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.
Similar content being viewed by others
Abbreviations
- 2,4-D:
-
2,4-dichlorophenoxyacetic acid
- pHc :
-
cytosolic pH
- pHe :
-
extracellular pH
- pHv :
-
vacuolar pH
References
Allison AC, Young MR (1964) Uptake of dyes and drags by living cells in culture. Life Sci 3: 1407–1414
Brawley SH, Wetherell DF, Robinson KR (1984) Electrical polarity in embryos of wild carrot precedes cotyledon differentiation. Proc Natl Acad Sci USA 81: 6064–6067
Buvat R (1979) Vacuole formation in the actively growing root meristem of barley (Hordeum sativum). Amer J Bot 66: 1219–1237
— (1989) Ontogeny, cell differentiation, and structure of vascular plants. Springer, Berlin Heidelberg New York Tokyo
Choi JH, Sung ZR (1989) Induction, commitment, and progression of plant embryogenesis. In: Kung S-D, Arntzen CJ (eds) Plant biotechnology. Butterworth, Boston, pp 141–160
Cole L, Coleman J, Evans D, Hawes C (1990) Internalisation of fluorescein isothiocyanate and fluorescein isothiocyanate dextran by suspension-cultured carrot cells. J Cell Sci 96: 721–730
De Vries SC, Booij H, Janssens R, Vogels R, Saris L, Lo Schiavo F, Terzi M, van Kammen A (1988) Carrot somatic embryogenesis depends on the phytohormone-controlled presence of correctly glycosylated extracellular proteins. Genes Dev 2: 462–476
Dorhout R, Kollöffel C (1992) Determining apoplastic pH differences in pea roots by use of the fluorescent dye fluorescein. J Exp Bot 43: 479–486
Felle H (1988) Cytoplasmic free calcium inRiccia fluitans L. andZea mays L.: interactions of Ca2+ and pH? Planta 176: 248–255
Gamborg OL, Miller RA, Ojiama K (1968) Plant cell cultures. Exp Cell Res 50: 151–158
Gluck S, Cannon C, Al-Awqati Q (1982) Exocytosis regulates urinary acidification in turtle bladder by rapid insertion of H+ pumps into the luminal membrane. Proc Natl Acad Sci USA 79: 4327–4331
Halperin W (1966) Alternative morphogenetic events in cell suspensions. Amer J Bot 53: 443–453
Köenig H (1963) Vital staining of lysosomes by acridine orange. J Cell Biol 19: 87A
Kurkdjian A, Guern J (1989) Intracellular pH: measurement and importance in cell activity. Annu Rev Plant Physiol Plant Mol Biol 40: 271–303
Matile P (1978) Biochemistry and function of vacuoles. Annu Rev Plant Physiol 29: 193–213
Nishimura M (1982) pH in vacuoles isolated from castor bean endosperm. Plant Physiol 70: 742–746
Norstog K (1972) Early development of the barley embryo: fine structure. Amer J Bot 59: 123–132
Oparka KJ (1991) Uptake and compartmentation of fluorescent probes by plant cells. J Exp Bot 42: 565–579
Padh H, Lavasa M, Steck TL (1989) Prelysosomal acidic vacuoles inDictyostelium discoideum. J Cell Biol 108: 865–874
Palmgren MG (1991) Acridine orange as a probe for measuring pH gradients across membranes: mechanism and limitations. Anal Biochem 192: 316–321
Pope AJ, Leigh RA (1988) Dissipation of pH gradients in tonoplast vesicles and liposomes by mixtures of acridine orange and anions. Plant Physiol 86: 1315–1322
Raven JA (1990) Sensing pH? Plant Cell Environ 13: 721–729
Reinert J (1958) Morphogenese und ihre Kontrolle an Gewebekulturen aus Karotten. Naturwissenshaften 45: 344–345
Robbins E, Marcus PI, Gonatas NK (1964) Dynamics of acridine orange-cell interaction. II. Dye-induced ultrastructural changes in multivesicular bodies (acridine orange particles). J Cell Biol 21: 49–62
Schiavone RM, Cooke TJ (1985) A geometric analysis of somatic embryo formation in carrot cell cultures. Can J Bot 63: 1573–1578
Smith DL, Krikorian AD (1990a) Somatic proembryo production from excised, wounded zygotic carrot embryos on hormone-free medium: evaluation of the effects of pH, ethylene and activated charcoal. Plant Cell Rep 9: 34–37
— — (1990b) Low external pH replaces 2,4-D in maintaining and multiplying 2,4-D initiated embryogenic cells of carrot. Physiol Plant 80: 329–336
Steward FC, Mapes MO, Mears K (1958) Growth and organized development of cultured cells. II. Organization in cultures grown from freely suspended cells. Amer J Bot 45: 705–708
Van Lammeren AAM (1986) Developmental morphology and cytology of the young maize embryo (Zea mays) L. Acta Bot Neerl 35: 169–188
Xu N, Bewley JD (1992) Contrasting pattern of somatic and zygotic embryo development in alfalfa (Medicago sativa L.) as revealed by scanning electron microscopy. Plant Cell Rep 11: 279–284
Yamamoto A, Takeuchi I (1983) Vital staining of autophagic vacuoles in differentiating cells ofDictyostelium discoideum. Differentiation 24: 83–87
Zimmerman JL (1993) Somatic embryogenesis: a model for early development in higher plants. Plant Cell 5: 1411–1423
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Timmers, A.C.J., Tirlapur, U.K. & Schel, J.H.N. Vacuolar accumulation of acridine orange and neutral red in zygotic and somatic embryos of carrot (Daucus carota L.). Protoplasma 188, 236–244 (1995). https://doi.org/10.1007/BF01280375
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF01280375