Indian citrus ringspot virus (ICRSV) is known to cause serious disease problem in Kinnow (Citrus nobilis Lour · C. deliciosa Tenora) plants. This work reports the elimination of ICRSV by using thermotherapy coupled with shoot tip grafting in vitro. Nodal segments from infected mother plants (indexed by indirect ELISA and RT-PCR) were treated both in water bath and moist hot air at different temperatures viz. 40, 45 and 50°C for 30, 60 and 120 min and cultured on MS medium containing 2-iP (1 mg/l) and malt extract (800 mg/l). Shoot tips were excised from the nodal sprouts and grafted on to rough lemon (C. jambhiri) rootstock under aseptic conditions. Water bath treatment was found to be more effective as compared to moist hot air treatment as maximum number of ICRSV free plants (36.84%) were obtained by grafting the tips (0.7 mm) taken from the nodal segments treated at 50°C in water bath for 2 h. In an alternate treatment regime, 1-year-old infected plants were kept at various temperatures viz.36, 38 and 40°C in a thermotherapy chamber. Maximum of 60% ICRSV free plants were obtained by grafting the tips (0.7 mm) from the plants placed at 40°C followed by the plants placed at 38°C (59.09%) and the least was observed in case of the plants placed at 36°C (40.74%). Only those plants/plantlets were considered virus free, which showed negative reaction both in Indirect ELISA and RT-PCR.
Production of Indian citrus ringspot virus (ICRSV)-free plants from an infected plant of kinnow mandarin (Citrus nobilis Lour 9 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.
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