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ThesisItem Open Access BIOCHEMICAL STUDIES ON RESISTANCE TO Fusarium WILT IN PIGEONPEA (Cajanus cajan (L.) Millisp.) SEEDLINGS(University of Agricultural Sciences, Dharwad, 2000) SRINIVAS, H; THIMMAIAH, S KABSTRACT NOT AVAILABLEThesisItem Open Access Candidate Gene Prediction in Qtl Regions and Expression Analysis in Field Evaluated Near Isogenic Lines (NILs) Carrying Stay-Green and Water use Efficiency Qty Combination in Three Recurrent Background of Rabi Sorghum(UAS, Dharwad, 2013) Gaurav N. Chaudhari; B.FakrudinStay-green (stg) and water use efficiency (wue) are important target traits considered for improving drought tolerance in sorghum. The zygosity of introgressed quantitative trait loci (QTLs) of stg and wue was determined in three recurrent backgrounds of NILs derived from SPV86, SPV570 and M35-1. The NILs were field evaluated to assess impact stg and wue QTLs. Significant positive correlations were observed between yield and stay-greeness of different stages in SPV 570 background indicating NILs possessing stg trait have significant yield advantage under post-anthesis drought stress condition. Three different ab initio gene prediction algorithms viz., FGENESH, GENSCAN, GENMARK indicated number of predicted genes anchored within targeted QTLs. Five genes (NSP, NAD, PHD, MADS, MLO) for stay-green QTL qSTG1A (1.82 Mbp), ten (IAA, SORBIDRAFT, CYP450, GAG/POL, PK, GENE X, UGTS, MTC, AGP16, VP25) for qSTG2 (2.54 Mbp) and one (SF CC1) for qSTG3 (2.18 Mbp) were predicted on sorghum chromosome No. 3 and 1, qCID2 (2.33 Mb) on chromosome 10. Based on the predicted features and functions of candidate genes, total of 24 predicted genes from different transcription factor families like MADS, PHD, NAD, EIF-4A and SPLICING FACTOR, CYP450, IAA were tested for their expressional analysis through quantitative real-time PCR in 15, 30 and 45DAF leaf tissue samples from introgressed line with M35-1 genomic background. Among all tested samples most of the candidate genes were found to be up regulation with fold change of 3 to 6 and 6 to 10 folds. Senescence related gene DIN1 was down regulated in M-35-1 introgressed lines. Result indicated the potential use of stg and wue QTL pyramided lines for improving sorghum performance under drought stress.ThesisItem Open Access Studies on Differentially Expressed Genes for Fibre Development in Diploid Cotton (Gossypium arboretum L.)(UAS, Dharwad, 2013) Atul Suresh Hande; I. S. KatageriA global gene expression profiling study at initiation and elongation stages of fibre development in Gossypium arboreum L. was undertaken to identify key genes using Fuzzylinted and Fuzzy-lintless lines. Scanning electron microscopy revealed no difference in fibre initials but showed difference at elongation stage. Overall 278 transcripts were differentially expressed during fibre initiation and elongation stage in fuzzy-lintless line. The network covers range of transcription factors like AP2-EREBP, C2H2, and WRKY up-regulated at 0 dpa and down-regulated at 10 in Fl line and similarly phytohormones like abscisic acid, auxin, brassinosteroid, ethylene, gibberellin, and salicylic acid expressed differentially. Transcripts coding TPS, UGE, EXPANSINs, AGPs, BGAL13, UGT74B1, TUBs, ACO and LTPs coding for energy and cell wall metabolism were down-regulated at 10 dpa in Fl line. Down-regulation of transcripts related to signal transduction like RLKs, LRR-family protein, Ca2+ and ROS resulted to the loss of co-ordination during the 0 dpa. Down-regulation of miscellaneous factors having role in stress response and cell growth like HSPs and SPDS3 at elongation stage suggested probable role in cell elongation. In SSH study, based on the putative functions of genes expressed in preferentially fibre elongation stage in forward library of fuzzy-linted line included ADF2, ACO1, -galactosidase, -glucanase, -tubulin, XTH32/XET32, EF1, CBL, CaM, PCY and PLC. About 155 novel genes having no similarity in the database were also found in this study. Attempt was made for validation of the same set of genes through quantitative real-time PCR which showed high correlations with the expression levels in morphology, microarray and SSH.ThesisItem Open Access Antiphytopathogenicity of Bioagents and Expression Anlysis of Selected Defence Genes in Rice in Presence of Actinobacteria and Rhizoctonia solani(UAS, Dharwad, 2013) Snehal Jagannath Shinde; S. K. PrashanthiA total of fifty bioagent isolates comprising Trichoderma, pseudomonad, PPFM and actinomycete spp., were screened against rice sheath blight pathogen Rhizoctonia solani under in vitro condition and potent isolates were selected for in vivo assay. Five potent isolates from each of the biocontrol agent was further evaluated against R.solani under glasshouse condition. Of the fifteen efficient bioagent isolates evaluated, combined treatment of Actinomycete isolate (IABT-A7) was promising with reduced disease parameters, lesion length and number of dried leaves and enhanced the plant growth parameters viz., the plant height, number of tillers, root length, root biomass. Potential actinomycete (IABT-A7) isolate was identified as Actinopolymorpha spp., through Amplified rDNA Restriction Analysis (ARDRA). Expression of the key genes involved in Induced Systemic Resistance (ISR) and Systemic Acquired Resistance (SAR) were analysed by real-time PCR. Gene expression and quantum of expression was profiled at different time intervals 24 hr, 48 hr,72 hr and 120 hr after infection of pathogen from treatments viz., seed treatment of IABT-A7, combined treatment of IABT-A7 (seed+soil+foliar),only bioagent (IABT-A7) treatment, treated control (only pathogen) and healthy plant. Jasmonic Acid (JA) pathway related genes, OsAOS2, OsJMT1, OsJAMYB showed the differential expression in all the treatments and at all time intervals over the treated control. ET biosynthesis gene, OsERF1 was up regulated in all treatments at 48 hr, 72 hr and 120 hr over treated control. The relative up regulation of these genes was highest in combined treatment over control. OsPR1b, a key gene for SAR pathway was up regulated in only pathogen treated samples and up regulated up to 12 fold at 48 hr. The master regulator defence gene OsNPR1 was constantly expressed at all intervals over untreated plant.ThesisItem Open Access Functional and Expressional quantitation of Selected MicroRnAs in Response to Low Temperature and NaCI Stress Conditions in and Root Tissues of Arabldopsis Thallana(UAS, Dharwad, 2013) Chaithra H.V.; B. FakrudinThe discovery of miRNAs has led to a fundamental change in the understanding of complex biological mechanisms involved in plant responses to stress tolerance. In this study, the expression of seven selected miRNAs was studied in Arabidopsis thaliana plants experiencing low temperature and salt stress separately. The selected miRNAs were in situ hybridized with Locked Nucleic Acid (LNA)-modified oligonucleotide probes. Among the tested miRNAs, expression of miR161, miR168, miR171 and miR397a in leaf tissues and miR171 and miR397a in root tissues was recorded in control plants. Elevated expression of miR171 and miR397a was recorded in both tissue types of low temperature treated plants. A set of four miRNAs viz., miR171, miR395b, miR399e and miR399 showed their up regulation in both tissue types upon NaCl (300 mM) treatment. Expression of miR168 was recorded only in leaf tissues, and on the other hand, down regulation of miR397a was recorded in both tissue types in response to NaCl stress. The miRNA stem-loop RT-PCR assay indicated gradual increase in the expression of miR171 and miR397 with the highest of 4.28 and 3.49 fold changes in leaf tissues of A. thaliana plants experiencing low temperature stress and 6.5 and 6.3 fold up-regulation of miR171, 0.8 and 0.9 fold down-regulation of miR397a and 3.4-3.5 fold up-regulation of miR399 and miR399e in leaf and root tissues, respectively, at 24 hrs of exposure to salt stress. The RT-qPCR assay recorded reduced levels of miRNA target gene transcripts viz., SCL6 III, SCL6 IV, LAC2, and LAC17 in response to low temperature and SCL6 III, SCL6 IV, APS1, APS4 and AGO1 transcripts in response NaCl treatment in both tissue types of Arabidopsis thaliana plants. The study points at the possibility of modulating low temperature and salt tolerance in plants through the down regulation of specific cognate genes.ThesisItem Open Access Development of Single Chain Fragment Variable Monoclonal Antibody Against Banana Bunchy Top Virus Coat Protein(UAS, Dharwad, 2013) Shilpa B.S.; Narayan MogerBanana bunchy top virus (BBTV) is a destructive pathogen in banana cultivating areas worldwide. The symptoms induced by BBTV are similar to those caused by abiotic factors and other vascular diseases. This lack of unambiguous symptoms necessitates early diagnosis of BBTV infections. The most commonly used diagnostic tool for BBTV detection is immunological assays, which is dependent on the availability of highly specific antibody to differentiate the viruses. Production of antibody using phage display technology needs pure protein therefore, BBTV/CP was bacterially expressed. A 531 bp PCR product containing coat protein coding region of BBTV was amplified using BBTVCPF and BBTVCPR primers and the amplified product was cloned into the pTZ57R/T and further subcloned in to the pQE30. After transformation the clones were confirmed through PCR and sequencing. Amplification with expected size of 531 bp and 100% homology with other isolates showed integrity of the clone. Further, the coat protein appeared to be expressed at 3hr after induction with 1 mM IPTG. A band of 21 kDa on the gel confirmed that coat protein was really fused to the His-tag. Further, 10mg/litre of the coat protein were purified using His-tag purification kit. Four round of biopanning was performed by coating purified BBTV/CP in to the immunotube using Tomlinson library. The fourth biopan reading (1.9) showed higher binding specificity to BBTV/CP. These were subsequently used for scFv monoclone for BBTV/CP. Finally, the randomly selected scFv clones were screened with ELISA. ELISA reading showed that two clones had higher binding affinity to BBTV/CP. The sequencing of the clones showed 85% homology with the scFv antibody gene (JN887438.1). The selected clone is highly specific to BBTV/CP as there was no cross reaction with banana CMV and groundnut GBNV. Further, the sensitivity test results imply that the developed scFv antibody can detect at the concentration of 20 ıg/ml.ThesisItem Open Access Biochemical Efficacy of Homa Organic Farming Practices in okra (Abelomoschus esculentus var. Arka Anamika L. Moench)(UAS, Dharwad, 2013) Rajeev Kumar; P.W. BasarkarA field experiment laid out in CRBD with 18 treatments replicated thrice was conducted during kharif 2012 to study the biochemical efficacy of Homa organic farming practices in okra (Abelomoschus esculentus var. Arka Anamika). The conventional control (CC) and control without homa (CWH) were maintained almost 1 km away. The soil type was red black. The non-homa ash was collected after burning the agricultural waste. Agnihotra homa (AH) was performed at sun rise and sun set and Om Tryambakam homa (OTH) was performed for 3-4 h daily during experimental period. Liquid organic manures viz, Panchgavya Jeevamruta and Gloria Biosol (GB) were prepared. The non-homa ash, AH ash, OTH ash and GB were used for soil and foliar application. Soil and foliar application of GB was significantly superior over organic control in yield attributes. Significant increase in microbial population in soil, ctivities of soil dehydrogenase and phosphatase was observed. Macro and micro nutrient status of soil and okra fruits in homa treatments was significantly superior over organic control. Glaring increase was observed in okra fruits in its ascorbic acid content and in P (58%), K (98%), Cu (52%), Zn (48%), Mn (17%) and Fe (23%) contents over organic control. Significant reduction was observed in the incidence of powdery mildew (36%), alterneria leaf spot (57%), insect attack by fruit borer (38%) and Spodoptera litura larvae per plant (68%) due to soil and foliar application of GB and different homa treatments.ThesisItem Open Access Expression of Serratia marcescens AUDS744 chiA in Escherichiq coil and Saccharromyces cerevisiae(UAS, Dharwad, 2013) Mohan N. Wawge; P.U. KrishnarajSerratia marcescens, a gram negative bacterium, classified in the large family of Enterobacteriaceae, is very efficient in degradation of chitin because of its ability to produce different chitinolytic enzymes which hydrolyze the b-1, 4 linkages in the chitin microfibril. In the present study, an attempt was made to isolate the variant chitinase gene from the bacterium and their expression in E. coli and yeast. One hundred and twenty isolates of S. marcescens from the culture collection of the Department of Biotechnology, University of Agricultural Sciences, Dharwad were screened for efficiency on the basis of their chitinolytic activity on colloidal chitin media, enzyme activity by DNSA method and chi profile in comparison with the reference strain Sm141. ARFLP profile revealed that SmAUDS794chiA, SmAUDS795chiA, SmAUDS796chiA and SmAUDS744chiA has different restriction fragments compared to the reference Sm141chiA. Based on the enzyme activity and difference in banding pattern, SmAUDS744chiA was selected further for cloning and expression studies. The variant chiA from S. marcescens AUDS744 was cloned in pTZ57R/T and further sub-cloned into pET32C+ and pYES2/CT. The SDS-PAGE analysis showed that the expression of chiA as a 57 kDa protein. To find out the functionality, the IPTG induced protein from E. coli BL21 clone was subjected for bioassay against fungal pathogens viz., S. rolfsii and R. solani and it showed crescent shaped growth inhibition of fungal pathogens compared to the control. The enzyme activity of galactose induced yeast clone was calculated and found that it was higher in supernatant than the lysate also it was 2.32 times higher than plain INVSc1 and 2.85 times higher than pYES2/CT which was used as a control.ThesisItem Open Access Development and Evaluation of Ds Tagged Mutants in Sorghum(UAS, Dharwad, 2013) Amrish H. Antre; Remesh BhatMaize Dissociator (Ds)-mediated insertional inactivation tagging and activation tagging were attempted in M 35-1, a popular rabi variety of Sorghum bicolor L. (Moench) for its gene discovery and functional genomics. Six and five transgenic events produced from pUR224NA and pNU435, respectively were used as the Ds starter lines for insertional inactivation tagging. Five Ds events obtained with pUbiDs were used for activation tagging. Five events of iAc plants obtained with pKU352NA were used as the source of transposase. Ds starter lines with single copy of the launch pad (T-DNA/Ds) were selected by segregation analysis and used for crossing with iAc plants following artificial emasculation and hand pollination. The seeds set on the female plant were harvested and sown in the green house. The plants showing the presence of both Ds and iAc as tested by various PCR were regarded as true F1s. The seeds borne on the true F1s were harvested to raise the F2 generation. Among the F2 plants, those with transposed Ds (excised out of launch pad and reinserted within the genome) and without iAc were identified by various PCR as Ds tagged stable mutants. In total, five and three insertionally inactivated mutants (IIM) were obtained from the launch pads of pUR224NA and pNU435, respectively. In addition, three activation tagged mutants (ATM) were recovered from the launch pad of pUbiDs. Majority of the mutants showed unlinked Ds transposition as they did not carry the empty launch pad (TDNA without Ds). Six mutants (IIM2, IIM3, IIM6, IIM7, ATM1 and ATM3) with unlinked transposition showed Ds insertion within genic regions, indicating tagging of six different genes of sorghum. Preliminary phenotyping of the Ds tagged mutants (without ascertaining their zygosity) under green house for various morphological traits did not reveal any gross morphological changes when compared to M 35-1 (wild type).