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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 Development of an in vitro regeneration system and validation of genetic stability in phalaenopsis hybrid winter spot with molecular marker(Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara, 2016) Asha Amal, Raj; KAU; Lissamma, JosephPhalaenopsis “Moth Orchids” are among the most beautiful flowers in the world. This genus has economic value for pot plant and cut flower production and is distributed throughout Southeast Asia. Most popular method of propagation for orchid is through in vitro propagation, as it produces large number of clones in relatively short duration. Despite its potential to produce numerous plants from a single leaf segment, it is liable to unpredictable mutations or somaclonal variation during the process of multiplication. Variation can arise due to many reasons such as type of media, plant growth regulators and its concentration, type of explants and number of subculture cycles. The percentage of the variation can range from 0-100% depending on varieties with an average of 10% among Phalaenopsis (Tokuhara and Mii, 1993). So the present investigation on “Development of an in vitro regeneration system and validation of genetic stability in Phalaenopsis hybrid Winter Spot with molecular marker” was taken up at the Center for Plant Biotechology and Molecular Biology, College of Horticulture from 2013-2016. Flowering mother plants of Phalaenopsis hybrid Winter Spot were used as explant source. Among the explants namely inflorescence node, transverse thin cell layer of leaf and root segments used for tissue culture study in this orchid, inflorescence node was the best with respect to culture response. The best surface sterilization treatment for leaf explants identified was treatment 0.1% bavistin + prill 2 drops (30 min) and 0.1 per cent HgCl 2 ( 8 min) which give maximum per cent of culture survival and minimum contamination rate. The best surface sterilization treatment for inflorescence node identified was treatment with 0.1% bavistin + 2 drops prill (30 min) , one minute dip in 70 per cent ethanol and 0.1% HgCl 2 (7 min).From different basal media (full MS and 1⁄2 MS) tried, response was observed only in the medium of Full MS for inflorescence node. Among the different growth regulators tried, MS medium supplemented with BA and TDZ was found to give good shoot regeneration from inflorescence node explants. MS +2mgl -1 TDZ recorded highest percentage (80%) of culture establishment, followed by MS + 4.5 mgl -1 of BA (55%) per cent of sprouting. Among the explants tried, only inflorescence node responded with sprouting. Root segment remained as such without any change, whereas leaf explants remained green up to 2 weeks, thereafter started drying in all the growth regulators combination. For induction of multiple shoot, MS medium supplemented with 4.5 mgl -1 BA resulted in the highest average number of multiple shoot (4.15). Elongation and rooting was observed in MS medium supplemented with BA 4.5mgl-1 +IAA 1mgl -1 with 80 % rooting. Root initials were observed 50 days after inoculation. The potting media, charcoal, brick pieces and sphagnum moss in the ratio of 1:1:1 was found ideal for hardening of Phalaenopsis hybrid winter spot with 100% survival. Genetic stability studies using RAPD marker were carried out with the mother plants along with three regenerants each. Six primers were selected based on DNA amplification pattern. In RAPD assay, M1 mother plant recorded the highest average polymorphism of 19.7% and M3 mother plant recorded the least average polymorphism of 8.18%. Using NTSYS software, the similarity coefficients for first, second and third plant between M1 mother plant, M2 mother plant and M3 mother plant and corresponding regenerants were 0.91, 0.92 and 0.93 respectively. In fourth plant, the similarity coefficient exhibited 100% similarity between mother plant, the first clone C1 and third clone C3. The established micropropagation protocol can be used with suitable modification for large scale production of other Phalaenopsis varieties.ThesisItem Open Access Molecular characterization of katte mosaic virus of cardamom ( Elettaria cardmomum Manton )(Centre for Plant Biotechnology and Molecular Biology, College of Horticulture,Vellanikkara, 2016) Abida, P S; KAU; Manglam AryaCardamom ( Elettaria cardamomum Maton) considered as “Queen of Spices” belongs to the family Zingiberaceae. Due to its aroma, cardamom is one among the most expensive spices in the world. Cardamom is infected by several fungal, bacterial and viral diseases. Katte or Cardamom mosaic disease is the most destructive viral disease affecting cardamom plantations worldwide. The disease is caused by Katte mosaic virus or Cardamom mosaic virus (CdMV) and spreads through infected suckers or through aphid Pentalonia nigronervosa Coq. The loss in yield due to katte disease ranges from 38 to 69 per cent and when infection occurs at early stage, the loss is complete. Management of katte disease totally depends on the use of disease free planting materials. As the infected plant often remains symptomless, identification and diagnosis of the virus becomes difficult at early stages. The present study was undertaken to develop serological and PCR based methods for identification and characterization of CdMV of cardamom. The infected and healthy samples were collected from four locations of Idukki and two locations of Wayanad districts of Kerala. The viral protein was isolated and purified from infected samples and SDS-PAGE analysis of purified protein from infected plants revealed the presence of 37 kDa band of viral coat protein. The purified protein from the infected plants was further used as antigen for the production of polyclonal antibody against CdMV in 6-9 month old rabbit. The rabbit was immunized with 5 mg of purified protein. The blood sample from the immunized rabbit was collected and the antibody was purified. The ODD (Ouchterlony Double Diffusion) assay was performed to standardize the titre of the antibody and results had shown that antigen-antibody complex was formed in 1:10, 1:100 and 1:150 dilutions of primary antibody. The indirect ELISA was carried out with 1:10, 1:100 and 1:150 dilutions of primary antibody and 1:200 dilution of secondary antibody. It was found that virus was easily detected in the crude extract of infected leaves with 1:100 dilution of primary antibody. Indirect ELISA was also performed for investigating the serological relationship of Katte mosaic virus with other viruses. The result of indirect ELISA revealed that the crude sap of infected cardamom leaves cross reacted with the antibody specific for Banana Bract Mosaic Virus (BBrMV) which also belongs to same potyvirus group. For the detection of CdMV through RT-PCR, the total RNA was isolated from the infected and healthy plants using Trizol and converted to cDNA. A total of 11 primers were designed for the amplification of coat protein gene of the virus using Primer 3 software. The primers designed were used for synthesis of second strand of cDNA and presence of virus was detected. Out of 11 primers, 8 primers were able to amplify the coat protein gene of the virus in infected plants. The band size of 250, 650, 750 and 950 base pairs were observed in infected plant but not in healthy plant samples. The viral amplicons of 250, 950, 750, 650 base pairs were generated with primers CP-2, CP-9, CP-10 and CP-11 respectively. These amplicons were further eluted, reamplified and sequenced. The nucleotide sequence annotated using bioinformatics tools BLASTn and BLASTx. The result of BLASTn showed 90 to 100 per cent nucleotide sequence similarity with CdMV whereas; the result of BLASTx revealed that the sequence had 55-100 per cent similarity with the coat protein of CdMV. The phylogenetic analysis was performed using 950 bp products generated with CP-9 primer. The phylogenetic tree was developed using MEGA.7 software by utilizing neibhourhood joining method. The result of phylogenetic analysis revealed that the isolates of Ambalavayal, Meppadi and Myladumpara are closely related and also related to isolates of Irettyar and Paravalam whereas, Pampadumpara isolate showed more variation to the above isolates. The methods developed in the present study are useful in detecting the Katte mosaic virus or Cardamom mosaic virus in the infected leaf samples of cardamom. The method will be useful in virus indexing, quarantine management in germplasm exchange, germplasm management, supply of disease free planting materials to the farmers and also for selecting the resistant line or cultivar for large scale production or incorporation in breeding programme for crop improvement.ThesisItem Open Access Molecular characterization of candidate gene for pungency in Capsicum spp.(Centre for Plant Biotechnology and Molecular Biology, College of Horticulture,Vellanikkara, 2016) Anju Viswanath, KAU; Deepu MathewChilli, also known as “Wonder spice”, has been cultivated since 3000 BC. Out of the 21 identified species of chilli, C. annuum, C. chinense, C. frutescens, C. baccatum and C. pubescens are the domesticated species. It is a major vegetable cum spice crop which can impart pungency, colour and aroma to the human foods. Pungency is one of the most important and peculiar character of all the species belonging to the genus Capsicum. Capsaicinoids are the alkaloid compounds which are responsible for pungency in chilli. Because of the nutraceutical properties possessed by these capsaicinoids, it has much importance in manufacturing several drugs. Though so many studies are conducted to understand the genetic mechanisms behind pungency, the gene action responsible for its production is still an enigma. This experiment was undertaken with the objective to assess the molecular mechanisms behind different levels of pungency in different species of Capsicum. The investigations were carried out in ten chilli genotypes namely, Ujwala, Anugraha, Byadagi Dabbi, Byadagi Kaddi, paprika Kt-Pl-19 and bell peppers Arka Gaurav and Arka Mohini (C. annuum), Vellayani Thejus (C. chinense) and White Khandari, Vellayani Samrudhi (C. frutescence). Among the genotypes Anugraha, Ujwala, Vellayani Thejus, Vellayani Samrudhi and White Kandari are pungent lines and Kt-Pl-19, Byadagi Dabbi, Byadagi Kaddi, Arka Mohini and Arka Gaurav are non-pungent lines. Good quality genomic DNA has been extracted from all the genotypes with an absorbance ratio ranging from 1.79 - 1.85 and concentration more than 1000 ng/μl. The DNA was screened with five pungency specific SCAR (Sequence Characterized Amplified Region) primers. Among the five SCAR primers used, three were specific for Pun1 locus (MAP1F/R, Pun1 1 fwd1/rev, Pun1 3 fwd/rev1) and two were specific for CS (Capsaicinoid synthetase) gene (CSF1/R2, BF7/R9). Pun1 and CS are the loci responsible for the synthesis of putative acyl- transferase and capsaicin synthase enzymes leading to the synthesis of capsaicinoids. The results revealed that MAP1F/R is the most significant primer which gave distinct amplifications in both pungent lines and non-pungent lines. A 15 bp deletion was clearly identified in the non-pungent lines compared to the pungent lines. This resultii revealed that the 15 bp deletion in the non-pungent lines is the reason for the absence of pungeny in them. The other two primers Pun1 1 fwd1/rev, Pun1 3 fwd/rev1 gave amplification only for pungent lines in C. annuum and C. frutescence respectively since Pun1 1 and Pun1 3 are the mutant alleles of Pun1 locus present in the respective species. The capsaicin, which is a capsaicinoid compound contributing about 69 per cent of pungency, is produced with the help of the capsaicin synthase enzyme produced from the CS gene. The primers specific for the CS gene have amplified only in the pungent lines. This result revealed that the nucleotide change in the primer binding region is the reason for the absence of pungency in them. The amplicon sequences of CS gene was subjected to insilico analysis such as BLASTn and Clustal Omega, which identified that the CS gene whose location was not yet confirmed also resides within the Pun1 locus. The insilico analysis has also proven that the 15 bp deletion identified in the non-pungent lines were located at the ORF3 in the Pun1 locus. This deletion in the coding region significantly affects the capsaicinoid formation for the pungency. Irrespective of the species, the deletions occurring in the coding regions of the Pun1 locus and CS gene, are the reasons for the variation of pungency levels in chillies. All the five primers attempted were promising and can be utilized to distinguish the pungent and non-pungent lines even in the seedling stage and hence in marker assisted selection (MAS). Identification of the location of CS gene inside the Pun1 locus is the most striking finding of this study. From this it can be infered that Pun1 locus, which is in the chromosome 2 of chilli is the major deciding locus for the production of capsaicinoids.ThesisItem Open Access Identification and characterization of Suppressor of Overexpression of Constans1 (SOC1) gene in Black Pepper (Piper nigrum L.)(Department of Plant Biotechnology, College of Agriculture, Vellayani, 2016) Manu, K Venu; KAU; Lekha SreekantanThe present study entitled“Identification and characterization of Suppressor of Overexpression of Constans1 gene in Black Pepper (Piper nigrum L.)”was conducted at the Integrated Biotechnology Block, College of Agriculture, Vellayani, during 2015-2016.The study envisagedisolation and sequencing ofSOC1, a flowering integrator gene in black pepper (variety - Karimunda) and functional characterization of the gene by studying the expression patterns. Degenerate primers were designed for the above said gene based on the gene sequences from NCBI database (SOC1 forward and reverse primers) which were used to isolate and identify the gene. Total RNA of black pepper was isolated using modified CTAB method followed by synthesis of cDNA using AMV RT (Avian myeloblastosis virus reverse transcriptase). PCR (Polymerase chain reaction) with degenerate primers was done using cDNA as the template. However no amplifications were observed after the first reactions. Therefore nested PCR reactions were done using the PCR products of the first reaction as the template. Two bands of size 640 bp and 330 bp were produced in the nested reactions. Sequencing of the product yielded four sequences with each of the sequence showing similarity to the SOC1 gene, when done sequence analysis, thus making it the first flowering integrator gene to be identified in black pepper. Microscopy studies were carried out to see the floral characters of black pepper in detail. Microscopy studies were done using FAA fluid as fixative, sectioning the tissues and staining with safranin and fast green were carried out to see the changes occurring in different development stages of spikes from immature spike to complete spike with berries.ThesisItem Open Access Characterization and validation of microsatelite markers for resistance to vascular streak dieback disease in cocoa (Theobroma cacao L.)(Centre for Plant Biotechnolgy and Molecular Biology, College of Horticulture, Vellanikkara, 2016) Waghmare Sandesh, Tulshiram; KAU; Deepu MathewCocoa is the third important plantation crop next to coffee and tea. The global production and consumption of cocoa is 27.00 lakh MT. Among the fungal diseases, Vascular Streak Dieback (VSD) caused by Ceratobasidium theobromae is the main constraint in cocoa growing countries, causing heavy losses in mature trees as well as seedlings. The VSD disease cannot be effectively controlled by chemicals and hence breeding for the development of resistant varieties is the best strategy to tackle the disease. In order to confirm the transfer of a desired gene into the offspring, conventional breeding methods rely on the field screening which will be highly influenced by the environmental factors. Marker assisted selection is an alternate where the tightly linked molecular markers will be employed to confirm the presence of the gene of interest in the selected plants. Five ISSR and one SSR markers linked to VSD resistance were identified at Kerala Agricultural University (Chandrakant, 2014). The present study was undertaken with the objective of validating the identified SSR and ISSR markers and to characterize the ISSR markers to identify the corresponding SSR markers. For validation and characterization, twenty VSD resistant hybrids and four susceptible clones were used. For molecular analyses, good quality genomic DNA was isolated from twenty four genotypes and ISSR markers UBC 811, UBC 815, UBC 826, UBC 857, UBC 866 and SSR marker mTcCIR 42 were screened. ISSR analysis had shown that all the primers are capable to differentiate resistant and susceptible genotypes. The SSR assay has also differentiated the resistant and susceptible genotypes. The distinct markers generated in resistant genotypes using UBC 811, UBC 826 and UBC 857 were eluted, cloned to pGEMT vector and sequenced. The nucleotide sequences were annotated using BLAST, ORF finder and SSR finder. The BLASTn of UBC811A and UBC811D nucleotide sequence have shown that this resistance locus lie in the chromosome V of Theobroma cacao genome. BLASTn of UBC826A, UBC826B and UBC857 has positioned these loci in chromosome III. ORF1 and ORF3 in UBC811D are shown to code for aflatoxin biosynthesis regulatory protein and NAD(P)H dehydrogenase quinine, respectively. ORF1 in UBC826B and ORF5 in UBC857-2 code for potassium transporter 27 (0sHAK-27) and structural polyprotein precursor of VP2, capsid protein VP2, respectively. All these proteins are identified to have definite roles in defence pathways. The frequency and distribution of SSR motifs, dimmers to decamers, in these ISSR markers and the corresponding primers were identified. The reported ISSR and SSR markers were validated and found to be successful in differentiating resistant and susceptible genotypes of cocoa; thereby these markers can be used in marker assisted breeding for VSD resistance.ThesisItem Open Access Validation of identified genes for water stress in rice (Oryza sativa L.) mediated by Pseudomonas fluorescens(Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara, 2016) Saakre Manjesh; Abida, P SRice is the staple food for an expansive part of the world, particularly Asia. It is grown under diverse water regimes ranging from poorly drained waterlogged situations to well drain or water stressed upland conditions. Drought is one of the major abiotic stresses that adversely affect crop plants limiting their growth and yield potential. It disrupts the ionic and osmotic equilibrium of the cell. Susceptibility to water stress in rice is more pronounced at the reproductive stage and causes the greatest reduction in yield when stress coincides with the irreversible reproductive processes. Transcriptome analysis was carried out in Matta Triveni (PTB 45) to understand the influence of PGPR under water stress tolerance. Matta Triveni is a popular short duration (100-105 days) rice variety of Kerala, recommended for rainfed ecosystem especially in lowlands during the first crop and irrigated conditions during summers (third crop). With growing emphasis on expanding area under upland rice in the state, the need for an early, profusely tillering, drought tolerant variety is on the rise. Matta Triveni though high yielding is found to be adversely affected by drought. Hence, the present study was performed to assess the impact of application of Pseudomonas fluorescens in inducing tolerance to drought in variety Matta Triveni. In the present study the already identified differentially expressed genes such as Cytochrome oxidase subunit I (CO I), Protein kinase domain protein (PKDP), bZIPl (basic Leucine Zipper), AP2-EREBP (APETALA2 and ethylene-responsive element binding protein), Hsp20 (Heat shock protein 20) and COC1 (Circadian oscillator component) which are involved in ABA dependent and independent signal transduction pathways under water stress were validated using real-time PCR. Seedlings of rice variety Matta Triveni were given three treatments. Unstressed plants were maintained as absolute control. Water was withheld in second treatment and in the third treatment water was withheld in plants for which Pseudomonas fluorescens Pf1(KAU strain) was applied. Plants were given two applications of Pseudomonas fluorescens(pf) i.e., seed treatment and foliar spray. Water was withheld continuously for 15 days during reproductive stage (panicle initiation stage) after foliar spray. Plants exhibited leaf rolling which was scored as per IRRI, SES and was used for total RNA isolation. Biometric parameters were recorded after harvest. Total RNA was isolated from 65 days old rice plants after drought induction using TRIzol reagent. The first strand cDNA was synthesized by reverse transcriptase PCR from the above RNA samples. The cDNA was normalized using actin, which is a housekeeping gene. The real-time PCR primers were designed for six differentially expressed genes such as COI, PKDP, bZIPl, AP2-EREBP, Hsp20 and COC1 using IDT PrimerQuest software and the designed primers were validated using IDT OligoAnalyzer 3.1 tool. The target genes were analyzed by keeping actin as endogenous control. Comparative Δ Ct method was used to analyze the expression levels of genes. Results revealed that all these genes were upregulated in water stressed + Pf treated plants. COI,PKDP, bZIPl, AP2-EREBP, Hsp20 and COC1 genes were found to have 2.3, 2, 6, 2.8, 4.5 and 4 fold increases in relative expression levels respectively in the same treatment as compared to control. In-silico analysis was performed to identify the Transcription Factor Binding Sites (TFBSs) and combinatorial cis-regulatory elements for COI, PKDP, bZIP, AP2- EREBP, Hsp20 and MYB genes which are expressed in treatments. TFBSs were identified by AthaMap database, a genome-wide map of TFBSs in Arabidopsis thaliana, and STIFDB2 database which is a comprehensive collection of biotic and abiotic stress responsive genes in Arabidopsis and Oryza sativa. The significant TFBSs were analyzed based on the parameters provided by databases and were cross validated. The results revealed that the WRKY, MYB, HSF, bZIP, ARF, AP2/EREBP, bHLH, Trihelix and ABI3/VP1 TF families and their respective regulatory elements were predicted as functionally significant. The combinatorial cis- regulatory elements for three genes viz, CO 7, AP2-EREBP and Hsp20 were also identified. The transcription factors belonging to MYB, WRKY, bHLH and Trihelix families which are mainly responsible for drought stress signals and were predicted as co-associated. The study had shown that gene expression in plants lacking adequate water can be remarkably influenced by microbial colonization. The activation of the ABA dependent signaling pathway induced by colonization of P. fluorescens might be a key element for induced systemic tolerance. The predicted TFBSs and combinatorial cis-regulatory elements would be responsible for the upregulation of genes under water stress.ThesisItem Open Access Molecular cloning and characterisation of coat protein gene of banana bract mosaic virus(Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara, 2016) Darshan Gowda, M R; KAU; Anita Cherian, KBanana (Musa spp.), identified as ‘tropical treasure’ is grown extensively in the tropical and sub tropical regions of the world. Diseases, especially those caused by viruses are major constraints for the profitable cultivation of banana. Among the viral diseases, banana bract mosaic is one of the most important, which leads to an yield reduction ranging from 52 to 75 per cent. This disease is caused by Banana bract mosaic virus (BBrMV) which is a member of genus Potyvirus and family Potyviridae. In case of any viral disease, early diagnosis is very important since symptomless hosts carry the viral inoculum. Development of molecular clones of viral genome has immense application in the field of disease diagnostics and management. Hence, the present study was carried out with the objective to develop molecular clones of coat protein (CP) gene of BBrMV and to characterize it.The infected suckers were collected from Banana Research Station (BRS), Kannara and maintained in the insect proof net house of Department of Plant Pathology. The symptoms developed on different plant parts under natural field conditions were documented which included longitudinal, irregular, reddish streaks of varying sizes on the base of pseudostem, mosaic pattern on bracts, fan like orientation of leaves, spindle shaped lesions on leaves, reduced bunch size and malformed fingers.The serodiagnostic technique namely, Direct Antigen Coating-Enzyme linked immuno sorbent assay (DAC-ELISA) was validated by determining the antibody titre with different dilutions of primary antibody viz., 1:100, 1:200, 1:300, 1:500 and it was found that BBrMV could be best detected at 1:200 dilution along with 1:500 dilution of secondary antibody. Later, the presence of virus particles in the samples were confirmed by DAC-ELISA using the standardized combination of primary and secondary antibody dilutions. Dot Immuno Binding Assay (DIBA) was validated to detect BBrMV and showed positive reaction for infected leaf sample which was detected by the development of purple coloured spots on nitrocellulose membrane.The genome of BBrMV is RNA and hence, molecular detection of virus was standardized by Reverse Transcription- PCR (RT-PCR). Total RNA was isolated by two different protocols using different reagents. Among the two methods, the one with Ambion Purelink RNA Reagent was the most appropriate for RNA isolation from banana since it provided highest quality and quantity of RNA compared to the protocol with TRIzol reagent. The isolated RNA was converted into complementary DNA (cDNA) using First Strand cDNA synthesis kit. RTPCR amplification of coat protein gene was standardized using gene specific reported primer (B1/B2) and designed primer (BCPF1/R1) which yielded amplicons of approximate size, 605 bp and 850 bp respectively. The molecular cloning of CP gene was done in Escherichia coli (DH5- alpha). The presence of gene insert in transformed colonies were confirmed by colony PCR using plasmid specific primer (T7 and SP6) which yielded amplicons of expected band size of 1150 bp. The amplified colony PCR products were sequenced to obtain CP gene sequence of BBrMV. The characterization of cloned CP gene of BBrMV was carried out by in silico analysis. The blast analysis revealed that the CP gene sequence of the virus showed maximum homology of 99 per cent to KER2 isolate from Kasargod, Kerala (Accession no. KF385491). The sequence exhibited significant nucleotide identity (99 to 96 per cent) and amino acid identity (95 to 83 per cent) with other nucleotide and protein sequences of BBrMV available in the database of Genbank. The phylogenetic analysis by the alignment of CP gene sequences of selected 22 isolates also revealed that the present isolate was more similar to KER2 isolate and the Indian isolates did not show any relationship based on geographical origin.The recombinant clones developed in the present study could be applied in serodiagnosis and genetic engineering. This could be also used as disease diagnostic probes for more sensitive molecular diagnostic techniques like Nucleic acid spot hybridization.ThesisItem Open Access Development of small interfering RNA (siRNA) mediated resistance in banana against banana bract mosaic virus(Department of Plant Biotechnology, College of Agriculture, Vellayani, 2016) Lekshmi, R S; KAU; Soni, K BThe present study entitled “Development of small interfering RNA (siRNA) mediated resistance in banana against Banana bract mosaic virus (BBrMV)” was carried out during 2012-2016 in the Department of Plant Biotechnology, College of Agriculture, Vellayani. The study was carried out with an objective to develop siRNA mediated technology for the development of banana plants resistant to Banana Bract Mosaic Virus (BBrMV). The study was conducted in banana cv. Nendran. A protocol for somatic embryogenesis in banana cv. Nendran was standardized by using immature male flowers as explants. Pale white friable callus with rich cytoplasm was initiated in Murashige and Skooge (MS) medium supplemented with BA (0.1 – 0.5 mgL-1) and picloram (0.5 – 2 mgL-1) incubated in dark with a maximum explant response of 30 per cent. For embryogenesis, the developed embryogenic calli were transferred to semisolid MS medium supplemented with BA 2 mgL-1 and IAA 0.5 mgL-1 which resulted in a maximum of 10 per cent embryogenesis. The glassy elongated monocot embryos were germinated in half strength semisolid MS medium (0.3 per cent Gelrite) supplemented with BA 2 mg L-1 and IAA 0.5 mg L-1 and incubated in dark. A maximum germination rate of 80 per cent was obtained in this medium. The germinated embryos were transferred to MS medium with BA 2 mg L-1 and NAA 1 mg L-1 resulted in 100 per cent Plant regeneration. The plantlets were transferred to coirpith compost in pot trays in mist chamber for one month for hardening and then transferred to polybags with soil and cowdung (1:1) mixture. To develop siRNA technology to silence the replicase gene of BBrMV, an intron hairpin RNA (ihpRNA) construct was developed. For this a partial mRNA sequence of replicase gene was isolated from BBrMV banana plants. Gene specific primers designed based on the whole genome sequence information retrieved from the GenBank, NCBI. Total RNA from infected banana leaves was isolated and cDNA was prepared using RT-PCR. The partial gene fragment isolated was sequenced and analysed using the bioinformatics tool BLAST. The sequence was subjected to miRNA target prediction and restriction mapping to select suitable region for the construct and further processing. Based on this information a fragment of 400 bp towards the 5’ end was amplified by designing a set of primers with anchored restriction sites. The primers anchored with BamHI and PacI sites were used for the amplification of sense strand and primers anchored with KpnI and SpeI sites were used for antisense strand amplification. The sense and antisense fragments amplified were cloned to pTZ57R/T cloning vector. In the next step the inserts were released from pTZ57R/T using the corresponding restriction enzymes and were integrated in pSTARLING (primary vector), on either side of the cre intron which facilitated the formation of the hairpin (ihpRNA) construct. Presence of the inserts was confirmed by restriction digestion and electrophoresis. The ihpRNA construct in pSTARLING now contained ubiquitin promoter, ubiquitin intron, sense strand of replicase gene, cre intron, antisense strand of replicase and termination sequence in the order with the NotI restriction sites. This construct was released from pSTARLING and ligated to the digested NotI site in the lacZ gene of the binary vector pART27 containing antibiotic resistance marker nptII and spec. The binary vector was confirmed for the insert by transferring to DH5α and colony selection by blue-white screening. The binary vector with the insert isolated from the white colony, was transferred to Agrobacterium tumefaciens strain LBA 4404 via freeze-thaw method. Transformed colonies were picked up and confirmed the presence of the vector and the ihpRNA insert by PCR. Somatic embryos were transformed with LBA 4404 carrying the ihpRNA construct the ihpRNA construct and the transformed embryos were selected with antibiotic pressure (Kanamycin 100 mg L-1). Transformed embryos were subjected to regeneration. A maximum regeneration of 25 per cent was obtained after transformation. The regenerants were confirmed for the presence of ihpRNA construct using PCR with specific primers for sense-intron-antisense fragment, npt II and cre intron. The study was successful in developing a siRNA construct for resistance against BBrMV and obtaining transformed Nendran banana plantlets.ThesisItem Open Access Photosynthesis and enzyme activities regulating starch biosynthesis in different varieties of sweet potato (Ipomoea batatas Lam.)(Department of Plant Biotechnology, College of Agriculture, Vellayani, 2016) Aswini Krishnan, N K; Ravi, VThe present study entitled “Photosynthesis and Enzyme activities regulating starch biosynthesis in different varieties of Sweet potato (Ipomoeabatatas Lam.)” was conducted at ICAR-CTCRI, Trivandrum during the period 2015-2016. The objective of the work was to the study the relation between photosynthesis, leaf area and enzyme activities involved in regulating starch biosynthesis in different genotypes of sweet potato and to identify the limiting factor(s) in low starch varieties of Sweet potato. The rate of photosynthesis, morphological and biochemical parameters and enzymes activities involved in the starch biosynthesis were recorded at monthly intervals in selected varieties. Morphological parameters such as total number of leaves per plant, leaf area and tuber yield per plant were recorded during 1 to 4 MAP. Rate of photosynthesis was measured using IRGA (infra-red gas analyzer). The leaf area and photosynthetic rate were directly increasing the productivity which was found to be higher in the Sree Arun and Kanhangad variety. Biochemical parameters such as sucrose and starch content were estimated in leaves and tubers of sweet potato varieties. The sucrose content was relatively higher in leaves of Kanhangad variety and in tubers of ST-13 variety. Starch content was higher in the variety Sree Arun which had a direct relation with the photosynthetic rate and tuber yield. Activities of enzymes such as AGPase, starch synthase (SS), sucrose phosphate synthase (SPS), sucrose synthase (SuSy) and invertase were measured in both the leaves and tubers of sweet potato. AGPase and SS were directly involved in the tuberization throughout the development which was observed in Sree Arun variety. SPS was highly active in leaves than tubers. SuSy and invertase are mainly involved in the sucrose metabolism in leaves and highly active during the day time. The increased activity of AGPase and SS in sweet potato tubers will open a new strategy for bioengineering the efficiency of enzymes to improve the starch content in low yielding sweet potato varieties. A detailed molecular study on enzymes involved in starch biosynthesis will be necessary for increasing or improving the starch content in tuber crops.
