Proline accumulation and its implication in cold tolerance of regenerable maize callus

Embryogenic callus of maize (Zea mays L.) inbreds B37wx, H99, H99(3)H95, Mo17, and Pa91 accumulated proline to levels 2.1 to 2.5 times that of control callus when subjected to mannitol-induced water stress, cool temperatures (19 degrees C) and abscisic acid (ABA). A combination of 0.53 molar mannitol plus 0.1 millimolar ABA induced a proline accumulation to about 4.5 times that of control callus, equivalent to approximately 0.18 millimoles proline per gram fresh weight of callus. Proline accumulation was directly related to the level of mannitol in the medium. Levels of ABA greater than 1.0 micromolar were required in the medium to induce proline accumulation comparable to that induced by mannitol. Mannitol and ABA levels that induced maximum accumulation of proline also inhibited callus growth and increased tolerance to cold. Proline (12 millimolar) added to the culture media also increased the tolerance of callus to 4 degrees C. The increased cold tolerance induced by the combination of mannitol and ABA has permitted the storage of the maize inbreds A632, A634Ht, B37wx, C103DTrf, Fr27rhm, H99, Pa91, Va35, and W117Ht at 4 degrees C for 90 days which is more than double the typical survival time of callus. These studies show that proline and conditions which induce proline accumulation increase the cold tolerance of regenerable maize callus.