TISSUE CULTURE OF PLANTS 



349 



TISSUE CULTURE OF PLANTS 



mental conditions do not disturb the 

 duration of mitosis. If such disturb- 

 ance occurs it must be compensated 

 for, as must also variation in the pro- 

 liferation rate with different periods in 

 the diurnal cycle. 



By time-lapse cinematography at low 

 magnifications for a period of hours, it 

 is possible to include 100 or more cells 

 in the field, and at the same time to 

 obtain detail adequate to recognize any 

 cell undergoing division. It is conse- 

 quently practical to determine the per- 

 centage of cells which undergo mitosis 

 per unit interval of time with a high 

 degree of accuracy. If at least two or 

 more optical systems are available a 

 control culture and one or more under 

 experimental treatment may be simul- 

 taneously recorded. While the method 

 has the disadvantage of requiring ex- 

 pensive equipment, such multiple op- 

 tical system time-lapse cinematographs 

 promise to be powerful instruments in 

 the quantitative study and comparison 

 of such phenomena as rates of cell 

 proliferation, migration and necrosis 

 in tissue culture preparations. 



Where the cells can be grown from a 

 cell suspension planted on the surface 

 of a substrate such as cellophane or 

 glass, the cells may be treated with a 

 special citric acid diluting solution and 

 the cell nuclei may be enumerated in 

 a hemocytometer (Sanford, K. K., 

 Earle, W. R., Evans, V. J., Waltz, H. 

 K. and Shannon, J. E., Jr., J. Nat. 

 Cancer Inst., 1951, 11, 773). While as 

 yet tested for only a few cell types, of 

 which the L strain of mouse cells of 

 single cell origin is the chief one, the 

 method should be applicable to other 

 cell types and should be tried. From 

 the results so far obtained the method is 

 slow, but is both practical and accurate 

 for measuring any change in the total 

 number of nuclei in the culture planted 

 from the cell suspension. It has the 

 advantage that it enumerates only ap- 

 parently healthy, living nuclei, and it 

 also allows distinguishing and dif- 

 ferentially enumerating cells actually 

 in mitosis if this is desired. 



Measurement of growth by increase 

 of weight of the culture is usually im- 

 practical with cultures of sizes and 

 types now available. If the cells are 

 within a plasma matrix they cannot be 

 separated from it for weighing. Even 

 when the cells are grown on cellophane 

 or glass substrates, difficulties of getting 

 the cells and substrate freed of media 

 and of fluid without changing the 

 weight of the cells makes weight deter- 

 minations difficult. Determinations of 

 dry weight are similarly complicated. 



While optical methods for determin- 

 ing the "growth" of a culture have been 

 tried, optical quality of cultures and of 

 culture media may vary so greatly due 

 to other changes (e.g., clouding or 

 precipitation of the culture media, 

 cell granulation, accumulation of fat 

 or granules; occurrence of necrosis) 

 that various methods suggested have 

 not proven practical under the widely 

 varied experimental conditions which 

 may prevail. 



Many chemical indices of "growth" 

 have been suggested and tried. Some 

 of these have been: rate of utilization 

 of glucose, lactic acid production, 

 change of pH of the medium, rates of 

 aerobic and anaerobic glycolysis, oxy- 

 gen consumption, increase in (Kjel- 

 dahl) nitrogen content of the culture, 

 increase in desoxyribose nucleic acid 

 or ribose nucleic acid, increase in 

 organic phosphorus and in lipid-free 

 organic phosphorus and the accumula- 

 tion of radioactive isotopes of some 

 metabolized substance. To a greater 

 or lesser degree these are useful as 

 indices of change of one or more com- 

 pounds or groups of compounds by the 

 cell or its enzyme systems. Such 

 chemical indices of change are par- 

 ticularly valuable when a number of 

 them can be simultaneously provided 

 in a single study so that results may be 

 correlated. In future work such chem- 

 ical studies will probably become in- 

 creasingly significant. However, under 

 the wide range of experimental condi- 

 tions which may be encountered in 

 tissue culture studies and with our 

 present limited knowledge, it is unwise 

 to rely on any one of these chemical 

 indices as an accurate, quantitative 

 measure of the proliferation of tissue 

 cells or nuclei. 

 Tissue Culture of Plants is also a fine art. 

 Fortunately an excellent account is 

 available in book form: White, P. R., 

 A Handbook of Plant Tissue Culture. 

 Lancaster: Jaques Cattell Press, 1943, 

 277 pp. The nutrient fluids used are 

 chiefly composed of pure chemicals, 

 blood plasma, embryo juice and so forth 

 are lacking. The temperature of incu- 

 bation ranges from about 30°C. down 

 to 5°C. The tissues are easily killed 

 by high temperatures. The special 

 techniques required in physiology, 

 pathology and morphogenesis are de- 

 scribed by White who also reviews the 

 literature. The technique of tissue 

 culture has proved useful in researches 

 on the disorderly growth of cells from 

 Crown-galls (White, P. R. and Braun, 

 A. C, Cancer Research, 1942, 2, 597- 

 617). 



