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This paper deals with problems of somatic embryogenesis (totipotency) of carrot cells in liquid culture and when dispersed in thin films of nutrient agar. The critical events that intervene between somatic cells and embryo plantlets during morphogenesis are delineated. Factors that affect the course of these events are described with special reference to the stimuli which permit the very smallest units to develop into organized structures. Special attention is directed to the importance of a protracted period of darkness and the use of an ‘embryo-conditioned medium’ as interacting stimuli that facilitate the early stages of development. Means of arresting and releasing the development of pro-embryonic units are discussed. Aspects of growth and form of the developing cultures are presented photographically at different levels. The behaviour of free protoplasts from pro-embryonic units is described and contrasted with that of intact totipotent cells. The significance of all these observations is examined in the light of practical applications and in relation to problems of development.

THE mature vesicle of Valonia ventricosa was an early source of information about the nature of cellulose1 and the configuration of a cellulose cell wall2,3. Later it was used in one of the first investigations with the electron microscope of the way a plant cell wall develops at a naked protoplasmic surface4. The opportunity to make such a study was provided by the formation, at will, of aplanospores and by their subsequent development5. Aplanospores are small spherical masses of protoplasm, each with a nucleus, and they may form in very large numbers from the multinucleate, non-septate, protoplasm within a single vesicle. This system, therefore, has also provided an opportunity to allow sporelings to develop their cell walls while in contact with 14C-proline in sea-water. Current methods of autoradiography and electron microscopy make possible determination of the distribution of the radioactivity fixed from 14C-proline. This should test whether a developing cellulose cell wall incorporates substantial amounts of “cell wall protein” as indicated by radioactivity bound in the form of 14C-proline. Also the cell wall of mature vesicles of V. ventricosa can easily be obtained free of cytoplasm so that it is possible to ascertain, by direct analysis, how much total protein each contains and its content of proline and/or hydroxyproline.

Publisher Summary This chapter relates to the means by which living cells from certain vascular plants can be obtained and propagated for experimental use. Many nonvascular plants are unicellular (as are bacteria), and their culture mainly requires a proper nutrition. Hence, much physiological use has been made of some fungi and algae (e.g., Saccharomyces, Chlamydomonas, Euglena, and Chlorella). Very large cells, especially suitable for certain investigations, can be obtained from some algae (Nitella, Chara Valonia) merely by severing them from the plant or its substratum, but these are strictly multinucleate coenocytic vesicles, and, when used, they are relatively mature, sluggish in their metabolism, and do not multiply. In some cases, however, a true “free cell culture” of aplanospores can be produced easily by causing the protoplasm of the vesicle to segregate around individual nuclei. Such cultures that really start as free protoplasts, have in fact been used to study the growth of the cell wall and for 14 C-labeling experiments and in studies of ion transport and membrane potentials, but their full use in membrane-related studies has not yet been exploited.

Earlier papers of this series relate to different growth-promoting substances and systems which, singly and in combination, have interacted with trace elements (Mn and Mo) and Fe to induce growth and to affect the metabolism of aseptic cultures of carrot. The solutes of cultured carrot cells (K(+), Na(+), Cl(-), total solutes) are also affected. Two clones were grown in 9 combinations of growth factors and under 4 trace-element regimes (a complete complement including Fe, and this complement lacking either Mn or Mo, or both Mn and Mo), a total of 36 treatments under otherwise standardized experimental conditions. Under the treatments applied the number of cells varied over a 35fold range and their average size over a 7fold range; the concomitant effects on their solutes are expressed in terms of concentrations and of total content per cell. Both growth and the solutes accumulated were variously affected by carrot growth-promoting system I (mediated by inositol), by system II (mediated by IAA), and by coconut milk in the presence of Fe, with and without Mn, Mo, or Mn and Mo.The greatest concentrations of total solutes occurred in tissue cultured in nutrient solutions which lacked the stimuli to rapid cell multiplication and were also limited by the trace elements Mn and Mo. Moreover, specific regulatory effects of the trace elements on solute content, not solely attributable to their effects on cell growth, have been noted. An imbalanced growth-factor regime (zeatin acting alone, i.e. without IAA) shifted the normal preference for K(+) over Na(+) strongly toward Na(+), a trend which could also be induced by certain trace elements and more balanced growth-factor regimes, e.g. in a basal coconut milk medium lacking only Mn.The data are interpreted in the context of views on the de-novo uptake of salts and solutes in cultured cells as they grow. These cells respond to a network, or matrix, of interacting factors by distinctive effects that are attributable to the component parts of the culture medium acting singly and in various combinations. These interactions (involving trace elements and exogenous growth factors) control growth (fresh weight, number and size of cells) and regulate the solutes (organic and inorganic; K(+) vs. Na(+); organic anions vs. Cl(-)) which the cells acquire as they grow and develop. The intensity of the response of the cultures to balanced, or imbalanced, growth factors creates the internal spaces accessible to solutes; and the metabolism, as it is also affected by growth factors and trace elements, determines how these spaces are to be filled at a given osmotic value. The evidence shows the range of factors that affect the accumulation of solutes in cells as they grow and is to be contrasted with conventional observations on mature cells held in steady states under conditions that preclude all growth and when only a single ionic species is followed over a very short interval of time.