Cereal Callus Cultures: Control of Headspace Gases Can Optimise the Conditions for Callus Proliferation

Abstract

The protective conditions under which callus cultures are grown to prevent microbial contamination and to reduce tissue desiccation cause the accumulation of volatiles in the vessel headspace and reduce the availability of oxygen for respiration. To demonstrate the importance of the gaseous atmosphere to culture growth a study was undertaken on non-morphogenic rice and wheat callus incubated under a number of environmental conditions. Changes in the gaseous atmosphere above rice (Oryza sativa L.) callus during routine culture in a petri dish suppressed growth and promoted necrosis. Incubating callus under a continuous flow of gas mixtures of known composition suggested that the inhibition of growth was caused by the accumulation of high levels of ethylene and to the rapid depletion of oxygen. In order to evaluate the importance of ethylene accumulation aminoethoxyvinyl glycine (AVG), I-aminocyclopropane-I-carboxylic acid (ACC) and silver nitrate (AgNO3) were added to the nutrient medium and ethylene was measured during callus culture. Ethylene restricted callus growth particularly under high (35°C) compared with moderate (25°C) incubation temperatures and under illuminated compared with dark incubation. Under illuminated incubation at 25°C, AVG ( 5 μM ) and AgNO3 (50 μM) improved rice callus growth by 69 and 54% respectively while ACC (100 μM) decreased growth by 15%. Furthermore, rice callus growth was better in large compared with small culture vessels since ethylene accumulation was reduced. In contrast, wheat (Triticum aestivum L.) callus grew well in the petri dish system and released very little ethylene into the culture vessel headspace. Growth was better under illuminated than darkened conditions and under moderate (25°C) compared with high (35°C) incubation temperatures. Furthermore, wheat callus growth was only marginally better in large compared with small culture vessels. Ethylene was not a restrictive factor of wheat callus growth since only low levels were detected in all conditions of incubation. Better control of ethylene and increased oxygen availability could be a way of increasing cell and tissue production for genetic engineering studies of otherwise recalcitrant species such as rice, and may be a way of improving manipulation of wheat.