Evidence is presented for the increased excretion of amylolytic enzymes into a sugarcane cell culture medium when starch was substituted for sucrose as an energy source. The excretion was further enhanced by the inclusion of 1 /AM gibberellic acid in the nutrient medium. The growth rate of the cells increased after they became adapted to starch relative to cells grown on sucrose, but the rate of amylolytic enzyme excretion remained unaltered. Amylolytic enzymes in the medium included aamylase but the identity of one or more other enzymes related to starch hydrolysis remains in doubt.The proliferation of sugarcane cells on a medium containing starch is as good as or better than on one containing sucrose (16). A similar effect has been shown in cell suspensions of sycamore (19). Starch has served as a satisfactory carbohydrate source for higher plant tissues of several (12-14), but not all species (5, 9). Amylases are excreted or released into the culture medium of Rumex (2), of tobacco crown gall (10), and of five other species, including maize endosperm, that were investigated by Straus and Campbell (20). The latter investigators also found phosphatase, peroxidase, and IAA oxidase activities in the spent nutrient medium, but IAA oxidase was released only in the presence of calcium ions. Gamborg and Eveleigh (4) have reported glucanases in suspension cultures of wheat and barley, and Veliky et al. (22) The standard medium contains 2% sucrose. Stocks were inoculated with 6.0 ml of a cell suspension transferred from a 3-week-old stock 3 to 4 weeks prior to their use in an experiment. The experimental medium referred to as "synth II" in this report included an amino acid mixture (M-2), 6 jug/g 2, 4-D, vitamins, and a modified White's mixture of inorganic salts (15), but instead of the usual 2% sucrose the carbohydrate source was either 1% sucrose or 1% starch. The starch was a preparation from corn, consisting of 85 to 90% amylopectin and the remainder as amylose, and was purchased from the Sigma Chemical Co. Starch concentrations greater than 1 % were undesirable, because the viscosity of starch interfered with aeration of cultures on the rotary shaker. Shaker speed was set at approximately 260 rpm. Cells from 20 ml of medium were used for inoculation, after washing the cells with sterile distilled water.Cell Sampling. For all rate experiments 5.0-ml samples were withdrawn and combined from each of at least two cultures per treatment. The samples of cell suspension were filtered, and the filtrates were dialyzed for 16 hr against 10 mm citrate-phosphate buffer prior to their use in enzyme assays; cells were washed on the filter, lyophilized, and weighed.Substrate Preparation. Preparation of 4-limit and a-, ,Blimit dextrins was based on a method described by Greenwood et al. (6). An aqueous 1% starch (as described under "Cell Propagation") solution was made 10 mm and pH 4.6 with respect to acetate. This solution was divided into three equal volumes of 100 ml each. One part, called amylopectin, was stored und...