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Subject: Biochemistry × End date: 2009 × Start date: 2009 ×
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    ThesisItemOpen 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, Sandeep
    Brassica 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.
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    ThesisItemOpen 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.