Proline: A biochemical indicator for the embryogenic potential of 'Swainsonia formosa' callus

An experiment was conducted to investigate the association between endogenous proline and the embryogenic potential of callus mass proliferated from the anther wall tissue of Swainsonia formosa. Callus proliferation was initiated from anthers containing microspores at early- to late-uninucleate stages, pre-treated with water (control) or mannitol starvation at 4 °C for 2 days, and cultured on double-phase B5 medium supplemented with vitamins, 2% sucrose and 49.3 p.M IBA and 4.61 1.1M zeatin. Fico11-400® was added at a 10% (w/v) concentration to enhance embryogenic callus formation. After 4 weeks in total darkness, cultures were placed under cool fluorescent light with an intensity of 50 1.1mol m-2 s-1 for a 16 hour photoperiod at 25 ± 1°C. Somatic embryos were successfully regenerated from embryogenic callus. Nonembryogenic callus continued to grow, and resulted in further callus proliferation when transferred to a regeneration medium supplemented with 1% sucrose and 4.63 tM kinetin. Microscopic observation combined with amino acid analysis of callus tissue using ethanol extraction and sulphosalicylic acid provided strong evidence that embryogenic potential was correlated with endogenous proline content; embryogenic callus contained proline whereas proline was absent in non-embryogenic callus. This suggests that proline quantification may provide an effective biochemical method to assess or to improve the embryogenic potential of callus mass.