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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 BIOPROSPECTING OF SILVER NANOPARTICLES SYNTHESIZING BACTERIA AND POTENTIAL APPLICATIONS(UHF,NAUNI, 2017) SHARMA, ANKITA; SHIRKOT, POONAMABSTRACT The synthesis of nanostructured materials, especially metallic nanoparticles, has accrued utmost interest over the past decade owing to their unique properties that make them applicable in different fields of science and technology. The biological method of nanoparticles synthesis is a relatively simple, cheap and environmentally friendly method than the conventional chemical and physical methods of synthesis. Therefore, isolation and identification of bacteria with ability of silver nanoparticles synthesis from natural sources is very important in terms of discovering new industrial products. Keeping in view, a silver mine located at Uchich village of Pulga valley, apple orchards located in Chong village and two hot water springs located in Manikaran and Vashisth in Kullu district of Himachal Pradesh were selected as a source for new silver nanoparticles synthesizing bacteria. Therefore, aim of present study was the isolation and characterization of silver nanoparticles synthesizing bacteria from these sites for biosynthesis of silver nanoparticles followed by applications of these biosynthesized silver nanoparticles to control various plant pathogens, textile dye degradation, phytotoxicity study and pesticide degradation. A total of 106 putative silver nanoparticles synthesizing bacterial isolates were obtained from 45 samples collected from different selected sites. Thirty bacterial isolates were selected on the basis of their ability to show maximum silver nanoparticles synthesizing activity which were characterized morphologically and biochemically. Four bacterial isolates exhibiting maximum silver nanoparticles synthesizing activity viz., UMAS1, UMBS1.1, UMBP1 and UMBP2 were selected for further, molecular characterization using 16S rrna gene technology. In silico analysis of 16S rrna gene sequences led to identification of these bacterial isolates and all the four were found to belong to genus Bacillus and were identified as Bacillus siralis strain UMAS1, Bacillus siralis strain UMBS1.1, Bacillus algicola strain UMBP1 and Bacillus tianmuensis strain UMBP2. On the basis of maximum silver nanoparticles synthesizing activity Bacillus siralis strain UMBS1.1 was selected for in vitro biosynthesis of silver nanoparticles. Maximum silver nanoparticles synthesis was achieved at 60°C, pH: 8.0 and after 24 hrs of incubation with 3.0mM silver nitrate, 3.0 % tryptone, 3.0 % yeast extract and 2.0% inoculum size. Central Composite Design was used to determine the optimal values of incubation time (A), incubation temperature (B), pH (C), tryptone concentration (D) and yeast extract concentration (E). The highest activity was obtained from Run number-22, which consisted of incubation time of 46.0 hrs; incubation temperature of 36.5°C; pH 8.5, 3g/l tryptone and 3g/l yeast extract leading to 16.06 fold increase in silver nanoparticles activity. In vitro biosynthesis of silver nanoparticles was carried out using optimum conditions which were characterized using UV-visible spectroscopy, Fourier transform infrared spectroscopy (FTIR), X-rays diffractions (XRD), Scanning Electron Microscopy (SEM) and Dynamic Light Scattering (DLS). In the present study, ability of silver nanoparticles and bacterial culture preparation of Bacillus siralis strain UMBS1.1 was assessed for degradation of eight textile dyes produced significant results. Both Bacillus siralis strain UMBS1.1 culture preparation as well as silver nanoparticles has significant potential for use in the detoxification of eight textile dyes and for treating textile waste waters including water recycling. Silver nanoparticles and bacterial culture preparation were also found to inhibit various fungal and bacterial pathogens under in vitro conditions appreciably. Silver nanoparticles and Bacillus siralis strain UMBS1.1 culture preparation were also found to degrade chloropyrifos significantlyThesisItem Open Access Molecular characterization of turnip mosaic potyvirus (TuMV) infecting radish (Raphanus sativus L.)(YSPU, 2012) Parmar, Nehanjali; Bhardwaj, S. V.Radish (Raphanus sativusL.) is an edible root crop of family Brassicaceaewhich is grown worldwide especially in Asia. Turnip mosaic potyvirus(TuMV; genus: Potyvirus, family: Potyviridae) is considered as one of the most important viruses in the world that infect field-grown vegetables and displays a large natural as well as experimental host range. TuMV was found to be prevalent in different regions of India. In all, nineisolates were collected on the basis of symptoms from areas comprising Himachal Pradesh (Mandi, Solan, Shimla and Kinnaur), Chandigarh, Punjab (Ludhiana), Haryana (Karnal), New Delhi (West Patel Nagar) and Rajasthan (Bharatpur). During serological detection, ELISA tests were conducted. All seven isolates except from New Delhi and Rajasthan reacted positively with monoclonal antibodies against TuMV. These studies were further confirmed through RT-PCR using specific primers for coat protein (CP) gene as a molecular detection procedure. A cDNA of approximately 1000 bp was amplified from all the seven TuMV Indian isolates. The RTPCR products were subsequently cloned and sequenced. The sequenced product of all the seven TuMV Indian isolates (IND1-IND7) was approximately 986 bp whichcomprised of 54 bp of the 3´ end of nuclear inclusion b (NIb) gene, the whole CP gene and 65 bp of the 3´ untranslated (UTR) region. CP gene of all the seven Indian isolates of TuMV was 867 bp long, encoding 288 amino acid residues which were submitted to NCBI. Accession numbers JQ246074 to JQ246080 and AFE55681to AFE55687 were assigned to seven TuMV Indian isolates IND1 to IND7 CP gene nucleotide and amino acid sequences, respectively. Conserved motif DAG (Asp-Ala-Gly) and NAG (Asn-Ala-Gly), which has beenreported to be important for potyvirustransmission by aphids, were found at positions 6-8 and 56-58 aa residues, respectively in the seven TuMV Indian isolates CP gene sequences. Another conserved motif, GDD (Gly-Asp-Asp) which has been identified as a hallmark of RNA dependent RNA polymerase was observed at 158-160 aa position in all the seven CP gene sequences of Indian isolates of TuMV. Percent homology of CP gene of seven Indian isolates among themselves and with other TuMV isolates retrieved from NCBI database was within the range of 87-99% and 92-100% at nucleotide and amino acid level, respectively. Phylogenetic analysis based upon nucleotide and amino acid sequences using UPGMA, NJ, MP and ME methods inferred classification of seven TuMV Indian isolates, tentatively into basalBR group, due to its occurrence nearest to those isolates of TuMV which have been earlier classified to this group. Conserved domain for TuMV CP gene was observed at 51-287aa position in all the seven test Indian isolates. Computational predictions for various restriction enzymes were also carried out. Alpha helixconsensus secondary structure was predicted and found to dominate in all the seven protein sequences of CP gene of Indian isolates of TuMV.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 STUDIES ON CHARACTERIZATION AND CLONING OF BOWMAN-BIRK TRYPSIN INHIBITOR GENE FROM A LOCAL BEAN (Phaseolus vulgaris L.) CULTIVAR(UHF,NAUNI, 2017) SUBHASH, CHAND; NATH, AMARJIT K.ABSTRACT Legumes are rich source of proteases inhibitors (PIs) and the particular are known as most promising weapons that confer resistance against insects by inhibiting proteases present in gut of insect larvae. In the present study, before selecting a cultivar for isolation of trypsin inhibitor gene, the local bean cultivars were characterized using EST-SSR markers. Out of 25 EST-SSR primers used for characterization, 19 primers were selected to analyze polymorphism present among bean cultivars. Total number of bands amplified was 26, out of which 24 (92.31%) were polymorphic and 2 (7.69%) were monomorphic. The numbers of unique bands obtained were 2 and the size of amplified fragments ranged from 70 bp to 800 bp. Dendrogram constructed using UPGMA method grouped the bean cultivars into two major clusters. Similarity matrix was constructed using Jaccard’s coefficients and the value of similarity coefficient ranged from 0.17 to 0.90. Maximum similarity coefficient (0.90) was obtained between cultivar Capsule and Kaju and minimum (0.17) between Chitra and Luxmi. Screening of Phaselous vulgaris L. cultivars for total and biological inhibitor activity revealed maximum activity in Baspa cultivar and further studies were conducted in this cultivar. Total trypsin inhibitor activity was detected at early stage of seed development, 10 days after flowering (DAF) and it increased progressively with seed development (10-60 DAF). The inhibitor activity decreased during seed germination as compared to dry seeds. Crude trypsin inhibitor extracted from developing and germinating seeds also inhibited larval midgut proteases of Pieries brassicae, Helicoverpa armigera and Spodoptera littoralis. Significant reduction in egg hatching was observed in egg masses treated with crude trypsin inhibitor protein extracted from mature seeds. The inhibitor protein was purified by ammonium sulfate precipitation, gel filtration chromatography and ion exchange chromatography on DEAE-Sephadex column. The purified inhibitor protein showed antimicrobial activity against fungus (viz., Alternaria sp., Fusarium oxysporium, Phytophtora nicotiana, Pythium sp. and Rhizoctonia solani) and bacteria (viz., Providencia stuartii, Bacillus subtilis and Pseudomonas aeroginosa). The trypsin inhibitor gene (372 bp) was amplified from cDNA synthesized from developing seeds (40 DAF) of Baspa cultivar using designed primers. Amplified PCR product was cloned and sequenced. The isolated gene was also integrated into plant and bacterial expression vector pBI121 and pET 23a, respectively. Sequence of Phaseolus vulgaris L. Baspa cultivar trypsin inhibitor gene has been submitted to NCBI with accession no. KX703026. Trypsin inhibitor gene and its deduced amino acid sequence showed homology with Bowman-Birk inhibitors of legumes crops. The predicted molecular weight of deduced amino acid sequence was ~ 13.4 kDa and it had N terminal signal peptide of 29 amino acid residues. The secondary structure of deduced amino acid sequence of trypsin inhibitor gene showed dominance of coils and sheets over alpha helix. Homology modeling was employed to predict the three dimensional structure of protein. Ramachandran plot for Phaseolus vulgaris L. cultivar Baspa trypsin inhibitor showed that the model generated by GENO3D2software had most residues in favorable region and had overall good quality.ThesisItem Open Access MOLECULAR INVESTIGATIONS ON GROWTH AND DEVELOPMENT OF IN VITRO RAISEDSOMACLONES OF Dianthus caryophyllus L. cv. ‘Master’(UHF,NAUNI,SOLAN, 2017) THAKUR, KALPNA; KANWAR, KAMLESHABSTRACT The present investigation aims at “Molecular investigations on growth and development of in vitro raised somaclones of Dianthus caryophyllus L. cv. ‘Master’”. A standard plant regeneration and in vitro selection protocol was developed for Dianthus caryophyllus L. cv. ‘Master’. Indirect organogenesis from leaf and nodal explant was carried out. Callus was induced from leaf explant on solid MS medium supplemented with 2.0 mg/l 2,4-D in combination with 0.5 mg/l NAA while in case of nodal explant the best treatment for callus induction was found out to be solid MS medium supplemented with 1.5 mg/l 2,4-D and 1.0 mg/l NAA. Highest percentage of callus was obtained from leaf (94.44%) explants followed by nodal (79.17%) explants. Shoots were induced from leaf and nodal derived calli on solid MS medium supplemented with 1.50 mg/l TDZ, 0.25 mg/l Kinetin and 0.25 mg/l NAA with 80.56% and 65.28% shoot bud induction. Solid MS medium supplemented with 2.00 mg/l Kinetin, 0.25 mg/l NAA and agar concentration of 1.0 per cent was found best for in vitro shoot multiplication, which resulted in 14.64 average number of microshoots per explant. The regenerated shoots were rooted in half strength MS basal medium supplemented with 1.5 mg/l IBA, 200 mg/l activated charcoal and 0.6% agar concentration. Gamma rays emitted by unstable nuclei of Cobalt-60 was used for physical mutagenesis of leaf callus of Dianthus caryophyllus L. cv. ‘Master’. Leaf derived calli were exposed to five different doses of gamma radiations (10, 20, 30, 40 and 50 Gy) from Cobalt-60 as a source of mutagen. Control showed 100% survival percentage at 4 and 8 weeks. A steady decrease in the survival was observed with increase in the dose of radiation with passage of time. Lethal dose value (LD50) calculated from percent survival of gamma irradiated calli at different treatments which was found close to 30.5 Gy. For chemical mutagenesis, four different percent (v/v) solutions of EMS were used for two time intervals (15 and 30 minutes) and untreated calli served as control. The highest survival percentage of EMS treated callus was recorded on the lower concentrations of EMS used for lesser time. The LD50 was approximately 0.16 % (v/v) for 15 minutes EMS treatment and in case of 30 minutes EMS treatment, LD50 was calculated to be 0.08 % (v/v). There was a decrease in the percent shoot bud induction, number of microshoots per explant, rooting percentage and decrease in the survival frequency after hardening with the increase in dose/duration of gamma and EMS treatment in all the treatments than the control. Five variants were obtained from gamma treated leaf callus induced plants, one mutant from 10 Gy, 2 from 20 Gy, 1 from 30 Gy and 1 from 40 Gy gamma treatment. Similarly, five variants were obtained from EMS treated leaf callus induced plants, obtained with 0.10%, 0.15% and 0.20% EMS treatment for 15 min and 0.10% and 0.15% EMS treatment for 30 min. A total of 20 Randomly Amplified Polymorphic DNA (RAPD) primers, 20 Inter Simple Sequence Repeats (ISSR) primers and 15 Simple Sequence Repeats (SSR) primers were used for genetic variation studies in selected variants. Only 16 RAPD, 14 ISSR and 10 SSR primers were able to amplify the genomic DNA of mother plant, control and selected variants raised through gamma and EMS treated leaf callus, respectively. In gamma treated variants, similarity value of 0.57-0.74, 0.50-0.79 and 0.39-0.76 were observed from RAPD, ISSR and SSR studies, respectively which showed high genetic difference. In EMS treated variants, RAPD studies showed 0.44-0.71 similarity value whereas 0.51-0.80 similarity value was observed in ISSR studies and SSR studies showed 0.32-0.739 similarity value. Gamma and EMS treated selected variants showed random grouping of selected variants by all the three markers used for the study which showed variation between the selected variants, mother plant and control. It is hoped that the in vitro EMS and gamma induced mutations can open up a new approach for the breeding of carnation cultivar ‘Master’. These results must be regarded as preliminary studies because of the small size analyzed, the low number of used primers and the low number of generated RAPD, ISSR and SSR bands.ThesisItem Open Access IN VITRO MUTATIONS, SELECTION AND MOLECULAR MARKERS BASED CHARACTERISATION OF Gerbera jamesonii Hook.(2012) GHANI, MINERVA; SHARMA, S.K.ABSTRACT In the present studies, entitled ‘In vitro mutations, selection and molecular markers based characterisation of Gerbera jamesonii Hook.’, the regeneration protocols from capitulum and petiole explants have been standardised. MS + 5.0 mg/l BA + 0.5 mg/l IAA and MS + 4.0 mg/l BA + 1.0 mg/l TDZ were found to be the best media for regeneration from capitulum and petiole explants, respectively. The best multiplication medium comprised of MS + 1.0 mg/l BA + 0.5 mg/l IAA and MS + 4.0 mg/l IBA was found to be best medium for root induction. Shoots were treated with eight doses on gamma rays (1.5, 2, 2.5, 5, 10, 15, 20, 30 Gy) and five doses of EMS (0.1, 0.2, 0.5, 0.8, 1.0%, v/v) for two durations (10 and 20 min). Following gamma irradiation maximum survival of plants both in vitro and in vivo was recorded with 1.5 Gy dose and with EMS treatment 0.1 %/ 10 min led to the maximum plant survival both in cultures and in the glasshouse. The LD50 value for gamma rays was 6.5 Gy and for EMS (10min) LD50 value was 6.5 % and less than 0.1 % in case of 20 min treatment of EMS. The increase in the content/activity of proteins, phenols and antioxidant enzymes (SOD, APOX, GR, CAT) and PPO was observed in both kinds of mutated plants. However, no significant difference in sugar content was observed. However, decrease in the total chlorophyll content were observed in both types of mutated plants. Morphologically, mutagenesis (physical and chemical) led to reduction in the number of leaves and leaf area of plants. However, a significant increase in the length of flower scape and flower head size was observed with both kinds of mutagens. Three flower colour variants, two with EMS (0.1%/10 min, 0.5 %/ 10 min) and one with gamma rays (5 Gy), were obtained. Also, a significantly early flowering was induced in the plants after mutagenesis. RAPD and SSR markers revealed 100 % and 96 % polymorphism and value of Jaccard’s similariy coefficient ranged from 0.11-0.80 in case of RAPD markers and 0.23-0.81 in case of SSR markers. All the plants were differentiated in to three major groups, after RAPD and SSR analysis, based on their similarity as represented graphically, from the dendrogram