The effect of conditioned medium on colony formation from ‘black mexican sweet’ corn protoplasts

Abstract

Protoplasts from suspension cultures of ‘Black Mexican Sweet’ corn ( Zea mays L.) exhibited increased colony formation frequency when cultured in the presence of conditioned medium isolated from Black Mexican Sweet (BMS) cultures. The increase in colony formation frequency of protoplasts plated at 5 × 10 4 protoplasts/ml was used as a bioassay for detecting conditioning factors and to investigate the response of protoplasts to conditioned medium. Conditioned medium was stable when stored frozen at −14°C, retained 80% activity when stored for 10 days at 24°C and was inactivated by heating to 100°C for 10 min. The most active isolates of conditioned medium were recovered during the exponential growth phase of the suspension cultures. Colony formation frequencies were greatest when conditioned medium was added to protoplasts within 12 h of plating and was not removed for 9 days thereafter. Addition of conditioned medium to protoplasts increased cell division frequency and did not increase protoplast viability or cell wall formation above the control cultures. Most of the conditioning activity was due to factors that were rejected by a 500 molecular weight (MW) cutoff ultrafiltration membrane. A mixture of threonine, glutamate, cysteine and methionine (0.25 μM each), which were the only amino acids detected in conditioned medium, and proline (1 mM) increased colony formation but to lower levels than 25% conditioned medium. Our results indicate that BMS culture cells produce conditioning factors that increase and sustain cell division from BMS protoplasts and that free amino acids are not the active factors in conditioned medium.