Jones, L. E. (Oregon State Coll., Corvallis), A. C. Hildebrandt, A. J. Riker, and J. H. Wu. Growth of somatic tobacco cells in microculture. Amer. Jour. Bot. 47(6): 468–475. Illus. 1960.—Somatic cells of hybrid tobacco (Nicotiana tabacum × N. glutinosa) grew more than 4 mo. in microcultures in which critical microscopic observations could be made. Microchambers were made aseptically by placing cells in a droplet of medium on a cover slip that was inverted onto a standard microscope slide with a ring of paraffin oil (U. S. P. Heavy Mineral Oil) and 2 coverslip risers. Growth of single cells was good in a medium previously “conditioned” by a mass of growing cells. The cells divided, enlarged, differentiated, and became senescent. Mitosis was timed in somatic cells in microcultures. During prophase the streaming cytoplasm formed distinctive strands suspending the nucleus and then entered a state of “fixed‐tension” in which there was no massflow of organelles. Reversion of the cytoplasm to the usual fluid‐flow condition followed cytokinesis.Senescence and impending death in undisturbed, mature, parenchyma cells were preceded by concatenation of the discrete round‐oval mitochondria into filiform aggregates. A few “giant” parenchyma cells rejuvenated by forming discrete, free‐floating, endogenous cells. When placed in microcultures, the endogenous cells grew into daughter populations that appeared normal.Tobacco cells in microcultures provided a unique experimental material for cytological and cytochemical studies of growth, differentiation, senescence, and rejuvenation in living, somatic, angiosperm cells that were free of artifacts and protected from reactions to shock.

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The ability to observe for extended periods of time individual tobacco cells growing in microculture has made it possible to describe the behavior of their nucleoli and contracting nucleolar vacuoles. Nucleoli typically disappeared in prophase and reappeared in telophase. If several nucleoli were present in telophase they generally fused to form only one or two during interphase. In one instance a nucleolus was seen to separate into two nucleoli prior to disappearance in late prophase. In aging and senescent cells the number of nucleoli or bodies similar to normal nucleoli often increased, and occasionally fragmentation of nucleoli was noted prior to death of cells. Budding of solid material from the nucleolus was also observed. The amount of nucleolar material decreased rapidly prior to death of tobacco cells. Nucleolar vacuoles were found to be a general and consistent component of tobacco cells in microculture. Nucleolar vacuoles typically formed and contracted repeatedly in interphase nuclei and apparently released a fluid material into the nucleus. Associated with the contraction of the nucleolar vacuoles was a corresponding decrease in diameter of the nucleolus. Nucleolar vacuoles were observed to occur in about 70% of the actively growing cells examined, whereas they were present in only 33% of the senescent or weakened cells. These data indicate a relationship between nucleolar vacuoles and the morphogenic status of the cells. Since it has been shown by others that the nucleolus is an active site of RNA metabolism, it is suggested that the contracting nucleolar vacuoles may be involved in the controlled release of a soluble product associated with RNA metabolism.

Spores of Equisetum telmateia Ehrh. retained 74% viability after two years of storage under glycerine at minus 10 C. Stored spores germinated, grew, and formed mature gametangia on mineral nutrient media in petri plates and in micro‐cultures where detailed cytological observations could be made. Growing gametophytes were favorable materials for studies of mitosis, chloroplast replication, gametogenesis, and senescence.

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