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Chaudhary Sarwan Kumar Himachal Pradesh Agriculture University, Palampur
Himachal Pradesh Krishi Vishvavidyalaya (renamed as Chaudhary Sarwan Kumar Himachal Pradesh Krishi Vishvavidyalaya in June, 2001) was established on 1st November, 1978.The College of Agriculture (established in May, 1966) formed the nucleus of the new farm University. It is ICAR accredited and ISO 9001:2015 certified institution. The Indian Council of Agricultural Research has ranked this University at eleventh place among all farm universities of the country.
The University has been given the mandate for making provision for imparting education in agriculture and other allied branches of learning, furthering the advancement of learning and prosecution of research and undertaking extension of such sciences, especially to the rural people of Himachal Pradesh.
Over the years, this University has contributed significantly in transforming the farm scenario of Himachal Pradesh. It has developed human resources, varieties and technologies and transferred these to farming community enabling the State to receive the “Krishikarman award” of Govt. of India four times in row for food grain production among small states of the country. Today, the State has earned its name for hill agricultural diversification and the farming community has imposed its faith in the University.
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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 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 studiesThesisItem Open Access GENE NETWORKS INVOLVED IN COLD STRESS RESPONSE IN CHICKPEA (Cicer arietinum L.) ANTHERSVARMA(CSK HPKV Palampur, 2020-01-07) VERMA, ANNU; Chaudhary, H.K.Cold stress (CS) coinciding with the onset of the flowering in chickpea poses a major threat for realizing high productivity in northern hill regions of India. CS induces pollen sterility in chickpea leading to flower abortion and consequently low productivity. Some genotypes of chickpea, though agronomically inferior, tolerate cold and produce viable pollen leading to seed set even under cold stress. Changes in carbohydrate content in contrasting genotypes of chickpea were observed in the present study under CS wherein, the genotype ICC 16349 (cold tolerant) maintained normal carbohydrate pool whereas genotype GPF2 (cold susceptible) failed to do so. Not only carbohydrate content but enzymes of the carbohydrate pathway e.g. invertase, sucrose synthase, alpha and beta amylase showed greater activity in cold tolerant genotype. Under recovery period, the levels of carbohydrates as well as carbohydrate metabolism enzymes shifted towards normal with a few modifications. The expression of carbohydrate metabolism genes that is UDP glucose pyro phosphorylase like, ADP glucose pyro phosphorylase, Beta amylase3 and Beta amylase1 changed significantly under cold stress in the cold tolerant as well as cold susceptible genotypes. The expression level of Isoamylase3 and Isoamlyase3x2 was higher in leaves of cold susceptible genotype GPF2. Starch degradation was lower in ICC 16349 (cold tolerant) as compared to GPF2 (cold susceptible) as evident from expression of genes; Beta amylase3 and Beta amylase1 and glucan water kinase. The expression of starch synthase increased in theICC16349.The expression of ABA metabolism genes was higher in GPF2 and the expression of GA biosynthesis genes Gibberellin20oxidase1 and Gibberellin20 oxidase3 reduced in the anthers of ICC 16349 and GPF2 with the exception of Gibberellin20oxidase1 that enhanced in GPF2. A combination of 5µM Sucrose and 5µM GA was the best treatment for mitigation of cold under pot as well as field conditions as it showed marked increase in chlorophyll content, relative leaf water content, cellular respiration and reduction in electrolyte leakage in both the genotypes. The study has implications in the development of cold tolerant chickpea varieties and devise other appropriate strategies for mitigation of cold, thereby resulting in increased productivity of chickpea.ThesisItem Open Access STUDIES ON CRYOPRESERVATION AND REGENERATION OF IN VITRO RAISED CULTURES OF PICRORHIZA KURROA(CSK HPKV Palampur, 2020-09-28) ANKITA; Shashi, BhushanPicrorhiza kurroa is an endangered Himalayan perennial herb and important ingredient of traditional medicinal systems of Asian region. It’s medicinal value primarily depends upon presence of picrosides in its roots. These bioactives are used for preparation of hepatoprotective phytopharmaceuticals. This herb is uprooted from wild for extraction of bioactive ingredients, which is a major reason for its depletion from natural habitat. Considering the high demand and low supply of P. kurroa population, efforts for its conservation through biotechnological tools is urgently required. In current research, cryopreservation protocol of high picroside producing cell lines of Picrorhiza kurroa were developed. P. kurroa shoot cultures incepted with 0.50 mg/L of Kn. Highest in vitro average shoot number was observed at 0.50 mg/L Kn strengthened MS medium with 3.18 shoots per shoot tip whereas 4.29 cm shoot length with 11.46 average number of leaves recorded on MS basal medium. Highest callus induction (100%) was found on MS medium augmented with 0.0125 mg/L IBA + 0.125 mg/L TDZ from leaf and root explants. Calli multiplied on the same medium with 6.89 fold increase in fresh weight. Leaf and root explants of in vivo and in vitro grown plants were analysed for identification of high metabolite producing cell lines. Among in vivo leaf and root explants, highest P-I content (5.65 %) was found in leaves whereas highest P-II content (8.37 %) was found in roots. In case of in vitro leaves and roots, leaves contained higher P-I (1.160 %) & P-II (0.280 %) content. Callus derived from leaves also showed highest total picroside content i.e 0.2 per cent, therefore, leaf derived callus was used for cryopreservation. Among all the cryopreservation methods used i.e encapsulation-vitrification, vitrification and desiccation, only encapsulation-vitrification showed 60 per cent regeneration at 15 minutes of dehydration time with PVS2 solution. Encapsulation-vitrification protocol would be useful for ex-situ conservation of P. kurroa callus cultures.
