1. KAUTIR (Kerala Agricultural University Theses Information and Retrieval)
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Item Gene expression analysis in relation to fusarium wilt resistance in banana (Musa spp.)(Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara, 2013) Jusna Mariya, P L; Keshavachandran, RBanana is one of the important fruit crops of India. Banana is susceptible to several fungal pathogens, nematodes, viruses and insect pests. The greatest threats to global banana production is Fusarium wilt or Panama wilt caused by Fusarium oxysporum f. sp. cubense. Control of the pathogen is difficult and mainly involves the use of disease free suckers. Although disease resistance exists in some banana cultivars, introducing resistance into commercial cultivars by conventional breeding is difficult due to its triploid nature and sterility factors of banana. The study entitled "Gene expression analysis in relation to Fusarium wilt resistance in banana (Musa spp.)" was carried out at the Centre for Plant Biotechnology and Molecular Biology, Vellanikkara during the period 2009-2013 with an objective to identify differentially expressed genes in disease resistant genotype of banana, Palayankodan using the molecular technique called suppression subtractive hybridization (SSH). Total RNA and mRNA were isolated from healthy and inoculated plants (with Fusarium oxysporum f.sp. cubense) and were used respectively as 'driver' and 'tester' in SSH reaction. The reactions were performed utilizing the PCR select" cDNA subtraction kit provided by CLONTECH, USA. Control subtraction was carried out first using PCR select" cDNA subtraction kit, which gave satisfactory and expected results. For experimental subtraction, the double stranded cDNAs synthesized from Zug mRNA from normal 'driver' and treated 'tester' were digested with RsaI enzyme. Two tester populations were created and each ligated to two different adaptors. This was followed by two hybridization reactions and finally a selective PCR amplification. Only differentially expressed cDNAs were amplified exponentially. This was confirmed by analyzing the PCR products on agarose gel, which showed a smear ranging from 0.9 to 1.3 kb in the subtracted sample and was different from smear pattern of unsubtracted ones. The cDNA fragments from subtracted sample were cloned in pJET and pGEMT vectors and sequenced. Fifty clones were sequenced and analysed after vector and adaptor editing. In silica analysis using bioinformatics tools revealed that some of the cloned sequences showed similarity with known sequences which play important roles during disease resistance conditions directly or indirectly. These included resistance gene candidate NBS type protein, mitogen activated protein kinase, phytoene desaturase, glycerol 3-phosphate dehydrogenase, neutral invertase, 1- aminocyclopropane-l-carboxylase synthase, superoxide dismutase, MADS-box protein, ubiquitin 2, actin, NADPH oxidase, phytoene synthase, ACC synthase, sucrose phosphate synthase, phosphatidic acid phosphatase-like protein, ORF III like polyprotein, bHLH transcription factor like protein, cytochrome oxidase, isochorismatase hydrolase, basic helix-loop-helix family protein, constitutive triple response I-like protein, granule bound starch synthase, alpha amylase precursor, rop protein, GTPase family protein, S-adenosyl-L-methionine synthase protein, ADP-glucose pyrophosphorylase glucose-l-phosphate adenylyl trans, ethylene signal transduction factor and ribosomal protein. Clones were classified into 6 major groups based on function of protein. Sequences had conserved domains for the above mentioned proteins. Genes involved in defense, signal transduction, metabolism, hypothetical protein, transcription factor and translation. For further exploitation of these sequences it is necessary to clone full length cDNA. ESTs thus generated in the present study will be of great use in future for further downstream applications.Item Morphological and molecular analysis of genetic stability in micropropagated banana (Musa spp) var. Nendran(Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara, 2012) Amar Ramesh Kadam; Nazeem, P AItem Molecular characterization of geminivirus causing yellow vein mosaic in pumpkin (Cucurbita moschata Duch. Ex Poir)(Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara, 2011) Sahna Hamsa, N H; Girija, DThe study entitled “Molecular characterization of geminivirus causing yellow vein mosaic in pumpkin (Cucurbita moschata Duch. Ex poir.)” was carried out in the Molecular Biology Laboratory of Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara during the period 2009-2011. The objectives of the study included the molecular characterization of geminivirus causing yellow vein mosaic disease in pumpkin and developing a PCR based diagnostic kit. Yellow vein mosaic infected pumpkin leaf sample was collected from the field of Olericulture Department, College of Horticulture, Vellanikkara and total genomic DNA was extracted by CTAB method. Specific primers for coat protein and movement protein genes were designed based on the sequences of geminiviruses infecting vegetables, obtained from the NCBI database. PCR amplification was carried out using these primers. Amplicons of size ~900bp and ~700bp were obtained for coat protein and movement protein genes respectively. The purified PCR products were ligated in pGEMT plasmid vector and cloned. The recombinant E. coli cells were selected based on blue white screening on LB agar containing ampicillin layered with X-gal and IPTG. After confirmation of the inserts by colony PCR, the clones were sequenced. Sequences of 891bp and 702 bp were obtained for coat protein and movement protein genes respectively. Sequence analysis was carried out with standard bioinformatics tools. On blastn analysis both coat protein and movement protein sequences showed maximum similarity to Squash leaf curl China virus (SLCCV) from Coimbatore. For coat protein gene, full length ORF of 771bp was obtained and the ORF of movement protein was partial. Primers MP1F and MP1R with expected amplicon size of 1363bp were designed to get full length ORF (846bp) of movement protein gene. The technique was validated with DNA from 15 PYVM infected and 4 healthy pumpkin leaf samples collected from Palakkad, Thrissur and Malappuaram districts. The virus was detected only in the diseased samples. Hence these primers could be used in developing a molecular diagnostic tool to detect the virus. PCR amplifications were carried out in weed plants like Emilia sonchifolia, Ageratum conyzoides, Hibiscus surattensis and Synedrella nodiflora and crop plants like okra and ash gourd with yellow vein symptom to check whether these plants serve as the collateral hosts of the virus. PCR amplification was also performed in bitter gourd with distortion mosaic symptom. No amplifications were obtained in plants other than pumpkin. Using the primers PYVMV was detected in apparently healthy mature leaves of infected pumpkin. Hence, these primers could be used to detect latent infection. Sequence and phylogenetic analysis of coat protein and movement protein gene sequences showed maximum similarity to bipartite Squash leaf curl china virus (SLCCV) from Coimbatore. Hence Pumpkin yellow vein mosaic virus (PYVMV) from Kerala can be considered as a strain of SLCCV.Item Isolation and characterization of water stress activated protein kinase gene from black pepper (Piper nigrum L.) var. Kalluvally.(Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara, 2011) Bankar Ashok; Nazeem, P AWater stress is identified as one of the main constraint for enhancing the productivity in black pepper. To survive stress, plants employ a complex set of distinct signaling pathways that trigger stress-specific tolerance or avoidance in the organism as a whole. An important biochemical mechanism for regulating such pathways is reversible protein phosphorylation which is mediated by protein kinases. Gaining an understanding of the mechanisms that regulate the expression of these genes and functional annotation of their transcripts will be necessary for the genetic improvement of plants cultivated in extreme environments. Genotypic variations for drought tolerance have been reported in black pepper and the variety Kalluvally is one of the drought tolerant genotype of black pepper (Thankamani, 2003). In the previous studies at the Centre, water stress specific cDNA library of Kalluvally was constructed by suppression subtractive hybridization (SSH) (Kushwah, 2008). Several protein kinase genes were found to be up-regulated during the study. The present investigation was undertaken to obtain full length coding sequence information on the partial clones of protein kinase genes available in the cDNA library by reverse transcriptase polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). The clones named PNK21 and PNK49 were selected from the library. The clones were revived on LB/ampicillin media and subjected to full length sequencing. Degenerate primers were designed based on protein kinase gene sequences of various crops, obtained from NCBI database to amplify the unknown ends of the transcripts by RT- PCR. Plants raised under greenhouse were screened for revival after water stress with different interval and frequency under open condition. The total RNA was isolated using TRI® reagent from stressed plants and subsequently cDNA was synthesized. The PCR amplification was carried out using degenerate primers designed for all three clones. Amplicons of size of ~600 bp for PNK21 clone and ~600 bp for PNK49 clone were obtained. Direct sequencing of PCR product of PNK21 clone was done. The sequence data obtained was merged and analysed using bioinformatics tools. Blastn analysis revealed ~50 per cent coverage with the cDNA sequences for protein kinase from database. So, further primers were designed to amplify the full length cDNA sequence by RNA ligase mediated-RACE. The 3’ end of the PNK21 cDNA was successfully amplified using RLM-RACE PCR with amplicon size of ~400 bp. The purified PCR product was ligated in pGEMT plasmid vector and cloned. The recombinant E. coli cells were selected based on blue white screening on LB agar containing ampicillin with X-gal and IPTG. After confirmation of the insert by colony PCR, the clones were sequenced. The finally enriched sequence was analysed using bioinformatics tools. Blastn and Blastx revealed maximum similarity with Ricinus communis APK1B protein kinase. The sequence indicated open reading frame and conserved domains for protein kinase and polyadenylation signal site TATAAA was found just upstream of the polyA tail at 3’ end when analysed with different Bioinformatics tools.Item Development of a molecular marker for bacterial wilt resistance in brinjal ( Solanum melongene L.) varieties Surya and Swetha.(Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara, 2011) Somya, P P; Valsala, P AThe study entitled ‘Development of a molecular marker for bacterial wilt resistance in brinjal (Solanum melongena L.) varieties Surya and Swetha’ was carried out at the Centre for Plant Biotechnology and Molecular Biology (CPBMB), College of Horticulture, Vellanikkara during the period 2009-2011. Bacterial wilt caused by Ralstonia solanacearum (Smith) Yabuuchi et al. is one of the important problems of brinjal cultivation in warm humid tropics. The loss due to this varies from 30-100 per cent. Use of chemicals and field sanitation are not sufficient for controlling the disease. Worldwide approach is to use resistant varieties. KAU has developed and released bacterial wilt resistant brinjal varieties for cultivation. The Surya and Swetha are two among them and have received bacterial wilt resistance from SM-6 an Annamalai collection. This investigation was taken up to develop a molecular marker for bacterial wilt resistance in Surya by RAPD through bulk segregant analysis as reported by Michelmore et al (1991). It also aimed to test the suitability of the same for identifying bacterial wilt resistance trait of resistant variety Swetha. The genotypes used for the study were Surya, Pusa Purple Long (susceptible variety released from IARI), Swetha and F2 population of the cross between Surya and Pusa Purple Long. To raise segregating F2 population F1 was raised by controlled crossing of Surya with pollen grains of Pusa Purple Long. Then F1 plant was selfed to get F2 population. Two different methods viz., stem puncturing and soil drenching with root wounding were compared for the delivery of inoculum of R. solanacearum for bacterial wilt incidence and stem puncture method was found as the best. So stem puncturing method was used for phenotyping of genotypes for bacterial wilt incidence. The F2 population along with Surya, Pusa Purple Long and F1 were phenotyped for bacterial wilt incidence. This was done through artificial inoculation with Ralstonia solanacearum by stem puncture method. Confirmation was done by ooze test. The genotypes were classified according to classification of Mew and Ho (1976). The variety Surya was resistant and Pusa Purple Long was susceptible. F1 population showed 90 per cent susceptibility while F2 population showed 83 per cent susceptibility. They were classified as susceptible. Five resistant and five susceptible genotypes from F2 were selected for bulk segregant analysis. Genomic DNA for RAPD analysis was isolated by Rogers and Bendich method (1994). Good quality DNA with an absorbance ratio of 1.8-2.0 was used for RAPD analysis.PCR reaction mixtures and conditions for DNA amplification were standardised. Ninty two, 10-12 bp primers belonging to OPA, OPB, OPC, OPF, OPE, OPU, OPH, OPAH, OPAG, OPL, OPM, RY, RN, RA, SC, RF, AG 8, WG 44, GLE11, RF, R10, R6, and PUC101 were initially screened with resistant genotype Surya and susceptible genotype Pusa Purple Long to select primers with polymorphism and good amplification. Out of ninty two primers tested thirty were reported as bacterial wilt specific. The PCR products were electrophoresed and twenty two primers were selected for BSA based on amplification power and polymorphism. They were RY 01, RY 02, RY 03, RN 19, OPF 5, OPL 04, OPA 04, OPA 6, OPA 9, OPA 24, OPA 26, OPA 34, OPA 36, OPS 9, OPS 10, OPS 16, OPS 17, PUC 101, RA 12-41, and RF. Among these only the primer RY 02 recorded polymorphism between resistant and susceptible variety with an amplicon of 1.2kb. In bulk segregant analysis DNA of Surya, Swetha, Pusa Purple Long and bulk DNA from resistant genotypes and susceptible genotypes were amplified with selected primers and products were electrophoresed. All primers produced only monomorphic band. None has produced unique polymorphic bands capable of differentiating resistant and susceptible genotypes. This may be due to low polymorphism at genomic level.Item Detection and quantification of piperine from in vitro cultures of black pepper (Piper nigrum L.)(Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara, 2011) Achintya Kumar Dolui; Sujatha, RItem Genetic analysis of plantain ecotypes of banana (Musa spp.) using RAPD and ISSR markers.(Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara, 2011) Choudhary Rakeshkumar Sheshrao; Kesavachandran, RThe present investigation on “Genetic analysis of plantain ecotypes of banana (Musa spp.) using RAPD and ISSR markers” was undertaken in the Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara during 2009-2011 with an aim to characterize the variability in plantain ecotypes of banana (Musa spp.) using Random Amplified Polymorphic DNA (RAPD) and Inter Simple Sequence Repeat (ISSR) markers. Twelve plantain ecotypes collected from BRS, Kannara was used for the study. Standardisation of DNA was done with the CTAB method. Optimum PCR conditions for both RAPD and ISSR were standardised with various quantities of DNA, dNTPs, MgCl2, primers and Taq polymerase. Initially 60 RAPD and 40 ISSR primers were screened against genomic DNA of two plantain ecotypes (Big Ebanga and Njockkon) for their ability to amplify DNA fragments. Of these, 16 RAPD and 14 ISSR primers were selected for further detailed RAPD and ISSR profiling. All selected primers produced robust amplification patterns. The PCR products obtained were separated on 1.4 per cent and 1.6 per cent agarose gel respectively stained with ethidium bromide. A total of 138 bands were obtained by using 16 RAPD primers. The number of bands produced by the primers varied from 5 (OPS 40) to 14 (OPS 31 and 37) and the molecular weight of bands varied from 2.876 to 0.564 Kb. The average number of bands was 8.63 and average percentage of polymorphism was 3.25. The total percentage of polymorphism was 37.68. The Polymorphic Information Content (PIC) value for 16 primers varied between 0.79 (OPS 40) and 0.92 (OPS 37) with mean of 0.87. The Resolving power (Rp) of the random primers ranged between 9.33 (OPS 12) and 24.33 (OPS 37) with an average of 15.49. The Marker Indices (MI) of primers varied from 0.83 (OPS 7) to 7.28 (OPS 31) with a mean of 2.86. In the dendrogram, the 12 plantain ecotypes were grouped into three major clusters. The ecotypes Njockkon and Changalikodan, occurring in the first cluster were the most closely related with 94 per cent similarity. The Principal Component Analysis (PCA) showed a similar result to that of clustering. A total of 111 bands were obtained by using 14 ISSR primers. The number of bands produced by the primers varied from 5 (UBC 835, 820) and 11 (UBC 857) and the molecular weight of bands varied from 1.584 to 0.564 Kb. The average number of bands was 7.93 and average percentage of polymorphism was 4.14. The total percentage of polymorphism was 52.25. The PIC value for 14 primers varied between 0.77 (UBC 835) and 0.90 (ISSR 6) with an average of 0.85. The resolving power of the ISSR primers ranged between 7.83 (UBC 820) and 16.83 (ISSR 6) with an average of 12.49. The Marker Indices (MI) of primers ranged from 0.77 (UBC 835) to 8.01 (UBC 857) with a mean of 3.55. In the dendrogram, the 12 plantain ecotypes were grouped into three major clusters. The ecotypes Changalikodan and Zanzibar; Manjeri Nendran (a) and Manjeri Nendran (b) occurring in the first cluster were the most closely related with 94 per cent similarity. The Principal Component Analysis (PCA) showed a similar result to that of clustering. The combined dendrogram was also derived from pooled data from RAPD and ISSR analysis and morphological data. The dendrogram generated revealed grouping of plantain ecotypes into clusters more or similar to earlier dendrogram with a few exceptions. The study revealed that variability exists among the plantain ecotypes of banana.Item Genetic transformation for hairy root induction and enhancement of secondary metabolites in Aswagandha (Withania somnifera (L) Dunal)(Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara, 2006) Smini Varghese; Keshavachandran, RItem Isolation and characterisation of B-1, 3-glucanase gene from Piper spp.(Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara, 2005) Mable Rose George; Nazeem, P AThe study entitled isolation and characterization of β-1, 3- glucanase gene in Piper spp was carried out at the Centre for Plant Biotechnology and Molecular Biology (CPBMB), College of Horticulture, Vellanikkara from December 2003 to September 2005. β-1, 3-glucanase is reported to be involved in the defense mechanism of Piper against the Phytophthora foot rot disease. Attempts were undertaken to isolate the gene from tolerant species P. colubrinum and susceptible species P. nigrum. The information of plant β-1, 3- glucanase gene sequence available in the public domain NCBI was collected and subjected to multiple sequence alignment to detect conserved boxes of the gene among different plant species. Based on the data, eight degenerate primers were derived and out of this, three sets of forward and reverse primers were synthesized for gene isolation. Genomic DNA isolated from Piper spp namely P. nigrum and P. colubrinum was subjected to PCR with the designed degenerate primers at various combinations at different annealing temperatures. The amplification with the primer combination GluF1R2 yielded multiple bands in both the selected Piper species, whereas the primer combination GluF2R1 yielded multiple bands in P. colubrinum only. The primer combination GluF2R2 and GluF3R3 yielded single intact band in both P. nigrum and P. colubrinum. The amplicons obtained were eluted and cloned in pGEM-T vector and transformed into competent cells. High level of recombination was observed on blue and white screening. Recombination of the insert was confirmed by PCR and restriction analysis of the plasmid isolated from white colonies. The cloned fragments were sequenced. The fragments Pnglu from P. nigrum and Pcglu from P. colubrinum sequenced and subjected to BLAST search revealed, significant level of homology to β-1, 3-glucanase genes reported from other plants deposited in the public domain. Two other fragments cloned and sequenced from P. nigrum did not show homology to β-1, 3-glucanase genes deposited in the public domain. The sequences of the fragment Pnglu and Pcglu were subjected to other sequence analysis utilizing bioinformatics tools including BCM Search Launcher, ORF finder, GENSCAN, Biology workbench, Conserved domain database, Interproscan and CATH. Multiple sequence alignment of nucleotide of Pnglu and Pcglu with the selected nucleotides of -1, 3- glucanase gene sequence from different plant species indicated several conserved regions. The cloned fragment Pnglu and Pcglu had largest open reading frame of size 450 and 372 bp respectively. Internal exons of size 423 bp and 348 bp respectively were detected in the fragment Pnglu and Pcglu respectively. Restriction analysis revealed that both Pnglu and Pcglu have restriction sites for the frequent cutter AluI and the rare cutter NaeI. The fragments had high GC content of 56.1 per cent and 56.9 per cent for Pnglu and Pcglu respectively. The major amino acid composition deduced from Pnglu were alanine, leucine, arginine and proline, while in Pcglu major amino acids were alanine, glycine, arginine, leucine and valine. The secondary structures predicted for the polypeptides deduced from Pnglu and Pcglu had a high proportion of helices. Conserved domains detected from deduced amino acids of Pnglu and Pcglu suggested that both belong to glycosyl hydrolase family 17, which is a single domain family. Both fragments lack transmembrane regions. Domain structure comparison indicated that domains of Pnglu and Pcglu had similarity to glycosidase, which is the sequence super family of -1, 3-glucanases. The sequence information obtained from Pnglu and Pcglu confirmed that they are genomic counter part of -1, 3- glucanase gene from Piper spp and can be further utilized for the isolation of full length gene from Piper spp.Item Genetic transformation of chilli (Capsicum annuum L.) with osmotin gene(Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara, 2005) Resmi Henry, T; Girija, DThe study entitled ‘genetic transformation of chilli (Capsicum annuum L.) with osmotin gene’ was carried out at the Centre for Plant Biotechnology and Molecular Biology (CPBMB), College of Horticulture, Vellanikkara, from December 2003 to September 2005. The study was undertaken to standardize in vitro regeneration and Agrobacterium mediated transformation of chilli with osmotin gene. Different explants, media and hormonal combinations (auxin and cytokinin) were tried in order to standardize in vitro regeneration in chilli var.Ujwala. The best explant for in vitro regeneration was hypocotyl. Only the buds originated from hypocotyl explants showed elongation. Shoot buds formed from cotyledon and leaf segments did not elongate in any of the media tested. Based on the percentage regeneration in cultures and no. of shoot-buds per explant, MS medium containing the hormonal combination; BA (5.0 mg l-1) + IAA (0.3 mg l-1) was found optimum. The regenerated plantlets were transferred to pots for acclimatization so that they can sustain and survive in the natural conditions. The hardened plantlets were planted out. Agrobacterium mediated transformation protocol was optimized considering all the factors for successful transformation. Optimum inhibitory concentration of selectable marker (Kanamycin: 100mg l-1) was established. The antibiotic cefotaxime (200 mg l-1) was selected for killing the bacteria. Agrobacterium strain EHA 105 harbouring the gus reporter gene was used for the standardization of transformation. Hypocotyl explants of chilli were co-cultivated with Agrobacterium strain (EHA 105). The inoculum density-0.1 OD600 nm, infection time-5minutes and co-cultivation period-2 days were found optimum based on GUS assay and survival rate 60 days after co-cultivation. The hypocotyl explants of chilli were co-cultivated with Agrobacterium strain (GV2260) harbouring osmotin gene in the plasmid pGV2260 tagged with 35S CaMV promoter. The transformed explants were regenerated on the selection medium optimized for regeneration of chilli. However, none of the transformed buds were elongated in the elongation medium containing selection agent. Hence the transformed plants were transferred to elongation medium containing no antibiotics. Even in this medium, no elongation of shoots was observed even after 60 days. So, further refinement of transformation protocol using an optimal selectable marker is needed for the production of transgenic chilli. After selection of the transformants, the putative transgenics were characterized employing molecular biology techniques viz. PCR-utilizing the gene specific primers of nptII. The presence of transgene was confirmed in the transformed plants.