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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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2000 - 200956
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Subject: Plant Biotechnology × Start date: 2000 × End date: 2009 ×
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Now showing 1 - 9 of 56
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    ThesisItemOpen Access
    GENETIC TRANSFORMATION FOR POD BORER RESISTANCE USING cry1A (b) IN PIGEONPEA [Cajanus cajan ( L ) Millsp.] cv. ICPL-8863 (MARUTI)
    (University of Agricultural Sciences, Dharwad, 2002) NISHANI, SANDHYARANI; BHAT, SUMANGALA
    A study was undertaken to standardize in vitro plant regeneration and Agrobacterium mediated transformation procedure for pigeonpea (Cajunus cajan) cv. ICPL-8863 (Maruti). For regeneration direct organogenesis was attempted using different explants viz., shoottip (ST), cotyledonary node (CN), half cotyledon with cotyledonary node (V2 CNC) and CNC. These were cultured on various levels of benzyl amino purine (BAP) (1, 2, 3, 4 mg W) and thidiazuron (TDZ) (0.01, 0.05, 0.1, 0.5 mg l1). CNC found to produce average of 1.69 shoots/explant and was better among all explants used. Among different levels of BAP and TDZ tried, BAP 2 mg l1 was found to be better for multiple shoot and shootbud induction. Shootbuds were cultured on MS with reduced levels of cytokinins and TDZ 0.05 mg l 1 gave better elongation compared to other levels, elongated shoots were rooted on MS with IBA (0.1-0.5 mg W). Among all the levels tried 0.2 mg l1 IBA gave good healthy roots. For transformation Agrobacterium strains EHA 105 harboring pBinBtl plasmid [cryl A{b)} and GV2260 harboring pCAMBIA1301 plasmid (gus) with nptll as selectable marker, which confers kanamycin resistance were used. Initially kanamycin sensitivity of control explants was tested at different growth stages. Inhibitory levels at different stages were used for selection of transformants. Precultivation of explants on MS with 2 mg l 1 BAP for two days prior to cocultivation resulted in increased survival. Explnats were cocultured for two days in dark and transferred to selection medium (with kanamycin and cefotaxime). Approximately 1.4 per cent shoots obtained were cry positive. In in planta approach plants were treated with Agrobacterium inoculum at different growth stages. Germinating seeds were injected with GV2260 strain and shoots were histochemically assayed and 0.9% of shoots were gus positive. In seedling dip and flower injection methods cryl A (b) gene was transferred and confirmed through PCR analysis (9/54, 11/26 plants were cry positive respectively). Thus efficient regeneration and transformation protocol has been standardized for pigeonpea cv. ICPL-8863 (Maruti).
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    ThesisItemOpen Access
    DEVELOPMENT AND CHARACTERIZATION OF SPORELESS MUTANTS OF LOCAL B.thuringiensis ISOLATES
    (University of Agricultural Science, Dharwad, 2000) Murugendra, S; Kuruvinashetty, M S
    "The focus of the present study was to isolate Bacillus turingiensis from soils of Western Ghat region in Uttar Kannada district of Karnataka, characterization of their insecticidal activity and development of sporeless mutants. Out of 32 Bacillus isolates, only eleven had endospores and crystals. Two isolates M3 and M7 were toxic to diamond back moth (Plutella xylostella) and Spodoptera litura causing 98.2 % and 97.4 % mortality, respectively. Isolates, M3 and M7 were subjected to MNNG mutagenesis for developing sporeless mutants. Sporeless mutants, Mut M3 and Mut M7 were obtained from M3 and M7 strains, respectively. Both mutants and wild type isolates were characterized for intrinsic antibiotic resistance, insecticidal activity, protein and plasmid profile. All the isolates including mutants were sensitive to tetracycline, chloramphenicol and kanamycin. Both the mutants were resistant to streptomycin but sensitive to nalidixic acid. On the other hand wild type strains were resistant to nalidixic acid, but sensitive to streptomycin. SDS-PAGE protein profile indicated the presence of Cry protein bands of more than 130 kda and 65 kda in M3, M7 and Mut M3. But Mut M7 had 67kda band The genetic distance (%) based on protein profile was 50.00 per cent between Mut M3 and M7 and the maximum of 86.667 per cent between Mut M3 and M3. All the isolates had a single plasmid of about 15kb. Toxicity of both wild type and mutant strain was tested against nematode. All of them were found to be toxic to nematode. However PCR with nematode specific primer did not yield any amplification product. Talc and starch based wettable powder (WP) formulations of mutants and wild type strains were tested against Spodoplera litura. Among talc and starch based formulations talc based formulation was found to be more effective and it was at par with the aqueous formulation of commercial preparation ( Dipel 8L)"
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    ThesisItemOpen Access
    MOLECULAR CHARACTERIZATION OF GENETIC MALE STERILE GENOTYPES IN COTTON (Gossypium Sp.)
    (UNIVERSITY OF AGRICULTURAL SCIENCES GKVK, BANGALORE, 2004) Mudaraddi, Bharati; Kiladi, B M
    "Cotton is the most important textile fibre and knowledge on genetic relatedness among the advanced lines is essential for crop improvement. The genetic diversity analysis of GMS genotypes through molecular markers will be useful in diversifying the genotypes for creation of hybrid combination. Application of molecular markers is an interesting alternative for selecting male sterile plants. Twenty near isogenic diploid GMS lines were screened using 119 random decamer primers of which 82 markers were found to polymorphic with 61.7 per cent polymorphism. Out of 314 amplicons amplified, 187 were found to be polymorphic, with an average of 3.83 fragments per primer of which 2.28 were polymorphic. The similarity among genotypes ranged from 70 to 98 per cent. Genetic diversity analysis among fourteen near isogenic tetraploid GMS lines was carried out using 88 random decamer primers. Out of 310 fragments amplified, 190 were found to be polymorphic with an average of 5.44 fragments per primer, 3.33 fragments per primer were polymorphic. OPY primers were found to be highly polymorphic. Presence of genetic diversity was evidenced by genetic similarity indices (0.76 - 0.98). The sterile and fertile plants of different genotypes made independent clusters indicating their divergence. RAPD markers are used as a tool for estimating genetic diversity and can be used on continuing basis to document the available variability in the cotton germplasm. Since G. hirsutum and G.arboreum groups have been improved independently, these form separate clusters, depicting enormous variation among them despite having same genome. Markers OPB04 and 0PZ14 showed genetic diversity between fertile and sterile plants of all the diploid GMS genotypes. Similarly the marker OPB04 also showed genetic diversity within fertile and sterile plants of the tetraploid GMS genotypes. Hence, they can be considered as putative markers for linkage studies and identification of male sterile and fertile plants."
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    ThesisItemOpen Access
    CLONING OF ENDOCHITINASE AND ENDOGLUCANASE GENES FROM NATIVE ISOLATES Trichoderma
    (University of Agricultural Sciences Dharwad, 2009-06) MANJUNATH SWAMY J. K.; Dr. SUMANGALA BHAT
    Eighty six Trichoderma isolates previously isolated and maintained in the Department of Biotechnology, were screened against Sclerotium rolfsii, Rhizoctonia solani and Colletotrichum capsici through dual plate assay. Based on growth inhibition, they were grouped into efficient, moderate and poor isolates. Further, endochtinase (ech42) and endoglucanase (, 1-6 endoglucanase) genes were cloned from efficient, moderate and poor isolates of T. harzianum using specific primers. Differences were not observed in the amino acid sequences of ech42 cloned from efficient, moderate and poor isolates of same species (T. harzianum). However, ech42 cloned from most efficient isolates of T. virens showed differences at amino acid level in 15 positions compared to ech42 cloned from T. harzianum. Further, the , 1-6 glucanase gene cloned from efficient, moderate and poor isolates showed differences at amino acid level. The gene cloned from efficient (IABT 1041) T. harzianum isolates differed from other two cloned from moderate and poor isolates (IABT 1046 and IABT 1054) in 10 amino acid positions and the , 1-6 glucanase cloned from IABT 1046 and IABT 1054 differed at 2 amino acid positions. The cloned genes can be further subjected for expression and bioassay studies to know their utility in development of transgenic plants.
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    ThesisItemOpen Access
    Genome-wide molecular mapping, introgression of stable QTLs and expressional quantitation of transcription factor genes in charcoal rot manifestation in sorghum bicolour (L) Moench
    (UAS, Dharwad, 2009) Ayyanagouda.M.Patil; B.Fakrudin
    Recombinant inbred lines derived from the cross IS22380 (susceptible) and E36-1 (resistant) were characterized for the morphological and biochemical components of charcoal rot resistance and yield related traits at three locations over four rabi seasons revealed significant differences among RILs and GxE interactions. A significant association among relevant traits was noticed at phenotypic and genotypic level with high heritability estimates. A total of 141 polymorphic DNA markers (48/275 SSRs, 65/533 EST-SSRs, 28/410 RAPD) were genotyped to construct a genetic linkage map spanning 2905 cM and used for Composite Interval Mapping of QTLs. Stable QTLs were detected lodging percent (xtxp176– xtxp312 (CR1) number of internodes crossed by the fungus (xtxp297-xiabt273 (CR2) and length of infection (xtxp275-xtxp241(CR3): the additive effect at all these loci was contributed by E36-1. Two major QTLs of plant height and three QTLs of plant yield accounted for 38% and 41% phenotypic variance respectively. On LG-I, the genomic region flanked by xtxp274–xiabt29 harbored a common QTL. Three stable QTLs, CR1, CR2 and CR3 collectively contributing 43% of phenotypic variation were introgressed into M35-1 and SPV86 backgrounds: at BC1F1 with 2285 and 2033 marker and at BC2F1 5989 and 6002 marker points in M35-1 and SPV86 backgrounds were screened to identify progenies harboring all the three QTLs. Expressional quantitation of 185 transcription factor genes was done in charcoal rot challenged and control tissue of E36-1 and SPV86 genotypes at 75 DAS and 90 DAS where 142 genes up regulated in both genotypes while eight genes of five family viz., AP2 (PTSb00019.1, PTSb00022.1, PTSb00024.1 WRKY (PTSb00331.1), HMG (PTSb00179.1, PTSb00185.1) ARF (PTSb00033.1) and bHLH (PTSb00349.1) were up regulated in pathogen challenged tissues. Two genes of WRKY family (PTSb00328.1 and PTSb00331.1) were significantly up regulated in resistant genotype alone: these understandings would help in developing strategies for resistance breeding in sorghum.
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    ThesisItemOpen Access
    Production of single chain fragment variable (ScFv) monoclonal antibodiy for Sclerotium rolfsii lectin using phage display technology
    (UAS, Dharwad, 2009) Satish. Kumar.Verma; Narayana Moger
    Sclerotium rolfsii is an omnivorous, soil borne fungal pathogen which causes disease on a wide range of agricultural and horticultural crops. About 500 species in 100 families are susceptible. It produces sclerotial bodies which are main source of primary inocolum for disease development. The lectin is one among those well known characterized protein from S. rolfsii and plays an important role in growth, germination and disease development of fungal. There are several methods for the detection of fungi. The conventional methods to detect S. rolfsii mainly depend on symptoms, biochemical or morphological identification which is inaccurate and unreliable because they require skilled taxonomical expertise for diagnosis. Molecular methods such as polymerase chain reaction (PCR) and immunological assay can be used to detect plant pathogens. The phage display techniques is rapid, simple and economic for the production of antibodies without sacrificing animals. Four rounds of biopanning was done using scFv library with SR lectin. The fourth round of panning showed higher binding specificity to SR lectin. These were subsequently used for production of scFV monoclone for SR lectin. Finally, the randomly selected scFv clones were screened with ELISA. ELISA reading showed that the two clones (pscFcSRL-20 and pscFvSRL-34) having higher binding affinity to SR lectin. These clones were tested with isolates obtained from different crops (groundnut, sesamum and potato). The clone pscFvSRL-20 shows higher binding specificity with all the isolates. Clone pscFvSRL-20 was cloned in to pTZ57R/T vector and sequenced. The sequence results indicated the presence of 923bp which code for 242 amino acids. BLAST results showed 100 per cent homology to Homo sapiens IGK mRNA for the immunoglobulin kappa light chain VLJ region.
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    ThesisItemOpen Access
    Co - transformation of Tomato with ech42 and bgn and construction of plant transformation vector carrying Both ech42 and bgn
    (UAS, Dharwad, 2009) Gaurav Sharma; Sumangala Bhat
    The present investigation was carried out to construct plant transformation vectors with ech42 ad bgn cloned from Trichoderma species and to transfer them to tomato to have better resistance to fungal diseases. A plant transformation vector (pGS1) carrying previously cloned b-1, 6 endoglucanase (T. virens) was constructed and transferred to Agrobacterium tumefaciens strain LBA 4404 carrying pGS1 has hygromycin resistance (hpt) gene as a selectable marker. This Agrobacterium clone was used with another Agrobacterium clone carrying pHS 100 (has kanamycin resistance gene as a selectable marker) with endochitinase (ech42) gene from T. virens for co-transformation of cotyledonary explants of tomato cv. Pusa Ruby. Putative transformants were selectable on MS medium with kanamycin (100 mg/ml) and hygromycin (6 mg/ml). Only 6 per cent of the survived plants were positive for ech42 and none of them were positive for bgn. Also attempts were made to construct plant transformation vector pRAGS carrying both ech42 and bgn under single T-DNA region. Initially, HindIII site at 5' end of earlier cloned bgn (T. harzianum) was removed using primers during reamplification of the gene. The amplicons were cloned into pTZ57R/T containing T overhangs at Eco321 site and transferred to E. coli DH5a and further to plant transformation vector pBI121 which was named as pRA121. In order to clone another gene (ech42) into pRA121, expression cassette from iHP vector was transferred to pRA121 and named as pRAG121. Further in order to gain XhoI site for cloning ech42 gene into pRAG121, ech42 (pSUM1) was cloned into pYES2/CT, named as pSAG1, ech42 from pSAG1 cloned with KpnI and XhoI in pRAG121 and named as pRAGS121. The vector constructed in the present study can be used to transform important crop plants to have enhanced resistance to fungal diseases since the genes encoding chitinase and glucanase act synergistically.
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    ThesisItemOpen Access
    Cloninig and expression of cry1, cry2 and cry9 genes from native Bacillus thuringiensis isolates
    (UAS, Dharwad, 2009) Mohd Safuddin; P.U.Krishnaraj
    The present study was conducted to clone cry1, cry2 and cry9 genes from native Bacillus thuringeinsis isolates of BR-Hill region and to study the expression of genes in E. coli BL21(DE3)pLysS and acrystelliferous B. thuringeinsis (IPS78/11). Using specific primers of cry1, cry2 and cry9 gene encoding insecticidal activity from Bacillus.thuringeinsis were cloned into cloning vector pTZ57R/T. The clones were confirmed through PCR and restriction analysis. The clones were sequenced and analyzed for the homology at the nucleotide and at the protein level for domain study. The complete sequence of cloned cry1 and cry2 showed 98% homology with the sequences belonging to same group in NCBI database whereas cloned cry9 showed 97% homology. Comparison of the deduced amino acid sequence of deduced Cry1 showed a maximum of 98% homology with known Cry1Ab toxins. Similarly, two and three per cent variability observed in the deduced amino acid sequence of Cry2 and Cry9 respectively suggest that the cloned genes are different. cry1, cry2 and cry9 genes cloned from different isolates were expressed in E. coli BL21(DE3)pLysS using pET43.1 vector and in acrystelliferous B. thuringeinsis (IPS78/11) using pSUP27/A vector. Further the bioassay activity of the recombinant clone using total cell lysate of E. coli BL21(DE3)pLysS containing cry gene were fed to DBM larvae and the per cent mortality was observed between 43-53 per cent. However, the with crude protein from acrystalliferous B. thuringiensis containing cry1, cry2 and cry9, the mortality ranged from 56-73 per cent suggesting the intact biological activity of the expressed Cry protein.
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    ThesisItemOpen Access
    Functional analysis of synthetic promoters and construction of expression cassettes with xynA
    (UAS, Dharwad, 2009) Sharanabasappa.Yeri; Ramesh Bhat
    Four synthetic promoters containing multiple copies of cis-elements (W2X, GCC2X, GCC3X and S2X) with -46 region of CaMV 35S promoter as the minimal promoter were analyzed for their inducibility upon exposure to salicylic acid (SA), methyl jasmonate (MeJ) and Erysiphe cichoracearum, which causes tobacco powdery mildew. These promoters cloned into a promoter-probe vector (pRR21) with SgfpS65T reporter gene were used for tobacco transformation. Of the totally obtained 39, 20, 22, 13 and 37 putative transgenic tobacco plants containing W2X, GCC2X, GCC3X, S2X and CaMV 35S promoters, respectively, 22, 9, 13, 8 and 20 were confirmed positive by PCR. Plants sprayed with 5 mM concentration of SA or MeJ showed SgfpS65T after 5 min, 1, 2, 3 and 7 hr. GCC2X and S2X were induced by both SA and MeJ, W2X was induced by SA, but not by MeJ. However, GCC3X promoter did not show any induction. With SA, the strength of induction of W2X and GCC2X promoters as measured by ImageJ software were comparable and relatively higher than S2X. Similarly, GCC2X and S2X had similar magnitude of induction with MeJ. In general, the synthetic promoters were more strongly induced by SA compared to MeJ, and their strength was less compared to that of CaMV 35S promoter. Though, plants carrying the synthetic promoters when infected with E. cichoracearum showed a compatible reaction, SgfpS65T expression was not observed even after 24 hr. A new promoter-probe vector (pYR74) with xynA reporter gene and rbcs T terminator was constructed in a binary vector (pCAMBIA1305.1) background. Two synthetic promoters (W2X and GCC2X) were cloned into the multiple cloning site of pYR74 to get pYR75 and pYR76, respectively. These constructs would help in determining promoter strength using xylanase reporter.