1. KAUTIR (Kerala Agricultural University Theses Information and Retrieval)
Permanent URI for this communityhttp://localhost:4000/handle/123456789/1
Browse
8 results
Search Results
Item Induction of embryogenic calli from immature ovaries in coconut (cocos nucifera L.)(Department of Molecular biology and biotechnology , College of Agriculture, Vellayani, 2024-02-01) Vyshnavi, K; Anuradha, TThe study entitled " Induction of embryogenic calli from immature ovaries in coconut (Cocos nucifera L.)" was conducted at the Department of Molecular Biology and Biotechnology, College of Agriculture, Vellayani, Thiruvananthapuram during 2022-23. The objective of the study was induction of embryogenic calli from immature ovary in coconut variety West Coast Tall (WCT) and effect of sodium butyrate in callus induction through histone deacetylation. In the present study, immature ovaries from explants of maturity indices 4 to -6 were extracted and surface sterilized using 3% sodium hypochlorite, 0.2% mercuric chloride, and 70% ethanol. They were inoculated into basal Y3 medium containing different plant growth regulators such as 2,4-D, TDZ, and picloram. The results showed that out of all the maturity indices tried (-4 to -6), inflorescence of maturity index -4 showed a maximum response in callus induction, and among different callus induction medium (CIM) tried CIM with 100 μM 2,4-D showed maximum response (47.28%) to embryogenic calli initiation. The time taken for embryogenic calli-like structure development was 45 days. To analyze the effect of sodium butyrate (SB) in callus initiation, the excised immature ovaries were inoculated into standardized CIM (Y3 + 100 μM 2,4-D) with different concentrations of SB. The results displayed that, compared with the CIM, the induction rates of embryogenic calli were significantly increased (66%) and the time taken for embryogenic calli-like structure development was significantly reduced (20 days) with the addition of 100 μM SB. The gene expression analysis shows that gene expression of the HDA6 gene was reduced in CIM with SB when compared to CIM. The increased embryogenic response of immature ovaries to CIM with SB as well as the reduced time taken for the embryogenic response can be correlated with the downregulation of the HDAC gene.Item Molecular cloning of key genes involved in piperine biosynthesis pathway from piper sp.(Department of Molecular biology and biotechnology , College of Agriculture, Vellayani, 2023-10-03) Devika Babu.The study entitled “Molecular cloning of key genes involved in piperine biosynthesis pathway from Piper sp.” was carried out at Department of Molecular Biology and Biotechnology, College of Agriculture, Vellayani during the year 2022-2023. The objective of the study was molecular cloning and sequence comparison of piperic acid CoA ligase and piperine synthase genes involved in piperine biosynthesis from selected Piper sp. Bush pepper plants of Piper nigrum varieties Panniyur 1 and Panniyur 4; Piper longum and Piper colubrinum were used for the study. The total RNA was isolated from the leaf and immature fruits of Panniyur 1, Panniyur 4, P. longum and P. colubrinum plants. Piperine synthase and piperic acid CoA ligase were amplified from each cDNA samples using designed primers of Piperine synthase and piperic acid CoA ligase gene. Amplicons of size approximately 1000 bp and 150 bp were obtained for leaves of Panniyur-1, Panniyur-4 and Piper colubrinum, whereas only 150 bp amplicon was obtained for leaves and immature fruit of Piper longum and spikes of Panniyur-1, Panniyur-4 and Piper colubrinum. Amplicon of approximately 1000 bp size was obtained from the leaves of Panniyur-1, and Panniyur-4. No bands were obtained for the leaves and immature fruits of Piper colubrinum and Piper longum and spikes of Panniyur-1, Panniyur-4 and Piper longum. Since intact bands of expected size was not obtained on the amplification of cDNA from Piper longum using designed primers, it is concluded that, there could be variation in the primer binding site of Piper longum genomic sequence, which was later confirmed by analysing the transcript sequence. PCR product was cloned into pGEM®-T vector and sequenced. The sequence obtained was subjected for in silico analysis. Nucleotide sequence of piperine synthase and piperic acid CoA ligase was found to contain 1018 bp and 970 bp respectively. The BLAST analysis of piperine synthase nucleotide sequence showed 99% similarity with piperine synthase reference gene in NCBI database. Amino acid sequence from Panniyur 1, Panniyur 4 and P. colubrinum was subjected for BLAST analysis and showed a similarity of 48 % with benzyl alcohol O-benzoyltransferase in Coffea arabica. BLAST analysis of amino acid sequence from Panniyur 1 and Panniyur 4 showed 65% similarity with acyl-CoA ligase of Macadamia integrifolia. Eventhough protein sequences of reference gene and query sequence of piperine synthase and piperic acid CoA ligase are similar, there is a substitution of amino acids at distinct regions. Meanwhile, P. colubrinum showed distinct sequence variation at the active sites of piperine synthase gene. The phylogenetic analysis of piperine synthase gene from different Piper sp. showed relation with Zingiber sp. and Rhodamnia sp. Phylogenetic tree with piperic acid CoA ligase from Piper nigrum varieties formed a separate lineage from CoA ligase enzyme from Camellia sp, Telopia sp and Macadamia sp. with a common branch point. Tertiary structure predicted were similar for Panniyur 1 and Panniyur 4, whereas differed for P. colubrinum due to sequence variation. Piperic acid CoA ligase protein from Panniyur 1 and Panniyur 4 were found to be similar. The stability of the predicted tertiary structure was determined using Ramachandran plot, which was found to be stable. The present study revealed the sequence information of piperine synthase gene from two cultivated varieties Panniyur 1, Panniyur 4 and a wild variety, P. colubrinum and piperic acid CoA ligase gene from Panniyur 1 and Panniyur 4. Although the sequences are similar to the reference sequence of Indonesian black pepper cultivar, distinct coding variants and single nucleotide polymorphisms that lead to substitution of certain amino acids was detected in both Indian cultivars and wild sp. As these variations affects the protein function and activity, future work will be directed towards the recombinant expression and further validation of the protein.Item Anticancer activity of clitoria ternateal through epigenetic regulation(Department of Molecular biology and biotechnology , College of Agriculture,Vellayani, 2023-09-21) Arya, C S.The study entitled “Anticancer activity of Clitoria ternatea L. through epigenetic regulation” was carried out in the Department of Molecular Biology and Biotechnology, College of Agriculture, Vellayani, Thiruvananthapuram during 2022 - 2023. C. ternatea is a perennial herbaceous plant belonging to the family Fabaceae and it is reported to have anti-oxidant, anti-proliferative, and cytotoxic effects. However, its role in modulation of key epigenetic mechanism namely methylation and the difference in expression of genes associated with methylation has not yet been studied. Hence the objective of this study was to investigate the in vitro anticancer activity of Clitoria ternatea L. and analysing its role on modulation of methylation and differential expression of genes involved in methylation. Leaves of C. ternatea were dried and extracted with methanol using both cold and soxhlet extraction methods. 640mg and 2510mg of crude extract were obtained from 10g of powdered leaves sample by cold and soxhlet extractions respectively. Cytotoxic effect of the leaf extract was evaluated by MTT assay in human colon cancer cell line (HCT-116), breast cancer cell line (MCF-7), and normal cell line (HEK-293). The extracts showed dose-dependent reduction in cell survival percentage and IC50 values of cold extract were found to be 189.19µg and 176.15µg for colon and breast cancer respectively. The IC50 values of soxhlet extracted methanolic leaf extract were found to be 146.62µg and 148.34µg for colon and breast cancer cell lines respectively. MTT assay on HEK-293 cell line revealed the extracts did not show cytotoxicity up to IC50 values. Soxhlet extracted methanolic leaf extract has higher cytotoxicity than cold extract. Hence soxhlet extract was used for further experiments. On analysing the global DNA methylation pattern of treated cells with respect to control it was found that there was hypermethylation of 1.20% and 0.76% in colon and breast cancer cell lines respectively. However, there was no change in the methylation pattern of mTOR gene on treated cells. Expression of the methylation specific genes DNMT1, DNMT3a, DNMT3b, MeCP2, and TET2 indicated that hypermethylating genes (DNMT1, DNMT3a, DNMT3b, and MeCP2) were upregulated and the demethylating gene (TET2) was downregulated in treated cells. The findings suggest that the extract is capable of modulating the methylation profile of cells by regulating the expression of methylation specific genes and C. ternatea can be considered as a good candidate for the development of cancer therapeutics.Item Nano-PCR for the detection of tomato leaf curl virus(Department of Department of Molecular biology and biotechnology , College of Agricultureand botechnology , College of Agriculture,Vellayani, 2023-09-21) Devika, P P.The study entitled "Nano-PCR for the detection of Tomato leaf curl virus (ToLCV)" was conducted at the Department of Molecular Biology and Biotechnology, College of Agriculture, Vellayani, Thiruvananthapuram during 2022-23. The objective of the study was to evaluate the improvement of detection efficiency of Tomato leaf curl virus (ToLCV) in Solanum lycopersicum L. by NanoPCR by the inclusion of magnesium oxide and silver nanoparticles. ToLCV – partial CP gene cloned in plasmid DNA (pMD 20-T) (Athira et al., 2022) and maintained in E. coli cells (strain DH5-α) was used for standardization of concentration of nanoparticles for viral detection in PCR. Plasmid DNA was isolated from overnight grown cultures of E. coli using alkaline lysis method (Birnboim and Doly, 1979), and the good quality was confirmed using nanodrop spectrophotometric analysis. A universal primer for geminiviruses designed using the CP gene was used for viral detection (Deng et al., 1994). The minimum concentration of plasmid DNA at which virus amplification is obtained using PCR was standardized as 1.5ng and was used for further treatments using nanoparticles in PCR. Silver nanoparticles (AgNPs) of size 20 nm (Sigma Aldrich, USA) and magnesium oxide nanoparticles (MgONPs) of size ≤ 50 nm (Sigma Aldrich, USA) was used for the study. In treatments 1-4, different concentration of AgNPs (1 ng/µL, 2.5 ng/µL, 3 ng/µL, 3.5 ng/µL and 4 ng/µL) alone was used in PCR. In treatments 5-8, different concentration of MgONPs (100 ng/µL, 150 ng/µL, 200 ng/µL, and 250 ng/µL) was used in PCR. Combinations of AgNPs and MgONPs (3 ng/µL +200 ng/µL, 3 ng/µL +150 ng/µL, 2.5 ng/µL +200 ng/µL, 2.5 ng/µL +150 ng/µL) were used for treatments 9-12. In treatments 13-16, different concentrations of MgONPs (100 ng/µL, 200 ng/µL, 250 ng/µL, 275 ng/µL, and 300 ng/µL) by replacement of magnesium chloride (MgCl2) in PCR buffer were tried. Combinations of AgNPs and MgONPs (3 ng/µL +275 ng/µL, 3 ng/µL +250 ng/µL, 2.5 ng/µL +275 ng/µL, 2.5 ng/µL +250 ng/µL) by the replacement of MgCl2 in PCR buffer were used for treatments 17-20. PCR was performed at 95⁰C for 3 min followed by 34 cycles of 95⁰C for 30s, 53⁰C for 90s, 72⁰C for 90s, and final extension at 72⁰C for 10 min. Control was kept without any nanoparticles. Three 100 replications were done. The efficiency and specificity of PCR were checked by analyzing the intensity of the expected amplicon (500bp) in agarose gel electrophoresis and comparing the band area with that of the control using Image Lab/ImageJ software. The inclusion of AgNPs and MgONPs in PCR reaction mix at concentrations of 3 ng/µL and 200 ng/µL respectively, exhibited a 4-fold and 7.6-fold increase in the intensity of the band. Simultaneous inclusion of both AgNPs and MgONPs at concentrations of 3 ng/µL and 200 ng/µL respectively in PCR exhibited a 4.5-fold increase in the intensity of the band. The inclusion of MgONPs (275 ng/µL) replacing the MgCl2 in PCR buffer resulted in a 13-fold increase in band intensity and the simultaneous inclusion of both AgNPs and MgONPs replacing the MgCl2 in PCR buffer exhibited a 12-fold increase in band intensity. The inclusion of nanoparticles in PCR resulted in the production of the visible band even at 25 cycles, thereby reducing the duration of PCR by 26%. The results were further confirmed by using genomic DNA isolated from ToLCV-infected tomato plants as the template for PCR, by the inclusion of MgONPs (275 ng/µL) replacing MgCl2 in PCR buffer with optimized PCR cycles (25 cycles). Leaf samples for the isolation of genomic DNA of ToLCV-infected tomato plants were collected from Instructional Farm, College of Agriculture, Vellayani. The modified cetyltrimethylammonium bromide (CTAB) method was performed for isolating genomic DNA and the good quality was confirmed using nanodrop spectrophotometric analysis. To conclude, the inclusion of MgONPs at a concentration of 275 ng/µL replacing MgCl2 in PCR buffer, exhibited maximum improvement (13-fold increase) in the sensitivity of PCR. The cycle number in PCR is reduced to 25 cycles, thereby decreasing the duration of PCR by 26%. Evaluation of ToLCV-infected samples at different stages can be studied by challenge inoculation to make detection possible at the earliest stage for diagnostic purposesItem Expression profiling of nitrogen responsive genes in rice varieties(Department of Molecular biology and biotechnology, College of Agriculture, Vellayani, 2023-09-25) Anupama Padmakumar.The study entitled “Expression profiling of nitrogen responsive genes in rice varieties” was conducted at the Department of Molecular Biology and Biotechnology, College of Agriculture, Vellayani during 2022-23 with an objective to compare the expression profiles of nitrogen responsive genes OsAMT1.2, OsAMT2.1, OsNRT2.3, and OsNAR2.2 in organic (Jaiva) and popular (Uma) rice varieties. The seeds of the rice variety Jaiva were collected from RARS, Pilicode, Kasaragod, and the seeds of the rice variety Uma were collected from RRS, Mancompu, Alappuzha for the study. The seeds were treated with Pseudomonas fluorescens through the wet seed treatment method. The sprouted seeds were transferred to a pot containing clayey soil, which was collected from the rice field of the College of Agriculture, Vellayani after checking pH and liming addition. The rice varieties Jaiva and Uma were provided with 2 nutrient treatment methods i.e., inorganic and organic management during the entire crop duration. The inorganic nutrient treatment included the application of urea, rajphos, and Muriate of Potash (MOP) in the ratio 90:45:45 kgNPKha-1 and the organic nutrient application included the application of neem cake (4:1:2 ratio NPK), and cow dung (0.5:0.2:0.5 ratio NPK), at nitrogen equivalent basis. The plant performance analysis was carried out by observing various growth and yield parameters. The expression profiles of four nitrogen responsive genes, namely OsAMT1.2, OsAMT2.1, OsNRT2.3 and OsNAR2.2 were analyzed at seedling and tillering stages by performing RT-qPCR with OsActin as reference gene. It was observed that the rice varieties Uma and Jaiva showed better performance when managed with inorganic nutrients except in the number of days to flowering and sterility percentage. The yield gap under the inorganic and organic nutrient management was 14.14% and 25.77% for Jaiva and Uma respectively. When managed with inorganic and organic nutrients, the number of days to flowering differed by 2 and 9 days in Jaiva and Uma rice varieties respectively. The early flowering considered to be beneficial for higher yield. The sterility percentage in Jaiva was on par under both nutrient management whereas in Uma there was a 0.95% decrease under inorganic management. The gene expression analysis showed elevated levels of OsAMT1.2 (ammonium uptake gene) in the leaf and root samples of Jaiva at seedling (1.85 and 4 folds) and tillering stages (1.69 and 1.57 folds) under organic nutrient management. In the case of Uma, these tissues showed low expression of OsAMT1.2 at both seedling and tillering stages under organic management. A similar trend was observed for OsAMT2.1 (ammonium transporter) expression in these rice varieties under organic nutrient management. Both OsAMT1.2 and OsAMT.2 showed maximum expression in roots (4 folds). The nitrate transporter gene OsNRT2.3 showed 7-fold expression in roots at the tillering stage of Jaiva when managed with organic nutrients. The variety Uma showed elevated expression of OsNRT2.3 in the leaves and roots at both stages under inorganic nutrient management. In the rice variety Jaiva, the nitrate assimilating gene OsNAR2.2 showed higher expression under inorganic nutrient management in the seedling stage, but in the variety Uma the expression was higher under organic management. The relative expression levels of the nitrogen responsive genes in the rice varieties Uma and Jaiva reveal that Jaiva maintains a higher level of expression of OsAMT1.2, OsAMT.2 and OsNRT2.3 under organic nutrient management, which may be the reason for its better yield. These genes after further verification can be used for selecting or breeding rice varieties for organic cultivationItem Identification of simple sequence repeat (SSR) markers linked to high temperature tolerance in rice (Oryza sativa L.) by bulked segregant analysis(Department of Molecular Biology and Biotechnology, College of Agriculture, Vellayani, 2023-08-24) Aparna, K.; Beena, RThe study entitled “Identification of Simple Sequence Repeat (SSR) markers linked to high temperature tolerance in rice (Oryza sativa L.) by bulked segregant analysis” was conducted at the Department of Molecular Biology and Biotechnology and the Department of Plant Physiology, College of Agriculture, Vellayani, Thiruvananthapuram during 2022-23. The objective of the study was to evaluate the F3 population of Uma X NERICA-L-44 for identifying SSR markers linked to high temperature tolerance in rice by bulked segregant analysis and to establish the role of the genes associated with them in heat tolerance. Seeds collected from 46 F2 segregants (23 tolerant and 23 susceptible) of Uma X NL-44 along with their parents were raised and transplanted to pots after 18 days of sowing in the Kharif season of 2022. These 48 lines were kept under normal conditions up to the maximum tillering stage and then transfered to polyhouse conditions where it was subjected to a high temperature of 38-40 ⁰C. Phenotypic evaluation was done for plant height, tiller number, productive tiller number, days to flowering, time of anthesis, Pollen viability, Spikelet fertility, and 100 seed weight. Based on spikelet fertility percentage, 10 extremely tolerant and 10 extremely susceptible lines were selected. DNA was extracted from the selected 10 heat tolerant and 10 susceptible lines along with the parents by the modified Cetyltrimethylammoniumbromide (CTAB) method of DNA extraction. The quality and quantity of extracted DNA were checked by using agarose gel electrophoresis and spectrophotometric analysis. The DNA samples were screened by using 55 SSR primers distributed across the rice genome. Out of 55 SSR primers, 18 of them showed polymorphism between the parents. Then equal quantity of DNA was pooled to make heat tolerant and susceptible bulks. The bulked DNA samples were screened using the 18 SSR primers that have shown polymorphism between parents. The polymorphic markers between the tolerant and susceptible bulks were used to study the segregation of the alleles in the individual lines constituting the tolerant and susceptible bulks. Through bulked segregant analysis (BSA), 10 SSR markers were found polymorphic between tolerant and susceptible bulks and their individual lines. It revealed the possible presence of genetic loci for heat tolerance in those locations. Out of the 10 SSR markers identified in the BSA (RM337, RM10793, RM242, RM5749, RM6100, RM490, RM3475, RM470, RM473, and RM556), nine of the markers have been previously reported for heat tolerance traits. RM337 is newly identified in the present study. The genes in the 200 kb vicinity of the RM markers were retrieved from the Rice Annotation Project database. To get a deeper insight into how these genes participate in heat tolerance, gene annotation, gene ontology (GO) enrichment analysis, and trait ontology (TO) were performed for all the significant markers. Upon screening of the loci in the proposed region, genes LOP1 (LOC_Os08g01330) and LOP2 (LOC_Os08g0112) were found to be associated with RM337. LOP1 is a NAC transcription factor that is reported to be involved in the regulation of cellulose synthesis, secondary wall biosynthesis (Os08t0103900-01), and inflorescence development. LOP2 is also known as OsMOT1, which is a molybdate transporter involved in the uptake and translocation of molybdate (Os08t0101500-01). Hence their expression was analyzed in the two rice varieties under both control and high temperature conditions. LOP1 was found to be significantly upregulated in the NL-44 variety under high temperature condition compared to the normal temperature conditions and susceptible variety, Uma. On the other hand, the gene LOP2 was found to be upregulated in both varieties under the higher temperature condition compared to their respective controls. However, the relative expression in Uma was higher than in NL-44. In the present study, the phenotypic evaluation and bulked segregant analysis using 55 SSR primers in F3 generation of Uma X NL-44 revealed that 10 SSR markers (RM222, RM242, RM337, RM470, RM473, RM490, RM556, RM5749, RM6100, RM10793) are linked to high temperature tolerance in rice. The newly identified SSR marker RM337 and associated gene LOP1 is also linked to high temperature tolerance in rice. The results demonstrate that BSA using SSR markers and gene annotation and enrichment analysis is useful in identifying genomic regions and genes that contribute to thermotolerance respectively. Also, these F3 lines can be used for the development of high temperature tolerant rice varieties and these markers can be used for marker assisted selection (MAS) in rice.Item Biotization using piriformospora indica for induction of in vitro floral primordia in saffron(crocus sativus L.)(Department of molecular biology and biotechnology, College of agriculture , Vellayani, 2023-09-25) Midhukrishna; Smitha BhasiThe study entitled "Biotization using Piriformospora indica for induction of in vitro floral primordia in saffron (Crocus sativus L.)" was conducted at the Department of Molecular Biology and Biotechnology, College of Agriculture, Vellayani, Thiruvananthapuram during 2022-23. The objective of the study was to evaluate the effect of biotization using Piriformospora indica for culture establishment and induction of in vitro floral primordia in saffron. The explants namely corms collected from farmer’s field, Jammu were used for the study. The pure cultures of P. indica were collected from the Department of Agricultural Microbiology, College of Agriculture, Vellayani. The effect of biotization of corms using P. indica was evaluated both in potting medium (Treatment 1) as well as in Murashige and Skoog (MS) medium (Treatment 2). Non-biotized corms were used as control (Treatment 3). Corms were planted in sterile potting medium (perlite and compost in 1:1 ratio) during June and kept in shade conditions for 3 months. However, no sprouting or root induction was noticed and later the corms got dried due to fungal contamination. Another strategy attempted was inducing in vitro rooting and further biotization. For this, the corms were kept at 16ºC during August-October. 100% sprouting was observed in 50 days of treatment and the sprouts thus induced in vivo were maintained at 18ºC for elongation. Further, 120-day-old elongated sprouts were cultured in basal MS medium for root induction. Healthy roots appeared in 3 days and in vitro rooted plants were transferred to potting media incorporated with P. indica mycelium. However, roots of all samples got dried in a week. For attempting biotization in vitro, full corms and 1cm3 cut corms were used for initiating sterile cultures. Sprouts were initiated within 15 days from 1cm3 cut corms cultured in MS medium supplemented with 1.5ppm BAP (6 benzylaminopurine). Further, the sprouts induced both in vitro and in vivo were subjected for elongation in basal MS medium. Significant increase in shoot length was noticed in sprouts induced in vivo (kept at 16ºC) compared to sprouts induced in vitro. Flower buds were induced from elongated sprouts by sub culturing in MS 81 medium and 50% sprouts that were induced in vivo, developed in vitro floral buds. However, no floral buds were observed in elongated sprouts induced in vitro. Biotization of in vitro cultures (Treatment 2) was carried out using P. indica agar discs and derivatives of P. indica. 130-day-old elongated sprouts having in vitro roots were inoculated with P. indica agar discs in MS medium for colonization. However, colonization was not observed under microscopic analysis. 165-day-old elongated sprouts were also treated with derivatives of P. indica and compared with control. Elongated sprouts treated with derivatives of P. indica exhibited a significant increase in plant height compared to control. These sprouts were further sub-cultured for a period of 7 weeks in MS medium for inducing multiple shoots. 165-day-old elongated plantlets treated for 80 days with derivatives of P. indica produced more number of multiple shoots per corm compared to control. Further, the multiple shoots developed were cultured in MS medium supplemented with 9% sucrose for inducing in vitro cormlets. Multiple shoots pre-treated with P. indica produced larger and more number of cormlets compared to control. To conclude, sterile potting media (perlite-compost) was found not suitable for the establishment and growth of corms under field conditions of Vellayani region. Storage of corms at 16ºC during dormant stage is found critical for in vitro growth, rooting and flowering in saffron. In vitro treatment using derivatives of P. indica in saffron showed positive responses with respect to plant height, number of multiple shoots, number, and size of in vitro cormlets. Hence, derivatives of P. indica can be used for better in vitro establishment of cultures, multiple shooting, and in vitro cormlet production in saffron.Item Induction of variability in Vanilla planifolia Andrews through intra/inter specific hybridisation and embryo culture technique(Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara, 2005) Blessy Paul; Valsala, P A