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Callus and Suspension Cultures of Melilotus alba Tissues and Cells¹

These studies were designed to define conditions for the satisfactory establishment and maintenance of sweet. clover (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 rag/liter) callus induction; other auxins and cytokinins were incl. fective. Best callus growth occurred at a 2,4-D concentration of about 2 rag/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.

Key Words: Auxin • Cytokinin • Sweetclover • Tissue culture

² Postdoctoral fellow, Dep. of Plant Science, Univ. of Manitoba, Winnipeg; Foundation Professor of Agronomy, Univ. of Nebraska-Lincoln; and research geneticist, ARS-USDA, and professor, Lincoln, NE 68583, respectively.

Received for publication August 19, 1976.