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Dr. Y. S. Parmar University of Horticulture & Forestry, Solan
Dr. Yashwant Singh Parmar University of Horticulture and Forestry, Solan, was established on 1st December, 1985 with the objective to promote education, research and extension education in the fields of Horticulture, Forestry and allied disciplines. Late Dr. Yashwant Singh Parmar, the first Chief Minister and the architect of Himachal Pradesh perceived the importance of Horticulture and Forestry to develop and improve the State economy which led to the establishment of this University. Its history lies in erstwhile Himachal Agricultural College, Solan, established in 1962 and affiliated to the Panjab University. It became one of the campuses of Agriculture Complex of Himachal Pradesh University on its formation in 1970. Consequent upon the establishment of Himachal Pradesh Krishi Vishvavidyalaya in 1978, this campus became its Horticulture Complex and finally in 1985, assumed the status of a State University, being the only University in the country engaged exclusively in teaching, research and extension in Horticulture and Forestry.
The University is located at Nauni in Solan District of Himachal Pradesh, 13 km from Solan on Solan-Rajgarh Road, at an elevation of 1300 metres above mean sea level. Solan town is situated on national highway (NH-22) and is well connected by train and bus services.
The University has four constituent colleges, out of which, two are located at the main campus Nauni, one for horticulture and the other for forestry, having 9 and 7 departments, respectively. The third College i.e., College of Horticulture & Forestry is located at Neri in Hamirpur District on Nadaun-Hamirpur state highway, about 6 Km from Hamirpur town and is well connected with bus service. The college offers three Undergraduate Degree Programmes i.e. BSc (Hons.) Horticulture, BSc (Hons.) Forestry and B. Tech. Biotechnology and MSc degree programme in a few subjects. The fourth college i.e. College of Horticulture and Forestry, Thunag (Mandi) is located at Thunag District Mandi. This college offer BSc (Hons.) Horticulture and BSc (Hons.) Forestry degree programme. In addition, there are five Regional Research Stations, 12 Satellite Stations and five Krishi Vigyan Kendras (KVKs) situated in different zones of the State.
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ThesisItem Open Access Studies on construction of frame work genetic linkage map of Stevia rebaudiana Bertoni using molecular markers(YSPU, 2013) Sharma, Neha; Kaur, RajinderThe present investigation on Stevia rebaudiana was carried out with the objective to construct frame work genetic linkage map of stevia by employing multiple marker systems. Before starting the work on linkage map construction, genetic diversity was analyzed amongst the available genotypes/ clones/ accessions/ morphotypes of Stevia rebaudiana to confirm two parents with differences in rebaudioside-A and stevioside content. This study represents the first genetic linkage study of Stevia rebaudiana comprising of multiple marker systems together. Among the collected 16 accessions polymorphism was studied using 27 RAPD, 26 ISSR and 50 EST-SSR. Results were analyzed in the form of dendrograms, similarity, disimmilarity matrices and polymorphism information content. For linkage map construction, F2 population was used as a mapping population. To survey the polymorphism among contrasting parents 170 RAPD, 26 ISSR and 89 EST SSR were employed and it was observed that 36 RAPD, 10 ISSR and 33 EST – SSR primers were found to be polymorphic. These primers were then used for the genotyping of the mapping population. Phenotyping was carried out by using High Performance Liquid Chromatography (HPLC) of parents as well as segregating population. Both genotypic and phenotypic data were used to construct a linkage map using MAPMAKER/EXP ver 3.0b. A total of four linkage groups were constructed spanning a distance of 927.3 cM with an average distance between loci as 16.29 cM. First linkage group (LG1) comprised of 33 markers, second linkage group (LG2) contained 6 markers and third (LG3) and fourth group (LG4) had 16 and two markers, respectively. On QTL identification, a total of 53 QTL locations were found for both trait 1 (rebaudioside-A) and trait 2 (stevioside). Among 53 QTL locations of trait 1, two major QTLs were found for rebaudioside-A viz., in the marker interval of L67- L71 (ISSR HB-11) - (IISRS-3-L) in LG1 and in the marker interval L38 - L40 (Sigma-5383-027)- (Sigma-5383-029) in LG3 at LOD of 2.5 and 2.7, respectively.ThesisItem Open Access Studies on Agrobacteriummediated insect resistance gene transfer in Cabbage (Brassica oleracea L. var. capitata) and molecular analysis of regenerated plantlets(YSPU, 2013) Gambhir, Geetika; Srivastava, D.K.Genetic transformation studies were carried out to standardize a protocol for insect resistance gene (cryIAa) transfer in cabbage (Brassica oleracea L. var. capitata cv. Pride of India). Agrobacterium tumefaciens strain containing npt-II and cryIAa genes in binary vector pBin-1Aa was used for genetic transformation studies. Plant regeneration studies were carried out using four different types of explants viz. cotyledon, hypocotyl, leaf and petiole Cotyledon and hypocotyl explants were used from seven to nine days old aseptically grown seedlings whereas, leaf and petiole explants were procured from glass house 20-25 days old grown seedlings of cabbage. Hypocotyl explants showed better shoot regeneration as compared to other explants. High efficiency shoot regeneration was obtained in cotyledon (91.11 %), hypocotyl (94.44 %), leaf (91.11 %) and petiole (88.88%) explants on MS medium supplemented with 0.33mg/l TDZ + 79.7 mg/l Adenine, 0.22mg/l TDZ + 0.088mg/l IAA, 0.22mg/l TDZ + 0.02mg/l NAA and 0.33mg/l TDZ + 0.02mg/l NAA, respectively.MS medium supplemented with 0.10mg/l NAA was found best for root regeneration from in vitro developed shoots. The regenerated plantlets were acclimatized successfully on cocopeat. Kanamycin sensitivity experiment was carried out to study the effect of antibiotic on relative growth of leaf and petiole tissues and to select transgenic shoots during transformation experiment. Kanamycin sensitivity (10-60mg/l) was checked by fresh weight of the explants which was measured at the interval of 7 days. From the relative growth of the explants it was found that the concentration as low as 10mg/l is toxic to the explants. Effect of different concentrations of cefotaxime was studied on the regeneration potential in cotyledon and hypocotyl explants of cabbage and found no much effect of cefotaxime on regeneration potential. Effect of different concentrations of cefotaxime and kanamycin (50mg/l) were studied on the growth of agrobacterial cells and regeneration potential of cotyledon and hypocotyl tissues after cocultivation. In both the explants the growth of agrobacterial cells were controlled at concentration of 400mg/l cefotaxime and maximum per cent shoot regeneration in cotyledon (35.55 %) and hypocotyl (48.15 %) was obtained on MS medium supplemented with 400mg/l cefotaxime, respectively. Effect of preculturing and co-cultivation was studied on the transformation frequency. Preculturing of cotyledon and hypocotyl explants for 72 hours and co-cultivation with agrobacterial cells for 48 hours worked out to be the best treatment as it gave the highest transformation frequency (4.66 %) and (14.50 %) in respective explants. Effect of different concentrations of acetosyringone was studied in cotyledon and hypocotyl explants to enhance the transformation frequency. The maximum percent shoot regeneration (18.66 %) and (32.00 %) was obtained from cotyledon and hypocotyl explants cultured on shoot regeneration medium containing 100μM acetosyringone at standardized preculturing and cocultivation time interval i.e. 72 hours and 48 hours. The presence/integration of transgene (cryIAa) into the genome of cabbage was confirmed by PCR using gene specific primers and Southern blot analysis using radioactive labelled DNA probe. The Southern blot analysis has also been used to confirm copy number of transgene into the genome of cabbage. For PCR analysis, 40 putative transgenic shoots were randomly selected and out of 40 putative transgenic shoots/plantlets, 20 shoots were found to be +ve for the presence/integration of transgene i.e. cryIAa into the genome of cabbage. For Southern blot analysis 10 RT-PCR +ve shoots were selected. Out of the 10 PCR +ve shoots, 5 shoots were confirmed +ve for integration of transgene cryIAa into the genome of cabbage with 1 to 3 copies of gene insertion. The confirmation of expression of the transgene cryIAa into the genome of cabbage at transcriptional level was confirmed by Reverse Transcriptase-PCR and Real Time-PCR and at translational level by Bioassay. A protocol for high frequency plant regeneration and insect resistance gene transfer in cabbage (Brassica oleracea L. var. capitata cv. Pride of India) has been standardized.ThesisItem Open Access Studies on isolation and characterization of trypsin inhibitor (TI) gene from Dolichos biflorus L. (Kulth)(DYSPU, 2013) Reena Kumari; Nath, A.K.Protease inhibitors are one of the most promising agents that confer resistance in plants against insect pests by inhibiting larval midgut proteases. Maximum extraction of trypsin inhibitor protein from seed flour of Dolichos biflorusL. was in 0.1 M phosphate buffer (pH 7.6) after four hours of extraction. Screening of Dolichos biflorus L. cultivars for trypsin inhibitor activity revealed maximum activity in HPK4 cultivar and further studies were conducted in this cultivar. Crude trypsin inhibitor of all cultivars inhibited midgut protease of P. brassicaelarvae. Inhibitor activity was detected at early stages of seed development (3 days after flowering (DAF)) and it increased progressively with seed development (21 DAF to 60 DAF). Trypsin inhibitor activity decreased during seed germination as compared to dry seeds. Crude trypsin inhibitor extracted from developing and germinating seeds also inhibited larval midgut protease of S. littoralis. Neonate larvae of P. brassicae fed on cabbage leaf discs coated with 0.025-2.50 mg crude trypsin inhibitor caused 10–80 % larval mortality. The calculated LC 50 value was 1.05 mg crude trypsin inhibitor and for 2.5 mg crude trypsin inhibitor the calculated LT 50 value was 3.2 days. Leaf area eaten and faecal matter produced by treated larvae were significantly lower as compared to untreated controls. Larvae fed on leaf discs coatedwith 2.5 mg crude trypsin inhibitor for 5 days had significantly less total soluble protein in faecal matter and midgut trypsin activity as compared to untreated control. Significant reduction in egg hatching (75%) was observed in egg mass treated with 5.3mg of crude trypsin inhibitor of mature seeds. Trypsin inhibitor gene (309 bp) was amplified from cDNA synthesized from mature seeds of Dolichos biflorus L. HPK4 cultivar using designed primers. The amplified PCR product was cloned and sequenced. Sequence of Dolichos biflorusL. HPK4 cultivar trypsin inhibitor (DbTI) gene hasbeen submitted to NCBI with Accession No. JQ259858. DbTI gene and its deduced amino acid sequence showed homology with Bowman-Birk inhibitors of Dolichos spp., Phaeolus spp., Vigna spp. and Glycine spp. The predicted molecular weight of deduced amino acid sequence was ~11.5 KDa and it had N terminal signal peptide of 19 amino acid residues. The secondary structure of deduced amino acid sequence of DbTI showed dominance of coils and sheets over alpha helix. Homology modelling was employed to predict the three dimensional structure of DbTI. Docking of trypsin enzyme and DbTI showed the inhibitor to be of non- competitive typeThesisItem Open Access Bioprospecting of bacteria for production and purification of laccase enzyme(YSPU, 2013) Dhiman, Karuna; Shirkot, PoonamLaccase enzyme has acquired the status of ‘green catalyst’ as it possesses remarkable bioremediation potential along with numerous applications in effluent detoxification, degradation of textile dyes, herbicide and insecticide degradation, wine clarification, enzymatic conversion of chemical intermediates, biosensors and organic synthesis. In the present study, significant high diversity of laccase producing bacteria from rhizosphere of rice plants from paddy fields of H. P. was assessed whereas medium diversity was obtained from the samples of paper mills of H.P. A total of 449 bacterial isolates were obtained from 198 samples using M162 and TY media containing 5mM guaiacol and 40 mg/l CuSO4. These were rescreened on the basis of their ability to oxidise tannic acid and dimethoxyphenol leading to selection of 67 bacterial isolates which were characterized both morphologically and biochemically alongwith the laccase activity and 14 bacterial isolates exhibiting maximum laccase activity of 10-19 U/l were selected. Molecular characterization of the selected isolates was carried out using RAPD-PCR and 16S rrna gene technology and in silico analysis of 16S rrna gene sequences lead to identification of these bacterial isolates as Pseudomonas putida strain LUA15.1 and LHB7.1, Pseudomonas umsongenesis strain LHB9.1., Pseudomonas mohnii strain LHN12.2, Pseudomonas chlororaphis strain LUD7.1, Pseudomonas jessenii strain LHN9.1, Pseudomonas lurida strain LB6.2, Pseudomonas graminis strain LHN8.1, Pseudomonas veronii strain LUA14.1, Pseudomonas fulva strain LR5.1, Lysnibacillus fusiformis strain LKM7.1, Lysnibacillus sphaericus strain LH3.4 and Bacillus subtilis strain LB6.1 and LR6.3 . On the basis of maximum laccase enzyme activity Pseudomonas putida strain LUA15.1 was selected for production and purification of the laccase enzyme. Maximum extracellular enzyme production was achieved at 28°C, pH 7 (24 hrs incubation) with 5mM guaiacol, 50 mg/l CuSO4, 5% tryptone and 3% yeast extract in combination as nitrogen source in Tryptone Yeast medium. The laccase crude extracellular enzyme preparation was purified by ammonium salt precipitation (50-90%) followed by gel filtration and ion exchange chromatography which showed 10.74 yield and 61.36 fold purification. The purified enzyme had optimal activity at pH 7.0 and 40°C and 0.80 mM Km value. The molecular weight of laccase in the present study was found to be 42.5 kDa. The activity was inhibited by sodium azide and DTT. Strain LUA15.1 as well as its enzyme preparations were studied for their ability to decolourize dyes which are the potential contributors of water pollution. All six different synthetic dyes were decolourized RBBR (48%), congo red (35%), indigo carmine (80%), brilliant green (97%), bromophenol blue (78%) and aniline blue (23%) when treated with the culture of Pseudomonas putida strain LUA15.1. However, the crude as well as partially purified enzyme preparation of Pseudomonas putida strain LUA15.1 showed greater decolourization of dyes comparatively congo red (98%), indigo carmine (99%), RBBR ( 96%), aniline blue (37%) bromophenol blue (70%) and brilliant green (60%). The purified enzyme was successfully immobilized using encapsulation method in calcium alginate beads with 76% immobilization percentage and immobilized laccase enzyme beads were studied for their ability to degrade dyes. The stability and reusability of the immobilized enzyme system has the potential to make the entire treatment process inexpensive. An extracellular laccase producing gene has been isolated using degenerate primer based on the copper I and II conserved site of laccase enzyme, from the rice rhizospheric bacteria, Pseudomonas putida strain LUA15.1 followed by determination of the nucleotide sequence of this gene and it showed 91% similarity with Pseudomonas putida strain mt-2 Mn(II)-oxidation-associated multicopper oxidase (cumA) gene, partial cds. This nucleotide sequence of laccase was translated into amino acid and encodes a polypeptide comprised of 113 amino acids which showed 85 % identity with the amino acid sequences of bacterial laccases i.e. Mn (II)-oxidation-associated multicopper oxidase [Pseudomonas putida]. Further multiple sequence alignment using MULTALIN and structure prediction using Phyre 1 & 2 revealed conserved histidine residues.ThesisItem Open Access Agrobacterium MEDIATED cry 1Aa GENE TRANSFER IN Punica granatum L. cv. Kandhari Kabuli(2013) VERMA, VIPASHA; KANWAR, KAMLESHAbstract The present investigation aims at “Agrobacterium mediated cry 1Aa gene transfer in Punica granatum L. cv. Kandhari Kabuli”. A valuable plant regeneration and genetic transformation protocol was developed for Punica granatum L. cv. Kandhari Kabuli using both mature and juvenile explants. Regeneration from mature explants (leaf and nodal segment) procured from 8 years old selected tree and juvenile (cotyledon and hypocotyl) explants excised from 14 to 15 days old in vitro germinated seedlings of Punica granatum L. cv. Kandhari Kabuli was carried out through indirect and direct method. Calli were initiated from mature leaf, cotyledon and hypocotyl explants. The best media for callus induction from mature (leaf) and juvenile explants were MS medium supplemented with 20.0 μM NAA + 10.0 μM BA and MS medium supplemented with 12.5 μM NAA+15.0 μM BA, respectively. The highest percentage of callus was obtained from cotyledon (84.56%) explants followed by hypocotyl (77.71%) and leaf (76.72%) explants. The calli thus obtained from mature (leaf) and juvenile (cotyledon and hypocotyl) explants showed highest differentiation on MS medium supplemented with 8.0 μM BA + 2.5 μM kinetin + 2.5 μM NAA and MS medium supplemented with 11.0 μM BA + 2.5 μM NAA. The highest percentage of direct adventitious shoot bud induction from mature explants such as leaf (42.95%) and nodal segment (57.80%) was observed on MS medium supplemented with 10.0 μM BA + 2.5 μM NAA and MS medium supplemented with 9.0 μM BA. Solid MS medium supplemented with 9.0 μM BA + 8.0 μM kinetin + 2.5 μM NAA resulted in highest per cent shoot regeneration from both cotyledon (69.60%) and hypocotyl (58.80%) explants. Cotyledon explants was found to be most responsive explants for regeneration through direct as wells as indirect method. The adventitious shoots obtained were rooted on ½ strength MS medium containing 500 mg l-1 activated charcoal. Sixty five percent of plantlets were successfully established in earthen pots containing soil and sand (1:1). Different explants such as leaf, nodal segment, cotyledon and hypocotyl were transformed using Agrobacterium tumefaciens strain EHA105 harbouring pBinAR-1Aa plasmid carrying cry1Aa (insect resistance gene) and neomycin phoshotransferase-II (nptII) marker through both indirect and direct standardized regeneration protocols. Factors affecting transformation frequency such as age of seedlings, pre-conditioning duration, wounding of explants, immersion duration, co-cultivation duration, presence of acetosyringone and osmoprotectants (betaine HCl and proline) and pH of infection and co-cultivation medium, explants type were studied. Both age of seedlings and pre-conditioning of explants affected putative transformation frequency. Co-cultivation of all the explants (wounded mildly with needle) for 2 days after immersion in Agrobacterium suspension for 5 minutes was found to be optimal for transformation. Inclusion of acetosyringone (100 μM) and osmoprotectants (12 μM betaine HCl and 10 μM proline) in the infection and co-cultivation medium (adjusted at 5.8 pH) led to an increase in putative transformation frequency. By applying optimized transformation conditions, the highest putative transformation frequency of 15.12% was obtained with cotyledon explants among all the explants through indirect organogenesis. On the other hand nodal segment showed highest putative transformation frequency of 9.08% as compared to all the other three explants through direct organogenesis. Efficient selection was obtained and escapes were prevented when kanamycin was used at 50 mg l-1 concentration. The putative transgenic shoots were rooted on selective rooting medium and hardened in plastic cups containing sterilized sand. PCR analysis showed integration of cry1Aa and npt-II gene in 55% of the putative transgenic plantlets. Subsequent RT-PCR analysis showed expression of cry1Aa gene in 100% of the PCR confirmed transformed plantletsThesisItem Open Access STUDIES ON Agrobacterium-MEDIATED INSECT RESISTANCE GENE TRANSFER IN CABBAGE (Brassica oleracea L. var. capitata) AND MOLECULAR ANALYSIS OF REGENERATED PLANTLETS(2013) GAMBHIR, GEETIKA; SRIVASTAVA, D.K.ABSTRACT Genetic transformation studies were carried out to standardize a protocol for insect resistance gene (cryIAa) transfer in cabbage (Brassica oleracea L. var. capitata cv. Pride of India). Agrobacterium tumefaciens strain containing npt-II and cryIAa genes in binary vector pBin-1Aa was used for genetic transformation studies. Plant regeneration studies were carried out using four different types of explants viz. cotyledon, hypocotyl, leaf and petiole Cotyledon and hypocotyl explants were used from seven to nine days old aseptically grown seedlings whereas, leaf and petiole explants were procured from glass house 20-25 days old grown seedlings of cabbage. Hypocotyl explants showed better shoot regeneration as compared to other explants. High efficiency shoot regeneration was obtained in cotyledon (91.11 %), hypocotyl (94.44 %), leaf (91.11 %) and petiole (88.88%) explants on MS medium supplemented with 0.33mg/l TDZ + 79.7 mg/l Adenine, 0.22mg/l TDZ + 0.088mg/l IAA, 0.22mg/l TDZ + 0.02mg/l NAA and 0.33mg/l TDZ + 0.02mg/l NAA, respectively.MS medium supplemented with 0.10mg/l NAA was found best for root regeneration from in vitro developed shoots. The regenerated plantlets were acclimatized successfully on cocopeat. Kanamycin sensitivity experiment was carried out to study the effect of antibiotic on relative growth of leaf and petiole tissues and to select transgenic shoots during transformation experiment. Kanamycin sensitivity (10-60mg/l) was checked by fresh weight of the explants which was measured at the interval of 7 days. From the relative growth of the explants it was found that the concentration as low as 10mg/l is toxic to the explants. Effect of different concentrations of cefotaxime was studied on the regeneration potential in cotyledon and hypocotyl explants of cabbage and found no much effect of cefotaxime on regeneration potential. Effect of different concentrations of cefotaxime and kanamycin (50mg/l) were studied on the growth of agrobacterial cells and regeneration potential of cotyledon and hypocotyl tissues after cocultivation. In both the explants the growth of agrobacterial cells were controlled at concentration of 400mg/l cefotaxime and maximum per cent shoot regeneration in cotyledon (35.55 %) and hypocotyl (48.15 %) was obtained on MS medium supplemented with 400mg/l cefotaxime, respectively. Effect of preculturing and cocultivation was studied on the transformation frequency. Preculturing of cotyledon and hypocotyl explants for 72 hours and co-cultivation with agrobacterial cells for 48 hours worked out to be the best treatment as it gave the highest transformation frequency (4.66 %) and (14.50 %) in respective explants. Effect of different concentrations of acetosyringone was studied in cotyledon and hypocotyl explants to enhance the transformation frequency. The maximum percent shoot regeneration (18.66 %) and (32.00 %) was obtained from cotyledon and hypocotyl explants cultured on shoot regeneration medium containing 100μM acetosyringone at standardized preculturing and cocultivation time interval i.e. 72 hours and 48 hours. The presence/integration of transgene (cryIAa) into the genome of cabbage was confirmed by PCR using gene specific primers and Southern blot analysis using radioactive labelled DNA probe. The Southern blot analysis has also been used to confirm copy number of transgene into the genome of cabbage. For PCR analysis, 40 putative transgenic shoots were randomly selected and out of 40 putative transgenic shoots/plantlets, 20 shoots were found to be +ve for the presence/integration of transgene i.e. cryIAa into the genome of cabbage. For Southern blot analysis 10 RT-PCR +ve shoots were selected. Out of the 10 PCR +ve shoots, 5 shoots were confirmed +ve for integration of transgene cryIAa into the genome of cabbage with 1 to 3 copies of gene insertion. The confirmation of expression of the transgene cryIAa into the genome of cabbage at transcriptional level was confirmed by Reverse Transcriptase-PCR and Real Time-PCR and at translational level by Bioassay. A protocol for high frequency plant regeneration and insect resistance gene transfer in cabbage (Brassica oleracea L. var. capitata cv. Pride of India) has been standardized.ThesisItem Open Access STUDIES ON CONSTRUCTION OF FRAME WORK GENETIC LINKAGE MAP OF Stevia rebaudiana Bertoni USING MOLECULAR MARKERS(2013) SHARMA, NEHA; RAJINDER KAURABSTRACT The present investigation on Stevia rebaudiana was carried out with the objective to construct frame work genetic linkage map of stevia by employing multiple marker systems. Before starting the work on linkage map construction, genetic diversity was analyzed amongst the available genotypes/ clones/ accessions/ morphotypes of Stevia rebaudiana to confirm two parents with differences in rebaudioside-A and stevioside content. This study represents the first genetic linkage study of Stevia rebaudiana comprising of multiple marker systems together. Among the collected 16 accessions polymorphism was studied using 27 RAPD, 26 ISSR and 50 EST-SSR. Results were analyzed in the form of dendrograms, similarity, disimmilarity matrices and polymorphism information content. For linkage map construction, F2 population was used as a mapping population. To survey the polymorphism among contrasting parents 170 RAPD, 26 ISSR and 89 EST SSR were employed and it was observed that 36 RAPD, 10 ISSR and 33 EST – SSR primers were found to be polymorphic. These primers were then used for the genotyping of the mapping population. Phenotyping was carried out by using High Performance Liquid Chromatography (HPLC) of parents as well as segregating population. Both genotypic and phenotypic data were used to construct a linkage map using MAPMAKER/EXP ver 3.0b. A total of four linkage groups were constructed spanning a distance of 927.3 cM with an average distance between loci as 16.29 cM. First linkage group (LG1) comprised of 33 markers, second linkage group (LG2) contained 6 markers and third (LG3) and fourth group (LG4) had 16 and two markers, respectively. On QTL identification, a total of 53 QTL locations were found for both trait 1 (rebaudioside-A) and trait 2 (stevioside). Among 53 QTL locations of trait 1, two major QTLs were found for rebaudioside-A viz., in the marker interval of L67- L71 (ISSR HB-11) - (IISRS-3-L) in LG1 and in the marker interval L38 - L40 (Sigma-5383-027)- (Sigma-5383-029) in LG3 at LOD of 2.5 and 2.7, respectively.ThesisItem Open Access DEVELOPMENT OF HERBAL FORMULATION AGAINST PLANT VIRUS(ES)(2013) SHARMA, PRATIMA; BHARDWAJ, S.V.ABSTRACT A strain of PVMV belonging to Potyviridae family was identified as a mechanically transmissible virus exhibiting characteristic symptoms of mottling, mild mosaic, blister formation and crinkling resulting in stunted growth of plants and fruit distortion. Host range was restricted to few hosts belonging to Solanaceae and Chenopodiaceae families. Serological detection through DAS-ELISA revealed a strong positive serological relationship with PVMV antiserum. Employing RT-PCR and using primers CVMV1037Pol and P9502, resulted in an amplified product of approximately 1.2 Kb. Asparagus adscendens Roxb., root extract exhibited maximum antiviral activity against the test virus. On characterization, the antiviral activity was found to be associated with acetone precipitated protein fractions. Pre-inoculation treatment resulted in 96.52% inhibition of virus with respect to control. Lesser percent inhibition of 65.07 % was observed in systemic host plant Nicotiana tabacum L. “White Burley”. Partial purification of roots by solvent fractionation using organic solvents concluded acetone fraction to exhibit antiviral properties. 67.43 % inhibition was achieved when 50 ppm of the extract was applied on the systemic host leaves. Phytochemical analysis showed presence of various constituents viz. alkaloids, saponins, carbohydrates, glycosides, phytosterols and flavanoids to be present in different solvent extracts. When evaluated under in vivo conditions, the test chemical (with Indtron) was capable of inhibiting PVMV to maximum extent at a concentration of 50 ppm when sprayed after 7 day intervals. Virus indexing through RT-PCR resulted in amplified products that showed intense bands of the correct size (approx. 1.2 Kb) in the positive control, but a light intensity faint band was visible in antiviral principle treated plants showing maximum inhibition. Root extract of Asparagus adscendens Roxb., fractionated in acetone, dried in a rotary evaporator and then dissolved in minimum volume of DMSO supplemented with Indtron, is the basic formulation developed, ready to be used as stock solution which can be used at 50 ppm concentration as a spray against PVMV infection under field conditions.ThesisItem Open Access BIOPROSPECTING OF BACTERIA FOR PRODUCTION AND PURIFICATION OF LACCASE ENZYME(2013) DHIMAN, KARUNA; SHIRKOT, POONAMABSTRACT Laccase enzyme has acquired the status of ‘green catalyst’ as it possesses remarkable bioremediation potential along with numerous applications in effluent detoxification, degradation of textile dyes, herbicide and insecticide degradation, wine clarification, enzymatic conversion of chemical intermediates, biosensors and organic synthesis. In the present study, significant high diversity of laccase producing bacteria from rhizosphere of rice plants from paddy fields of H. P. was assessed whereas medium diversity was obtained from the samples of paper mills of H.P. A total of 449 bacterial isolates were obtained from 198 samples using M162 and TY media containing 5mM guaiacol and 40 mg/l CuSO4. These were rescreened on the basis of their ability to oxidise tannic acid and dimethoxyphenol leading to selection of 67 bacterial isolates which were characterized both morphologically and biochemically alongwith the laccase activity and 14 bacterial isolates exhibiting maximum laccase activity of 10-19 U/l were selected. Molecular characterization of the selected isolates was carried out using RAPD-PCR and 16S rrna gene technology and in silico analysis of 16S rrna gene sequences lead to identification of these bacterial isolates as Pseudomonas putida strain LUA15.1 and LHB7.1, Pseudomonas umsongenesis strain LHB9.1., Pseudomonas mohnii strain LHN12.2, Pseudomonas chlororaphis strain LUD7.1, Pseudomonas jessenii strain LHN9.1, Pseudomonas lurida strain LB6.2, Pseudomonas graminis strain LHN8.1, Pseudomonas veronii strain LUA14.1, Pseudomonas fulva strain LR5.1, Lysnibacillus fusiformis strain LKM7.1, Lysnibacillus sphaericus strain LH3.4 and Bacillus subtilis strain LB6.1 and LR6.3 . On the basis of maximum laccase enzyme activity Pseudomonas putida strain LUA15.1 was selected for production and purification of the laccase enzyme. Maximum extracellular enzyme production was achieved at 28°C, pH 7 (24 hrs incubation) with 5mM guaiacol, 50 mg/l CuSO4, 5% tryptone and 3% yeast extract in combination as nitrogen source in Tryptone Yeast medium. The laccase crude extracellular enzyme preparation was purified by ammonium salt precipitation (50-90%) followed by gel filtration and ion exchange chromatography which showed 10.74 yield and 61.36 fold purification. The purified enzyme had optimal activity at pH 7.0 and 40°C and 0.80 mM Km value. The molecular weight of laccase in the present study was found to be 42.5 kDa. The activity was inhibited by sodium azide and DTT. Strain LUA15.1 as well as its enzyme preparations were studied for their ability to decolourize dyes which are the potential contributors of water pollution. All six different synthetic dyes were decolourized RBBR (48%), congo red (35%), indigo carmine (80%), brilliant green (97%), bromophenol blue (78%) and aniline blue (23%) when treated with the culture of Pseudomonas putida strain LUA15.1. However, the crude as well as partially purified enzyme preparation of Pseudomonas putida strain LUA15.1 showed greater decolourization of dyes comparatively congo red (98%), indigo carmine (99%), RBBR ( 96%), aniline blue (37%) bromophenol blue (70%) and brilliant green (60%). The purified enzyme was successfully immobilized using encapsulation method in calcium alginate beads with 76% immobilization percentage and immobilized laccase enzyme beads were studied for their ability to degrade dyes. The stability and reusability of the immobilized enzyme system has the potential to make the entire treatment process inexpensive. An extracellular laccase producing gene has been isolated using degenerate primer based on the copper I and II conserved site of laccase enzyme, from the rice rhizospheric bacteria, Pseudomonas putida strain LUA15.1 followed by determination of the nucleotide sequence of this gene and it showed 91% similarity with Pseudomonas putida strain mt-2 Mn(II)-oxidation-associated multicopper oxidase (cumA) gene, partial cds. This nucleotide sequence of laccase was translated into amino acid and encodes a polypeptide comprised of 113 amino acids which showed 85 % identity with the amino acid sequences of bacterial laccases i.e. Mn (II)-oxidation-associated multicopper oxidase [Pseudomonas putida]. Further multiple sequence alignment using MULTALIN and structure prediction using Phyre 1 & 2 revealed conserved histidine residues.