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...

The metabolism and the in vitro effects of the cytokinin N6‐(3‐hydroxybenzyl)adenine (meta‐topolin, mT) were compared with those of N6‐benzyladenine (BA), N6‐benzyl‐9‐(2‐tetrahydropyranyl)adenine (BPA) and N6‐benzyladenine‐9‐riboside ([9R]BA) in micropropagated Spathiphyllum floribundum Schott cv. Petite. In vitro, BA, BPA and [9R]BA were mainly converted into the stable derivative N6‐benzyladenine‐9‐glucoside ([9G]BA), located at the basal part of the plant. N6‐(3‐O‐β‐D‐glucopyranosyl)benzyladenine‐9‐riboside {(OG)[9R]mT} was the main derivative of mT. This new cytokinin‐O‐glucoside, which was present in all plant parts, was metabolized much faster than [9G]BA during acclimatization. The effect of BA and mT on in vitro shoot and root production and post vitro rooting was compared. Only mT combined a good shoot production with in vitro root formation. The plants that developed on medium with 10 µM or more mT rooted better during acclimatization than those developed on medium with equimolar concentrations of BA.

Survival of rice (Oryza sativa) upon an extreme rise of the water level depends on rapid stem elongation, which is mediated by ethylene. A genomic clone (OS-ACS5) encoding 1-aminocyclopropane-1-carboxylic acid (ACC) synthase, which catalyzes a regulatory step in ethylene biosynthesis, has been isolated from cv IR36, a lowland rice variety. Expression was induced upon short-and long-term submergence in cv IR36 and in cv Plai Ngam, a Thai deepwater rice variety. Under hypoxic conditions, abscisic acid and gibberellin had a reciprocal opposite effect on the activity of OS-ACS5. Gibberellin up-regulated and abscisic acid down-regulated OS-ACS5 mRNA accumulation. Growth experiments indicated that lowland rice responded to submergence with a burst of growth early on, but lacked the ability to sustain elongation growth. Sustained growth, characteristic for deepwater rice, was correlated with a prolonged induction of OS-ACS5. In addition, a more pronounced capacity to convert ACC to ethylene, a limited ACC conjugation, and a high level of endogenous gibberellin 20 were characteristic for the deepwater variety. An elevated level of OS-ACS5 messenger was found in cv IR36 plants treated with exogenous ACC. This observation was concomitant with an increase in the capacity of converting ACC to ethylene and in elongation growth, and resulted in prolonged survival. In conclusion, OS-ACS5 is involved in the rapid elongation growth of deepwater rice by contributing to the initial and long-term increase in ethylene levels. Our data also suggest that ACC limits survival of submerged lowland rice seedlings.

In Spathiphyllum floribundum 'Petite', which was cultured on medium containing benzyladenine (BA), uptake of this cytokinin and its conversion to 9-ß-D-ribofuranosyl-benzyladenine (9R-BA) or 9-ß-glucopyranosyl-benzyladenine (9G-BA) was monitored. BA and extremely large quantities of 9G-BA were exclusively located in the basal part of the plant (callus and meristems). 9R-BA was found in the basal part, the petioles and the leaf blades. After an acclimatisation period of 9 weeks the plants still contained high levels of 9G-BA, but BA and 9R-BA could no longer be detected after one week. The possible role of BA and its derivatives on inhibition of root initiation or irreversible chloroplast deficiency is discussed.