Callus and Suspension Cultures of \u3ci\u3eMelilotus alba\u3c/i\u3e Tissues and Cells

These studies were designed to define conditions for the satisfactory establishment and maintenance of sweetclover (Melilotus alba Desr.) callus and cell suspension cultures. Callus was derived from cotyledons and hypocotyls of germinated sweetclover seeds of various genotypes. Under the conditions used, 2,4-dichlorophenoxyacetic acid (2,4-D) was effective (optimal concentration ca I mg/liter) for callus induction; other auxins and cytokinins were ineffective. Best callus growth occurred at a 2,4·D concentration of about 2 mg/liter and a sucrose concentration of 2%. Callus growth was improved by casein hydrolysate (0.9 g/liter). Glutamic acid appeared to be the most important single amino acid in the hydrolysate. Suspension cultures failed to survive in the absence of 2,4-D. Highest cell numbers and final/initial cell-count ratios were observed at a 2,4-D concentration of 0.2 mg/liter. The callus type that yielded highest cell counts in suspension culture was friable, but friable growth type on agar was not always associated with highest cell numbers in suspension cultures. Inoculum size was important in determining the performance of suspension cultures, possibly because of the influence of conditioned medium. Attempts to regenerate intact plants from the cultured callus tissues were not successful