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ThesisItem Open Access In vitro establishment of Polygonatum verticillatum (L.) All. and augmentation of its phytochemical profile using elicitors(G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand), 2017-11) Tiwari, Tanuja; Chaturvedi, Preetilants as a valuable and safe natural source of medicine have drawn much scientific and commercial attention in past few decades. Polygonatum verticillatum (L.) All., an Asparagaceae member, is an important and highly valued ‘Astavarga’ medicinal herb of North West Himalaya. Regeneration of the plant through seeds is a slow process in nature owing to epicotyl dormancy. Therefore, in the present study, an effort was made to break seed dormancy, increase seed germination potential and micropropagate P. verticillatum using epicotyl and sprouted buds. Among all storage conditions and PGRs, cold moist stratification of seeds (4±10C for 4 weeks) together with GA3 (100 mg/L) pretreatment followed by germination on GA3 (50 to 100 mg/L) supplemented MS medium was found most effective to improve seed germination. Maximum per cent germination (78±3.74) was achieved through elicitation using 50µM sodium nitroprusside (SNP). Maximum per cent bud induction (90±6.1) with maximum number of buds (7.0±0.45) and shoot length (7.10±3.18cm) was achieved from epicotyl explants inoculated in MS+TDZ(1.0mg/L)+NAA(0.5mg/L)+GA3(2.0mg/L). Microshoots inoculated in MS+KN (1.0mg/L)+IAA(0.5mg/L) and MS+IBA(1.0 mg/L) respectively showed maximum per cent microrhizome induction (95±5.0) and root induction (85±6.12). Sprouted bud explants inoculated in MS+TDZ (2.0mg/L)+NAA(1.0mg/L)+GA3(2.0mg/L) showed maximum per cent shoot induction(85±10.0). However, maximum per cent rooting (70±5.0) was achieved in MS+IBA (1.0 mg/L) and MS+NAA (1.0 mg/L)+KN(0.5mg/L). Plants raised through epicotyls and sprouted buds showed 80% and 56% survival rate respectively in garden soil. An effort was also made to investigate the effect of various chemical elicitors on phytochemical profiling of the plant. Maximum total chlorophyll content (1.67±0.05 mg g-1 fresh wt.), total phenolic content (14.71±0.47mg GAE g-1), total flavonoid content (14.66±0.17 mg QE g-1) and %DPPH radical scavenging activity (60.23±0.77) were achieved in SA (300 µM), JA (300 µL/L), SA (300 µM) and SNP (50µM) elicited plants respectively. Protein content (2.40±0.06 mg g-1 fresh wt.), peroxidase activity (1.33±0.03 unit min-1mg-1protein) and catalase activity (1.32±0.05 unit min-1mg-1protein) were augmented maximum in underground parts of JA (300 µL/L) and SNP (50 µM and 100 µM) elicited plants respectively, whereas SOD activity (4.79±0.13 unit min-1mg-1protein) was reported maximum in aerial parts of SA (300 µM) elicited plants. GC-MS analysis of differently elicited plants divulged remarkable (%) increase in medicinally important compounds. Diosgenin (anticancerous, antidiabetic), 9,12Octadecadienoic acid (z,z) (anti-inflammatory and antiarthritic), Gamma-sitosterol (antidiabetic) and Squalene (anticancerous, anti-tumor) increased to maximum viz., 68.11%, 89.67%, 78.87% and 81.82% respectively in SA treated plants. Guanosine (antiprotozoal), Palmitic acid (antioxidant), Vitamin E (antiinflammatory), 1,2-Benzenedicarboxylic acid (anti-inflammatory) increased by 53.25%, 51.68%, 83.04%, 66.50% respectively in SNP elicited plants. 2,3-dihydro-3,5-dihydroxy-6-methyl-4H-pyranone (anticancerous), Deoxy-d-mannoic lactone (antibacterial) increased by 23.56% and 66.67% respectively in JA elicited plants. The present investigation gave a complete reproducible protocol for in vitro establishment of P. verticillatum. Furthermore, elicitation study on the plant helped in augmentation and authentication of neutraceutical status of the plant.ThesisItem Open Access Diversity of bryophytes and pteridophytes in Pantnagar(G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand), 2017-08) Panthari, Divyansh; Chaturvedi, PreetiDuring past few decades, Pantnagar region in U. S. Nagar district (Uttarakhand) has faced lot of industrialization and urbanization activities. Change in land use patterns due to industrialisation and intensive agricultural practices, led to loss of large portion of microhabitats of the lower plants from the region. In order to prepare a complete database of the floristic diversity of the region, there is an urgent need for the conservation of these plants and their habitats. Conservation can be done by many methods viz., in vitro techniques, monitoring and assessment of rare and endemic plants. Taxonomical studies do play an important role in conservation practices by providing useful information regarding biodiversity and changing dynamics of the diversity with development. Lower plants were ignored for a long period of time, due to their small size and ignorance toward their ecological and economical value. Although, there are various previous attempts regarding taxonomical studies of bryophytes and pteridophytes across the world including India but the Indian studies are mainly confined to the hills. Detailed taxonomical studies of bryophytes and pteridophytes have been highly neglected in the agrarian landscapes of Tarai region. Therefore, the present study is an attempt to survey, collect and prepare check list of the bryophytic and pteridophytic flora of Pantnagar region including university campus, as well as various research centers. Total 34 species were reported, out of which 6 were liverworts, 27 were mosses and 1 was hornwort. Liverworts belonged to 4 genera and 4 families, mosses belonged to 17 genera and 10 families while hornworts belonged to 1 genera and 1 family. In mosses, 2 familis viz., Pottiaceae and Bryaceae were found to dominate in the study area by representing 10 and 7 species respectively. In liverworts, Ricciaceae was found to dominate with 3 species followed by Marchantiaceae, Aytoniaceae and Cyathodiaceae with 1 species each.7 species of pteridophytes belonging to 7 genera representing 7 families, 4 orders and 2 classes were reported from the study area. Equisetum sp. and Salvinia sp. were found to dominate the area. The other aspect of this study was to test total phenolic content, total flavonoid content and antioxidant activity via DPPH and TAA assay in methanolic extract of three selected bryophytes. DPPH radical scavenging activity (%) and total antioxidant activity were found to be highest in Marchantia papillata (61.77±0.94) and Philontis hastate (8.27±0.38) respectively. Maximum phenolics (15.58±0.12unit) and flavonoid content (108.175±1.13unit) were reported from Marchantia papillata.ThesisItem Open Access Bryophyte diversity of Pithoragarh and Munsiyari regions of Western Himalaya(G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand), 2017-08) Raturi, Ashish; Chaturvedi, PreetiBryophytes, a group of species of smaller and apparently simpler stature are the oldest known land plants.The three major groups of bryophytes namely mosses, liverworts, and hornworts comprise the earliest lineages of land plants derived from green algal ancestors.Yet despite their huge diversity, phylogenetic importance and key function in the world’s ecosystem, study of various aspects of bryophytes lagged behind in comparison to larger land plants. It is probably because of their small size, apparently simpler and less exciting evolutionary stories and limited scientific consideration. Being primitive and cosmopolitan in nature bryophyte diversity have been adversely affected due to the growing industrialization, urbanizationand introduction of new agriculture practices. Hence, there is an emerging need of its conservation against manmade ecosystem. Present study was an attempt to survey and collect the bryoflora of Pithoragarh and Munsiyari region in Uttarakhand. Additionally, qualitative and quantitative screening of phytoconstituents of three selected bryophytes (Leucodon secundus, Plagiomnium integrum and Thuidium cymbifolium) and analysis of soil of bryophytes collection sites were also done. Total 36 species were collected; out of them 31 were mosses belonging to 18 families, 4 liverworts belonging to 4 families and single hornwort. Biochemical characterization was done in term of total phenolic content (TPC), total flavonoid content (TFC)and antioxidant activity via DPPHand TAA assay. DPPH radical scavenging activity (%) was found highest in L.secundus(79.82±0.65) and lowest in P. integrum(56.63±0.28). Maximum total antioxidant activity (TAA) was reported in T. cymbifolium (88.57±0.70) while minimum in L. secundus(12.55±0.07). Total phenolic content was seen highest in T. cymbifolium(38.50±0.69mg GAE g-1) and least in L. secundus(6.23±0.17mg GAE g-1). Total Flavonoid Content was found highest in L.secundus(94.025±0.94mg QE g-1) and least in T. cymbifolium(28.2±0.29mg QE g-1).Soil analysis showed maximum pH (6.5) at site 4 and maximum Electric Conductivity (0.17kg/ha) at site 5.Total available nitrogen (292.69 ± 15.65kg/ha),total available phosphorous(52.12 ± 2.58kg/ha)and total available potassium (589.49 ± 2.30kg/ha)werefound highestat site 5.ThesisItem Open Access Influence of plant growth promoting rhizobacteria (PGPR) on wheat (Triticum aestivum L.) and cowpea [Vigna unguiculata (L.) Walp.] and their impact on rhizospheric microbial diversity(G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand), 2017-08) Anand Kumar; Sharma, A.K.ThesisItem Open Access Process development for the production of bioethanol from banana peels (Musa sapientum L.) using thermo-alkali-stable depolymerising enzymes(G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand), 2017-07) Heena Prakash; Sharma, A.KUse of cheap lignocellulosic biomass, such as banana peels, for bioethanol production, can simultaneously provide a solution to the major key global issues of the energy crisis and environmental pollution. In present study, the possibility of using waste banana peels for the production of ethanol has been investigated. To minimize the cost of ethanol production using enzymatic hydrolysis, the hydrolytic enzymes that are necessary for cellulose and hemicelluloses hydrolysis were produced in-house using a bacterial strain, Geobacillus stearothermophilus HPA19, isolated from an alkaline soil sample. The bacterium was able to produce cocktail of the thermo-alkali-stable xylano-cellulo-pectino enzymes using wheat bran (an agro-residue) as carbon source within 24 h peroid. These enzymes showed excellent stability at high temperature (80 ºC) and pH (10.0). The optimal temperature and pH for xylanase, FPase, pectinase are 80, 70, 80 ºC and 9.0, 8.0, 9.0, respectively. The metal ions Ni+2, Zn+2 promoted the relative activities of xylanase and FPase whereas Na+, Ca+2 and K+ promoted pectinase activity in the enzymes cocktail. After that application of this cocktail was assessed in the saccharification of banana peel. Reducing sugar obtained (37.06 mg/ml) after OVAT method is greatly influenced by enzyme dose [ratio 1:1:2], substrate concentration (10.0%), hydrolysis time (1 h) and hydrolysis temperature (70 ºC). Further Response Surface Methodology (CCD) was used to optimize saccharification. This led to a 2 fold increase in reducing sugar (54.14 mg/ml) giving a high saccharification efficiency of 54.14%. Under the optimum conditions of saccharification, ethanol was produced using yeast which gives highest ethanol production of 21.10 g/l and fermentation efficiency of 76.50% within 30 h. Thus, the study presented proved to be a comprehensive study, where in-house enzymes cocktail by a thermophilic bacteria G. stearothermophilus HPA19 was produced for bioconversion of banana peels into second generation bioethanol.ThesisItem Open Access Taxonomic pattern of elevational distribution of seed plants in Uttarakhand(G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand), 2017-07) Tarafdar, Sumant; Rawat, D.S.ThesisItem Open Access Antioxidant profiling in ten selected wild leafy vegetables of Kumaun region(G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand), 2017-07) Hemdani, Ruchi; Rao, P.B.In recent times, considerable increase in concern regarding the harmful effect of oxidants on human health, a great deal of attention is being given to the exploration of such sources that might play a major role in neutralizing these damaging species. Antioxidants have acquired an excellent position in this regard as they known to provide protection against the free radicals. Therefore, natural antioxidants are considered comparatively safer and healthier than synthetic ones. To ascertain the potential of wild leafy vegetables as an important source of natural antioxidants, the antioxidant activity in ten different wild leafy vegetables were collected from Kumaun region of Uttarakhand in methanolic extract and evaluated by using three different assays namely, 2, 2-diphenyl-1- picrylhydrazyl (DPPH), ferrous chelating ability (FCA), total antioxidant activity (TAA) . In addition, total phenolic content (TPC) and total flavonoid content (TFC) are also measured by using Folin Ciocalteu reagent and flavonoid assay. In the present study, all ten wild leafy vegetables exhibited the presence of phytochemical components i.e., carbohydrates, protein, fat, terpene and flavonoids. The mean values (mean of five conc.) ranged from 91.11 (I.cairica ) to 41.74 (S.americanum) in DPPH (%), 93.98 (I.cairica) to 46.62 (S. americanum) in FCA (%); 9.04 (I. cairica) to 2.9 (S. americanum) in TAA (µg AAE/mg extract); 9.52 ± 1.0 (O. debilis) to 2.5 ± 2.0 (S. americanum) in TPC (µg GAE/mg extract) and7.8 ± 0.12 (I. cairica) to 1.9 ± 0.16 (S. americanum) in TFC (µg QE/mg extract). Among species, comparatively higher values in DPPH, FCA, TAA and TFC are in I. cairica whereas TPC is in O. debilis. Thus, the species I. cairica and O. debilis have high amount of antioxidants thus, they are recommended for consumption. Significant and positive correlation was found between all possible combinations with a highly significant between DPPH vs. FCA, DPPH vs.TAA, TAA vs. FCA and TPC vs. TFC except between TPC vs. TAA, where it was negatively correlated. Thus, the results of the present study, supports the view that all selected wild leafy vegetables are promising source of natural antioxidants.ThesisItem Open Access Assessment of morphological diversity and biochemical parameters in some selected genotypes of Trigonella foenum-graecum L.(G.B. Pant University of Agriculture and Technology, Pantnagar (Uttarakhand), 2017-01) Nisha; Rao, P.B.ThesisItem Open Access Isolation, characterization and growth promoting attributes of plant growth promoting bacteria (PGPB) on field crops under irrigated and drought conditions(G.B. Pant University of Agriculture and Technology, Pantnagar (Uttarakhand), 2017-01) Dinesh Chandra; Sharma, A.K.Plant growth promoting bacteria (PGPB) containing 1-aminocyclopropane-1-carboxylate (ACC) deaminase has been reported to play an important role in alleviation of drought stress. In present study, eight ACC deaminase producing tolerant bacteria were isolated from a rainfed agriculture field of Kumaun region of Central Himalaya, Uttarakhand, India and evaluated for their efficiency in improving the growth of finger millet (Eleusine coracana (L.) Gaertn.), rice (Oryza sativa L.) and wheat (Triticum aestivum L.) under irrigated and drought conditions. These bacteria withstand a substrate metric potential of -1.0 MPa (30% PEG 8000) and therefore are considered to be drought-tolerant. The selected bacterial isolates were identified as Ochrobactrum sp., Pseudomonas sp., and Variovorax sp. by fatty acid methyl ester (FAME) analysis and 16S rRNA gene sequencing. The presence of acdS, the structural gene for ACC deaminase in selected bacterial strains was confirmed by the polymerase chain reaction (PCR). Glass house experiment showed that enhanced level of nutrients and antioxidant properties with the consortium of different bacterial strains. The consortium of Ochrobactrum anthropi DPC9 + Pseudomonas palleroniana DPB13 + Pseudomonas fluorescens DPB15 + Pseudomonas palleroniana DPB16 significantly increased the 1000 grain weight (9.3%, 28.9%), number of grains/panicle or spike (31.3%, 32.4%), grain yield (39.6%, 42.7%), and straw yield (21.1%, 26.9%) of rice and wheat, respectively as compared to their respective non-inoculated plants under organic field conditions. Variovorax paradoxus RAA3 treated plants maximally increase the total tillers/plant (40.0%, 31.7%), number of grains/spike (30.3%, 28.2%), spike length (26.4%, 25.8%), 1000 grain weight (17.4%, 22.0%), grain yield (28.2%, 47.4%), straw yield (11.2%, 26.0%) and harvest index (10.6%, 15.3%) under irrigated and rainfed conditions, respectively as compared to non-inoculated plant. Quantitative RT-PCR (qRT-PCR) based expression analysis of drought responsive genes (helicases and aquaporin) was also performed in response to V. paradoxus RAA3 inoculation in two varieties of wheat (HD 2967 and PBW 660) under irrigated and rainfed conditions The relative expression of aquaporin (TaTIP1;1) is significantly higher (7.26-fold) in the wheat variety PBW 660(drought tolerant), as compared to drought sensitive HD 2967 where expression level of aquaporin is down-regulated. The transcript level of WDH45 is down-regulated in both the varieties under irrigated conditions. In rainfed conditions, the relative mRNA level of wheat DNA helicase (WDH45) is significantly higher in HD 2967 (1.39-fold) than the PBW 660, whereas the expression pattern of aquaporin in PBW 660 is higher (2.70-fold) than the variety HD 2967 under rainfed conditions. This finding may imply PGPB having ACC deaminase activity could turn out to be effective inoculants for improving the growth of plants. Such promising strains could possibly be used under field conditions in rainfed environment where agriculture is exclusively dependent on rainfall.