Characterization of Conditioning Factors that Increase Colony Formation from «Black Mexican Sweet Corn» Protoplasts

Abstract

Summary Colony-formation frequency of protoplasts isolated from suspension cultures of «Black Mexican Sweet (BMS) corn» ( Zea mays L.) is increased about ten-fold when conditioned medium (CM) isolated from BMS suspension cultures is supplied to protoplasts plated at 50,000 protoplasts/ml. The objective of this study was to characterize the factor(s) (CMF) from conditioned medium responsible for the increased colony formation. Colony-formation frequency was a linear function of the amount of CM, indicating that either a single CMF is present in CM or that several CMF are present but each acts independently. Removal of polyvalent cations stabilized CMF at elevated temperatures. CMF was most stable at pH 5, losing activity rapidly at pH = 8 or pH = 2. Based on its partitioning between water and ethyl acetate or butanol, and its solubilities in mixtures of water and isopropanol, CMF is very hydrophilic. Neither anion-exchange (NO 3 - form, in H 2 O) nor cation-exchange (Na + form, in H 2 O) resins at pH 5 retained CMF, suggesting that CMF is uncharged. Ultrafiltration showed that the molecular weight of CMF is slightly less than 1350. Subsequent gel filtration with Biogel P-2 and Biogel P-6 resolved CMF into a major peak with an apparent molecular weight of ca. 1200. Thus, CM appears to contain a single major CMF, which has characteristics similar to that of an oligosaccharide. Although the final structure of CMF is unresolved, CMF is chemically unlike the reported conditioning factor(s) of either Hordeum vulgare anther cultures or of dicot suspension cultures, or the cell-division-inducing phytohormones typically used in the culture of protoplasts.