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2012 - 20172
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Subject: Plant Pathology × Author: Asha, B Nair × Author: KAU × End date: 2017 × Start date: 2012 ×
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    ThesisItemOpen Access
    Identification of graft transmissible resistant factors and development of si RNA mediated resistance in cassava against cassava mosaic geminivirus
    (Department of Plant Pathology, College of Agriculture, Vellayani, 2017) Asha, B Nair; KAU; Umamaheswaran, K
    The present study entitled ‘Identification of graft transmissible resistant factors and development of siRNA mediated resistance in Cassava against Cassava mosaic virus’ was carried out during the period 2012-2017 at the Department of Plant Pathology, College of Agriculture, Vellayani. The study was carried out with the objective of identification of transferability of resistance factor from resistant cassava, Sree Padmanabha to susceptible, Vellayani Hraswa by grafting and to develop siRNA mediated technology for the development of cassava plant resistant to Cassava mosaic geminivirus. Grafting experiments were conducted using resistant Sree Padmanabha as root stock and susceptible Vellayani Hraswa as scion. Symptoms like leaf mosaic, chlorotic spots, reduction in leaflet size and stunting of plants were noticed in susceptible variety. Virus concentration was found to be less in grafted plants. Grafting experiments showed the expression of an extra protein by SDS-PAGE and Coomassie staining in grafted plants which is around 38 kDa. Molecular weight of the new protein revealed the presence of extracellular protein in grafted samples. The extra proteins found in the grafted plants are assumed to be transferred from Sree Padmanabha to Vellayani Hraswa by the process of grafting. The study also involved the development of an intron hairpin RNA vector against replicase gene of SriLankan cassava mosaic virus and introduction of this construct into embryogenic cells via Agrobacterium mediated transformation. A protocol for somatic embryogenesis in cassava variety, Vellayani Hraswa was developed by using immature leaf lobes as explants. The young leaf lobes from tissue culture plantlets produced through meristem culture was used for embryogenic callus formation. Cremish white calli was initiated in Murashige and Skooge (MS) medium supplemented with picloram 12 mg L-1 in dark. For embryogenesis, the calli were transferred to MS medium supplemented with BA 2µM and NAA 1µM which resulted in the production of glassy elongated somatic embryos. The germinated cotyledonary embryos were then regenerated into plantlets by culturing in MS medium supplemented with BA 1mg L-1. Effort was taken to construct an intron hairpin RNA vector and the gene targeted for silencing was the replicase gene of SriLankan cassava mosaic virus (SLCMV). Total DNA was extracted from virus infected plants and the whole replicase gene was isolated using gene specific primers. Sequencing of the whole gene was done. BLAST analysis showed 98% similarity to replicase gene of various isolates of SLCMV. The sequence was then subjected to miRNA target prediction and restriction mapping to select suitable region for the construct. Based on this information, a fragment of 397 bp towards the 5’ end was amplified by designing a set of primers with anchored restriction sites. The primers anchored with Xho and Kpn 1sites were used for the amplification of sense strand and the primers anchored with Xba and Cla 1sites were used for amplification of antisense strand. Selected region was amplified to form sense and anti-sense fragments and cloned to pTZ57R/T cloning vector. Inserts were then released from pTZ57R/T using the corresponding restriction enzymes. The sense and anti-sense fragments were then integrated in the primary vector pHANNIBAL on either side of the pdk intron which facilitated the formation of intron hairpin RNA construct. The intron hairpin RNA construct in pHANNIBAL contained CaMV35S promoter, sense strand, pdk intron, antisense strand and OCS terminator in the order with Not 1 restriction sites. After confirmation of integration by restriction digestion, the Not1 fragment with sense and anti-sense strand were released from pHANNIBAL and ligated to the digested Not1 site in the lacZ gene of binary vector pART27 containing antibiotic resistant marker nptII and spec. the binary vector was confirmed for the presence of insert by transferring to DH5α cells and colony selection by blue white screening. Plasmid DNA isolated from transformed colonies grown on Luria agar medium supplemented with 100 mg L -1 spectinomycin were confirmed for the presence of insert. After confirmation of insert in the binary vector, it was transformed to Agrobacterium tumefaciens strain LBA4404 via freeze thaw method. Transformed colonies were selected on kanamycin selection medium at 100 mg L -1 and confirmed for the presence of binary vector and ihpRNA insert using nptII primers and primer for sense and antisense strands by PCR reaction. Cotyledons excised from the somatic embryos were transformed with LBA4404 having pART 27 by co-cultivation and the transformed embryos were selected with antibiotic pressure (Kanamycin 100 mg L-1). DNA was isolated from the transformed somatic embryos and confirmed for the presence of insert using forward primer of sense fragment and reverse primer of antisense fragments. Transformed embryos were subjected to regeneration.
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    ThesisItemOpen Access
    Serodiagnosis and standardization of techniques for production of virus free planting materials of Cassava variety,Vellayani Hraswa
    (College of Agriculture, Vellayani, 2012) Asha, B Nair; KAU; Umamaheswaran, K
    Cassava (Manihot esculenta Crantz), the staple or subsidiary food for about one fifth of the world’s population, is an important source of dietary calories. Among the diseases and pests of cassava, cassava mosaic disease (CMD) is a serious factor limiting the productivity of cassava and the variety, Vellayani Hraswa is found to be highly susceptible to the disease. CMD is caused by viruses belonging to the genus Begomovirus of the family Geminiviridae. Since the crop is vegetatively propagated, the CMD is carried from one crop cycle to the next, through the use of infected cuttings used as planting material. Studies were conducted for the diagnosis of cassava mosaic geminivirus and production of disease free planting material of cassava variety, Vellayani Hraswa causing the mosaic disease of cassava in Kerala. The characteristic symptoms observed include chlorotic areas on the leaf and bright mosaic pattern of yellow or pale green patches. In severely affected plants, leaf distortion and twisting, reduction in size of leaf lamina and stunting of plant growth were observed. Host range studies were conducted and the virus was found to infect only Datura stramonium belonging to the Solanaceae family. The sap transmission of the virus using 0.1 M sodium phosphate buffer (pH 7.0) was found not successful from cassava to cassava. Chlorotic lesions were produced on the local lesion host, Datura which later turned necrotic. Insect transmission studies revealed that only whiteflies (Bemisia tabaci) were able to transmit the virus whereas the spiralling whiteflies (Aleurodicus dispersus) and mealy bugs (Ferrisia virgata) associated with cassava could not transmit the virus. Graft transmission of the virus was also found successful. The pathophysiological studies revealed that the total carbohydrate and phenol contents in graft inoculated plants were significantly higher compared to the uninoculated cassava plants. The content of chlorophyll a, b and total chlorophyll were lower in inoculated cassava plants. There was a decrease in total protein content in inoculated plants compared to the healthy plants but was found to be increasing at different stages of inoculation. The activity of the defense related enzymes were higher in case of inoculated plants than that of the control plants. Protein profile study of virus infected cassava plants using SDS- PAGE showed an extra novel protein with approximate molecular weight of 40 KDa corresponding to the coat protein of the virus. Isozyme analysis of polyphenol oxidase produced three isoforms in both healthy and inoculated plants with relative mobility (Rm) values of 0.68, 0.75 and 1. Two isoforms with Rm values 0.63 and 0.74 were observed in case of peroxidase isozyme analysis in inoculated plants whereas only one in healthy plants. The activities of the isoforms were more in the inoculated plants. Serological and molecular characterization of the virus was also done. The virus was partially purified from the infected leaves and antiserum with a titre of 1: 512 was produced. Serological characterization of the virus using ELISA showed the close relationship of ACMV and ICMV with the disease. Presence of the virus in the whitefly vector B. tabaci was confirmed through DAC- ELISA. Detection of the virus was also done using Dot Immunobinding Assay (DIBA). A field level diagnostic kit using DIBA was developed. Multiplex PCR using ICMV and SLCMV specific primers produced an amplicon of size 600 bp corresponding to the DNA- A fragment of SLCMV. Sequencing of the PCR product obtained 584 bp long nucleotide sequences. The cluster dendrogram constructed by multiple alignment of sequences showed three major clusters and the isolated viral sequence shared the same cluster with SLCMV isolates collected from Kerala. The meristem from the infected cassava plants were regenerated into complete plantlets within 30 days in a suitable MS media supplemented with benzyladenine (0.5 ml l-1), naphthalene acetic acid (0.1 ml l-1) and gibberellic acid (0.1 ml l-1) of millimolar concentration. Root induction was better in the basal MS media and was tested for the presence of the virus by subjecting it to DAC- ELISA. The absorbance of the plantlet regenerated from healthy and infected meristem were found to be 0.22 and 0.31 respectively which was on par with the healthy field sample (0.28) but much lower than that of the infected field sample (1.23) which was used as the positive control. The virus free plantlets were transferred to pots containing sand, soil and compost in 1:1:1 ratio and were grown in a green house. Meristem culture and virus indexing by use of protein and nucleic acid based detection thus, can be used as a viable strategy for the early detection and elimination of the virus and hence for the production of virus free planting materials.