GROWTH AND ORGANIZED DEVELOPMENT OF CULTURED CELLS. V. THE GROWTH OF COLONIES FROM FREE CELLS ON NUTRIENT AGAR

When angiosperm cells are cultured in a liquid medium they may grow in the form of free, single cells and form small to large groups of cells. This has been shown in earlier papers. This paper deals with the growth of strains of cells (Daucus carota and Haplopappus gracilis cells were used), washed and filtered free from the larger groups, on nutrient agar media in Petri dishes, thus simulating familiar microbiological technique. Each discrete member of a suspension is referred to as a "unit." On the order of 1-10% of the separate units of a suspension may be induced to grow into viable colonies, depending on the strain and the conditions employed. Whereas at least 80% of the free single carrot cells were shown to be capable of division, only up to about 4% continued their growth to form macroscopically visible colonies when they were widely dispersed. Coconut milk promotes the growth of carrot cells into colonies. Both coconut milk and napthaleneacetic acid, which interact synergistically, are required for the growth of Haplopappus cells. Various techniques which affect viability (the frequency with which units grow into colonies) were investigated. The viability of carrot uniits was found (1) to increase with their density on the plates; (2) to decrease upon washing the suspensions prior to plating; (3) to increase with increasing initial size; and (4) to decrease to a vanishingly low value in rigorously filtered suspensions which consist principally of single cells, although the single cells were found to grow with appreciable frequency when the larger units were also present; and (5) to increase dramatically (100-fold) when a rigorously filtered suspension was plated oni a medium uponl which pieces of growing cultured tissue were placed. Thus, the induction of growth in free cells is enhanced, even in an environment nutritionally optimal for the growth of the larger cell masses, by as yet unknown factors which are contributed by the cells themselves, or by adjacent cells or groups of cells. It is suggested that within a group of cells growing in culture, and perhaps also in the organized growing regions of intact plants, the dividing cells are nourished or stimulated by adjacent but less frequently dividing cells. The implications of these results are discussed. A FEATURE of modern biology is the extent of our knowledge about self-duplicating structures and the precision with which likenesses are transmitted genietically from generation to generation, from cell to cell, alnd from one organelle to another. By contrast, very little is really known about the devices which permit cells with the same genetic information to achieve a variety of form and composition during their differentiation and to perform such a variety of functions. Indeed, as knowledge of genetics and genetically determined biochemistry becomes even more precise and as more and more of the biochemical 1 Received for publication November 14, 1963. Acknowledgments: The work reported here and in the 2 subsequent papers of this series was supported by grants C-1357 and RG 9609 to one of us (F.C.S.) from the National Institutes of Health, Department of Health, Education, and Welfare. 2 The work recorded here and in the 2 subsequent papers of this series formed part of a thesis submitted by one of us (L.M.B.) for the degree of Doctor of Philosophy at Cornell University, and during part of the period of work he was supported by a National Science Foundation predoctoral fellowship, and he also served as a research assistant to Prof. F. C. Steward. The present address of L. M. Blakely is Biological Sciences Department, California State Polytechnic College, Pomona, California. 3 Laboratory of Cell Physiology, Growth and Development, Cornell University, Ithaca, New York. attributes of living cells yield to work on isolated cell-free preparations, the problems of growth, differentiation and morphogenesis becomes even more obtrusive. In this latter area it is apparent that the whole cell is the minimum level of organization at which the phenomenea can be observed and interpreted. The problem here is to know how all the physiological and biochemical attributes of cells work together in a controlled way and produce, within the same genetic framework, the familiar but nevertheless mysterious differentiation of cells, tissues and organs of the plant body from the meristematic cells of its growing regions. This is the backgrounid that provides both the stimulus and the challenge for work on the culture and growth of free cells. The present group of 3 papers4 is, therefore, a sequel to previous ones on this general theme (Steward, Mapes, and Mears, 1958; Steward, MVapes, and Smith, 1958; Steward, 1958; Mitra, Mapes and Steward, 1960 [on carrot cells]; and Blakely and Steward, 1961; Mitra and Steward, 1961 [oln Haplopappus]). If the zygote receives all the genetic information which is essential for the whole organism, and if 4The first two (V and VI) appear in this issue; the third (VII) follows in the September issue.