Flooding tolerance of rice

The possibility was examined that the flooding tolerance of (Oryza rice sativa L.) might be improved through use of somaclonal variants regenerated from callus cultures. The response was also studied of cell cultures to gaseous environments which mimic submergence conditions. Scutellum callus was induced from a range of rice cultivars by placing seed for 2 weeks on MS medium supplemented with 2,4-D (10 µM), casein hydrolysate (3 gl-1) and sucrose (20 gl-1). The scutellum was then excised and further callus growth took place for 4 weeks on the same medium but with a lower 2,4-D (5 µM) concentration. Embryogenic callus was isolated under a dissection microscope and transferred to a regeneration medium. Callus production, growth, and regeneration ability was strongly affected by genotype. Regeneration was obtained on media with or without kinetin, but high levels of kinetin were toxic. The immediate ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) was successfully used at 5 or 10 µM, as a plant growth regulator to stimulate regeneration of plantlets. A technique which allowed callus to be exposed to gradually increasing levels of plant growth regulators was devised by inserting a silicone tubing well into the centre of the agar medium in a 90 mm plastic petri dish and adding filter sterilised plant growth regulators to the well. A total of 503 regenerated plants (R1 generation) were potted out and their seed collected. Seedlings from each self-pollinated R1 plant (referred to as an R2 line) were germinated then placed on polystyrene "rafts" for testing for submergence tolerance in aquaria in a glasshouse. Conditions simulating flooding were achieved by use of appropriate nutrient solutions at 30°C, covering the tank with shade cloth to cut down the light intensity to 50 µmolm-2s-1 and submerging plantlets natural to 50 cm depth. The period of submergence for each line was that which was expected to kill 90% of the parent plants. Seedlings were then desubmerged and the rafts refloated on the nutrient medium to allow for plant recovery for 12 days. The R2 lines were assessed for submergence tolerance by the number of seedlings that survived, and chlorophyll contents of the third and/or fourth leaf. To determine if any lines had enhanced tolerance to flooding these data were compared with data for the parental lines which were also submerged at same time. A total of 231 lines of R2 and R3 generations seedlings have been screened and it was found that 74 lines (32.0%) were less tolerant of submergence than parents, 29 lines (12.6%) showed slightly better tolerance, and 7 lines (3.0 %) showed much better tolerance than their parents. The promising lines were sent to Thailand and IRRI for further research, and several of them proved their superiority in submergence tolerance in field testing. Somaclonal variation in other morphological and physiological characterustcus was observed in the R1 plants but could not be followed through to the R2 generation as most of the plants were deliberately killed when submerged. One somaclone that may have commercial value is a white-seeded line of the submergence tolerant line FR 13A, which normally has a red seed coat. The gaseous phase above cultured rice callus was studied by analysing carbon dioxide, ethanol, ethylene, and oxygen. An apparatus was developed whereby growing callus could be exposed to flowing sterile gas mixtures. When callus was exposed to a gas with composition similar to that found in submerged plants (2% (v/v) oxygen, 8% (v/v) carbon dioxide, and 10 ppm ethylene), callus from rice cultivars with low submergence tolerance grew more slowly, utilised more oxygen, produced more ethylene, and showed higher necrosis than callus lines from submergence tolerant rice. Ethylene released from the callus during culture resulted in early necrosis of the callus in some submergence sensitive lines. This correlates with the difficulty of keeping callus in good condition during the sequence of subcultures necessary to induce regeneration