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ThesisItem Open Access Genomics-proteomics transition approaches for dissection of complexity involved in molecular pathogenesis of karnal bunt(G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand), 2017-08) Pandey, Vishakha; Anil KumarThesisItem Open Access Preparation and characterization of finger millet prolamin based nano-delivery vehicle for lipophilic substances(G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand), 2015-07) Subin Raj, C.K.; Pandey, DineshBioactives such as omega-fatty acids, phytosterols, vitamins, carotenoids are all known to reduce the risk associated with a number of chronic diseases and help in improving public health. However, these bioactive compounds /drugs are exhibit low bioavailability due to their low permeability, biostability, solubility, limited retention time and poor uptake or absorption in human body when they are consumed through food items such as fruits, vegetables etc. These constraints limit their use in development of functional foods and nutraceuticals. In order to overcome the above mentioned disadvantages, the present study was undertaken in the ”Immunobiologicals & Proteomics lab (Dept. of Molecular Biology & Genetic Engineering)” during the II-semester, 2014-15. This is the first report of a nanodelivery vehicle prepared using finger millet prolamin as the encapsulating matrix over oil in water nanoemulsion of sesame oil containing dissolved β-carotene, which was used as a model bioactive compound. The nanoparticles were prepared in a single step ultrasonication process and did not involve the use of organic solvents and cross-linking agents. Effects of different parameters on prolamin based nanoparticle were investigated. Optimal nanoparticles with regular shape, small size (230- 320 nm), high zeta potential and narrow size distribution were obtained at protein contents upto 2% and oil content upto 4% as characterized by electron microscopy and Zetasizer. The delivery system was shown to have high encapsulation and loading efficiencies. During simulated in vitro digestion, sustained release of β-carotene was observed from the prolamin stabilized nanoemulsion. The cytotoxicity and influence of the nanoparticles was assessed on Caco-2 cells, and the results demonstrated that the nanoparticles were biocompatible and non-toxic to the Caco-2 cells even at concentration as high as 0.5 mg mL-1 . There was observed differentiation in the cells challenged with higher concentrations of the nanoparticles. The use of food grade protein derived from a low agri-input requiring crop, finger millet could provide a platform for value addition, creating a new market for the agri-produce and thereby helping the farming sector. Such products are also beneficial to consumers, providing a better alternative to pharmaceuticals. This delivery oriented food derived nanoparticle system has applications as an oral agent for the controlled release and enhanced absorption of lipophilic bioactives and drugs. It has the potential to be incorporated and developed into various forms for nutraceuticals for general health benefits of the populace.ThesisItem Open Access A study of 45S and5S rRNA genes in some species of Allium(G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand), 2017-07) Lenka, Amit Kumar; Sundip KumarThesisItem Open Access Functional validation of down regulation of Dof1 transcription factor using RNA interference in Arabidopsis thaliana(G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand), 2017-08) Saha, Arnab; Singh, B.R.Nitrogen (N) is one of the most important mineral elements which directly affect on crop yield. Due to this reason, in the last 40 years, the amount of synthetic nitrogen (N) applied to crops has risen dramatically, from 12 Tg/year to 104 Tg/year, resulting in increase in yield but with considerable impacts on the global environment because plants consume much less than half of the fertilizers applied, means more than half of the fertilizer are leach into surrounding lake, ponds, river and cause environmental problem. To solve this problem, various studies of different regulatory genes were carried out in recent years. One of the regulatory gene is Dof1 (DNA Binding with one Finger domain 1). Dof1 gene was first recognized in Maize and by its over expression in Arabidopsis thaliana it was concluded that, it regulates the enzymes which participate in Carbon/Nitrogen assimilation like nitrate reductase, phosphoenolpyruvate carboxylase (PEPC), pyruvate kinase (PK), citrate synthase (CS), and isocitrate dehydrogenase (ICDH), GS(Glutamate Synthetase) and GOGAT (Glutamine oxoglutarate aminotransferase) enzyme at transcription level. Most of the studies for defining its role have been carried out by over expression of this (Dof1) gene in different plants, but still its proper role is yet to be validated and for that another approach can be used i.e. its down regulation study which could be done by gene silencing. For this, pRNAi golden gate vector present within agrobacterium cell suspension was transformed in Arabidopsis plant through floral dip method. Selection of transgenic line was done in kanamycin selection medium. After that different morphological, biochemical and molecular assays were done to validate the functions of dof TF in NUE. It was clearly seen that upto 20-32 % decrease has been observed in case of different biochemical analysis of enzyme activities which are directly controlled by this Dof1 transcription factor. Total chlorophyll content has also reduced which clearly indicated that photosynthetic activity is hampered. 25-40% down regulation of gene has been observed in real time PCR analysis of Dof 1 transcription factor. This transformation technique clearly indicated the functional validation of dof1 transcription factor which directly and indirectly control the enzyme of carbon and nitrogen metabolism.ThesisItem Open Access Evaluation of immunomodulatory and antioxidative potential of Nycatanthes arbor-tristis Linn. and Moringa oleifera Lam. in chicken lymphocytes culture system(G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand), 2017-08) Ruj, Anamitra; Ambwani, SonuIndia is among those countries in the world where ancient system of medicine, such as Ayurveda, Siddha, Unani, Tribal medicine and naturopathy have been in practice for many years. Antioxidants are important substances that have the ability to protect the organism from the damage caused by the oxidative stress. From the ancient times many diseases were treated by modulating the immune system through medicinal plants. Poultry industry is highly susceptible to biotic and abiotic stresses that are incurring heavy economic losses. Immunomodulation of poultry is one measure to prevent the poultry diseases. Keeping in view of the above, present study was planned to explore immunomodulatory and antioxidative potential of Nyctanthes arbor-tristis Linn. and Moringa oleifera Lam. in chicken lymphocytes culture Extracts of these plants were prepared and evaluated for the presence of various phytoconstituents as well. The extraction yield of Nyctanthes arbor-tristis and Moringa oleifera was found to be 10.69% and 8.16%, respectively. Biochemical analyses revealed the presence of various phytoconstituents in both NAE and MOE. The total phenolics content of NAE and MOE was estimated to be 180.55mg GAE/g and 173.6mg GAE/g, respectively while total flavonoids content of the NAE and MOE was 125mg RE/g and 127.7mg RE/g, respectively. The antioxidant property of both the extracts was also evaluated by DPPH assay. Noncytotoxic doses of both the extracts were determined in the lymphocytes culture system which was found to be 300µg/ml and 250µg/ml for NAE and MOE, respectively. NAE displayed significant proliferation of 19.94% and 32.24% in T cell proliferation in case of PHA and Con A stimulated cells, respectively. There was 11.09% increase in B cell proliferation in LPS stimulated cells as compared to control. In case of MOE there was significant increase in T cells proliferation of 7.65% and 9.20% in case of PHA and Con A stimulated cells, respectively. There was 7.90% increase in B cell proliferation in case of LPS stimulated cells due to in vitro exposure of MOE. There was significant decrease in the percent Lipid peroxidation and Nitric oxide after the treatment with the plant extracts. However, GSH, SOD and Catalase levels increased in the cells treated with the extracts in comparison to control cells. Both the extracts, viz. NAE and MOE exhibited presence of antioxidants and displayed immunomodulatory effects in chicken lymphocytes culture system and thus could be used for development of immunomodulatory herbal preparations for poultry. However intensive research should be done to identify the specific phytochemicals, which are responsible for immunopotentiating activity of both plants by various biochemical and molecular biology techniques.ThesisItem Open Access Study on natural allelic diversity in drought responsive Myb gene in germplasm of Eleusine coracana collected from different regions of Uttarakhand(G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand), 2017-08) Sinha, Akash; Lohani, PushpaAbiotic stresses like drought, salinity and cold cause huge losses to crop yield. MYB transcription factors have been found to be regulated by ABA signaling and modulate the expression of various downstream abiotic stress related genes. In the present study the effect of 11 day drought stress treatment on various genotypes of finger millet was evaluated at the vegetative stage. Drought stress was found to have an effect on various morpho-physiological and biochemical characters. DNA isolation of all the 12 genotypes was done and PCR amplification using gene specific primers was performed. The amplicons were sequenced and the sequences hence obtained were subjected to further analysis using various bioinformatics tools. Multiple Sequence Analysis results indicated that considerable nucleotide diversity for the drought responsive Myb gene is present in the studied finger millet germplasm. PRM 6107 was found to be the most drought tolerant genotype, amongst all the genotypes under study based on the various morpho-physiological and biochemical tests conducted. PCPGR 8115 was observed as the most sensitive genotype. Single nucleotide polymorphisms and insertions in the Myb allele of PRM-6107 were studied. PRM-6107 Myb allele was found to be highly similar to Saccharum barberi Myb 18 in terms of nucleotide sequence, protein sequence, protein structure and function and phylogeny. Saccharum barberi Myb 18 has a role in imparting dehydration and salinity tolerance. Hence, it was concluded from this study that finger millet genotype PRM-6107 harbours a superior drought tolerant allele for drought responsive Myb gene.ThesisItem Open Access Optimization of explants for Agrobacterium mediated gene transfer in Eleusine coracana (PES-400)(G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand), 2017-08) Raingam, Masochon; Lohani, PushpaFinger millet is the primary food source for millions of people living in tropical dry lands.It posses many desirable properties in terms of adaptability and nutrition. Finger millet can become a hub for fishing out vital genes involved in adaptation to biotic and abiotic stress which can be used for improvement and development of superior crop varieties. For genetic improvement of this important crop, development of efficient protocol for transformation is necessary. In this study, an effort was made to optimize the explants for Agrobacterium mediated gene transfer. Several experimental factors influencing the transformation efficiency were evaluated for four explants; seed, coleoptile, mesocotyl and callus. Among different hormone concentrations and combinations studied, the best callus induction rate for all the explants was observed in 3(2,4 D)+1.5 NAA while the size of the callus was biggest in 2 mg/l NAA .The best proliferation was observed in 1mg/l NAA, 1.5mg /l NAA, 2mg/l NAA and 1mg/l 2,4 D + 1mg/l NAA. Antibiotic sensitivity test for kanamycin showed callus induction and survival in presence of 400 mg/l kanamycin. Hygromycin above 20 mg/l was found to be lethal for finger millet explants. A good Callus induction of explants was observed in presence of 200mg/l-500mg/l cefotaxime. Cefotaxime enhanced the formation of shoot at the concentration of 200 and 250mg/l. Agrobacterium strain LBA4404 harboring pCAMBIA1301 and optimized callus induction media(3mg/l 2,4 D+1.5 mg/l NAA) and antibiotics (300mg/l cefotaxime and 25 mg/l hygromycin for selection) and were used for further optimization of acetosyringone concentration, infection time, co-cultivation time. Infection time of 30 minutes, acetosyringone concentration of 200µM and co-cultivation for three days were found to be optimum for Agrobacterium mediated transformation. Therefore, it can be concluded that callus is the best target explants for Agrobacterium transformation with the efficiency of 15.2 %.ThesisItem Open Access Molecular cloning, expression and characterization of methionine-rich prolamin seed storage protein geneE (fimP2) of finger millet (Eleusine coracana)(G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand), 2017-08) Namdeo, Jadhav Roshankumar; Anil KumarNutrition is very much important for healthy human being. Content and composition of diet is crucial for preventing protein energy malnutrition. Any significant alteration of nutrients due to improper dietary practices affects the health outcome. Finger millet (Eleusine coracana) is one of the economically and agriculturally important crops of arid and semi arid regions and it possesses high nutritional value. Finger millets can be proposed as nutraceutical crops after identification of quality proteins of high biological value and enriched with essential amino acid (EAA). It can be explored for the development of preventive modalities and food supplements. Amino acids like methionine as an EAA has been suggested as EAA and is one of the four main dietary sources of methyl groups, its deficiency can be associated with fatty liver, atherosclerosis, neurological disorder and tumorigenesis-all are methylation related disorders. Therefore, having methionine rich protein in our diet or taking it as neutraceuticals is one of the important modes of enrichment of our diet. Attempts were made in our laboratory to report for the first time de novo assembly of transcriptome data from two finger millet genotypes differing in their seed protein content. The annotation of the transcriptome data has made it possible to annotate novel and nutritionally important genes. One of such gene fimP2 (finger millet Prolamin-2 encoding gene) was cloned and characterized for its physicochemical properties and enriched with 14.8% methionine amino acid. The fimP2 gene was cloned in bacterial system for heterologous expression. The recombinant protein was further isolated and purified. In silico studies were employed for physico-chemical characterization as well as the structure interpretation of the protein. The biological roles of the protein were also predicted by homology searching, conserved domain analysis etc. The presence of two internal repetitive conserved domain of the protein is suggestive of seed storage protein, indicating its roles in fulfillment of nutritional need. In order to determine the tissue specificity of fimP2 gene, transcript profiling was performed in two genotypes of finger millet differing in seed protein content i.e. GE-1437 and GE-3885. The expression analysis of the gene by real-time PCR showed an increasing order of expression in developing spikes from S1 to S4 however the gene was abundantly expressed in GE-3885-a high protein genotype indicating that higher grain protein content is also linked with higher expression of prolamin protein. The results of present investigation led to identify and characterize a novel FIMP-2 protein envisaged to have nutritional role for serving methyl donor in many biological processes. Thus can be further validated using various nutritional studies and molecular biology approaches to prove its nutraceutical and biological function.ThesisItem Open Access Evaluation of anti-bacterial and anti-proliferative potential of Tinospora cordifolia in In-vitro models(G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand), 2017-07) Pandey, Nikita; Joshi, G.C.