PG Thesis
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Item Characterization of selected curcuma species germplasm using morphological and molecular markers(Department of Plant Biotechnology, College of Agriculture, Vellayani, 2018) Bimal Thomas; Asha, K ICurcuma L., a perennial rhizomatous herb, is gaining global importance as a source of starch besides its medicinal property and use as a spice. Characterization of germplasm is very essential in crop plants and it is the basis for selection of accessions for use in crop improvement programmes. This research work was an attempt to characterize the fifteen selected accessions in eight species of Curcuma collected from different parts of India and maintained in the field gene bank of ICAR-CTCRI using morphological and molecular markers. Two accessions in each of C. amada, C. angustifolia, C. aromatica, C. decipiens, C. malabarica, C. raktakanta, C. zedoaria and one of C. longa were selected. These 15 accessions were morphologically characterized using 13 qualitative and 15 quantitative traits and a wide variability was observed. Dendrogram based on the morphological characters grouped the genotypes into four clusters. PCC analysis revealed that the accessions of the same species have shown more than 83% similarity except C. angustifolia. C. raktakanta accessions have shown a highest intra-specific similarity of 94%. C. decipiens accessions were found to be the highly variable from the most commonly exploited species C. longa while C. aromatica has shown highest similarity. PCA showed that the characters such as leaf midrib colour, rhizome flesh colour, leaf texture and aroma of rhizome have contributed mostly to the variability. Molecular characterization was done using 10 ISSR and 7 SSR markers. The total percentage polymorphism obtained by ISSR characterization was 94.31 while it was 91.11 percentage in the SSRs. C. angustifolia-1 was found to be highly variable from C. angustifolia-2 suggested the occurrence of intraspecific variability. The intra-specific similarity among C. raktakanta accessions were found to be highest than all other accession pairs. Clustering based on ISSR markers grouped the genotypes into five clusters while SSRs into six clusters. Mantel’s test showed a positive correlation between the morphological and molecular data. The results of the present study indicated that the morphological as well as the molecular tools were found to be very effective in the characterization of germplasm of Curcuma species for the developement of core collections and for further use in the crop improvement programmes.Item Development of recombinant coat protien for immunodetection of cucumber mosaic virus infecting banana(Department of Plant Pathology, College of Horticulture, Vellanikkara, 2019) Alan C Antony; Vimi LouisBanana (Musa spp.) is infected by four well characterised plant viruses viz., Banana bunchy top virus, Cucumber mosaic virus (CMV), Banana bract mosaic virus and Banana streak virus. Among these, CMV causes devastating effect on tissue culture banana plants. The study entitled “Development of recombinant coat protein for immunodetection of Cucumber mosaic virus infecting banana” was carried out using existing facilities of Department of Biochemistry, Indian Institute of Science, Bangalore, Division of Plant Pathology, Banana Research Station, Kannara and Department of Plant Pathology, College of Horticulture, Vellanikkara, Thrissur during 2018- 2019. The present study was carried out to produce recombinant coat protein, which can be utilised later for producing high quality antiserum for the detection of CMV infecting banana. Cucumber mosaic virus infected samples were collected based on various characteristic symptoms and screened by direct antigen coating immunosorbent assay using commercially available CMV polyclonal antiserum. Isolate namely, KANC- 4, KANC- 2 and NDRNS- 4 showed maximum absorbance at 405 nm and hence selected for molecular detection using reverse transcriptase polymerase chain reaction with CMV- CP specific primer. The PCR product was purified and CMV- CP amplicon of NDRNS- 4 isolate was ligated to pGEM- T linear plasmid vector, which was later transformed into E. coli DH5α cells. Positive clones were selected according to blue-white screening. Cloning i.e., E.coli DH5α/pGEM- T/CMV- CP was confirmed through colony PCR using coat protein specific primer, restriction digestion of recombinant plasmids using EcoR1 enzyme followed by sequencing. The vectors viz. pRSET- C and pET28a were selected for the expression of CMV- CP gene in E. coli. Coat protein specific forward (5’GGG GCT AGC ATG GAC AAA TCT GAA TCA ACC3’) and reverse primers(5’CCC GGA TCC TTA CTC TCC ATG GCG TTT AG 3’) were designed along with recognition sites of restriction enzymes BamH1 and Nhe1.The annealing temperature of designed primer was standardised as 55°C using gradient PCR. The coat protein gene of CMV was amplified at 750 bp using designed primers and high fidelity Pfu DNA polymerase enzyme. Expression vectors as well as amplicon were subjected to ligation and the recombination in expression plasmids (pRSET- C/ CMV- CP and pET28a/CMV- CP) were confirmed through PCR and sequencing. The plasmid with maximum homology i.e., pRSET-C/CMV- CP was selected for further studies. The recombinant plasmid was transformed into E. coli BL21(DE3)pLysS cells for the expression of CMV- CP gene and the expression of 25 kDa recombinant CMV coat protein was confirmed in 12 per cent sodium dodecyl sulphate - polyacrylamide gel electrophoresis (SDS- PAGE). Tris- NaCl buffer of pH 8.0 was selected for solubilising the recombinant protein using ExPASy - protein translation tool. The recombinant protein was further purified through Nitrilotriacetic acid column purification, in which the 6X histidine tagged recombinant protein was bound with agarose coated nickel beads. Buffers containing imidazole were used for the elution of histidine tagged recombinant protein, since imidazole competes with histidine for the binding site in nickel beads. Each fraction viz., cell pellet, supernatant, flow through, wash and elution were collected and later detected for protein using SDS- PAGE. Absence of 25 kDa protein in cell pellet indicated that the recombinant coat protein completely soluble in Tris- NaCl buffer (pH 8.0). Confirmation of recombinant coat protein was carried out through DAC- ELISA and western blotting using commercially available polyclonal CMV antiserum (1: 2000; NRCB, Trichy). The recombinant coat protein developed through this study could be utilised for large scale production of antiserum for immunodetection of CMV.Item Genetic diversity analysis of phytophthora colocasiae using SSR markers(Department of Plant Biotechnology, College of Agriculture, Vellayani, 2017) Akshara George; Jeeva, M L