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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 IN VITRO SELECTION OF CELL LINES IN Punica granatumL. (DARU) AGAINST BACTERIAL BLIGHT(UHF,NAUNI, 2013) GARIMA, KUMARI; KANWAR, KAMLESHABSTRACT The present investigation aims at “In vitro selection of cell lines in Punica granatum L. (Daru) against bacterial blight. Cotyledon and hypocotyl segments of 3 weeks old in vitrogerminated seedlings and mature leaf were used as explants. For leaf explants the sterilization protocol was standardized with 0.2 % bavistin treatment for 10 minutes and 0.5% sodium hypochloride treatment for 5 minutes. Callus induction and plantlet regeneration varied with explant type and growth regulators. The best callus induction medium for all the explants was MS medium supplemented with 2.0 mg/l BA and 4.0 mg/l NAA. Hypocotyl and cotyledon were more responsive explant and showed 96.67 and 85.00 per cent callus induction. The best medium for callus differentiation and shoot bud induction fromcotyledon derived callus was MS medium supplemented with BA (2.0 mg/l), Kinetin (1.5 mg/l) and GA 3 (3.0 mg/l), while best medium for shoot bud induction from leafderived callus was MS medium containing BA (2.0 mg/l) and Kinetin (0.5 mg/l). Cotyledon showed better regeneration (81.67 percent) as compared to leaf explant (48.33per cent). No regeneration was observed in hypocotyl derived callus explants. Rooting of in vitro raised shoots was done on half strength MS medium supplemented with 0.04 % charcoal. The well rooted plantlets were acclimatized in autoclaved sand. Cell line selection was done by using bacterial culture filtrate of Xanthomonas axonopodis pv. punicae as a selective agent. Resistant lines were selected at 40 per cent level of culture filtrate after two cycles of selection. Multiplication and shoot regeneration from selected calli was obtained on previously standardized medium. Fresh weight of callus increased progressively upto third subculture passage while shoot bud induction from selected calli was observed only after third subculturing. The selected microshoots were rooted on the rooting medium. After in vitro testing of shoots regenerated from selected calli 4 resistant plantlets were obtained.ThesisItem Open Access In vitro PROPAGATION AND CONSERVATION OF Swertia chirayita(UHF,NAUNI, 2013) SHAILJA; KANWAR, KAMLESHAbstract A protocol for in vitro propagation and conservation was developed for Swertia chirayita, an endangered medicinal plant. The sterilized explants (leaves) cultured on MS medium supplemented with 0.1 mg/l NAA and 3.0 mg/l BA gave best results for in vitro callus induction. Shoot regeneration was obtained from the callus on the same medium. The in vitroshoots cultured on MS medium supplemented with 2.5mg/l BA and 0.1 mg/l Kinetin gave best results for in vitro shoot multiplication. The MS medium supplemented with 0.1 mg/l NAA and 3.0 mg/l BA medium was found to be thebest for direct shoot regeneration from in vitroleaves. 80.30% root induction from in vitro grown shoots was obtained on half strength MS medium supplemented with 400 mg/l activated charcoal and 0.1 mg/l NAA. In vitro conservation was carried out by using two different approaches namely slow growth by changing media composition (sucrose and abscisic acid), at low temperature and cryopreservation following vitrification.With increase in concentration of sucrose and ABA decrease in growth of in vitroshoots was observed. No shoot multiplication with average leaf size of 0.35 cm and shoot length 0.67 cm was observed on half MS containing 90 g/l sucrose. Similarily, in case of media containing half strength MS salts and 3.0 mg/l ABA showed no shoot multiplication,0.83 cm average leaf size and 0.83 cm shoot length. At low temperature the in vitro shoots incubated at 4 o C, showed 100% retrieval, with 1.00 cm average number of shoots, 0.86 cm shoot length and 0.34 cm leaf size. In vitro shoots incubated at 10 o C, showed 100% retrieval, with 1.00 cm average number of shoots, 0.76 cm shoot length and 0.23 cm leaf size. During studies the vitrified shoot gave retrieval of 42.33% when precooled at 4 o C while only 22.37% vitrified shoots were retrieved from those precooled at 10 o C.ThesisItem Open Access GENETIC TRANSFORMATION STUDIES IN LETTUCE (Lactuca sativaL.) WITH npt-IIAND gusGENES(UHF,NAUNI, 2014) SHAUNAK, ISHANI; SRIVASTAVA, D.K.ABSTRACT Genetic transformation studies were carried out to standardize a protocol using Agrobacterium-mediated gene transfer technique. High efficiency plant regeneration protocol is a prerequisite of genetic transformation, hence the plant regeneration protocol was also standardized. Two types of explants viz leaf and petiole were used for plant regeneration studies. High percentage shoot regeneration in petiole (70.73%) and leaf (70.10%) explants were obtained on MS medium containing 0.25mg/l BAP+0.10mg/l NAA and MS medium containing 1.00 mg/l BAP+0.10mg/l NAA respectively. MS medium supplemented with 0.50mg/l IBA was found to be best for root regeneration (68.00%). Kanamycin sensitivity experiment was carried out to study the effect of antibiotic on relative growth of leaf and petiole explant and to select transgenic shoots during genetic transformation experiment. Kanamycin concentration above 30mg/l was toxic to the explants on selective shoot regeneration medium. Disarmed Agrobacterium tumefaciens LBA 4404 strain containing a reporter β- glucuronidase [uidA (gus)] gene in binary vector (pBI 121) system along with kanamycin resistance gene (npt-II) for selection in both bacteria and plant was used for genetic transformation studies in lettuce. After co-cultivation only the transformed cells/tissues were able to grow on selective shoot regeneration medium (25mg/lKanamycin and 400mg/l cefotaxime) whereas non-transformed cells/tissues turned brown and died on the selective medium. Transformation experiment could be scored as early as 5-6 weeks after selection. The effect of pre-culturing and cocultivation was studied on transformation frequency. Explants pre-cultured for 48 hours and cocultivated for 48 hours resulted in shoot regeneration. Putative transformed shoots obtained from leaf and petiole explants, were able to grow on the selective medium containing 25mg/l Kanamycin. The presence of npt-IIand uid A (gus)was confirmed by PCR using designed primers in thetransgenic shoots. Out of 5 putative transformed shoots, 3 have shown the amplification of npt-IIand 4 shown the amplification of uid A (gus)genes. Expression of uid A (gus) gene was studied in the PCR positive shoots by β-glucuronidase assay using biochemical and histochemical methods. The transformed shoots were GUS positive. A protocol for genetic transformation in lettuce with npt-IIand gusgenes has been standardized.ThesisItem Open Access EVALUATION OF GENETIC RELATEDNESS AMONG TEMPERATE POME FRUIT CROPS OF FAMILY ROSACEAE USING PCR BASED MOLECULAR MARKERS(UHF,NAUNI, 2014) SHARMA, HIMANI; SHARMA, RAJNISHABSTRACT The family Rosaceaeis third most economically important plant family that include temperate pome fruit crops having a wide genetic diversity. For a rationale use of the genetic resources in breeding programs it is necessary to understand relationship within and among related crop varieties and to assess their transferability to the species with little genomic information making them significant for genetic research. Thus, in the present study genetic relatedness and SSRs transferability among fifty genotypes of temperate pome fruit crops were tested using (RAPD, ISSR and SSR) molecular marker analysis. The level of polymorphism across genotypes was 97.29% and 97.45% by RAPD and ISSR markers, respectively. The dendrogram derived from RAPD and ISSR analysis grouped temperate pome fruit crops into two major cluster comprising of 40 (cluster A) and 10 (cluster B) genotypes which branched at similarity value of 0.31 and 0.33, respectively. Estimates of pooled similarity coefficient ranged from 0.30 to 0.94. It was concluded from the pooled analysis of both the molecular marker that 50 genotypes of temperate pome fruit crops were grouped in two clusters A and B comprised 45 and 5 genotypes, respectively which branched at similarity value of 0.30 with the maximum similarity of 94% was found between EC-02520 and EC-024530 quince genotypes. Further, out of 13 and 15 screened apple and pear genomic SSR, 10 (76.92%) and 11 (73.33%) produced amplification in 20 apple and pear genotypes, respectively. Highest transferability of apple and pear SSR, 61.53% and 73.33% was observed in closely related quince and apple genotypes, respectively. This high level of polymorphism and good transferability of apple and pear SSRs to other temperate pome fruit crops indicated their promise for application to future molecular screening, map construction, and comparative genomic studies, etc.ThesisItem Open Access Refinement of technology for micropropagation of carnation (Dianthus caryophyllus L.) cv. ‘Master’(UHF,NAUNI, 2015) THAKUR, KANIKA; KANWAR, KAMLESHABSTRACT The present investigation aims at “Refinement of technology for micropropagation of carnation (Dianthus caryophyllus L.) cv. ‘Master’”. Surface sterilized shoot buds cultured on control (MS + 3% sucrose + 0.8% agar-agar) supplemented with 2.0 mg/l BA which showed 77.77% establishment. Best medium for multiplication was MS medium supplemented with 2.0 mg/l BA producing 4.50 number of microshoots of shoot length of 2.44 cm, but as it resulted in hyperhydricity of shoots so medium supplemented with kinetin (2.0 mg/l) was used which produced 3.67 number of microshoots and 4.55 cm shoot length. Low cost medium containing 30 g/l table sugar, 50 g/l starch and aquaguard water produced 3.52 shoots with 3.01 cm shoot length. It was also observed that subculturing passage affected the average number of shoots per explant and also the shoot length which was highest in case of fifth subculturing resulting in 5.58 and 5.27 number of shoots per explant on control and low cost medium and shoot length of 4.66 cm and 3.53 cm , respectively. 62.49% rooting and 4.50 average roots per shoot with 1.61cm was found on 1/2 MS + 0.04% activated charcoal and 48.58% rooting with 3.50 average roots per shoot and 1.75 cm was observed on 1/2 MS + 50g/l starch + 0.04% activated charcoal + 15 g/l table sugar. The study has resulted in the reduction cost of the medium by substituting agar-agar and sucrose with starch and table sugar.ThesisItem Open Access BIOPROSPECTING OF THERMOTOLERANT BACTERIA FROM HOT WATER SPRINGS OF HIMACHAL PRADESH FOR PRODUCTION OF THERMOSTABLE PULLULANASE ENZYME(UHF,NAUNI, 2015) MANJUL, ANSHUL SHARMA; SHIRKOT, POONAMABSTRACT Thermophiles are the organisms which are adapted tolive at high temperatures. The survival of these organisms at high temperature is possible due to the thermostability of their enzymes. Many thermostable enzymes such as Taq DNA polymerase, pullulanase, amylase, lipase, protease, cellulase, RNA polymerase etc. find a number of commercial applications because of their thermostability. Therefore, the isolation of thermophilic bacteria from natural sources and their identification are very important in terms of discovering new industrial enzymes. Keeping in view, hot water springs of Himachal Pradesh could serve as a good source for new thermophilic microorganisms with novel industrially important properties. Therefore aim of the present study was the isolation and characterization of thermotolerant bacteria from hot water springs of Himachal Pradesh for production of thermostable pullulanase enzyme. Six hot water springs viz., Manikaran, Vashisht, Khirganga, Kasol, Kalath and Tattapani were purposely selected for the present studies. Samples consisted soil, water, rock matting and pebbles were collected from each hot water spring. The pH and temperature of the six thermal springs ranged from 5.7-9.5 and 35-106 o C respectively. The chloride, sulphate, total hardness, calcium hardness, CaCO 3 and magnesium content ranged from 578-1978, 09-54,32.9-141.3, 22-134, 52-283 and 3.15-26.75 mg/l respectively. Fifty eight bacterial isolates were isolated from the samples using nutrient agar medium. Bacterial population in water was highest (3.45×10 5 cfu/ml) at S1 site of Tattapani and minimum (0.76 ×10 5 cfu/ml) bacterial population was noted at S2 site of Kalath. Similarly Bacterial population in soil was highest (3.54×10 5 cfu/g) at S3 site of Vashisht and minimum (0.54 ×10 5 cfu/g) at S3 site of Kalath. All the bacterial isolates were studied for various morphological characters and on the basis of ability to grow on KWF medium seven bacterial isolates were selected, which were further investigated for biochemical characters. Quantitative screening was done to select one bacterial isolate showing maximum thermostable pullulanase activity. KS2W bacterial isolate was found to show maximum thermostable pullulanase activity of 3.96 U/l and was selected further for molecular characterization. Genomic DNA was isolated from the selected bacterial isolate KS2W. PCR amplification of the isolated DNA was carried out using universal primers for 16S rrnagene and an amplicon of 1500 bp was obtained. Sequencing of the PCR product was done using same primers. Sequence of the KS2W bacterial isolate so obtained was found to be 556 bp. In silico analysis using BLASTn showed 96% homology of the query sequence with Bacillus licheniformisstrain BCRC 11702. A total of 20 sequences were mined and these sequences were used to compare the 16S rrnagene sequence of the test isolate KS2W. Alignment score was highest for Bacillus licheniformisstrain BCRC 11702, 16S ribosomal RNA, partial sequence (NR_116023.1).Phylogenetic analysis based on nucleotide sequences using NJ method was achieved via MEGA 5.0. Bacillus licheniformis strain ASM3 was studied further for enzymatic studies. Optimization of culture conditions for growth and thermostable pullulanase activity of isolate Bacillus licheniformisstrain ASM3 was carried out. The crude extracellular extract was produced using optimized conditions and further partially purified using ammonium sulphate precipitation, gel chromatography and ion exchange chromatography. The procedure yielded 3.96 g protein with 29.04 folds of purification with a percent yield 15.48%. Molecular weight of the partially purified enzyme was found to be ~96 kDa using SDS-PAGE gel.ThesisItem Open Access STUDIES ON GENETIC DIVERSITY IN Rubus ellipticus (SMITH) USING MOLECULAR MARKERS(UHF,NAUNI, 2014) SAMRITI; KAUR, RAJINDERABSTRACT Rubus ellipticus Smith. commonly known as ‘Yellow Himalayan raspberry’ is an important member of Rosaceae family with high medicinal importance having high antioxidant and antibacterial properties. In the present study EST-SSR markers were datamined for R. ellipticusand were used for polymorphism studies. EST sequences of R.ellipticus /Rubus species were downloaded from NCBI website (www.ncbi.nlm.nih.gov/nucest). Seven EST sequences for R.ellipticusand 3184 for R.ulmifoliusand R.idaeuswere obtained. ESTs containing SSR motifs were extracted out using an online tool, SSRIT (www.gramene.org/db/searches/SSRtool). None of the R.ellipticusESTs contained any SSR motif, so EST sequences obtained for R.ulmifoliusand R.idaeuswere used for SSR extraction. SSR primers were designed from the EST-SSR containing sequences using PRIMER 3 software (www.frodo.wi-mit.edu/primer3/) and 20 primers were custom synthesized. Polymorphism study was carried out using 35 ISSR and 20 SSR primers among 21 collections of Rubus ellipticus. For polymorphism, DNA was isolated from young leaves of all the 21 collections using CTAB method (Doyle and Doyle, 1987). All EST-SSR and ISSR primers showed amplification, revealing 100% polymorphism. Jaccard’s similarity matrix was developed and dendrograms were generated using NTSYSpc ver.2.02h to establish the percent similarity among the 21 collections of Rubus ellipticus. With EST-SSRs, two clusters ‘A’ and ‘B’ were obtained. Cluster A contained 19 collections whereas cluster B contained two collections and Cluster A was further divided into sub clusters i.eA1 and A2 at similarity value of 3%. Maximum similarity i.e 67% was found between ‘Badhu-2’ and ‘Khadiana’. For ISSRs, 21 collections were divided into two cluster i.e ‘C’ and ‘D’. Cluster C contained six collections whereas cluster B contained 15 collections and Cluster C and D were further subdivided into clusters C21, C22 and D21, D22 at 9% similarity. Maximum similarity i.e 56% was found between ‘Kharkog-1’ and ‘Nagangi’ showing that these two are relatively closely related. Thus EST-SSRs and ISSRs used in the present study revealed a high level of polymorphism in the 21 colllections of Rubus ellipticus, revealing their efficiency for diversity analysis studies.ThesisItem Open Access STUDIES ON DEVELOPMENT OF GENIC-SSRs IN RASPBERRY (Rubus ellipticusSmith.) AND THEIR TRANSFERABILITY ACROSS RELATED SPECIES(UHF,NAUNI, 2013) THAKUR, RASHMI; KAUR, RAJINDERABSTRACT Rubus ellipticus Smith. commonly known as ‘Yellow Himalayan raspberry’ is an important member of Rosaceae family with high medicinal importance having high . In the present study EST-SSR markers were datamined for R. ellipticusand were used for crosstransferability studies. EST sequences of R. ellipticus /Rubus were downloaded from NCBI website (www.ncbi.nlm.nih.gov/nucest). Seven EST sequences for R.ellipticusand 3184 for other Rubusspecies, R. ulmifoliusand R.idaeuswere obtained. ESTs containing SSR motifs were extracted out using an online tool, SSRIT (www.gramene.org/db/searches/SSRtool). None of the R.ellipticusESTs contained any SSR motif, so EST sequences obtained for R.ulmifoliusand R.idaeuswere used for SSR extraction. SSR primers were designed from the EST-SSR containing sequences using PRIMER 3 software (www.frodo.wimit.edu/primer3/) and 20 primers were custom synthesized. SSR studies was carried out using ten Rubus species (four R. ellipticus collections of different geographical origin, R. ulmifolius, R. hypargyrus, R. panniculata, R.nutans, R.macilentusand R.strigosus). To study polymorphism and transferability among the ten Rubus species, DNA was isolated from young leaves of all the ten species using CTAB method (Doyle and Doyle, 1987). The polymorphism study among ten Rubus accessions was carried out with the 20 custom synthesized Rubus primers. All 20 primers showed amplification, with polymorphism of 98.36%. The transferability studies were also carried out using already used 20 polymorphic peach primers, which had shown transferability of 95% to four genotypes of apple and rose each, all belonging to Rosaceae family. Jaccard’s similarity matrix was developed and dendrogram was generated using NTSYSpc ver.2.02h to establish the percent similarity among the ten Rubus accessions. Two clusters ‘A’ and ‘B’ were obtained. R. strigosusin cluster ‘A’ was found to diverge from rest of the nine accessions, all grouped under cluster ‘B,’ revealing high percentage of variability of R. strigosus from rest of the nine species. Maximum similarity was found between R.ellipticusIII and R.macilentus. Thus EST-SSRs used in the present study revealed a high level of polymorphism in the ten Rubusaccessions. Also interspecific and intergeneric cross transferability was established among these accessionsThesisItem Open Access STUDIES ON CROSS-TRANSFERABILITY OF EST-SSRS OF Stevia rebaudiana BERTONI IN RELATED GENERA(UHF,NAUNI, 2014) POONAM; KAUR, RAJINDERABSTRACT In the present investigation EST–SSR markers were developed for Stevia rebaudiana and were further used for cross-transferability studies in 20 species of different genera of Asteraceae family viz. Spilanthes acmella, Eclipta alba, Echinacea angustifolia, Achillea millefolium, Artemisia annua, Matricaria recutita, Silybum marianum, Callistiphus chinensis, Calendula officinalis, Chrysanthemum coronarium, Tagetes erecta, Helichrysum bractiatum, Arctotis stoechodifolia, Dimorphothica sinuata, Acroclinum roseum, Brachyscome dichromosomatica, Centaurea cyanus, Bellis perennis,Anthemis coluta and Lactuca sativa. 5548 EST sequences of Stevia rebaudiana available on NCBI website (www.ncbi.nlm.nih.gov/nucest) were screened for SSR motifs using an online tool, SSRIT (www.gramene.org/db/searches/SSRtool). 317 EST sequences were detected to contain SSRs, out of which 45 EST-SSRs were designed using PRIMER 3 software (www.frodo.wi-mit.edu/primer3/). In the wet lab experimentation, DNA from fresh, young leaves of 21species of different genera of Asteraceae family was isolated by CTAB method (Doyle and Doyle, 1987) for polymorphism and transferability studies. These 45 EST-SSRswere tried for amplification on 16 collections of Stevia rebaudiana. All these primers proved polymorphic. After this these primers were tried for cross-genera portability in 20 species of different genera of Asteraceae family. 26.6 per cent, 44.4 per cent, 20 per cent, 33.3 per cent, 51.1 per cent,64.4, 20 per cent, 26.6 per cent, 8.8, 37.7 per cent, 75.5 per cent, 44.4 per cent, 33.3 per cent, 6.6 per cent, 20 per cent, 40 per cent, 31.1 per cent, 15.5 per cent, 35.5 per cent and 46.6 per cent transferability was observed in Spilanthes acmella, Eclipta alba, Echinacea angustifolia, Achillea millefolium, Artemisia annua, Matricaria recutita, Silybum marianum, Callistiphus chinensis, Calendulaofficinalis, Chrysanthemum coronarium, Tagetes erecta, Helichrysum bractiatum, Arctotis stoechodifolia, Dimorphothica sinuata, Acroclinum roseum, Brachyscome dichromosomatica, Centaurea cyanus, Bellis perennis,Anthemis coluta and Lactuca sativa, respectively. Similarity matrix and dendrogram was generated using NTSYS ver.2.02h. All the 21 species of different genera under study were grouped into two main clusters. Thus it is concluded from the present study that ESTs of Stevia rebaudianaproduced high polymorphism in different genera of Asteraceae family indicating their cross-transferability and use across different members of Asteraceae family.
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