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The responses of carrot explants to various growth-promoting agents and to certain trace elements with which they interact have been investigated. A great range in the metabolic behavior of the tissue may be brought about in this way. The responses to the exogenously applied substances are described in terms of the growth of the carrot explants in fresh weight and number of cells and also in terms of their metabolism, as shown by the final content and composition of the non-protein N compounds, by the relations between protein and non-protein (alcohol-soluble) N and by the content of nucleic acid in the cultured tissue.The growth-promoting agents employed consisted of (1) the balanced complex of factors found in coconut milk, (2) an active isolate from Aesculus (AFaesc), which is one of a class of growth factors (AF1) that interact with inositol (AF1+inositol) and which in this sense comprise growth-promoting System I, (3) the substance zeatin (Zeat) which is typical of a class of active factors (AF2) that interact with indoleacetic acid (AF2+IAA) and which, therefore, function as a growth promoting complex termed System II in the culture of carrot tissue.The carrot explants stimulated by coconut milk grew better than those stimulated by the other combinations of growth factors and they converted their soluble N more effectively to protein. The growth, whether it was induced by coconut milk or by System I or II, and other specific effects attributable to the growth factors employed were markedly affected also by the elements iron and molybdenum.The carrot explants that had responded to coconut milk emphasized alanine in their soluble, non-protein, nitrogenous pool, whereas those subjected to the active components of System I or of System II as clearly emphasized glutamine as the prominent non-protein, nitrogen-rich compound.The partial effects due to the component parts of System I (AFaesc or inositol) and to the component parts of System II (Zeat. or IAA), as these interacted also with iron and molybdenum in an otherwise trace element free basal medium (B (**)), revealed a pattern of interlocking effects, due to trace elements and to growth factors, upon the metabolism (especially the nitrogen metabolism) of the aseptically cultured carrot explants. These effects show that the individual growth factors do not act alone and that their implications are far reaching. The interactions between growth promoting Systems I and II and their component parts, with each other, with various environmental factors, and especially with trace elements constitute a network, or a matrix, of parameters that will merit further investigation to reveal all that is required to control the growth and metabolism of carrot cells.

The induction and maintenance of growth in small, standard explants of carrot root exposed to a trace-element-free basal nutrient medium (B (**)) have been investigated. The organic growth factors that induce the growth are complex as represented by a trace-element-limited coconut-milk preparation (CM(**)) or the component parts of distinct growth promoting systems mediated by inositol or by 3-indoleacetic acid (IAA). In System I inositol interacts with growth factors from Aesculus, of which a known example is an IAA-rhamnoseglucose compound; in System II IAA interacts with adenyl compounds, of which zeatin is a known example. But the organic growth-promoting substances also interact with trace elements; therefore, the effects of the component parts of the growth promoting systems, separately and in combination, have been investigated with respect to their interactions with Mo, with Fe and also with these trace elements in combination. The growth so induced has been measured in terms of the fresh weight of the explants, the number and average size of their cells, as well as their total content of protein and nucleic acids. The metabolic responses of the cultured explants to the combined effects of growth factors and of trace elements are also described in terms of the principal soluble nitrogenous compounds. Each basis is informative, and suitable graphical devices are adopted so that the interactions of the multivariate factors that affect the behavior of the cultured tissue may be seen with respect to the various parameters selected. The study shows the range of complexity to be understood before the exogenous factors that determine cell growth and metabolism are both known and controllable; it also shows the limitations when attention is directed, simplistically, to one parameter or to the controlling influence of any single factor, or class, of growth factors.

The effects of Fe, Mo and Mn on the growth of explants drawn from different carrot clones were investigated. The explants were cultured aseptically on purified basal media supplemented with coconut milk. The two components of growth, i.e. cell division and cell enlargement, seem to respond differentially to the trace elements in question. Fe plays the key role and acts as a "trigger" of the action of coconut milk in stimulating cell division. Neither Mo nor Mn could replace iron in this respect. Mo and Mn acting separately tend to foster growth by cell enlargement to a different degree and at different concentrations. However, when Mo and Mn were added together to a medium containing iron, they seem to interact and stimulate growth by cell division and cell, enlargement still further. The suggestion is made that the behavior of the explants from different clones may have been responsive to differences in the functional Fe/Mn ratio which also involves endogenous levels of trace elements as well as the exogenous levels furnished in the medium.Thus, the element Fe emerges as the key trace element which interacts with the factors present in coconut milk to induce growth in the otherwise quiescent carrot tissue.

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.