• Research in Plant Disease
• /
• v.18 no.3
• /
• pp.231-235
• /
• 2012

Indian citrus ring spot virus (ICRSV) is known to cause a serious disease to citrus, especially to Kinnow mandarin, the popular cultivated citrus species in India. In this study, we developed diagnostic systems based on enzyme-linked immunosorbent assay (ELISA). In order to generate antibodies against ICRSV coat protein, we overexpressed the coat protein in Escherichia coli using the pET15b expression vector containing an optimized ICRSV coat protein gene. The recombinant ICRSV coat protein was overexpressed as soluble form at $37^{\circ}C$ upon IPTG induction. The protein was purified to 95% in purity by Ni-NTA column chromatography. The purified protein was immunized to rabbit for the generation of polyclonal antibody (PAb). The PAb showed a specific immunoreaction to recombinant ICRSV coat protein in western blot analysis and ELISA. Diluted rabbit antisera (10,000 fold) could detect less than 10 ng and 5 ng of the target protein in western blot and ELISA analysis, respectively.

• Plant Biotechnology Reports
• /
• v.2 no.2
• /
• pp.137-143
• /
• 2008

Production of Indian citrus ringspot virus (ICRSV)-free plants from an infected plant of kinnow mandarin (Citrus nobilis Lour ${\times}$ C. deliciosa Tenora) is reported. The shoot apices of different sizes (0.2-1.0 mm) excised from the ICRSV-infected plant were micrografted onto decapitated rootstock seedlings of rough lemon (C. jambhiri). Micrograft survival depended on the size of shoot apex and the sucrose concentration of the culture medium. Increase in scion size from 0.2 to 0.7 mm resulted in an increase in micrografting success rate from 30.55 to 51.88%. Further, micrograft survival obtained with 0.2 mm was improved from 30.55 to 38.88% by increasing sucrose concentration in the culture media from 5 to 7.5%. The micrografted plants were tested for ICRSV using ELISA and RT-PCR. All plants raised from 0.2-mm scion were found negative with both ELISA and RT-PCR whereas only 20% of the ELISA negative plants raised from 0.3-mm scion were found negative for ICRSV with RT-PCR. The outcome of this research is the successful establishment, acclimatization and virus testing of micrografted plants.

• Journal of Plant Biotechnology
• /
• v.7 no.4
• /
• pp.259-265
• /
• 2005

Indian citrus ringspot virus (ICRSV)-free plants of Kinnow mandarin (Citrus nobilis Lour x C. deliciosa Tenora) were raised from virus-infected plants using unfertilised ovules as explants. Plants were tested by indirect ELISA and RT-PCR before using their explant. An amplified product of 539 bp was obtained by RT- PCR in ICRSV infected plants. Unfertilized ovules were excised from unopened flower buds of plants tested postive for virus and were cultured on Murashige and Skoog's (MS) basal medium supplemented with various concentrations of kinetin (KN) or malt extract (ME). Maximum induction (31.94%) of embryogenic callus was observed on MS medium supplemented with KN ($9.29\;{\mu}M$). Transfer of embryogenic calli to similar media composition resulted in somatic embryogenesis in all cultures, with an average number of 60.36 globular, 17.39 heart and 7.71 cotyledonary-shaped somatic embryos per culture. All cotyledonary shaped embryos developed into complete plantlets within 60 days on transfer to similar medium. Embryogenic callus induction, somatic embryo formation, maturation, germination and plantlet formation were achieved on MS medium supplemented with KN ($9.29\;{\mu}M$) alone. The plantlets derived from somatic embryos were transferred to sterilized soil, sand and vermiculite (3:1:1) mixture. After acclimatization, the plantlets were transferred to screen house and were indexed for ICRSV employing indirect ELISA and RT-PCR and found free of virus. A distinct feature of this study is the induction of somatic embryogenesis from unfertilised ovules to produce virus-free plants.