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University of Agricultural Sciences, Bengaluru
University of Agricultural Sciences Bangalore, a premier institution of agricultural education and research in the country, began as a small agricultural research farm in 1899 on 30 acres of land donated by Her Excellency Maharani Kempa Nanjammanni Vani Vilasa Sannidhiyavaru, the Regent of Mysore and appointed Dr. Lehmann, German Scientist to initiate research on soil crop response with a Laboratory in the Directorate of Agriculture. Later under the initiative of the Dewan of Mysore Sir M. Vishweshwaraiah, the Mysore Agriculture Residential School was established in 1913 at Hebbal which offered Licentiate in Agriculture and later offered a diploma programme in agriculture during 1920. The School was upgraded to Agriculture Collegein 1946 which offered four year degree programs in Agriculture. The Government of Mysore headed by Sri. S. Nijalingappa, the then Chief Minister, established the University of Agricultural Sciences on the pattern of Land Grant College system of USA and the University of Agricultural Sciences Act No. 22 was passed in Legislative Assembly in 1963. Dr. Zakir Hussain, the Vice President of India inaugurated the University on 21st August 1964.
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ThesisItem Open Access IDENTIFICATION OF GENOMIC REGIONS HARBORING QTLs FOR STAY-GREEN AND RELATED TRAITS IN SORGHUM [Sorghum bicolor{L) Moench](University of Agricultural Sciences GKVK, Banglore, 2007-08-30) SUNDARESHA; M. S. KURUVINASHETITIStay-green feature in cereals, the ability of the plant to retain significant green leaf area until complete grain maturity, is considered important as it allows proper grain fill and such genotypes are often drought tolerant. In sorghum, stay-green feature permits complete grain fill during terminal moisture stress, enables the plant to resist charcoal rot {stalk rot) disease and produces quality fodder. Being a quantitative character it is difficult to breed for this character. Present technology permits construction of detailed genetic map with the help of molecular markers and to identify genomic regions responsible for any quantitative trait. In the present investigation, a recombinant inbred population (226) derived from N 13 (senescent) x E 36-1 (stay-green) was phenotyped for one season {rabi 2005-06) and genotyped with 28 polymorphic sorghum E5T-SSR markers, recently developed in our lab. The genetic data for 32 SSR markers already mapped using the same population, kindly provided by ICRISAT. India, was used to anchor the EST-SSR markers on the map. These 32 markers were distributed on LG1-0, LG2-3, LG3-5, LG4-5, LG5-1, LG6-4, LG7-2, LG8-6, LG9-3 and LG10-3. Seven QTLs were detected at a cut off LOD 2.5. Three of them were located on linkage group B. Except one QTL on linkage group D, others were found at new regions, previously not reported. Five QTLs contributed by the stay-green parent E 36-1 accounted for 33.34 per cent of the phenotypic variation.ThesisItem Open Access MOLECULAR MAPPING AND LOCALIZATION OF STABLE QTLs FOR CHARCOAL ROT RESISTANCE IN SORGHUM {Sorghum bicolor{L,) Moench)(University of Agricultural Sciences GKVK, Banglore, 2007-08-30) PALAMREDDY S. REDDY; B. FAKRUDINThe genetic architecture of charcoal rot resistance and drought tolerance through the construction of molecular linkage maps and identification of QTLs can expectedly hasten the development of charcoal rot and drought tolerant sorghum cultivars. A set of 93 Recombinant Inbred Lines (RlLs) derived from 1822380 and E36-1 were evaluated for charcoal rot incidence and its related traits in sick plot at Main Agricultural Research Station, Dharwad and Regional Agricultural Research Station, Bijapur during rabi 2004. The RlLs displayed highly significant differences in their mean performance for all traits. A total of 240 RAPD, 262 genomic SSR and 20 genie SSR markers were used and of which 19 RAPD, 62 genomic SSRs and 4 genie SSR markers were found to be polymorphic between parents. All 85 markers genotyped across RlLs and a genetic linkage map was constructed by using MAPMAKER/EXP b 3.0 in which 80 markers were assigned to A, B, C, D, E, F, G, H, 1 and J linkage groups. A total genomic length of 650.3 cM was covered in this map. QTL analysis by composite interval mapping using QTL Cartographer (v2.5) indicated five and four QTLs for charcoal rot component traits at Dharwad and Bijapur locations respectively on A, B, D and 1 linkage groups. Three QTLs, one each for length of infection [xtxp297 on B), number of internodes crossed by the fungus (AC13 on A) and per cent lodging (xtxp343) explaining a phenotypic variance of 9.29, 12.54 and 15.2 per cent respectively were detected consistently between two locations and are considered to be the stable across environments.ThesisItem Open Access MOLECULAR MAPPING AND LOCALIZATION OF STABLE QTLs FOR CHARCOAL ROT RESISTANCE IN SORGHUM {Sorghum bicolor{\..) Moench)(University of Agricultural Sciences GKVK, Banglore, 2007-08-30) PALAMREDDY S. REDDY; B. FAKRUDINThe genetic architecture of charcoal rot resistance and drought tolerance through the construction of molecular linkage maps and identification of QTLs can expectedly hasten the development of charcoal rot and drought tolerant sorghum cultivars. A set of 93 Recombinant Inbred Lines (RlLs) derived from IS22380 and E36-1 were evaluated for charcoal rot incidence and its related traits in sick plot at Main Agricultural Research Station, Dharwad and Regional Agricultural Research Station, Bijapur during rabi 2004. The RlLs displayed highly significant differences in their mean performance for all traits. A total of 240 RAPD, 262 genomic SSR and 20 genie SSR markers were used and of which 19 RAPD, 62 genomic SSRs and 4 genie SSR markers were found to be polymorphic between parents. All 85 markers genotyped across RILs and a genetic linkage map was constructed by using MAPMAKER/EXP b 3.0 in which 80 markers were assigned to A, B, C, D, E, F, G, H, I and J linkage groups. A total genomic length of 650.3 cM was covered in this map. QTL analysis by composite interval mapping using QTL Cartographer (v2.5) indicated five and four QTLs for charcoal rot component traits at Dharwad and Bijapur locations respectively on A, B, D and 1 linkage groups. Three QTLs, one each for length of infection (xtxp297 on B), number of internodes crossed by the fungus (AC13 on A) and per cent lodging (xtxp343) explaining a phenotypic variance of 9.29, 12.54 and 15.2 per cent respectively were detected consistently between two locations and are considered to be the stable across environments.ThesisItem Open Access CLONING AND CHARACTERIZATION OF NPRl GENE FROM MUSTARD (Brassica naous)(University of Agricultural Sciences GKVK, Banglore, 2007-08-30) AKHILESH RAWAT; M. S. KURUVINASHETTIThe overexpression of non-expressor of PR proteins [NPRl] gene has proved effective in providing resistance to a broad spectrum of pathogens in different plant species, indicating its functionality across a wide taxonomic range. In the present study. NPRl gene from mustard [Brassica napus) was cloned by using gene specific primers. The gene encoding NPRl protein was amplified and cloned in pTZ57R/T vector, sequenced and analyzed in sUico. Cloned NPRl gene had 98 per cent homology with reported NPRl gene of mustard at both nucleotide and protein level. It has four exons with three stretches of internal introns (642-851.1588-1801,1970-2055). The NPRl protein of mustard has an ankyrin repeat and a BTB/POZ (Broad-complex, Tramtrack, and Bric-abrac/ pox virus and zinc finger) domain and codes for 579 amino acids. Limited homology is also detected to the mammalian transcription factor inhibitor IkBa, This implies a close evolutionaiy relationship between these proteins. To facilitate transformation of crop plants, NPRl gene was subcloned into a plant transformation vector pHSlOO at Xbal and BarhHl restriction sites. The recombinant vector pHSAM was mobilized into Agrobacterium tumefaciens LBA4404 by tri-parental mating. A. tumefaciens with recombinant clone pHSAM was used for transforming tobacco. A number of kanamycin resistant plants were generated. Of the 15 plants PGR checked with NPRl gene specific primers, 5 were positive. These positive plants need to be analyzed further for the expression of NPRl gene.ThesisItem Open Access CLONING AND EXPRESSION OF A NOVEL vip GENE FROM B. thuringiensis(University of Agricultural Sciences GKVK, Banglore, 2007-08-30) ROOPADEVI .H; P. U. KRISHNARAJBacillus thuringiensis, a gram positive spore forming bacterium with many several subspecies, serovars and strains, is a valuable source of 5-endotoxins and vegetative insecticidal proteins (VIPs) that are active against a wide range of insect pests. VIPs, known to be produced during the vegetative stage of the cell growth have insecticidal property. Of the sixty-one B. thuringiensis strains used to analyze the vip profile using viplA, vip2A and vip3A specific primers, three of them showed amplification for viplA (4K1, 401 and 4N1), six for mp2A (4C2, 4G1, 4K1, 401, 4N1, 4T1) and seven strains for vip3A (HDl, 401, 4C2, 4G1, 4T1, 4J4, 4L3). In order to identify possible u genes present in the isolates, five pairs of degenerate primers w designed using Vip3 conserved domains. These amplified fragmen expected size in majority of B. thuringiensis strains were analyze' RFLP analysis of the PCR produce led to the identification c vip2A (in strain 4G1, 4T1 and 4C2) and mp3A (4C2). T' amplicons of vip2A (1389 bp) from B. thuringiensis subsp HD8 (4G1) and B. thuringiensis subsp. wuhanensis HD52f cloned into pTZ57 R/T and transformed into E. coli DHf analysis of vip2A showed 82.2 per cent and 82.5 per ' with the published Vip2A sequence. The sequence e presence of two conserved domains of ADP-ribosyltra' toxins. The vip2A amplicons were subcloned i prokaiyotic expression vector. The SDS-PAG^ recombinant clones showed expression of a' protein.ThesisItem Open Access ISOLATION AND EXPRESSION OF rafpl AND rafp2 ENCODING ANTIMICROBIAL PEPTIDES FROM A LOCAL CULTIVAR OF Raphanus sativus(University of Agricultural Sciences GKVK, Banglore, 2007-08-30) ASHOK V. KULKARNI; M. S. KURUVINASHETTIPathogens are known to develop varying levels of resistance to the applied chemicals, resistant varieties and biological molecules. Antimicrobial peptides that act mainly through disintegrating the cell membrane of the pathogen offer a new option for creating resistance to the pathogens. In this study, a working database with reported AMPs was constructed, which could be queried and updated. 350 bp and 300 bp DNA fragment corresponding to rafpl and rafp2 genes were amplified using specific primer pairs, designed based on their published nucleotide sequence in Raphanus sativus. The rafpl and rafp2 from a local cultivar of R. sativus were cloned in pTZ57R/T and transferred to E. coli DH5a. The presence of the inserts was confirmed by sequence analysis and nucleotidenucleotide BLASTn search. The sequence comparison showed that the cloned rafpl and rafp2 had 99 per cent homology with respective, published sequences of R. sativus. The defensin genes rafpl and rafp2 were subcloned in a prokaryotic expression vector pET28a(+). The SDS-PAGE analysis of total protein from recombinant clones pKKK166A and pKKK166B in E. coli BL21pLysS showed the expression of 12.6 kDa and 10.6 kDa protein, respectively. The crude protein extract from transformed E. coli reduced the growth of Sclerotium rolfsii, Rhizoctonia bataticola and Fusarium solani. The rafpl and rafp2 inserts were further subcloned into a plant transformation vector pHSlOO and mobilized into Agrobacterium tumefaciens LBA4404 by triparental mating. The A. tumefaciens with recombinant clone pKKK207B containing rafp2 was used to transform tobacco. The putative transformants were subjected to PGR analysis and four plants were found to be PGR positive.ThesisItem Open Access COMPARATIVE STRUCTURAL AND FUNCTIONAL ANALYSIS OF NBS-LRR TYPE RESISTANCE (R) GENES FROM SORGHUM [Sorghum bicolor (L.) Moench](University of Agricultural Sciences GKVK, Banglore, 2007-08-30) MANJU N. KITTUR; B. FAKRUDINSorghum [Sorghum bicolor (L.) Moench) is an important crop of semiarid regions where it suffers from several biotic stresses. Identification and characterization of resistance genes is a long term solution towards development of disease resistant cultivars. Here, we report for the first time, the isolation and characterization of full-length CC-NBS-LRR type disease resistance gene homologue [SbRGAlM] from sorghum. Among a set of 13 NBS-LRR sorghum resistance gene analogue [SRGAs) elucidated previously in our lab, SRGA114 was found to be unique among the characterized RGAs. Based on the short sequence information of SRGA114, a 3,220bp composite cDNA including 371bp of 5' untranslated region (UTR) and 88bp of 3' UTR was amplified using 5' and 3' rapid amplification of cDNA ends (RACE) technique. Further, the entire coding region of 2,733bp was cloned by nested PGR using specific primers designed from 5' and 3' RACE products. It encodes a predicted polypeptide of 910 amino acids with a computed molecular weight of 104.1kDa. The deduced SbRGAlM protein consists of a N-terminal coiled-coil (CC) domain, nucleotide binding site (NBS), three imperfect leucine rich repeats (LRR) and nine potential N-linked glycosylation sites. These domains are known to participate in protein-protein interaction and signal transduction during plant response to pathogen. Sequence analysis of SbRGAlM showed high homology with reported NBS-LRR type resistance genes of maize, rice and barley. Functional validation of SbRGAlM by its transfer in susceptible cultivars of sorghum will have a significant impact as a strategy in translational genomics for disease management.ThesisItem Open Access CLONING AND FUNCTIONAL CHARACTERIZATION OF ENDOCHITINASE GENE FROM TRICHODERMA SPP.(University of Agricultural Sciences GKVK, Banglore, 2007-08-30) GANAPATI BHAT; SUMANGALA BHATThe present study was conducted to isolate Thchoderma spp. from different soil samples, to screen them for chitinase activity using glycol chitin plate assay, to clone full-length genes encoding endochitinase from Trichoderma species and expression of cloned endochitinase gene in Saccharomyces cerevisiae. During the study 44 Trichoderma isolates were obtained from 48 soil sample collected. Among the thirty three isolates tested for chitinase activity (glycol chitin plate assay), isolates T. virens lABT 1010, T. konlngiil^Ell^l^ and T. poiysporumlk^l were efficient producers of chitinase enzyme. Further, using specific primers, genes encoding endochitinase chiMS (1.6kb) from T viride and T. harzianum (1.5kb) were cloned into pTZ57R/T vector. The clones were confirmed through PGR amplification and restriction analysis. The clones were sequenced and analyzed for homology at nucleotide and protein level to find out conserved domain of protein. Genes encoding endochitinase from both the species have 95 % and 99.5% homology with reported sequence both at nucleotide and protein level. Phylogenetic analysis of cloned genes indicated that both the genes fall in same cluster and closely related to T harzianum endochitinase gene. Endochitinase gene cloned from T. v/re/75 (cloned earlier) was expressed in S. cerevisiae using pYES2/cr vector. Substrate conversion assay using cell lysate indicated 9.8 times more activity in recombinant clones compared to control. Further, cell lysates of recombinant clones significantly inhibited the growth of Scierotium roifsiL In addition, reduced expression of the same gene was observed when 173bp upstream sequence was removed.ThesisItem Open Access POST-TRANSCRIPTIONAL GENE SILENCING (PTGS) OF TOMATO LEAF CURL VIRUS (ToLCV) IN TOMATO(University of Agricultural Sciences GKVK, Banglore, 2007-08-30) K. KRISHNAMURTHY; P. U. KRISHNARAJTomato, an economically important crop in many countries is plagued by many viral diseases including leaf curl caused by Tomato leaf curl virus (ToLCV) belonging to the genus begomovirus. Begomoviruses are small circular, single stranded DNA plant viruses. Yield losses usually vary from 28-92% making tomato cultivation unprofitable. Genetically engineering resistance is a viable alternative is to genetically engineer tomato for protection against ToLCV. PTGS/RNAi is a novel gene regulatory mechanism that limits the transcript level by either suppressing transcription or by activating a sequence-specific RNA degradation process. We have cloned and characterized ToLCV-coat protein (TCP), replicase (TRP) and suppressor of PTGS (TRS) genes from a Dharwad local isolate. Constructs have been developed using all the three genes for the available gene silencing strategies viz., sense (s), antisense (as), ihp (sas) and HUTR (heterologous 3'-untranslated region). Coat protein (TCP) gene has been cloned and expressed in a prokaryotic system. As a comparative study plant expression vectors carrying TCP, TRP and TRS gene following different strategies were used for transgenic development through Agrobacterium. Analysis of putative To-transgenics showed positive for PCR, GUS. Dot blot and Southern blot analysis. Semi-quantitative PGR analysis of plants from TRP constructs showed drastic reduction in the virus inoculum compared to non-transgenic plants. The Ti-generataion transgenic plants obtained from TRP constructs were positive for PCR and Dot blot analysis. Among different strategies tested for resistance to ToLCV in transgenics, those with sas/ihp construct showed significant resistance against ToLCV followed by HUTR, antisense (as) and sense (s). Similarly, among the three different genes tested, silencing was more in TRS constructs followed by TRP and TCP.