Genetics 6:433-444 (Sept. 1921)
Connecticut Agricultural Experiment Station, New Haven, Connecticut
Received March 10, 1921

A striking variation in agricultural varieties of tobacco (Nicotiana) has been frequently noted, in which the plants fail to complete vegetative growth and produce seed but go on growing in an indefinite manner until killed by frost in the fall. Varieties of tobacco commonly grown are characterized by a fairly constant average number of leaves. The new indeterminate-growth type produces a variable number of leaves according to the time of planting and length of season. Such plants have never been observed to flower in the field. When the plants are transplanted to the greenhouse at the end of the growing season they flower and produce seed during the winter months but the same plants kept alive and transplanted to the field the second year again fail to flower during the summer season. Plants started very early in the greenhouse and set in the field do not flower. Neither does restriction of growth by keeping the plants in small pots or pruning the roots favor seed production. GARNER and ALLARD (1920) have demonstrated that the blooming of this type of tobacco as well as of many other plants is governed by the relative length of daylight and sunlight. When the day is artificially shortened the new type of tobacco flowers and sets seed normally in the summer. The shortened day in the winter therefore is responsible for the change in the habit of growth when grown in the greenhouse.

ALLARD (1919) has reviewed the occurrence of this aberrant form in many agricultural varieties. HAYES and BEINHART (1914) report the appearance of indeterminate plants in the shade-grown Cuban tobacco in Connecticut. The original plants were found in the proportion of about one to a million. The variant has bred true to its type since that time and large numbers have been grown. It is one of the best examples on record of a mutation, as the change, although possibly a simple one, is visibly pronounced; the plants are sharply differentiated from the original stock and they were found in a naturally self-fertilized and uniform species and in a family which had been previously artificially self-fertilized under bags for five generations.

The change involved principally the number of leaves and habit of growth without altering the general characters of the variety. See figures 1 and 2. A thinner leaf and slightly different shape and color of the leaf are probably physiologically correlated with the indeterminate habit of growth. From this and its mode of origin it appeared probable that the change involved only a single locus. The plants used in the experiments reported here are descendants of the material described by HAYES and BEINHART.

FIGURE 1.—The original type of tobacco at the left, the indeterminate, non‑flowering type which originated from this by mutation at the right, and their first generation hybrid in the center.

When the cross of the mutant with the original type was made, the normal habit of growth was dominant in both reciprocal combinations. All the first-generation plants flowered and produced seed. The results obtained in the second generation did not at first indicate a simple mode of inheritance. Table 1 gives the numbers of the two kinds of plants obtained in ten segregating progenies. The appearance of only 61 recessives in a total of 5062 individuals giving a ratio of 82 to I certainly did not look like a monohybrid ratio. However, these plants were handled in the usual manner of tobacco field culture. The seed was sown rather thickly in beds, the plants pulled as soon as they reached suitable size and set in the fields. The counts were made late in the season after the normal plants had been in flower for more than a month. The recessive plants were easily seen on account of their taller growth and absence of flowers and seed pods (figure 3). In this method of handling, an inequality in the germination of the seed or in the rate of growth of the young seedlings would not give a representative sample of the different kinds of plants present.

FIGURE 2.—Comparative height, spread of inflorescence and condition with regard to flowering of the determinate and indeterminate types at the left, and the first generation hybrid at the right.

The number of determinate and indeterminate plants in ten segregating progenies.

1 510 9
2 502 4
3 490 11
4 444 6
5 491 8
6 504 9
7 510 5
8 532 2
9 488 4
10 530 3
Total 5001 61

Table 2 shows the numbers and ratios obtained when attempts were made to secure a more nearly random sample. In 1917 and 1918 at Bloomfield the plants were handled as noted above, giving ratios of 82.0 to 1 and 31.4 to 1 in the two different years. In 1917 at Mt. Carmel the plants were handled differently. The seeds were sown in a flat and later transplanted to another flat before setting in the field. Competition was less severe and a ratio of 7.4 to 1 was obtained. The next year at Mt. Carmel the seed was sown very thinly, not transplanted before setting in the field and all the seedlings from a given area in the seed flat were set in the field hoping thereby to get a fair sample of all the plants which had remained alive up to that time. A few plants died in the field after setting. A ratio of 9.8 to I resulted, which was not as close to a monohybrid ratio as was found the previous years.

FIGURE 3.—The second generation of the cross showing the recessive plants compared with the normal habit of growth late in the season.

It was noted that the seedlings of the pure recessive type were smaller and slower in starting than the original variety and in 1919 two lots of seedlings were taken from an F2 population shortly after they had produced several leaves. One lot consisted of the largest seedlings, the other the smallest seedlings, to be found in a large number of the progeny of one plant. These were carefully transplanted into flats and so handled that the mortality was low. Later these were all set in the field and gave a ratio of 30.7 to 1 for the large seedlings and 6.1 to 1 for the small seedlings A difference in initial rate of growth of the dominant and recessive plants is probably the principal reason for the wide divergence of the ratios from each other and from a simple monohybrid ratio. Differences in viability of the seed may also be partly responsible. The germination of F2 lots of seed was compared with both pure types without any consistent results. When tobacco seed is saved under bags there is usually a large amount of infertile seed. The germination of all lots was low.

ALLARD (1919) reports data obtained from a large number of crosses between different varieties in which this character was involved and found altogether 439 indeterminate plants in a total of 1820 F2 plants where 455 were theoretically expected, which was a reasonably close agreement. None of his crosses included the Connecticut Cuban strain of non‑flowering tobacco. Either his plants were handled in such a way as to secure a more representative sample or else the Connecticut strain differs from others in viability and initial rate of growth.

The number and ratio of determinate and indeterminate plants under different treatments.

1917 Bloomfield 5001 61 82.0 : 1
1917 Mt. Carmel 348 47 7.4 : 1
1918 Bloomfield 2984 95 31.4 : 1
1918 Mt. Carmel 98 10 9.8 : 1
1919 Mt. Carmel 92 3 30.7 : 1
1919 Mt. Carmel 86 14 6.1 : 1

Although the numbers obtained in the segregating progenies do not prove that the original variation was due to a change in a single factor, the data on leaf number given in table 3 are more convincing. The original determinate type of tobacco is designated A and its non‑flowering, indeterminate offspring as B in the tables. In leaf number A ranges from 17 to 29 and averages from 20 to 23 leaves. The data for leaf number of the B type are not given, as there is no basis for comparison. The leaf numbers at the close of the season usually run above 50 and in some cases more than 100 leaves have been noted. The first-generation reciprocal hybrids are identical in leaf number and are considerably advanced in number of leaves above the position of the determinate parent.

Leaf number and variability of the determinate parent and the first, second* and third* generation hybrids.

* The indeterminate plants in these progenies are not included in the tabulations.
† In addition to these 60 plants there were 7 indeterminate plants which can not be accounted for and are not included.

In the second generation there is clear-cut segregation into two types like the parental types. The non‑flowering recessive segregates reduplicate the one type in appearance, in leaf number, and in habit of growth. The data from these plants are omitted in table 3 as the figures are given to show only the segregation taking place in the flowering plants. The F2 plants of determinate growth are clearly of two types as the figures for leaf number show. One lot returns to the homozygous dominant condition and the other reduplicates the F1 generation. There is overlapping of the distributions but the bimodal condition is clearly apparent.

The relation between leaf number of the parental F2 plants and their zygotic condition in respect to indeterminate growth as shown by their progenies.

(A X B) -1 -1      30 390 10
3      30 536 15
4      26 497 19
6      27 417 15
(B X A) -1 -1      26 87 4
2      25 78 5
3      28 564 12
4      22 609 0
5      22 90 0
6      29 580 24

The modes of the two F2 groups agree closely with the modes of the flowering parent and first-generation hybrid, particularly in the plants grown in 1917 when all the plants were handled alike. Moreover, the monohybrid segregation which is taking place is clearly shown by the F3 progenies grown in 1919. Part of these give an exact return to the normal grandparental type in appearance, size and habit of growth. The distribution of leaf number of the one progeny upon which leaf counts were made, coincides so closely with the parental distribution in range, mode, variability and average leaf number, that there can be scarcely a doubt that genetically it is an exact copy of the original recovered from the cross. The other progenies are as clearly a continuation of the heterozygous condition. Moreover, there is a close agreement between the leaf number of the parental F2 plants and the type of the progeny which they produce as brought out in table 4. Ten F3 families are given here of which 8 came from F2 plants with high, and 2 from plants with low leaf number. All plants tested, having 25 or more leaves, gave segregating progenies while the two with 22 leaves gave uniform offspring.

Figure 4 shows the distribution of all the F2 and segregating F3 progenies when combined. Before adding the figures the leaf numbers of the plants grown in 1917 were all reduced two places. A comparison of the results obtained that year with the two following years shows that the leaf numbers average more for all the plants grown, by about two leaves. This variation in average leaf number from year to year is a change due largely to the time of transplanting in relation to the age and growth of the seedlings so that the shifting of all the distributions for any one year up or down is justifiable.

FIGURE 4.—Graph showing the distribution of leaf number based on the combined data from the determinate plants of all F2 and segregating F3 progenies.
Leaf classes 18 19 20 21 22 23 24 25 26 27 28 29 30
No. of plants 1 14 47 87 109 79 65 101 120 108 58 13 5

The graph shows that the distribution is distinctly bimodal and that the areas of the two parts are unequal. However, that part of the figure which is taken to represent the heterozygous plants should be twice as large in area as that which represents the dominant homozygotes. Such is clearly not the case but it is believed that the same factor which is causing the recessive plants to be few in number is also discriminating against the hybrid plants. The intermediate nature of the hybrid is shown in time of flowering as given in table 5.

There are reasonable grounds, therefore, for considering that the original mutation was a single-factor change and that the segregation simulates a monohybrid ratio. The segregation is of two kinds, qualitative and quantitative. As an illustration of Mendelian inheritance of a quantitative character based upon a single-factor difference it is almost unique.

The foregoing presentation of tedious and for the most part well known facts is an introduction to the bringing out of a novel situation exemplified in the first‑generation plants with regard to the writer's hypothesis of hybrid vigor as being due to the complementary action of dominant favorable growth factors (JONES 1917). A comparison of the F1 plants with their two parents at that stage of growth when the determinate plants have just reached their full development (figure 2 and table 6) shows that the hybrid plants are taller than the flowering parent. The leaf number is greater, the inflorescences are larger and the plants produce more seed. Since the other parent is unable to propagate itself in the same environment the hybrid is superior to either parent in respect to reproductive capacity which plays such a large part in survival. Here, apparently, is what might be taken as an instance of hybrid vigor where the parents differ in only a single gene. According to the theory of dominant factors at least two genes must differentiate the parents so that heredity favorable to growth may be contributed by each.

A comparison in time of flowering between the parents and their reciprocal F1 hybrids.

60 21.3 0 0 1.8
61 33.3 0 0.9 6.3
68 72.2 0 48.1 53.6
70 90.7 0 67.6 79.5
72 91.7 0 83.3 83.9
81 97.2 0 92.5 98.1
100 100.0 0 100.0 100.0

Rate of growth of the determinate and indeterminate parents and their reciprocal F1 hybrids.

30 1.9 ± .07 1.3 ± .05 2.0 ± .07 2.2 ± .06 -2.2
40 5.7 ± .19 2.8 ± .12 5.3 ± .20 5.8 ± .21 -1.7
50 24.7 ± .63 13.4 ± .41 21.7 ± .56 23.4 ± .49 -2.3
60 54.6 ± .54 31.7 ± .61 49.0 ± .84 48.2 ± .79 +0.7
70 76.8 ± .48 58.1 ± .74 78.9 ± .65 79.5 ± .56 -0.7
80 80.2 ± .36 69.1 ± .78 84.1 ± .48 83.0 ± .39 +1.8
90 82.5 ± .70 82.5 ± .78 88.5 ± .30 86.5 ± .32 +4.5
100 82.6 ± .30 88.4 ± .86 88.9 ± .32 86.6 ± .31 +5.1

A study of the growth curves (figure 5) of height of plant of these two types and their reciprocal hybrids brings out the real situation. The data upon which the curves are based are given in table 6. The height of plant was measured from the ground to the tip of the highest leaf held to its furthest upward extent. When the flowering shoot appeared and exceeded the tallest leaf it was measured from then on. There is therefore a somewhat arbitrary break in the taking of the data on the flowering parent and the hybrids. However, the growth curves obtained in this manner show no abrupt change and it is believed that this method of measurement is the most practicable one that could be used to compare the plants throughout the season.

FIGURE 5.—Growth curves of the determinate, A, and indeterminate, B, parental types and their reciprocal hybrids.

The non‑flowering type is consistently slower in its upward development and does not surpass the flowering parental type until the latter has reached its full development and stopped. Both hybrids hold practically the same rate of growth as the flowering parent which they closely resemble until the latter stops growing, but the hybrid does not stop at that level but goes on growing until an appreciably taller plant is produced. It also has an average of 5 more leaves and consequently is able to produce a larger inflorescence with more seed. But there is no hybrid vigor shown in so far as this is represented by an accelerated rate of development. A larger growth is attained because the plants take a longer time to complete their growth. This is also shown in table 5 in respect to time of blooming.

The two reciprocal crosses show a significant difference in height at the close of the growing season although this is not true of leaf number. It is the F1 combination in which the indeterminate type is the seed parent which attains the greater height. Table 6 shows that there is no significant difference between the two hybrids until 90 days after transplanting. From then on the B x A plants are taller by a difference which is 4.5 and 5.1, respectively, times the probable error of the differences of the last two averages of the F1 plants.

The two reciprocals also differ in time of flowering (table 5). Here also it is the offspring from the flowering maternal parent that blooms earlier. This inequality may be considered as due to the same combinations of genes acting with different cytoplasms or, what may amount to saying the same thing in another way, as an effect carried over in the seed from one generation to the next.

The non-blooming plants are much shorter throughout the first of the season. Up until 80 days the growth curve is of same type as for the flowering plants. There is a slackening in the rate of growth and the line bends over like the usual curve of an autocatalytic reaction characteristic of growth processes. But soon there is a noticeable change. Growth in height is resumed at very nearly the same rate as before even though it is now in the later and cooler part of the season. Finally there is a gradual slowing down in the rate of growth as the end of the season approaches.

That the abrupt change is due to readjustments within the plant and not to a seasonal check is indicated by the even growth of the other plants in adjoining rows. We have therefore in this new type, when compared with its original form, a good illustration of the controlling action of a single Mendelian factor upon the development of a plant. Throughout the season the rate of growth is reduced, but growth processes go on normally until the usual onset of the blooming period at which time vegetative growth is checked, just as it is in the original type from which it came, but when no flowers are formed vegetative growth is resumed until stopped by the end of the growing season.

The hybrid between the mutant and the original stock, which upon fairly good evidence is heterozygous in only a single factor, differs in an appreciable manner from either parent. Normal growth and reproductive processes are dominant but the intermediate nature of the hybrid delays the time of flowering and permits a larger growth and therefore greater reproduction capacity. In a short-season environment the later maturity would be a disadvantage but otherwise the hybrid is superior to either parent in size and ability to produce seed. We must therefore note that the haploid condition may, in particular instances, be advantageous even on a purely inheritance basis and this fact must be taken into consideration in comparing heterozygous and homozygous combinations.

The fact that the indeterminate plants function normally when there is a suitable adjustment of the light relations, as shown by GARNER and ALLARD, makes this variation a good illustration of an hereditary change which wholly unfits an organism for one situation but does not in another. Although there is probably no place on the globe where this new type of tobacco would have an advantage over the original stock, still, from this it is not difficult to conceive of a variation occurring which would fill these conditions. If a mutating race producing such variations were taken to the new environment and the mutation occurred this might easily be taken as an adaptive change in response to the altered environment. Yet a variation quite similar to this hypothetical case is now occurring in practically all kinds of tobacco where it entirely unfits the plants for further propagation under natural conditions.