Browsing by Author "Vimi Louis"

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Biological characterization of bud necrosis virus disease in watermelon(Citrullus lanatus T.)
(Department of Plant Pathology, College of Horticulture, Vellanikkara, 2014) Aswathi, K K; Vimi Louis
Cataloguing and management of major diseases of monopodial orchids
(Department of Plant Pathology, College of Horticulture,Vellanikkara, 2012) Meera, T M; Vimi Louis
Disease is one of the major production constraints in orchid cultivation. Hence an investigation has been undertaken to study the symptomatology, etiology and management of various diseases of important monopododial orchids viz., Phalaenopsis, Vanda (Basket Vanda), Mokara and Arachnis. A survey was conducted in different locations of Thrissur District viz., orchidarium of AICRP on Floriculture Improvement, Department of Pomolgy and Floriculture, COH, Vellanikkara, nurseries at Cheroor, Thrissur, Perinjanam, Madakkathara and Kanimangalam. Different fungal and bacterial diseases were observed and isolation of pathogen yielded eleven fungal pathogens and two bacterial pathogens. The pathogenicity of these organisms was proved by artificial inoculation.Symptomatology of various diseases was studied in detail under natural and artificial conditions. Fusarium sp. caused wilt symptom in Phalaenopsis and caused leaf spot in Arachnis. Sclerotium sp. caused collar rot in Phalaenopsis and dry rotting in Basket Vanda, Anthracnose pathogen, Colletotrichum sp. produced different symptoms in Phalaenopsis, Mokara and Arachnis. In Phalaenopsis, the symptom was irregular sunken spot, in Mokara leaf blighting and in Arachnis, round sunken spot. Heart rot was the symptom of Phytophthora infection in Basket Vanda. Botryodiplodia leaf spot of Basket Vanda was characterized by greyish white coloured spot with black margin and pycnidia at the centre. Alternaria leaf spot of Mokara, was round to oval shaped with a vertical splitting through the centre of the spot. Soft rot of Phalaenopsis by Erwinia sp. and bacterial wilt of Basket Vanda by Burkholderia sp. were the bacterial diseases observed during the survey. Water soaking and rotting of leaves were the symptoms of soft rot while yellowing, wilting and leaf detachment were the symptoms of bacterial wilt.Cultural characters and morphological characters of fungal pathogens were studied and they were identified at species level. Cultural, morphological and gram reaction of bacterial pathogens were studied and they were identified at species level by molecular techniques.For the management of fungal pathogens, an in vitro evaluation was conducted with fungicides and liquid formulation of Pseudomonas fluorescens. Most of the fungicides revealed cent per cent inhibition on the growth where as P. fluorescens showed 53 - 86 per cent inhibition. For the management of bacterial pathogens, an in vitro evaluation was conducted with cowdung, liquid formulation of P. fluorescens, cowdung + P. fluorescens and Streptocycline and differences were observed in their inhibitory properties. Seasonal influence on the incidence of diseases of monopodial orchids was studied for one year. Influence of temperature, humidity and rainfall was prominent in the incidence of Fusarium wilt, collar rot and soft rot of Phalaenopsis whereas not very prominent in the incidence and severity of Botryodiplodia, Alternaria and Fusarium leaf spot diseases of Basket Vanda, Mokara and Arachnis respectively . Incidence of Fusarium wilt was more in the month of November, collar rot in the month of December whereas bacterial soft rot was prominent in rainy months.
Cyclea peltata - a new host of phytophthora palmivora (butler) butler
(Kerala Agricultural University, 1996) Estelitta, S; Sukumara Varma, A; Vilasini, T N; Vimi Louis; Raji, P
Development of recombinant coat protein for immunodiagnosis of banana bunchy top and bract mosaic diseases
(Department of Plant Pathology, College of Agriculture, Vellanikkara, 2021) Darsana Dilip, K C; Vimi Louis
The present investigation was undertaken to develop recombinant coat protein (rCP) of Banana bunchy top virus (BBTV) and Banana bract mosaic virus (BBrMV) for immunodetection of the viruses. The experiments were conducted at the Virology Lab, Banana Research Station, Kannara; Department of Plant Pathology, College of Agriculture, Vellanikkara, Kerala Agricultural University and Indian Institute of Science, Bengaluru during the period of 2016-2020. A roving survey in 10 districts of Kerala, divided into population subsets viz., North, Central and Southern zones were conducted for sample collection. After a preliminary DAC-ELISA, 17 and 12 representative samples respectively were selected and carried forward for further evaluations. The CP gene of BBTV was amplified from the total DNA isolated using reported primers by Polymerase Chain Reaction (PCR) and that of BBrMV by Reverse Transcriptase-PCR (RT-PCR). The CP gene sequences of these isolates were determined and submitted in the NCBI-GenBank Database. The 17 BBTV isolates were designated as MT174314-MT174330 and the 12 BBrMV isolates as MT818176- MT818187. It was inevitable to evaluate the molecular diversity of the viruses prior to devising nucleic- acid based and serological detection methods. The phylogeographic analysis depicted a clear demarcation of BBTV Kerala isolates based on geography whereas no such clustering was observed in the case of BBrMV isolates. Being an RNA virus, the molecular diversity of BBrMV (ranging between 1-12 %) was higher than BBTV. However, the 5’ and 3’ terminal of BBrMV CP gene was hypervariable and found unsuitable to be targeted for nucleic-acid based detection. Hence, forward primer was designed from the NIb region of ssRNA genome of BBrMV and reverse primer from 3’ UTR region upstream and downstream to the CP gene respectively. For nucleic-acid based detection of BBTV, highly conserved non-coding region of DNA-S upstream and downstream to the CP ORF was targeted. The primers were validated by detecting virus from the field samples collected from various parts of the state. The rCPs were chosen as a potential antigen for raising antibodies in order to develop serodiagnostic assays for the early detection of the viruses. The BBTV CP gene was clonedin to three expression vectors viz., pRSET-C, pGEX-4T-2 and pET32a(+) and transformed to expression hosts like BL21 (DE3) pLysS, Rosetta (DE3) pLysS and C41 strains of E. coli after amplification in DH5α. The 20 kDa recombinant BBTV CP (rBBTV CP) cloned in to pRSET-C, and overexpressed in various E. coli hosts had a hexa histidine (6X His) tag at the N terminal. Similarly, a 37 kDa fusion protein (pET/rBBTV CP) was overexpressed from pET/BBTVCP clone had a thioredoxin (Trx) tag (17 kDa) along with the 6X His tag. Whereas, a 45 kDa fusion protein (pGEX/rBBTV CP) with GST tag was overexpressed from pGEX/BBTVCP clone. These affinity tags in the fusion rCP enabled purification from other E. coli proteins. Although pRSET/rBBTV CP was soluble, the 20 kDa protein was highly unstable and partially degraded during purification at 4 °C. Curiously, pGEX/rBBTV CP dissociated from its GST affinity tag and the rCP without the tag degraded. On evaluating the protease cleavage sites in the fusion protein, trypsin cleavage sites were present between the C terminal of GST and N terminal of BBTV CP which might be the reason for cleavage of the ~20 kDa protein from its affinity tag. Thus, it was impossible to purify the protein from the pool of E. coli proteins. Restriction free (RF) cloning of BBTV CP to pGEX-4T-2 was attempted not only to replace these trypsin cleavage sites but also the thrombin cleavage site present in the vector with Tobacco etch virus (TEV) NIa protease site. Thrombin is a specific enzyme used to cleave off the tag from the fusion protein after purification. However, its specificity is not universal. Furthermore, the commercially available enzyme is costly. TEV protease on other hand was produced in the laboratory and was highly specific. However, the cleavage using TEV protease was unsuccessful apparently because of a steric hindrance contributed by the two extremely ordered regions flanking the TEV cleavage site present in the disordered region of the fusion protein. pET/rBBTV CP was highly soluble like ΔpGEX/rBBTVCP. Likewise, BBrMV CP gene was cloned into pRSET-C and pGEX-4T-2 to obtain pRSET/rBBrMV CP and pGEX/rBBrMV CP of size 34 kDa and 60 kDa respectively. The 34 kDa pRSET/rBBrMV CP was insoluble. Overexpression and purification of the protein was standardized in various conditions to increase solubility. On the contrary, pGEX/rBBrMV CP was highly soluble and was purified by GSH Sepharose affinity column chromatography. 360 μg/ml of untagged protein was obtained from 1 l culture. However, like any other potyviral CP, the exposed N and C terminal of BBrMV CP was also prone to proteolytic cleavage. It partially degraded when incubated with thrombin atroom temperature for GST tag cleavage. All these bands were detected by potyviral CP specific antibody in Western blot. Further on storage complete degradation of the protein was observed. Further standardisation of the protocol is necessary to either stabilise monomeric CP or develop BBrMV VLPs in vitro for immunising animal in order to raise the antiserum. The immunogenicity of the antigens (rBBTV CP and rBBrMV CP) was confirmed by Western blot using BBTV CP specific and potyvirus CP specific antibody procured from NRC, Banana and IISc, Bangalore respectively. The rCPs were also characterized by fluorescence spectroscopy, sucrose gradient ultra centrifugation and electron microscopy. The fluorescent spectra of tagged and tag less rBBrMV CP deviated from 330 nm which is typical for a partially disordered protein. However, the spectra of pET/rBBTV CP and ΔpGEX/rBBTV CP were different. The former depicted the spectra of a mostly globular protein. There were two λmax for the fluorescence spectra of ΔpGEX/rBBTV CP. The epitope prediction of BBTV CP with Trx tag gave interesting insights. A single linear epitope of 80 residues were detected in pET/rBBTV CP comprising of C terminal of the affinity tag and the N terminal of BBTV CP. This was expected to increase the immunogenicity of the antigen and administered for production of antiserum. The titre value of polyclonal antiserum produced against the 37 kDa pET/rBBTV CP was evaluated by DAC-ELISA and was found to be 1:128000. Titre value for serological assays of field samples was standardized as 1:10000 to be more inclusive for detecting virus even at early stages of infection. A total of 247 tissue culture samples and 10 field samples were screened for the presence of the virus using the antiserum and was compared with the procured antiserum. Seemingly, the latter non-specifically reacted with plant proteins which gave a higher absorbance value in negative control and correspondingly high absorbance in the infected samples. The polyclonal antiserum raised against rBBTV CP was used to standardize serological detection assays like IC-PCR, DIBA and TAS-ELISA apart from DAC-ELISA. DIBA and TAS-ELISA were the most sensitive assays which could detect up to 1:80 dilution of the antigen. In conclusion, due to the higher nucleotide variability of the CP gene, serological detection is preferred over nucleic acid based assays. However, the quality of antigen used for raising the antibody plays a major role in serodiagnostics. Hence, high quality rCPs of both BBTV and BBrMV were developed in the laboratory in various vector/host systems. ThepET/rBBTV CP overexpressed in C41 strain of E.coli (1.1 mg/ ml obtained from 1 L culture) was used for immunisation of the animal. A highly sensitive antiserum specific to BBTV with a titre ten fold higher than that of the commercially available antiserum was obtained. Using this antiserum raised against rBBTV CP, various serodiagnostic assays were standardised in the laboratory. Among these, TAS-ELISA was the most sensitive, detecting antigen even at higher dilution.
Development of recombinant coat protien for immunodetection of cucumber mosaic virus infecting banana
(Department of Plant Pathology, College of Horticulture, Vellanikkara, 2019) Alan C Antony; Vimi Louis
Banana (Musa spp.) is infected by four well characterised plant viruses viz., Banana bunchy top virus, Cucumber mosaic virus (CMV), Banana bract mosaic virus and Banana streak virus. Among these, CMV causes devastating effect on tissue culture banana plants. The study entitled “Development of recombinant coat protein for immunodetection of Cucumber mosaic virus infecting banana” was carried out using existing facilities of Department of Biochemistry, Indian Institute of Science, Bangalore, Division of Plant Pathology, Banana Research Station, Kannara and Department of Plant Pathology, College of Horticulture, Vellanikkara, Thrissur during 2018- 2019. The present study was carried out to produce recombinant coat protein, which can be utilised later for producing high quality antiserum for the detection of CMV infecting banana. Cucumber mosaic virus infected samples were collected based on various characteristic symptoms and screened by direct antigen coating immunosorbent assay using commercially available CMV polyclonal antiserum. Isolate namely, KANC- 4, KANC- 2 and NDRNS- 4 showed maximum absorbance at 405 nm and hence selected for molecular detection using reverse transcriptase polymerase chain reaction with CMV- CP specific primer. The PCR product was purified and CMV- CP amplicon of NDRNS- 4 isolate was ligated to pGEM- T linear plasmid vector, which was later transformed into E. coli DH5α cells. Positive clones were selected according to blue-white screening. Cloning i.e., E.coli DH5α/pGEM- T/CMV- CP was confirmed through colony PCR using coat protein specific primer, restriction digestion of recombinant plasmids using EcoR1 enzyme followed by sequencing. The vectors viz. pRSET- C and pET28a were selected for the expression of CMV- CP gene in E. coli. Coat protein specific forward (5’GGG GCT AGC ATG GAC AAA TCT GAA TCA ACC3’) and reverse primers(5’CCC GGA TCC TTA CTC TCC ATG GCG TTT AG 3’) were designed along with recognition sites of restriction enzymes BamH1 and Nhe1.The annealing temperature of designed primer was standardised as 55°C using gradient PCR. The coat protein gene of CMV was amplified at 750 bp using designed primers and high fidelity Pfu DNA polymerase enzyme. Expression vectors as well as amplicon were subjected to ligation and the recombination in expression plasmids (pRSET- C/ CMV- CP and pET28a/CMV- CP) were confirmed through PCR and sequencing. The plasmid with maximum homology i.e., pRSET-C/CMV- CP was selected for further studies. The recombinant plasmid was transformed into E. coli BL21(DE3)pLysS cells for the expression of CMV- CP gene and the expression of 25 kDa recombinant CMV coat protein was confirmed in 12 per cent sodium dodecyl sulphate - polyacrylamide gel electrophoresis (SDS- PAGE). Tris- NaCl buffer of pH 8.0 was selected for solubilising the recombinant protein using ExPASy - protein translation tool. The recombinant protein was further purified through Nitrilotriacetic acid column purification, in which the 6X histidine tagged recombinant protein was bound with agarose coated nickel beads. Buffers containing imidazole were used for the elution of histidine tagged recombinant protein, since imidazole competes with histidine for the binding site in nickel beads. Each fraction viz., cell pellet, supernatant, flow through, wash and elution were collected and later detected for protein using SDS- PAGE. Absence of 25 kDa protein in cell pellet indicated that the recombinant coat protein completely soluble in Tris- NaCl buffer (pH 8.0). Confirmation of recombinant coat protein was carried out through DAC- ELISA and western blotting using commercially available polyclonal CMV antiserum (1: 2000; NRCB, Trichy). The recombinant coat protein developed through this study could be utilised for large scale production of antiserum for immunodetection of CMV.
Effect of selected medicinal plant extracts on the incidence of pumkin mosaic
(Department of Plant Pathology, College of Horticulture, Vellanikkara, 1994) Vimi Louis; Balakrishnan, S
Evaluation of expression clones for recombinant coat protein of cucumber mosaic virus infecting banana
(Department of Plant Pathology, College of Agriculture Vellanikkara, 2022) Sowmya, R; Vimi Louis
The present study was carried out to evaluate recombinant expression clones for recombinant coat protein of CMV infecting banana, which can be utilized for producing high quality antiserum for the detection of CMV infecting banana. The study was conducted using existing facilities at Banana Research Station, Kannara and College of Agriculture, Vellanikkara during the academic year 2019-2022. The confirmation of recombinant expression clones with pRSET-C and pET28a expression vectors were carried by restriction digestion using Nhe1 and BamH1 restriction enzymes. The insert having 750 bp CMV-CP gene was released from pET28a plasmid. The confirmed pET28a expression clone along with CMV-CP insert was transformed into E. coli DH5α cells to amplify the clone. The recombinant pET28a/CMV-CP plasmid was cloned into E. coli BL21 pLysS cells for over expression of the protein and presence of recombinant clone was confirmed by colony PCR by using specific CMV-CP primers. The E. coli BL21 pLysS cells harbouring pET28a/CMV-CP was grown in LB broth until reached the log phase and induced with 0.3 mM, 0.5 mM and 1 mM IPTG. Induced culture along with uninduced culture were incubated at varying temperature and time. The incubated bacterial culture was lysed by using Tractor buffer (Protein purification kit)/Sonicator. The cell lysate of induced and uninduced culture were loaded on to 12 per cent SDS-PAGE for separation and to understand the protein profile. In the gel, even though slight expression of CMV coat protein was observed, no band was observed in Western blotting which was the confirmation test. As production of recombinant coat protein was not successful in pET28a expression clone, experiments were conducted to develop new expression clone using expression vector pET32a. Infected leaf samples of banana showing typical symptoms, along with healthy control were tested for CMV infection by DAC-ELISA using specific antiserum purchased from the National Research Centre for Banana, Trichy, Tamil Nadu. Isolate namely, NDRNS-4, KANM1 and KAAS-2 showed maximum absorbance at 405 nm and hence selected for molecular detection using reverse transcriptase polymerase chain reaction with CMV- CP specific primer. The PCR product was purified and CMV- CP amplicon of NDRNS-4 isolate was ligated to pGEM- T linear plasmid vector, which was later transformed into E. coli DH5α cells. Positive clones were selected according to blue-white screening. Cloning i.e., E.coli DH5α/pGEMT/CMV- CP was confirmed through colony PCR using T7 and Sp6 primers of that plasmid, followed by sequencing. Coat protein specific forward (5’GGG GAA TTC ATG GAC AAA TCT GAA TCA ACC 3’) and reverse primers(5’CCC CTC GAG AAC TGG GAG CAC CCC AGA TG 3’) were designed along with recognition sites of restriction enzymes EcoR1 and Xho1.The annealing temperature of designed primer was standardized as 55 °C using gradient PCR. The coat protein gene of CMV was amplified at 750 bp using designed primers and phusion DNA polymerase enzyme. Expression vector as well as amplicon were subjected to ligation and the recombination in expression plasmid (pET32a/CMV- CP) was confirmed through colony PCR and followed by sequencing. The recombinant pET32a/CMV-CP plasmid was transformed into E. coli BL21 pLysS cells for overexpression of the protein and presence of recombinant clone was confirmed by colony PCR by using specific CMV-CP primers. The E. coli BL21 pLysS cells harbouring pET32a/CMV-CP was grown in LB broth until reached the log phase and induced with 0.3 mM IPTG. Induced culture along with uninduced culture were incubated at 37 °C for 3 h, 4h, 5h and 20 °C 16 h. The incubated bacterial culture was lysed by using Tractor buffer (Protein purification kit)/Sonicator. The cell lysate of induced and uninduced culture was loaded on to 12 per cent SDS-PAGE for evaluating the over expression of coat protein. Expression was observed on gel at 25 kDa corresponding to the CMV coat protein.
In vitro screening of some medicinal plant extracts against Phytophthora capsici, incitant of foot rot of black pepper (Piper Nigrum L.)
(Kerala Agricultural University, 1996) Vimi Louis; Balakrishnan, S; James Mathew
Management of bitter gourd mosaic by enhancing host resistance
(Department of Plant Pathology, College of Horticulture, Vellanikkara, 2015) Ashwini, K N; Vimi Louis
Management of pumpkin mosaic using selected medicinal plant extracts
(Department of Plant Pathology, College of Horticulture,Vellanikkara, 2003) Vimi Louis; Sukumara Varma, A
"Management of pumpkin mosaic using selected medicinal plant extracts" was undertaken in the College of Horticulture, Kerala Agricultural University, Vellanikkara, Thrissur during 1998-2002. Selection of suitable medicinal plant having antiviral property to pumpkin mosaic virus (PMV), isolation of the inhibitory principle present in the medicinal plant, management of pumpkin mosaic using the plant extract and partial purification and serological studies of PMV were the objectives of the study. Symptomatology, transmission, host range and electron microscopy of PM V were also studied. The symptomatology of pumpkin mosaic was studied by observing the development of symptoms in naturally infected as well as artificially inoculated pumpkin plants. The symptoms appeared as typical mosaic mottling with light and dark green patches in the leaf lamina. This was followed by blistering and malformation of leaves into filiform or some other shapes and resulted in reduction of leaf area. The infected plants were stunted, flowered very sparingly with less number of female flowers and reduced fruit setting. The fruits were often malformed . The virus could be transmitted mainly through sap and vector, Aphis gossypii. The virus found to be weakly transmitted also through seeds. The inoculation of PMV on host plants of four families viz., cucurbitaceae, solanaceae, fabaceae and caricaceae showed systemic infection in water melon, snake gourd, bitter gourd, winter squash,' wild ash gourd (cucurbitaceae) chilli, datura (solanaceae), soybean, cow pea (fabaceae) and papaya (caricaceae). Electron microscopic studies revealed the presence of flexuous virus particles (700-800 x 11 nm) in infected leaf sample. Antiserum was raised against the virus and used for serodiagnostic work. The antiserum showed serological relationship with poty viruses infecting snake gourd, bitter gourd, wild ash gourd, cowpea, soybean, chilli and papaya. DAC- ELISA procedure was standardized and used for detection of PM V from pumpkin. The inhibitory property of extracts of five medicinal plants namely Basella alba, Glycyrrhiza glabra, Phyllanthus fraternus, Plumbago rosea 'and Thespesia populnea were studied-against PMV by pre-inoculation application on pumpkin seedlings. The medicinal plant extracts were prepared using different extraction media viz., chloroform, distilled water, ethyl acetate and petroleum ether at different dilutions. The inhibitory property varied with extraction media and dilution used. The PMV inhibitory property of different parts of Plumbago viz., tender leaf, mature leaf, tender stem, mature stem and root were studied at different temperatures and found that all parts showed inhibitory property which varied with temperature. The root extract which showed the maximum inhibitory property at 30°C (near to room temperature) was used for further studies. The effect of Plumbago on vector transmission was studied by applying the extract before acquisition feeding and inoculation feeding of Aphis gossypii, the vector of PMV. Application before inoculation feeding was found to be effective than acquisition feeding and the inhibitory effect decreased with time after application. Distilled water extract of Plumbago was separated through silica gel column to isolate the inhibitory fraction and found that individual fractions were not effective as plant extract as such against PMV. The inhibitory effect of Plumbago water extract one per cent, on artificially inoculated and healthy pumpkin seedlings was tested by weekly, fortnightly, monthly, bimonthly and single application. Weekly spray was effective to reduce disease severity of artificially inoculated and naturally infected pumpkin seedlings. Delayed incidence of the mosaic and enhanced yield of infected plants was also resulted due to weekly spray of the extract. Enzyme, protein, chlorophyll and phenolics estimation revealed that Plumbago extract spray favoured the resistance and thereby suppression of symptoms. The DAC-ELISA of field samples showed the lower concentration of the virus in Plumbago treated plants.
Molecular characterization of Envinia species causing rhizome rot in banana
(Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikara, 2017) Geethu Gokul,G; Vimi Louis
RNA mediated resistance against banana bunchy top virus (BBTV) in banana
(Department of Plant Pathology, College of Agriculture, Vellanikkara, 2024-02-17) Aparna, K Gokul; Vimi Louis
The study entitled “RNA mediated resistance against Banana bunchy top virus (BBTV) in banana” was conducted at the Department of Plant Pathology, College of Agriculture, Vellanikkara and National Research Centre for Banana, Trichy during the period from 2018 to 2023. The study aimed at developing banana lines that have resistance against Banana bunchy top virus (BBTV) through RNAi, using the ligation independent cloning (LIC) method. The RNAi approach targeted the replicase gene of BBTV. The process began with the amplification of replicase gene fragment that contains the dicer substrate siRNA region. Gene specific primers with adaptor sequences were used to amplify the DNA fragment, creating the sense and antisense fragments of the RNAi construct. The LIC vector which specifically contained four adaptor sequences (LIC1 to LIC4) was linearized with SmaI followed by treatment with T4 DNA polymerase and dTTP. Simultaneously, the amplified gene fragments were treated with T4 DNA polymerase in the presence of dATP, facilitating the development of 5' extending single stranded tails. Subsequently, the T4 DNA polymerase treated vector and gene fragments with sticky ends were mixed and incubated. The splicing of the cohesive ends on the insert fragments and the vector resulted in the circularization of the vector. The prepared vector, housing the desired sense and antisense fragments was transformed into E. coli DH5α. All the transformed colonies, obtained on antibiotic selection medium underwent initial screening via colony PCR, demonstrating cent per cent transformation. Further confirmation involved plasmid isolation, verification of intron, restriction digestion and sequencing. The next phase was transforming Agrobacterium EHA105 strain with the prepared vector by modified freeze-thaw method. The success of transformation was confirmed by verifying the presence of sense and antisense fragments through colony PCR. For plant transformation, embryogenic calli of banana cv. Nendran were developed from the immature male inflorescence of 0.5 cm in size, by culturing on MS medium supplemented with different combinations of hormones viz., 2,4 dichlorophenoxyacetic acid (2,4-D), indole-3-acetic acid (IAA), naphthalene acetic acid (NAA) and picloram. The embryogenic calli development was observed after five months, with the highest per cent in MS medium with 2,4-D (2 mg l-1) and IAA (1 mg l-1). In order to minimize the time delay, the embryogenic cell suspension (ECS) obtained from NRCB, Trichy was used for plant transformation. The co-cultivation of Agrobacterium carrying the desired construct and ECS was done in the presence of acetosyringone for three days, in the dark at 24 to 25 ⁰C, followed by washing of excess Agrobacterium with cefotaxime. The co-cultivated embryogenic cells were transferred to a selection medium containing kanamycin (100 mg l-1). The transformed selected embryogenic cells were then transferred to the embryogenesis medium for 35 to 40 days, followed by culturing on embryo germination medium, under light. The germinated embryos were cultured in Petri plates for one and half months, followed by regular subculturing and maintenance in tissue culture bottles, till they attained the three to four leaf stage. The plants were rooted in a rooting medium for 30 to 40 days, and then acclimatized in a containment facility. The PCR assay with the primers for amplification of sense and antisense fragments confirmed the presence of RNAi construct in the acclimatized plants. These plants were then tested for BBTV resistance by aphid (Pentalonia nigronervosa) challenge inoculation. The transformed plants remained healthy, while the typical bunchy top symptoms appeared within 25 days on the non-transgenic control plants. However, it was observed that two out of the forty eight transgenic lines gradually developed yellowing symptoms, indicating varying levels of resistance among the transformation events. Therefore, further evaluation of these transgenic lines is crucial to better understand the resistance levels. The study has successfully demonstrated the efficacy of transgenic lines in conferring BBTV resistance through RNAi approach. By utilizing the ligation independent cloning, the hairpin construct preparation was done in more efficient manner. The transgenic lines developed in the study represent a promising tool, offering the scientific community a proactive defense against unmanageable BBTV epidemics in the absence of resistant lines.
Studies on transmission, host range and management of ash gourd mosaic disease.
(Department of Plant Pathology, College of Horticulture, Vellanikkara, 2011) Divya, M; Vimi Louis