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Kerala Agricultural University, Thrissur
The history of agricultural education in Kerala can be traced back to the year 1896 when a scheme was evolved in the erstwhile Travancore State to train a few young men in scientific agriculture at the Demonstration Farm, Karamana, Thiruvananthapuram, presently, the Cropping Systems Research Centre under Kerala Agricultural University. Agriculture was introduced as an optional subject in the middle school classes in the State in 1922 when an Agricultural Middle School was started at Aluva, Ernakulam District. The popularity and usefulness of this school led to the starting of similar institutions at Kottarakkara and Konni in 1928 and 1931 respectively.
Agriculture was later introduced as an optional subject for Intermediate Course in 1953. In 1955, the erstwhile Government of Travancore-Cochin started the Agricultural College and Research Institute at Vellayani, Thiruvananthapuram and the College of Veterinary and Animal Sciences at Mannuthy, Thrissur for imparting higher education in agricultural and veterinary sciences, respectively. These institutions were brought under the direct administrative control of the Department of Agriculture and the Department of Animal Husbandry, respectively. With the formation of Kerala State in 1956, these two colleges were affiliated to the University of Kerala. The post-graduate programmes leading to M.Sc. (Ag), M.V.Sc. and Ph.D. degrees were started in 1961, 1962 and 1965 respectively.
On the recommendation of the Second National Education Commission (1964-66) headed by Dr. D.S. Kothari, the then Chairman of the University Grants Commission, one Agricultural University in each State was established. The State Agricultural Universities (SAUs) were established in India as an integral part of the National Agricultural Research System to give the much needed impetus to Agriculture Education and Research in the Country. As a result the Kerala Agricultural University (KAU) was established on 24th February 1971 by virtue of the Act 33 of 1971 and started functioning on 1st February 1972. The Kerala Agricultural University is the 15th in the series of the SAUs.
In accordance with the provisions of KAU Act of 1971, the Agricultural College and Research Institute at Vellayani, and the College of Veterinary and Animal Sciences, Mannuthy, were brought under the Kerala Agricultural University. In addition, twenty one agricultural and animal husbandry research stations were also transferred to the KAU for taking up research and extension programmes on various crops, animals, birds, etc.
During 2011, Kerala Agricultural University was trifurcated into Kerala Veterinary and Animal Sciences University (KVASU), Kerala University of Fisheries and Ocean Studies (KUFOS) and Kerala Agricultural University (KAU).
Now the University has seven colleges (four Agriculture, one Agricultural Engineering, one Forestry, one Co-operation Banking & Management), six RARSs, seven KVKs, 15 Research Stations and 16 Research and Extension Units under the faculties of Agriculture, Agricultural Engineering and Forestry. In addition, one Academy on Climate Change Adaptation and one Institute of Agricultural Technology offering M.Sc. (Integrated) Climate Change Adaptation and Diploma in Agricultural Sciences respectively are also functioning in Kerala Agricultural University.
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ThesisItem Open Access Salicylic acid and Piriformospora indica induced modulation of stress resistance mechanism in Tomato (Solanum lycopersicum L.)(Department of Plant Biotechnology, College of Agriculture, Vellayani, 2021) Deepthi Mohan; KAU; Roy StephenThe study entitled “Salicylic acid and Piriformospora indica induced modulation of stress resistance mechanism in tomato (Solanum lycopersicum L.).” was conducted at College of Agriculture, Vellayani, Thiruvananthapuram, during 2020 – 2021. The primary objective of this study was to analyse the salicylic acid induced NPR1 gene expression as well as Piriformospora indica induced resistance modulation to stress and its persistence and transmission through seeds. The induced resistance in plants can be classified as Systemic Acquired Resistance (SAR), mediated by the signalling molecule salicylic acid and Induced Systemic Resistance (ISR) which was induced by the presence of non-pathogenic plant growth promoting fungi or bacteria. In the present study two treatments, plant growth promoting endophytic basidiomycete fungus Piriformospora indica (biological inducer) and salicylic acid (chemical inducer) were applied on bacterial wilt resistant (Vellayani Vijay, KAU) and susceptible (Pusa Ruby, IARI) varieties of tomato and the expression rate of Non-expresser of pathogenesisrelated genes1 (NPR1) gene was evaluated using q-RTPCR. In first generation, one set of seed were inoculated with P. indica (1%) by incorporating it into the potting mixture before filling the pot-tray cavities. After confirming endomycorhizal root colonization, leaf samples were collected for analyzing NPR1 expression. Seeds from these plants were collected and progeny seedlings were maintained for analysing transgenerational expression of NPR1 gene. Second set of seeds were sown in the nursery and two weeks old seedlings were transplanted in to pots. Salicylic acid(100ppm) was sprayed at flowering stage and NPR1 gene expression was analysed on 5th day and 7th day after treatment in leaves and compared to next generation (in two weeks old seedlings) for understanding transgenerational expression. The evaluation of stress tolerance of the treated (P. indica and S.A) plants was done on progeny seedlings of Vellayani Vijay by treating it with a low density inoculum of virulent Ralstonia solanacearum (106 cells/ ml). Similarly abiotic stress tolerance evaluation was carried out by subjecting the plants to a higher temperature in polyhouse. Physiological and biochemical observations showed that application of P. indica and salicylic acid can induce resistance by regulating the enzyme activities and other physiological parameters. The resistant variety (Vellayani Vijay) gave a relative change of 7 fold and 4 fold for NPR1 expression (on 5th day and 7th day respectively) on the application of salicylic acid. The susceptible variety (Pusa ruby) showed a relative change of 6 fold and 2 fold for NPR1 expression on the application of salicylic acid. A relative change of 7 fold and 5 fold was shown respectively for first generation and second generation on treatment with salicylic acid, which confirms a transgenerational expression of the defence related gene. Similarly P.indica also showed transgenerational expression with a relative change of 5 fold and 2 fold respectively for first and second generation. P. indica treatment and exogenous application of salicylic acid can induce resistance in plants and thus induced resistance can be transferred to progenies. Transgenerational induction might be vital to the long-term survivorship of plant species on novel environments or adapt to other forms of environmental change.ThesisItem Open Access Detection of Banana bunchy top virus (BBTV) through colorimetric closed tube loop medicated isothermal amplification (Lamp)(Department of Plant Biotechnology, College of Agriculture, Vellanikkara, 2021) Nahla Binth, T; KAU; Smita, NairBanana is an important food and cash crop world wide. India leads in world banana production with 31.5 million tons from 0.9 million ha area. The Banana Bunchy Top Disease caused by Banana bunchy top virus (BBTV) is considered to be the most economically destructive of the viral diseases. Primary transmission of disease is through infected planting material. Virus indexing of suckers and tissue culture plantlets is essential to ensure the availability of virus free planting materials. The available detection methods for the BBTV are ELISA and PCR. The ELISA is time consuming while PCR requires thermal cycling and the post amplification sample handling predisposing to sample cross contamination. A nucleic acid amplification method termed Loop Mediated Isothermal Amplification (LAMP) enables rapid, sensitive and specific amplification of template DNA under isothermal condition using a strand displacing polymerase like Bst DNA polymerase and 4 to 6 primers producing 109 copies of the target in less than 1 h. Combined with simple visual detection of amplicons using the Hydroxy Naphthol Blue (HNB) dye, closed tube detection can be made hence avoiding sample cross contaminations. Hence, the present study entitled “Detection of Banana bunchy top virus through colorimetric closed tube Loop Mediated Isothermal Amplification (LAMP)” was undertaken at the Center for Plant Biotechnology and Molecular Biology, College of Agriculture, Kerala Agriculture University, Thrissur, during the period from 2019 to 2021 with the objective to develop a colorimetric closed tube LAMP assay for rapid and sensitive detection of BBTV. As the initial step, samples were collected from 15 BBTV symptomatic banana plants, two symptomless plants and from plants showing symptoms of other viruses infecting banana such as Banana bract mosaic virus (BBrMV), Banana streak virus (BSV) and Cucumber mosaic virus (CMV). The samples were collected from Banana Research Station, Kannara. One sample was taken from healthy tissue culture raised plant at the Center for Plant Biotechnology and Molecular Biology, College of Agriculture, Kerala Agriculture University, Thrissur. Good quality DNA was obtained with Doyle and Doyle method followed by RNase treatment. For LAMP primer designing, coat protein gene, replicase gene and movement protein gene sequences of BBTV were selected as the target. The LAMP external and internal primers could be picked from all the targets, but loop primers were not available for replicase gene and movement protein gene sequences. Hence, six LAMP primers were designed targeting BBTV coat protein gene using the software Primer Explorer version 5.0. The concentration of different components in the LAMP reaction like dNTPs (0.8 to 1.8 mM), MgSO4 (4 to 8 mM), HNB (120 µM and 150µM) and betaine (0.8 to 1M) was optimized. The final reaction mixture contained 50 ng template DNA, 1.4 mM each dNTP, 0.8 μM each primer BBTVFIP and BBTVBIP, 0.2 μM each primer BBTVF3 and BBTVB3, 0.4 μM each primer BBTVLF and BBTVLB, 0.8 M betaine, 6 mM MgSO4, 1X Thermopol buffer with 2 mM MgSO4, 8U Bst polymerase large fragment and 120 μM HNB dye in 25 μl reaction volume. Isothermal amplification was set at 65 oC and end point detection made using 120 µM HNB dye in the reaction mixture where the BBTV positive samples showed colour change from violet to blue. The LAMP positive amplicons appeared as ladder like bands on agarose gel. Molecular characterization of LAMP amplicon was made with sequencing and restriction analysis. A restriction enzyme having cut site in between the LAMP internal primer flanking region was identified. When the LAMP amplicons were digested with the restriction enzyme Sau3AI having single cut site, fragments of expected size (62 bp and 89 bp) were obtained. The optimized LAMP assay was validated using 15 BBTV symptomatic samples, three healthy samples and samples showing symptoms of BBrMV, CMV and BSV, as observed by the agarose gel profile and the colour change with HNB dye. All the 15 BBTV symptomatic samples showed amplification in the LAMP assay while the healthy samples and the samples showing symptoms of other viruses remained unamplified. Comparison of LAMP assay with conventional PCR in the detection of BBTV was also made. Only 13 samples out of the 15 BBTV symptomatic samples tested positive in the PCR assay, thus showing higher sensitivity of the LAMP method. Moreover, the closed tube visual detection in LAMP avoids the post amplification sample handling. Since LAMP reaction can be carried out on a dry bath or water bath, it is a rapid and simple alternative to the available PCR based detection method for BBTV.ThesisItem Open Access Reverse transcription loop medicated isothermal amplification(RT-Lamp) for diagnosis of Banana bract mosaic virus in banana (Musa spp.)(Department of Plant Biotechnology, Centre for Plant Biotechnology and Molecular Biology, College of Agriculture, Vellanikkara, 2021) Midhuna Madhu, K; KAU; Smita NairBanana (Musa spp.) is one of the most important and widely cultivated crops around the world. Banana is cultivated on 0.9 million hectares in India with a production of 30.8 million tonnes (NHB, 2020). Despite its importance, growers are not in a position to achieve high productivity due to the presence of various biotic and abiotic stresses. Viruses are a major concern to banana production causing up to 100 per cent yield reduction. The Banana bract mosaic virus (BBrMV), a ssRNA virus of the genus Potyvirus is reported to cause 40 per cent yield reduction in banana. The virus is transmitted through vegetative propagules and insect vectors like Pentalonia nigronervosa, Aphis gossypi and Rhopalosiphum maidis. Internal quarantine is necessary to prevent the spread of the virus. We need a platform for virus indexing, so that farmers can get good quality virus-free planting materials. The available detection methods for BBrMV are the Enzyme-Linked Immunosorbent Assay (ELISA) and Reverse Transcriptase Polymerase Chain Reaction (RT-PCR). The ELISA is time consuming while the RT- PCR needs post PCR sample handling predisposing to sample cross contamination. Hence the study entitled “Reverse Transcription Loop Mediated Isothermal Amplification (RT-LAMP) for diagnosis of Banana bract mosaic virus in banana (Musa spp.)” was undertaken during the period from 2019 to 2021 at the Department of Plant Biotechnology, College of Agriculture (CoA), Vellanikkara, Thrissur, with an objective to develop a rapid and efficient molecular diagnostic method for BBrMV. The LAMP is an auto-cycling strand displacement isothermal DNA synthesis method using a strand displacement DNA polymerase. The RTLAMP is a specific, highly sensitive and relatively faster diagnostic method for RNA viruses. Initially, leaf samples of BBrMV symptomatic banana plants were collected from Banana Research station (BRS), Kannara, Kerala. Healthy leaf samples were collected from asymptomatic plants in the field and from tissue culture plants produced at CPBMB, CoA, Vellanikkara. Samples from banana plants showing symptoms of other viruses such Banana bunchy top virus (BBTV), Banana streak virus (BSV) and Cucumber mosaic virus (CMV) as were also collected from BRS, Kannara, to validate the results of the study. Three different methods using TRI reagent, Purelink Plant RNA reagent and RNeasy plant mini kit were tried for isolation of total RNA. Good quality RNA with intact bands could be obtained with RNeasy plant mini kit (Qiagen). For the RT-LAMP reaction, a set of six primers were designed using the software Primer Explorer version 5.0. Initially, three targets in the BBrMV genome namely, coat protein gene, replicase gene and movement protein gene were selected for picking LAMP primers. Due to unavailability of loop primers for BBrMV replicase gene and movement protein gene, six primers targeting the BBrMV coat protein gene were selected. All the primers were validated using BLASTN. Both one-step and two-step RT-LAMP assay was optimized for diagnosis of BBrMV. For two-step RT-LAMP assay, RNA was converted to cDNA and was used as template for isothermal amplification. Optimisation of the assay was done by varying the concentration of the reagents like MgSO4 (4-8 mM), dNTP (1.4-1.6 mM each), betaine (0.8-1 M) and Bst polymerase (4-8 U). The final optimised reaction mixture contained 40 ng cDNA, 1.6 mM each dNTP, 0.2 µM each primers F3 and B3, 0.8 µM each primers BrFIP and BrBIP and 0.4 µM each primers BrLF and BrLB, 1 M betaine, 4 mM MgSO4, 1x Thermopol buffer with 2 mM MgSO4, 4 U Bst polymerase large fragment and 120 µM HNB in 25 µl reaction volume. Incubation temperature at 65° C was found optimum. One step RT-LAMP is the method suited for routine diagnostics as the RNA sample can be directly used as the template for amplification. One step RT-LAMP reaction mixture contained reverse transcriptase and RNase inhibitor along with the other components of the LAMP reaction and RNA was used as the template instead of cDNA. The positive amplicons showed ladder like bands on agarose gel and showed a colour change from violet to sky blue in the presence of HNB dye in the reaction mixture. Molecular typing of RT-LAMP products was done using sequence analysis and restriction digestion. For restriction profiling, the enzyme Sau3AI having internal cut site in the RT-LAMP internal primer flanking region was used. The enzyme having single cut site produced fragments of size 100 bp and 45 bp. The RT-LAMP assay was validated using 12 BBrMV symptomatic samples, healthy samples and samples showing symptoms of other banana viruses like BBTV, BSV and CMV. All the 12 BBrMV symptomatic samples amplified in the RT-LAMP assay while no amplification was observed for healthy samples and samples showing symptoms of other viruses. Hence, in the current study, a rapid, sensitive and specific RT-LAMP based detection method for Banana bract mosaic virus was developed.ThesisItem Open Access Editing of rice transcription factor OsMADS26 for drought tolerance through CRISPR/Cas9 system(Department of Plant Biotechnology, College of Horticulture, Vellanikkara, 2021) Anjala, K; KAU; Rehna, AugustineRice (Oryza sativa L.) is the most widely consumed staple food of world’s human population belonging to Asia and Africa. Being a semi-aquatic annual plant, rice is highly prone to losses due to various environmental stresses. Many studies regarding this had revealed the need for developing varieties tolerant to abiotic and biotic stresses. Various methods like Marker Assisted Breeding, mutation breeding, RNAi, Antisense technology, ZFNs and TALENs were in use to develop elite traits for abiotic stress tolerance in crops like rice. But very recently, CRISPR/Cas9 system had come into the limelight as an efficient tool for the genetic manipulations of crops. Studies have identified OsMADS26 transcription factor as a negative regulator of drought tolerance in rice. Hence the current study, ‘Editing of rice transcription factor OsMADS26 for drought tolerance through CRISPR/Cas9 system’ was undertaken during the period from 2019 to 2021 at the Centre for Plant Biotechnology and Molecular Biology, CoA, Vellanikkara, Thrissur with an objective to develop drought tolerance in rice. The rice cultivar Nipponbare was selected for the study due to its competence in genetic transformation and regeneration. For CRISPR/Cas9 mediated targeted editing of OsMADS26 gene, guide RNAs (gRNAs) were designed using online software CRISPR-P v2. Genome sequence information of OsMADS26 gene available from rice genome annotation project was used for the study. Genomic region of OsMADS26 gene, flanking the gRNA target (~ 450 bp) was amplified using gene specific primers and sequence of the target region was confirmed using BLASTn and ClustalW analysis. The CRISPR/Cas9 binary vector pRGEB32 was used to clone the guide RNAs using BsaI restriction sites. Three gRNAs were selected for cloning based on features like on score value (higher the value better the editing efficiency), GC content, (40-60%), no. of off-target sites (Minimum number of off-target sites preferred), presence of secondary structure, location on the genome (towards 5' end of gene in exonic region is preferred) etc. The CRISPR/Cas9 construct for cloning was developed by annealing and ligating the gRNAs to the pRGEB32 vector followed by cloning in E. coli strain DH5α. The putative positive clones were identified by colony PCR and further confirmed by Sanger sequencing. The plasmids isolated from PCR positive colonies were sequenced using universal M13 Reverse primer which is present on pRGEB32 vector. The sequences of the clones were confirmed using multiple sequence alignment tool ClustalW. One colony of gRNA 1 construct (OsMADS26 #G1-1) and two colonies of gRNA 3 (OsMADS26 #G3-3 and OsMADS26 #G3-4) were found positive. The CRISPR/Cas9 constructs of OsMADS26 were then mobilized into Agrobacterium tumefaciens strain EHA105 following the Freeze-thaw method. The positive clones were identified using plasmid PCR using hygromycin gene specific primers. Positive colonies of OsMADS26 #G1-1 and OsMADS26 #G3-3 constructs in EHA105 were then used for rice genetic transformation. The seeds of Oryza sativa sub species japonica cultivar Nipponbare were inoculated into N6 medium supplemented with 3.0 mgL-1 2,4-D for callus induction. After five days, the calli were infected with Agrobacterium cultures harboring desired gRNA constructs for 1.5-2 min. Along with the gRNA constructs, an empty vector was also transformed to rice as vector control and a set of untransformed culture were also maintained. After around two days of co-cultivation, the excess Agrobacterium growth was washed-off thoroughly from the calli using the bacteriostatic agent Augmentin. The calli were then placed on selection medium containing Augmentin and Hygromycin. The hygromycin resistant calli showed proliferation after 14 days of incubation. The proliferating microcalli were then transferred to regeneration medium after 21 days. Proliferation of microcalli was observed in vector control, wild type as well as OsMADS26 #G1-1 and OsMADS26 #G3-3 co-transformed plates. The vector control and untransformed calli showed greening and shoot primordia initiation in regeneration medium. The regenerated shoots will be analyzed for mutation in future. Hence, in the current study, gRNA constructs for targeted editing of OsMADS26 gene was successfully developed and transformed in to rice cultivar Nipponbare. Rice genetic transformation suitable to our lab conditions were also optimized. Rice plants with mutations in the OsMADS26 gene is expected in future which can confer drought tolerance.ThesisItem Open Access Impact of water stress on sucrose metabolism in rice (oryza sativa L.)(Department of Plant Biotechnology, College of Agriculture, Vellayani, 2021) Anie, Thomas; KAU; Beena, RThe study entitled “Impact of water stress on sucrose metabolism in rice (Oryza sativa L.)” conducted at Department of Plant Biotechnology and Department of Plant Physiology, College of Agriculture, Vellayani during 2020-21. The objective was to study the impact of water stress on sucrose metabolism by analyzing the physiological and biochemical parameters and gene expression in selective drought tolerant and susceptible rice genotypes. In this study, two rice varieties, drought tolerant variety, PTB 7 (Parambuvattan) and drought susceptible, PTB 23 (Cheriya Aryan) were grown in pot culture and after the panicle initiation stage, crops were subjected to water stress by withdrawing irrigation until the plants experienced the symptoms of stress (leaf rolling). Then the various physiological parameters were studied five days after the induction of water stress. Extraction and estimation of sucrose metabolizing enzymes such as invertase, α- and β-amylase were done five days after induction of stress spectrophotometrically. Expression levels of sucrose synthase (SuSy) and sucrose transporter gene (SUC2) were analyzed from both root and leaf seven days after induction of water stress. Under water stress, physiological parameters such as cell membrane stability index, relative water content and yield trait like number of productive tillers were significantly reduced, activity of enzymes such as invertase, α- and β-amylase were increased and gene expression level of sucrose synthase (SuSy) and sucrose transporter (SUC-2), which are associated with the sucrose metabolism were upregulated. Water stress enhanced the sucrose content and reducing sugar content in rice plant. There was significant increase in root traits in PTB 7 but they were reduced in PTB 23. Also, there was a reduction in shoot biomass than the root biomass in tolerant rice variety, which leads to an increase in root to shoot (R/S) ratio. In this study, sucrose metabolism and transport were increased in both drought tolerant and susceptible variety under water stress condition. However, PTB 7 (drought tolerant) showed an improved sucrose metabolism than PTB 23 (drought susceptible) during water stress condition.ThesisItem Open Access Molecular characterization of gynoecy in bitter gourd (Momordica charantia L.)(Department of Plant Biotechnology, College of Agriculture, Vellanikkara, 2021) Nivethitha, B; KAU; Pradeepkumar, TBitter gourd (Momordica charantia L.) is an important vegetable of tropics and sub-tropics of Asia belonging to Cucurbitaceae family. The immature fruits of bitter gourd are valued for its culinary and medicinal importance. Having highest amount of ascorbic acid and iron content, it is considered to be the most nutritive among all the cucurbitaceous vegetables. Heterosis is well exploited in bitter gourd for early maturity, increased yield and other agronomic traits through development of hybrids. However, the production of hybrids is labour-intensive involving manual bagging and hand pollination, thereby increasing the cost of seed production. The predominant sex form of bitter gourd is monoecious which bears separate male and female flowers on the same plant. However, gynoecious type bearing only female flowers were also reported in few locations of India, Japan and China. These gynoecious bitter gourd lines can be exploited as female parent to make hybrid seed production economical and easier, as it eliminates the need for emasculation and assisted pollination. It also aids in maintaining the genetic purity of hybrids and helps in harnessing the benefit of hybrid vigour including early maturity and high yield. Usually, the sex expression in cucurbits is highly influenced by environment and hormones, which makes the early phenotypic identification of gynoecy challenging. Identification of molecular markers tightly linked to gynoecy trait would ease the identification of gynoecious line in breeding programmes. In this study, 20 putative candidate genes governing sex expression were selected from different literatures and the gene sequences were retrieved from bitter gourd and cucumber genome assembly in NCBI GenBank database. Four ethylene biosynthesis genes (ACO1, ACS2, ACS3. ACS4 and ACS7), three MADS-box transcription factor encoding genes (AG6, MADS-boxTF23 and McAG2), one auxin related gene (CsARF5) and 12 WRKY transcription factor encoding genes comprise the list. A total of 20 gene-specific primer sets were designed from the selected region of each of the 20 genes. Genomic DNA was isolated from monoecious genotype, MC-136 and gynoecious, KAUMCGy-101. Efforts were made to amplify all the 20genes, however, only seven gene-specific primer sets designed from bitter gourd produced PCR amplification. The amplicons of expected product size from both the samples were sequenced. Sequence variation analysis was done by comparing the monoecious and gynoecious sequences generated to the reference sequence (monoecious) available in NCBI database for bitter gourd. Six valid variations including three SNPs and three In/Dels were found in AG6 and McAG2. All the variations except SNP1 of AG6 were present in non-coding regions. In/Del of 48 bp ([TC] 24 ) in AG6 gene caused a significant difference in the number of ‘TC’ repeats in the sequences which was used for the development of SSR marker. The marker showed clear polymorphism between monoecious and gynoecious genotypes used. It was further validated in three other monoecious lines namely, Priya, Priyanka and the wild type M. charantia var. muricata. Expected banding pattern showing polymorphism for gynoecious line was obtained for all the three monoecious lines screened. Thus, the study identified two potential candidate genes, AG6 and McAG2 for sex expression in bitter gourd. The SSR marker developed needs to be validated in large number of population and more number of varieties to confirm its use as a reliable polymorphic marker in marker-assisted selection of gynoecious lines of bitter gourd.ThesisItem Open Access Molecular mechanism of virulence in the bacterium, Photorhabdus luminescens (Thomas and Poinar) against Tetranychus truncatus Ehara (Prostigmata: Tetranychidae)(Department of Plant Biotechnology, College of Agriculture, Vellanikkara, 2021) Ashwini, M N; KAU; Haseena, BhaskarSpider mites (Tetranychidae) are a major group of sucking pests of many agricultural and horticultural crops worldwide. Tetranychus truncatus Ehara is the predominant mite species infesting economically important crops of Kerala. Novel acaricides are being extensively used for managing mite infestation, which has led to development of significant level of resistance in mite populations to these acaricides. This, along with other adverse effects of synthetic acaricides to the environment, calls for alternative strategies in mite pest management. The entomopathogenic bacteria Photorhabdus luminescens (Thomas and Poinar), a natural symbiont of the entomopathogenic nematode, Heterorhabditis spp. has potential virulent properties and the ability to infect a wide range of insect pests and a few mite pests. An array of toxins produced by the bacteria are responsible for this appreciable insecticidal activity. Most of the orally active toxins are encoded by tca and tcd loci of TC genes. These toxins might also be responsible for the acaricidal activity of the bacterium. In this context, the present study was undertaken to identify the virulent strain of P. luminescens against T. truncatus and to elucidate the molecular mechanism of virulence against T. truncatus. Eleven bacterial isolates associated with the entomopathogenic nematodes, Heterorhabditis spp. were isolated in the study. Cultural characterization of the bacterial isolates showed that the bacterial colonies were circular to irregular, entire, opaque, smooth colonies on NBTA medium. All the isolates were found to be Gram negative and rod shaped. For molecular characterization, the 16S rRNA gene of the isolates was amplified, sequenced and subjected to BLASTn for homology search. One species of EPN symbiotic bacteria, P. luminescens and eight nonsymbiotic/ associated bacteria namely, two isolates of Pseudomonas aeruginosa, five isolates of Ochrobactrum and one isolate of Stenotrophomonas maltophila were identified. Phylogenetic tree was constructed based on the gene sequences of 16S rRNA to validate the bacterial identification.Laboratory bioassays were carried out to evaluate the efficacy of the both cell suspension and cell free supernatant (CFS) of five bacterial isolates (1 isolate of P. luminescens, 2 isolates of P. aeruginosa, and 2 isolates of Ochrobactrum sp.) separately against the eggs and adults of T. truncatus. Though the isolates did not show any significant ovicidal action, they recorded significant adulticidal action against the mite. The P. luminescens isolate was superior over other isolates recording 64.67 and 60.67 per cent mortality for CFS and cell suspension, respectively at 96 h of treatment The virulent P. luminescens isolate was subjected to characterization of the toxin complex (TcdA gene). The full length gene (7.5 kb) was split into eight regions and the primers were designed to amplify each regions separately. Among the eight regions, only six got amplified and were sequenced. Toxin gene sequence of the isolated P. luminescens bacteria involved in the virulence against T. truncatus was partially sequenced and the variations in the sequence were analyzed. The study identified a potential isolate of P. luminescens with significant adulticidal action against T. truncatus and the major gene TcdA encoding the toxin complexes which possess appreciable insecticidal activity was sequenced and characterized. The P. luminescens isolate obtained in the study can be evaluated for efficacy against major insect pests in the region in order to identify the potential in pest management.ThesisItem Open Access Editing of rice transcription factor OsMADS26 for drought tolerance through CRISPR/Cas9 system(Department of Plant Biotechnology, College of Horticulture, Vellanikkara, 2021) Anjala, K; KAU; Rehna, AugustineRice (Oryza sativa L.) is the most widely consumed staple food of world’s human population belonging to Asia and Africa. Being a semi-aquatic annual plant, rice is highly prone to losses due to various environmental stresses. Many studies regarding this had revealed the need for developing varieties tolerant to abiotic and biotic stresses. Various methods like Marker Assisted Breeding, mutation breeding, RNAi, Antisense technology, ZFNs and TALENs were in use to develop elite traits for abiotic stress tolerance in crops like rice. But very recently, CRISPR/Cas9 system had come into the limelight as an efficient tool for the genetic manipulations of crops. Studies have identified OsMADS26 transcription factor as a negative regulator of drought tolerance in rice. Hence the current study, ‘Editing of rice transcription factor OsMADS26 for drought tolerance through CRISPR/Cas9 system’ was undertaken during the period from 2019 to 2021 at the Centre for Plant Biotechnology and Molecular Biology, CoA, Vellanikkara, Thrissur with an objective to develop drought tolerance in rice. The rice cultivar Nipponbare was selected for the study due to its competence in genetic transformation and regeneration. For CRISPR/Cas9 mediated targeted editing of OsMADS26 gene, guide RNAs (gRNAs) were designed using online software CRISPR-P v2. Genome sequence information of OsMADS26 gene available from rice genome annotation project was used for the study. Genomic region of OsMADS26 gene, flanking the gRNA target (~ 450 bp) was amplified using gene specific primers and sequence of the target region was confirmed using BLASTn and ClustalW analysis. The CRISPR/Cas9 binary vector pRGEB32 was used to clone the guide RNAs using BsaI restriction sites. Three gRNAs were selected for cloning based on features like on score value (higher the value better the editing efficiency), GC content, (40-60%), no. of off-target sites (Minimum number of off-target sites preferred), presence of secondary structure, location on the genome (towards 5' end of gene in exonic region is preferred) etc. The CRISPR/Cas9 construct for cloning was developed by annealing and ligating the gRNAs to the pRGEB32 vector followed by cloning in E. coli strain DH5α. The putative positive clones were identified by colony PCR and further confirmed by Sanger sequencing. The plasmids isolated from PCR positive colonies were sequenced using universal M13 Reverse primer which is present on pRGEB32 vector. The sequences of the clones were confirmed using multiple sequence alignment tool ClustalW. One colony of gRNA 1 construct (OsMADS26 #G1-1) and two colonies of gRNA 3 (OsMADS26 #G3-3 and OsMADS26 #G3-4) were found positive. The CRISPR/Cas9 constructs of OsMADS26 were then mobilized into Agrobacterium tumefaciens strain EHA105 following the Freeze-thaw method. The positive clones were identified using plasmid PCR using hygromycin gene specific primers. Positive colonies of OsMADS26 #G1-1 and OsMADS26 #G3-3 constructs in EHA105 were then used for rice genetic transformation. The seeds of Oryza sativa sub species japonica cultivar Nipponbare were inoculated into N6 medium supplemented with 3.0 mgL-1 2,4-D for callus induction. After five days, the calli were infected with Agrobacterium cultures harboring desired gRNA constructs for 1.5-2 min. Along with the gRNA constructs, an empty vector was also transformed to rice as vector control and a set of untransformed culture were also maintained. After around two days of co-cultivation, the excess Agrobacterium growth was washed-off thoroughly from the calli using the bacteriostatic agent Augmentin. The calli were then placed on selection medium containing Augmentin and Hygromycin. The hygromycin resistant calli showed proliferation after 14 days of incubation. The proliferating microcalli were then transferred to regeneration medium after 21 days. Proliferation of microcalli was observed in vector control, wild type as well as OsMADS26 #G1-1 and OsMADS26 #G3-3 co-transformed plates. The vector control and untransformed calli showed greening and shoot primordia initiation in regeneration medium. The regenerated shoots will be analyzed for mutation in future. Hence, in the current study, gRNA constructs for targeted editing of OsMADS26 gene was successfully developed and transformed in to rice cultivar Nipponbare. Rice genetic transformation suitable to our lab conditions were also optimized. Rice plants with mutations in the OsMADS26 gene is expected in future which can confer drought tolerance.ThesisItem Open Access Metagenomic analysis of bacterial diversity in the rice rhizosphere of kole lands of Thrissur(Department of Plant Biotechnology, College of Agriculture, Vellanikkara, 2021) Athira Krishnan, L R; KAU; Girija, DKole wetlands of Kerala are a complex ecological system and are known for the higher productivity of rice. The Kole lands remain submerged under flood water for about six months in a year and this seasonal alteration gives it both terrestrial and water related properties which determine the ecosystem structure. Though several studies have been conducted for exploring the diversity of fishes, birds, flora, butterflies, etc., in Kole lands, no systematic studies have been made on rhizosphere microbial diversity. This study was intended to analyze the bacterial diversity in the rice rhizosphere ecosystem of Kole lands of Thrissur. Rhizosphere soil samples were collected from three locations of Kole lands of Thrissur viz. Puzhakkal (Pzk), Mullassery (Mls) and Cherpu (Chr) and analyzed for physico-chemical and biological properties. Culturable microflora was enumerated using serial dilution plate method for bacteria, fungi, actinomycetes, N- fixers, P, K and Zn-solubilizers. Twenty four predominant bacterial isolates were purified and screened for PGP activities including production of IAA and ammonia and phosphate solubilization. The bacterial diversity of the rhizosphere samples was analyzed by metagenomic library construction and sequencing of V3-V4 regions of the 16S rRNA gene, using Next Generation Sequencing (NGS) technology. The sequences thus obtained were analyzed for the Operational Taxonomic Units (OTUs) using MEGAN and MG- RAST server. The analysis of physico-chemical parameters showed a comparatively low pH in all the samples. An extreme low pH can reduce the availability of major and secondary nutrients in the soil. The sample Pzk showed higher content of organic C. Culturable microflora and microbial biomass C analysis also showed a slight increase in the sample Pzk. The soil organic C content and microbial biomass C are reported to be positively correlated. The microbial biomass C is the measure of the weight of the organisms present.The predominant bacterial phyla in the rice rhizosphere of Kole lands of Thrissur included Proteobacteria, Chloroflexi, Acidobacteria, Actinobacteria, Bacteroidetes and Nitrospirae. The bacterial population was found higher in the sample Puzhakkal and comparatively lower in the sample Chr. Phylum Proteobacteria was found to be the most predominant bacterial phylum in Pzk while, Chloroflexi was more predominant in Mls and Chr. The classes Acidobacteria and Ktedonobacteria were found dominant in the samples Mls and Chr and the Pzk sample was dominated by Acidobacteria and Deltaproteobacteria. The phylum level bacterial diversity was found highest in the sample Chr with 21 phyla while the genus level bacterial diversity was highest in the sample Mls. The abundance of genera Desulfobacca, Thermoanaerobaculum, Thioalkalispira, Anaerolinea, Ktedonobacter, Gemmatimonas, Puedolabrys, Sulfuricurvum, Syntrophobacter, Haliangium, Geobacter and Syntrophorhabdus was observed in the Kole land rice rhizosphere samples. Many of these genera are involved in geo-cycling of nutrients like Fe, S and Mn and a few are used in waste water treatment. The species- level bacterial diversity was found to be highest in the sample Mls as indicated by the Chao1 and observed species indices. The predominant archaeal phyla in the rice rhizosphere of Kole lands of Thrissur included Euryarchaeota, Crenarchaeota and Thaumarchaeota. Archaea are still an under- detected and little-studied part of the soil, so their full influence on global biogeochemical cycles remains largely unexplored. This study has thrown light on the diversity of bacterial and archaebacterial communities in the peculiar ecosystem of Kole lands of Thrissur. Many of the biofertilizer organisms like Azospirillum, Paenibacillus, Cellulosimicrobium and biocontrol agents like Bacillus and Pseudomonas could be detected, which could be cultured and used as potential acid tolerant biofertilizers and PGPR. Many of the ‘Unclassified’ genera could be novel bacteria and more research is needed to identify their taxonomic position and functional role in the ecosystem.