Nature 188(4746): 242-243 (Oct 15, 1960)
Influence of Light from an Infra-red Bulb on the Mutagenic Effect of Colchicine on Sorghum

TRUE-BREEDING diploid mutants have been found to arise from colchicine-treated Sorghum seedlings of the true-breeding lines, Experimental 1 and Experimental 31-5. Their true-breeding nature is similar to that obtained after many generations of selfing so that they appear to be immediately homozygous for the new characteristics. Some non-true-breeding mutants also occur. It was proposed that the true-breeding condition might arise by a somatic reductional division of the chromosomes, similar to that observed by Huskins6, with subsequent restoration to the diploid number, and that the mutant condition might arise through concentration of chromatin from one of the original ancestors from which Experimental 3 had been derived. However, from cytogenetic studies of hybrids between true-breeding mutants and their untreated full sibs, the genetic changes appear to be due to gene mutations4,7-9. Tests of the somatic reduction hypothesis using chromosome markers (recessive genes and reciprocal translocations) in the heterozygous condition in sorghum and other materials have not been decisive1,7,10 except for one case in flax (gene markers) which indicated its seeming validity11.

The definition of a laboratory method for the production of true-breeding mutants after colchicine treatment has been found to be of paramount importance from the point of view of testing the hypothesis of somatic reduction since variable success results under greenhouse conditions. For this purpose the influences of environmental factors on the induction of mutants are being determined. Recent investigations involving more than 1,000 colchicine-treated seedlings have been carried out using aseptic cultures kept under light and temperature conditions with various degrees of control12. Only four mutated plants, none true-breeding, were obtained. The problem, therefore, was to identify the particular factors responsible for the difference in results between sand jars in the greenhouse, which had given as high as 42 per cent true-breeding mutants2, and aseptic cultures under light and temperature conditions controlled to varying degrees, which gave very few mutants, none true-breeding. Light was a possible factor since most of the seedlings in aseptic culture were grown in a box with artificial light, or in the laboratory, or under shades in the greenhouse, while those in sand jars were grown in the greenhouse without shades. Infra-red light was investigated since those rays penetrate glass, have been found to modify chromatin re-arrangements induced in Drosophila13 and in Tradescantia14, and have been shown to play an active part in the control of many aspects of growth and differentiation15.

In experiments in Table I, untreated and treated seedlings of line Experimental 3 were grown in glass-covered jars on sand with tap-water and in test-tubes on agar both with full nutrient and with distilled water. Treatment consisted of application of 0.5 per cent colchicine in lanolin to the coleoptiles immediately after germination. Comparisons were made between shaded and unshaded conditions in the greenhouse and continuous illumination from a 'Heat Ray' bulb (infra-red, 250 W) at approximately 78°F. for seven days immediately after treatment. Progeny tests were made of surviving plants from these experiments.

Light conditions and medium influenced both survival of treated seedlings and induction of mutants. Infra-red light cannot be considered the only factor precipitating the appearance of true-breeding mutants since changing the culture from sand to agar prevented their induction. It maybe of importance that mutants appeared only under conditions which gave high mortality. This observation agrees with previous findings that although agar with or without nutrients increased survival of treated seedlings over that in sand jars, the number of mutants induced by colchicine treatment is decreased by this and other improved growing conditions12. The high rate of mortality associated with the induction of true-breeding diploid mutants suggests that the balance between stress conditions causing death and those causing origin of a mutated shoot, which displaces original tissues, is delicate and subject to many factors difficult to control.

Table 1. INCIDENCE OF TRUE-BREEDING DIPLOID MUTANTS AND POLYPLOID PLANTS, AS INDICATED BY PHENOTYPE AND VERIFIED BY PROGENY TESTS, FROM
UNTREATED AND COLCHICINE-TREATED SORGHUM SEEDLINGS (LINE EXPERIMENTAL 3) WITH DIFFERENT ILLUMINATION AND MEDIA

Medium Greenhouse Infra-red bulb (7 days)
Shaded Unshaded
Treated Untreated Treated Untreated Treated Untreated
  1957 Experiment
Sand, tap-water            
    Original number 20 20 20 60
    Survivors 19 7 13 9
    Polyploids 0 0 0 0
    True-breeding diploid mutants 0 0 0 4
  1958 Experiments
Sand, tap-water            
    Original number 20 60 10 20 30 80
    Survivors 19 32 10 5 27 6
    Polyploids 0 0 0 1 0 0
    True-breeding diploid mutants 0 0 0 0 0 1
Agar, full-nutrient*            
    Original number 10 20 10 20
    Survivors 10 20 10 15
    Polyplolds 0 2 0 4
    True-breeding diploid mutants 0 0 0 0
Agar, distilled-water            
    Original number 10 20 10 20
    Survivors 10 20 5 13
    Pelyploids 0 1 0 0
    True-breeding diploid mutants 0 0 0 0
*5 x major minerals, 1 x minor elements, 2 per cent sucrose and 0.05 p.p.m. indole-3-acetic acid (ref. 12).

The true-breeding diploid mutants which arose after infra-red illumination were similar to those obtained previously from treated seedlings grown in an unshaded greenhouse. Both groups exhibited obvious changes in height, leaf, midrib, panicle, glumes, awns and seed colour, and bred true immediately. These results provide a clue to the variability in occurrence of mutants in greenhouse experiments since light conditions change with the amount of sunshine. They are particularly significant since it is indicated that the phenomenon of the induction of true-breeding mutants can be produced under laboratory conditions. There are therefore external conditions such as light, medium and temperature which determine the occurrence of this phenomenon as well as internal conditions determined by the genotype as indicated by Atkinson et al.2.

Induction of true-breeding mutant types by colchicine treatment of germinating seedlings in the greenhouse has been used with great success in the sorghum-breeding programme at South Dakota State College both to secure new variability and to obtain fixed genotypes immediately. The significance of this phenomenon for currently held theories regarding the behaviour of chromosomes within somatic tissues would seem to be far-reaching, particularly in our understanding of growth processes and, perhaps, of the origin of abnormal growths such as cancer.

This research was supported by funds from the National Science Foundation.

CLIFFORD J. FRANZKE
MARY E. SANDERS
JAMES G. ROSS

Agronomy Department,
South Dakota State College,
Brookings, South Dakota.

  1. Atkinson, G. F., Ms. thesis, South Dakota State College (July 1956).
  2. Atkinson, G. F., Franzke, C. J., and Ross, J. G., J. Hered., 48, 259 (1957).
  3. Franzke, C. J., and Ross, J. G., J. Hered., 43, 107 (1952).
  4. Franzke, C. J., and Ross, J. G., J. Hered., 48, 46 (1957).
  5. Ross, J. G., Franzke, C. J., and Schuh, L. A., Agron. J., 46, 10 (1954).
  6. Huskins, C. L., J. Hered., 39, 310 (1948).
  7. Foster, A. E., M.S. and Ph.D. theses, South Dakota State College (March 1956 and August 1958).
  8. Foster, A. E., Ross, J. G., and Franzke, C. J., Agron. J. (in the press).
  9. Harpstead. D. D., Ross, J. G., and Franzke, C. J., J. Hered., 45, 255 (1954).
  10. Hanson, G. P., MS. thesis, South Dakota State College (June 1958).
  11. Dirks, V. A., Ross, J. G., and Harpstead, D. D., J. Hered., 47, 229 (1956).
  12. Sanders, M. R., Franzke, C. J., and Ross, I. G., Amer. J. Bot., 46, 119 (1959).
  13. Kaufmann, B. P., Hollaender, A., and Gay, H., Genetics, 31, 249 (1946).
  14. Swanson, C. P., and Hollaender A., Proc. U.S. Nat. Acad. Sci., 32, 295 (1946).
  15. Hendricks, S. B., Amer. Scientist, 44, 220 (1956).

Somatic Segregation Biblio