Nutrient modifications for improved growth of Brassica nigra cell suspension cultures

Cell pellet yield of two Brassica nigra suspension cultures was stimulated by amino acid supplements in the growth medium. This could confound the interpretation of amino acid feeding studies involved in characterizing amino acid metabolism mutants. The nutritional requirements of one of the Brassica nigra suspension cultures growing in modified Murashige & Skoog medium were therefore reviewed. Sucrose at 2% w/v was growth limiting and amino or organic acid supplements stimulated growth rate and yield. Increasing sucrose to 6% and supplementing with 15 mM sodium succinate increased maximum cell pellet volume by 2.7 times and maximum dry weight by 2.8 times, stimulated cell enlargement and produced similar maximum numbers of cells per culture. The further addition of an amino acid supplement of 4 mM alanine, 4 mM glutamine and 1 mM glutamate produced no further improvement. The revised medium was more strongly buffered, supported cell growth for a longer period and permitted a 30-fold reduction in the minimum cell inoculum. Cells grown in the revised medium are 10-fold more resistant to growth inhibition by the tryptophan analogue 5MT. These advantages recommend the revised medium for amino acid feeding, mutant isolation and similar studies.