Loading...
Theses
Browse
Search Results
ThesisItem Open Access Identification, characterization and expression analysis of calmodulin and calmodulin-like protein genes in chickpea (Cicer arietinum L.)(CSK HPKV, Palampur, 2022-12-16) Verma, Avnika; Sharma, Kamal DevChickpea (Cicer arietinum L.), is an important legume crop grown widely for its nutritious seeds. It is believed to be originated in South-West Asia. Chickpea productivity is severely hampered by both abiotic (cold, heat, draught, salt) and biotic stresses (wilt & blight). Following stress, the plant cells exhibit Ca2+ fluxes. The Ca2+ binds to the calcium binding proteins such as Calmodulin and Calmodulin-like leading to Ca2+ induced signaling and plant’s responses to stress. In the present investigation, Ca2+ binding genes, Calmodulin (CaM) and Calmodulin-like (CML) in chickpea were identified and characterized. Four calmodulin and thirty-eight calmodulin-like genes were identified in the chickpea genome. The Calmodulin and Calmodulin-like genes were distributed in all the eight chromosomes of chickpea while chromosomal location of four Calmodulin-like genes could not be ascertained. A new nomenclature for these genes in chickpea was also proposed because the previous nomenclature was erroneous and confusing. The gene, mRNA, protein and coding sequences were identified for each genes using appropriate database and software, and the gene identities were confirmed using NCBI, SMART and Pfam tools. Gene structure, phylogenetic relationships and protein-protein interaction were also deduced by using appropriate bioinformatics tools. Expression of four CaM (Calmodulin) and thirty-eight CML (Calmodulin-like) genes was studied in five different organs of chickpea (leaf, stem, root, anther and ovule). CaM and CML gene families in chickpea expressed differentially in different organs. Expression analysis showed that maximum number of CaM and CML genes expressed at very high levels in roots and ovules showing root and ovule specificity. One CaCaM and 14 CaCMLs genes overexpressed in ovule, 2 CaCaM and 13 CaCMLs genes overexpressed in root, 4 CaCMLs genes overexpressed in leaf, 3 CaCMLs genes overexpressed in anther and 2 CaCMLs genes overexpressed in anther. The study revealed the existence of large number of CaCaM and CaCML genes in chickpea and that these genes express in organ specific manner suggesting organ specific function of each gene.ThesisItem Open Access Cryopreservation studies in Callus culture of Arnebia euchroma(palampur, 2022-05-09) Rai, Sourav; Bhushan, ShashiArnebia euchroma commonly known as Ratanjot belongs to Boraginaceae family. Arnebia species is known for rich source of shikonin and their derivatives have antibacterial, antifungal, anti-HIV, anti-inflammatory and several other medicinal properties. The genus Arnebia has been over exploited as a result of these characteristics, and it is now classified as a endangered species. These resources must be protected in order to be used on a large scale in the future, and this valuable treasure must be preserved. Callus was induced from the leaves with (75%) induction when cultured on MS medium augmented with IBA (1.02 mg/l) and BAP (2.25 mg/l) and multiplied on same medium. Leaf derived callus were used for the cryopreservation Therefore, a protocol is optimized for cryopreservation of callus using vitrification, encapsulation vitrification and desiccation technique. Callus was precultured on sucrose enriched medium containing 3% (w/v) and 13.7% (w/v) sucrose. Among all these techniques of cryopreservation highest survival rate (65%) was found in desiccation when precultured on 13.7% sucrose followed by 90 minutes desiccation time. Callus used for the cryopreservation was not revived after cryogenic treatment and necrosed after eight weeks of culture in encapsulation vitrification method. Hence, this method of cryopreservation was not found suitable for preserving the callus tissues of A. euchroma. Non cryopreserved callus showed the highest total Deoxyshikonin content i.e. 369.28 (µg/gm) and 301.31 (µg/gm) shikonin content was observed and Cryopreserved callus showed the highest Deoxyshikonin content i.e. 319.23 (µg/gm) and total shikonin content of 296 (µg/gm). In UPLC chromatography only deoxyshikonin content i.e. 65.05 (µg/gm) was observed in non cryopreserved callus and 39.07(µg/gm) content was found in cryopreserved callus.ThesisItem Open Access Elucidating the role of MYC2 transcription factor in Cucumber mosaic virus infection(palampur, 2019-07-30) Eshwar, Jadhav; Hallan, VipinCucumber mosaic virus (CMV) has the broadest host range among known plant viruses, infecting more than 1,200 species of plants from monocotyledons to dicotyledons. As the effects of CMV is very diverse in nature causing epidemics in diverse crops, it is important to understand the host- pathogen interactions in order to develop plants which can be resistant to plant virus infections. The expression of stress-responsive genes is intimately dependant on its transcriptional control which directly leads to plant response to various stresses. The MYC2 class of transcription factors has recently emerged as a master regulator of the signalling pathways of jasmonic acid (JA) and those of other phytohormones such as abscisic acid (ABA), gibberellins (GAs), and auxin (IAA). Jasmonic acid is the plant hormone that regulates the plant growth, development and defense responses. The MYC2-3 mutant line of Arabidopsis thaliana was used for the analysis of the role MYC2 transcription factor in CMV virus infection. Characterization of MYC2-3 mutant line through jasmonic acid and abscisic acid treatments revealed that the wild type showed reduced root length as compare to MYC2-3 mutant. It was also revealed that the MYC2-3 mutant line has resistance to CMV virus as compared to the wild type plant. Also, SDS PAGE and MALDI-TOF analysis showed that the expression of enzymes and proteins involved in the photosynthetic pathway to be directly affected in the MYC2-3 mutant line on CMV infection. This study could be directly used for development of CMV resistant plant varieties through genome editing in futureThesisItem Open Access Studies on chickpea (Cicer arietinum L.) defence to blight pathogen Ascochyta rabiei(palampur, 2019-07-30) Gaikwad, Dinesh Subhash; Sharma, Kamal DevChickpea (Cicer arietinum L.), a self-pollinated diploid (2n=2x=16) with haploid genome size of ∼740 Mbps, is an important food legume of subtropical and tropical regions and is believed to be originated in the Mediterranean region. In cool and humid regions of the world, chickpea blight caused by Ascochyta rabiei causes huge yield losses. Some accession of C. arientinum and wild Cicer species possess resistance to the disease, however molecular mechanism governing host resistance are poorly understood. With the aim to understand regulation of expression of genes involved in defence to A. rabiei, the expression of six defence genes [Phenylalanine ammonia lyase (PAL), Isoflavone reductase (IFR) Flavanone 3- hydroxylase (F3H), Pathogenesis related protein - 2b (PR-2b), Basic leucine zipper 24 (bzip24) and SNAKIN-2 (Antimicrobial peptides)] was elucidated in resistant and susceptible genotype of chickpea after inoculation (2 h, 12 h, 24 h, 36 h, 72 h) with A. rabiei. Gene expression was studied using quantitative Real-Time PCR and the expression was normalized with two reference genes, Clathrin adaptor complexes (CAC) and ATP-binding cassette transporter (ABCT). The sequences of the mRNAs were retrieved from National Center for Biotechnology Information database followed by identification of coding sequences (CDS) and design of primers. The genes (PAL, IFR, F3H) of the phenylpropanoid pathway that govern phytoalexin production expressed within 2 h after landing of the spores on chickpea surface and expression was considerably higher in resistant HC-1 then susceptible GPF2. The genes for pathogenesis related protein (PR-2b), antimicrobial peptide (SNAKIN-2) and transcription factor (bZIP24) had peaks at 24 h in the resistant genotype indicating maximum expression in resistant genotype at the time of host penetration and subsequent pathogen spread. The susceptible host (GPF2), on the other hand was slow in its response as is evident from low or delayed overexpression as compared to resistant host. It appears that defence to Ascochyta blight in chickpea is a precisely coordinated reaction of the host, where phytoalexin accumulation appears to occur within hours of inoculation, whereas antimicrobial peptides accumulate at the time of host invasion and pathogen spreadThesisItem Open Access Influence of cold stress on expression of invertase and calcium-dependent protein kinase genes in chickpea (Cicer arietinum L.)(Palampur, 2021-11-12) Shree, Bharti; Sharma, Kamal DevChickpea (Cicer arietinum L.) is an important food grain legume. Chickpea is sensitive to cold and suffers substantial yield losses due to cold stress. Invertases hydrolyse sucrose into glucose and fructose and play an important role in plant growth and development as well as plants’ responses to various stresses including cold. In addition to invertases, calciumdependent protein kinases (CDPKs) modify gene expression via transcription factors to achieve systematic plant growth/development and reaction to stresses. Information on invertases and calcium-dependent protein kinases as well as the role of these genes in stress tolerance/susceptibility in chickpea is unavailable. In this study, 19 invertase genes (11 cellwall invertase, one vacuolar invertase and seven alkaline/neutral invertase genes) and 31 CDPKs genes were identified in the chickpea genome. These genes were located on 7 chickpea chromosomes. A comprehensive analysis of invertase as well as CDPK genes and proteins were performed, including gene structure, mRNA structure, cis-acting elements in the promoter regions, phylogeny, evolutionary relationships, gene duplication events, protein structure, motifs, domains, physiochemical properties, sub-cellular localization and interactions of invertases as well as CDPKs with other proteins. Phylogenetic analysis revealed that chickpea invertases were comprised of five major lineages whereas CDPKs had four lineages. The members within the same sub-groups shared conserved domains. Expression analysis revealed that all the invertase genes were functional in chickpea however, these genes expressed differentially in contrasting chickpea genotype under cold stress and Ascochyta blight infection. Expression analysis revealed that cell wall invertases were associated in cold tolerance whereas majority of the CaCDPK genes were involved in low temperature responses by tolerant as well as sensitive genotypes of chickpea. Invertase genes associated with Ascochyta blight resistance in chickpea were also identified. The study laid the foundation for unravelling the complexity of chickpea responses to cold and Ascochyta rabiei infection and develop protocols for mitigation of cold stress in chickpea.ThesisItem Open Access Introgression of blast resistance and semi dwarfing gene sd1 in rice using marker assisted backcross breeding(Palampur, 2021-10-31) Diliprao, Pote Tushar; Rathour, RajeevIn present study two blast resistance genes Pi9, Pi54 and one semi-dwarfing gene sd1 were incorporated into a tall traditional basmati rice variety, ‘Ranbir Basmati’ from donor genotypes Pusa1637 (Pi9+sd1) and DHMAS164 (Pi54) using marker-assisted backcross breeding (MABB). Molecular marker based background analysis was combined with stringent phenotypic selection to achieve a maximum of 92.63% and 96.15% recovery of recurrent parent genome in progenies of crosses Ranbir Basmati*3 /Pusa1637 and Ranbir Basmati*3 /DHMAS164 after two backcrosses. Altogether sixteen pyramid lines homozygous for three target genes (Pi9+Pi54+sd1) were identified by foreground analysis of 1219 progenies derived from the intercross of elite BC2F1 recombinants of two crosses. These 16 lines were further advanced through pedigree selection in the field to select 39 stable ICF3 lines showing phenotypic similarity to Ranbir Basmati. All the lines were found to have reconstituted the recurrent parent alleles for genes and QTLs related to aroma and amylose content. Analysis of variance revealed sufficient variation among pyramid lines for different traits except panicle length, percent spikelet fertility and thousand grain weight. As many as 14 pyramid lines viz., RPL1-11-2, RPL1-90-1, RPL1-90-2, RPL1-90-4, RPL1-262-1, RPL1- 262-2, RPL1-495-1, RPL1-495-2, RPL1-559-1, RPL1-934-2, RPL1-1075-1, RPL1-1075-3, RPL1-1086-4 and RPL1-1121-2 were found to be significantly superior to Ranbir Basmati for yield. All the pyramid lines were highly resistant to leaf and neck blast compared to recurrent parent Ranbir Basmati on which a high incidence of leaf and neck was recorded. Five high yielding semi-dwarf pyramid lines, viz., RPL1-559-1, RPL1-934-2, RPL1-1075-1, RPL1- 1075-3 and RPL1-1121-2, showing high degree of resistance to blast and superior basmati quality attributes were identified as a possible substitute to Ranbir Basmati.ThesisItem Open Access Mapping of genomic regions associated with dwarfing and determinate growth habit in horsegram (Macrotyloma uniflorum)”(Palampur, 2021-10-26) RAM, MALA; Chahota, R.K.Macrotyloma uniflorum is an important, self pollinated diploid (2n=2x=20) food legume with probable genome size of 400Mbps. Limited genomic resources and lack of genetic variation are major constrains in its genetic improvement. Further, horsegram production is hampered due to twining growth habit, longer days to maturity, photosensitivity and indeterminate growth habit. The present study was aimed to mining the new microsatellite markers and construct linkage map of an intraspecific F8 RILs population of 157 individuals derived from HPKM249×HPK4 of horsegram and identification of genomic regions linked to dwarfing and determinate growth habit related traits. 2395 molecular markers were screened for parental polymorphism and 600 (25.05 %) were found to be polymorphic among the parents. Of these, 287 were mapped on ten linkage groups at LOD 3.5 spanning 796.76 cM with an average marker density of 2.78 cM. Analysis of variance of 157 RILs revealed significant differences for all the measured traits. Phenotypic data from the RILs were used to identify QTLs for plant height and growth habit related traits by composite interval mapping (CIM). A total of 4 QTLs (LOD ≥ 2.5) were detected across the ten linkage groups for 4 traits. All these QTLs were major QTLs with PVE greater than ten per cent. In conclusion, it is envisaged that the present linkage map, fortified with 287 SSR markers and 4 QTLs for dwarf plant height and determinate growth habit related traits would provide genomics tools to breeders for further genetic enhancement of this crop species. Thus, the current study will serve as a strong foundation for further validation and fine mapping of QTLs for utilization in horsegram breeding programs.ThesisItem Open Access Association analysis of molecular markers with various agronomic traits of horsegram (Macrotyloma uniflorum)(Palampur, 2021-11-30) SHARMA, ANKITA; Chahota, R.K.Macrotyloma uniflorum is an important, self pollinated diploid (2n=2x=20) food legume with probable genome size of 400Mbps. Limited genomic resources and lack of genetic variation are major constrains in its genetic improvement. Further, horsegram production is hampered due to twining growth habit, longer days to maturity, photosensitivity and indeterminate growth habit. The present study was aimed with the objective(s) to estimate genetic diversity among a diverse panel of horsegram accessions and to analyze the association of molecular markers with various agronomic traits. To dissect the association of molecular markers with various agronomic traits in horsegram and identification of genomic regions linked to morphological, phenological, biochemical and yield related traits 88 diverse horsegram germplasm lines were evaluated for various agronomic traits for four years at two locations. Lines were analysed using 791 SNPs and 37 SSRs polymorphic markers. Of these 704 SNPs and 37 SSR were mapped with known chromosomal location and were found to be significantly associated with traits of interest. Analysis of variance of germplasm population revealed significant differences for all the measured traits except days to maturity. Phenotypic and genotypic data were used to identify QTLs for various agronomic traits by using compressed Linear model (CMLM), Generalized linear model (GMLM) and Mixed linear model (MLM). A total of 146 QTLs were detected across the five environments for 12 agronomic traits. Among these, 25 were major and stable QTLs across locations and years with heritability more than 80 percent. In conclusion, it is envisaged that the present association study fortified with 704 SNP markers, 37 SSR markers and 25 QTLs for various agronomic traits, which would provide genomics tools to breeders for further genetic enhancement of this crop species. Thus, the current study will serve as a strong foundation for further validation and fine mapping of QTLs for utilization in horsegram breeding programs.ThesisItem Open Access Bacterial strain improvement for biomolecules through chemical and physical mutagens(Palampur, 2021-03-30) THAKUR, AKRITI; Singh, DharamMicroorganisms are crucial life form present on earth, inhabiting all climatic zones including high altitude Himalayan niches. Microbes can thrive in harsh environmental conditions due to their ability to produce biomolecules such as enzymes and metabolites that perform specialised biological functions. Biomolecules produced by microbes are in minute quantities. Therefore, there is a need to increase the production for large scale applications through strain improvement. Hence, the current study was focussed on the strain improvement of unique bacterium Iodobacter sp. PCH194 through the application of chemical mutagens MMS, EMS, and NMU and physical mutagen in the form of UV radiation. The isolate PCH194 coproduces PHA and violacein, which has wide industrial applications. Through systematic applications of mutagens on wild-type PCH194, mutants with desired features were obtained and designated as IN1, IN2, IN3, IN4, and IN5. Their growth kinetics at alleviated temperature were observed. It was found that their growth temperature increased from 20 to 25C, and slow growth was also observed at 28C. However, the application of thermo protectants glycine betaine and glutamate could not significantly enhance the growth at 28C. There was a marked increase in growth, PHA and violacein production of the mutants at 20C. The PHA production was 1.24 mg/ml for IN5 and violacein production was 1.63 mg/ml for IN2, whereas wild strain produced 0.42 mg/ml PHA and 0.20 mg/ml violacein, respectively. In conclusion, the present study successfully increased the growth temperature of Iodobacter sp. PCH194 from 20C to 25C and also enhanced the production of PHA and violacein. Hence, generated mutants can further be used for process optimisation and scale-up studies
