The Role of Auxin, pH, and Stress in the Activation of Embryogenic Cell Division in Leaf Protoplast-Derived Cells of Alfalfa

Abstract

Culturing leaf protoplast-derived cells of the embryogenic alfalfa (Medicago sativa subsp. varia A2) genotype in the presence of low (1 m) or high (10 m) 2, 4-dichlorophenoxyacetic acid (2,4-D) concentrations results in different cell types. Cells exposed to high 2,4-D concentration remain small with dense cytoplasm and can develop into proembryogenic cell clusters, whereas protoplasts cultured at low auxin concentration elongate and subsequently die or form undifferentiated cell colonies. Fe stress applied at nonlethal concentrations (1 mm) in the presence of 1 m 2,4-D also resulted in the development of the embryogenic cell type. Although cytoplasmic alkalinization was detected during cell activation of both types, embryogenic cells could be characterized by earlier cell division, a more alkalic vacuolar pH, and nonfunctional chloroplasts as compared with the elongated, nonembryogenic cells. Buffering of the 10 m 2,4-D-containing culture medium by 10 mm 2-(N-morpholino)ethanesulfonic acid delayed cell division and resulted in nonembryogenic cell-type formation. The level of endogenous indoleacetic acid (IAA) increased transiently in all protoplast cultures during the first 4 to 5 d, but an earlier peak of IAA accumulation correlated with the earlier activation of the division cycle in embryogenic-type cells. However, this IAA peak could also be delayed by buffering of the medium pH by 2-(N-morpholino)ethanesulfonic acid. Based on the above data, we propose the involvement of stress responses, endogenous auxin synthesis, and the establishment of cellular pH gradients in the formation of the embryogenic cell type.One of the characteristics of plant development is that somatic cell differentiation is reversible. This can be best demonstrated in in vitro systems where somatic plant cells can regain their totipotency and form embryos through the developmental pathway of somatic embryogenesis. Somatic embryo formation resembles zygotic embryogenesis in many aspects (for review, see Dodeman et al., 1997). However, beside the similarities, there are obvious differences: For example, whereas zygotes formed by the fusion of the egg and sperm cells are clearly determined to follow embryogenic development, somatic cells have to acquire competence to be able to respond to embryogenic signals and initiate embryogenesis. In carrot (Daucus carota), embryogenic cells of the proembryogenic cell mass are small, densely cytoplasmed, and full of starch grains, whereas nonembryogenic callus cells are large and highly vacuolated. This can be generalized for most embryogenic systems, including alfalfa (Medicago sativa), where protoplast-derived cells cultured at different 2, 4-dichlorophenoxyacetic acid (2,4-D) concentrations can develop into embryogenic or nonembryogenic cell types with the above characteristic morphologies (Bö gre et al., 1990; Dudits et al., 1991). Komamine (1985, 1995) showed that isolated, small, cytoplasm-rich carrot cells have the ability to develop to somatic embryos and go through an unequal first divisio...

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