Development of protocols for introducing mature seeds of rice (Oryza sativa L. subsp. indica and subsp. japonica) into aseptic conditions and obtaining callus culture

Background. Rice (Oryza sativa L.) is considered a model monocotyledonous plant, which makes it one of the most studied crops. Despite this, geneticists, breeders, and biotechnologists face difficulties such as early necrosis and poor callus formation in subsp. indica cultivars and lines. While high callus formation ability (40%) was observed in subsp. japonica cultivars, the response to callus induction was 0% in cultivars and lines of subsp. indica.

Materials and methods. The studied material included 40 rice accessions from the VIR collection (11 accessions of subsp. indica, and 29 of subsp. japonica). Chu (N6), Gamborg B5 and MS nutrient media were used for seed germination and callus mass production. For callus formation, mature rice seeds cleaned of the seed coat were selected and then cultured on nutrient media with different compositions. The plant material was maintained on light installations in a 16/8 h daylight mode at a temperature of 23–24°C and 25,000 lux illumination.

Results. A protocol for sterilization of seeds for subsequent introduction into aseptic in vitro conditions was developed. Plants of all 40 rice accessions germinated in aseptic culture. Callus culture was obtained from plants of subsp. indica (41% of callus formation) and subsp. japonica (38%). Protocols for nutrient media were obtained to maintain and proliferate callus mass cells, and to produce regenerated plants.

Conclusion. Protocols for obtaining callus culture from two rice subspecies were developed and modified. The Chu (N6) nutrient medium supplemented with 2 mg/L of 2,4D and 0.5 g/L of casein was selected for subsp. indica accessions. The most suitable medium for inducing callus formation in subsp. japonica accessions was the ½MS medium supplemented with 0.2 g/L of proline, 0.4 mg/L of kinetin, and 0.8 mg/L of 2,4D. Addition of proline (0.5 g/L) and casein hydrolysate (0.3 g/L) to the MS nutrient medium helped to initiate the starting stage of shoot and root regeneration.

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