Production of phytoplasma-free plants in sugarcane (Saccharum spp.) using temporary immersion bioreactor

Lack of high quality planting materials, specifically phytoplasma-free stem cuttings, is the major constraints for sugarcane production in Thailand. The objective of the research was to establish a masspropagation system in sugarcane. Temporary immersion bioreactor (TIB, SETIS^TM) has been used for 'Khon Kaen 3', a Thai commercial cultivar. Two techniques, oxytetracycline hydrochloride application and shoot tip culture, were used for phytoplasma elimination. The use of 2.5 mg L^-1 oxytetracycline hydrochloride supplemented in the media was the optimum concentration for phytoplasma elimination. Concentration increment of this antibiotic above this dose resulted in lethal effect of microshoots. Phytoplasma investigation has been tested by nested-polymerase chain reaction (nested-PCR). Microshoots obtained from both types of elimination techniques resulted in 63-72% phytoplasma-free plants. Phytoplasma-free microshoots were used as starting materials for masspropagation in TIB system. The highest multiplication rate (29.4-31.2 microshoots per starting microshoot within 4 weeks) was obtained from MS liquid media supplemented with 0.5 µM paclobutrazol, 0.2-0.4 µM benzyladenine (BA) and 3% (w/v) sucrose. The optimum frequency of liquid media for starting microshoots was 6 or 8 times day^-1 (every 4 or 3 h, respectively). Healthy microshoots were able to root on ½MS media supplemented with 4 µM indole-3-butyric acid (IBA) and 6% (w/v) sucrose. The plantlets were planted in vermiculite with high survival percentage. The rapid production of these planting materials could scale up the industrial sugarcane production in Thailand.