Chapter 17

Moreover the progeny must be numerous, since neither constancy, nor the exact proportions in the case of instability, can be determined with a small lot of plants.

Finally, and in order to come to a definite choice of research material, we should keep in mind that the chief object is to ascertain the relation of the offspring to their parents. Now in nearly all cases the seeds are separated from the fruits and from one another, before it becomes possible to judge of their qualities. One may open a fruit and count the seeds, but ordinarily nothing is noted as to their characters. In this respect no other plant equals the corn or maize, as the kernels remain together on the spike, and as it has more than one variety characterized by the color, or constitution, or other qualities of the grains. A corn-grain, however, is not a seed, but a fruit containing a seed. Hence the outer parts pertain to the parent plant and only the innermost ones to the [288] seedling and therefore to the following generation. Fruit-characters thus do not offer the qualities we need, only the qualities resulting from fertilizations are characteristic of the new generation. Such attributes are afforded in some cases by the color, in others by the chemical constitution.

We will choose the latter, and take the sugarcorn in comparison with the ordinary or starch producing forms for our starting point. Both sugar- and starch-corns have smooth fruits when ripening. No difference is to be seen in the young ripe spikes. Only the taste, or a direct chemical analysis might reveal the dissimilarity. But as soon as the spikes are dried, a diversity is apparent. The starchy grains remain smooth, but the sugary kernels lose so much water that they become wrinkled. The former becomes opaque, the latter more or less transparent. Every single kernel may instantly be recognized as belonging to either of the types in question, even if but a single grain of the opposite quality might be met with on a spike. Kernels can be counted on the spike, and since ordinary spikes may bear from 300-500 grains and often more, the numerical relation of the different types may be deduced with great accuracy.

Coming now to our experiment, both starchy [289] and sugary varieties are in this respect wholly constant, when cultivated separately. No change is to be seen in the spikes. Furthermore it is very easy to make the crosses. The best way is to cultivate both types in alternate rows and to cut off the staminate panicles a few days before they open their first flowers. If this operation is done on all the individuals of one variety, sparing all the panicles of the other, it is manifest that all the plants will become fertilized by the latter, and hence that the castrated plants will only bear hybrid seeds.

The experiment may be made in two ways; by castrating the sugary or the starchy variety. In both cases the hybrid kernels are the same. As to their composition they repeat the active character of the starchy variety. The sugar is only accumulated as a result of an incapacity of changing it into starch, and the lack of this capacity is to be considered as a retrogressive varietal mark. The starch-producing unit character, which is active in the ordinary sorts of corns, is therefore latent in sugar-corn.

In order to obtain the second generation, the hybrid grains are sown under ordinary conditions, but sufficiently distant from any other variety of corn to insure pure fertilization. The several individuals may be left to pollinate [290] each other, or they may be artificially pollinated with their own pollen.

The outcome of the experiments is shown by the spikes, as soon as they dry. Each spike bears two sorts of kernels irregularly dispersed over its surface. In this point all the spikes are alike. On each of them one may see on the first inspection that the majority of the kernels are starch-containing seeds, while a minor part becomes wrinkled and transparent according to the rule for sugary seeds. This fact shows at once that the hybrid race is not stable, but has differentiated the parental characters, bringing those of the varietal parent to perfect purity and isolation. Whether the same holds good for the starchy parent, it is impossible to judge from the inspection of the spikes, since it has been seen in the first generation that the hybrid kernels are not visibly distinguished from those of the pure starch-producing grains.

It is very easy to count the number of both sorts of grains in the spike of such a hybrid. In doing so we find, that the proportion is nearly the same on all the spikes, and only slight variations would be found in hundreds of them. One-fourth of the seeds are wrinkled and three-fourths are always smooth. The number may vary in single instances and be a little more or a little less than 25%, ranging, for [291] instance, from 20 to 27%, but as a rule, the average is found nearly equal to 25%.

The sugary kernels, when separated from the hybrid spikes and sown separately, give rise to pure sugary race, in no degree inferior in purity to the original variety. But the starchy kernels are of different types, some of them being internally like the hybrids of the first generation and others like the original parent. To decide between these two possibilities, it is necessary to examine their progeny.

For the study of this third hybrid generation we will now take another example, the opium poppies. They usually have a dark center in the flowers, the inferior parts of the four petals being stained a deep purple, or often nearly black. Many varieties exhibit this mark as a large black cross in the center of the flower. In other varieties the pigment is wanting, the cross being of a pure white. Obviously it is only reduced to a latent condition, as in so many other cases of loss of color, since it reappears in a hybrid with the parent-species.

For my crosses I have taken the dark-centered "Mephisto" and the "Danebrog," or Danish flag, with a white cross on a red field. The second year the hybrids were all true to the type of "Mephisto." From the seeds of each artificially self-fertilized capsule, one-fourth (22.5%) [292] in each instance reverted to the varietal mark of the white cross, and three-fourths (77.5%) retained the dark heart. Once more the flowers were self-pollinated and the visits of insects excluded. The recessives now gave only recessives, and hence we may conclude that the varietal marks had returned to stability. The dark hearted or dominants behaved in two different ways. Some of them remained true to their type, all their offspring being dark-hearted. Evidently they had returned to the parent with the active mark, and had reassumed this type as purely as the recessives had reached theirs. But others kept true to the hybrid character of the former generation, repeating in their progeny exactly the same mixture as their parents, the hybrids of the first generation, had given.

This third generation therefore gives evidence, that the second though apparently showing only two types, really consists of three different groups. Two of them have reassumed the stability of their original grandparents, and the third has retained the instability of the hybrid parents.

The question now arises as to the numerical relation of these groups. Our experiments gave the following results: [293]

Cross 1. Generation 2. Generation 3. Generation

Mephisto 4- 100% Mephisto | / | / | 77.5 % Dom. | / \ > --All Mephisto \ | \ 9- all hybrids with 83-68% | 22.5 % Rec. dominants and 17-32% | recessives. 100% Danebrog. Danebrog

Examining these figures we find one-fourth of constant recessives, as has already been said, further one-fourth of constant dominants, and the rest or one half as unstable hybrids. Both of the pure groups have therefore reappeared [293] in the same numbers. Calling A the specimens with the pure active mark, L those with the latent mark, and H the hybrids, these proportions may be expressed as follows:

1A+2H+1L.

This simple law for the constitution of the second generation of varietal hybrids with a single differentiating mark in their parents is called the law of Mendel. Mendel published it in 1865, but his paper remained nearly unknown to scientific hybridists. It is only of late years that it has assumed a high place in scientific literature, and attained the first rank as an investigation on fundamental questions of heredity. [294] Read in the light of modern ideas on unit characters it is now one of the most important works on heredity and has already widespread and abiding influence on the philosophy of hybridism in general.

But from its very nature and from the choice of the material made by Mendel, it is restricted to balanced or varietal crosses. It assumes pairs of characters and calls the active unit of the pair dominant, and the latent recessive, without further investigations of the question of latency. It was worked out by Mendel for a large group of varieties of peas, but it holds good, with only apparent exceptions, for a wide range of cases of crosses of varietal characters. Recently many instances have been tested, and even in many cases third and later generations have been counted, and whenever the evidence was complete enough to be trusted, Mendel's prophecy has been found to be right.

According to this law of Mendel's the pairs of antagonistic characters in the hybrid split up in their progeny, some individuals reverting to the pure parental types, some crossing with each other anew, and so giving rise to a new generation of hybrids. Mendel has given a very suggestive and simple explanation of his formula. Putting this in the terminology of to-day, and limiting it to the occurrence of only [295] one differential unit in the parents, we may give it in the following manner. In fertilization, the characters of both parents are not uniformly mixed, but remain separated though most intimately combined in the hybrid throughout life. They are so combined as to work together nearly always, and to have nearly equal influence on all the processes of the whole individual evolution. But when the time arrives to produce progeny, or rather to produce the sexual cells through the combination of which the offspring arises, the two parental characters leave each other, and enter separately into the sexual cells. From this it may be seen that one-half of the pollen-cells will have the quality of one parent, and the other the quality of the other. And the same holds good for [296] the egg-cells. Obviously the qualities lie latent in the pollen and in the egg, but ready to be evolved after fertilization has taken place.

Granting these premises, we may now ask as to the results of the fertilization of hybrids, when this is brought about by their own pollen. We assume that numerous pollen grains fertilize numerous egg cells. This assumption at once allows of applying the law of probability, and to infer that of each kind of pollen grains one-half will reach egg-cells with the same quality [297] and the other half ovules with the opposite character.

Calling P pollen and O ovules, and representing the active mark by P and O, the latent qualities by P' and O', they would combine as follows:

P + 0 giving uniform pairs with the active mark, P + 0' giving unequal pairs, P' + 0 giving unequal pairs, P' + 0' giving uniform pairs with the latent mark.

In this combination the four groups are obviously of the same size, each containing one-fourth of the offspring. Manifestly they correspond exactly to the direct results of the experiments, P + O representing the individuals which reverted to the specific mark, P' + O' those who reassumed the varietal quality and P + O' and P + O' those who hybridized [298] for the second time. These considerations lead us to the following form of Mendel's,

P + O = 1/4 Active or 1A,

P + O' > = 1/2 Hybrid or 2 H, P' + O

P' + O' = 1/4 Latent or 1 L,

Which is evidently the same as Mendel's empirical law given above.

To give the proof of these assumptions Mendel has devised a very simple crossing experiment, [299] which he has effected with his varieties of peas. I have repeated it with the sugar-corn, which gives far better material for demonstration. It starts from the inference that if dissimilarity among the pollen grains is excluded, the diversity of the ovules must at once became manifest and vice versa. In other terms, if a hybrid of the first generation is not allowed to fertilize itself, but is pollinated by one of its parents, the result will be in accordance with the Mendelian formula.

In order to see an effect on the spikes produced in this way, it is of course necessary to fertilize them with the pollen of the variety, and not with that of the specific type. The latter would give partly pure starchy grains and partly hybrid kernels, but these would assume the same type. But if we pollinate the hybrid with pollen of a pure sugar-corn, we may predict the result as follows.

If the spike of the hybrid contains dormant paternal marks in one-half of its flowers and in the other half maternal latent qualities, the sugar-corn pollen will combine with one-half of the ovules to give hybrids, and with the other half so as to give pure sugar-grains. Hence we see that it will be possible to count out directly the two groups of ovules on inspecting the ripe and dry spikes. Experience teaches us [298] that both are present, and in nearly equal numbers; one-half of the grains remaining smooth, and the other half becoming wrinkled.

The corresponding experiment could be made with plants of a pure sugar-race by pollination with hybrid pollen. The spikes would show exactly the same mixture as in the above case, but now this may be considered as conclusive proof that half the pollen-grains represent the quality of one parent and the other half the quality of the other.

Another corollary of Mendel's law is the following. In each generation two groups return to purity, and one-half remains hybrid. These last will repeat the same phenomenon of splitting in their progeny, and it is easily seen that the same rule will hold good for all succeeding generations. According to Mendel's principle, in each year there is a new hybridization, differing in no respect from the first and original one. If the hybrids only are propagated, each year will show one-fourth of the offspring returning to the specific character, one-fourth assuming the type of the variety and one-half remaining hybrid. I have tested this with a hybrid between the ordinary nightshade with black berries, and its variety, _Solanum nigrum chlorocarpum_, with pale yellow fruits. Eight generations of the hybrids were cultivated, [299] disregarding always the reverting offspring. At the end I counted the progeny of the sixth and seventh generations and found figures for their three groups of descendants, which exactly correspond to Mendel's formula.

Until now we have limited ourselves to the consideration of single differentiating units. This discussion gives a clear insight into the fundamental phenomena of hybrid fertilization. It at once shows the correctness of the assumption of unit-characters, and of their pairing in the sexual combinations.

But Mendel's law is not at all restricted to these simple cases. Quite on the contrary, it explains the most intricate questions of hybridization, providing they do not transgress the limits of symmetrical unions. But in this realm nearly all results may be calculated beforehand, on the ground of the principle of probability. Only one more assumption need be discussed. The several pairs of antagonistic characters must be independent from, and uninfluenced by, one another. This premise seems to hold good in the vast majority of cases, though rare exceptions seem to be not entirely wanting. Hence the necessity of taking all predictions from Mendel's law only as probabilities, which will prove true in most, but not necessarily in all cases. [300] But here we will limit ourselves to normal cases.

The first example to be considered is obviously the assumption that the parents of a cross differ from each other in respect to two characters. A good illustrative example is afforded by the thorn-apple. I have crossed the blue flowered thorny form, usually known as _Datura Tatula_, with the white thornless type, designated as _D. Stramonium inermis_. Thorns and blue pigment are obviously active qualities, as they are dominant in the hybrids. In the second generation both pairs of characters are resolved into their constituents and paired anew according to Mendel's law. After isolating my hybrids during the period of flowering, I counted among their progeny:

128 individuals with blue flowers and thorns 47 individuals with blue flowers and without thorns 54 individuals with white flowers and thorns 21 individuals with white flowers and without thorns ---- 250

The significance of these numbers may easily be seen, when we calculate what was to be expected on the assumption that both characters follow Mendel's law, and that both are independent from each other. Then we would have three-fourths blue offspring and one-fourth individuals with white flowers. Each of these [301] two groups would consist of thorn-bearing and thornless plants, in the same numerical relation. Thus, we come to the four groups observed in our experiment, and are able to calculate their relative size in the following way:

Proportion Blue with thorns 3/4 X 3/4 = 9/16 = 56.25% 9 Blue, unarmed 3/4 X 1/4 = 3/16 = 18.75% 3 White with thorns 1/4 X 3/4 = 3/16 = 18.75% 3 White, unarmed 1/4 X 1/4 = 1/16 = 6.25% 1

In order to compare this inference from Mendel's law and the assumption of independency, with the results of our experiments, we must calculate the figures of the latter in percentages. In this way we find:

Found Calculated Blue with thorns 128=51% 56.25% Blue unarmed 47=19% 18.75% White with thorns 54=22% 18.75% White unarmed 21= 8% 6.25%

The agreement of the experimental and the theoretical figures is as close as might be expected.

This experiment is to be considered only as an illustrative example of a rule of wide application. The rule obviously will hold good in all such cases as comply with the two conditions already premised, viz.: that each character agrees with Mendel's law, and that both are wholly independent of each other. It is clear that our figures show the numerical composition [302] of the hybrid offspring for any single instance, irrespective of the morphological nature of the qualities involved.

Mendel has proved the correctness of these deductions by his experiments with peas, and by combining their color (yellow or green) with the chemical composition (starch or sugar) and other pairs of characters. I will now give two further illustrations afforded by crosses of the ordinary campion. I used the red-flowered or day-campion, which is a perennial herb, and a smooth variety of the white evening-campion, which flowers as a rule in the first summer. The combination of flower-color and pubescence gave the following composition for the second hybrid generation:

Number % Calculation Hairy and red 70 44 56.25% Hairy and white 23 14 18.75% Smooth and red 46 23 18.75% Smooth and white 19 12 6.25%

For the combination of pubescence and the capacity of flowering in the first year I found:

Number % Calculated Hairy, flowering 286 52 56.25% Hairy, without stem 128 23 18.75% Smooth, flowering 96 17 18.75% Smooth, without stem 42 8 6.25%

Many other cases have been tested by different writers and the general result is the [303] applicability of Mendel's formula to all cases complying with the given conditions.

Intentionally I have chosen for the last example two pairs of antagonisms, relating to the same pair of plants, and which may be tested in one experiment and combined in one calculation.

For the latter we need only assume the same conditions as mentioned before, but now for three different qualities. It is easily seen that the third quality would split each of our four groups into two smaller ones in the proportion of 3/4 : 1/4.

We would then get eight groups of the following composition:

9/16 X 3/4 = 27/64 or 42.2% 9/16 X 1/4 = 9/64 " 14.1% 3/16 X 3/4 = 9/64 " 14.1% 3/16 X 1/4 = 3/64 " 4.7% 3/16 X 3/4 = 9/64 " 14.1% 3/16 X 1/4 = 3/64 " 4.7% 1/16 X 3/4 = 3/64 " 4.7% 1/16 X 1/4 = 1/64 " 1.6%

The characters chosen for our experiment include the absence of stem and flowers in the first year, and therefore would require a second year to determine the flower-color on the perennial specimens. Instead of doing so I have taken another character, shown by the teeth of the capsules when opening. These curve outwards [304] in the red campion, but lack this capacity in the evening-campion, diverging only until an upright position is reached. The combination of hairs, colors and teeth gives eight groups, and the counting of their respective numbers of individuals gave the following:

Teeth Hairs Flowers of capsules Number % Calculated

Hairy red curved 91 47 42.2% Hairy red straight 15 7.5 14.1% Hairy white curved 23 12 14.1% Hairy white straight 17 8.5 4.7% Smooth red curved 23 12 14.1% Smooth red straight 9 4.5 4.7% Smooth white curved 5 2.5 4.7% Smooth white straight 12 6 1.6%

The agreement is as comprehensive as might be expected from an experiment with about 200 plants, and there can be no doubt that a repetition on a larger scale would give still closer agreement.

In the same way we might proceed to crosses with four or more differentiating characters. But each new character will double the number of the groups. Four characters will combine into 16 groups, five into 32, six into 64, seven into 128, etc. Hence it is easily seen that the size of the experiments must be made larger and larger in the same ratio, if we intend to expect numbers equally trustworthy. For [305] seven differentiating marks 16,384 individuals are required for a complete series. And in this set the group with the seven attributes all in a latent condition would contain only a single individual.