The Reproductive Cells of Hybrids.

The results of the previously described experiments induced further experiments, the results of which appear fitted to afford some conclusions as regards the composition of the egg and pollen cells of hybrids. An important matter for consideration is afforded in Pisum by the circumstance that among the progeny of the hybrids constant forms appear, and that this occurs, too, in all combinations of the associated characters. So far as experience goes, we find it in every case confirmed that constant progeny can only be formed when the egg cells and the fertilising pollen are of like character, so that both are provided with the material for creating quite similar individuals, as is the case with the normal fertilisation of pure species[38]. We must therefore regard it as essential that exactly similar factors are at work also in the production of the constant forms in the hybrid plants. Since the various constant forms are produced in one plant, or even in one flower of a plant, the conclusion appears logical that in the ovaries of the hybrids there are formed as many sorts of egg cells, and in the anthers as many sorts of pollen cells, as there are possible constant combination forms, and that these egg and pollen cells agree in their internal composition with those of the separate forms.

In point of fact it is possible to demonstrate theoretically that this hypothesis would fully suffice to account for the development of the hybrids in the separate generations, if we might at the same time assume that the various kinds of egg and pollen cells were formed in the hybrids on the average in equal numbers[39].

In order to bring these assumptions to an experimental proof, the following experiments were designed. Two forms which were constantly different in the form of the seed and the colour of the albumen were united by fertilisation.

If the differentiating characters are again indicated as A, B, a, b, we have:

AB,	seed parent;	ab,	pollen parent;
A,	form round;	a,	form angular;
B,	albumen yellow.	b,	albumen green.

The artificially fertilised seeds were sown together with several seeds of both original stocks, and the most vigorous examples were chosen for the reciprocal crossing. There were fertilised:

1. The hybrids

with the

pollen of

AB.

2. The hybrids

ab.

3. AB

the hybrids‍.

4. ab

the hybrids‍.

For each of these four experiments the whole of the flowers on three plants were fertilised. If the above theory be correct, there must be developed on the hybrids egg and pollen cells of the forms AB, Ab, aB, ab, and there would be combined:—

1. The egg cells AB, Ab, aB, ab with the pollen cells AB.

2. The egg cells AB, Ab, aB, ab with the pollen cells ab.

3. The egg cells AB with the pollen cells AB, Ab, aB, ab.

4. The egg cells ab with the pollen cells AB, Ab, aB, ab.

From each of these experiments there could then result only the following forms:—

1. AB, ABb, AaB, AaBb.
2. AaBb, Aab, aBb, ab.
3. AB, ABb, AaB, AaBb.
4. AaBb, Aab, aBb, ab.

If, furthermore, the several forms of the egg and pollen cells of the hybrids were produced on an average in equal numbers, then in each experiment the said four combinations should stand in the same ratio to each other. A perfect agreement in the numerical relations was, however, not to be expected, since in each fertilisation, even in normal cases, some egg cells remain undeveloped or subsequently die, and many even of the well-formed seeds fail to germinate when sown. The above assumption is also limited in so far that, while it demands the formation of an equal number of the various sorts of egg and pollen cells, it does not require that this should apply to each separate hybrid with mathematical exactness.

The first and second experiments had primarily the object of proving the composition of the hybrid egg cells, while the third and fourth experiments were to decide that of the pollen cells[40]. As is shown by the above demonstration the first and second experiments and the third and fourth experiments should produce precisely the same combinations, and even in the second year the result should be partially visible in the form and colour of the artificially fertilised seed. In the first and third experiments the dominant characters of form and colour, A and B, appear in each union, and are also partly constant and partly in hybrid union with the recessive characters a and b, for which reason they must impress their peculiarity upon the whole of the seeds. All seeds should therefore appear round and yellow, if the theory be justified. In the second and fourth experiments, on the other hand, one union is hybrid in form and in colour, and consequently the seeds are round and yellow; another is hybrid in form, but constant in the recessive character of colour, whence the seeds are round and green; the third is constant in the recessive character of form but hybrid in colour, consequently the seeds are angular and yellow; the fourth is constant in both recessive characters, so that the seeds are angular and green. In both these experiments there were consequently four sorts of seed to be expected—viz. round and yellow, round and green, angular and yellow, angular and green.

The crop fulfilled these expectations perfectly. There were obtained in the

1st

Experiment,

exclusively

round

yellow

seeds‍;

3rd

In the 2nd Experiment, 31 round and yellow, 26 round and green, 27 angular and yellow, 26 angular and green seeds.

In the 4th Experiment, 24 round and yellow, 25 round and green, 22 angular and yellow, 27 angular and green seeds.

A favourable result could now scarcely be doubted; the next generation must afford the final proof. From the seed sown there resulted for the first experiment 90 plants, and for the third 87 plants which fruited: these yielded for the—

1st Exp.	3rd Exp.
20	25	round yellow seeds	AB
23	19	round yellow and green seeds	ABb
25	22	round and angular yellow seeds	AaB
22	21	round and angular green and yellow seeds	AaBb

In the second and fourth experiments the round and yellow seeds yielded plants with round and angular yellow and green seeds, AaBb.

From the round green seeds plants resulted with round and angular green seeds, Aab.

The angular yellow seeds gave plants with angular yellow and green seeds, aBb.

From the angular green seeds plants were raised which yielded again only angular and green seeds, ab.

Although in these two experiments likewise some seeds did not germinate, the figures arrived at already in the previous year were not affected thereby, since each kind of seed gave plants which, as regards their seed, were like each other and different from the others. There resulted therefore from the

2nd Exp.	4th Exp.
31	24	plants	of	the form	AaBb
26	25	"		"	Aab
27	22	"		"	aBb
26	27	"		"	ab

In all the experiments, therefore, there appeared all the forms which the proposed theory demands, and also in nearly equal numbers.

In a further experiment the characters of floral colour and length of stem were experimented upon, and selection so made that in the third year of the experiment each character ought to appear in half of all the plants if the above theory were correct. A, B, a, b serve again as indicating the various characters.

A,	violet-red flowers.	a,	white flowers‍.
B,	axis long.	b,	axis short‍.

The form Ab was fertilised with ab, which produced the hybrid Aab. Furthermore, aB was also fertilised with ab, whence the hybrid aBb. In the second year, for further fertilisation, the hybrid Aab was used as seed parent, and hybrid aBb as pollen parent.

Seed parent, Aab.	Pollen parent, aBb.
Possible egg cells, Abab.	Pollen cells, aBab.

From the fertilisation between the possible egg and pollen cells four combinations should result, viz.:—

AaBb + aBb + Aab + ab.

From this it is perceived that, according to the above theory, in the third year of the experiment out of all the plants

Half

should

have

violet-red flowers (Aa),

Classes

1, 3

white flowers (a)

2, 4

a long axis (Bb)

1, 2

a short axis (b)

3, 4

From 45 fertilisations of the second year 187 seeds resulted, of which only 166 reached the flowering stage in the third year. Among these the separate classes appeared in the numbers following:—

Class.	Colour of flower.	Stem.
1	violet-red	long	47	times
2	white	long	40	"
3	violet-red	short	38	"
4	white	short	41	"

There consequently appeared—

The

violet-red

flower

colour

(Aa)

in 85

plants.

white

(a)

in 81

long stem

(Bb)

in 87

short "

(b)

in 79

The theory adduced is therefore satisfactorily confirmed in this experiment also.

For the characters of form of pod, colour of pod, and position of flowers experiments were also made on a small scale, and results obtained in perfect agreement. All combinations which were possible through the union of the differentiating characters duly appeared, and in nearly equal numbers.

Experimentally, therefore, the theory is justified that the pea hybrids form egg and pollen cells which, in their constitution, represent in equal numbers all constant forms which result from the combination of the characters when united in fertilisation.

The difference of the forms among the progeny of the hybrids, as well as the respective ratios of the numbers in which they are observed, find a sufficient explanation in the principle above deduced. The simplest case is afforded by the developmental series of each pair of differentiating characters. This series is represented by the expression A + 2Aa + a, in which A and a signify the forms with constant differentiating characters, and Aa the hybrid form of both. It includes in three different classes four individuals. In the formation of these, pollen and egg cells of the form A and a take part on the average equally in the fertilisation; hence each form [occurs] twice, since four individuals are formed. There participate consequently in the fertilisation—

The pollen cells A + A + a + a
The egg cells A + A + a + a.

It remains, therefore, purely a matter of chance which of the two sorts of pollen will become united with each separate egg cell. According, however, to the law of probability, it will always happen, on the average of many cases, that each pollen form A and a will unite equally often with each egg cell form A and a, consequently one of the two pollen cells A in the fertilisation will meet with the egg cell A and the other with an egg cell a, and so likewise one pollen cell a will unite with an egg cell A, and the other with egg cell a.

Pollen cells
Egg cells

The result of the fertilisation may be made clear by putting the signs for the conjoined egg and pollen cells in the form of fractions, those for the pollen cells above and those for the egg cells below the line. We then have

A/A + A/A + a/a + a/a.

In the first and fourth term the egg and pollen cells are of like kind, consequently the product of their union must be constant, viz. A and a; in the second and third, on the other hand, there again results a union of the two differentiating characters of the stocks, consequently the forms resulting from these fertilisations are identical with those of the hybrid from which they sprang. There occurs accordingly a repeated hybridisation. This explains the striking fact that the hybrids are able to produce, besides the two parental forms, offspring which are like themselves; A/A and a/a both give the same union Aa, since, as already remarked above, it makes no difference in the result of fertilisation to which of the two characters the pollen or egg cells belong. We may write then—

A/A + A/A + a/a + a/a = A + 2Aa + a.

This represents the average result of the self-fertilisation of the hybrids when two differentiating characters are united in them. In solitary flowers and in solitary plants, however, the ratios in which the forms of the series are produced may suffer not inconsiderable fluctuations[41]. Apart from the fact that the numbers in which both sorts of egg cells occur in the seed vessels can only be regarded as equal on the average, it remains purely a matter of chance which of the two sorts of pollen may fertilise each separate egg cell. For this reason the separate values must necessarily be subject to fluctuations, and there are even extreme cases possible, as were described earlier in connection with the experiments on the form of the seed and the colour of the albumen. The true ratios of the numbers can only be ascertained by an average deduced from the sum of as many single values as possible; the greater the number the more are merely chance elements eliminated.

The developmental series for hybrids in which two kinds of differentiating characters are united contains among sixteen individuals nine different forms, viz., AB + Ab + aB + ab + 2ABb + 2aBb + 2AaB + 2Aab + 4AaBb. Between the differentiating characters of the original stocks Aa and Bb four constant combinations are possible, and consequently the hybrids produce the corresponding four forms of egg and pollen cells AB, Ab, aB, ab, and each of these will on the average figure four times in the fertilisation, since sixteen individuals are included in the series. Therefore the participators in the fertilisation are—

Pollen cells

AB + AB + AB + AB + Ab + Ab + Ab + Ab +

aB + aB + aB + aB + ab + ab + ab + ab.

Egg cells

AB + AB + AB + AB + Ab + Ab + Ab + Ab +

aB + aB + aB + aB + ab + ab + ab + ab.

In the process of fertilisation each pollen form unites on an average equally often with each egg cell form, so that each of the four pollen cells AB unites once with one of the forms of egg cell AB, Ab, aB, ab. In precisely the same way the rest of the pollen cells of the forms Ab, aB, ab unite with all the other egg cells. We obtain therefore—

AB/AB + AB/Ab + AB/aB + AB/ab + Ab/AB + Ab/Ab + Ab/aB + Ab/ab +
aB/AB + aB/Ab + aB/aB + aB/ab + ab/AB + ab/Ab + ab/aB + ab/ab,

AB + ABb + AaB + AaBb + ABb + Ab + AaBb + Aab +
AaB + AaBb + aB + aBb + AaBb + Aab + aBb + ab = AB +
Ab + aB + ab + 2ABb + 2aBb + 2AaB + 2Aab + 4AaBb[42].

In precisely similar fashion is the developmental series of hybrids exhibited when three kinds of differentiating characters are conjoined in them. The hybrids form eight various kinds of egg and pollen cells—ABC, ABc, AbC, Abc, aBC, aBc, abC, abc—and each pollen form unites itself again on the average once with each form of egg cell.

The law of combination of different characters which governs the development of the hybrids finds therefore its foundation and explanation in the principle enunciated, that the hybrids produce egg cells and pollen cells which in equal numbers represent all constant forms which result from the combinations of the characters brought together in fertilisation.