1. KAUTIR (Kerala Agricultural University Theses Information and Retrieval)
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Item Effect of histone deacetylation in the regulation of somatic embryogenesis related genes in coconut (Cocos nucifera L.)(Department of Molecular Biology and Biotechnology, College of Agriculture,Vellayani, 2025) Beema, Y Basheer.The study entitled “Effect of histone deacetylation in the regulation of somatic embryogenesis-related genes in coconut (Cocos nucifera L.)” was conducted at the Department of Molecular Biology and Biotechnology, College of Agriculture, Vellayani, during 2023-2024. The objective of this study was to know the effect of histone deacetylation in the regulation of somatic embryogenesis related genes (SERK, BBM, WUS) and histone deacetylation gene (HDAC) in coconut (Cocos nucifera L.) in presence of HDAC inhibitor Trichostatin. Coconut somatic embryogenesis holds significant promise for cultivating superior coconut plants. Currently, no repeatable and efficient protocol exists for inducing somatic embryogenesis in this crop. Epigenetic regulators have been found to enhance cell differentiation and their role in promoting embryogenic induction has been observed in various recalcitrant crops (Abrahamsson et al., 2017). In many such species, histone acetylation modification of genes that control somatic embryogenesis has been reported to increase gene expression and subsequently improve the rate of somatic embryogenesis (Martinez et al., 2021). The acetylation of genes can be enhanced by using histone deacetylase inhibitors (HDAs). Trichostatin A (TSA), the most used histone deacetylase inhibitor (Görisch et al., 2005), blocks HDAC activity in cultured cells, leading to a significant increase in embryogenic growth (Wójcikowska et al., 2018). By specifically inhibiting HDAs, TSA causes an accumulation of acetylated histones, a corresponding reduction in DNA methylation, and an increase in gene activity (Wójcikowska et al., 2018). However, how HDAC inhibitors affect the acetylation of genes related to somatic embryogenesis in coconut is still not well explored. Keeping this in view, the present study was planned to find out the effect of Trichostatin in embryogenic callus induction. For analysing the effect of Trichostatin on callus induction, plumules from 11-month-old West Coast Tall (WCT) coconuts were scooped out, surface sterilized, and pre-cultured in Y3 basal medium for one month. The pre-cultured plumules were then inoculated into Callus Induction Medium (Y3 + 2,4-D (74.6μM), TDZ (4.5μM), spermine (50μM)) with varying concentrations of TSA (0.5μM to 2μM). The results showed no change in the rate of callus induction with Trichostatin treatment. In the control, a 20% embryogenic callus induction was achieved in 50 days. However, in the treatments with TSA, instead of callus development, enlargement of plumules was observed at 50 days of inoculation, with no change in status even after that period. The percentage of enlargement of plumules varied with TSA concentration and maximum was observed in 0.5μM TSA. Following the Trichostatin treatment, an analysis of the expression of somatic embryogenesis (SE) related genes and HDAC gene in control and treated samples was conducted. For gene expression analysis, RNA was isolated from plumules inoculated in 0.5μM TSA and control cultures using Trizol. Complementary DNA (cDNA) was synthesized by reverse transcription mix and subjected to quantitative real-time PCR (RT-qPCR) to analyze the expression levels of somatic embryogenesis marker genes (SERK, WUS, BBM) and histone deacetylase genes (HDAC). The results showed no significant change in the expression of the HDAC gene in both the control and the CIM with 0.5μM TSA. Additionally, the results revealed no expression of somatic embryogenesis-related genes in Trichostatin-treated samples. This finding suggests that Trichostatin treatment does not influence embryogenic callus induction, indicating a need to fine-tune the concentration and conditions of the treatment to optimize its effects. Conclusively, further optimization is required to achieve desirable results in somatic embryogenesis induction in coconut.Item Elucidation of role of small RNA mediated gene regulation in secondary metabolite pathway of black pepper (Piper nigrum L.)(Department of Molecular Biology and Biotechnology, College of Agriculture, Vellayani, 2024-07-19) Shruthy, N S; Asha, SThe study entitled “Elucidation of role of small RNA mediated gene regulation in the secondary metabolite pathway of black pepper (Piper nigrum L.)” was conducted in the Department of Molecular Biology and Biotechnology, College of Agriculture, Vellayani, Thiruvananthapuram during 2023-2024. Piperine, an amide alkaloid that contributes the unique spicy flavor is the major economic secondary metabolite of black pepper which is renowned for its pharmacological activities and therapeutic efficacies. The biosynthetic pathway of various secondary metabolites was highly influenced by a regulatory factor, microRNA- ‘the master regulators of gene expression’. They are small, non-coding RNAs of typically 18-24 nucleotides and can be a useful tool in metabolic engineering. Therefore, a better understanding of the mode of action of miRNA is very essential to exploit them in improving the plant for enhanced secondary metabolite production. Hence, the present study focuses on ‘identification and characterization of small RNAs involved in piperine biosynthesis from black pepper (Piper nigrum L.) In this study, the microRNAs targeting the key genes involved in the piperine biosynthesis pathway were analyzed by combined in silico-experimental method. From the 19 million reads of small RNA transcriptome, 303 conserved families of MiRNA were reported in black pepper. Among these, potential candidates of small RNAs were predicted to target the key genes involved in piperine biosynthesis such as Piperine synthase, piperamide synthase, and Piperoyl CoA Ligase genes. From these candidates, we characterized conserved and novel microRNAs. Precursor miRNAs for each candidate were also predicted based on their distinctive features such as length, ability to form hairpin stem-loop structure, and minimal folding energy. Among the potential candidate miRNAs, ‘Pni_miR5654’ and novel miRNAs such as ‘Pni_miR19’ and Pni_miR33’ were predicted for experimental validation. The tissue-specific expression of these miRNAs and their cognate targets in different parts of the black pepper variety Panniyur-1 were evaluated. The result indicates that the immature spike has the highest expression of microRNAs compared to leaf and mature berries whereas the highest expression of cognate target genes was observed in mature berries compared to leaf and immature spikes, that is, the miRNA: target pairs were inversely correlated. Further to analyse the expression of miRNA: target pairs in bio-elicitated callus cultures, initially the callus initiated from berries were treated with the culture filtrate of Pirimorsphora indica and R/NA was isolated at 0, 24, and 48 hrs after treatment. The study sowed and upregulation in the expression of both the miRNAs and their cognate pairs in 24 hour treatment. In this present study, the microRNA candidates Pni_miR5654, Pni_miR19’ and ‘Pni_miR33’ were identifies to have gene regulatory role in piperine biosynthesis by targeting the key genes in the pathway. Although miRNA medicated regulation was detected in the spike and berry stages, the inverse correlation of miRNA: target pairs was not found in in vitro cultures of black pepper, indicating the cellular reprogramming of miRNAs during callogenesis. The data generated from this study could be helpful in modifying the target genes, constructing artificial miRNAs, and performing inhibition of miRNA in order to enhance the production of piperine in black pepper.Item Copper nanoparticles green synthesized using leaf extract of Piper colubrinum for the management of foot rot disease in black pepper (Piper nigrum L.)(Department of Molecular Biology and Biotechnology, College of Agriculture, Vellayani, 2025) Aashima Rahim.The study entitled " Copper nanoparticles green synthesized using leaf extract of Piper colubrinum for the management of foot rot disease in black pepper (Piper nigrum L.)" was conducted at College of Agriculture, Vellayani during 2023-24. The objectives of the study were green synthesis of copper nanoparticles using leaf extracts of P. nigrum, P. colubrinum (foot rot-resistant,) their characterization; testing the efficacy of these copper nanoparticles in managing foot rot disease in black pepper caused by Phytophthora capsici. Copper nanoparticles (CuNPs) were synthesized using leaf extracts of P. nigrum and P. colubrinum for the management of foot rot of black pepper. The CuNPs from P. nigrum characterized using UV-visible spectroscopic analysis, X-ray diffraction patterns, and FTIR spectrum revealed an absorption spectrum typical of copper nanoparticles at 800 nm, the cubic lattice structure of copper nanoparticles and distinct characteristic bands at 3315.28 and 1635.50 cm-1. The CuNPs synthesized using P. colubrinum leaf extract had an absorption spectrum typical of copper nanoparticles at 800 nm, the cubic lattice structure of copper nanoparticles with prominent characteristic bands at 3264.52 and 1636.62 cm-.1. Field Emission Scanning Electron Microscopy (FESEM) and Transmission Electron Microscopy (TEM) revealed that CuNPs synthesized using both P. nigrum and P. colubrinum had sizes 59.86 and 64.52 nm, respectively. The pathogen was isolated and identified as P. capsici followed by proving the pathogenicity. In vitro bioassay (poisoned-food assay) demonstrated significant inhibition of the mycelial growth of pathogen when treated with green synthesized CuNPs using leaf extract of P. colubrinum (PC-CuNPs) at concentration 250 and 500 ppm. PC-CuNPs showed the highest per cent inhibition (PI) against P. capsici compared to those synthesized using P. nigrum leaf extract (PN-CuNPs) and commercial copper nanoparticles (C-CuNPs). The mycelial inhibition of 90% and 92% was exhibited by green synthesized PC- CuNPs in PDA medium amended with 250 ppm and 500 ppm, respectively. Thus, the green synthesized PC- CuNPs at 500 ppm had the maximum inhibition, significantly higher than the chemical control. Mycelial inhibition was absent in treatments with leaf extracts alone. Detached leaf assay was performed on black pepper leaves sprayed with the green synthesized PC-CuNPs, followed by challenge inoculation of the pathogen. The treatment reduced lesion development when compared to leaves treated with green synthesized CuNPs from P. nigrum (PN-CuNPs), C- CuNPs and both leaf extracts. Leaves treated with green synthesized PC-CuNPs at 250 ppm failed to develop symptom after three days of inoculation when compared to the other treatments. In vivo experiment gave results similar to in vitro studies. Application of GSCuNP from P. colubrinum at 250ppm deferred the symptom development in the varieties of black pepper viz., Karimunda, Panniyur-1. While the application of leaf extract of P. nigrum and P. colubrinum alone was ineffective in managing the disease. CuNPs synthesized using leaf extract from disease-resistant P. colubrinum at 250ppm effectively suppressed foot rot disease incidence in susceptible black pepper varieties. This study pioneers the demonstration of enhanced pathogen-suppression activity against Phytophthora capsici, a major pathogen of black pepper using copper nanoparticles green synthesized using the leaves of a disease-resistant Piper colubrinum genotype.Item Bioprospecting of lignocellulolytic microorganisms for management of agricultural residues(Department of Molecular Biology and Biotechnology, College of Agriculture, Vellayani, 2025-01-29) Athira, N S.The study entitled “Bioprospecting of lignocellulolytic microorganisms for management of agricultural residues” was conducted at College of Agriculture, Vellayani during 2023 – 2024. The objective of the study was to isolate, screen and characterize microorganisms with lignocellulolytic properties for management of agricultural residues. A total of 43 bacterial isolates were isolated from decaying wood, spent mushroom substrate, saw dust, and garden soil rich in decaying plant matter by serial dilution and plating on Hans carboxymethyl cellulose (CMC) medium. These isolates were screened for cellulolytic activity on Hans CMC medium. Cellulolytic index was estimated for 17 isolates with measurable clearance zones around their colonies indicating cellulolytic activity. Isolates with cellulolytic index above 2.0 were selected for further analysis. Among the isolates thus selected, isolate CLSM02 recorded the highest cellulolytic index of 5.83. All the selected isolates except CLDW10 demonstrated growth on lignin basal medium indicating potential ligninolytic activity. The cellulase activity of all the selected isolates was estimated by DNS method using CMC and filter paper as substrates. The bacterial isolate CLSD07 exhibited highest cellulase activity of 8.49 IU mL-1 min-1 when CMC was given as substrate. Among the isolates tested, only CLDW10, CLSG06, CLSD04, and CLSM02 were capable of degrading filter paper. The isolate CLSD04 recorded the highest filter paper cellulase (Fpase) activity of 2.50 IU mL-1 min-1. Estimation of ligninolytic enzymes, viz., laccase, lignin peroxidase (LiP) and manganese peroxidase (MnP) was done using substrates 2, 2′-azino-bis-(3-ethylbenzothiazoline-6-sulfonic) (ABTS), pyrogallol and guaiacol respectively. The bacterial isolate CLSM03 recorded highest laccase activity of 1.7 U mL-1. The highest LiP activity was observed in CLSD08 (15.49 U mL-1), which was on par with CLDW04 (15.37 U mL-1) on 72h after incubation. Three isolates exhibited 80 manganese peroxidase activity. The bacterial isolate CLSM03 recorded highest manganese peroxidase activity of 2.27 U mL-1. Based on their enzyme activity profiles, the best five isolates, CLSD04, CLSD07, CLSD08, CLSM02, and CLSM03, were selected for further analyses. All the selected isolates were gram positive, rod shaped and endospore formers. All the bacterial isolates were identified as Bacillus sp. by morphological and biochemical characterization. Molecular identification by 16S rRNA gene sequencing revealed that the isolates CLSD04 were Calidifontibacillus erzurumensis, CLSD07 were Bacillus velezensis, CLSD08 were Bacillus haynesii, CLSM02 were Bacillus stercoris and CLSM03 were Bacillus licheniformis respectively. The presence of cellulase and laccase genes was confirmed by gene-specific amplification. On PCR amplification with specific primer for cellulase, all five isolates exhibited bands corresponding to the cellulase gene. Bands corresponding to the laccase gene was detected in CLSM02 and CLSM03 on PCR amplification with primer for laccase. The selected isolates viz., Calidifontibacillus erzurumensis CLSD04, Bacillus velezensis CLSD07, Bacillus haynesii CLSD08, Bacillus stercoris CLSM02, and Bacillus licheniformis CLSM03, were found to be compatible with each other. A consortium of the compatible isolates was prepared. The efficacy of the lignocellulolytic consortium to degrade raw coir pith was assessed. The prepared consortium was sprayed on to thoroughly washed and dried raw coir pith and incubated for 15, 30 and 45 days. Tomato var. Anagha was raised in pro trays with coir pith subjected to various treatments as substrate. The highest plant height was recorded for T2 (raw coir pith sprayed with consortium and incubated for 30 days) at 19.30 ± 0.85 cm, followed by T5 (raw coir pith sprayed with water and incubated for 30 days) at 14.20 ± 0.31 cm. The treatments T2 and T5 exhibited the maximum dry weight (31.53 ± 0.98 mg and 28.84 ± 5.23 mg, respectively). The lignocellulolytic bacterial consortium consisting Calidifontibacillus erzurumensis CLSD04, Bacillus velezensis CLSD07, Bacillus haynesii CLSD08, Bacillus stercoris CLSM02, and Bacillus licheniformis CLSM03, exhibited 81 degradation of raw coir pith and plant growth promotion in tomato, suggesting its potential for agricultural residue management.Item Bio- elicitation of secondary metabolites in callus cultures of black pepper (Piper nigrum L.)(Department of Molecular Biology and Biotechnology, College of Agriculture , Vellayani, 2024-02-21) Edupulapati Nandini; Asha, SThe present study on “Bio-elicitation of secondary metabolites in callus cultures of black pepper (Piper nigrum L)” was carried out at the Department of Molecular Biology and Biotechnology, College of Agriculture, Vellayani, during the period from 2021-2023. The objective of the study was development of callus culture of black pepper (Piper nigrum L.) for secondary metabolite production and its bio-enhancement using derivatives of Piriformospora indica. Callus cultures were successfully initiated from the leaves and mature ripe berries of the Panniyur 1 variety. The use of 0.1% mercuric chloride for 3 minutes proved effective in achieving healthy, contamination-free callus initiation from leaf explants, while a duration of 5 minutes was found to be effective for healthy callus initiation from berry explants. Callus induction was carried out using Murashige and Skoog's (MS) medium, incorporating various combinations and concentrations of plant growth regulators (PGR). The percentage of callus induction varied depending on the type of explant and media composition used. In this study, it was observed that leaf tissue exhibited a higher responsiveness to callus induction compared to berry. Out of the four treatments, the callus induction was observed on MS media supplemented with 5.0 mg/L Napthelene Acetic Acid (NAA) and MS media supplemented with 1.0 mg/L 6benzyladenine (BA). Among these, the best response and friable callus was noted from MS media with 5.0 mg/L NAA, with 75% callus induction percentage from leaf explant within 4weeks. Better Callus proliferation with mean diameter of 1.094cm was also found in this media. Callus cultures from leaves were subjected to 24 hrs, 48 hrs and 72hrs of elicitation with Piriformospora indica cell wall extract (PiCWE) (1:1) and culture filtrate (PiCF) (1:1) and the effect of elicitation on secondary metabolite production was studied by detecting the presence of piperine by High Performance Liquid Chromatography. The maximum improvement in the accumulation of piperine was observed in callus cultures at early stages (at 24hrs) of elicitation with P. indica cell wall extract and P. indica culture filtrate. Meanwhile, P. indica CWE and CF treatment at 48hrs and 72hrs treatment was found lower performance compared with 24hrs of bioelicitation. The expression profiling of piperine synthase and Pip CoA ligase genes showed upregulation of the piperine synthase (2.49fold), and pip CoA ligase genes (63.87fold) in the 24hrs treatment with PiCF in leaf derived callus, Berry derived callus showed upregulation of piperine synthase gene with PiCWE treatment at 24 hrs with a fold change of (21.06) while pip coa ligase showed fold change of 5.13. The expression of genes were found to be decreased in later stages. The results of the study indicated that P. indica culture filtrate and cell wall extract acts as effective elicitors in boosting the production of secondary metabolites in the leaf and berry derived callus cultures of black pepper (Piper nigrum L.) respectively. But the expression of key genes and subsequent production of piperine decreased in the leaf callus, indicating the possibility of site-specific regulation of metabolites. Future studies will be directed towards the elucidation of tissue specific regulation of metabolites in black pepper.Item Pink pigmented facultative methylotrophs (PPFMs) from chilli(capsicum frutescens) for biotic and abiotic stress migration and plant growth promotion(Pink pigmented facultative methylotrophs (PPFMs) from chilli(capsicum frutescens) for biotic and abiotic stress migration and plant growth promotion, 2025) Jasna Sherin, P T.; Anurajan, SChilli, often referred to as the "wonder spice," is one of the most commercially valuable vegetable crop worldwide. Drought, a significant abiotic stressor, can cause substantial reductions in chilli crop yields and quality, resulting in significant economic losses. PPFMs have been shown to promote plant growth by producing plant growth regulators, altering agronomic traits, and improving water status, photosynthetic rates, and antioxidant enzyme activities. The study entitled “Pink Pigmented Facultative Methylotrophs (PPFMs) from chilli (Capsicum frutescens) for biotic and abiotic stress mitigation and plant growth promotion " was carried out during the period from 2023-2024 at College of Agriculture, Vellayani, Thiruvananthapuram, with the objective of isolation, screening and characterization of Pink Pigmented Facultative Methylotrophs (PPFMs) from chilli (Capsicum frutescens) and their evaluation for plant growth promotion with special emphasis on biotic and abiotic stress management. Thirty-five PPFM colonies were isolated from chilli phyllosphere using leaf imprint and serial dilution methods on AMS agar medium supplemented with 0.5% methanol and 10 μg cycloheximide and amended with 5% PEG 6000. These isolates were tentatively identified based on their pink pigmentation and further characterized by colony morphology, Gram reaction, and biochemical tests. Twenty-three PPFM isolates were selected for in vitro screening to assess their plant growth promoting abilities. IAA production among the isolates exhibited significant variability, ranging from 0.97 μg mL⁻¹ to 18.26 μg mL⁻¹ of culture filtrate. Isolate CF7 demonstrated the highest IAA production (18.26 μg mL⁻¹), followed by CF6 (13.60 μg mL⁻¹). All isolates produced varying amounts of gibberellic acid, ranging from 1.71 to 22.07μg mL-1. The highest gibberellic acid production was recorded in CF7 (22.07μg mL-1) followed by CF6 (21.35μg mL-1). Extracellular ammonia yield varied significantly, ranging from 0.27 to 10.06 μmol mL⁻¹. The isolate CF15 demonstrated the highest ammonia production (10.06 μmol mL⁻¹). ACC deaminase activity by different isolates ranged from 0.94 to 14.08 μg mL⁻¹, PPFM 38 (19.35 μg mL⁻¹) showed the highest activity followed by CF7 (14.08μg mL⁻¹). While all isolates grew in N-free malate bromothymol blue media, Waksman Base No. 77, and Jensen’s media, none demonstrated phosphate solubilization or antagonistic activity against phytopathogens. Based on Index ranking eight isolates were selected for primary assessment of drought tolerance potential in vitro. To evaluate drought tolerance potential in vitro, isolates were grown in media containing varying concentrations of PEG 6000. All isolates tolerated up to 30% PEG 6000. The isolates CF6, CF7, and CF15 were ranked based on drought tolerance in vitro for further studies. PCR amplification confirmed the presence of mxaF gene in these isolates, with an expected band size 550 bp indicating their ability to oxidise methanol using the methanol dehydrogenase enzyme (MDH). Molecular identification based on 16S rRNA sequencing of the isolates CF6, CF7, and CF15 showed maximum percentage identity to Methylorubrum populi, Methylorubrum thiocyanatum, and Methylorubrum podarium respectively. A pot culture experiment was conducted with chilli plants to assess the effects of PPFM isolates on plant growth promotion and drought tolerance. The experiment was a completely randomized design (CRD) with eight treatments and three replications which included four PPFM isolates (CF6, CF7, CF15, and reference culture PPFM 38), a consortium, and three control treatments (sterile water, AMS broth, and absolute control). Treatments were applied through seed treatment and foliar spray at 25th day after transplanting. The study demonstrated that application of PPFM isolates significantly enhanced plant growth parameters of chilli. The biometric parameters like maximum number of leaves (133.67), number of branches (4.50), shoot length (56.88 cm), dry weight of shoots (17.04 g), number of fruits (14.17) and yield per plant (53.45g) were recorded with CF6 treatment. Number of flowers (16), root length (35.42cm), fresh weight of roots (30.99g), and dry weight of roots (4.89g) were greater for CF15 treatment. PPFM 38 (81.83g) and CF6 (76.25g) recorded maximum fresh weight for shoots. The least parameters were recorded with either one or more of controls. Cell membrane integrity (82.3%), relative water content (85.52%), proline (86.51μg g-1tissues), SOD activity (0.44 activity g-1 min-1) and peroxidase (38.81activity g-1 min-1), were highest with PPFM 38 and CF6. The results suggest that Application of PPFMs through seed treatment and foliar spray (1%), can effectively improve growth and drought tolerance in chilli plants. Further research under field conditions is required before these can be developed as a bioformulation for sustainable agriculture.Item Controlled abiotic stress induction in tomato (Solanum lycopersicum L.) and its influence on the phenylalanine ammonia- lyase (Pal) gene expression(Department of Molecular Biology and Biotechnology, College of Agriculture, Vellayani, 2023-11-06) Bhagya Shibu.; Manju, R VThe study entitled “Controlled abiotic stress induction for biofortification in tomato (Solanum lycopersicum L.) and its influence on the Phenylalanine ammonia lyase (PAL) gene expression” was conducted at the College of Agriculture, Vellayani, Thiruvananthapuram, during 2022-2023. The objective of this study was to know the impact of application of mild soil moisture stress and elevated CO2 associated high temperature for biofortification of tomato and its influence on the expression levels of the key enzyme PAL, involved in the biosynthetic pathway of nutritionally relevant secondary metabolites in tomato. The experiment was conducted in a completely randomized design in pot culture with four replications with tomato, variety “Vellayani Vijay”. Plants were maintained under open field conditions (390 ppm) and under the elevated CO2 of 500 ppm in the Open Top Chamber (OTC) facility at the Department of Plant Physiology. The study consisted of four treatments (i) exposure to mild moisture stress [withdrawal of irrigation for 2 days 1 month after transplanting] (T1), (ii) exposure of vegetative phase to eCO2 (500 ppm) associated high temperature for 1 week, [1 month after transplanting] (T2), (iii) exposure of the entire vegetative phase to eCO2 (500 ppm) associated high temperature (T3) and (iv) control (T4) conditions. Observations on physiological and biochemical parameters were taken 60 days after transplanting. Growth parameters, yield, and quality were observed at the time of harvest. The expression analysis of the PAL gene was done in leaf tissues of experimental plants using Real-Time PCR after exposure to both stresses. Plants subjected to mild moisture stress and eCO2 exposure showed a significant reduction in RWC and % leakage. Exposure to mild moisture stress resulted in a significant increase in all the biochemical parameters like reducing sugar (52.55%), phenol content (68.7%), carotenoid (24.4%), alkaloid content (19.1%) and ascorbic acid (12.6%) in leaf tissues of tomato. Exposure to mild moisture stress resulted in a significant increase in all the biochemical parameters like reducing sugar (25.74%), phenol content (76.9%), carotenoid content (84.2%%), alkaloid content (20.8%), lycopene content (32.1%) and ascorbic acid (47.8%) in fruits. Elevated carbon dioxide exposure to a period of 1 week also had a positive impact on phenol, lycopene, and ascorbic acid in fruits though carotenoid and alkaloid contents showed a reducing trend with this stress factor. Both stress factors resulted in a reduction in root weight, shoot weight, and total dry matter. The root/shoot ratio was found to increase under mild moisture stress (14.28%). Phenylalanine ammonia-lyase (PAL) is a key enzyme involved both in the phenylpropanoid and flavonoid pathways. In the present study, PAL gene expression showed 1.64, 1.96, and 3.39 fold increase under mild moisture stress, eCO2(1 week), and eCO2 (vegetative phase) exposure respectively compared to control. Exposure of tomato plants to mild moisture stress and elevated carbon dioxide showed an upregulated PAL gene expression. The result of the study showed that the application of mild moisture stress and exposure to eCO2 for a short period can result in biofortification in tomato variety Vellayani Vijay. Stress-induced overexpression of PAL can be associated with increased accumulation of secondary metabolites. The findings of the current study will help to develop low-cost strategies for biofortification in tomato which can make high quality farm products available to low-income consumers.Item Screening of Schizophyllum commune strains for laccase and peroxidase production(Department of Molecular Biology and Biotechnology, College of Agriculture , Vellayani, 2024-05-16) Arya, S Raj.; Krishnapriya, PJThe study entitled “Screening of Schizophyllum commune strains for laccase and peroxidase production” was conducted at College of Agriculture, Vellayani during 2023-2024. The objectives of the study were to evaluate three strains of S. commune for their laccase and peroxidase activity during submerged and solid-state cultivation; and expression profiling of the best strain for its lignolytic potential at different developmental stages using quantitative Real-Time PCR. The three strains of S. commune viz., DMRX-2156, DMRX-2157 and DMRX-2160 were procured from All India Coordinated Research Project (AICRP) on Mushrooms, Department of Plant Pathology, College of Agriculture, Vellayani and maintained on potato dextrose agar medium. Among the S. commune strains, DMRX-2160 took the minimum period of 9.23 days for complete coverage on petridish with maximum radial mycelial growth (0.97 cm/day). The mycelial growth was white, thick and fluffy with smooth margins. Hyphae of Schizophyllum strains were septate, branched, hyaline, of average size 2.1μm with distinct clamp connection and spicules. All the three tested strains reacted positively to the qualitative plate assay for laccase and peroxidase activity. DMRX-2160 showed the highest efficiency of laccase production in growth media supplemented with 0.04% guaiacol and gave the maximum ratio of reddish zone diameter (Z) to colony diameter (C) value as 0.86. Addition of 1% pyrogallol and 0.4% hydrogen peroxide mixture to fully grown cultures of Schizophyllum strains resulted in development of yellowish brown colour, thus confirming its peroxidase activity. Quantitative estimation of laccase and peroxidase enzymes from mycelia and broth of S. commune submerged cultures in three different media viz., Stock Basal Media (SBM), Potato Dextrose Broth (PDB) and Potato Malt Peptone Broth (PMPB) was done using substrates viz., guaiacol and pyrogallol for laccase and peroxidase, respectively. DMRX-2156 recorded the highest intracellular laccase activity (30.55±3.66 U/g) in PMPB and peroxidase activity (242.34±36.66 U/g) in PDB at 5 days after inoculation (DAI) whereas, DMRX-2160 recorded the highest extracellular laccase activity (15.58±0.74 U/ml) in SBM and peroxidase activity (52.10±1.34 U/ml) in PMPB at 15 DAI. DMRX-2160 took the minimum time for mycelial run in paddy grains (13.70 days) and paddy straw (3.10 days) when used as spawn and bed substrates, respectively. DMRX-2160 took the minimum time for pin head formation (4.10 days) and first harvest (15.33 days), with maximum yield (11.29 g/Kg) and biological efficiency (1.13%), when paddy straw was used as bed substrate. The pinheads of S. commune strains were formed 3-6 days after spawning and the sporocarps took an average of 12 days from the day of pinhead formation to complete maturity. When the mushroom attained full maturity the basidiocarp became differentiated into pilear region, gills on underside were split into midregion and the cap was multistratous. Its stipe became rudimentary or even absent. Quantification of laccase and peroxidase activity during solid state cultivation revealed that DMRX-2160 showed the maximum intracellular laccase (36.39±0.78 U/g) and peroxidase activitiy (605.67±40.82 U/g) at mycelial stage however, the activities declined over the subsequent stages. DMRX-2160 showed the maximum extracellular laccase (15.16±2.59 U/ml) and peroxidase (121.13±5.40 U/ml) activities at young fruiting body stage. Thus, DMRX-2160 was identified as the best S. commune strain, in terms of maximum extracellular enzyme activities during submerged cultivation and solid-state cultivation, as well as maximum yield. The change in lignolytic activities over the growth stages starting from mycelial run to sporophore formation was further analyzed by gene expression studies of DMRX-2160 cultivated in paddy straw using Real time PCR. Laccase and peroxidase genes were found maximally expressed in the mycelium of DMRX- 2160 (42.52-fold for laccase and 4.86-fold for peroxidase than young fruiting body stage). The expression of these genes showed downregulation over the subsequent developmental stages with its lowest expression in young fruiting body stage. Gene expression data showed a similar trend to enzyme assays. Thus, the present study identified S. commune strain DMRX-2160 as a promising strain which can utilise paddy straw as substrate for fruiting body production with maximum biological efficiency, as well as a prospective enzyme source for the extraction of hydrolytic enzymes having biotechnological and industrial applications.Item Controlled abiotic stress induction in tomato (Solanum lycopersicum L.) and its influence on the phenylalanine ammonia- lyase (Pal) gene expression(Department of Molecular Biology and Biotechnology, College of Agriculture ,Vellayani, 2023-11-06) Bhagya ShibuThe study entitled “Controlled abiotic stress induction for biofortification in tomato (Solanum lycopersicum L.) and its influence on the Phenylalanine ammonia lyase (PAL) gene expression” was conducted at the College of Agriculture, Vellayani, Thiruvananthapuram, during 2022-2023. The objective of this study was to know the impact of application of mild soil moisture stress and elevated CO2 associated high temperature for biofortification of tomato and its influence on the expression levels of the key enzyme PAL, involved in the biosynthetic pathway of nutritionally relevant secondary metabolites in tomato. The experiment was conducted in a completely randomized design in pot culture with four replications with tomato, variety “Vellayani Vijay”. Plants were maintained under open field conditions (390 ppm) and under the elevated CO2 of 500 ppm in the Open Top Chamber (OTC) facility at the Department of Plant Physiology. The study consisted of four treatments (i) exposure to mild moisture stress [withdrawal of irrigation for 2 days 1 month after transplanting] (T1), (ii) exposure of vegetative phase to eCO2 (500 ppm) associated high temperature for 1 week, [1 month after transplanting] (T2), (iii) exposure of the entire vegetative phase to eCO2 (500 ppm) associated high temperature (T3) and (iv) control (T4) conditions. Observations on physiological and biochemical parameters were taken 60 days after transplanting. Growth parameters, yield, and quality were observed at the time of harvest. The expression analysis of the PAL gene was done in leaf tissues of experimental plants using Real-Time PCR after exposure to both stresses. Plants subjected to mild moisture stress and eCO2 exposure showed a significant reduction in RWC and % leakage. Exposure to mild moisture stress resulted in a significant increase in all the biochemical parameters like reducing sugar (52.55%), phenol content (68.7%), carotenoid (24.4%), alkaloid content (19.1%) and ascorbic acid (12.6%) in leaf tissues of tomato. Exposure to mild moisture stress resulted in a significant increase in all the biochemical parameters like reducing sugar (25.74%), phenol content (76.9%), carotenoid content (84.2%%), alkaloid content (20.8%), lycopene content (32.1%) and ascorbic acid (47.8%) in fruits. Elevated carbon dioxide exposure to a period of 1 week also had a positive impact on phenol, lycopene, and ascorbic acid in fruits though carotenoid and alkaloid contents showed a reducing trend with this stress factor. Both stress factors resulted in a reduction in root weight, shoot 84 weight, and total dry matter. The root/shoot ratio was found to increase under mild moisture stress (14.28%). Phenylalanine ammonia-lyase (PAL) is a key enzyme involved both in the phenylpropanoid and flavonoid pathways. In the present study, PAL gene expression showed 1.64, 1.96, and 3.39 fold increase under mild moisture stress, eCO2(1 week), and eCO2 (vegetative phase) exposure respectively compared to control. Exposure of tomato plants to mild moisture stress and elevated carbon dioxide showed an upregulated PAL gene expression. The result of the study showed that the application of mild moisture stress and exposure to eCO2 for a short period can result in biofortification in tomato variety Vellayani Vijay. Stress-induced overexpression of PAL can be associated with increased accumulation of secondary metabolites. The findings of the current study will help to develop low-cost strategies for biofortification in tomato which can make high quality farm products available to low-income consumers.Item Effects of egg treatment with antioxidants/antiseptics on the larval immunological indices of Trachinotus blochii and characterization of relevant immune gene(Department of Molecular Biology and Biotechnology, College of Agriculture , Vellayani, 2023-11-09) Sharavana, K S.; Sumithra, T GThe Silver Pompano, Trachinotus blochii, is a promising marine species for aquaculture. This study entitled "Evaluation of the effects of egg exposure to antioxidant/antiseptic solution on the immunological indices of Trachinotus blochii larvae and characterization of relevant immune gene " aimed to analyze the impact of egg treatment with glutaraldehyde and hydrogen peroxide on the egg hatching rates, larval immunology, growth, and survival of T. blochii with a final objective of finding out an optimal strategy for larviculture practices. Results showed that 40 ppm glutaraldehyde for 5 min, 400 ppm hydrogen peroxide for 10 min, and 20 ppm iodophor for 9 min were optimal for T. blochii. The optimal concentration was evident through the hatching rates, decreased microbial counts, and survival rates. The results showed a significantly higher hatching rate with glutaraldehyde treatment, whereas all other treatments were remarkably similar. The survival rates were 27.46%, 25.75%, 20.59%, and 19.19% in hydrogen peroxide, glutaraldehyde, iodophor, and negative control tanks. Evaluation of innate immune parameters showed that all treatments caused significantly higher protein content, catalase activity, and superoxide dismutase activity compared to the control. Among the evaluated treatments, iodophor- treated fish showed the maximum protein concentration, catalase activity, and SOD activity, followed by hydrogen peroxide and glutaraldehyde-treated fish. In brief, this research showed that significant improvements in hatching rates and larval survival could be obtained by using optimal chemicals at optimal concentration and exposure duration for egg disinfection of marine fish. Toll-like receptor 21 (TLR21) is a non-mammalian TLR playing an essential role in the innate immunity of fish, amphibians, and birds. The study's second objective was to get the complete sequence of the TLR21 gene from T. blochii. The entire length of TLR21 cDNA was 3171 bp, containing a 5' untranslated region and a 3' poly tail. The ORF length was 2931 bp, encoding 976 amino acid residues. The nucleotide and amino acid sequence showed maximum similarity with Trachinotus ovatus. Phylogenetic tree analysis showed the clustering of the T. blochii TLR21 gene with the other fish TLR21 genes. The sequence showed a typical TLR protein domain structure, including leucine-rich repeat motifs, a transmembrane domain, and a toll/interleukin-1 receptor domain. Strikingly, the TLR21 gene was found as an intron-less gene in T. blochii. The results reference further immune and disease management studies on T. blochii.
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