1. KAUTIR (Kerala Agricultural University Theses Information and Retrieval)
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Item Development of infectious clones of cassava mosaic virus and their validation(Department of Plant Biotechnology, College of Agriculture, Vellayani, 2018) Vishnu Narayanan; Makeshkumar, TThe study entitled “Development of infectious clones of cassava mosaic virus and their validation” was conducted at the ICAR- Central Tuber Crop Research Institute (ICAR-CTCRI), Sreekariyam, Thiruvananthapuram during 2017- 2018. The major objectives of the study were cloning and characterisation of SLCMV/ICMV infected leaf samples, construction of infectious clones of SLCMV/ICMV and agroinoculation of Nicotiana benthaminana with the partial dimers constructed in order to check the infectiousness of the viral clones. The whole genome amplification of SLCMV/ICMV DNA samples were done and cloned in pUC19 vectors to obtain pSLCMV A7 (2746 bp), pSLCMV B2 (2738 bp) and pICMV A5 (2739 bp) full length clones. The sequence of pSLCMV A7 showed maximum similarity of 99 % with ‘SLCMV-[TVM1]’ sequence in NCBI blast. While the sequence of pSLCMV B2 showed maximum similarity of 99 % with ‘SLCMV-[Ker20]’ sequence in NCBI blast. The sequence of pICMV A5 showed maximum similarity of 95 % with ‘ICMV-[Mah]’ sequence in NCBI blast. In order to develop infectious clones, partial dimers were constructed for SLCMV DNA-A and SLCMV DNA-B and cloned in binary vector pPZP201. These infectious clones were successfully transformed into wild type A. tumefaciens, Ach5 strain by triparental method. Then agroinoculation of N. benthamiana with the constructed partial dimers was found to be successful. After 14 days post inoculation, plants infected with DNA-A + DNA-B partial dimers showed severe symptoms like leaf curling, stunting. The plants infected with partial dimer of DNA-A alone showed mild symptoms like upward leaf curling which confirmed its monopartite lineage. While those plants agroinoculated with partial dimer of DNA-B alone did not show any symptoms. These efficient infectious clones of cassava mosaic virus and their subsequent inoculation technique would provide a major advancement to the resistance development in cassava.Item Standardization of virus inoculation method for cassava mosaic disease(Department of Plant Biotechnology, College of Agriculture, Vellayani, 2016) Geethu S Nair; Maheshkumar, TThe study entitled “Standardization of virus inoculation method for cassava mosaic disease”was carried out at the Division of Crop Protection, ICAR-Central Tuber Crops Research Institute, Sreekariyam, Thiruvananthapuram during 2015-2016. The objective of the study was to optimize the virus inoculation procedures for cassava mosaic disease using different methods viz., agro-inoculation, biolistic delivery of rolling circle amplification (RCA) product and whitefly transmission.In vitro derived virus free cassava plants (H226) was used for the study.Two infectious clones namely SLCMV (TVM 1) DNA A PD (pkkc24) and SLCMV (TVM 2) DNA B PD (pkkc25), were successfully transformed into three different Agrobacterium strains C58, GV3103 and LBA4404. The virus inoculation method for screening of cassava mosaic disease resistance was standardized and the factors that affect the efficiency of agroinoculation and whitefly transmission were investigated. Consequently, an optimal protocol was obtained by the syringe-infiltration method in the leaves of N. benthamiana. The protocol involved inoculation of 4-5 leaf stage N. benthamianaplants with the Agrobacterium strain C58 inoculum, having an initial OD600 0.5-0.8 and final OD600of 4.0 and incubation at 28°C for 6-7 days showed high cassava mosaic virus transmission efficiency in N. benthamiana. Viral symptoms were observed on the leaves of N. benthamianaplants 6 days after inoculation, which indicated that this protocol could be used to screen germplasm for their resistance to cassava mosaic disease.