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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 INDUCTION OF POLYPLOIDY IN GINGER (Zingiber officinale Rosc(UHF,NAUNI, 2024-01-11) KESHAV KUMAR; MANISHA THAKURABSTRACT The present study reports the successful in vitro tetraploid induction in ginger (Zingiber officinale Rosc.) cv. Himgiri using colchicine (0.1, 0.2, 0.3 and 0.4%) for different time durations (6, 12, 18, 24, 30, 36, 42 and 48 hrs). Maximum per cent bud survival (73.33%) was achieved with 0.1% colchicine treatment for 6 hrs which decreased with the increase in colchicine concentration and treatment duration. Shoot multiplication rate and average shoot length increased with the subculture and was maximum in shoots regenerated from buds treated with 0.2% colchicine for 24 hrs. LT50 calculated after 16 weeks was 22.13, 15.26, 4.53 and 1.59 hrs for colchicine treatment of 0.1, 0.2, 0.3 and 0.4%, respectively. The maximum induction of polyploidy (50%) was achieved with 0.2% colchicine treatment for 6 and 24 hrs whereas, no polyploidy was induced with 0.4% colchicine treatment. Flowcytometric analysis of regenerated shoots reported different ploidy levels (triploid, tetraploid and hexaploid). These identified polyploids were multiplied through repeated subculturing for four months and analyzed again for stability of ploidy level through flowcytometry. It was observed that the triploid and hexaploid plants reverted back to diploid and tetraploid state whereas, the tetraploids remained stable. The chromosome number of the tetraploids were again confirmed microscopically and was 44 in comparison to 22 observed in diploids. The confirmed tetraploids were multiplied in vitro for four months and the rooted plantlets were hardened with 100% survival. Morphological parameter such as plant height (73.33 cm), leaf length (25.50 cm), leaf width (3.50 cm), leaf area (58.92 cm2) were maximum in tetraploids induced after 24hrs treatment with 0.2% colchicine whereas, maximum number of tillers (7.60) was observed in tetraploids induced after 30 hrs treatment with 0.2% colchicine however, no significant difference in stem girth was observed in tetraploids and control plants. The number of leaves per tiller was maximum (14.33) in control plants. All the physiological parameters such as photosynthetic rate, stomatal conductance and transpiration rate were observed to be maximum in tetraploids. On biochemical analysis the tetraploids induced from 0.1, 0.2 and 0.3% colchicine treatment for 36, 24 and 30 hrs showed maximum chlorophyll a (6.25 μg/ml), chlorophyll b (4.40 μg/ml) and total carotenoid content (1.38 μg/ml), respectively. Maximum oleoresin (4.81%) and low crude fibre (8.60%) content was reported in rhizomes of tetraploids induced from 0.2% colchicine treatment after 30 hrs and tissue culture propagated control plants. Whereas, maximum total protein (112.22 μg/ml) and sugar content (40.86 μg/ml) was observed in plants regenerated after 0.2% colchicine treatment for 30 hrs. HPLC analysis depicted maximum gingerol content (498.36 μg/g) in harvested rhizomes of tetraploid induced with 0.2% colchicine treatment for 24 hrs. On molecular analysis maximum polymorphism of 10.87% and 35.67% was depicted by SCoT and CBDP respectively, showing alteration in the genomeThesisItem 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 GERMPLASM CHARACTERIZATION, IDENTIFICATION AND IN VITRO PROPAGATION OF ELITE GENOTYPES OF Saussurea costus (FALC.) LIPSCH - AN ENDANGERED MEDICINAL HERB(UHF,NAUNI, 2023-12-16) KAMAL THAKUR; RAJNISH SHARMAABSTRACT Saussurea costus (Falc.) Lipsch (Asteraceae), commonly known as kuth is a perennial, medicinally important endangered medicinal herb of the Indian Himalayan region. The roots are known for their abundant metabolic compounds with medicinal attributes, rendering them highly valuable for numerous therapeutic applications. Its natural propagation through seeds is slow, taking 3-4 years for a plant to grow and mature. Due to the high demands of roots, overexploitation and illiterate harvesting has become a major concern for its endangered status in the Himalayan region. Therefore, biotechnological approaches may be beneficial for conserving, restoring and sustainable production of secondary metabolites in this herb to a greater extent. In the present investigation, the EST-SSRs related to the bioactive compounds biosynthesis were developed from the root trancriptome data of S. costus and subsequently subjected to determine genetic diversity and population structure. Molecular markers analysis classified S. costus genotypes into two major groups i.e., cluster A (≥ 2500 m amsl) and cluster B (<2500 m amsl) according to their respective elevations. Due to the restricted gene flow and ability to adapt to their local environment, the population genetic structure showed the admixture of two genetic pools among the S. costus genotypes procured from Himachal Pradesh and Uttarakhand. The biochemical composition of two diversified locations was further evaluated using leaf and root tissues of S. costus with different solvents (methanol, ethanol and ethylacetate) and found that the methanolic root extract from higher elevation exhibited maximum accumulation of compounds qualitatively and quantitatively. In addition, cell suspension culture technique also revealed the maximum biomass accumulation and costunolide production in in vitro root induced calli. From the cell suspension culture and its potential to produce costunolide in vitro, it was concluded that suspension culture could be a suitable alternate to meet its market demand by overcoming unrestricted trading, relentless harvesting, and reckless management of this valuable endangered medicinal plant.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 Development of EST-SSR markers and assessment of genetic diversity, relationships and population structure in aonla (Phyllanthus emblica L(UHF,NAUNI, 2023-06-05) MEGHA SHARMA; RAJNISH SHARMAABSTRACT Phyllanthus emblica L. (Aonla) is an important edible and non-timber forest product species which have numerous medicinal and therapeutic uses. In the present study, commercial and wild aonla genotypes were characterized on physico-chemical parameters and molecular parameters. EST-SSRs were developed from fruit transcriptome data as well as data retrieved from NCBI database and further subjected to assess the genetic diversity and population structure. The significant variations were observed in physico-chemical parameters (fruit size, weight, colour, TSS, acidity, ascorbic acid and total phenolic contents) among all studied aonla genotypes. There was a significant positive correlation observed among most of the studied fruit attributes. Principal component analysis (PCA) divided the studied traits into two principal components i.e. PC1 and PC2 that accounted for 71.69 of the total variability. The cluster analysis was performed using Ward’s method that divided the aonla genotypes into two major clusters i.e. A and B where cluster A grouped all the wild genotypes, while cluster B comprised of all the commercial varieties. Under molecular characterization, out of total 102 EST-SSR primers, 46 were found to be polymorphic and further used in diversity analysis. The allele number was found to be varied from 2 to 26 with an average of 11.28 allele per locus. PIC, EMR, MI and Rp values ranged from 0.24 to 0.94, 0.50 to 25.00, 0.12 to 23.50 and 1.45 to 18.09, respectively. The mean value of He and Ho was 0.64 and 0.74. The dendrogram divided the aonla genotypes into two main clusters at a similarity coefficient of 0.43. The two wild genotypes ‘HPU5’ and ‘HPU6’ had the maximum similarity coefficient i.e. 0.82. However, the genotypes ‘NA-6’ and ‘HPSo3’ were found to be highly diversified to each other. Population structure analysis showed admixture of four different genetic pools. Although the correlation between physicochemical and molecular parameters found to be moderate but both these methods revealed the high genetic diversity among studied aonla genotypes which can be explored in breeding programmes for selection of superior genotypes. Furthermore, developed polymorphic EST-SSRs in this study can be utilized in future to study the population genetics and genetic resource management of the Phyllanthus emblica and its closely related species.
