Vacuolar accumulation of acridine orange and neutral red in zygotic and somatic embryos of carrot (Daucus carota L.)

Summary The accumulation of neutral red and acridine orange, to indicate differences in vacuolar pH, was studied during embryogenesis of carrot. Neutral red accumulated barely in proembryogenic masses, but was present conspicuously in globular-shaped somatic embryos. From the late globular to the torpedo-shaped stage, it was mainly found in the root side of the somatic embryo. Here, neutral red was predominantly present in large dark-red to purple stained vesicles. In the cotyledons neutral red was found in small orange vesicles. In zygotic embryos of carrot, the dye was uniformly distributed with no specific localization in organelles. During germination, however, neutral red accumulated mainly in regions in the root side and the hypocotyl of the germling. Acridine orange was dispersed erratically in proembryogenic masses with a great variety in intensity. It was quite obviously present in early stages of somatic embryogenesis and restricted to the root side in late globular to torpedo-shaped embryos. Confocal images revealed the vacuolar presence of the fluorescence and the predominant presence in the protoderm. During germination of zygotic embryos the signal changed from uniform to localized, with sharp borders between fluorescent and non-fluorescent regions. Two to three days after the beginning of germination, acridine orange accumulated preferentially in the root tip of the germling. Differences between somatic and zygotic embryos and similarities between somatic embryogenesis and zygotic embryo germination are discussed.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - pH_c cytosolic pH - pH_e extracellular pH - pH_v vacuolar pH