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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 CLONING OF ENDOCHITINASE AND ENDOGLUCANASE GENES FROM NATIVE ISOLATES Trichoderma(University of Agricultural Sciences Dharwad, 2009-06) MANJUNATH SWAMY J. K.; Dr. SUMANGALA BHATEighty 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.ThesisItem Open Access Analysis of Transgenic Tomato Carrying Remusatia vivipara Lectin Gene (rvl1)(UAS, Dharwad, 2012) Rohini Mahaling Kolekar; Ramesh S. BhatTransgenic 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.ThesisItem Open Access Cloning of ech33 and ech36 From Trichoderma spp. and Expression in Saccharomyces cerevisiae (INVSc1)(UAS, Dharwad, 2011) Avishek Chatterjee; Sumangala BhatThe 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%).ThesisItem Open Access Characterization of Antifungal Activity and Molecular Diversity of Trichoderma Isolates(UAS, Dharwad, 2011) Sagar Patil; Sumangala BhatEighty 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.ThesisItem Open Access Functional and Molecular Characterization of Native Isolates of Actinomycetes From Soil and Endorhizosphere of Medicinal Plants(UAS, Dharwad, 2011) Gayatree Mohapatra; Narayan MogerMicrobial 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.