Regeneration in alfalfa tissue culture: stimulation of somatic embryo production by amino acids and N-15 NMR determination of nitrogen utilization

The production of somatic embryos in alfalfa (Medicago sativa L., cv Regen S) is increased 5- to 10-fold by alanine and proline. However, utilization of nitrogen for synthesis of protein from alanine, proline, glutamate, and glycine is not qualitatively different, even though the latter two amino acids do not increase somatic embryo formation. These determinations were made by ¹⁵N labeling with detection by nuclear magnetic resonance. Overall metabolism of the nitrogen of proline, alanine, glutamate, and glycine is also similar in two regenerating and nonregenerating genotypes with similar germplasm, except that the levels of free amino acids are consistently higher in the nonregenerating line. In addition, when regeneration is suppressed in either of the two regenerating lines, the level of intracellular free amino acids increases. This increased level of metabolites is the only direct evidence provided by analysis of nitrogen metabolism of differences between the regenerating and nonregenerating states in alfalfa.