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Subject: Biotechnology × End date: 2003 × Start date: 2003 × Type: Thesis × Thesis Type: M.Sc ×
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    ThesisItemOpen Access
    GENETIC ANALYSIS OF WILT RESISTANCE AND IDENTIFICATION OF LINKED DNA MARKERS IN pigeon pea [Cajanus cajan (L.) Millsp.]
    (University of Agricultural Sciences, Dharwad, 2003) H, KOTRESH; KURUVINASHETTI, M S
    ABSTRACT NOT AVAILABLE
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    ThesisItemOpen Access
    CLONING AND EXPRESSION OF crylA(b) GENE AND FIELD EVALUATION OF NATIVE Bacillus thuringiensis (Berliner) ISOLATES
    (University of Agricultural Science, Dharwad, 2003) Balegar, Sanjeev S; Kuruvinashetty, M S
    ABSTRACT NOT AVAILABLE
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    ThesisItemOpen Access
    GENETIC TRANSFORMATION FOR FRUIT BORER RESISTANCE USING cry1Ab GENE IN TOMATO (Lycopersicum esculentum MILL.)
    (University of Agricultural Science, Dharwad, 2003) Paramesh, H; Fakrudin, B
    Genotypic response for in vitro callus induction and regeneration was studied in six-tomato genotypes viz., Pusaruby, Megha (LI5), PKMl, Dharwad local, SI40 and SI42. Different concentrations and combinations of 2,4-D, kinetin and lAA were used to induce callus and plantlet regeneration. Highly significant differences among genotypes and hormonal concentrations were noticed for callus induction and regeneration. A 2,4-D concentration of 2 mg/1 was found to induce highest frequency of good callus among genotypes. Different calli quality parameters were significantly influenced by the genotype, explant and 2,4-D concentration in the medium. Highest regeneration frequency from callus was obtained in MS basal supplemented with 3 mg/1 of kinetin and 0.3 mg/I of lAA. Megha found to be highly responsive for regeneration through callus. However, kinetin @ 5mg/l induced highest frequency of direct plantlet regeneration in cotyledonary leaf and hypocotyl explants which responded differently across genotypes. Sufficient and healthy i/i vitro root induction was achieved on MS basal supplemented with NAA (0.1 mg/1) and lAA (0.3rag/l). A plantlet survival of more than 95% was recorded during hardening process. Agrobacterium EHA 105 carrying pCAMBIA 1301 with uidA (GUS) and hygromycin selectable gene and pBinBtl with cryl Ab along with nptll gene, both driven by CaMV 35S promoter were used for transformation. Different parameters of transformation process were optimized using i« gene. Cefotaxime @ 300 mg/1 was used to eliminate Agrobacterium. A repeatable average transformation frequency of 2.32% was recorded in Megha. PCR assay using nptll specific primers and insect bioassay studies confirmed the transgenic status of Megha. RT-PCR analysis indicated the expression of transgene in tomato leaves.
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    ThesisItemOpen Access
    MOLECULAR CHARACTERIZATION OF INSECTICIDAL GENES IN NATIVE Bacillus thuringiensis ISOLATES
    (University of Agricultural Science, Dharwad, 2003) Athani, Vasantkumar V; Krishnaraj, P U
    The present study was conducted to assess the molecular diversity and to characterize the cry gene profile of native Bacillus thuringiensis isolates. Nine isolates of B. thuringiensis and three reference strains were characterized by PCR-product profiles using random primers obtained from Operon Technologies, USA. The nine primers used for assessing the molecular diversity among the isolates generated 169 bands, all polymorphic (100 % polymorphism). Among the nine primers, the primer OPC-18 had the lowest minimum (0.00) and least average (0.308) genetic similarity values. Hence, it can be used to distinguish the isolates and fingerprint the B. thuringiensis isolates used in this study. The isolates were also characterized by PCR-product profiles using cry specific primers to understand the distribution of cry genes in the isolates. cry\ and cryl were found in HDl, PI, Dl, PPIO, PPl 1 and PP12; cry% and cry9B were present in HDl, Dl and PP12; crv9A was found in Bt42, T2 and PP14; cry9C in HDl, PI, Dl, T2 and PP12; cryW in PP14 and SB. The nem and cyt genes were found in T2 and SI3 respectively. PCR amplification with crylA(c) specific primers revealed the presence of the gene in HD, PI, Dl, PPIO, PPll and PP12. The isolates PI and Dl had two distinct plasmid bands of size 18.3kb and 12.8kb and the reference strain HDl had seven distinct plasmid bands of size 25.33, 17.84, 11.57, 7.08, 4.91, 3.5 and 1.506kb. Plasmid DNA library of isolate PI was generated in pSK+ vector. Of the 40 clones PCR tested with a pair of cryl general primers, 26 showed the presence of cryl gene
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    ThesisItemOpen Access
    MOLECULAR CHARACTERIZATION OF cry1A(a) IN NATIVE Bacillus thuringiensis
    (University of Agricultural Science, Dharwad, 2003) Patwari, Shankar; Krishnaraj, P U
    "The focus of the present study was to characterize native Bacillus thuringiensis isolates for cry!A (a) through bioassay, specific PCR for crylA(a) , plasmid profile analysis and to clone the c rylA (a) specific amplicon (1.2 kb) into the pTZ57R/T vector having T overhangs. Among the ten isolates (PI, 513, D1, D21, PP9, PP7, PP6, HD1, HD73, Bt42) which were used in the study, isolates D1 (LC^o 1124.27 ppm), PP9 (LC^^ 1283.76 ppm) and PI (LC^^ 1541.22 ppm) were found to have least LCg^ value for Spodoptera litura and hence can be used for direct field applications. Among the isolates which are characterised for presence or absence of cry1A (a) through specific PCR, only HD1, D1, PP9, SI3 and PI were found positive for c r y lA (a). Isolates HD1, D1, HD73, PP9, PPG, SI3, PI and PP10 showed plasmid bands varying in number (1-10) and size (1.5 kb - > 29 kb). The c ry lA (a) specific amplicon (1.2 kg) from B. thuringiensis PP9 plasmid DNA was cloned into linear pTZ57R/T with T overhangs. The recombinant clone pSP500 was positive for specific amplification of c ry lA (a), which was further confirmed by the restriction of recombinant pSP500 w ith EcoRI and Psti that released insert of size 1.2 kb. However, SDS-PAGE studies and bioassay studies showed that there was no expression of recombinant fusion protein. The sequence data of pSP500 does not match with already established c r y lA (a) sequence, but it is found to have 93 per cent homology with B. thuringiensis serovar israelensis pGILOl and 90 per cent homology with B. cereus ATCC 14579 plasmid pBC lin 15. The functions of pGILOl and pBC Iin15 is not known."
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    ThesisItemOpen Access
    CLONING AND EXPRESSION OF crylA(c) GENE FROM NATIVE Bacillus thuringiensis (Berliner) ISOLATES
    (University of Agricultural Science, Dharwad, 2003) Biradar, Mangala S; Krishnaraj, P U
    The focus of the present study was to clone the full length and truncated CrylA(c) gene from native Bacillus thuringiensis isolates. Bioassay of certain native isolates of B. thuringiensis along with reference strain HD-1 showed that a native isolate, D-1 showed the maximum mortality of 90.6 per cent at 72 hours. Among the 21 isolates tested for presence of CrylK gene D-1, PP-6, PP-9 possessed CrylA (a), CrylA (b), CrylAic). PP-7, SI-3, D-21 did not possess any of these genes. Primers were designed to pickup full-length 3.5Kb Crv'IA(c) and truncated 2Kb Cry IA(c) gene, using the conserved regions of Cry gene. The amplified products using these p rimers were c loned into pTZ57R at EcoZll s ite containing T- o verhangs through T/A cloning strategy. Of the several clones obtained, two clones pKM1308 and pKM1508 harbouring 3.5Kb and 2Kb respectively were analysed further. The presence of insert in the constructs has been confirmed by restriction analysis, PCR amplication and plasmid preparation. Restriction of pKM1308 recombinant clone with BamUl resulted in production of two fragments one of 3500bp corresponding to full length CrvlA(c) and another 2868bp corresponding to vector. The restriction of 2.0kb recombinant with BamUl yielded a single band of 4868 bp indicating the presence of the insert. The recombinants pKM1308 and pKM1508 were sequenced. The obtained sequences v/ere used for nucleotide-nucleotide BLASTn search to know homology with different B thuringiensis genes. The gene fragments showed 98% homology to many of the Cry genes including c/7lA(c), crylA{a), cryIA(b) and cryllS . Blast search also revealed that the 3.5Kb was cloned in reverse orientation in pKM1308 and the 2.0 Kb insert was in correct orientation in pKM1508.
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    ThesisItemOpen Access
    STUDIES ON IN VITRO AND IN PLANTA TRANSFORMATION PIGEONPEA (Cajanus cajan L.) cv. ICPL-8863 (Maruti)
    (University of Agricultural Science, Dharwad, 2003) Basavanna, S; Bhat, Sumangala
    In vitro and in planta transformation of pigeonpea was achieved using Agrobacterium tumefaciens strain EHA105 harboring pBinBtS plasmid having crylAc gene linked to the cauliflower mosaic virus (CaMV) 35 S promoter and neomycin phosphotransferase (npt II) gene under the control of nopaline synthase (nos) promoter and terminator. Initially, protocol was developed for in vitro multiple shoot regeneration. Using this protocol in vitro transformation were attempted. Cotyledonary node with cotyledon were pre-cultured on MS medium with 2 mg 1-' BAP for two days, co-cultured for two days in dark, washed with MS broth containing cefatoxime transferred to shoot induction (MS + 2 mg 1' BAP) followed by elongation (MS + 0.1 mg BAP) and rooting medium (MS + 0.5 mg 1-' IBA). Rooted shoots were established in greenhouse. This protocol for pigeonpea transformation using crylAc gene was repeated 8 times through all the steps. In one batch 2 per cent of established plants showed PCR amplification for npt 7/and crylAc primer. In in planta approach, different methods such as simple dipping injury followed by dipping, injection and vaccum infiltration were used at different developmental stages like germination, seedling and at flowering stage. Among different methods vaccum infiltration of the germinating seeds and flower injection were found to be suitable for transformation.