Abstract
Mungbean yellow mosaic virus (MYMV) causes massive crop losses in green gram. MYMV is a member of begomovirus with bipartite genome comprising DNA-A and DNA-B components, which is transmitted by whiteflies. Cloning and preparation of infectious clone is very much essential for screening germplasm or transgenic material of pulse crops since viruliferous whiteflies may not be available throughout the year. In the current work, we have amplified rolling circle mediated viral genome of MYMV using Φ29 DNA polymerase. The amplified products was digested and cloned into the plant expression vector pCAMBIA2301.The cloned constructs was then transformed into Agrobacterium LBA4404 through freeze thaw method. Further, three viral transmission techniques including mechanical rubbing, Agroinfiltration and Agroinoculation, were employed for assessing the mosaic symptoms in green gram. The molecular confirmation through polymerase chain reaction (PCR) indicated that the yellow mosaic symptoms were formed due to infectivity of MYMV in the green gram.
Highlights
Mungbean yellow mosaic virus (MYMV) causes massive crop losses in green gram
MYMV is a member of begomovirus with bipartite genome comprising DNA-A and DNA-B components, which is transmitted by whiteflies
We report a simple procedure for construction of agro-infectious genomic clones of MYMV
Summary
The greengram plants exhibiting mosaic symptoms with irregular green and yellow spots were collected from greengram fields in Acharya N. Genomic DNA was extracted from infected leaf samples and control plants by following CTAB method. The genomic DNA from infected and control samples was subjected to RCA using Ö 29 DNA polymerase (Thermo Scientifics, USA) according to the instructions given by manufacturer. The ligated products that contain pCAMBIA2301 vector and MYMV genome (2.6 kb and 2.7 kb) fragments separately was transferred into E. coli (DH5á) by heat shock method[21]. The genomic DNA was extracted from infected leaves and analyzed through the PCR using AC1 gene specific primers. Occurrence of yellow mosaic symptoms (4 to 8 days), the genomic DNA was extracted from infected leaves and analyzed through the PCR using AC1 gene specific primers (Figure 3). The uninoculated seeds of each line were maintained as control
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