Can. J. Res. 9: 261-264.

The effect of aging and heat on the chromosomal mutation rates in maize and barley1
F. H. Peto2

Abstract

1 Manuscript received August 29, 1933.
Contribution from the National Research Laboratories, Ottawa, Canada. Issued as Paper No. 38 of the Associate Committee on Grain Research.
2. Junior Research Biologist, Associate Committee on Grain Research.

   The recent discovery by Navashin (3), that the chromosomal mutation rate in Crepis was influenced by aging of the seed, has been corroborated by observations on the mutation rate of corn plants grown from seed of various ages.
   A very high chromosomal mutation rate in barley was induced by heat treatments of seed under various conditions of humidity. The most common type of aberration resulting from these treatments appeared to be fracture of the chromosomes either at the attachment constriction, the secondary constriction or the point of attachment of the trabants. The reattachment of fragments to other chromosomes was observed in two instances.
   Considerable importance is attached to the discovery that a large proportion of the mutant cells are eliminated during the growth of the plant. The principle that only the fittest survive seems equally true of cells as of individuals and groups of plants or animals.

The Effect of Aging on the Mutation Rate

Navashin (3) reports that over 80% of the plants from seven-year-old seed of Crepis tectorum L. were chromosomal mutants of one sort or another while only 0.1% of the plants from fresh seed exhibited the same phenomenon.

The work outlined here was done in order to determine whether the above phenomenon was present in cereals grown from aged seed. Several inbred lines of maize were obtained from Professor L. C. Raymond of Macdonald College. Seed aged from one to six years of line II of this material was grown. In general there was a gradual decrease in germinability with the increase in age of the seed. There was a marked decrease in plant size and vigor in the plants from seed over a year old, and in addition a number of plants from seed lots over two years old were morphologically abnormal. Some were dwarfed and intensely green and died after reaching a height of a few inches; others were so heavily striped with chlorotic areas that they also died. A few of the more lightly striped plants survived but it is doubtful whether they will produce seed. A number of the chlorophyll-deficient plants resemble those produced when X-ray treatment was applied to corn by Stadler (5). After the plants had been growing for two or three weeks root tips were obtained from 20 of each of six lots of seed of different ages. These were examined for chromosomal abnormalities after fixing with La Cour's 2BE fixative and staining with gentian violet. To date, results are available from three lots only. These are given in Table I and appear to corroborate Navashin's findings in Crepis. The remaining lines are being studied and any data collected will be presented in a later publication.

TABLE I
Effect of aging on the mutation rate in maize

Age of seed Germination Days from
seeding to
sampling of
root tips
No.
plants
studied
Plants with
mutated cells
No. cells
observed
Mutated cells
No. % No. %
6 months 92 22 20 0 0 120 0 0
5 years 76 32 19 3 16 112 5 4
6 years 32 33 20 5 25 155 13 8

Eighteen mutated cells were observed in plants from seed five and six years old. Seven of these cells contained 21 chromosomes, seven 19, one 18, two contained one fragment in addition to the normal number and one contained two complete sets of 20 chromosomes in the metaphase condition. These two nuclei were separated in the cytoplasm but within the same cell wall probably as a result of failure in wall formation after the previous somatic division. If this binucleate cell was also induced by aging it would indicate that all the mutants do not occur in the embryonic stage but throughout the life of the plant. The abnormal cells were located at random throughout the root tips, and it was impossible to find a large sector with all the cells exhibiting the same aberration. This indicates that most of the mutations were of relatively recent occurrence in the development of the root. Other mutations, however, may have occurred earlier in the embryonic and early seedling stage and subsequently have been eliminated. The reasons for suspecting this elimination will be considered later in this paper.

The Effects of Heat on the Mutation Rate

Crocker and Groves (1, 2) investigated the effect of storage and heat on seeds, and from the relation between the time-temperature formula for the coagulation of proteins and the temperature-life-duration formula for seeds concluded that the loss of viability of the seeds was largely a matter of the coagulation of the cell proteins. Since it has been shown that loss of germinability in seeds is associated at least in its more advanced stages with an increase in the mutation rate, it is logical to suspect that any abnormal environmental factor that would favor the denaturation or coagulation of the nuclear proteins might be expected to result in an increased mutation rate. Consequently a series of experiments involving the treatment of O.A.C. No. 21 barley seeds under various conditions of temperature and moisture have been undertaken and the preliminary cytological findings are given in Table II.

TABLE II
Effect of heat on the mutation rate in barley

Heat treatment Germination Material examined No. plants
studied
Plants with
mutated cells
No. cells
observed
Mutated cells
No. % No. %
Untreated 99% in germinator Root tips taken 30 days after seeding 12 0 0 41 0 0
95° C. for 25 min. 67% in germinator Young seminal root tips 19 13 68 143 38 27
95° C. for 25 min. 67% in germinator, 32% in soil Root tips taken 40 days after seeding 20 4 20 129 12 9
40° C. for 30 days 4% in germinator Young seminal root tips 3 3 100 49 15 31

The barley was 1932 seed containing 9% moisture at the outset. In the treatment in a drying oven at 95° C. the seed was enclosed in a sealed glass capsule. There was a slight decrease in germinability after 15 min. heating. Further heating induced greater reduction in germination until at 35 min. the seed had lost its viability. There was a marked difference in the germination rate of seed placed in a Hearson’s germinator and of that sown in soil. In soil the germination was lower owing probably to the less vigorous of the seedlings dying prior to emergence.

Of the seed treated at 95° C., only that exposed for 25 min. has been subjected to cytological examination. In one lot the seminal root tips were taken from the germinating seeds within a day or two after germination had commenced. In the other lot the root tips were taken from the plants 40 days after seeding. The difference in the mutation rate between these lots from seed treated in the same manner is very striking. There is a reduction in mutants from 68% in the seminal root tips to 20% in the plants 40 days after seeding. This indicates that there must be an elimination of the mutant cells during the development of the plant.

This is of great significance since it suggests that Darwin's principle of the survival of the fittest applies to cells as well as to individuals or groups of plants and animals. It suggests further that in plants grown under disadvantageous environmental conditions, there may occur a great profusion of mutant types, a large number of which for various reasons fail to survive for any length of time. The amount of initial variation which is induced is important, however, and while elimination of unfavorable mutants may take place earlier in the life of the plant than previously expected, any dominant or favorable mutants which might occur would undoubtedly have survival value.

No definite sectors carrying the same type of mutation could be found in the young seminal root tops. However, in the root tops from the older plants of the similarly treated seed, one large sector was found in which the same mutation occurred throughout, proving that this particular mutation had survived longer than those in the younger roots.

The chromosomal mutants observed in the barley heated at 95° C. consisted almost entirely of fragments. Approximately half of them were less than one-fifth of the average chromosome length. Some of these very probably had been trabants that had become disconnected from their chromosomes. The fractures resulting in the longer fragments appeared to have occurred in about equal numbers at the attachment constriction and secondary constrictions. In one instance a large fragment had become reattached to a normal chromosome.

According to Navashin's (4) dislocation hypothesis regarding the evolution of chromosome numbers, a fracture cannot occur at the exact point where the kinetic constriction (attachment constriction) is located. Nevertheless preliminary studies of the heat treated material indicates that fractures do occur quite frequently at this point. Further studies should supply more definite information.

Preliminary results are also given on barley seed treated at 40° C. in an open dish in an unventilated incubator. A large pan of water was placed in the bottom of the incubator so that the air must have remained close to the saturation point. The incubator was opened daily to prevent the air from becoming unduly stagnant. The percentage germination was reduced after five days treatment, and after 30 days treatment only 4% of the seeds remained viable. It was from three of these germinating seeds that the seminal root tips were taken. This was the most severe treatment applied and as would be expected the effects on the chromosomes were most pronounced. In addition to the types of fragmentation observed in the barley, whole chromosomes were occasionally missing, one cell contained only 11 chromosomes and two very small fragments. These results show that the effect of heat on the chromosomal mutation rate is equally striking at low and high temperatures, providing adequate time is allowed for the treatment at the lower temperatures. This leads one to suspect that certain adverse environmental conditions in nature may be as effective in inducing changes in the chromosomal constitution of plants as is aging under natural conditions.

The evidence presented would appear to strengthen the mutationist's concept of evolution, but it indicates that the environment may play a large part in influencing the rate of mutation.

References

  1. CROCKER, W. and GROVES, F. J. A method of prophesying the life duration of seeds. Proc. Nat. Acad. Sci. 1: 152-155. 1915.
  2. GROVES, F. J. Temperature and life duration of seeds. Botan. Gaz. 63: 169-189. 1917.
  3. NAVASHIN, M. Origin of spontaneous mutations. Nature, 131: 435. 1933.
  4. NAVASHIN, M. The dislocation hypothesis of evolution of chromosome numbers. Z. indukt. Abst. Vererb. 63: 224-231. 1932.
  5. STADLER, L. J. Some genetic effects of X-rays in plants. J. Heredity, 21: 3-19. 1930.