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Open Access
IN VITRO INDUCTION OF POLYPLOIDY IN GINGER (Zingiber officinale Rosc
(UHF,NAUNI, 2024-01-11) KESHAV KUMAR; MANISHA THAKUR
ABSTRACT 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 genome
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 SHARMA
ABSTRACT 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.
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 SHARMA
ABSTRACT 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.
Open Access
MORPHOLOGICAL, BIOCHEMICAL AND MOLECULAR CHARACTERIZATION OF SOME OYSTER MUSHROOM spp. (Pleurotus spp.)
(UHF,NAUNI, 2023-05) ANSHU MALA; THAKUR, MANISHA
Mushrooms are the fruiting bodies of macro-fungi commonly belonging to Basidiomycota and Ascomycota. The genus Pleurotus mushroom generally is called oyster, abalone, tree mushroom and ‘dhingri’ in India constituting about 40 species. There is limited knowledge about genus Pleurotus and the cellular processes involved in the growth of its fruiting bodies at molecular level, hence the study was conducted to assess the morphological, biochemical and molecular characterization of oyster mushroom (Pleurotus species) viz. Pleurotus ostreatus, Pleurotus florida, Pleurotus sajor-caju, Pleurotus citrinopileatus and Pleurotus djamor. At macroscopic level, P. sajorcaju and P. citrinopileatus exhibited largest pileus and longest stipe whereas P. florida and P. ostreatus showed largest basidia and basidiospore at microscopic level. P. florida was found to have the highest total fresh yield (941.35g) on a substrate of wheat straw with the longest cropping time (47 days). Maximum protein (31.64%), crude fat (3.23%), β-carotene (0.144 g/g), lycopene (0.046 g/g), and DPPH scavenging activity (85.24%) were observed in P. djamor; however maximum fibre (16.25 g/g), ash content (9.42 g/g), and total phenolic (0.073 mg/g) content were observed in P. citrinopileatus. These Pleurotus species were further analyzed at molecular level using ISSR primers. Out of 21 ISSR primers, 18 primers were successful in DNA amplification resulting in 136 amplicons, out of which 8 were monomorphic and 128 were polymorphic resulting in 93.08% polymorphism with highest polymorphic information content (PIC) value as 0.432. The ITS and RPB2 DNA barcode regions were investigated in Pleurotus species with 700 bp and 1200 bp, respectively. All of the Pleurotus species had significantly higher levels of 5.8s rDNA and ostreolysin expression in fruiting bodies than in primordia, according to the gene expression analysis. The current study reveals that molecular markers, DNA barcoding and gene expression analysis are helpful techniques for characterizing various Pleurotus species of mushrooms.
Open Access
AGROBACTERIUM MEDIATED DEFENSE RELATED AdSGT1 GENE TRANSFER STUDIES IN TOMATO (SOLANUM LYCOPERSICUM L.)
(UHF,NAUNI, 2022-09) NEHA BHARTI; SHARMA, RAJNISH
ABSTRACT Tomato (Solanum lycopersicum L) is a commonly cultivated and highly consumed vegetable crop that is economically appealing to medium-scale farmers due to its high yield and short growing season. This crop is susceptible to several biotic stresses that greatly limit its production and productivity. Consequently, plants codes for a set of proteins called pathogenesis-related proteins (PR proteins) which play a key role in plant defense against various pathogens. Plant defense mechanisms have also been enhanced through genetic manipulations by Agrobacterium mediated defense related genes transfer approach. Therefore, the present study was focused on developing the transgenic tomato cv. Solan Lalima by Agrobacterium mediated transformation using the defense related AdSGT1 gene. Agrobacterium cell density of 0.4 at O.D600 and infection time of 10 min was found to be best for optimum agroinfection. The 48 hrs of pre-culturing followed by 48 hrs of cocultivation showed maximum average shoot regeneration in both cotyledon and hypocotyl explants using two different Agrobacterium strains (LBA4404 and C58C1). 300 mg/L concentration of timentin was found to be best to eliminate excessive growth of Agrobacterium (LBA4404) for cotyledon explants while, 400 mg/L timentin was found sufficient for hypocotyl explants. Augmentin at a concentration of 500 mg/L was found efficient in controlling the overgrowth of Agrobacterium (C58C1). Augmentin at 100 mg/L along with 200 mg/L of timentin concentration was found sufficient to control excessive growth of Agrobacterium (LBA4404) in cotyledon explants while, 100 mg/L augmentin with 300 mg/L timentin was found best for hypocotyl explants. 100 mg/L augmentin along with 400 mg/L timentin concentration was found for reducing the excessive growth of Agrobacterium strain (C58C1) using both of the explants. Out of total 21 recovered putative transformants, 7 putative transformants were confirmed for transgene integration by PCR analysis (NPT-II, CaMV35s and AdSGT1). Among the total 7 PCR positive transformants, 4 transformed tomato lines showed expression of AdSGT1 gene through semi-quantitative RT-PCR. The transformation efficiency was obtained to be 0.20% using C58C1 strain and 0.50% using LBA4404 Agrobacterium strain, respectively. Further, these transgenic lines are yet to be confirmed for other molecular and expression analysis as well as for bioassays, etc. However, these findings could be helpful in inducing resistance against various devastating biotic stresses (fungal and bacterial pathogens) of this economically important horticulture crop through genetic manipulation procedures.
Open Access
IN VITRO PROPAGATION AND SECONDARY METABOLITE PRODUCTION IN Podophyllum hexandrum ROYLE (INDIAN MAYAPPLE)
(UHF,NAUNI, 2021-12) SHARMA, NEHA; THAKUR, MANISHA
ABSTRACT The present study reports an optimized protocol for high frequency in vitro propagation through seeds and rhizome buds, molecular analysis and podophyllotoxin production in Podophyllum hexandrum. Maximum percent uncontamination of seeds (89%) was achieved on treatment with 0.1% HgCl2 for 3 minutes and highest percent surviving rhizome buds (82.62%) were obtained after surface sterilization with 0.1% HgCl2+1.0% NaOCl for 4 minutes with maximum in vitro establishment (95.97%) on MS medium fortified with 0.2mg/l BA and 0.1mg/l GA3. Maximum in vitro establishment through seeds (89.00%) and buds (90%) was achieved during spring, followed by 68 and 79% in winter season. The proliferated shoots from seeds showed highest multiplication (1:11) on MS medium fortified with 2.0mg/l BA + 0.2mg/l NAA+ 0.7mg/l CaCl2. Addition of CaCl2 (0.7mg/l) into the medium was done to prevent shoot chlorosis. For shoot regenerating from rhizome buds highest multiplication rate of 1:5 was achieved on MS medium fortified with 2.0mg/l BA +0.2mg/l NAA. Molecular analysis was done using SCoT and CBDP markers. SCoT markers showed 71% polymorphism in samples from different altitudes in comparison to 50.90% polymorphism depicted by CBDP markers. However, both the makers showed 100% monomorphism among mother plants and their tissue culture raised progeny. In vivo leaves and petioles as well as in vitro roots and leaves were used as explants for callus induction. Highest callus induction was observed under dark incubation in in vivo leaves (81.07%), petioles (87.19%), in vitro roots (90.67%) and leaves (89.20%).HPLC analysis revealed maximum (0.267%) production of podophyllotoxin from callus initiated from in vitro roots procured from experimental material of district Kinnaur followed by 0.258% from in vitro root callus of Lahaul – Spiti. For enhancing podophyllotoxin yield callus induced from in vitro roots of P. hexandrum plants of district Kullu were subjected to elicitation by incorporating different concentrations (0.5-1.5 mM) of elicitors (methyl jasmonate and chitin) in production medium. Highest podophyllotoxin content (0.33%) was observed after elicitation with 1mM methyl jasmonate followed by its higher concentration of 1.5 mM (0.31%).
Open Access
HOST INDUCED GENE SILENCING IN APPLE CULTIVAR TO CONFER DISEASE RESISTANCE
(UHF,NAUNI, 2021-09) NEHA KUMARI; MODGIL, MANJU
ABSTRACT Apple ‘Oregon Spur’ is a commercial cultivar recommended in India, but it is susceptible to Marssonina blotch, a fungal disease which causes premature leaf fall thus affects the fruit production. In order to avoid the excessive use of fungicides, a novel innovation Host Induced Gene Silencing (HIGS) approach, which is RNAi based gene silencing has been used to impart resistance to ‘Oregon Spur’ in the present studies. Before transformation trials, an efficient system for in vitro regeneration of shoots from leaf explants has been established by examining the type and concentration of plant growth regulators, dark-light incubation. Upto 95% of leaves could be induced to form direct and healthy adventitious shoots on MS medium supplemented with 4mg/l BA and 0.2mg/l NAA. Though the frequency of shoot regeneration increased to 100% on 2mg/l TDZ and 0.5mg/l NAA. All concentrations of TDZ produced abnormal and vitrified shoots. The effect of different concentration of antibiotics was also observed on shoot regeneration from shoot explants. Kanamycin at higher concentrations above 5mg/l inhibited shoot induction, whereas cefotaxime at 100-300mg/l promoted organogenesis. To eliminate bacterial overgrowth after co-cultivation, 500mg/l cefotaxime was found optimal. Agrobacterium tumefaciens strain LBA4404 harbouring the binary vector pRI101-AN hpRNA:CaM containing targeted region of calmodulin (CaM) gene of M. coronaria in sense and antisense orientation under CaMV 35S promoter and nptII gene under NOS promoter was used for transformation experiment. Putative RNAi transformants were selected on shoot regeneration medium supplemented with 5mg/l kan and 500 mg/l cef after a pause to kan for 2 wks. 10min infection and 2 d co-cultivation periods were found effective. A total of 15 putative RNAi shoots/lines from 18 events were obtained which were multiplied and rooted. Eleven lines out of 15 putatively created RNAi lines successfully integrated CaM sense-antisense region and nptII genes, yielding 381 and 725 bp respectively with specific primers. Gene integration was further confirmed by reverse transcriptase PCR analysis. Semi quantitative RT-PCR revealed low to medium level of expression of CaM off-targeted region in eleven transgenic RNAi lines which confirmed that gene is expressing in each line. Four lines E10a, E1b and E1a showed higher relative expression in qRT-PCR analysis and in these lines fungus was not able to colonize the leaf explants during bioassay experiment. In vitro detached leaf assay revealed lesion development and disease progression in wild type after 20 dpi which were not visible in eleven lines. Microscopic examination showed fully developed, septate mycelium and necrosis of whole tissue in wild type while not found in transformants which is associated with down regulation of CaM gene expression. Thus, it is concluded that deciphering the role of CaM gene using hpRNAi construct through HIGS approach has provided resistance to Marssonina blotch in RNAi transgenic lines of ‘Oregon Spur’. Signature of Major Advisor Signature of the Student Countersigned
Open Access
STUDIES ON GENE TAGGING FOR RESISTANCE AGAINST PHYTOPHTHORA BLIGHT IN BELL PEPPER (Capsicum annuum L.)
(UHF,NAUNI, 2021-06) MANU VIVEK; THAKUR, MANISHA
ABSTRACT The present investigation was carried out to tag genes for resistance against Phytophthora blight in bell pepper (Capsicum annuum L.). Three varieties of capsicum ‘Solan Bharpur’ (Tolerant), ‘California Wonder’ (susceptible) and ‘DKC-8’ (resistant) were used as parental genotypes. Phytophthora nicotianae var. nicotianae was isolated from infected fruit samples of Capsicum annuum L. on corn meal agar medium and confirmed on the basis of morphological characters and microscopic examination following standard keys. Through in vivo testing, ‘Solan Bharpur’, ‘California Wonder’ and ‘DKC-8’ were found moderately resistant, susceptible and resistant to Phytophthora blight, respectively. Out of 20 RAPD, 45 SSR, 10 EST- SSR and 36 SCoT primers used for molecular analysis of parents, polymorphism was depicted by 9 RAPD, 15 SSR, 3 EST-SSR and 27 SCoT primers. Dendrogram based on RAPD, SSR and SCoT markers data grouped ‘Solan Bharpur’ and ‘California Wonder’ in one group whereas ‘DKC-8’ was partitioned singly. F1 population raised from ‘Solan Bharpur’ and ‘California Wonder’ was found to be moderately resistant while population developed from ‘California Wonder’ and ‘DKC-8’ was found to be resistant for Phytophthora blight. Phenotypic data of F2 population raised from cross between ‘Solan Bharpur’ and ‘California Wonder’ depicted polygenic nature of inheritance whereas screening of F2 population of cross California Wonder’ and ‘DKC-8’ clearly supported the involvement of single dominant gene for resistance. Three SSR primers showing polymorphic banding pattern between both parents and bulks were used for genotyping of whole F2 population of cross ‘California Wonder’ and ‘DKC-8’. Both phenotypic and genotypic data were used to construct a linkage map using MapDisto ver.1.0 software which revealed that marker CAMS420 is at 19.1 cM and CAMS072 is at 45.2 cM distance from the target locus showing resistance. For thorough mapping of genes for Phytophthora resistance, many more markers need to be utilized so as to obtain whole genome coverage and generate high density linkage maps.
Open Access
DECIPHERING ROLE OF HEAT SHOCK TRANSCRIPTION FACTORS (HSFs) IN TOMATO AND POTATO: A COMPARATIVE ANALYSIS IN RESPONSE TO DROUGHT AND HIGH TEMPERATURE STRESS
(UHF,NAUNI, 2021-06) ANSHUL SHARMA MANJUL; ANSHUL SHARMA MANJUL; NATH, A K; NATH, A K
ABSTRACT Potato and tomato are important global crops; however, biotic and abiotic stresses have greatly limited their production and quality. Abiotic stresses are further expected to have even more production and quality. Abiotic stresses in the face of changing global climate. Plant HSFs are the terminal components of a signal transduction chain that helps in the expression of various abiotic stresses genes. Fifteen HSF genes from potato and 27 HSF genes from tomato were identified from PLAZA 4.0 Dicots. HSF genes from both tomato and potato showed differential locations on the 12 chromosomes. Molecular weight of all the HSFs were found to range between 12.20 to 94.24 kDa. All the HSF proteins were found to be hydrophilic in nature. Comparative phylogenetic analysis of HSF genes in both tomato and potato revealed 10 major groups with several orthologous and paralogous genes. Five HSF genes were cloned, their sequences were submitted to NCBI database. Semi-quantitative expression analysis indicated them to play prominent role in imparting resistance against heat and drought stress. The vector pCAMBIA3301+TRV2 was used in the present studies for the preparation of VIGS construct. Two genes viz., HP1 and HP2 were inserted into the VIGS construct. The developed agro constructs, pTRV2:HP1 and pTRV2:HP2 mobilized into Agrobacterium, were used for the VIGS experiment along with pTRV1 mobilized in Agrobacterium in potato cultivar K. Surya. It could be concluded from the results of the VIGS experiments that the downregulation of HP1 and HP2 genes resulted in the stunted growth of the potato plants as well as no or very less tuber formation. The results indicated both the genes HP1 and HP2 to play an important role in conferring heat and drought resistance to potato plants.

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