GROWTH AND ORGANIZED DEVELOPMENT OF CULTURED CELLS. VI. THE BEHAVIOR OF INDIVIDUAL CELLS ON NUTRIENT AGAR

Freely suspended cells of a long continuously cultured strain of carrot were dispersed in nutrient agar medium in Petri dishes. After inoculation, cells in randomly chosen fields were photographed at low magnification. The same cells were photographed at intervals over the next 23 days. The fate of 678 units, 24% of which grew visibly, was determined from the photographic record. Cells and units displayed a marked degree of individuality. In some units, there was a progressive increase in cell number; in others, after a few divisions, growth ceased; in still other units, cells enlarged without dividing. Although the evidence of cell division in free cells is conclusive, its incidence and maintenance increased with the size of the initial unit. In general, growth started after an "induction period" of about 5 days, but some cells remained apparently unchanged considerably longer after inoculation, before they started to grow. Although single cells of all sizes were observed to grow by one means or another, it was generally the shorter ones that continued to grow into viable colonies. Elongated cells predominated in the carrot strains used; these grew into colonies first by septation, with little or no over-all increase in size, followed by a stage of cell enlargement. Growth by both cell enlargement and division then ensued. During growth by septation within single cells, some derived cells were seen to burst and die. The most frequent divisions were observed during septation when the average cell generation time was 24 hr. Lacking the controls that normally operate in the intact plant body, there was much variation in the forms assumed during growth, a number of which are illustrated. The importance of epigenetic factors, or external controls, that determine the growth of cells in the plant body is stressed. THE PRECEDING paper (Blakely and Steward, 1964) concentrated upon the factors that permit isolated cells, or small groups of cells, to grow successfully into macroscopically visible colonies; this one concentrates upon the behavior of individual cells during the early stages of this growth. By suitable devices that are described, the fate of a large number (678) of single cells and small groups was followed through a growth period of 23 days. The different kinds of behavior thus observed are classified and form the basis of the interpretations which are made. A few selected cells were repeatedly observed with the phase microscope. MATERIALS AND METHODs-Repeated microscopic observations, with an inverted microscope, were made on carrot cells as they grew on nutrient agar medium containing 10% coconut milk and 200 ppm casein hydrolysate. The plating procedure was as described in the previous paper (Blakely and Steward, 1964). Observations were made at low magnification of a large random sample of units plated on agar in Petri dishes. Other observations were made at higher magnification, using the phase microscope, on selected cells growing in small plastic "Cooper" tissue1 Received for publication November 14, 1963. For acknowledgments see footnote 1 to Part V. 2 For acknowledgments and permanent address see footnote 2 Part V. culture dishes.3 Petri dishes were not well suited for phase microscopy. Observations at low magnification-A suspension of carrot cells from a long-maintained strain (304) was plated at a density of 31,000 units (discrete members of a suspension) per plate. Five fields, chosen at random, were outlined in ink on the bottoms of each of 3 plates. The cells within these fields were photographed at low magnification (4X objective; 6X ocular) 1, 5, 9, 15, and 23 days after inoculation. Each field contained about 40 units and covered about 12 square mm. Because of the low viability of these units at low densities, they were plated at a relatively high density to ensure that enough growth would be observed. After all the photographs had been taken, prints were made and arranged into sequences. A total of 678 units were visible on the 15 first photographs of each sequence. Each unit which was visible on a photograph 1 day after inoculation was followed through the rest of the time sequence. The initial length, or broadest dimension, of each unit was measured and recorded. Estimates were made of the number of cells in each unit or colony, and when cell enlargement occurred in the absence of cell division, this was noted. A few of the units which were seen at day 1 could not be followed through 3 Manufactured by Falcon Plastics, 5500 West 83rd Street, Los Angeles 45, Calif.