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University of Agricultural Sciences, Dharwad

The University of Agricultural Sciences, Dharwad was established on October 1, 1986. The University has 5 Colleges, 27 Research Stations, 6 Agriculture Extension Education Centers, 6 Krishi Vigyan Kendras and ATIC. The University has its jurisdiction over 7 districts namely Bagalkot, Belgaum, Bijapur, Dharwad, Gadag, Haveri, and Uttar Kannada in northern Karnataka. Greater diversity exists in soil types, climate, topography cropping and farming situations. The jurisdiction includes dry-farming to heavy rainfall and irrigated area. Important crops of the region include sorghum, cotton, rice, pulses, chilli, sugarcane, groundnut, sunflower, wheat, safflower etc. The region is also known for many horticultural crops. Considerable progress has been registered in the field of education, research and extension from this University.

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2010 - 201225
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Subject: Plant Biotechnology × End date: 2012 × Start date: 2010 ×
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Now showing 1 - 9 of 25
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    ThesisItemOpen Access
    Analysis of Transgenic Tomato Carrying Remusatia vivipara Lectin Gene (rvl1)
    (UAS, Dharwad, 2012) Rohini Mahaling Kolekar; Ramesh S. Bhat
    Transgenic tomato plants carrying Remusatia vivipara lectin (rvl1) gene were generated, characterized and analyzed for resistance to root knot nematode (Meloidogyne incognita) and white fly (Bemisia tabaci). Co-cultivation of 1000 cotyledonary leaf-disc explants from Pusa Ruby variety of tomato with Agrobacterium tumefaciens LBA 4404 carrying pNR73 binary vector with rvl1 resulted in 100 independent regenerants. The transgene-specific PCR amplified 771 bp product from 32 plants. Segregation analysis in T1 generation of two selected events indicated single copy integration of T-DNA/rvl1 in RVL1-T0(28) and multicopy integration in RVL1-T0(11). TAIL-PCR analysis in RVL1-T0(28) showed T-DNA integration between 1,07,295 bp and 1,07,296 bp on the clone C05_HBa0078I05 of chromosome 5. Proteins from both the events agglutinated rabbit erythrocytes. The total lectin activity for RVL1-T0(11) was 1.6 × 103 and the specific activity was 2.6 × 102. Likewise, for RVL1-T0(28), the total lectin activity was 3.2 × 103 and the specific activity was 4.2 × 102 which was relatively higher than that of RVL1-T0(11). Both the transgenic lines showed an expected band of 26.2 kDa on SDS-PAGE. Bioassay with M. incognita showed infection of juveniles (J2) in RVL1- T0(11), RVL1-T0(28) and the control plants on 3rd day of inoculation. On 6th day, 55.55 per cent of the juveniles developed into sausage form in control plants, but it was reduced to 47.05 per cent and 41.66 per cent in RVL1-T0(11) and RVL1-T0(28), respectively. On 70th day, the average number of galls on control plants was 62.85 per plant; and it was reduced by 36.36% and 45.46% in RVL1-T0(11) and RVL1-T0(28), respectively over the control. In general, non-transgenic plants showed the gall index of 7, and were rated as “highly susceptible”. Transgenic lines with the gall index of 3 were classified as “moderately resistant”. Nematode infection also resulted in significantly reduced average shoot and root length, and biomass in control plants compared to transgenic plants. Transgenic plants also caused white fly mortality. Control plants showed the mortality rate of 18.6%, and RVL1-T0(11) and RVL1- T0(28) exhibited an increased mortality of 69.35% and 92.47%, respectively over the control.
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    ThesisItemOpen Access
    Cloning of ech33 and ech36 From Trichoderma spp. and Expression in Saccharomyces cerevisiae (INVSc1)
    (UAS, Dharwad, 2011) Avishek Chatterjee; Sumangala Bhat
    The present study was conducted to isolate and clone ech36 and ech33 coding for endochitinase from Trichoderma harzianum and Trichoderma virens respectively and to study the expression of these genes in Saccharomyces cerevisiae (INVSc1 strain). Using specific primers, gene encoding ech36 (1.2 kb) and ech33 (1.2kb) were cloned into pTZ57R/T vector. The clones were confirmed through PCR and restriction analysis. BLAST analysis of ech36 showed 98 per cent homology at nucleotide level and 99 per cent homology at amino acid level. BLAST analysis of ech33 showed 99 per cent homology at nucleotide level and 95 per cent at amino acid level with sequence available in the NCBI databases. The cloned ech36 and ech33 were further cloned into yeast expression vector pYES2/CT and expressed in Saccharomyces cerevisiae INVSc1. They were named as pYESB36 and PYESB33 respectively. The expression of the cloned genes were confirmed by SDS-PAGE, substrate conversion assay and bioassay against Sclerotium rolfsii and it was compared with two previously cloned genes chit18-13 and ech42 to find out the most efficient endochitinase genes for further use in plant transformation. Chitinolytic activity was highest in pYESR18-13 and it was 3.15 times higher than control (yeast with plain vector pYES2/CT). Minimum chitinolytic activity was shown by pYESB36 and it was 1.44 times higher than the control (yeast with plain vector pYES2/CT). PDA plates with 1000 ıg of crude protein from recombinant yeast clone with ech42 (pYEGB42) showed maximum of 66.67 per cent inhibition of S. rolfsii where as yeast clone with ech36 (pYESB36) showed minimum inhibition of S. rolfsii (43.99%).
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    ThesisItemOpen Access
    Characterization of Antifungal Activity and Molecular Diversity of Trichoderma Isolates
    (UAS, Dharwad, 2011) Sagar Patil; Sumangala Bhat
    Eighty six Trichoderma isolates belonging to five species were used for antagonism test using dual culture assay against three fungal plant pathogens viz., Sclerotium rolfsii, Rhizoctonia solani and Cercospora capsici. Per cent inhibition of mycelial growth and time taken by Trichoderma isolates to overgrow the pathogen were recorded. Trichoderma isolates showed variation in their antagonistic property against S. rolfsii, R. solani and C. capsici. Further, these 86 isolates were grouped into five classes viz., most efficient, efficient, moderately efficient, poor and very poor. Three isolates IABT1242, IABT1243 and IABT1252 were grouped in most efficient class and took less time (8 days) to overgrow on S. rolfsii, R. solani and C. capsici. Isolate IABT1243 (T. harzianum) was most efficient against all three pathogens and showed the highest (91.83 pmol/μg/min) chitinolytic activity at 48 hours after induction on colloidal chitin containing medium and was significantly different from other isolates. Molecular diversity of twenty nine Trichoderma isolates belonging to five different species was assessed by twenty seven RAPD primers. Genetic similarities ranged from 49 to 83 per cent indicating substantial diversity present in the Trichoderma isolates. Twenty nine isolates were grouped into nine clusters, among which cluster IX was solitary. Isolates from the same region showed some similarities in RAPD clustering in UPGMA analysis. The diversity shown by the isolates did not correlate with the levels of antagonism shown against the pathogen. However, in few cases the correlation was observed between the cluster formed based on RAPD and the antagonistic property of Trichoderma isolates. The ITS region of eleven potent Trichoderma isolates were sequenced and confirmed that the isolates belonged to species T. harzianum, T. asperellum, T. viride and T. atroviride. The similar clustering of Trichoderma isolates was observed between dendrograms constructed using RAPD data and ITS sequences.
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    ThesisItemOpen Access
    Functional and Molecular Characterization of Native Isolates of Actinomycetes From Soil and Endorhizosphere of Medicinal Plants
    (UAS, Dharwad, 2011) Gayatree Mohapatra; Narayan Moger
    Microbial metabolic repertoire represents the primary resources from which new biomolecules are derived. Recently, the rise of resistance in pathogens renewed the interest on actinobacteria. In the present study, a total of 248 actinomycetes colonies were recovered from rhizosphere, endorhizosphere of 27 medicinal plants, an aquatic weed and road side run off soil. Randomly 114 actinobacteria isolates were picked for functional characterization against Rhizoctonia solani, Sclerotium rolfsii, Colletotrichum capsici, Ralstonia solanacearum and 40 actinobacteria against an insect Plutella xylostella. Out of which, 25, 85.5, 16 and 10 per cent of total isolates displayed an antagonism of (90-100) per cent against Rhizoctonia solani, Sclerotium rolfsii, Colletotrichum capsici, Ralstonia solanacearum respectively, and 20 isolates displayed more than 50 per cent mortality against Plutella xylostella. 26 isolates were selected for identification due to its functional significance through ARDRA. They were found to be members of Streptomyces, Microbispora, Nonomuraea and Amycolatopsis genera. Similarly, 16S rDNA sequence analysis of 11 extra potent isolates out of 26 isolates clustered them into Streptomyces genera. The REP PCR fingerprinting of whole genome of all these 26 isolates displayed 75 per cent identity among themselves. Supplementing to functional capability of these isolates 76 and 52 per cent total isolates were positive for NRPS and PKS I genes respectively. Cloning of partial sequence of PKS I gene of AUDT 248 exhibited an identity to the antibiotics, stambomycin gene cluster which was further supplemented with LC-MS analysis. Similarly, partial PKS I gene of AUDT 217 showed homology to the modular PKS of unknown biosynthetic identity.
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    ThesisItemOpen Access
    Analysis of cry Contents in Native Bacillus thuringiensis Isolates and Cloning of cry Gene
    (UAS, Dharwad, 2011) Johnson Luwang W.; Narayan Moger
    The present investigation was carried out to analyse cry gene content in 100 native isolates of B. thuringiensis randomly chosen from the Bacillus thuringiensis culture collection centre of the Department of Biotechnology, UAS Dharwad, and to clone cry gene based on variation in amplicon restriction fragment length polymorphism (ARFLP). From 100 native isolates tested for the presence of crystals, the most predominant crystal type was spherical (58%) followed by irregular (26%), bipyramidal (5%), rhomboidal (10%) and triangular (1%). Bioassay against third instar larvae of Plutella xylostella revealed 13 potent isolates. Two isolates DBT 2336 and DBT 2510 exhibited 100 per cent mortality at 72h of exposure whereas the reference strain HD1 exhibited 93.33 per cent mortality. SDS-PAGE profile revealed the presence of 135 kDa in 7 potent isolates. Most of the isolates screened for cry showed amplification with at least one cry type. The most predominant gene was cry1 (30%), followed by cry2 and cry6 (28% each), cry3 (23%), cry9 (18%), cry20 (17%), cry23 (16%), cry4 (11%) and cry8 (4%). None of the isolates amplified for cry10, cry34 and cry35. The variant full length cry1 gene from the isolates DBT 2510 was cloned into pTZ57R/T and labelled as pIJK101. The nucleotide sequence showed 99 per cent homology with the reference cry1Ac22 (EU282379.1). The 3-dimensional structure of variant Cry1 (pIJK101) toxin was deduced and validated with Ramachandran plot analysis with 94.3 per cent residues in the favoured region, 4.7 per cent in allowed region and 1 per cent in outlier region. Changes in the electrostatic potential distribution of the surface of Cry1 (pIJK101) toxin molecule were observed in relation to reference Cry1Ac22 toxin predominantly at domain III region.
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    ThesisItemOpen Access
    Generation and Characterization of Transformation Events in Tomato Carrying Sclerotium rolfsii Lectin Gene (srl1) for Root Knot Nematode and White Fly Resistance
    (UAS, Dharwad, 2011) Ashlesha C. Patil; Ramesh Bhat
    Transgenic tomato plants carrying Sclerotium rolfsii lectin gene (srl1) were generated by Agrobacterium-mediated transformation. Of the 90 independent regenerants, 40 could amplify 429 bp product with srl1-specific PCR. Segregation analysis in three selected events indicated single copy insertion in SRL1-T0(7) and multi-copy integration in SRL1-T0(10) and SRL1-T0(21). In SRL1-T0(7), the TDNA/ srl1 insertion was in a non-genic region between 85,375 and 85,376 bp of the clone C11_HBa0054I23 (GenBank Acc. No. AC212431.2) of chromosome 11. Crude extract of SRL1-T0(7), SRL1-T0(10) and SRL1-T0(21) showed an extra band of 17 kDa on SDS-PAGE. This protein along with their partially purified forms could agglutinate rabbit erythrocytes, indicating lectin activity. Specific activity of the lectin was more in the partially purified samples compared to protein from the crude extracts. Bioassay with second stage juveniles (J2s) of Meloidogyne incognita, the root knot nematode showed that on 3rd day of inoculation the infection rate was 66.66, 83.33, 91.66 percent in SRL1-T0(7), SRL1-T0(10), SRL1-T0(21), respectively compared to 100 per cent infection in the non-transgenic plant. On 6th day, the percentage of vermiform J2s developed into sausage-form was higher in nontransgenic plants compared to transgenic lines. After 70 days of inoculation, nontransgenic plants showed an average gall number of 59.6 per plant whereas, it was reduced to 33.6, 43.2 and 40.8 galls/plant, respectively in SRL1-T0(7), SRL1-T0(10), SRL1-T0(21). In general, non-transgenic plants showed a gall index of 7, hence they were rated as “highly susceptible”, whereas transgenic lines with a gall index of 3 were classified as “moderately resistant”. Nematode infection also resulted in significantly reduced average shoot and root length, and biomass in non-transgenic plants compared to transgenic plants. Transgenic plants were evaluated for white fly (Bemisia tabaci) mortality. It was 24%, 22% and 16% in SRL1-T0(7), SRL1-T0(10), SRL1-T0(21), respectively, which were higher than that found in non-transgenic plant (10%).
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    ThesisItemOpen Access
    Molecular Characterization of Mineral Phosphate Solubilization In Serratia marcescens AND Methylobacterium sp.
    (UAS, Dharwad, 2011) Archana Kumari; P.U. Krishnaraj
    The major goal of the present investigation was to study the mechanism of mineral phosphate solubilization in Serratia marcescens and Methylobacterium sp. All the 157 isolates of S. marcesens and 73 isolates of pink pigmented facultative methylotrophs were screened for their mineral phosphate solubilization phenotype on solid media viz., Modified Sperber’s and TCP agar media. Eight most potent isolates along with one reference strain Serratia marcescens ER2 were further screened for pH drop and Pi release in NBRIP-BPB and TCP broth. Both pH drops and Pi release were highly correlated upto 15 days. AUDS-151 and PPFM87 were the most potent ‘P’ solubilizing isolates. Gluconic acid was detected in the culture supernatants of these isolates through thin layer chromatographic technique. Effect of buffering and phosphate stress on MPS phenotype of these isolates were observed by external supply of tris buffer and K2HPO4 to the MSM agar medium. It indicates the metabolic control of mineral phosphate solubilization by external factors which either resist the change in pH or alters the gene expression. Tn5 mutants were generated from the most potent P solubilizing isolate AUDS-151 by biological (Tn5) mutagenesis. Mutants failed to show MPS activity even after 5 DAI. Strainal identity of AUDS-151 and PPFM-87 was confirmed as S. marcescens and Methylobacterium mesophilicum respectively through 16S rDNA sequencing. All the pqq operon gene (s) were amplified from AUDS-151 and PPFM-87 and the most conserved pqqE gene was cloned into E. coli DH5a from both the isolates. Both the clones pAMK101 and pASK101 were sequenced and showed 97 per cent homology with the Methylobacterium sp. (CPOOOO367) and 94 per cent homology with Serratia marcescens (DQ868536), respectively.
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    ThesisItemOpen Access
    Metagenomic Analysis of Forest and Farm Soils
    (UAS, Dharwad, 2011) Sachin A. More; P.U. Krishnaraj
    Metagenomics is an emerging field of science mainly aims at studying the activity of uncultured microorganisms through the nucleotide sequences. Standard culture techniques allow only 1% of microorganisms to grow in defined laboratory conditions. Thus, culture dependent study of microbial diversity unravels only part of the microbial populations present in the environment. The present study was done to analyze the soil microbial diversity between forest and farm soils through the metagenomic approach. The success of any metagenomic study depends on the method of DNA extraction. So considering the significance and need, method was optimized for extraction of microbial community DNA from forest and farm soils. Extracted DNA was purified with the aid of chemical flocculation using FeCl3 as chemical flocculent. Purity of DNA was confirmed by effective PCR amplification using 16S rDNA primers. The purified DNA from respective soil was PCR amplified using DGGE specific 16S rDNA primers. The amplified PCR products were then separated on DGGE gel to analyze fingerprinting pattern in each soil types. Soil microbial diversity analyses of fingerprints were done by using NTSYS software. DGGE analysis was shown that forest soil microbial community shares more similarity with organic farming soil microbial communities. The sequence based metagenomics approach was used to know the microbial community composition. This was done by constructing PCR based 16S rDNA library of metagenome of each soil using T/A cloning method. About 17 clones of each soil were randomly selected from respective 16S rDNA library and sequenced. Blast analyses of each clone form respective soil were done in RDP database to know the taxonomic affiliation of each clone. Further the processed 51 sequences were then submitted to NCBI database. Blast results obtained shown that majority of the microbial communities in forest and farm soil contained uncultured microorganisms.
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    ThesisItemOpen Access
    Studies on biochemical quality parameters of wheat as influenced by location
    (UAS, Dharwad, 2010) Shabana Nadaf; P.W.Basarkar
    Three different wheat varieties that are popular and recently released from each of the three species T. aestivum, T. durum and T. dicoccum grown during rabi 2007-2008 at MARS, Dharwad and ARS, Arabhavi were studied for biochemical parameters. Total carbohydrate content of T. aestivum varieties registered higher value at Dharwad, whereas T. durum varieties registered higher value at Arabhavi. Starch content of T. aestivum was comparable at both the locations. T. dicoccum varieties had higher nitrogen, crude protein and oil content at Dharwad location. T. durum varieties registered increase in -carotene content at Dharwad over Arabhavi. Soluble proteins were higher in T. dicoccum varieties at both the locations. Wet gluten content of T. durum varieties exhibited higher values at Arabhavi, whereas T. dicoccum contained higher phenol content at both the locations. Micronutrient content of T. durum varieties was high at Dharwad. DWR-1006 had good micronutrient content. Specific activities of hydrolytic enzymes, -amylase and acid invertase exhibited increasing trends in their activities up to 72 h of germination as compared to ungerminated seeds. The hydrolytic enzymes registered higher activities in germinated T. aestivum varieties at both the locations. Fractionation of HMW-GS and LMW-GS of protein carried out by SDS PAGE revealed different patterns. T. dicoccum and T. aestivum genotypes were more suitable for Dharwad environmental conditions, whereas T. durum varieties were suitable for Arabhavi.