Development of transgenic cucumber mosaic virus (CMV) resistant gerbera plants expressing CMV coat protein gene

Gerbera (Gerbera jamesonii L.) has its immense importance to the floriculture industry worldwide. The gerbera flower production has been hampered by various viruses, among them cucumber mosaic virus (CMV) has shown considerable damage.As natural resistance to CMV is absent in gerbera, here, we have made an attempt to develop transgenic gerbera plants expressing coat protein (CP) gene of CMV via Agrobacterium mediated transformation of base petiole explants for genetic resistance to CMV infection. Among the 44 putative transgenic gerbera plant acclimatized, 39 were found positive for integration of CP gene by polymerase chain reaction and southern hybridization assay using their specific primer and probe respectively. Northern hybridization assay using CP gene specific probe confirmed the transcription of transgene in all 39 transgenic plants. These plants showed translation of CP during DAS-ELISA when tested with antiserum specific to CP of CMV. These 39 plants when challenged by mechanical inoculations with CMV gerbera isolate showed virus resistance in 53% (21 out of 39) plants, virus tolerance (delayed mild symptom) in 33% (13/39) plants, while rest 12.8% (5/39) plants showed severe disease symptoms. The CP mediated resistance of CMV in transgenic gerbera is being reported for the first time from India.

Coat protein-mediated resistance against an Indian isolate of the Cucumber mosaic virus subgroup IB in Nicotiana benthamiana.

Coat protein (CP) -mediated resistance against an Indian isolate of the Cucumber mosaic virus (CMV) subgroup IB was demonstrated in transgenic lines of Nicotiana benthamiana through Agrobacterium tumefaciens-mediated transformation. Out of the fourteen independently transformed lines developed, two lines were tested for resistance against CMV by challenge inoculations. The transgenic lines exhibiting complete resistance remained symptomless throughout life and showed reduced or no virus accumulation in their systemic leaves after virus challenge. These lines also showed virus resistance against two closely related strains of CMV. This is the first report of CP-mediated transgenic resistance against a CMV subgroup IB member isolated from India.

Association of cucumovirus and potyvirus with betelvine (Piper betle L.) as evidenced by ELISA and RT-PCR.

An attempt was made to detect various viruses of Piper betle grown at Mahoba and Banthara in India. DAC-ELISA and RT-PCR tests were performed in leaf sap samples of betelvine for detection of a cucumovirus (Cucumber mosaic virus) and potyvirus (Bean yellow mosaic virus) using specific antibodies and universal primers of respective viruses. DAC-ELISA could detect only CMV. However, RT-PCR detected both cucumovirus and potyvirus infection in betelvine samples. Association of CMV with betelvine was observed for the first time in the present study.

Role of satellite RNA of an Indian isolate of cucumber mosaic virus in inducing lethal necrosis of tobacco plants.

Lethal necrosis or systemic stem necrosis followed by death of Nicotiana benthamiana, severe leaf deformations of N. tabacum cv. white burley and blister formations on N. tabacum cv. samsun NN symptoms were induced by experimental inoculations of CMV RNA preparations containing satellite RNA (sat-RNA). Inoculations of RNA preparations without sat-RNA did not induce that severe symptoms on these plants, only late mild mosaic was observed. It is suggested that sat-RNA of CMV isolate has a certain role for enhancing severity of symptoms in tobacco plants. Local and systemic lethal necrosis of N. benthamiana is due to sat-RNA present with genome of CMV isolate. It is the first report of lethal necrosis induced in N. benthamiana by CMV satellite.

Detection of plant viruses--biotechnological and molecular advances.

Recent advances in biotechnology and molecular biology have played a significant role in development of rapid, specific and sensitive assays for detection of plant viruses. Production of monospecific polyclonal antibodies, monoclonal antibodies have enabled to group isolates of viruses and distinction of closely related strains. In cDNA hybridization applications, there is an increasing interest to employ non-radioactive probes for detection of nucleic acids. Detection limit of nucleic acid is remarkably comparable to those of radioactive labelled probes. Application of polymerase chain reaction (PCR) has made it possible to amplify the low numbers of viral RNA/DNA molecules and their subsequent detection. Underlying principles, their advantages and disadvantages for application of monospecific polyclonal antibodies, hybridoma technology, molecular hybridization and PCR technology with reference to detection of plant viruses have been discussed in this review.

Some Indian strains of cucumber mosaic virus (CMV) lacking satellite RNA.

Virus strains isolated from Ocimum sanctum and Zinnia elegans were identified as cucumber mosaic virus (CMV) strains on the basis of non-persistant aphid transmission, 28 nm particles, 26 kDa coat protein subunits and serological relationships with CMV and chrysanthemum aspermy virus. The strains showed some biological, serological and satellite RNA based differentiation with other CMV strains isolated earlier from chrysanthemum, petunia and tobacco.

Association of geminivirus infection with yellow green mosaic disease of Cucumis sativus: diagnosis by nucleic acid probes.

Geminivirus association with yellow mosaic disease of C. sativus has been investigated by dot/slot blot hybridization tests using nucleic acid probe derived from DNA-B of Indian Tomato Leaf Curl geminivirus. The disease was transmitted experimentally on C. sativus by whitefly, Bemisia tabaci. Some weeds were also found to harbour geminivirus infection in dot/slot blot hybridization tests.

Association of a cucumber mosaic virus strain with mosaic disease of banana, Musa paradisiaca--an evidence using immuno/nucleic acid probe.

Virus causing severe chlorosis/mosaic disease of banana was identified as a strain of cucumber mosaic virus (CMV). Association of CMV with the disease was established by Western immunoblot using polyclonal antibodies to CMV-T and slot blot hybridization with nucleic acid probe of CMV-P genome.

Molecular cloning of coat protein gene of cucumber mosaic virus, CMV-U strain.

RNA isolation from purified cucumber mosaic virus, CMV-U strain and cDNA synthesis was carried out. The coat protein gene (RNA4) region was amplified selectively by polymerase chain reaction (PCR) with CMV RNA4-specific primers. Double-stranded cDNA was cloned in PRT103 vector at Xho1/Kpn1 site and about 1kb insert obtained. The insert was partly sequenced which showed 50% sequence homology with CMV-Q, C and WL strains.

Elimination of eggplant mottled crinkle virus using virazole in explant cultures of Solanum melongena L.

Virazole (1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamide; 100 and 150 mg/l) incorporated into brinjal explant culture medium induced in complete elimination of eggplant mottled crinkle virus - Indian isolate (EMCV-1) from infected explant cultures of S. melongena L. and production of virus free plant progeny. Acridine orange and ethidium bromide (150 ml/l) were also potent antiviral agents to some extent.