PG Thesis
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Item Anther culture of Capsicum L. for doubled haploid production(Department of Plant Biotechnology, College of Agriculture,Vellayani, 2022-04-13) Ninitha Vijayan; Swapna AlexThe study entitled “Anther culture of Capsicum annuum L. for doubled haploid production” was carried out at the Department of Plant Biotechnology, College of Agriculture, Vellayani, Thiruvananthapuram during 2019 - 2021. The objective of the study was to develop haploids of Capsicum annuum var. Arka Meghana by anther culture. Standardisation of the suitable stage of bud of Capsicum annuum var. Arka Meghana for anther culture was carried out by staining the anthers of different bud stages with 1% aceto-orcein and calculating the percentage of microspores at late uninucleate and early binucleate stages. Buds collected at six days after bud initiation had 57% of microspores in the late uninucleate stage and 34% in the early binucleate stage. Buds collected at nine days after bud initiation had 21% of microspores in the late uninucleate stage and 49% in the early binucleate stage. Hence the buds collected at six and nine days after bud initiation were found to be most suitable for anther culture. The optimum surface sterilization condition for buds was standardised by treating the buds with 70% of ethanol and 4% of sodium hypochlorite for different time intervals. Surface sterilization with 70% ethanol for 30 seconds followed by 4% sodium hypochlorite for 15 minutes was found to be most effective. Anther culture was carried out in nine media compositions for optimization. Anthers were inoculated onto full strength Murashige and Skoog (1962) medium (MS) and Dumas de Vaulx (1981) medium (CP) with different concentrations of plant growth regulators Kinetin, BA, NAA, IAA and 2,4-D with AgNO3 and activated charcoal as additives. The anthers were incubated at 25°C for two and eight days in darkness. The treatment T5 - MS + 4.00 mg/L NAA + 0.10 mg/L BA + 0.25% activated charcoal + 10 mg/L AgNO3 incubated at 25°C for two days in darkness showed callogenesis (3.17%) at six weeks after inoculation. All the treatments incubated at 25°C for eight days in darkness showed response at sixth week of inoculation with callogenesis varying from 3.75% to 15.38%. Among the nine treatments, highest callogenesis of 15.38% was observed in the treatment T5 at sixth week of inoculation. Globular embryo initiation (1.25%) was observed in the treatment T4 - MS + 4.00 mg/L NAA + 1.00 mg/L BA at sixth week of inoculation. The six treatments that showed good response at 25°C incubation temperature and eight days darkness were incubated at 35°C in two days darkness. All the treatments showed response varying from 4% to 15.54% of callogenesis at second week of inoculation. Callogenesis varying from 10.66% to 34.48% and embryonic calli induction varying from 3.17% to 17.24% was observed in the fourth week of inoculation. At sixth week of inoculation callogenesis varied from 11.11% to 37.93% and embryonic callus induction varied from 3.17% to 19.54%. Among the six treatments, the maximum callogenesis of 37.93% and embryonic calli induction of 19.54% was observed in the treatment T5 at fourth week of inoculation. Embryogenesis was observed in the treatment T7 – MS + 4.00 mg/L NAA + 0.50 mg/L BA+ 0.25% activated charcoal + 15 mg/L AgNO3 (1.82%) and treatment T6 - MS + 4.00 mg/L NAA + 0.10 mg/L BA + 0.25% activated charcoal + 15mg/L AgNO3 (1.33%) at fourth week of inoculation. At sixth week of inoculation, embryo induction (1.15%) was observed in the treatment T5 – MS + 4.00 mg/L NAA + 0.10 mg/L BA + 0.25% activated charcoal + 10mg/L AgNO3. Highest embryogenesis of 2.72% was observed in the treatment T7 – MS + 4.00 mg/L NAA + 0.50 mg/L BA+ 0.25% activated charcoal + 15 mg/L AgNO3. To conclude, among the treatments tested in Capsicum annuum var. Arka Meghana the best treatment for indirect embryogenesis and direct embryogenesis was MS + 4.00 mg/L NAA + 0.10 mg/L BA + 0.25% activated charcoal + 10 mg/L AgNO3 and MS + 4.00 mg/L NAA + 0.50 mg/L BA + 0.25% activated charcoal + 15 mg/L AgNO3 respectively at culture conditions of 35°C initial incubation temperature and two days darkness.Item Cytomorphological and chemical studies on intervarietal crosses of Capsicum annuum, L.(Division of Agricultural Botany, Agricultural College & Research Institute, Vellayani, 1970) Manikantan Nair, P; Mary K George1. The cytomorphological and chemical aspects of four F1 hybrids of crosses involving five varieties of Capsicum annuum L. were studied . The variety ' Local blue' which possessed mosaic resistance , prolific bearing habit , long life span and high pungency was selected as the common seed parent. The selected pollen parents were, Russian, Indian long red , Chinese giant and Oskosh which were gifted with a higher content of ascorbic acid and sucrose and larger fruit size , but lacking the qualities of the common seed parent. 2. All the four F1 hybrids manifested a marked degree of heterosis in many economically important attributes like , carliness in blooming, number of leaves, number of branches, leaf area and chemical constituents like ascorbic acid and sucrose. 3. An intermediate condition was observed with regard to height , spread, number of fruits, fruit size and number of F2 seedsItem Molecular cloning and characterization of virus causing leaf curl disease of capsicum spp.(Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara, 2019) Niranjana Menon, C; Anita Cherian, KChilli is one of the most important crops cultivated across the globe, as vegetable, spice and for industrial purposes. According to the statistics of National Horticulture Board (2017), the crop covers an area of 1860 ha in the state of Kerala with an average production of 12470 tonnes. During the last decade, the threats posed by the emerging begomoviruses infecting solanaceous crops have affected the economic cultivation of chilli. Chilli leaf curl disease caused by Chilli leaf curl virus belonging to the genus Begomovirus and family Geminiviridae is a serious constraint to chilli production in India which causes upto 100 per cent yield loss especially when infected at an early stage of the crop. Considering the importance of the disease, the present study was undertaken with the objective to study the incidence and symptomatology of chilli leaf curl disease and to characterize and clone the coat protein gene of the Chilli leaf curl virus isolates. The project initiated with purposive sampling surveys conducted in eleven different locations of Thrissur district, Kerala to document the incidence and symptomatology of leaf curl disease on chilli plants. The disease incidence recorded during the survey ranged from 43.30 to 85.00 per cent under open field conditions and from 45.75 to 79.40 per cent under protected conditions while the disease severity ranged from 43.60 to 81.54 per cent and from 49.40 to 87.50 per cent, respectively under open field conditions and protected conditions. The symptomatology of chilli leaf curl disease on different parts of the plant such as leaves, internodes, fruits and the whole plant under natural conditions was documented during the survey. The symptoms observed on the leaves of infected chilli plants under natural conditions include upward curling, crinkling, puckering, vein banding, interveinal chlorosis, size reduction of leaf lamina and leaf malformation. The fruits produced by the infected plants showed size reduction and deformation. The infected plants were stunted and bushy in appearance. The transmission of the virus by insect vector, Bemisia tabaci and grafting was studied and the symptoms were documented. The newly emerged leaves after artificial inoculation expressed symptoms such as curling, puckering and crinkling along with stunting of plant growth. Molecular characterization of the four virus isolates collected from various locations of Thrissur district viz., VKA1 VKA2, KAR1 and KOD4 and two isolates viz., VLNY1 and PKD1 collected from Vellayani, Thiruvanathapuram district and from Vithinasseri, Palakkad district, respectively were undertaken. The total genomic DNA from virus infected chilli leaf samples was isolated and subjected to PCR amplification of viral coat protein gene to confirm the presence of virus infection. PCR amplification of the isolated DNA was carried out using two Begomovirus specific degenerate (universal) primers, namely, AV494 / AC1048 (Wyatt and Brown, 1996) and Deng 540 / 541 (Deng et al., 1994). The amplicons of size 550 bp were obtained and were sequenced. The partial coat protein gene of size 550bp were also cloned into the vector pTZ57R/T and transformed into DH5α strain of Escherichia coli and the true recombinants with desirable insert were confirmed by colony PCR. The sequence data obtained in the study were subjected to in silico analysis to assess the diversity of the isolates. The nucleotide BLAST (BLASTn) analysis revealed more than 90 per cent sequence identity with Chilli leaf curl Vellanad virus isolate (Accession No. NC038442.1) from Vellanad region of Thiruvanathapuram district, Kerala. The translated nucleotide - protein BLAST (BLASTx) analysis of the viral sequences revealed more than 96 per cent sequence identity with Chilli leaf curl Vellanad virus coat protein sequence (accession no. YP_009506391.1). The coat protein sequences of all the six isolates were translated into corresponding amino acid sequence by ExPASy Translate tool and were used for further analysis and interpretation. The phylogenetic analysis revealed that, the isolates VKA2, KAR1 and KOD4 had very distinct sequence alignment when compared to other Chilli leaf curl virus isolates from India. The results indicated that, the three isolates viz., VKA2, KAR1 and KOD4 could be new strains of Chilli leaf curl virus infecting chilli. Three, possibly new strains of Chilli leaf curl virus infecting chilli have been identified and hence the study highlights the need for monitoring the emergence of new strains of plant viruses especially begomoviruses infecting solanaceous crops grown in Kerala. As this disease is one of the most important challenges to chilli cultivation, the information generated from the study could also be applied for the timely detection and effective disease managementItem Interspecific hybridization in capsicum(Department of Olericulture, College of horticulture, Vellanikkara, 1990) Pradeepkumar, T; Gopalakrishnan, T RThe present investigation "Interspecific hybridization in Capsicum" was carried out at the College of Horticulture, Vellanikkara, Thrissur during September 1988-April 1990 to study cross compatibility among five Capsicum species and to exploit heterosis in interspecific hybrids. Eighty four chilli accessions, when subjected to the modern taxonomic treatments were found to fall under C. annum L. (62), C. frutescens L. (7), C. chinense Jacq. (14) and C. baccatum L. (1) Protein electrophoretic focussing revealed species specific protein bands in C. chinense, C. baccatum and C. chacoense. Fruit set was obtained in all the 28 crosses made among C. annuum.C. frutescens, C. chinense, C. baccatum and C. chacoense. Viable F1 and F2 seeds were obtained in eight crosses viz., C. annuum x C. chinense (P), C. annuum x C. chinense (NP), C.frutescens x C. annuum, C.chacoense x C. annuum, C. frutescens x C. chinense (P), C. chinense (P) x C. frutescens, C. frutescens x C. chinense (NP) and C. chinense (NP) x C. frutescens.Item Triazole,strobilurin and its combination fungicides for the management of anthracnose and fruit rot of chilli(Department of Plant Pathology, College of Agriculture, Vellayani, 2018) Anjana, R S; Joy, MItem Elicitation mediated carotenoid prodouction and capsanthin capsorubin synthase gene expression in byadagi chilli (capsicum annuum L.)(Centre of Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara, 2018) Pooja, S L; Shylaja, M RByadagi chilli is well-known for its deep red colour and zero pungency. The fruits of Byadagi chilli are brown to deep red at full maturity. The red colour of chilli fruits are due to several carotenoid pigments. They are good source of natural colourants used in food, feed, textile and cosmetic industries. The chilli type is best suited for oleoresin extraction and exported as a substitute for paprika oleoresin. The present study ‘Elicitation mediated carotenoid production and Capsanthin capsorubin synthase gene expression in Byadagi chilli (Capsicum annuum L.)’ was undertaken at Centre for Plant Biotechnology and Molecular Biology (CPBMB), College of Horticulture, Kerala Agricultural University, Thrissur during 2016-2018. The study was carried out using two genetically distinct chilli genotypes based on their colour at fully ripe fruit stage namely Byadagi Dabbi and variety Anugraha. Callus cultures were produced from tender leaves of both the genotypes using MS media augmented with different plant growth regulators viz., Indole-3- acetic acid (IAA), Kinetin (Kin) and Benzyl adenine (BA) at different concentrations as reported by Kintzios et al. (1996), Kehie et al. (2012), Kabby et al. (2015) and Santos et al. (2017). Though, callogeneses were obtained in all the media tried, early callusing and higher callus index were achieved in MS medium supplemented with 1 mgL-1 2,4-D and 1 mgL-1 Kin. Elicitation of two month old calli was done with two different chemical elicitors viz Salicylic acid and Methyl jasmonate at different concentrations like 20, 40 and 60 mgL-1 for 72h. The carotenoids were extracted and β-carotene was quantified using HPLC. Elicitor treatment with salicylic acid increased β-carotene content significantly. The highest β-carotene was recorded in Byadagi calli elicited with salicylic acid 20 mgL-1 for 72 hours. In this treatment Byadagi Dabbi recorded 6.75 times higher β-carotene (42.85 μgg-1 fresh weight) than the control (6.34 μgg-1 fresh weight). In methyl jasmonate elicitation, both the genotypes were found on par with respect to β-carotene production in the different concentrations studied. Expression of Capsanthin capsorubin synthase (Ccs) gene was studied in the highest β-carotene yielding treatment viz elicitation with salicylic acid 20 mgL-1 for 72 hours using real time PCR. The total RNA extracted from elicited calli were converted to cDNA. The Capsicum annuum L. β-tubulin gene was used as the endogenous control. Dissociation curves were obtained as a single dominant peak denoting that there was specific gene amplification for both endogenous control and Ccs gene in different treatments. Relative quantification of the Ccs gene expression was done using the Comparative CT method reported by Livak and Schmittgen, (2001). The Ccs gene was found up-regulated 2.35 fold in Byadagi Dabbi elicited calli with salicylic acid 20 mgL-1 for 72 hours. The expression of Ccs gene in the variety Anugraha was down-regulated (0.906 fold) when elicited with salicylic acid 20 mgL-1 for 72 hours. The major outcome of the present investigations are the suitability of leaf and calli induced from leaves for carotenoid production, scaling up of carotenoid production through salicylic acid elicitation and upregulation of Ccsgene expression in highest carotenoid yielding elicited calli. Another significant finding is the response of calli to salicylic acid elicitation which is a cheaper elictor as compared to Methyl jasmonate and the highest content of β-carotene at lower concentration of elicitor which are plus points as far as commercial exploitation of carotenoids is concerned. However, much more elaborate studies are required on explant stage, culture systems, age of cultures, culture conditions, duration and concentration of elicitors and activity of regulatory enzymes and elicitor mediated expression of genes involved in carotenoid metabolic pathway for commercial exploitation of the system for carotenoid production. A more in-depth understanding of the underlying biological mechanisms will enable to fully harness the potential of cell cultures and to enhance carotenoid production on an industrial scale.Item Economic feasibility of vegetable production under polyhouse cultivation(Department of Agricultural Economics, College of Horticulture, 2016) Swathylakshmi, P V; Prema, AItem Analysis of capsanthin capsorubin synthase gene in byadagi chilli (Capsicum Annuum L.) and elucidation of carotenoid metabolic pathway(Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara, 2017) Naresh, S; Shylaja, M RByadagi chilli is famous for its deep red colour and negligible or zero pungency. Demand for Byadagi chilli has increased enormously as a source of natural red colour in food industry, confectionaries, cosmetics, beverages, pharmaceuticals and even as a dye in textile industries. Byadagi chilli is mainly exported as oleoresin which serves as a substitute for paprika oleoresin.The red color of chilli fruits is due to several related carotenoid pigments. The most important pigments are capsanthin and its isomer capsorubin. The present study was conducted to analyze Capsanthin-capsorubin synthase gene (Ccs) in Byadagi chilli and to elucidate the carotenoid metabolic pathway for production of capsanthin and capsorubin. The studies were focused on seven genetically distinct chilli varieties /accessions of three different Capsicum spp. based on colour at fully ripe fruit stage. The accessions selected were ByadagiKaddi, Byadagi Dabbi, Ujwala, Anugraha (Capsicum annuum), Vellayani Samrudhi (Capsicum frutescens), Vellayani Thejus and CC8-1 (Capsicum chinense). Genomic DNA was isolated from tender leaves of one month old plants by CTAB method. Two chilli Ccs gene specific SSR primers viz. Ccs Cds and Ccs promoter were used to amplify the Ccs gene.The amplified PCR products obtained with Ccs Cds and Ccs promoter were sequenced by outsourcing and sequence data analyzed using bioinformatics tools. The Ccs gene was found amplified in all the genotypes including the yellow fruitedaccession CC8-1. Size of amplified product was 1.5kb with Ccs cds primer in all the genotypes. For Ccs promoter, amplified product was920bp inByadagiKaddi, Byadagi Dabbi, Ujwala, Anugraha, Vellayani Samrudhi and 1200bp in Vellayani Thejus and CC8-1 BLASTn analysis of the Ccs gene amplified with Ccs cds primer showed 99 -100 per cent similarity with the reference nucleotide sequence in all the genotypes. BLASTx analysis of Ccs gene sequence amplified with Ccs cds primer showed 99-100 per cent similarity with the reference amino acid sequence in the seven genotypes studied. Analysis of conserved domains revealed that lycopene beta cyclase was the conserved domain in Capsicum annuum and C. chinense genotypes while in C. frutescens NADB super family protein was the conserved domain. The number of ORFs in the Ccs sequence amplified with Ccs cds primer ranged from six to seven in the genotypes studied and the number of amino acids coded ranged from 463-469 in C. annuum, 298 in C. frutescens and 217 in C. chinense. Multiple sequence alignment of the sequences revealed SNP variations in the genotypes studied and SNP variation caused change in amino acid coded. SNP variations were observed in five genotypes viz. Byadagi Kaddi, Byadagi Dabbi, Vellayani Samrudhi, Vellayani Thejus and CC8-1 while no SNP variations were seen in the varities Ujwala and Anugraha Byadgi kaddi had two SNPs leading to change in amino acids at 43rd and 425th position of Capsanthin capsorubin synthase peptide. Tyrosine (Y) was found replaced by Phenyl alanine (F) in the 43rd position and Lysine (K) was found replaced by Glutamic acid (E) in the 425th position. Byadagi dabbi also had the same amino acid change at 425th position, Lysine (K) was replaced by Glutamic acid (E). PrematureStop codon UAG was observed in yellow fruited variety CC8-1 at 200th position BLASTn analysis of Ccs gene sequence amplified with Ccs promoter primer in seven genotypes showed 90-99 per cent similarity with the reference nucleotide sequence. Multiple sequence alignment of the promoter region could see structural changes in the sequences. Several SNPs in the sequences, a tandem repeat structure, insertion, deletions and various cis regulatory elements like heat stress related cis-elements (HSE), Myb binding site (MYBPZM) and light responsive elements, TATA box, and CAAT box could be observed in the promoter region. Ccs gene was located in Chromosome six of Capsicum annuum and in the genome map of chilli it was seen in between 9497216 – 9500911kb. Ccs cds gene specific primer was seen to bind 18bp downstream region of the sequence. The Ccs promoter was seenupstream of the protein coding region. Elucidation of carotenoid metabolic pathway in Capsicum annuum revealed that 17 enzymes were present in the carotenoid biosynthesis pathway. Gene regulatory network analysis, using cytoscape showed that network contained 94 nodes and many of the genes were associated with carotenoid biosynthesis processes. The main seventeen carotenoid metabolic pathway genes, some transcription factors and transferase/transport proteins were densely connected. Among the pathway genes, Phytoene synthase had the highest number (30 No.) of interactive proteins. The identified SNPs in the present study have to be further characterized and validated, transcriptome analysis of Ccs gene in the different genotypes, homology modeling the Ccs enzyme and prediction of active sites could derive more information on the identified SNPsItem Screening of spice chilli (Capsicum annuum L) genotypes suitable for Kerala(Plantation Crops and Spices, College of Horticulture, Vellanikkara, 2017) Nabeela, K; Krishnakumary, KItem Standardisation of fertigation schedule and spacing for bell pepper (Capsicum annum L.var.grossum sendt) in polyhouse(Department of Agronomy, College of Agriculture, Vellayani, 2017) Athira, R C; Sajitha Rani, T