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ThesisItem Open 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, SUMANGALAA 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).ThesisItem Open Access MOLECULAR CHARACTERIZATION OF NATIVE Bacillus thuringiensis(University of Agricultural Sciences, Dharwad, 1999) YARADONI, SHRINIVAS N; KRISHNARAJ, P UABSTRACT NOT AVAILABLEThesisItem Open 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)"ThesisItem Open 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."ThesisItem Open Access Testing promoter trapping activity of a new vector (pNU435) in tomato(UAS, Dharwad, 2008) S.S.Biradar; Ramesh BhatThis study aimed at transforming PUSA-RUBY, a cultivar of tomato with a new promoter trapping vector pNU435 using Agrobacterium-mediated transformation. The Agrobacterium strain LBA4404 carrying pNU435 when co-cultivated with tomato leaves, sixteen plantlets (T1) were obtained of which 6 were found to be PCR positive and among them two PCR positive plants (PT4 and PT5) upon RB TAIL-PCR, produced amplicon of ~400bp which were cloned into pTZ57R/T by T/A cloning and sequence of the RB TAIL-PCR product with M13 F/R primers upon BLAST search showed homology to right border of T-DNA. Progenies of PCR confirmed T1 plants were found to be resistant to Basta at 10ppm of glufosinate ammonium. Progenies of PT4 and PT5 were gus-specific PCR positive and LB TAIL-PCR for T2 plants from these two T1 plants (PT4 and PT5) produced amplicon (~400bp), which were directly sequenced with primer, RB57_LBTAIL3. The flanking sequences from two plants were found to be one and the same, indicating that those plants resulted from the same transformation event and the BLAST search of these sequences against the tomato genome showed multiple hits, indicating that the query sequence has many homologous regions in the genome. A close search of the BLAST result showed that none of the homologous regions were genic, instead matched to retrotransposons like Tork-1 and Jinling-2. Since the flanking sequence showed homology to a genome-wide repeat of the non-genic region, possibility of promoter trap could be very remote. This study demands the need for generating and testing large number of independent transgenic events for promoter trapping. At the same time, these lines can be tested for promoter trapping under special conditions such as biotic and abiotic stresses.ThesisItem Open Access Molecular cloning and expression of lectin gene (srl) from sclerotium rolfsii sacc.(UAS, Dharwad, 2007) T.M.Chandrashekar; Ramesh BhatIn the present study, an effort was made to clone srl gene encoding Sclerotium rolfsii lectin, and express it in Escherichia coli and Saccharomyces cerevisiae. The presence of lectin in sclerotial bodies was confirmed by haemagglutination assay. Hapten inhibition assay indicated that it had sugar specificity for Mucin and Asialofetuin. A PCR product of ~450bp amplified from S. rolfsii DNA using degenerate primers was cloned into pTZ57R/T. The sequence showed an open reading frame (ORF) of 426bp, encoding 142 amino acids. BLASTp with deduced protein of srl (DP-srl) showed high homology with SRL, ABL (Agaricus bisporus lectin) and XCL (Xerocomus chrysenteron) confirming that it is a fungal lectin. DP-srl showed a maximum of 75% identity and 89% similarity with the SRL (Acc. No. 2OFC_A). Structural comparison between deduced protein of srl (DP-srl) and SRL at primary (Tyr27, Ala28, Ser47, Gly48, His70, Asn71, Tyr72, Arg105) and secondary (Asp77, Ile78, Thr80, Arg101, Tyr112, Val114) carbohydrate-binding sites showed no difference for primary structure. Of the 35 mismatches between DP-srl and SRL, 7 were conservative and 6 were ambiguous substitutions. Amino acid sequence alignment of related fungal lectins identified two conserved regions (Ser47 to Gly51 and Gly68 to Lys73). DP-srl had higher similarity (73%) with XCL than SRL (72%). Coding sequence of srl gene was cloned into pET-32b(+), the resulting vector (pCR29) was transferred to BL21(DE3)pLysS. Similarly, pCR14 yeast expression vector containing the SRL coding region in pYES2/CT and transferred to S. cerevisiae strain, INVSc1. Quantity of heterologous protein produced in E. coli and yeast was 7.50 and 0.78μg/μl respectively. SDS-PAGE analysis of the purified heterologous proteins from pCR29 and pCR14 showed protein bands of corresponding sizes. Hemagglutination assay and hepten inhibition assay confirmed that the expressed protein is Sclerotium rolfsii lectinThesisItem Open Access Expression and antifungal activity of trichoderma virens ech42 in tobacco(UAS, Dharwad, 2007) B.M.Murali; Sumangala BhatEndochitinase is a principal fungal phytopathogen cell wall chitin degrading enzyme utilized to develop transgenic crops resistance to diseases. The full length cDNA coding for endochitinase gene (ech42) was cloned from Trichoderma virens total RNA isolated from induced fungal mycelium. The amplicon obtained through RT-PCR using gene specific primers was cloned into pTZ57R/T vector and confirmed through PCR amplification, restriction analysis, and sequencing. Analysis of sequence has shown 99 per cent homology with the reported endochitinase gene at nucleotide and protein levels. Further, the endochitinase gene sequence was in silico modified and artificially synthesized to overcome codon bias and other undesirable regulatory coding sequences for its improved expression in tobacco. The purified endochitinase was obtained by expressing the modified gene in Escherichia coli with His-tag fusion sequence facilitating Nickel agarose column chromatography. The transgenic tobacco plants with endochitinase gene (ech42g) were analyzed for its expression. Transgenic plants were PCR screened with marker (nptII) and endochitinase gene specific primers. Expression of ech42g at transcription level was confirmed through RT-PCR. Chitinase activity was analyzed through glycol chitin plate assay and reducing sugar estimation across three different growth stages. All the tested transgenic plants showed higher level of chitinase activity compared to untransformed control tobacco plants, and the variation was observed among the progenies of different transgenic lines. Transgenic expression of ech42g tobacco showed resistance against foliar pathogen Alternaria spp. causing leaf spot and soil borne pathogen Sclerotium rolfsii causing root rot.ThesisItem Open Access Comparative structural analysis and allele mining of dreb2 transcription factor in sorghum [sorghum biocolor (L.) moench](UAS, Dharwad, 2007) Shivanand Lathe Rahul; M.S.Kuruvinashettygenetic diversity. Identification of novel regulatory genes involved in abiotic stress tolerance and allele mining would lead to better and quicker solution for improving stress tolerance in crop plants. Structural and functional analysis of genomic DNA of DREB2 transcription factor including allele mining was done in sorghum. Sorghum DREB2 (SbDREB2) genomic clone was found to be 1591 bp long with an internal intron of 747 bp between 68 and 815 bp. SbDREB2 encodes a putative protein of 262 amino acids with a predicted molecular mass of 28.88 KDa and has potential four casein kinase II phosphorylation sites, five protein kinase C phosphorylation sites and one tyrosine kinase phosphorylation site. Multiple sequence alignment of SbDREB2 with other DREB genes revealed presence of single, highly conserved, functional 58 amino acids long AP2/ERF domain. Its features show that it could act as a phospoprotein. The comparative modeling of AP2/ERF domain revealed a 3 stranded -sheet and an -helix antiparellel to -sheet. The 14th valine and 19th glutamic acid positioned on -sheet were conserved within AP2/ERF domain indicating their critical role in DNA binding. Phylogenetically SbDREB2 is closely related to DREBs of monocotyledonous. The CaMV35SThesisItem Open Access Molecular characterization of cry/vip genes and efficacy of native bacillus thuringiensis isolates(UAS, Dharwad, 2007) Bisveswara Prasad Yadav; M.S.KuruvinashettyBacillus thuringiensis, a widely used biocontrol agent against insect pests, produces crystal proteins, which are toxic to many insects. 406 isolates were freshly made from soils of North Eastern hill region of Sikkim and Tripura. The predominant crystal type was spherical (41%) in Sikkim isolates, whereas in Tamil Nadu and Tripura isolates, bipyramidal (88.6%) and irregular (50%) types, respectively, were predominant. The presence of different cry/vip genes was determined in 106 isolates. One or more cry genes were detected in every isolate. Prevalent genes were cry2 in Sikkim (56.66%) and Tripura (59.37%) and cry4 in Tamil Nadu isolates (77.27%). Among vip genes; vip3A was most frequent (24.52%). Bioassay against Plutella xylostella, Crocidolomia binotalis, Spodoptera litura, Tribolium castenium and Atholia lugens proxima was done and none of the Sikkim and Tripura isolates were better than reference strain HD1 against 3rd instar larvae of P. xylostella. Many Tamil Nadu isolates were comparable to HD1 causing complete mortality. Against C. binotalis 3rd instar larvae, four Sikkim isolates were similar to the reference strain HD1 with 90 per cent mortality. But, three Tamil Nadu isolates causing 100 per cent mortality were superior. Against, T. castenium, S. litura and A. lugens proxima none of the isolates was effective. All the isolates, which were effective against DBM and cabbage leaf webber (>80% mortality) consisted of at least one known lepidopteran specific cry/vip gene, except TX 136 which had cry4 and cry28. cry10 specific primer gave a larger (615 bp) than expected (404 bp) amplicon in reference strain 4Q1. Cloning and sequencing showed that it is 98 per cent homologous to another reported cry10 partial cds (EF182766.1). Isolation, crystal identification and molecular characterization of cry/vip genes of different B. thuringiensis strains and their insecticidal bioassay is necessary to detect both known and novel cry genes.