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It is not yet known whether higher plants, which have evolved in a gravity oriented environment, can reproduce normally or develop via single cells (somatic or zygotic) and pass normally through all phases of development, tissue differentiation and reproduction through consecutive generations at near-zero g. Most published work deals with directional gravitational stimuli (geotropisms) as they operate on the subsequent growth of preformed plant organs that have acquired the ability to perceive and respond to gravity during development at 1 g. But it should be established whether plant development can proceed normally in the weightless state, especially that critical stage in which single cells produce multicellular units leading to embryos with the growing regions of shoot and root which, in turn, give rise to all the tissues of the plant body. We consider here an experiment that tested whether carrot embryos, capable of developing from cultured somatic cells, could do so under conditions of near-weightlessness. Space flight seemed to have no adverse affects on the induction of such embryos or on the initiation of their organs. But there are still reservations which are discussed. Other systems adaptable to the study of embryogenesis and development in space and to resolve the current reservations are described and their place in future space research is indicated.

An experiment designed to test whether embryos capable of developing from isolated somatic carrot cells could do so under conditions of weightlessness in space was performed aboard the unmanned Soviet biosatellite Kosmos 782 under the auspices of the joint United States-Soviet Biological Satellite Mission. Space flight and weightlessness seem to have had no adverse effects on the induction of embryoids or on the development of their organs. A portion of the crop of carrot plantlets originated in space and grown to maturity were not morphologically different from controls.

Morphogenetically competent proembryonic cells and well-developed somatic embryos of carrot at two levels of organization were exposed for 18.5 days to a hypogravity environment aboard the Soviet Biosatellite Cosmos 1129. It was confirmed that cultured totipotent cells of carrot can give rise to embryos with well-developed roots and minimally developed shoots. It was also shown that the space hypogravity environment could support the further growth of already-organized, later somatic embryonic stages and give rise to fully developed embryo-plantlets with roots and shoots.

Cultured carrot explants, stimulated to grow rapidly in a medium containing coconut milk, were labeled with radioactive proline. After an initial period of absorption (8 hr for proline-(3)H; 24 hr for proline-(14)C) the tissue was allowed to grow for a further period of 6 days in a similar medium free from the radioactivity. Samples were prepared for electron microscopy and radioautography at the end of the absorption period and also after the further growth. The distribution of the products from the radioactive proline in the cells is shown by high-resolution radioautography and is rendered quantitative for the different regions of the cells. The results show that the combined label, which was present in the form of proline and the hydroxyproline derived from it, was all in the protoplasm, not in the cell walls. Any combined label that appeared to be over the cell walls is shown to be due to scatter from adjacent cytoplasmic sites. Initially the radioactivity was concentrated in nuclei, even more so in nucleoli, but it subsequently appeared throughout the ground cytoplasm and was also concentrated in the plastids. The significance of these observations for the general concept of a plant cell wall protein and for the special problem of growth induction in otherwise quiescent cells is discussed.

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.

The distribution of the radioactivity from [(14)C]proline that is bound in cultured cells of Acer has been determined by electron microscope autoradiography. In this way proline may be related to the cell wall as a morphological entity rather than as a fraction in a biochemical separation of a heterogeneous crop of cells. The cells in culture may vary greatly. Some are active growing, turgid cells, with thin protoplasts tightly pressed against their walls; in others the protoplasts may spontaneously withdraw from the wall; in still others the protoplasts disorganize, and walls thicken and become sculptured as the cells differentiate and even senesce. Different culturing practices may affect the status of the cells, and this, in turn, affects the distribution of radioactivity from proline in the cells. Cells which are actively growing, turgid, and nucleated have the highest grain density in their protoplasts and nuclei; as the protoplasts of such cells withdraw from their walls, they retain the bulk of the radioactivity. On the other hand, in cells which have thickened walls and sparse protoplast contents, the radioactivity is accumulated in their walls. A high content of proline and hydroxyproline-rich protein is, therefore, not a necessary or invariable feature of the cell walls of cultured Acer cells but depends on the state of development of these cells.