α -T3 and δ-TOC were found to be major contributors to total vitamin E content while in whole grain these were γ-T3 and δ- TOC, irrespective of colour of genotypes. The free phenolics were more susceptible to degradation on parboiling compared to bound phenolics. These findings contributed towards further understanding of the distribution of nutritional and phytochemical components in rice grain fractions of PR and NP rices, and may assist rice breeders and rice growers with new opportunities to promote production of rice with enhanced level of phytochemicals to augment human health.ThesisItem Embargo Evaluation of leaf composition and glyoxylase pathway in barley genotypes in response to leaf blight disease(Punjab Agricultural University, Ludhiana, 2022) Prabhjot Kaur; D. Bhardwa, RachanaThe present investigation was carried out to study the leaf composition and methylglyoxal pathway in four barley genotypes on infection with Bipolaris sorokiniana. Disease data indicated PL 891 as highly resistant, PL 807 as resistant whereas genotypes PL 426 and RD 2786 were found to be susceptible to spot blotch disease. Total soluble sugars and proteins increased in PL 807 and PL 891 at growth stage (GS) 55 but remained unaffected at GS 87. Glyoxalase I activity increased slightly in the leaves of PL 807 and PL 891 on infection with Bipolaris sorokiniana while it increased in their lower leaf at a later stage. Glyoxalase II activity increased in the infected leaves of PL 891 at GS 55 stage while a decrease was reported in the susceptible genotype RD 2786. Glyoxalase III activity increased in the flag and lower leaves of resistant genotypes at the GS 87 stage while declined or remained unaffected in the susceptible genotypes. Methylglyoxal reductase activity remained unaffected in the leaves of all the barley genotypes at GS 55 stage but at a later stage, it increased significantly in the flag as well as lower leaves of tolerant genotypes. Glutathione content increased in leaves of PL 807 and PL 891 and might be responsible for lowering MG levels. Methylglyoxal content was reported to be minimum in the leaves of PL 891 at GS 55 stage and it further reduced on infection with Bipolaris sorokiniana.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 Embargo Biochemical and molecular characterization of nutritionally important starch fractions and in vitro digestibility of parboiled rice(Punjab Agricultural University, Ludhiana, 2023) Sonakshi; Sharma, NeerjaParboiling of rice is a hydrothermal treatment that includes soaking, steaming and drying, which affects the milled rice recovery and nutritional properties of rice. In the present study, biochemical evaluation of eleven rice genotypes was carried out to assess the variability in proximate composition, physicochemical profile, nutritionally important starch fractions along with in vitro starch and protein digestibility of parboiled and non-parboiled rice. The variation in coding regions of GBSSI, SSIIa and OsGELP1 was also studied. Parboiling increased milled and head rice recovery by ~1.02 and ~1.16-folds, respectively. L/B ratio before and after cooking decreased significantly by ~1.06 and ~1.08-fold respectively. Ash content increased by ~2.27-fold while crude fat and crude protein decreased significantly by ~2.06- and 1.09-folds, respectively. Crude fibre, amylose content and gel consistency of the milled rice were unaffected. The pasting temperature increased significantly (~1.14-fold) with parboiling while peak viscosity (1.96-fold), breakdown viscosity (3.59-fold) and final viscosity (~1.83-fold) decreased. Total starch and rapidly digestible starch decreased significantly by ~1.04- and 1.08-folds, respectively, while slowly digestible starch and resistant starch increased by ~1.11- and 1.18-folds, respectively. In vitro protein digestibility, hydrolysis index (HI) of starch and predicted glycaemic index (pGI) decreased significantly. Parboiling decreased the whiteness (~1.16-fold) of rices. In vitro amplification of 5‘UTR region of GBSSI enabled classification of genotypes based upon the product size (120, 110, 100 bp). PCR amplification around GC/TT functional SNP in exon 8 region of SSIIa demonstrated that PB1718, PB1509, PB1121 and PR114 carried ‗TT‘ allele while amplification of exon 2 to exon 4 region of lipase gene produced a1275 bp product. It is therefore concluded that parboiling is a good alternative to conserve and increase milling, nutritional and processing quality of rice genotypes.
