Browsing by Author "Shylaja, M R"
Now showing 1 - 18 of 26
Results Per Page
Sort Options
ThesisItem Open Access 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; KAU; 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 SNPsThesisItem Open Access Anatomical biochemical Investigations on Phytophthora foot rot disease reaction in piper spp.(Department of Plantation Crops and Spices, College of Horticulture, Vellanikkara, 1999) Suresh Baban Dagade; KAU; Shylaja, M RAnatomical and biochemical investigations on Phytophthora foot rot disease reaction in Piper spp. were carried out in the Department of Plantation Crops and Spices and Biochemistry Laboratory of the College of Horticulture, Vellanikkara during 1996 to 1998. The study revealed that the two species viz., Piper colubrinum L. and P. nigrum L. and two genotypes of P. nigrum viz., Panniyur I and Kalluvally differed significantly in the various anatomical and biochemical parameters studied. The immune genotype was characterised by compact arrangement of cells, small epidermal, mesophyll and spongy parenchyma cells, thick palisade and collenchyma tissues, large vascular bundles and mucilage canals, thick lower epiderm with large cells and less number of stomata per unit area in the leaves. The stem was characterised by thin cuticle, epiderm and hypoderm, small sized epidermal cells, thick cortex, large cortical cells with more inter- cellular spaces, thick chlorenchyrna and sclerenchyma, more number of large peripheral and medullary vascular bundles, large mucilage canals and small indistinct pith. It had thick root epiderm,less number of root hairs, small cortical cells, thick endoderm, big stele, pericycle cells and vascular bundles. In contrast, the susceptible genotype was characterised by thin palisade and collenchyma tissues, small vascular bundles and mucilage canals, thin lower epiderm with small ceIls and more number of stomata per unit area. The stems of the susceptible genotype exhibited thicker cuticle with large epidermal cells, more epidermal appendages, thick hypoderm with small cells, thin cortex with small cortical cells arranged with less interceIlular spaces and thin sclerenchymatic tissues. It had thin root epiderm, more number of epidermal appendages, small cortical cells with large stele, thin pericycle with small cells and distantly placed small vascular bundles. The tolerant genotype Kalluvally exhibited somewhat intermediate values to P. colubrinum and Panniyur 1 in all the anatomical characters tested. In the biochemical parameters studied, I). colubrinum registered high content or total phenol, reducing sugar, total free amino acid and higher activities of peroxidase and polyphenol oxidase enzymes. The susceptible genotype: Panniyur I was marked by low of total and OD phenols, reducing sugars, total free amino acids, peroxidase and polyphenol oxidase activities and 'high glucanase and IAA oxidase activities. The tolerant genotype Kalluvally registered medium values for total phenols, reducing and non reducing sugars, total free amino acid" and enzymes like peroxidase, glucanase and polyphenol oxidase. However, the content of OD phenol was high and IAA oxidase activity was low in Kalluvally. On inoculation with Phytophthora capsici, the content of total phenol, reducing sugar, total free amino acid and enzyme activities like glucanase and peroxidase increased in Panniyur 1 whereas the contents of OD phenol, non reducing sugar and activities of IAA oxidase and polyphenol oxidase decreased. In P. colubrinum the contents of total free amino acids and peroxidase enzyme activity increased at a higher rate than in the other genotypes while the content of OD phenol, reducing and non reducing sugars decreased drastically on inoculation. Kalluvally exhibited similar trend as that of P. eolubrinum and registered somewhat intermediate values between P. colubrinum and Panniyur 1 in almost all the biochemical parameters tested. However, decrease in glucanase activity and a greater reduction in polyphenol oxidase activity were observed in Kalluvally on inoculation. The anatomical and biochemical differences observed in Piper spp. can effectively be used for screening the genotypes for tolerance I resistance to Phytophthora capsici and for the management of the disease.ThesisItem Open Access Characterization of antioxidant fractions in curry leaf (Murraya koenigii L.) and molecular docking of selected bioactive compounds(Centre for Plant Biotechnology and Molecular Biology, College of Horticulture,Vellanikkara, 2019) Deepak Prashant, Bhamare; KAU; Shylaja, M RCurry leaf (Murraya koenigii L.) belonging to the family Rutaceae is one of the extensively used spices in traditional Indian medicine against variety of ailments. Curry leaf is reported to possess several pharmaceutical properties such as antioxidant, anticancerous, antidiabetic, anti-inflammatory, antidiarrheal, analgesic and hepatoprotective. The therapeutic potential of curry leaf is due to several chemical constituents such as carbazole alkaloids, phenols, flavonols, tannins, terpenes, and lipids. The overproduction of reactive oxygen species (ROS) serve as the initiation point for many diseases like cancer, diabetes, arthritis and Alzheimer’s. The ROS can be scavenged by antioxidants but side effects have been reported for synthetic antioxidants like butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT). Hence, natural antioxidants are gaining scientific attention now-a-days. Though, the pharmacological potential of curry leaf is well understood, very few reports are available on the role of bioactive phytocompounds for curing diseases by interacting with target proteins. The study entitled “Characterization of antioxidant fractions in curry leaf (Murraya koenigii L.) and molecular docking of selected bioactive compounds” was undertaken with the objective to characterize antioxidant fractions in curry leaf through in vitro assays and to identify the most potent bioactive compound through LC-MS/MS and molecular docking analyses. Oleoresin was extracted from curry leaf (var. Suvasini) and further subjected to in vitro antioxidant assay. Antioxidant fractions from curry leaf were separated by silica gel column chromatography using hexane: ethyl acetate solvent system in various proportions (100:0, 80:20, 60:40, 40:60, 20:80 and 0:100) and further subjected to antioxidant assay. Sub-fractionation of the fraction exhibiting the highest antioxidant activity was done at five minutes interval and sub-fractions were also subjected to antioxidant assay. Sub-fractions exhibiting maximum antioxidant activity were analyzed by LC-MS/MS. Compounds identified through LC-MS/MS analysis were docked against eight target proteins for cancer, seven for diabetes, four for arthritis and four for Alzheimer’s. Mature leaves of curry leaf recorded higher oleoresin recovery of 9.16 per cent and possessed high antioxidant activity with a DPPH inhibition of 85.19 per cent. Fraction eluted with hexane: ethyl acetate (60:40) recorded the highest yield of extract (707.4 mg) and showed the highest antioxidant activity with 88.68 per cent inhibition of DPPH. Sub-fractionation of the fraction with the highest antioxidant activity has yielded 47 sub-fractions. Of the sub-fractions, 28th fraction showed the highest DPPH inhibition (91.51%) followed by 26th (91.08%), 34th (91.08), 38th (89.53%) and 40th (89.53%). The DPPH inhibition potential of sub-fractions 28th, 26th and 34th was similar to synthetic antioxidant BHA (91.89%). The LC-MS/MS analysis of these fractions revealed presence of 52 compounds in whole fraction (hexane: ethyl acetate 60:40), 62 in 26th sub-fraction, 51 in 28th and 34th sub-fraction, 49 in 38th sub-fraction and 45 in 40th sub-fraction. Seven compounds of curry leaf viz. alpha-aminodiphenylacetic acid, doxylamine, flucoxetine, histidinol, pheniramine, prometon and valylmethionine were found to interact with different targets for cancer. Maximum number of curry leaf phytocompounds interacted with targets for breast cancer, 17β hydroxysteroid dehydrogenase (17β HSD) and Polo-like kinase 1 (PLK1). valylmethionine inhibited 17β HSD and PLK1 with good binding energy of -66.7903 and -122.5233 kcal/mol respectively. Eight phytocompounds of curry leaf viz. alpha-aminodiphenylacetic acid, DL-2- aminooctanoic acid, doxylamine, flucoxetine, histidinol, pheniramine, prometon and valylmethionine interacted with seven different targets for diabetes. Maximum number of compounds interacted with the target fructose 1,6-bisphosphatase and valylmethionine inhibited the target with good docking score with a binding energy of -81.143 kcal/mol. Six compounds of curry leaf viz. alpha-aminodiphenylacetic acid, flucoxetine, norpropoxyphene, histidinol, pheniramine and valylmethionine interacted with four different targets of arthritis studied. Maximum number of compounds interacted with the target Nitric oxide synthase and it was inhibited by histidinol with good binding energy - 109.5131 kcal/mol. Five phytocompounds viz. alpha-aminodiphenylacetic acid, flucoxetine, norpropoxyphene, histidinol and valylmethionine interacted with four different targets for Alzheimer’s. Maximum number of compounds interacted with targets human beta- secretase 1, tau protein kinase and human butyrylcholinesterase. Good docking score was recorded for interaction of human beta-secretase 1 with histidinol. The study could bring about the potential of curry leaf as a natural antioxidant and could identify three safe phytocompounds viz. alpha-aminodiphenylacetic acid, DL-2- aminooctanoic acid and valylmethionine which could interact with targets for cancer, diabetes, arthritis and Alzheimer’s.ThesisItem Open Access 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; KAU; 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.ThesisItem Open Access Commercial micropropagation of banana (Musa spp.) using a temporary immersion bioreactor system(Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara, 2020) Waghmare Vaibhav, Gautam; KAU; Shylaja, M RBanana (Musa spp.) is an important fruit crop cultivated worldwide. Tissue culture banana plants are now widely used as planting materials for commercial banana cultivation. High cost of production, poor multiplication rate and less survival of plantlets during acclimatization are some of the problems experienced in conventional micropropagation of banana. As there is high demand for tissue culture banana plants, the conventional tissue culture techniques need to be modified to increase the multiplication rate and to reduce the unit cost of production of plantlets. Use of temporary immersion bioreactor (TIB) is a very good option to increase multiplication rate and reduce cost of production. The investigations on ‘Commercial micropropagation of banana (Musa spp.) using a temporary immersion bioreactor system’ was hence taken up at Centre for Plant Biotechnology and Molecular Biology (CPBMB), College of Horticulture from 2018- 2020. The aim of the investigations was to develop an efficient commercial micropropagation protocol in banana using a temporary immersion bioreactor system. Established cultures of the cultivar Nedunendran (AAB) received from the commercial micropropagation unit of CPBMB at the 5 th subculture cycle were utilized for the study. The micropropagation protocol standardized at CPBMB for the cultivar Nedunendran was followed for production of plantlets. The protocol was optimized for bioreactor micropropagation and at each stage of propagation, it was compared with conventional micropropagation system. Number of clumps/ 500 ml. of medium to initiate shoot multiplication, media for multiplication and rooting were standardized for TIB and compared with conventional micropropagation system. Immersion duration of one minute at three hourly intervals were fixed for all the experiments. The root and shoot characters, survival of plantlets after hardening and growth of plants after hardening were also compared for plants from both the culture systems. The clonal fidelity analysis standardized at CPBMB for banana using specific ISSR marker was used to analyze the fidelity of rooted plants from the two culture systems after 8th subculture passage. Higher clump size (20 clumps/ 500 ml media) was found good to initiate shoot multiplication in TIB with multiplication of 6.2 shoots/clump while in conventional system 15 and 20 clumps/ 500 ml of medium were found on par and recorded multiplication of 6.7 and 6.2 shoots/ clump respectively. Temporary immersion bioreactor system exhibited significantly higher shoot proliferation (13.00 shoots/clump) than the conventional system (7.67 shoots/ clump). Murashige and Skoog (MS) multiplication medium supplemented with 5mg L-1 BA recorded highest shoot proliferation in the both culture systems, recording 14.71 shoots/ clump in TIB and 8.35 shoots/ clump in conventional system. In both the culture systems, in the three different rooting media tried (MS + 2mg L-1 IBA+ 2% Sucrose, MS + 2mg L-1 IBA+ 3% Sucrose, MS + 1mg L-1 IBA+ 2% Sucrose), 99-100 per cent rooting was observed. Plantlets from TIB recorded significantly higher number of roots (10.29) than the conventional system (4.70). The root length of plantlets was more in conventional system (8.82 cm) as compared to TIB system (7.93 cm). Plantlets from bioreactor recorded significantly higher shoot length (10.88 cm) and more number of leaves (4.84) while plantlets from conventional system recorded less shoot length (8.94 cm) and less number of leaves (4.39). Survival of the plantlets after hardening was 90-91 per cent in both the culture systems. After sixty days of secondary hardening, plants from TIB exhibited better growth recording more plant height and more number of leaves than the conventional system. Plants from the rooting medium MS + 2mg L-1 IBA+ 2 per cent sucrose recorded plant height of 36.05 cm in TIB while plants from conventional system recorded plant height of 29.80 cm and the number of leaves was 6.3 in bioreactor plants and 5.7 in conventional system. Clonal fidelity analyzed using specific ISSR marker, UBC857 revealed that there was no polymorphism in the ISSR amplification profiles of rooted plants after the 8th subculture cycle when compared with the amplification profile of source mother plant in both the culture systems and the plants produced are true to type. An efficient commercial micropropagation protocol for banana using a TIB was thus developed in the present study. Temporary immersion bioreactor system exhibited high shoot multiplication, better shoot and root characters for plantlets and better growth of plants after hardening. Due to the high rate of multiplication in TIB, there is more production of plantlets and use of liquid media considerably reduced the media cost. Clonal fidelity analysis revealed that the plantlets produced after 8th subculture stage were true to type. Field evaluation of plants from both the culture systems and comparison of yield and quality of plants have to be taken up further.ThesisItem Open Access Commercial production of ginger (Zingiber officinale Rosc.) microrhizomes using temporary immersion bioreactor system(Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara, 2020) Rasha Fathima, A A.; Shylaja, M RGinger (Zingiber officinale Rosc.), is an important commercial spice crop grown in India from very ancient times. High seed rate of ginger (1500 kg/ha) and desiccation of seed rhizomes during storage are the problems faced by farmers in ginger cultivation. The tissue culture plants are not commercially distributed in ginger as they require an additional one more season for rhizome formation. Microrhizomes of ginger induced in vitro if used as planting materials, rhizomes can be harvested in the same season as conventional seed rhizomes and year round availability of seed material can be ensured. Hence, in vitro induced microrhizomes are included in the seed chain of ginger and there is high demand for microrhizomes for clean ginger production. The study entitled “Commercial production of ginger (Zingiber officinale Rosc.) microrhizomes using Temporary Immersion Bioreactor (TIB) system” was conducted at Centre for Plant Biotechnology and Molecular Biology (CPBMB), College of Horticulture, Vellanikkara during 2018 to 2020. The objective of the study was to develop an efficient commercial production protocol for ginger microrhizomes using a TIB system. The study was conducted in the ginger variety Athira using Plantform TIB purchased from Sweden. The multiple shoot cultures in the 5th subculture stage received from the commercial micropropagation unit, of CPBMB were used for the study. The protocol for in vitro induction of microrhizomes reported by Shylaja et al. (2016) was optimised for bioreactor production and at each stage it was compared with the conventional microrhizome production. The number of clumps/ 500ml of medium to initiate multiple shoot production, media for shoot multiplication and microrhizome induction were optimised for TIB and compared with conventional microrhizome production system. The microrhizome, root and shoot characters in microrhizome plantlets and growth of microrhizome plants after hardening were evaluated in the two culture systems. The clonal fidelity analyses of microrhizome plants derived from 8th subculture cycle were done using the specific ISSR marker as reported by Gavande, (2013). The clump size of 15 clumps/ 500 ml of medium showed higher shoot multiplication in both TIB and conventional system. The shoot proliferation in bioreactor (7.71 shoots/clump) was significantly higher than the conventional microrhizome production system (5.24 shoots/clump). Early induction of microrhizomes was observed in TIB system. In both the culture systems, microrhizome induction was faster in MS medium with 90 gL-1 sucrose. The number of microrhizomes produced in the medium in TIB varied from 87.75 to 96.75/ 500ml medium and in conventional system it varied from 84 to 88/ 500 ml medium. The microrhizome plantlets produced in TIB recorded significantly higher weight of microrhizomes, better shoot and root growth and more number of leaves compared to the microrhizome plantlets produced in the conventional system. The weight of microrhizomes, number of roots and root length were higher in microrhizome plantlets in MS medium with 90 gL-1 sucrose. The mean weight of microrhizome in the medium was 0.29 g in bioreactor and 0.18 g in conventional system. The survival of microrhizome plants after primary hardening was 94.34 per cent and after secondary hardening was 99.29 per cent in TIB. Microrhizome plants from bioreactor recorded significantly higher plant height (18.60 cm) compared to conventional system (14.80 cm). The number of leaves recorded in microrhizome plants of both the culture systems were on par and ranged from 6.97 to 7.18. Clonal fidelity analyses using specific ISSR marker revealed that there were no polymorphism in the ISSR amplification profiles in microrhizome plants produced after the 8th subculture cycle with the source mother plant and hence plants produced from both the culture systems are true to type. The protocol developed in the present study can be further modified by early bulking of the clumps, employing TIB for multiple shoot production in early culture phase and reducing the number of culture cycles so that the entire protocol period can be reduced. Evaluation of TIB microrhizome plants in high tech poly house or field and comparison of yield and quality with microrhizome plants from conventional production system also can be focused for further research.ThesisItem Open Access Development of a nanobiosensor for detection of banana bunchy top virus(Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara, 2016) Vinusri, S; KAU; Shylaja, M RThe investigations on ‘Development of a nanobiosensor for detection of Banana bunchy top virus’ were conducted at the Centre for Plant Biotechnology and Molecular Biology (CPBMB), College of Horticulture, Kerala Agricultural University, Thrissur and Centre for Materials for Electronics Technology(C-MET), Thrissur during August, 2014 to June, 2016. The objective of the study was to develop a nanobiosensor for easy and quick detection of Banana bunchy top virus (BBTV). Biosensors based on Localised Surface Plasmon Resonance (LSPR) have gained much attention now-a-days. Gold nanoparticles are ideal for optical sensing due to their better light absorption and scattering properties, chemical stability, high surface to volume ratio and high surface energy to provide stable immobilization of large amount of biomolecules. In the present study, a solution phase LSPR biosensor using gold nanorods (GNRs) was developed for detection of BBTV. Gold nanorods were synthesized using seed mediated growth method. The concentration of silver nitrate and volume of seed solution required to synthesize stable GNRs were optimized in the present study. The characterization of GNRs was done using UV-Vis spectrophotometry and Transmission Electron Microscopy. Two absorption bands were observed in the absorption spectrum of GNRs. The longitudinal plasmon band was observed at 679 nm and the transverse plasmon band was observed at 515 nm. The mean length, width and aspect ratio of the synthesized GNRs recorded using transmission electron microscopy were 35.84 nm, 13.57 nm and 2.64 respectively. Surface modification of CTAB capped GNRs was done by ligand exchange method. Functionalization of GNRs with BBTV specific antibody was undertaken by conjugating the antibody with GNRs to make a GNR probe. Dilution of antibody required for conjugation with GNRs was standardised as 1:100. Development of LSPR based biosensor using GNRs was attempted as chip based and solution phase based. As immobilization of GNRs on glass surface was not successful, studies on chip based biosensor could not be continued and further focus was made on solution phase LSPR biosensor. The BBTV antigen was isolated from the BBTV infected samples of banana collected from Banana Research Station of Kerala Agricultural University. The isolated antigen was allowed to interact with GNR probe and it was found that LPB was more sensitive to the interaction. Different concentrations of antigen were allowed to interact with GNR probe solution to identify the minimum detection limit. The efficacy of solution phase LSPR biosensor in detecting the antigen was further checked with BBTV infected leaf samples of six different banana cultivars viz. Yangambi Km5, Chenkadali, Nendran, Palayamkodan, Grand Naine and Kunnan. The kinetics of interaction of GNR probe with antigen from different banana cultivars and characterization of the interaction with UV-Vis spectrophotometry revealed that the developed biosensor was able to detect very low concentration of antigen (0.02 mg/ml) within a period of five to ten minutes. Colour change was also noticed due to interaction of GNR probe with antigen. In the infected samples, colour change from pinkish red to pale grey was evident while no such colour change was noticed in healthy samples. Due to the colour change, the developed solution phase sensor as such can be used for field level applications. The efficacy of developed solution phase LSPR based GNR biosensor was compared with Enzyme Linked Immuno Sorbent Assay (ELISA) for detecting BBTV antigen. The detection limit of antigen in ELISA was 0.08 mg/ml (80 ppm) while in the developed biosensor the detection limit was 0.02 mg/ml (20 ppm). The solution phase LSPR based GNR biosensor developed in the present study is thus effective for easy and quick detection of BBTV. Conversion of solution phase based GNR biosensor to chip based sensor will open up more applications and will help for easy commercial fabrication. Multiplexing is also possible in chip based sensor by controlling the aspect ratios of GNRs and functionalising with different antibodies specific to different pathogens so that the sensor can detect multiple pathogens using a single chip. The technology generated from the present investigations for the development of LSPR based GNR biosensor could be applied in other crops/ other pathogens with modificationsThesisItem Open Access DNA fingerprinting of released varieties and selected superior somaclones of ginger (Zingiber officinale Rosc)(Centre for plant biotechnology and molecular biology, College of horticulture, Vellanikkara, 2013) Pujaita Ghosh; KAU; Shylaja, M RGinger (Zingiber officinale Rosc.), one of the widely cultivated and consumed spices worldwide, is well known for its medicinal properties also. As the natural variability stands limited in the crop, induction of variability through tissue culture techniques was attempted at College of Horticulture, Vellanikkara from 1996 onwards. After indepth investigations on the somaclones regenerated, two varieties viz., “Athira” and “Karthika” were released during 2010 and four clones viz. B3, 292R, 88R and 478R were selected as superior somaclones. For the newly released ginger varieties and selected superior somaclones in pipeline for release, no fingerprint data are available for genotype identification and protecting the plant varieties / clones. The investigations on “DNA fingerprinting of released varieties and selected superior somaclones of ginger (Zingiber officinale Rosc.)” were carried out at the Centre for Plant Biotechnology and Molecular Biology (CPBMB), College of Horticulture, Kerala Agricultural University, Thrissur during the period from January 2012 to March 2013. The objectives of the study were to characterize two released varieties and four selected superior somaclones using molecular markers and to develop a DNA fingerprint specific to each variety / somaclone. Morphological characters like growth habit and size and shape of the rhizomes were found to vary in the varieties / somaclones studied. The somaclone 292R could be distinguished based on its dwarf plant stature and dark green leaves. The variety Athira has bold and flat rhizomes while the variety Karthika has medium bold and round rhizomes. Quantitative clustering for vegetative and rhizome characters attempted as per Mahalanobis D2 analysis could group the varieties and somaclones into three separate clusters. Of the seven vegetative characters analysed, plant height and number of tillers showed more divergence. The number of fingers, girth of primary and secondary fingers, thickness of flesh and inner core were the characters which exhibited more divergence for the rhizome characters. For molecular characterization, good quality genomic DNA extracted from ginger varieties / somaclones using CTAB (Rogers and Bendich, 1994) method was used. Thirty five RAPD and thirty ISSR primers were screened for amplification of genomic DNA and ten RAPD and eleven ISSR primers were selected based on the amplification pattern. DNA fingerprints of the varieties / somaclones were developed utilizing the clear, distinct bands generated in RAPD and ISSR profiles and size of the bands. Different colour codes were assigned for sharing of bands between varieties / clones to generate specific fingerprints. The RAPD marker system could bring out unique bands in the variety Karthika and somaclones B3, 292R and 478R. The RAPD primer, OPA 12 produced unique band in Karthika and B3, the primer OPA 04 in 292R and the primer OPA 28 in 478R. ISSR marker system could also bring out unique band in the variety Athira with primer ISSR 06. The RAPD, ISSR and combined fingerprints developed for each variety / somaclone were unique. Variability in the somaclones and the extent of variability from source parent cultivars were analysed using cluster analysis. The dendrogram seperated Maran and Rio-de-Janeiro somaclones in two separate clusters. Somaclones derived from cultivar Maran exhibited more variability than somaclones from Rio-de- Janeiro. The variety Athira was more diverse from the source parent cultivar Maran. Similarly, the somaclone 292R was more diverse from the source parent cultivar Rio-de-Janeiro. The Resolving Power (Rp) of RAPD and ISSR primers ranged from 6.00 to 16.25, indicating the ability of the selected primers to distinguish the varieties / clones most efficiently. The Polymorphic Information Content (PIC) ranged from 0.67 to 0.88, indicating the suitability of the selected primers for DNA fingerprinting. RAPD, ISSR and combined fingerprints developed specific for the ginger varieties / somaclones could be utilized for registration, documentation of varieties and for settling IPR issues.ArticleItem Open Access Effect of toxic metabolite(s) of phytophthora capsici on various stages of morphogenesis of black pepper calli(Kerala Agricultural University, 1997) Shylaja, M R; Sreekandan Nair, G; KAUThe effect of toxic metaholite(s) of Phytophthora capsici on various stages of morphogenesis of black pepper calli was studied. The survival rate of the call! in toxin medium was influenced by the varieties / cultivars used for the study. Once the calli survived in the toxin medium, the toxic tnetabolite(s) did not inhibit further growth of the calli. The shoot proliferation and elongation were also not influenced by the metabolite(s) in the media. However, the root growth was affected adversely.ThesisItem Open Access 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.ThesisItem Open Access Expression of chalcone synthase gene in ginger (Zingiber officinale Rosc.) as influvenced by various management practices(Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara, 2019) Archita Unnikrishnan; KAU; Shylaja, M RGinger (Zingiber officinale Rosc.), a rhizomatous spice crop is known for its nutraceutical potential due to the presence of non-volatile pungent principles, gingerols. The pungent principles in ginger are derived via “stilbenoid, diarylheptanoid and gingerol biosynthesis” pathway and the key enzyme involved in gingerol biosynthesis is Chalcone synthase. The present research work was undertaken at Centre for Plant Biotechnology and Molecular Biology (CPBMB), College of Horticulture, Kerala Agricultural University, Thrissur from 2017 to 2019 to analyse the influence of nutrient management and spraying of elicitors on Chalcone synthase gene expression in ginger. The study was conducted under two situations, in high tech polyhouse available at CPBMB and in open conditions. Various treatments viz., T1 (Nutrient management as per PoP, KAU), T2 (PoP soil test based nutrient management), T3 (Adhoc PoP organic, KAU), T4 (PoP nutrient management, KAU + salicylic acid foliar spray - 100 µM) and T5 (PoP nutrient management, KAU + methyl jasmonate foliar spray, 100 µM) were imposed. The KAU released ginger variety Karthika, known for its high gingerol content was used for the experiment. The morphological observations such as length of pseudostem, number of tillers per plant, number of leaves per tiller and leaf area were recorded at 60, 90 and 120 Days After Planting (DAP). All the treatments except T4 (PoP nutrient management KAU + salicylic acid foliar spray, 100 µM) and T5 (PoP nutrient management, KAU + methyl jasmonate foliar spray, 100 µM) recorded higher pseudostem length irrespective of the growing conditions. The plants under polyhouse recorded higher pseudostem length than the plants raised under open condition. The tiller production was higher in plants grown in open condition. The number of leaves per tiller was less in treatment T5 (PoP nutrient management, KAU + methyl jasmonate foliar spray - 100 µM) both under polyhouse and open conditions. In polyhouse condition, the leaf area was higher than in open condition. The relative expression of Chalcone synthase gene was studied from the leaves of various treatments at 120 DAP. All the treatments in polyhouse and open conditions recorded higher gene expression over the control treatment T1 (Nutrient management as per PoP, KAU). The treatment T2 (PoP soil test based nutrient management) recorded the highest expression of Chalcone synthase gene both under polyhouse and open conditions with a fold increase of 1.346 and 1.166 respectively. The plants under polyhouse recorded higher fresh yield of the rhizomes than plants under open condition. The treatment T1 (Nutrient management as per PoP, KAU) was the best irrespective of the growing conditions with regard to fresh yield. The rhizome characters such as number, length and girth of primary, secondary, tertiary fingers, thickness of inner core, colour and plumpiness of rhizomes were recorded. Rhizome characters were good in the treatments T1 (Nutrient management as per PoP, KAU), T2 (PoP soil test based nutrient management) and T3 (Adhoc PoP organic-KAU). There was no significant difference in the dry ginger recovery for the rhizomes harvested from both polyhouse and open conditions. The treatment T2 (PoP soil test based nutrient management) recorded higher recovery of both oleoresin and gingerol. The increased recovery of oleoresin in the treatment T2 (PoP soil test based nutrient management) was 71.16 per cent over the control in open condition and 32.11 per cent in polyhouse over the control. Similarly, the total gingerol content recorded an increase of 15.28 per cent over the control in polyhouse and 31.98 per cent over the control in open condition. The major outcome of the present investigations is the high recovery of total gingerol in soil test based nutrient management in ginger. The abiotic elicitors like salicylic acid and methyl jasmonate sprayed could not improve the recovery of gingerols. The soil test based nutrient management recorded higher recovery of both oleoresin and gingerol and was found cost-effective when compared to the other treatments.ThesisItem Open Access Gingerol yield in microrhizome derived rhizomes and clonal fidelity analysis at different subculture passages of microrhizome production in ginger (Zingiber officinale Rosc.)(Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara, 2020) Aswathy, Prabhakaran; Shylaja, M RGinger (Zingiber officinale Rosc.) is an important spice crop bestowed with numerous therapeutic properties. The therapeutic properties are attributed by various bioactive compounds, the most potent of which are the gingerols. The investigations entitled “Gingerol yield in microrhizome derived rhizomes and clonal fidelity analysis at different subculture passages of microrhizome production in ginger (Zingiber officinale Rosc.)” were carried out at the Centre for Plant Biotechnology and Molecular Biology (CPBMB), College of Horticulture, Vellanikkara, Thrissur, Kerala during 2018-2020. The objectives of the study were to assess gingerol yield in elicited microrhizome derived ginger rhizomes and to assess genetic fidelity at different subculture passages of microrhizome production using specific ISSR marker. Microrhizomes received from previous doctoral study (Manjusha, 2018) of the variety Aswathy were evaluated in the present investigations. The study was conducted as two separate experiments one in high tech polyhouse available at CPBMB and the other in open condition. Various planting materials viz. T1 (Microrhizomes in MMS medium), T2 (Microrhizomes in MMS medium with 5 % salicylic acid elicitation), T3 (Microrhizome in MS medium) and T4 (Conventional seed rhizomes-20g seed bit) were evaluated in high tech polyhouse as per the production technology standardized at CPBMB (Shylaja et al., 2018). The experiment in open condition was done as per PoP recommendations of KAU (KAU, 2016). The plant growth, yield of fresh rhizomes, oleoresin recovery and gingerol content were compared in both the experiments. For clonal fidelity analyses, multiple shoot cultures of three ginger varieties viz. Athira, Karthika and Aswathy received from CPBMB in the 5th subculture stage were advanced up to 9th subculture passage and fidelity was analysed using specific ISSR marker as per Gavande (2013). Microrhizome derived plants and conventional seed rhizome plants exhibited the same growth performance in characters like height of the pseudostem and number of leaves per longest tiller. The area of the longest fully opened leaf recorded at 120 DAP was more for microrhizome derived plants compared to conventional seed rhizomes. The fresh yield of rhizome per plant was on par in the different planting materials studied in both the experiments and ranged from 114.84g to 148.59g/ plant in poly house. The rhizome characters studied were on par in different planting materials except for the girth of secondary and tertiary fingers in which the microrhizome derived plants in MMS medium exhibited significantly higher values. The oleoresin yield was found higher in the microrhziome derived rhizomes in MS medium in polyhouse. Oleoresin yield was found higher in various treatments in the open condition. Conventional seed rhizomes showed the highest total gingerol content of (16.45%) in polyhouse. Among the microrhizome derived rhizomes, the microrhizomes in MMS medium recorded the highest total gingerol content (14.73%) in polyhouse. Highest gingerol content was recorded in conventional seed derived rhizomes followed by microrhizome derived rhizomes from MMS medium in poly house. The same result was observed in open condition experiment also but the recovery of gingerol was more in poly house experiment. The microrhizome derived rhizomes from MMS medium without elicitation recorded 14.5 per cent increase in gingerol yield compared to microrhizome derived rhizomes from MMS medium with elicitation in the polyhouse experiment.In clonal fidelity analysis, amplification with the ISSR primer (ISSR 05) generated clear amplicons in the size range 350- 1100 bp. Monomorphic amplicons were observed in all the three ginger varieties viz. Athira, Karthika and Aswathy for the 7th, 8th and 9th subculture passages. When compared with the source mother plant, polymorphism was not observed up to the 9th subculture stage for all the three ginger varieties and hence the plants produced are true to type. The study revealed that microrhizome plants have the potential to give high yield and quality rhizomes as evident by the same growth parameters exhibited by microrhizomes and conventional seed rhizome plants and higher leaf area and better rhizome characters exhibited by microrhizome plants. The yield realized in microrhizome plants are also on par with conventional seed rhizomes. The high gingerol content in the induced microrhizomes was seen exhibited in the harvested microrhizome derived rhizomes also. Microrhizomes perform better in high-tech poly house with precision agriculture. Clonal fidelity analyses with specific ISSR marker revealed that the regenerants produced up to 9th subculture cycle were true to type.ArticleItem Open Access In vitro production of toxic metabolite(s) by phytophthora capsici and partial purification of the metabolite(s)(Kerala Agricultural University, 1997) Shylaja, M R; Sreekandan Nair, G; Augustine, A; James Mathew; KAUPhytophthora capsici, the causal organism of Phytophthora foot rot disease in black pepper produces toxic metaholite(s) under in vitro conditions. Maximum accumulation of toxic metabolite(s) was observed in shake cultures of 15 days incubation in Ribeiro's medium. The symptoms induced by toxic metabolite(s) were quite typical to symptoms of natural and artificial infection by the pathogen. The toxic metabolite(s) accumulated in the in vitro culture was found to be heat stable aim non-specific. The toxic metabolite(s) could not be separated using organic solvent fractionation since it is present in the aqueous fraction of the culture filtrate. However, ion exchangers like Dowex 1 and Dowex 50 could be used for separating the metabolite(s) from the aqueous fraction.ThesisItem Open Access In vitro synthesis of gingerol and analysis of expressed sequence tags for gingerol production in ginger(Zingiber officinale Rosc.)(Centre for plant biotechnology and molecular biology,College of Horticulture, Vellanikkara, 2020) Manjusha, Rani; KAU; Shylaja, M RBlack pepper (Piper nigrum L.), often described as the ‘King of spices’ is the most important spice crop, grown for its berries in the world. Indian pepper is preferred across the globe due to its intrinsic qualities. Foot rot is a devastating disease of black pepper. In the changing climate, drought can be a major threat in black pepper production. Hence, the present study was taken up at College of Horticulture, Vellanikkara and ICAR-IISR, Kozhikode to characterise and to identify superior accessions of black pepper for yield, quality and tolerance to biotic and abiotic stresses. Fifty accessions of black pepper in the bearing stage maintained in the National Active Germplasm Site of ICAR-IISR, Kozhikode formed the base material for the study. The accessions were characterised for fifty qualitative and fifty quantitative characters following the descriptor developed by IPGRI (1995). Wide variability was observed among the accessions for ten qualitative characters. Quantitative characters of shoot, leaf, spike and fruit also showed wide variability. Field tolerance to foot rot disease and pollu beetle infestation was observed among the accessions. Twenty accessions were selected from the base collection based on superiority of yield (> 450g green berries/vine) , field tolerance to foot rot disease infection (biotic susceptibility score 1) and pollu beetle infestation (biotic susceptibility score 1-3). They were further evaluated for biochemical principles of quality, tolerance to foot rot disease under artificial inoculation and tolerance to drought by physiological and biochemical analyses. Piperine, essential oil and oleoresin ranged from 3.61 - 6.96 per cent, 3.00 - 5.87 per cent and 7.10 - 11.18 per cent, respectively, across the accessions. The accessions with high value of piperine, essential oil and oleoresin were identified as 7293, 7211 and 7289 respectively. The two accessions identified viz. 7293 and 7252 contained more piperine than the highest of Panniyur 2 (6.6 per cent) reported among the released varieties . Artificial inoculation of selected accessions using Phytophthora capsici culture for screening for foot rot disease resistance based on over all disease severity index of both stem and leaf lesions showed that accession 7259 was moderately resistant. The selected accessions did not exhibit significant variation for various physiological and biochemical parameters at field capacity. However higher value of photosynthesis, chlorophyll content, chlorophyll stability index, relative water content and membrane stability index and low leaf temperature were observed for accessions viz. 7215, 7240, P 5 and 7241 after five days and ten days of moisture stress induction following field capacity compared to other accessions. Higher values of proline, SOD, catalase and peroxidase were also observed for these accessions. The visual scoring showed that accessions with higher values for most of physiological and biochemical parameters of drought tolerance viz. 7215, 7240, P5, and 7241 had lesser number of fallen leaves and more number of leaves retained at permanent wilting point (PWP). The accessions 7215 and 7240 took twenty days to reach PWP compared to eleven accessions which took only 16 days to reach PWP. Foliar nutrition with sulphate of potash, IISR - Power mix and Pink Pigmented Facultative Methylotrophs (PPFM) had positive effect on drought tolerance for the accessions (7215, 7240, P5 and 7241) having natural tolerance. The identified accessions with high yield , quality and tolerance to biotic or abiotic stress can be used for further breeding programme.ThesisItem Open Access Influence of micro meteorological factors on flowering in vanilla (Vanila Planifolia Andrews)(College of Horticulture, Vellanikkara, 2008) Ramya, R; KAU; Shylaja, M RInvestigations on “Influence of micro meteorological factors on flowering in vanilla” were carried out at the Department of Plantation Crops and Spices, College of Horticulture, Kerala Agricultural University during 2005-2007. The objective of the study was to find out the effect of soil moisture stress and micro - meteorological factors on flowering in vanilla. The studies were carried out in five year old vanilla plants maintained in Department of Plantation Crops and Spices farm and in a selected farmer’s field at Thrissur district. Moisture stress was induced in vanilla gardens by withholding irrigation at four levels viz. one month, 1½ month, two months and 2½ months. The influence of soil moisture stress on flowering in vanilla and the changes in physiological and biochemical parameters in vanilla due to moisture stress and influence of micro meteorological parameters on flowering in vanilla were studied in the present investigations. Soil moisture stress induced flowering in vanilla. Maximum flowering of 80 per cent was observed in plants stressed for moisture for a period of one month followed by 60 per cent flowering in 1½ month stress period. Hence soil moisture stress for a period of 1-1½ month during November-December is sufficient to induce flowering in vanilla. Soil moisture stress altered various physiological and biochemical parameters in vanilla. Leaf thickness, relative leaf water content, membrane stability, soluble protein and total chlorophyll content decreased due to soil moisture stress. The content of epicuticular wax, total free amino acids, accumulation of proline, activity of peroxidase enzyme, total sugar content and K concentration in tissues increased due to stress. The changes in physiological and biochemical parameters were more pronounced as the intensity of stress increased. The micro-meteorological parameters of the garden also influenced flowering in vanilla. Flower opening in vanilla was found positively correlated with maximum temperature and negatively with relative humidity and minimum temperature. Flower opening showed highly significant positive correlation with light received from North and South directions and percentage of light infiltration. Soil moisture stress for a period of 1-1 ½ month during November- December is sufficient to induce flowering in vanilla. Changes in physiological parameters due to moisture stress could be used to visually assess the extent of moisture stress in the garden. The biochemical parameters recorded at ideal stress period could be used as indices to assess the extent of moisture stress in plants more precisely in high tech / precision farming systems. Manipulation of microclimate with respect to temperature, light and relative humidity is essential for getting proper flowering in vanilla.ThesisItem Open Access Low cost alternatives in commercial micropropagation of banana(musa spp.)(Centre for Plant Biotechnology and Molecular Biology,College of Horticulture, Vellanikkara, 2018) Faiza Mohamed; KAU; Shylaja, M RTissue culture banana plants have become an integral part of commercial banana production. Banana micropropagation is hampered by high unit cost of production, poor multiplication and low survival rates during acclimatization. To make a commercial micropropagation unit viable, cost of production of plantlets should be brought to minimum. The investigations on ‘Low cost alternatives in commercial micropropagation of banana’ was hence taken up at Centre for Plant Biotechnology and Molecular Biology, College of Horticulture from 2016-2018. The aim of the investigations was to reduce the cost of production in commercial micropropagation of banana. Studies were conducted in different cultivars like Attunendran (AAB), Nedunendran (AAB), Chengalikodan (AAB), Poovan (AAB), Njalipoovan (AB) and Grand Naine (AAA) which were commercially produced at CPBMB in the micropropagation unit. Established cultures of six cultivars in the 5th subculture cycle received from the commercial micropropagation of unit CPBMB were utilized for the study. The micropropagation protocols optimized for the six different cultivars at CPBMB were followed for production of plantlets. The clonal fidelity analysis standardized using specific ISSR marker at CPBMB was used to analyse the clonal fidelity of regenerants from S7 to S11 stage. Sucrose was substituted with common sugar @ 30gL-1 in MS multiplication and rooting media and shoot proliferation, rooting and root characters in different cultivars were studied. Common sugar was found equally good to sucrose and it did not influence shoot proliferation in different banana cultivars. Common sugar was also found equally good to sucrose in the rooting media recording 100 per cent rooting in all the six cultivars studied. Substitution of sucrose with common sugar had no significant influence in the days taken for root initials to appear. For number of primary roots produced, there was no significant difference in all the cultivars in the two media except Attunendran in which medium with common sugar produced more number of primary roots (8.41) than sucrose (6.75). Root length was higher in MS rooting medium with common sugar in cultivars like Chengalikodan, Njalipoovan and Grand Naine. Medium with common sugar produced more number of secondary roots in cultivar Chengalikodan while medium with sucrose produced more number of secondary roots in cultivars like Attunendran and Njalipoovan. When fifty per cent of agar was substituted with other solidifying agents like sago or isabgol, there was good solidification of MS medium. There was no significant variation in shoot proliferation in different cultivars when half of agar was substituted with isabgol or sago in the MS multiplication medium. Fifty per cent substitution of agar with isabgol or sago was found equally good to agar (100%) in rooting medium. Hundred per cent rooting was observed in the three different media combinations in all the cultivars. Substitution of 50 per cent agar with isabgol or sago showed no significant difference in days taken for root initials to appear, number of primary roots, number of secondary roots and root length in cultivars like Attunendran, Njalipoovan and Grand Naine. There was no difference in survival of plantlets in treatments with different low cost additives. When low cost carbon source and gelling materials were used instead of standard additives, there was 87 per cent reduction in media cost. Clonal fidelity was analysed using specific ISSR marker optimized at CPBMB as reported by Rajitha et al. (2015). Polymorphic amplicons were observed as subculture progressed from S9 – S11 in cultivars like Nedunendran, Attunendran and Grand Naine. In Chengalikodan, up to 10th subculture passage no polymorphic bands were observed. In cultivars like Poovan and Njalipoovan which exhibited low multiplication rate, no polymorphic bands were observed in regenerants up to S11 stage. For highly multiplying cultivars like Attunendran, Nedunendran and Grand Naine subculturing for multiplication up to 8th subculture stage is recommended. In Chengalikodan, subculturing for multiplication can be advanced up to 9th subculture passage and in Poovan and Njalipoovan multiplication can be advanced up to 10th subculture passage in the protocol standardized at CPBMB. However, the commercial feasibility of the findings and working out the economics of production is possible only by large scale adoption of media components and subculture pattern in commercial micropropagation protocol.ThesisItem Open Access Metabolite Profiling and gene expression analysis for gingerol production in selected somaclones of ginger (zingiber officinale rosc.)(Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara, 2017) Sreeja, S; KAU; Shylaja, M RGinger (Zingiber officinale Rosc.) is one of the oldest known spices and is much valued for its medicinal properties. Ginger rhizome is a source of active biological compounds which are responsible for its medicinal properties. Gingerol is the major pungent polyphenol in ginger which has got a wide array of pharmacological properties. Research on somaclonal variation done at Kerala Agricultural University could locate ginger somaclones with high gingerol content and in the course of investigations high somaclonal variation was observed for quality components. Ginger has a very big genome of 23,618 Mbp which is little exploited and reports on the genomic base of gingerol biosynthesis are scanty. The present investigations hence aim at profiling the metabolites in selected ginger somaclones using high throughput analytical platforms and to analyze the gene expression with respect to gingerol production. One released ginger variety from KAU (Athira), two selected ginger somaclones (B3 and 132M) and parent cultivar (Maran) formed the experimental materials for the study. Studies were carried out at Centre for Plant Biotechnology and Molecular Biology, Distributed Information Centre of College of Horticulture and Arjuna Natural Extracts Pvt. Ltd., Aluva during August 2013 to July 2017. The profiling of aroma principles using Gas Chromatography-Mass Spectrometry (GC-MS) and pungency principles using High Performance Liquid Chromatography (HPLC) at various growth stages viz. five months after planting (5MAP), six months after planting (6MAP) and seven months after planting (7MAP), revealed that aroma and pungency principles accumulated in ginger rhizomes at the rhizome formation stage (5MAP). Clone to clone variation was observed in the number and quantity of aroma and pungency principles accumulated in the rhizome. Total gingerol content in somaclone B3 (19.07%) was high when compared to the control cultivar Maran (17.49%) irrespective of the growth stages. Gene expression for Chalcone synthase in selected somaclones done using real time PCR assay showed highest gene expression in somaclone B3 when the control cultivar Maran was set as calibrator. Somaclone B3 recorded 54 per cent increase in Chalcone synthase gene expression over the control cultivar Maran. Suppression subtractive hybridization done to identify differentially expressed genes in somaclone B3 and control cultivar Maran could prepare Expressed Sequence Tag (EST) libraries both for rhizome and leaf. Analysis of EST sequences (25 rhizome ESTs and 19 leaf ESTs) using various bioinformatic tools revealed that there were no differentially expressed genes for gingerol production in rhizome ESTs. But eleven differentially expressed proteins involved in signaling response, protein trafficking, photosynthesis, ATP formation and transposon mediated mutation were observed in rhizome ESTs. The analysis of leaf ESTs showed differential gene expression in somaclone B3 for 3-ketoacyl CoA thiolase (ACAA1) gene which is involved in gingerol biosynthetic pathway. Hence the higher expression of 3-ketoacyl CoA thiolase gene is responsible for the high gingerol content in somaclone B3 as compared to control cultivar Maran. Eighteen other differentially expressed proteins involved in biological processes like transportation of plant secondary metabolites and their intermediates, mobilization of sucrose into pathways involved in metabolism, lipid biosynthesis, transportation of cellular material to microtubules, biogenesis of metabolic pathways in Calvin cycle were observed in leaf ESTs. The differentially expressed gene (ACAA1) can be further validated using northern blotting and quantitative real time PCR by designing specific primers from the ESTs. Expressed sequence tags and corresponding differentially expressed proteins can be used as molecular markers. Post translational modification in differentially expressed proteins can be used to study the mechanism of gingerol production. Forty four sequences deposited at NCBI form the base sequences for further research.ThesisItem Open Access Micropropagation of gerbera (Gerbera jamesonii Bolus) and assessment of genetic stability of plantlets using ISSR assay(Centre for plant biotechnology and molecular biology, College of horticulture, Vellanikkara, 2014) Awchar Datta, Manikrao; KAU; Shylaja, M RGerbera jamesonii Bolus commonly known as African daisy is an important cut flower ranking fifth in the global cut flower trade. Gerbera is also used as potted plant due to its attractive flowers of varying colours. It is generally propagated by division of suckers or clumps, but the multiplication rate is found very slow. New varieties are introduced every year in large numbers for commercial cultivation through high-tech system. Development of an efficient micropropagation protocol is of great significance for meeting the large scale demand of quality planting material and to popularise the new varieties. As there is high demand for new variants in gerbera, tissue culture induced variability also could be exploited in breeding programmes. The investigations on micropropagation of gerbera (Gerbera jamesonii Bolus) were hence taken up at Centre for Plant Biotechnology and Molecular Biology, College of Horticulture from 2012 to 2014. The present study aims to micropropagate gerbera using flower buds and leaf explants and to test the genetic stability of the micropropagated plants using ISSR assay. The IIHR variety Arka Krishika and three other varieties procured from AVT, Kochi viz., Dubai, Shania and Hotpsring were utilised for the study. Micropropagation protocols were attempted in different gerbera varieties using two different pathways viz., direct and indirect organogenesis with explants like flower bud and leaves. The micropropagation protocol developed at CPBMB by Shylaja et al. (2014) was used to regenerate plants from flower bud explants. Regeneration from flower bud explants was achieved through direct organogenesis in three gerbera varieties viz., Arka Krishika, Dubai and Shania. An efficient protocol was standardised from in vitro leaf explants through indirect organogenesis and from initial cultures to establishment of micropropagated plants it took only six months. High genotypic difference was observed in propagule multiplication in different gerbera varieties studied. The white flowered variety Dubai showed high rate of multiplication in both the routes of micropropagation viz., indirect organogenesis using leaf explants and direct organogenesis using flower bud explants. The potting media for hardening micropropagated plants were standardised and plantlets produced were successfully acclimatized. Genetic stability studies using ISSR assay were carried out in three groups of plants viz., mother plants, flower bud regenerants and leaf calli regenerants. Of the seven gerbera specific ISSR primers tested, five primers showed monomorphic banding pattern. In variety Dubai, primers ISSR 18 and ISSR 21 exhibited polymorphism to the extent of 25 to 33.33 per cent. In variety Shania, the primers ISSR 15 and ISSR 25 exhibited polymorphisms to the extent of 25 to 33.33 per cent. Genetic stability studies showed that mother plant and regenerants derived from flower buds were uniform to the extent of 80 per cent. Variation at DNA level observed was more (40%) for plantlets regenerated through indirect pathway. The established micropropagated plants (200 Nos.) and plants showing variation at DNA level are to be evaluated further for flower production and floral characters before recommending the protocol for commercial micropropagtion. Genotype specific optimisation should be done in micropropagation protocol for effective large scale multiplication of different varieties.