Browsing by Author "Asha, S"

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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, S
The 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.
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, S
The 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.
Elucidation of the role of tetraspanin(TET) gene family in stress response of piper nigrum L.
(Department of Molecular Biology and Biotechnology, College of Agriculture, Vellayani, 2024-08-08) Athira Prasad.; Asha, S
The study entitled “Elucidation of the role of Tetraspanin (TET) gene family in stress response of Piper nigrum L.” was conducted in the Department of Molecular Biology and Biotechnology, College of Agriculture, Vellayani, Thiruvananthapuram from 2023-2024. Tetraspanins are evolutionary conserved transmembrane proteins present in all multicellular organisms and they act as key modulators of signaling cascades and respond to cold, drought, and pathogen stress. Tetraspanins also play a crucial role in the biogenesis of extracellular vesicles (EVs) that facilitate the transport of various biomolecules such as secondary metabolites, small RNAs, short peptides, etc. They are the key players in the stress response of plants, as they interact with signaling molecules and target intracellular locations. The objective of the study was the characterization of the tetraspanin (TET) gene family in the stress response of Piper nigrum L. In this study, twelve tetraspanin gene candidates were identified from the P. nigrum via genome wide analysis. The P. nigrum tetraspanin genes identified belong to six distinct classes namely TET1, TET2, TET3, TET6, TET8, and TET10. The length of the genes ranged from 798 to 1491 bp, with encoded protein of 265- 496 amino acids. The deduced PnTET proteins were basic in nature with molecular weight ranging from 29.3 to 53.5 kDa. The characteristic motifs and residues were detected in all the identified P. nigrum tetraspanin proteins. Phylogenetic analysis segregated the Piper nigrum tetraspanins into six distinct paralogous clades. A detailed in silico analysis revealed cis-regulatory elements involved in stress response in the promoter region of PnTET genes. The expression profiling of selected PnTET genes such as PnTET8.1, PnTET3.1, and PnTET10 were carried out from the biotic and abiotic stressed P. nigrum variety Panniyur1 plants. The biotic stress was induced by the artificial inoculation of Phytophthora capsici in the leaves and roots of the plants and total RNA was isolated from the infected plants at 24hpi, 48hpi (early response), and 72hpi, 96hpi, 120hpi (late response). PnTET3.1 and PnTET8.1 were upregulated from 48 to 120 hours post-infection (Day 5) in the leaf and root of P. capsici-infected plants. The expression of PnTET10 showed a late upregulation in the root of the infected plant after 96hpi (1.71fold) meanwhile, an early upregulation at 24hpi (14.07 fold) was observed in the infected leaf. Although the upregulation of PnTET10 was observed in the subsequent stages in the infected leaf, the expression was found to be low compared to its early (24hpi) induction. Expression profiling of the selected three genes ie. PnTET3.1, PnTET8.1, and PnTET10 were also carried out from the leaves and roots of black pepper plants subjected to abiotic stress by 105 withholding irrigation for 10 days. PnTET3.1 showed the highest upregulation in leaves (2.53 fold) and roots (3.05 fold) of the abiotic stressed plant; while PnTET8.1 showed an upregulation in the leaves and roots with a fold change of 1.25 and 1.63 respectively. An upregulation of PnTET10 was observed in leaves and roots with a fold change of 1.47 and 1.32 respectively. The sequence characterization of Indian cultivated variety, Panniyur-1 PnTET3.1, PnTET8.1, and PnTET10 revealed the three-dimensional structure of their protein and typical magnoliid characteristics via phylogenetic analysis. The present study is the first report on the identification of tetraspanin (TET) gene family from black pepper. The genome-wide analysis found six distinct classes of tetraspanin genes in black pepper. The differential expression of PnTET3.1, PnTET8.1, and PnTET10 genes in stress conditions as well as the presence of stress-responsive regulatory components in the PnTET promoter regions indicated its functional role in black pepper. Future studies will be directed towards the elucidation of their role in defense responses in the susceptible and resistant varieties of black pepper.
Evaluation of non symbiotic nitrogen fixation by brady rhizobium in cowpea
(Department of Plant Pathology, College of Horticulture, Vellanikkara, 1997) Asha, S; Rajendran Pillai, M V
A study was conducetd at the College of Horticulture, Vellanikkara during the year 1993-95 on the evaluation of non-symbiotic nitrogen fixation by Bradyrhizobium in cowpea. Twelve isolates collected from different agroclimatic regions of Kerala, two exotic isolates and one KA U isolate were used for the study. First, a pot culture experiment was conducted to screen the above 15 isolates for their efficiency in non symbiotic nitrogen fixation. Two media and two methods of application were also tried. A second pot culture experiment was also conducted to find out the most suitable method of application of the best performer in terms of foliar nitrogen fixation. The results of the screening experiment showed that no particular isolate was effective in improving any of the parameters observed. However,out of the ten characters studied, isolate liS (KAU isolate) ranked best in improving five characters namely, plant height, number of leaves, dry weight of nodule, fresh weight of plants and nitrogen content. Thus 115 was selected as the promising isolate for the second experiment. Isolate 114 (CB-7 56), which showed next better performance in improving number of leaves, dry weight of nodules and fresh weight of plants was also selected. Moreover, it was one of the known exotic isolates supporting non-symbiotic nitrogenase activity. The results of the second experiment clearly established the superiority of seed (soil) inoculation (AI) of Bradyrhizobium over different methods of foliar application. However, foliar applications also showed improved performance over control plants in improving the different parameters observed. Spraying the bacteria grown in broth shake culture (A3) was found effective in improving the number of leaves, dry weight of roots, chlorophyll content and nitrogen content of plant. The study show that, eventhough less efficient, the foliar application of Bradyrhizobium can also be resorted to in improving the performance of cowpea plants in situations where seed (soil) inoculation is not possible.
In planta transformation via pollen tube pathway in black pepper(Piper nigrum L.)
(Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara, 2009) Asha, S; Rajendran, P C