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ThesisItem Open Access Assessment of genetic stability of in vitro regenerated plantlets of Chlysanthemum morifolium L.(College of Horticulture & Forestry Dr YSP UHF, Neri, Hamirpur(H.P.), 2024-04-08) Sharma, Neeraj; Reena KumariThe present investigation was carried out on "Assessment of genetic stability of in vitro regenerated plantlets of Chlysanthemum morifolium L". Nodes, leaves, shoot tips and ray florets from maintained mother plants of chrysanthemum were used as the source of explants. These explants were treated with 5percent bavistin for l min followed by 0.3 percent sodium hypochlorite for 60 sec proved to be best as it gave 87.0 percent survival rate of nodal explant. 0.2 percent mercuric chloride for 30 sec proved to be best as it gave 83.0 percent survival rate of leaf explant. 0.1 percent mercuric chloride for 30 sec proved to be best as it gave 87.0 percent survival rate of shoot tip explant and 0.1 percent mercuric chloride for 60 sec proved to be best as it gave 85.0 percent survival rate of ray floret explant. Surface sterilized explant was culture on MS medium supplemented with different growth regulator and maximum in vitro shoot regeneration 92.0 percent from nodal explant was observed on MS medium supplemented with 1 .0 mg/I BAP and 1.0 mg/I NAA. However, Maximum direct shoot regeneration 85.0 percent was observed using shoot tip explant on MS medium containing 2.0mg/I NAA + 0.5mg/1 TDZ. Further MS medium fortified with 0.5mg/I BAP + 2.0mg/l IAA proved to be the best and showed 86.0 percent indirect shoot regeneration in case of ray floret explant. Whereas, MS medium fortified with l .5mg/1 BAP + 0.3mg/l 2, 4-D was reported to show 98.0 percent indirect shoot regeneration frequency from leaf explant. In vitro raised rnicroshoots were multiplied further on MS medium containing l .Omg/1 NAA+1 .0mg/1 BAP. Microshoots cultured on half strength MS media supplemented with 0.5mg/1 IBA showed 90.0 percent rooting after 3-4 weeks. Well developed in vitro raised plantlets were hardened in potting mixture containing cocopeat: perlite in 1:2 ratio with 85.0 percent survival rate. The genetic fidelity of Chrysanthemum morifolium L. clones was assessed by using SCoT (Start codon targeted) marker. Among 33 SCoT primers, 21 primers showed good scorable bands. The average number of monomorpbic bands per primer was 3.33 and with overall 96.16 percent monomorphism . The similarity coefficient values was ranged from 0.96 to 1.00 for SCoT markers. The results revealed that SCoT primer showed overall good monomorpbism (96.19) and six in vitro raised plants out of nine were found to be 100 percent similar to mother plant showing true to type nature of in vitro regenerated Chrysanthemum Morifolium L. plants. Hence, this protocol could be used for the large-scale production of true to type planting material of Ch1ysanthemum morifolium L.ThesisItem Open Access IN VITRO ROOT REGENERATION IN KIWIBERRY [Actinidia arguta (Siebold & Zucc.) Planch. ex. Miq. var. Arguta](UHF,NAUNI, 2023-12-30) KRITIKA KHAGTA; PARUL SHARMAABSTRACT During the present investigation, the root regeneration in four cultivars viz. Annanasya, Dumbarton Oaks, Kens Red and Meader Male of kiwiberry (Actinidia arguta) via two approaches i.e., in vitro and ex vitro were followed. In vitro root regeneration was studied using two methods (onestep and two-step). In the former method, the best response for root regeneration was recorded in treatment RMO4 (1.00 mg/L IAA) as it produced callus free, good and healthy roots in all the four kiwiberry cultivars.The hardening of plantlets raised from one-step method was best in potting mixture, PMO5 [cocopeat + perlite + vermiculite in (3:1:1)] with respect to all the recorded parameters in case of all the four kiwiberry cultivars. In the two-step method, the best treatment for root regeneration was RMT2 (100.00 mg/L IBA) as it produced highest average number of roots per shoot in all the four kiwiberry cultivars. Meanwhile, for the hardening of plantlets raised from twostep method again the potting mixture PMO5 was the best with respect to all the recorded parameters in case of all the four kiwiberry cultivars. On the other hand, in the second root regeneration approach i.e., ex vitro, the treatment RME2 (100.00 mg/L IAA) resulted in maximum average per cent root regeneration and average per cent survival of the plantlets in all the four kiwiberry cultivars, respectively. Out of two root regeneration approaches followed, in vitro (one-step method) was proved better than ex vitro as it resulted in maximum average per cent root regeneration and maximum average per cent survival of plantlets. Overall, cv. Dumbarton Oaks performed well for root regeneration followed by cv. Meader Male. Thus, the present study developed an efficient and reliable method for rooting and hardening of in vitro raised microshoots of kiwiberry (Actinidia arguta).ThesisItem Open Access Biosynthesis and characterization of silver nanoparticles using S. mukorossi extract possessing antimicrobial potential(College of Horticulture and Forestry Dr YSP UHF, Neri, Hamirpur(H.P.), 2023-11-28) Thakur, Sapna; Sharma, SnehThe present study on the “Biosynthesis and characterization of silver nanoparticles using S.mukorossi extract possessing antimicrobial potential” were carried out in Department of Biotechnology, College of Horticulture and Forestry (Dr. Y.S. Parmar University of Horticulture and Forestry), Neri, Hamirpur (H.P) during 2022-2023. Sapindus mukorossi is an extremely valuable cultivated medicinal plant which is mostly found in hilly regions of India. In the lists of herbs, it is a popular herb in Ayurveda and is used as an important ingredient in cleansers and shampoos. It is well known for its folk medicinal values and known by several names like washnut, soapnut, soapberry, dodan and ritha. The use of this plant promoted considerable attention because of their various biological and pharmacological activities. The major compound isolated from genus Sapindus are saponins, triterpeniods, fatty acids and flavonoids are well known for their antimicrobial, antidiabetic, cytotoxic, fungicidal, and anti-inflammatory activities. The study was undertaken for biosynthesis and characterization of silver nanoparticles derived from S. mukorossi pericarp extract. The sample was collected from village Sheglagalu, Chail Chowk, Distt. Mandi (H.P). Synthesis was carried out using different solvents viz., methanol, chloroform and aqueous. It was found that distilled water acts as a better solvent for the synthesis of silver nanoparticles. The characterization of prepared silver nanoparticles was assessed using distinct techniques namely UV-vis spectroscopy, X-ray diffraction (XRD), Fourier transmission infrared (FTIR) and High resolution- transform electron microscopy (HR-TEM). The antioxidant activity of aqueous extract and biosynthesized silver nanoparticles was evaluated using DPPH (2,2-diphenyl-1-picrylhydrazyl) method. The different concentrations of sample ranging from (100-1000 µg/ml) was prepared and mixed with 3 ml of DPPH solution. The maximum antioxidant activity was confirmed at 1000 µg/ml of concentration that is 91 % and 88 %, respectively. The antimicrobial property of silver nanoparticles derived from S. mukorossi extract was evaluated against four strains viz., P. aeruginosa, E. coli, B. subtilis and S. aureus. The maximum antimicrobial property was seen against gram-negative bacterium P. aeruginosa, E. coli then gram-positive bacterium B. subtilis and S. aureus. These results suggest that silver nanoparticles made from S. mukorossi extract may beused as antimicrobial and antioxidant agents in several fields, including medicine, agriculture, and personal care products.ThesisItem Open Access Green Synthesis, Characterization and Application of Silver Nanoparticles using Cauliflower (Brassica oleracea var. botrytis L.) extract(College of Horticulture and Forestry Dr YSP UHF, Neri, Hamirpur(H.P.), 2023-11-28) Prashar, Ankita; Sharma, SnehThe present investigation on the “Green Synthesis, Characterization and Application of Silver Nanoparticles using Cauliflower (Brassica oleracea var. botrytis L.) extract.” were carried out in the Department of Biotechnology, College of Horticulture and Forestry (Dr. Y.S. Parmar University of Horticulture and Forestry), Neri Hamirpur (HP) during 2022-2023. Silver nanoparticles have gained significant attention in various fields due to their unique physical, chemical, and biological properties. Nanotechnology offers the possibility of modifying the materials used in conventional packaging for food and enables the introduction of fresh ideas for quality control, food safety, identification of pathogens by mechanical means, and barrier properties. Nanoparticles are also carried out in relation to food, its manufacturing, processing, and specially packaging, which is intimately tied to the shelf-life of food production. The cauliflower is a rich source of predominantly S-methylcystein sulfoxide, sinigrin, and glucobrassisin, which have predominant anti-carcinogenic properties. The planting material were collected from experimental farm in Department of Vegetable science at College of Horticulture and Forestry Neri, Distt. Hamirpur, Himachal Pradesh. The aim of this study was to synthesize silver nanoparticles from cauliflower extract and investigate the antibacterial activity and evaluation of biosynthesized nanoparticles in post-harvest. The UV-vis spectrum shows the absorption peak at 412 nm which confirms the synthesis pf AgNPs. FTIR spectroscopy confirms the presence of bio-compound functional groups on the surface of AgNPs. The particle size of AgNPs were found to be 10-50 nm in HR-TEM. In XRD, AgNPs determined to be cubic crystal structure with face centered. The synthesized silver nanoparticles from cauliflower showed antibacterial activity against B. subtilis, E. coli, P. aeruginosa and S. aureus. These properties utilized to reduce the bacterial load on vegetables, thereby inhibiting the spoilage and extending the shelf life of vegetables. The biosynthesized silver nanoparticles can act as barrier against moisture loss, gas exchange, and microbial contamination, thus preserving the freshness of vegetables and increasing their shelf life. The antioxidant activity of biosynthesized silver nanoparticles and aqueous extract of B. oleracea var. botrytis was carried out by using 2,2-diphenyl-1-picryhydrazyl (DPPH) assay. The different concentrations of AgNPs and aqueous extract (50, 100, 150, 200, 250, 300 µg/mL) mixed with 3 mL of DPPH. The result revealed that the antioxidant activity of biosynthesized silver nanoparticles and aqueous extract of B. oleracea var. botrytis was increased at 200 µg/mL and maximum antioxidant of solutions observed 80.04 % and 69.07 % at 300 µg/mL due to the presence of polyphenols, vitamin A, beta carotene and sulphrophane.ThesisItem Open Access Isolation and purification of proteins from Tinospora cordifolia against Pseudomonas aeruginosa(College of Horticulture and Forestry Dr YSP UHF, Neri, Hamirpur(H.P.), 2023-05-14) Parul; Sharma, SnehThe present investigation on the “Isolation and purification of proteins from Tinospora cordifoliaagainst Pseudomonas aeruginosa.” were carried out in the Department of Biotechnology, College of Horticulture and Forestry (Dr. Y.S. Parmar University of Horticulture and Forestry), Neri Hamirpur (HP) during 2021-2022. Tinospora cordifolia is a deciduous shrub. It is a well-recognized for its medicinal properties in Indian Ayurveda system. It is one of the most versatile shrub commonly known as “giloy”. The plant gained attention due to its various biological activities such as anti-diabetic, anti-microbial, anti-allergic, anti-oxidant and anti-cancer activities. The plant parts contains various chemical constituents such as alkaloids, steroids, glycosides and polysaccharides. The study was undertaken for isolation and purification of antibacterial protein of Tinospora cordifolia collected from Hamirpur district (Neri), Himachal Pradesh. Analysis was done by stem ethanolic, methanolic, extract of samples taken from Neri village. It was found that methanol act as better solvent for extraction of proteins. Ammonium sulphate precipitation of methaolic crude extract showed that 60-70% precipitation was found best for precipitation of proteins. Further, 60-70% precipitates was subject for dialysis followed by ion exchange chromatography. The fractions obtained after ion exchange chromatography was assayed by antibacterial activity against test organism. The active fractions were pooled together and carried out for SDS-PAGE. The total protein content present in methanolic crude extract was 2.056 mg/ml. The ammonium sulphate precipitation showed 10.833±0.928 mm zone of inhibition and after purification the antibacterial activity increased to 15±0.289 mm. The partial characterization of proteins revealed that the antibacterial proteins was stable at optimal pH 8, heat stable at 40 ℃ temperature and stable at low salt concentration. For future aspects, the stem extract and protein can be further exploited for its other properties such as its sequencing.ThesisItem Open Access IN VITRO REGENERATION AND PHYTOCHEMICAL STUDIES IN KAKOLI (Fritillaria roylei HOOK.) – HIGH VALUE CRITICALLY ENDANGERED HIMALAYAN HERB(UHF,NAUNI, 2023-09-04) SHAGUN SHARMA; PANKAJ KUMARABSTRACT Fritillaria roylei Hook. (Liliaceae), commonly known as kakoli or janglilahsun, is a bulbous perennial herb that thrives in the Indian Himalayan region at altitudes of 2800-4600 m above mean sea level. The plant's bulbs are known for their abundant metabolite compounds with medicinal properties, making them valuable for various therapeutic uses. However, natural propagation through seeds and bulbs is slow, taking approximately 5-6 years for a plant to grow and mature. Due to the high demand for its bulbs, overexploitation has become a concern. Therefore, biotechnological intervention is crucial for conserving, restoring, and sustainably producing these valuable bioactive metabolites. In the present study, in vitro cultures were established using bulb scales as explants. The highest survival percentage (88.77%) was achieved by treating the explants with 0.5% bavistin for 20 minutes, followed by 70% ethanol for 45 seconds, and HgCl2 (0.1%) for 14 minutes. By using different combinations and concentrations of plant growth regulators, a high-efficiency callus (100%) was obtained using 4.0 mg/L Picloram and 2.0 mg/L TDZ at 25°C and 15°C, respectively, after 30 and 21 days. Transferring the callus from dark to light conditions and utilizing a high sucrose concentration led to the formation of more bulblets. For bulblet production, different plant growth regulators were tested, and the highest number of bulblets per explant (10.07) with a regeneration response of 90.98% was achieved using NAA and KIN at 1.0 mg/L each after 25 days. Phytochemical analysis was performed using crude extracts from different in vivo (leaf, stem and bulb) and in vitro (callus and bulblets) samples. Among the in vivo samples, the highest total phenol content was observed in leaves (63.64 mg GAE/g), while in vitro callus exhibited the highest phenolic content (54.31 mg GAE/g). Flavonoid content was found to be highest in leaves among in vivo samples (133.0 mg RE/g) and in bulblets among in vitro samples (38.38 mg RE/g). Regarding antioxidant activity, leaves showed the highest activity among the in vivo samples, while in vitro callus and bulblets displayed antioxidant activity statistically at par in comparison to the in vivo samples. UHPLC-based quantification was done for targeted steroidal alkaloids namely peimisine, sipeimine and peimine. Among in vivo sample extracts, highest peimisine, sipeimine and peimine were quantified as 119.27 μg/g DW, 394.62 μg/g DW and 25.46 μg/g DW respectively in bulbs. In contrast, in vitro, highest peimisine, sipeimine and peimine were quantified as 26.46 μg/g DW, 0.60 μg/g DW and 13.92 μg/g DW, respectively in bulblets. Thus, rapid in vitro regeneration protocol enables large-scale production of these targeted bioactive compounds in a shorter period and throughout the year. Consequently, it offers a reliable and sustainable source for the development of new drugs or natural remedies without the need to uproot the entire plantThesisItem Open Access In vitro propagation and phyotochemical profiling of Rauwolfia serpentina (Sarpagandha)(College of Horticulture and Forestry, Dr YSP UHF, Neri, Hamirpur (H.P.), 2022-04-13) Sharma, Shipra; Dhiman, KarunaIn the present investigation a technique for in vitro propagation and phytochemical profiling ofRauwolfia serpentina (Sarpagandha) has been developed. Axillary buds, shoot tips and leaves from maintained mother plants of Sarpgandha were used as the source of explants. Treatment of different explants with 1 % HgCl2 for 30 seconds proved to be best as it gave maximum 80 % uncontaminated explants with 65 % culture survival rate. Maximum in vitro axillary bud proliferation (76%) on MS medium supplemented with 1.0 mg/l Zeatin and 2.0 mg/l IBA whereas MS medium fortified with BAP (2.0 mg/l) + Kn (2.0 mg/l) proved to be best for shoot tips regeneration with 87.55% maximum regeneration frequency. Maximum indirect shoot regeneration (89 %) was observed using leaf as explants on MS medium containing 2.0 mg/l BAP and 1.0 mg/l NAA. Shoots were multiplied further on MS medium containing2.5 mg/l NAA + 1.0mg/l BAP. Microshoots were cultured on half strength MS media for in vitro rooting and 82% root induction was achieved when IBA (1.5mg/l) and IAA (1.5mg/l) was used in combination. Well developedin vitro raised plantlets were hardened in potting mixture containing soil: sand: vermiculite in 1:1:1 ratio. Phytochemical profiling of in vitro raised plantlets as well as mother plants was carried out qualitatively, quantitatively and by TLC analysis. Alkaloid , flavonoids, tannin and phenolic compounds, phytosterols and saponins were present in leaves and roots of in vitro raised plant. TLC analysis also showed presence of reserpine in root samples ofin vitroraised plants.ThesisItem Open Access GERMPLASM CHARACTERIZATION OF KAGZI AND ORNAMENTAL LIME USING MOLECULAR MARKERS(UHF,NAUNI, 2022-11) GILL, KOMAL JEET; PANKAJ KUMARABSTRACT In the present study, twenty-seven accessions of kagzi and ornamental lime were evaluated for morphological (based on IPGRI citrus descriptor), biochemical and molecular characterization (using RAPD, SCoT and SSR markers). Under morphological characterization, significant variation was recorded in all the studied parameters viz. leaf lamina attachment, leaf lamina shape, leaf lamina margins, fruit shape, shape of fruit base, shape of fruit apex and fruit texture, respectively. Likewise, minimal differences were also recorded in kagzi and ornamental lime accessions for biochemical studies i.e. total soluble solids (TSS), titratable acidity and TSS: TA ratio. These findings were further validated and confirmed using PCR-based molecular markers viz., RAPD, SCoT and SSR markers. Analysis of PCR products of 22 RAPD, 16 SCoT and 40 SSR primers showed 115, 74 and 64 amplicons. The polymorphic percentagewere ranged from 33.33% to 100% for RAPD and SCoT markers, while SSR markers showed 50% to 100% polymorphism among the studied accessions. Likewise, polymorphic information content (PIC) was ranged from 0.20 to 0.90 for RAPD markers, 0.41 to 0.99 for SCoT markers and 0.07 to 0.67 for SSR markers, respectively. The analysis of molecular variance (AMOVA) studiesamong 27 accessions of kagzi and ornamental lime proved the significant genetic differencesi.e. for RAPD (PhiPT = 0.267, P = 0.010); for SCoT (PhiPT = 0.351, P = 0.010) and for SSR (PhiPT = 0.107, P = 0.010), respectively. With the help of STRUCTURE software 2.3.4 and structure harvester ΔK was observed at k=2 indicating the genetic admixture between the two studied populations. Thus, the present study showed clear and distinct variation among kagzi and ornamental lime accessions. Therefore, it could be concluded that the use of RAPD, SCoT and SSRmolecular markers are efficient to identify diversity or closeness in genotypes which may contribute to speed up the breeding programs and aid in citrus crop improvementThesisItem Open Access Quantification of bioactive molecules and evaluation of anti-microbial potential of Tinospora cordifolia Miers(College of Horticulture and Forestry Neri, Hamirpur (H.P.), 2021-12-04) Rajvanshi, Ashish Kumar; Sharma, SnehThe present investigation on the “Quantification of bioactive molecules and evaluation of antimicrobial potential of Tinospora cordifolia Miers.” were carried out in the Department of Biotechnology, College of Horticulture and Forestry (Dr. Y.S. Parmar University of Horticulture and Forestry), Neri Hamirpur (HP) during 2020-2021. Tinospora cordifolia is an angiosperm belonging to the Menispermaceae family and is a division of Magnoliophyta, class Magnoliopsida, and order of Ranunculaceae. It is a wide deciduous, glabrous, rapidly ascending shrub with several coiling branches extending approximately 3-4 feet in height and roughly 1 foot long. Tinospora cordifolia is a well-recognized and widely distributed traditional plant that is used successfully in Indian Ayurveda medicine. It has shown many promising biological activities such as antioxidative, antimicrobial, antihyperglycemic, anti-inflammatory, osteoprotective, hepatoprotective, antidiarrheal and antistress effects. It also contains many secondary plant metabolites, such as terpenes, alkaloids, flavonoids, steroids, and glycosides. The study was undertaken for evaluating the bioactive molecules and antimicrobial potential of Tinospora cordifolia collected from different districts of Himachal Pradesh. Analysis was done by using HPTLC of each root, stem and leave ethanolic, methanolic, chloroform and aqueous extract of samples taken from two villages of 3 districts. On the basis of RF value the districts Kangra sample was found to posses highest RF value i.e., 0.97 and the sample with highest RF value was selected for LC -MS. In result found that 7 number of compounds were present. Those seven compounds are berbrine, magnoflorine, menisperine, 20b-hydroxyecdysone, palmatine, 2deoxy-20b-hydroxyecdysone and columbaine which was responsible for the medicinal properties of Tinospora cordifolia. Considering the vast potentiality of Tinospora cordifolia as a source for antimicrobial drugs with reference to antibacterial, antifungal and anticandida agents, a systematic investigation was undertaken to screen roots, stem and leaves samples of Tinospora cordifolia collected from different Districts of Himachal Pradesh for its activity against various pathogens. Ethanolic, methanolic, aqueous and chloroformic extracts were tested but methanolic leaf extract of Tinospora cordifolia showed the maximum inhibitory activity against tested pathogens i.e S. aureus, E. coli, P. aeruginosa and Aspergillus niger. MIC of leaf methanolic extract against Staphylococcus aureus and Pseudomonas aeruginosa was observed and found to be 4.7mg/ml and 3.2mg/ml. For future aspects, the isolation and purification of bioactive molecules and evaluation of their pharmalogical activity from the crude sample are recommended as future research