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ThesisItem Open Access Screening of tea cultivars grown in Kumaon region for their total phenolics, pigments and individual catechin contents(G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand), 2005-08) Pandey, Sudhanshu; Mishra, D.P.Total chlorophylls, chlorophyll a , chlorophyll b, total carotenoids and total phenolics contents and HPLC profiling of various catechins and quantitative estimation of (-)EGCG,(-)EC and (-)ECG were carried out in fresh tea leaf leaves (dried immediately after sampling), of 6 varieties grown in Kumaon region and a black orthodox tea sample processed locally. The main objective of the study was to screen these varieties for their catechin contents and other flavanoid constituents. Dried leaf powder was used to estimate chlorophylls, carotenoids and total phenols. For catechins, dried leaf powder was first extracted with aqueous methanol. This extract was subjected to separation of catechins on HPLC and identification and quantitative estimation were done using standard catechins. Contents of various parameters were variable among varieties and there was no set pattern. Kangra Jat had highest content of chlorophyll a, and total carotenoids while chlorophyll b, total chlorophyll and total phenols were highest in UPAS1-9. T-78 exhibited lowest content of all these parameters. Black orthodox tea had pretty high content of chlorophylls and carotenoids but its phenolics content was lowest, perhaps due to oxidation of phenolics during processing. The HPLC profiles gave eight peaks, out of which first two peak, were probably of some non-catechin compounds, hence ignored. Out of remaining 6 peaks, 3 were of (-)EGCG,(-)EC and (-)ECG. Other three were named as U1, U2 and U3.Out of them U1 was supported to be (-)EGC. By comparison of peak areas,retention times etc., quantitative estimation of (-)EGCG, (-)EC and (-)EC was performed, which revealed these three catechins comprised nearly 0.11-0.14% of dried solids (110-140 mg g-1) and (-)EGCG was nearly (69-82 mg g-1),half of all catechins estimated. On the basis of increasing catechin contents, order of varieties was: Black Orthodox tea(commercial)ThesisItem Open Access Biochemical evaluation of genotypes of Jatropha curcas L., and optimization of micropropagation schedule(G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand), 2005-07) Munagapati, Sandhya; Gaur, A.K.Five genotypes of Jatropha curcas viz. shu03001, shu03002, shu03004, shu04001 and shu04004 were selected for their further identification and biochemical characterization. Identification of genotypes had been done on the basis of banding pattern on SDS-PAGE of total seed storage protein and its fractions viz. globulins and albumins. Based on the Jaccard‟s similarity coefficient obtained from the banding patterns of SDS-PAGE, the five genotypes are grouped into two categories. Group1 consisting of shu03001, shu03004 and shu04004 and Group2 consisting of shu03002 and shu04001. These results were based in accordance with the percentage protein content of the respective genotypes taken for study and the oil contents of these genotypes were almost comparable. However, the lipid/protein ratio was highest in shu04001. In view of optimizing micropropagation schedule, the experiments were also conducted on three genotypes of J.curcas viz. shu03001, shu03002 and shu03004 to study the effect of various parameters viz. genotypes, growth regulators and explants and their interactions on callus induction frequency. Two explants viz., leaf and petiole from all the three genotypes were placed on different combinations of growth regulators. Calluses were induced from the two explants on all types of callus induction media containing four levels of 2,4-D (1, 2, 2.5and 3mg/l) with BAP (0.1,0.2,0.25 and 0.3mg/l) and two levels of NAA (1 and 2 mg/l) with two levels of BAP (0.1 and 0.2 mg/l). Maximum callus induction frequency of 92.8, 93.1; 97.4, 98.7 and 88.8, 96.1% was observed for leaf and petiole explants of shu03001, shu03002 and shu03004 genotypes respectively. In a generalized way, it was observed that shu03004 genotype showed highest callus induction frequency followed by shu03002 and shu03001. Media with NAA (1mg/l) and BAP (0.1mg/l) showed highest callus induction frequency with both leaf and petiole explants in all the three genotypes. Amongst explants, petiole showed higher callus induction frequency followed by leaf explant on all types of callus induction media.ThesisItem Open Access Biochemical and molecular tools for screening ashwagandha genotypes combining phenotypic and chemotypic attributes for elite germplasm(G.B. Pant University of Agriculture and Technology, Pantnagar (Uttarakhand), 2006) Punetha, Himanshu; Gaur, A.K.ThesisItem Open Access Genetic Delineation Of Molecular Basis Of Resistance Against Isoproturon In Phalaris Minor Retz.(Govind Ballabh Pant University of Agriculture and Technology;Pantnagar, 2005) Kachare, Dilipkumar; Mishra, D. PThesisItem Open Access Characterization And Cloning Of Alpha-Amylase Inhibitor Gene From Indian Wheat And Millet(Govind Ballabh Pant University of Agriculture and Technology;Pantnagar, 2004) Singh, Annika; Mishra, D.P.ThesisItem Open Access Biochemical, physiological and molecular aspects of drought and cold tolerance in finger millet (Eleusine coracana L.) germplasm(G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand), 2008-08) Rai, Shubha; Agarwal, SanjeevFinger millet (Eleusine coracana L.) is considered a drought-tolerant crop species; however, within this species, there are considerable genotypic differences in tolerance to this environmental stress. In the present work, the effect of mannitol induced drought stress on germination, plant growth, proline and hydrogen peroxide content in ten finger millet genotypes was analyzed. These genotypes were further explored for their biochemical adaptation mechanism of drought and cold tolerance in the glasshouse condition. Different biochemical parameters viz. MDA content, hydrogen peroxide content, chlorophyll content APX activity, ascorbate content and proline were determined under drought and cold stress. PRM-6107, VL-283 and VL-328 registered minimum reduction in per cent germination, shoot length and root length; however, genotype PES-400, VR-708, VL-149 and VL-146 registered maximum reduction in per cent germination, shoot length and root length, under mannitol induced drought stress. Accordingly they were classified as relatively tolerant and susceptible group, respectively. Relatively tolerant genotypes registered lower hydrogen peroxide content and higher proline content compared to susceptible ones under mannitol induced drought stress. These genotypes also recorded lower MDA and hydrogen peroxide content and higher APX activity and proline content compared to susceptible one under cold and drought stress in the glasshouse condition. Biochemical responses of all the genotypes in terms of MDA, H2O2, ascorbate, proline content and APX activity were more pronounced in drought as compared to cold stress. In addition to this total leaf protein profile of all the ten genotypes under drought stress was studied. In tolerant genotypes A4 protein band with molecular weight of less than ~66 KDa was more pronounced under drought stress. During this study, efforts have been made to explore these genotypes for ascorbate peroxidase and cold shock domain protein gene(s).The APX and CSDP gene(s) are successfully amplified. Nucleotide sequence data have been submitted to GeneBank database (Accession nos. EU795006 and EU795007). These nucleotide sequences could be used as probe to fishout the corresponding full length gene in E. coracana plant.ThesisItem Open Access Engineering antioxidant defense mechanism to combat salinity stress in Brassica juncea(G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand), 2009-06) Saxena, Saurabh Chandra; Arora, SandeepBrassica juncea is a widely distributed and economically important oil seed crop, grown throughout the Indian sub-continent. The production and yield statistics of the crop has recorded wide fluctuations because of several environmental factors. Salinity stress is one of the major constraints limiting the overall yield potential of Brassica. An underlying mechanism for reduction in crop yield under salinity stress is the excessive production of reactive oxygen species (ROS) that can potentially damage lipids, nucleic acids and proteins, leading to disruption of essential physiological & biochemical processes. Plants possess a complex antioxidative defense system, comprising of enzymatic and non enzymatic molecules for scavenging these ROS. Ascorbate peroxidase (APX,E.C. 1.11.1.11), an important and widely distributed antioxidant enzyme is a part of Ascorbate –Glutathione pathway in the cell. It plays a crucial role in the detoxification of hydrogen peroxide in plants. The efficient scavenging of ROS is needed for increased tolerance to salinity stress. In the present studies, experiments were conducted to determine whether over-expression of APX could provide protection against salinity stress. Cytosolic ascorbate peroxidase (apx1) gene, isolated from Arabidopsis thaliana, was chosen as the candidate gene for strengthening the antioxidative defense system of Brassica juncea. An efficient and reproducible protocol for Agrobacterium mediated transformation of Brassica juncea (var. pusa jaikisan) was developed using hypocotyl explants. Transgenic plants over-expressing apx1 gene were developed and the successful integration of apx1 gene insert was confirmed through PCR using nptII and apx1 cDNA specific primers. The growth & physiological status of the transgenic plants was assessed using parameters like photosynthetic efficiency, stomatal conductance, chlorophyll fluorescence and SPAD values. The results indicate that the physiological and metabolic status of the transgenic plants was comparable with that of the wild type plants. Leaf disc assay was performed to evaluate the salinity stress tolerance potential of transgenic plants, using standardized biochemical parameters. Under 200 mM NaCl stress, the transgenic plants decisively performed better than the wild type plants; as revealed through greater Proline accumulation, increased Chlorophyll stability index, lower chlorophyll a/b ratio, higher activities of Ascorbate peroxidase, Guaiacol peroxidase & Total peroxidase along with lower H2O2 levels and lesser membrane damage as measured through MDA contents. From the present studies it can be concluded that cytosolic ascorbate peroxidase (apx1) can be used to strengthen the anti-oxidative defense system in plants and the transgenic Brassica juncea plants developed during the study have improved tolerance against salinity stress.ThesisItem Open Access Molecular typing of Salmonella isolated from Gangetic water(G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand), 2009-01) Balwant; Mishra, D.P.The present study was conducted at the Animal Biotechnology Center, Pantnagar to check the level of Salmonella contamination in the river Ganga. Total 500 samples were collected from 10 stations (50 samples from each station) viz; Gangotri, Uttarkashi, Rishikesh, Haridwar, Hastinapur, Garhmukteshwer, Narora, Kanpur Allahabad and Varanasi. These samples were subjected to Salmonella specific ‘his’ gene PCR. Samples were then Biochemically screened and serotyped at National Salmonella Research Centre IVRI, Izatnagar U.P. These isolates were serotyped as S. Abuja, S. Lagos. S. Pontypridd, S. Chinkual, S. Zwickau, S. Goldenberg and S. Oritamerin. DNA from these samples were extracted. ERIC-PCR, REP-PCR and OMPs profiling were conducted for serovar identification and strain differentiation of Salmonella rare serovars. For molecular typing ERIC- PCR, REP- PCR and OMPs analysis were conducted and it was found that REP- PCR was more efficient in strain differentiation. Therefore the data of ERIC PCR and REP PCR and OMPs profiling were used in Combined Molecular Typing and it was found that combined molecular typing was best typing method. Discriminating ability of ERIC PCR, REP PCR, OMPs profiling and Combined Molecular Typing methods were calculated by Simpson’s Index of Diversity. ERIC PCR showed D value of 0.92 and REP PCR gave 0.99 Value and of OMPs profiling it was found to be 0.94, while it was found 1 in Combined Molecular Typing. A Dendrogram was constructed by using Tree Con software by feeding data of Combined Molecular Typing then similarity and dissimilarity of isolates was checked. Twenty rare isolates showed maximum similarity. Therefore it can be concluded that combined molecular typing by using ERIC-PCR, REP-PCR and OMPs profiling gave better identification of Salmonella rare isolates.
