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Govind Ballabh Pant University of Agriculture and Technology, Pantnagar

After independence, development of the rural sector was considered the primary concern of the Government of India. In 1949, with the appointment of the Radhakrishnan University Education Commission, imparting of agricultural education through the setting up of rural universities became the focal point. Later, in 1954 an Indo-American team led by Dr. K.R. Damle, the Vice-President of ICAR, was constituted that arrived at the idea of establishing a Rural University on the land-grant pattern of USA. As a consequence a contract between the Government of India, the Technical Cooperation Mission and some land-grant universities of USA, was signed to promote agricultural education in the country. The US universities included the universities of Tennessee, the Ohio State University, the Kansas State University, The University of Illinois, the Pennsylvania State University and the University of Missouri. The task of assisting Uttar Pradesh in establishing an agricultural university was assigned to the University of Illinois which signed a contract in 1959 to establish an agricultural University in the State. Dean, H.W. Hannah, of the University of Illinois prepared a blueprint for a Rural University to be set up at the Tarai State Farm in the district Nainital, UP. In the initial stage the University of Illinois also offered the services of its scientists and teachers. Thus, in 1960, the first agricultural university of India, UP Agricultural University, came into being by an Act of legislation, UP Act XI-V of 1958. The Act was later amended under UP Universities Re-enactment and Amendment Act 1972 and the University was rechristened as Govind Ballabh Pant University of Agriculture and Technology keeping in view the contributions of Pt. Govind Ballabh Pant, the then Chief Minister of UP. The University was dedicated to the Nation by the first Prime Minister of India Pt Jawaharlal Nehru on 17 November 1960. The G.B. Pant University is a symbol of successful partnership between India and the United States. The establishment of this university brought about a revolution in agricultural education, research and extension. It paved the way for setting up of 31 other agricultural universities in the country.

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  • ThesisItemOpen Access
    Response of iron and zinc nano oxides in Picroside I biogenesis during in vitro growth and development of Picrorhiza kurroa Royle ex Benth.
    (G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand), 2022-01) Rawat, Geeta; Gaur, A.K.
    Picrorhiza kurroa Royle ex Benth is one among human and animal health associated valuable herbs. Several iridoid glycosides, as well as their derivatives with diverse pharmacophore, have been documented in this plant which have been undertaken for detail studies for their potential in a variety of modern and traditional healthcare systems toward managing a variety ailment since ancient times. Commercially available formulations for cure of disorders are Livocare, Livomap, Livplus, Katuki and Arogya. Due to business potential, an increasing global demand of this plant, this important plant resource has received much attentions of various researchers. Focus in this study have been provided with special reference to optimize protocols of in vitro regeneration of Picrorhiza kurroa. The influence of nanoparticles (ZnO-NPs and Fe2O3- NPs) during callus cultures, upon secondary metabolites specially on picroside I and levels of antioxidative enzymes namely superoxide dismutase (SOD), Catalase (CAT), Guaiacol peroxidase (GPOX), Ascorbate peroxidase (APOX). Biological assays against potent harmful microbes were also undertaken. Laboratory scale parameters were established for developing optimum suspension cultures for picroside I in order to develop large-scale production parameters. Elicitation response for picrosides production and related genes expression analysis of picroside metabolic pathway in suspended microcalli were observed. Zinc oxide nanoparticles (ZnO NPs) and Iron oxide nanoparticles (Fe2O3 NPs) responses were noticed to recognise specific identification of pathway genes with special reference to Hydroxy-methyl glutaryl CoA reductase (HMGR), Mevalonate diphosphate synthase (MVDD), Isopentyl diphosphate isomerase (IPPI), 3-Deoxy- D-arabino heptulosonate 7-phosphate synthase (DAHPS), 5-enolpyruvylshikimic acid-3-phosphate synthase (EPSPS), Dehydroquinate dehydratase (DQS), Geraniol diphosphate synthase (GDPS), 1-deoxy- D-xylulose-5-P-synthase (DXPS), Hydroxy methyl butenyl-4-diphosphate reductase (ISPH), Cytochrome P-450 monooxygenase (CPM), 2-hydroxy isoflavanone dehydratse (2HFD), Chorosmate mutase (CM) and Phenylalanine ammonia lyase (PAL) for picroside I only as an attention for its biogenesis. These observations imply that such an induced callus cultures under response of nanoparticles are suitable for picroside I production from Picrorhiza kurroa. Exploration of mass manufacturing particles at least of picroside I is possible by establishing suitable parameters in a simulated bioreactor system based on laboratory scale parameters identified from this particular study.
  • ThesisItemOpen Access
    Characterization of novel mutants of hexaploid wheat (Triticum aestivum L.) for isolation of mutants with higher content of micronutrients and lodging tolerance
    (G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand), 2021-03) Bisht, Darshana; Sundip Kumar
    Producing nutritious and safe foods sustainably and sufficiently is the ultimate goal of modern agriculture. Mutagenesis is one of the powerful genetic strategies for crop improvement programmes. A chemically induced mutated genetic resource for detecting novel variations has been developed in bread wheat (Triticum aestivum) cultivar DPW-621-50. 1536 EMS-induced wheat mutant lines along with 4 checks grown during the Rabi season of 2019/2020 were evaluated for variability parameters, correlations and path coefficients for thirteen quantitative traits i.e., plant height, number of tillers per plant, flag leaf length, flag leaf width, flag leaf area, ear length, peduncle length, number of spikelets per spike, number of grains per spike, grain length, grain width, 1000 grain weight and grain yield per plant. Significant genotypic differences were observed for all the traits studied indicating considerable amount of variation among mutant lines for each character. The cluster analysis grouped these mutant lines into 40 different clusters. Grain yield per plant showed highly significant positive correlation with number of tillers per plant, number of grains per spike, thousand grain weight, number of spikelets per spike, plant height, grain width, grain length, ear length peduncle length. Path coefficient analysis revealed that number of tillers per plant had the highest direct effect on grain yield per plant. Prior efforts have focused on raising crop yields, but enhancing the concentrations of mineral micronutrients has become an urgent task because about half of the world population suffers from the malnutrition of iron and zinc. The overall impact of this high concentration of micronutrients is directly based on their bioavailability which is influenced by antinutritional (tannins and phytic acid) and nutritional (ascorbic acid) factors. This mutant population was used to isolate the mutants containing higher contents of minerals (Fe and Zn) in grains. Mutant lines namely, BSM 410, BSM 531, BSM 659, BSM 580, BSM 531, BSM 5, BSM 804, BSM 485, BSM 518, BSM 644, BSM 611, BSM 1004, BSM 390, BSM 990, BSM 609, BSM 97, BSM 567 and BSM 380 were found to possess high Fe and Zn and low phytic acid content. BSM 379, BSM 11, BSM 462, BSM 558, BSM 647, BSM 344, BSM 49, BSM 54, BSM 44, BSM 269, BSM 348, BSM 560, BSM 386, BSM 354, BSM 556, BSM 373, BSM 3, BSM 583, BSM 419 and BSM 580 were found to contain high Fe and Zn and low tannic acid content. BSM 549, PMW-2016-1, BSM 348, BSM 483, BSM 455, BSM 558, BSM 591, BSM 523, BSM 74, BSM 31, BSM 863, BSM 347, BSM 360, BSM 531, BSM 532, BSM 24, BSM 373 and BSM 408 were found to contain high Fe and Zn and high ascorbic acid content in comparison to parent. From the newly developed mutant population, we have also identified a putative lodging tolerant plant (PMW-2016-1). Comparative anatomical studies of the culm of lodging tolerant plant (PMW-2016-1) and parent (DPW-621-50) were carried out by means of various microscopic techniques coupled with chemical studies. It was found that the culm of PMW-2016-1 possessed greater stem diameter, thicker width, higher thickness-diameter ratio and higher percentage of mechanical tissue, comparing with its parent. Furthermore, the lignin content was determined by Klason’s method. The result showed that Klason lignin content in the novel mutant (PMW- 2016-1) was higher than that in its parent genotype (DPW-621-50). Estimation of mechanical stem strength with Universal Tensile Machine exhibited that the force required to break the straw of lodging tolerant mutant wheat PMW-2016-1 is greater as compared to the straw of control (DPW-621-50). Bending of stems of mutant PMW-2016-1 and control (DPW-61-50) was analyzed by using the prostate tester. Stem strength of mutant PMW-2016-1 was found to be significantly higher than the control (DPW-61-50). Therefore, it is suggested that newly identified mutant PMW-2016-1 can be used as variety or as a donor parent for the development of lodging tolerance in wheat cultivars. This mutant population may serve as new genetic resource for functional genomics studies and novel variants for different traits in elite germplasm can be made available to the plant breeders for wheat improvement.
  • ThesisItemOpen Access
    Effect of differential nitrogen fertilization and nano zinc seed priming on yield and seed protein quality in finger millet genotypes
    (G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand), 2020-02) Gururani, Kavita; Pandey, Dinesh
    The “green revolution” based modern agriculture helped in increasing crop production and averting the hunger with the use of Nitrogenous fertilizers mainly. However, excessive use of fertilizers has threatened agriculture and sustainability of environment; and reduced the diversity of food, which is a major cause of malnutrition. Agronomic biofortification through direct application of fertilizer is an effective method to overcome the problem of malnutrition; however, fertilizers should be used with caution for both commercial and environmental reasons. Therefore there is need to optimize the dose of fertilizer and to use naotechnological interventions so as to produce nutritionaly rich food without compromising yield and damaging the environment. Such approaches will not only biofortify the crops but also ensure environment-friendly agriculture by promoting low-input sustainable crop cultivation. Finger millet (Eleusine coracana), holds immense agricultural and economic importance due to its high nitrogen use efficiency and richness in proteins being good source of essential amino acids and minerals. Two genotypes of finger millet viz. GE-1437 and GE-3885 were taken for the study to observe the effect of differential nitrogen fertilization without and with nano zinc seed priming (5ppm) on agro-morphological parameters, seed protein content, seed zinc content, expression analysis of seed storage proteins, regulatory genes of essential amino acid metabolism and accumulation of seed storage proteins. Results of agro-morphological data analysis showed the maximum biomass, grain yield and zinc content was obtained at 60kg/h urea without nano zinc seed priming; and 40kg/h urea with nano zinc seed priming. Nine regulatory genes involved in metabolism of essential amino acids have been identified from the transcriptome of finger millet spike. Promoter analysis revealed the presence of various cis-regulatory elements in the regulatory genes i.e. GCN4 motif, O2-site, G-box and GATA-motif, P-box etc. which are found to play an important role in N metabolism, SSP synthesis, light response, hormone response or stress response and directly or indirectly may regulate the biosynthesis of essential amino acids. Among the treatments without nano zinc seed priming the maximum expression of all the seed storage proteins was obtained with 40kg/h urea at later stages of spike development. On the other hand, for the treatments with nano zinc seed priming, the maximum expression of all the SSPs was obtained with control and 80kg/h urea at later stages of spike development. Both the genotypes showed higher expression of most of the regulatory genes with farm yard manure without and with nano zinc seed priming. The comparative analysis showed the higher expression of globulin and prolamin with the treatments without nano zinc seed priming and albumin with the treatments with nano zinc seed priming at later stages of spike development. SDS-PAGE further confirmed the increase in globulin and prolamin with the treatments without nano zinc seed priming and albumin with the treatments with nano zinc seed priming in mature seeds. Results of present investigation have suggested that high productivity/yield along with high Zn content of Finger millet can be obtained upon application of 40kg/h urea with nano zinc seed priming. Though this treatment can be used to solve Zn malnutrition problem among the masses yet it compromises with quality of protein and protein content in grains. In order to solve protein malnutrition problem, however, one can apply 40kg/h urea without nano zinc seed priming to enhance the seed protein content and quality in terms of essential amino acids.
  • ThesisItemOpen Access
    Isolation, cloning and in silico analysis of drought responsive EcMyb1 transcription factor gene from Eleusine coracana
    (G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand), 2021-01) Bhatt, Megha; Lohani, Pushpa
    Myb genes are members of a family of transcription factors that play an important role in cell division, differentiation, response to abiotic stress etc. It has been reported that MYB transcription factor play an important role as a master regulator and modulates the expression of various downstream genes involved in drought stress response. The partial sequence of Ecmyb1 gene was isolated and submitted to NCBI with accession number JN107890.1. In present study, full length Ecmyb1 gene was isolated and cloned. The cloned fragment was sequenced and submitted to NCBI with accession number MT312253. For expression analysis of Ecmyb1 gene, finger millet drought tolerant variety PRM 6107 was given three different abiotic stress treatment i.e. drought, salt and heat stress. The expression of the gene increased from 12 to 28 fold under drought stress as compared to control. Similarly, in salt and heat treated plants, expression increased from 7 to 29 fold and 19 to 46 fold respectively. The cloned gene was introgressed into Arabidopsis thaliana plant by floral dip method. The transformed plants were selected by hygromysin screening and confirmed by PCR using hygromycin primers. Insilco analysis of Ecmyb1 gene was carried out using various softwares. Promoter analysis of Ecmyb1 gene revealed the presence of various regulatory elements like ABA responsive elements (CACT GG), stress responsive elements (AAGG GG), Myb recognition sites (CAACAG), Myb binding sites (CAAC TG), CpG island and tandem repeats. Homology and phylogenetic analysis of Ecmyb1 gene and amino acid sequences showed similarity with other MYB proteins such as Sateria italica, Hordeum vulgare, Saccharum barberi and Oryza sativa etc. Motif analysis predicted the presence of HTH domain in EcMYB1 protein. Subsequently, it was computed that EcMYB1 protein is water soluble, thermostable and slightly acidic in nature. Additionally, it was observed that EcMYB1 protein contains 28% alpha helix, 5.43% extended strand, 2.57% beta turn and 64% random coil. 3-D structure of EcMYB1 using swiss model predicted that 90% aminoacid residues were in Ramachandran favored region with no bad angles. It indicated that EcMYB1 protein may exist in nature.
  • ThesisItemOpen Access
    Functional validation of the potential EcCAM, EcCAX1 and EcCAX 3 genes in grain calcium accumulation through over-expression studies in Arabidopsis thaliana: Development of an efficient plant regeneration protocol towards calcium biofortification in finger millet
    (G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand), 2019-02) Jamra, Gautam; Anil Kumar
    Mineral malnutrition is one of the major problems crippling by one-half world population mostly women’s and pre-schooled children. Calcium deficiency is also considered as major problem which may lead to defects in bones, teeth, and osteoporosis. Finger millet (Eleusine coracana) is an orphan crop, rich in calcium with high nutritional significance and antioxidant properties to grow under harsh drought conditions. Multi-pronged molecular approaches were used in our Lab to identify the candidate genes of calcium sensor and transporter families associated with differential grain calcium in different genotypes due to induction of differential calcium signaling and transport machinery. The identified genes were functionally validated in the present investigation through over-expression studies in a model plant of Arabidopsis thaliana. Complete CDS of EcCAM, EcCAX1 and EcCAX3 genes were retrieved from transcriptomic data of pooled spikes of high calcium containing genotype; GP-45. In-silico molecular characterization of these genes based on protein sequences showed the presence of conserved domains as characteristic features. The phylogenetic analysis showed the genes are closely related to Siteria italic and Oryza sativa while distinctly related with Arabidopsis. To decipher the function of EcCaM, EcCAX1 and EcCAX3 genes, the gain-offunction approach was used to generate the transformed lines in Arabidopsis thaliana and expression analysis by semi-quantitative RT-PCR of such genes carried out in T3 homozygous transgenic lines showed higher expression as compared to no expression in wild type. Phenotypic assays were performed under various abiotic conditions such as EcCAM under PEG induced drought stress, IAA induced homeostasis, NaCl induced salt stress and calcium induced ionic stress; EcCAX1 and EcCAX3 under calcium & magnesium ionic stress, and IAA induced homeostasis. The observations recorded in 7 days old seedlings suggested that over-expressed transgenic lines were more tolerant as compared to wild type as evident from root elongation with lateral growth and better physiology. The anti-oxidative potential of over-expressed transgenic lines was higher in transgenic lines showing less ROS accumulation while more ROS accumulation in wild type plants as indicated by NBT and DAB staining method. Thus, more oxidative damage was observed in wild type as compared to over-expressed transgenic lines. Finger millet is highly adaptive in harsh conditions and nutritionally superior necessitate its further improvement using transgenic technology. In order to develop superior genetically modified plants, an efficient plant tissue culture protocol is essentially required taking innate nutritional, biochemical and hormonal attributes of finger millet genotypes as indices of plant tissue culture responsiveness. In order to determine the influence of stress tolerant behavior and inherent composition on plant regeneration, four genotypes of finger millet (GP-45, GP-1 GE-1437 and GE-3385) were taken in the present study. The results indicate that GP-45 was found to show maximum stress tolerance whereas GP-1 was the least tolerant. Further estimation of endogenous total calcium, carbohydrates, protein, total phenols, total flavonoids and phytohormones (ABA and GA3) showed genotype dependent variations and high calcium is related with stress tolerance and in turn plant tissue culture responsiveness. The results of the present study clearly elucidate the importance of selection of genotypes based on biochemical indices such as innate phytonutrients, phytochemicals and phytohormones for the development of an efficient regeneration protocol in finger millet to introgres the potential genes whose functions validated through genetic transformation studies in Arabidopsis could further be harnessed for crop improvement especially for improving calcium nutrition and stress tolerance.
  • ThesisItemOpen Access
    Genotyping-by-sequencing and functional genomics based approaches for identification and nano-minerals (Fe and Zn) mediated modulation of candidate genes involved in iron and zinc homeostasis in finger millet
    (G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand), 2020-08) Chandra, Ajay Kumar; Pandey, Dinesh
    Micronutrient deficiencies of iron and zinc are mainly responsible for significant amount of malnutrition among children and women across the world. One of the approaches to solve this problem is through biofortification of staple food crops provided that key candidate genes and markers associated with iron and zinc accumulation are identified. Being very good accumulator of minerals such as iron and zinc, finger millet is an ideal crop for identifying the molecular mechanism of iron and zinc homeostasis and molecular markers linked to high iron or zinc content in finger millet. With an aim to identify potential markers linked to grain iron and zinc content, initially phenotyping of 202finger millet genotype was done for fourteen morphological traits along with grain iron and zinc content at two different environments viz. Pantnagar (L1) and Almora (L2). Analysis of variance for both the environments resulted in highly significant differences among accessions for most of the traits, which showed the existence of sufficient variability. Also the wide range of variation in the agronomic performance of the accessions suggested that these traits could be considered as good candidates for marker-trait associations. The grain iron content varied from 26.72 to 78.59 mg.kg-1 seed with an average of 44.34 mg.kg-1 seed at L1 whereas, the GIC at L2 varied from 29.83 to 76.40 mg.kg-1 seed with an average of 46.14 mg.kg-1 seed. Similarly, the grain zinc content varied from 16.28 to 45.17 mg.kg-1 seed with an average of 32.18 mg.kg-1 seed at L1 whereas, the GZC at L2 varied from 12.40 to 47.18 mg.kg-1 seed with an average of 32.36 mg.kg-1 seed which indicates presence of sufficient variability for grain iron and zinc trait amongst the selected finger millet genotypes. Further, Genetic diversity and population structure analysis was conducted on a natural collection of 202 finger millet genotypes. Neighbour joining cluster analysis grouped the population into 3 clusters ‘A’, ‘B’ and ‘C’ based on their geographical location and grain iron and zinc content. The major cluster A consisted broadly of all the Indian genotypes whereas cluster B and C comprised of exotic genotypes. Thus, there was good congruence between the phylogenetic tree and the population structure. SNPs through GBS were used for association mapping to identify reliable marker(s) linked to grain iron and zinc content. A total of five SNP markers, three for grain iron [TP413281 associated with ferritin (Fer1), TP566120 with iron-regulated transporter-like protein (IRT2), and TP1431395 associated with yellow stripe-like 2 proteins (YSL2)] and two for grain zinc [TP1316808 associated with zinc transporter ZIP1 protein and TP784188 associated with zinc transporter ZTP29-like protein], showed homology to candidate genes of Oryza sativa and Setaria italica, which might play an important role in grain iron and zinc homeostasis process in finger millet. Further, a total of fifteen genes (EcDMAS1, EcFER1, EcIRT2, EcNAAT6, EcNAC2, EcNAS1NAS2, EcNRAMP2, EcNRAMP6, EcPDR9, EcTOM1, EcTOM2, EcYSL1, EcYSL2, EcZIP1, and EcZTP29) orthologous to CGs of Oryza sativa and Setaria italica putatively involved in iron and zinc homeostasis in finger millet were identified through Genome-Transcriptome transition approaches. Structural and functional annotation of identified genes further highlights its significance that the genes are putatively involved in iron and zinc homeostasis process in finger millet. Furthermore, it was studied whether nano-treatments have any influencing role in epigenetic behavior so that it could enhance the molecular machineries of Fe and Zn enrichment in the plants. Molecular modeling and docking potential of identified proteins with nano-minerals (Fe3O4 NPs and ZnO NPs) confirmed that these nano-minerals may significantly influence the binding potential and modulation of regulatory genes. In addition, comparative In vitro studies for nano-minerals augmentation using seed priming approach resulted that the lower concentrations of Fe3O4 NPs (100 ppm) and ZnO NPs (5 ppm) play a significant and promotory effect on fourteen quantitative traits. Besides, the mean iron content in nano seed primed harvested grains, significantly increased by 12.26% and, the zinc by 13.96% concerning controlled harvested grains. Thus to confirm the genetic and molecular basis of identified genes and their modulation in response to nano-minerals, the Transcriptome based expression profiling revealed that of these 15 genes, EcFER1, EcIRT2, EcYSL2, EcZIP1, and EcZTP29 are the major genes which might play an important and direct role while the rest genes may indirectly involved in differential iron and zinc homeostasis in finger millet. The result was further validated by quantitative real-time PCR analysis. Structural and functional validation of set of genes involved in strategy-I (EcIRT2, EcFER1, and EcZIP1), strategy-II (EcYSL2), combined i.e., strategy-I and strategy-II (EcIRT2, EcFER1, EcYSL2, and EcZIP1) and the expression of EcZTP29 (stress-responsive gene) in finger millet tissues suggested that being a stress-resilient crop finger millet utilizes a combination of strategies I and II of iron and zinc homeostasis pathway. Since, there is not yet available any information on genetic and molecular basis of Fe and Zn homeostasis in this crop, therefore the results of present investigations have unlocked new avenues for effective utilization of finger millet in future iron and zinc biofortification programs.
  • ThesisItemOpen Access
    Assessment of transgenic Brassica juncea cv. Varuna harbouring MAPK3 gene against Alternaria brassicae after preconditioning with phytohormones and MAPK inhibitor
    (G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand), 2019-09) Srivastava, Snigdha; Gohar Taj
    Alternaria blight is one of the most devastating fungal disease which hampers the productivity of Brassica juncea in India. The disease is caused by a semi-biotrophic pathogen, Alternaria brassicae. The absence of resistance source against Alternaria blight has made the plant researchers shift their interest towards de-novo sources of resistance. The MAPK 3 gene is an important gene expressed in plants during biotic stress for strengthening the defense system of plant. The MAPK 3 gene was overexpressed in Brassica. juncea cv.Varuna for the evaluation of resistance provided by transgenic lines against Alternaria blight. The two important plant hormones salicylic acid and jasmonic acid plays a major role in transducing the activation of plant defense systems against pathogen attack. These phytohormones regulate the expression of MAPK cascade genes that are the key role player of defense signaling in plants. The codon usage bias is an evolutionarily conserved phenomenon that affects the expression level of genes. The knowledge regarding the codon usage biasness pattern of genes helps us to know whether the proteins will be expressed in the heterologous system or not that guides in the development of transgenic plants. In the present investigation the copy number of MAPK 3 gene in transgenic B.juncea cv. Varuna plants were calculated using real-time PCR and Southern blotting. The effect of salicylic acid, jasmonic acid, and MAP kinase inhibitor was studied on the transgenic and non-transgenic B.juncea cv.Varuna plants at different stages of disease progression. The antioxidant activities of APX, GPX, CAT, proline was higher in phytohormones treated transgenic plants. The MAPK cascade genes viz. MAPKKK 1, MAPKK 4, MAPKK 9, WRKY 33, WRKY 22, OASTL-B, ACD-2, CSD-2 that are involved in defense pathway were observed to be expressed higher in transgenic plants as compared to the non-transgenic plants. The codon usage bias analysis of MAPKs and WRKY genes of A. thaliana and B. rapa was carried out by using the bioinformatics approaches. The studies revealed that the pattern of CUB in these genes are affected by natural selection and mutational pressure. The clustered heat map grouped the MAPKs and WRKY genes having similar RSCU values and similar functions under the same cluster. The codon AGA coding for arginine was found to be preferred in most of the MAPKs and WRKY genes. The codon usage bias in A.thaliana and B.rapa MAPKs and WRKY genes was observed to be low based on ENc values. The expression level of MAPKs and WRKY genes was observed to be low based on the RSCU values. These studies will give direction for the engineering of defense pathway so that the Alternaria blight resistant B. juncea plants could be developed.
  • ThesisItemOpen Access
    Bioinformatics analysis of camalexin biosynthetic pathway and studies on defense responses of MAP2K4/MAP2K9/MAPK3 mutants during pathogenesis of Alternaria blight in Arabidopsis thaliana
    (G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand), 2018-08) Gaur, Manu; Pandey, Dinesh
    Alternaria blight is one of the most destructive fungal diseases which seriously hamper productivity of Brassica oilseeds in India. Alternaria brassicae is the main pathogen of the disease which produces chlorotic, necrotic toxins and phytohormones to cause the disease. These disease determinants have been known to affect various genes/ proteins of cell cycle and cell death pathways in susceptible Brassica plants to cause the disease and enable the pathogen to follow a semibiotrophic life style. It is being speculated that Alternaria brassicae pathogen affects highly conserved MAP kinase signal transduction pathway to cause the disease. In Arabidopsis , some of the MAP kinases including MPK3, MKK4, MKK9, are responsible for synthesis of ‘Camalexin’ which is one particular phytoalexin involved in mediating defense response against the necrotrophic fungal pathogen viz. ‘Botrytis cinerea’. In the present investigation, role of MPK3, MKK4 and MKK9 in triggering Camalexin based defense response of Brassica plant towards Alternaria brassicae was studied by using Arabidopsis mutants for these kinases and Bioinformatics analysis. Following the infection of Alternaria brassicae pathogen, disease index of mkk4, mkk9 and mpk3 mutant plants was observed to be more than that of wild type plants of Arabidopsis thaliana. These observations suggested increased susceptibility of mpk3, mkk4, mkk9 mutants of Arabidopsis for Alternaria blight due to decreased camalexin biosynthesis or differential expression of proteins. In order to study differential expression of proteins, the proteins were extracted from infected leaf samples of both wild type and mutant Arabidopsis plants and protein profiles were compared at initial, middle and late stage of infection by performing one dimensional SDS PAGE analysis. In wild and mutant plants of Arabidopsis, the protein extractability decreased as the disease progressed from early to middle stage due to degradation of host proteins and increased from middle to late stage due to synthesis of new proteins. SDS PAGE based analysis of proteins extracted from leaves of wild type and mutant Arabidopsis plants indicate differential expression of proteins in form of expression of unique prroteins and downregulation or upregulation of proteins during disease progression from early to late stage of pathogenesis in Arabidopsis mutants and wild type plants. MKK4/MKK9/MPK3 signalling module involved in biosynthesis of camalexin during defense response of B. rapa towards, Alternaria brassicae pathogen was elucidated with the help of Molecular modelling, docking, and protein-protein interaction analysis of MAP kinases retrieved from Brassica rapa genome. Network Biology based in silico approach was followed to identify linkages of MKK4, MKK9 and MPK3 with 51 known defense related genes through construction of PPI network. MPK3 was observed as major hub to which large numbers of defense regulated genes/proteins are connected. This suggests that MPK3 is largely responsible for activation of large number of defense related genes besides camalexin biosynthetic genes. These studies will open up new avenues for engineering defense pathway in Brassica for preventing losses caused by Alternaria blight disease.
  • ThesisItemOpen Access
    Delineating the redox signaling network under oxidative stress through over expression of Ecapx1 in Eleusine coracana
    (G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand), 2018-12) Pande, Anjali; Arora, Sandeep
    Global warming has emerged as one of the biggest threats to agriculture in the last two decades. In arid and semi-arid regions, where temperatures are already close to the physiological maxima for crops, higher temperatures may increase the incidence of heat stress in crops; negatively affecting the growth and productivity. Under such conditions, development of climate resilient crops is a dire need. As reactive oxygen species are a common denominator in the effect of various environmental stresses, therefore it is important to understand the signaling events in plants that are implicated in activating the anti-oxidative defense in plants and providing redox homeostasis. Towards this objective, we successfully developed Exapx1 over-expressing E. coracana plants. The transgenic lines were developed by using Agrobacterium mediated transformation method. The transgenic lines with increased ascorbate peroxidase activity where used to delineate the redox sensing mechanisms that are involved in providing protection from oxidative stress. The transgenic lines were morphologically similar to the wild type plants and had similar PS-II activity. Increased expression of apx1 gene in the transgenic lines could induce higher expression of superoxide dismutase and monodehydroascorbate reductase genes, under stress; but no significant increase in dehydroascorbate reductase and glutathione reductase gene expression was recorded. Over-expression of Ecapx1 gene could selectively (and not universally) induce the expression and activity of other anti-oxidant enzymes of the AsA-GSH pathway, indicting the existence of a cross talk or signaling mechanism in plants, for coordinated expression of these enzymes. On exposure to stress, the transgenic lines accumulated lesser H2O2 as compared to the wild type plants and also had lower proline levels. An effective control over H2O2 levels (through Ecapx1 over-expression) prevented undue activation of specific antioxidative enzymes like DHAR and GR, while helping to maintain the redox balance of the cells under stress. Incidentally, the transgenic lines had higher reduced to total ascorbate ratio, than the wild type plants, that was responsible for sensing the increased influx of ROS and activate the cellular anti-oxidative defense network, through down-stream signaling.