PG Thesis
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Item Efficacy of silver nanoparticles in eliminating systemic contamination in the in vitro culture of black pepper (Piper nigrum L.)(Department of Plant Biotechnology, College of Agriculture , Vellayani, 2023-04-05) Savio Franklin; Swapna AlexThe study entitled “Efficacy of silver nanoparticles in eliminating systemic contamination in the in vitro culture of black pepper (Piper nigrum L.) was carried out at the Department of Plant Biotechnology, College of Agriculture, Vellayani, Thiruvananthapuram during 2019 to 2021. The objective of the study was to evaluate the efficacy of silver nanoparticles for eliminating the systemic contamination and improving the efficacy of micropropagation protocol in black pepper Piper nigrum L. Single nodal cuttings of Piper nigrum L. var. Panniyur 1 collected from Instructional Farm, College of Agriculture, Vellayani were used as explants. Among the five surface sterilization treatments tried with different concentrations of mercuric chloride, ethyl alcohol and pretreatments with bavistin, surface sterilization with 70 % ethyl alcohol for 1 min followed by 0.2 % mercuric chloride for 10 min showed maximum percentage of uncontaminated cultures for a period of two weeks (26.6 %), Phenolic exudation was observed in all the cultures within a period of two weeks. No contamination was observed when the uncut surface sterilized nodes were stabbed on Nutrient Agar (NA) and basal Murashige and Skoog (MS) media, thereby confirming that surface sterilization treatment was effective and contamination observed in the cut nodes after a period of two weeks was endophytic in nature. All the cultures were incubated on the illuminated racks at 25 ◦C with 16 h photoperiod. Tissue indexing by pricking the tissue with a sterile needle at three different positions (portions below medium, above the medium, and also at the tip of the shoot) followed by stabbing the needle on NA and MS media did not show any contamination. Plant tissue homogenate indexing by grinding one-gram tissue homogenate in one millilitre of sterile distilled water followed by serial dilution and plating showed the presence of 5.84 x 106 cfu in NA medium on the second day of inoculation and 4.88 x 106 cfu colonies in MS medium on the fourth day of inoculation. On medium indexing by placing the two vertical halves of the node in horizontal position on NA and MS media, endophytic contamination was observed on seventh day and fourteenth day of inoculation on NA and MS media respectively. Serial dilution of the endophytic contamination was done and the colonies picked from 10-6 dilution were used for bacterial in vitro inhibition assay. In vitro inhibition assay was carried out using sterile discs loaded with different concentrations (10 mgL-1 and 20 mgL-1) of 20 nm and 100 nm silver nanoparticles alone and in combination with streptopencillin, 150 mgL-1 of streptopenicillin alone, and sterile distilled water as control. No inhibition zone was observed on treatments with silver nanoparticles alone whereas an inhibition zone of 28 ± 2 mm was observed for streptopenicillin (150 mgL-1), 21 ± 1 mm for streptopenicillin with 20 nm silver nanoparticles and 18 ± 1mm for combination of streptopenicillin with 100 nm silver nanoparticles. To conclude, surface sterilization with 70 % alcohol for 1 min followed by 0.2 % mercuric chloride for 10 min was effective in controlling surface contaminants in black pepper var. Panniyur 1. Endophytic contaminants could not be effectively removed by 20 nm and 100 nm of silver nanoparticles at concentrations of 10 and 20 mgL-1 and hence is not recommended as an alternative to incorporation of 150 mgL-1 of streptopencillin in controlling the systemic contamination of black pepper in tissue culture.Item Alleilic difference in the putative gene ipk1 sequence and phytic acid (INSP6) content in Black Pepper(Department of Plant Breeding & Genetics, College of Agriculture, Padannakkad, 2019) Gladish Mary Joy; Sujatha, RBlack pepper (Piper nigrum L.) is one of the world’s highly demanded and most traded spices with high medicinal and therapeutic values. A unigene pnc135 (995 bp) was developed by the Expressed sequence tags data obtained by next generation sequencing. This unigene was found to show similarity with ipk1 gene reported in other crop species which encodes for inositol pentakisphosphate-2 kinase enzyme (Unpublished data, Sujatha,R.). This enzyme is involved in the phosphorylation of inositol pentakisphosphate to inositol hexakisphosphate or phytic acid (InsP6), last step in the biosynthetic pathway of phytic acid. This unigene was later partially sequenced (1072 bp) towards the 3’ end by directional genome walking by Giridhari (2017). Phytic acid functions as the major storage form of phosphorus in seeds, cereals and legumes possessing significant benefits including signalling, plant communication, messenger RNA transport etc. However, phytic acid also acts as an anti-nutrient in animals as its chelating property will cause malnutrition in organisms and also leads to environmental pollution due to phosphorus excretion by non-ruminant animals. Therefore researchers are finding ways to create ipk1 mutants for either to decrease or increase the phytic acid content in organisms. However the genetic information about the black pepper crop remains very limited and the metabolic pathways and the genes related to it are also poorly understood. So in this study entitled “Allelic difference in the putative gene ipk1 sequence and phytic acid (InsP6) content in black pepper (Piper nigrum L.)”, the objective was to find out the flanking region towards the 5’ region of pnipk1 gene fragment (1072 bp) reported earlier by Giridhari (2017), to identify the allelic differences in pnipk1 gene in 10 black pepper genotypes and to estimate and quantify the phytic acid content in these 10 black pepper genotypes using polyacrylamide gel electrophoresis (PAGE). Genomic DNA was isolated from Panniyur 1 variety of black pepper and used it for the whole genome amplification by Rolling Circle Amplification method using Phi 29 DNA polymerase and walker adaptors (WA1, WA2, WA3 and WA4) reported by Reddy et al. (2008). After whole genome amplification, genome walking using primer combinations of walker primers, locus specific primers and nested locus specific primers were performed to find out the flanking region towards the 5’ region of pnipk1(1072 bp) gene fragment of black pepper. The walker primers (WP1 and WP2) used for genome walking were same as that of reported in Reddy et al. (2008) and the locus and nested locus specific primers were designed on the basis of pnipk1 gene fragment (1072 bp) sequenced by Giridhari (2017). From the nested PCR amplification four products, two amplicons, A1R3 and A4R3, each at two different temperatures viz., 51.6⁰Ϲ and 56.8⁰Ϲ were obtained and sequenced. On assembling the sequences a contig of length 523 bp was obtained towards the 5’ region of pnipk1 gene fragment and this showed similarity to ipk1 gene in other crops. This 523 bp contig was assembled with ipk1 gene fragment (pnipk1-1072 bp) to get a total length of 1535 bp. The newly assembled ipk1 gene sequence (pnipk1-1535 bp) was analysed in ORF finder for the coding region and found an Open Reading Frame (ORF) with 645 bp encoding for 214 amino acids. Phylogenetic analysis of the sequence and translated amino acid sequence showed closer evolutionary relationship with that of Dendrobium catenatum. Primers were designed based upon the pnipk1 gene sequence (1535 bp) to amplify the genomic DNA of Panniyur 1 and other 10 black pepper genotypes. The selected 10 genotypes were Panniyur 5, Panniyur 7, Chettanvally, Kottanadan, Karimunda 7, Thottamundy, Karimunda kuttyatur, Payyanganam 2, PRS 160 and Chumala. Amplification of pnipk1 gene (1535 bp) was obtained from genomic DNA of Panniyur 1, Panniyur 5, Panniyur 7 and Karimunda 7 with the expected of amplicon size indicating a similar sequence among these genotypes. Whereas amplification was not obtained in genomic DNA in rest of the genotypes showing allelic variation is present for ipk1 gene in these genotypes. To estimate and quantify the phytic acid content in Panniyur 1 and 10 black pepper genotypes, polyacrylamide gel electrophoresis (PAGE) was performed. Phytic acid was extracted from black pepper varieties same as that of selected for allelic difference analysis in ipk1 gene (pnipk1-1535 bp). The samples were loaded with phytate standards and band intensities of each concentration were determined with Gelquant.NET. The values of phytic acid in black pepper genotypes were estimated by the standard curve. The quantity of phytic acid in samples are: Panniyur 5 with 502.5nmoles/g, Panniyur 7 with 367.5nmoles/g, Chettanvally-511.5nmoles/g, Kottanadan-463.5nmoles/g, Karimunda7- 387nmoles/g, Chumala-201nmoles/g, Karimunda kuttyatur-637.5nmoles/g, Payyanganam 2-196.5nmoles/g and Panniyur 1- 275 nmoles/g, Thotamundy- 198nmoles/g, PRS 160-697.5nmoles/g. Based on the phytic acid content in the black pepper genotypes, they can be classified into low (<210 nmoles/g), medium (210-510 nmoles/g) and high (>510 nmoles/g) phytic acid content. The ipk1 gene fragment (pnipk1-1535 bp) was amplified in the genotypes Panniyur 1, Panniyur 5, Panniyur 7 and Karimunda 7. These genotypes all came under the category of medium phytic acid content group. The study resulted in sequencing a total of 1535 bp long segment of ipk1 gene black pepper variety Panniyur 1 and analysing the presence of allelic variation in ipk1 gene and phytic acid content in selected black pepper genotypes.Item Identification and characterization of suppressor of over expression of constans1 (SOC1) gene in black pepper (Piper nigrum L.)(Department of Plant Biotechnology, College of Agriculture, Vellayani, 2016) Manu K Venu; Lekha SreekantanThe present study entitled“Identification and characterization of Suppressor of Overexpression of Constans1 gene in Black Pepper (Piper nigrum L.)”was conducted at the Integrated Biotechnology Block, College of Agriculture, Vellayani, during 2015-2016.The study envisagedisolation and sequencing ofSOC1, a flowering integrator gene in black pepper (variety - Karimunda) and functional characterization of the gene by studying the expression patterns. Degenerate primers were designed for the above said gene based on the gene sequences from NCBI database (SOC1 forward and reverse primers) which were used to isolate and identify the gene. Total RNA of black pepper was isolated using modified CTAB method followed by synthesis of cDNA using AMV RT (Avian myeloblastosis virus reverse transcriptase). PCR (Polymerase chain reaction) with degenerate primers was done using cDNA as the template. However no amplifications were observed after the first reactions. Therefore nested PCR reactions were done using the PCR products of the first reaction as the template. Two bands of size 640 bp and 330 bp were produced in the nested reactions. Sequencing of the product yielded four sequences with each of the sequence showing similarity to the SOC1 gene, when done sequence analysis, thus making it the first flowering integrator gene to be identified in black pepper. Microscopy studies were carried out to see the floral characters of black pepper in detail. Microscopy studies were done using FAA fluid as fixative, sectioning the tissues and staining with safranin and fast green were carried out to see the changes occurring in different development stages of spikes from immature spike to complete spike with berries.Item Somatic embryogenesis in black pepper (Piper nigrum L.)(Department of Plant Biotechnology, College of Agriculture, Vellayani, 2018) Afnamol, O P; Soni, K BItem Formulation of a key for identification of the different types of pepper, Piper nigrum L.(Department of Agricultural Botany, College of Horticulture, Vellanikkara, 1982) Kanakamany, M T; Luckins C BabuThe studies reported herein were carried out in the Department of Agricultural Botany, College of Horticulture, Vellanikkara, during the year 1980-82 with a view to formulating a key for identification of different varieties of pepper. From the germ plasm collection maintained in the Pepper Research Scheme of the College, 45 types of uniform age were earmarked. Observations on twentyeight quantitative and seventeen qualitative characters were recorded from all the fortyfive types and the variability among the types was assessed. The study revealed that the material was highly variable with reference to many of the characters.Item Genome walking for putative phytic acid (InsP6)unigene in black pepper (Piper nigrum L.)(Centre for Plant Biotechnology and Molecurar Biology, College of Horticulture, Vellanikkara, 2017) Ananduchandra Giridhari; Sujatha, RItem Characterization of PR proteins in selected calliclones of black pepper in relation to phytophthora foot rot disease(Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara, 2017) Debashis Sahoo; Shylaja, M RBlack pepper (Piper nigrum L.), the king of spices is severely affected by Phytophthora foot rot disease caused by Phytophthora capsici. The disease results in severe crop loss, and valuable genotypes are lost every year due to this dreadful disease. The local cultivars and released varieties of black pepper are susceptible to the pathogen, but variation exists in genotypes in the degree of tolerance and mechanism of defense to the disease. The plants resist the pathogen infection by accumulating a number of defense related proteins in the intercellular spaces which are collectively known as pathogenesis related (PR) proteins. The study was aimed to characterize PR proteins in selected eleven calliclones of black pepper along with susceptible variety Panniyur-1 after challenge inoculation with Phytophthora capsici so as to get more insight on the defense mechanism of Phytophthora foot rot. Investigations on disease reaction of the selected calliclones and variety Panniyur-1 after challenge inoculation with Phytophthora capsici, β-1,3-glucanase activity and protein analysis by SDS-PAGE was carried out at 0, 24, 48 and 72 hours after inoculation. Protein profiling by 2D-gel electrophoresis and protein identification by MALDI-TOF MS / MS in the most tolerant and susceptible calliclone and in silico analysis for characterization of proteins were also attempted in the present study. In leaf symptom bioassay, variety Panniyur-1 showed susceptible reaction as compared to calliclones of Cheriakanyakkadan and Kalluvally. Based on β-1,3-glucanase activity and expression of 16.5 kDa band in SDS-PAGE, clone KLCC 89 was selected as the tolerant calliclone. 2D-gel electrophoresis was attempted in the selected tolerant calliclone, KLCC 89 and susceptible variety Panniyur-1. Proteome analysis by 2D-gel electrophoresis could locate 167 differentially expressed protein spots in KLCC 89, 24 hours after inoculation. Analysis by PDQUEST software could select four protein spots (Spot 1, Spot 2, Spot 3 and Spot 4) from 167 spots based on higher degree of differential expression. The selected spots were sent to Rajiv Gandhi Centre for Biotechnology, Thiruvananthapuram for protein identification by MALDI-TOF. Analysis by MALDI-TOF and MASCOT search could identify 15 hit peptides from the selected four protein spots. Analysis and functional characterization of the 15 hit peptides by BLAST2GO revealed the defense response of tolerant calliclone KLCC 89 against Phytophthora foot rot disease. The enhanced expression of plastocyanin protein, TRAF-like family proteins, RUBISCO dependent glycolate and glyoxylate metabolism, light dependent ROS production during photorespiration, F-box proteins, synthesis of antimicrobial metabolites and retrotransposition activity were observed in the tolerant calliclone KLCC 89 as defense related responses. Plastocyanin is involved in regulation of photosynthesis to meet the requirements of nutrient competition by the P. capsici whereas the TRAF-like family proteins is involved in regulation of programmed cell death. The increment in RUBISCO, regulates the glycolate and glyoxylate metabolism for H2O2 production. F-box proteins are involved in regulation of jasmonate regulated defense-related pathways. The study could characterize PR proteins in the selected calliclones and investigate in depth the Phytophthora capsici interaction in black pepper at proteome level. The future research should focus on validation of identified proteins in defense mechanism, characterization of novel protein in KLCC 89, in-depth investigations on retrotransposons in defense mechanism of Phytophthora foot rot tolerance in black pepper, metabolic engineering of the pathways triggering transcription of PR-genes and transgenic / cisgenic research for Phytophthora foot rot resistance.