A) The Appearance of New Vital Phenomena|
I - Practical Part
After having observed mutation among plants for several years, I came to the conclusion that in order to have any chance of provoking it systematically, it was necessary to produce and develop the plant individual (in the state of sexual elements or fecundated ovule) in surroundings different from those offered by nature.
That is the subject developed in this work. I thought it might be interesting before proceeding to the theoretical part, to relate some experiments, the account of which I hope will induce the reader to study the rest.
Seedling bred from seeds obtained by premature fertilisation (after operation direct in the ovary).
One day at the beginning of 1922, my little boy of ten wanted to make me believe that he had changed the male flowers of the Impatiens sultani into female flowers and that he had successfully fertilised them. Highly amused (the flowers of Impatiens are hermaphrodites), but wishing to see what the little fellow meant, I made him repeat his experiment before me. He took a flower and with a penknife scratched the covering of the ovary surmounted by the stamens, thus exposing the ovary bearing a rudiment of pistil. I understood: by this slight operation he simply took off the covering a few days before its normal fall, thus allowing of the premature fertilisation. I myself repeated the experiment twenty times and each time the fertilisation took place perfectly.
I however ascertained that it was almost impossible to take off this covering by force without injuring the pistil slightly, and it even sometimes happened that the pistil and the upper part of the ovary were cut, but that in spite of this, the fertilisation took place normally. At first I was very astonished; is not the part played by the pistil in the process of fertilisation considered very important? I reflected, however, that if the possibility of fertilising the ovary was really independent of the existence of a pistil, it would follow that the maturity of the ovules would not necessarily correspond with the complete development of the pistil; now, all fertilisation produced successfully after having taken off the covering by force, becoming premature, confirmed these suppositions. And I ventured to experiment on buds of Impatiens still much younger and at different degrees of development. I injured several ovaries, but, nevertheless, the success was complete.
|United ovaries of Impatiens showing flowers of peculiar conformation. Seedlings bred from seeds obtained by premature fertilisation (after operation direct in the ovary).|
By experimenting on buds, by fertilising thus direct in the imperfectly developed ovary, conditions are established which nature does not realize: the pollen not passing through the pistil comes directly on the ovules, which are in a state of comparatively imperfect maturity. Thus there are necessarily other physiological influences which, theoretically, seem to be able to manifest themselves by the appearance of new phenomena or by morphological results different from those in normal fertilisation.
In fact, I saw the seeds of the Impatiens sultani germinate by growing two adjacent leaves instead of opposite ones. (Fig. 1). Certain seedlings showed among the normal flowers, here and there, flowers with united ovaries (two and even three), thus showing corollas of strange formation (Fig. 2 and 3). Other seedlings had leaves more or less abnormal; one of them even had all its leaves different in shape (Fig. 4).
It is an interesting plant in many respects.
The young cuttings put into pots develop at the beginning of their growth less abnormal leaves than afterwards. Placed right in the ground, that is to say, under the greenhouse shelves, where conditions of growth for the Impatiens are especially advantageous, this plant produces more regular leaves than it does in the cultivation in pots. In short, it presents a collection of troubles, physiological, hereditary, varied and very sensitive to differences of cultivation.
The flowers also exhibit an anomaly: they are of a brick-red colour, darker in places, which gives them a strange aspect. They get paler with age, the colour becoming white, slightly pinkish, a thing that I have never noticed with the flowers of the Impatiens.
The results obtained on this plant are all the more important that these anomalies continue in the offspring bred by self-fertilisation.
I gave an account of these varied results as they occurred to my friend Rabaud who, although highly interested, made objections several times, the principal one being the possibility of having operated, in spite of precautions taken, with flowers already accidentally fertilised before my intervention.
Although this accident seemed to me impossible I resolved to do better.
To begin the experiment again on more easily controllable material (the ovary of the Impatiens being very minute), I chose as subjects the flowers of Clivia. I took the precaution to remove the anther long before the opening of the corolla and did not keep any other Clivia either in bud or in flower in the greenhouse. On the opening of the flowers, the fertilisation after operation direct in the ovary was a great success. Rabaud still remained sceptical and demanded experiments with dioecious flowers. I had none at hand. I endeavored to dispel his doubts by another method. I repeated the experiment on the buds of the Clivia when they were still quite green. (I saw later that in a hothouse at 15° centigr. that means ten or twelve days before the opening of the flower, thus thirteen or fourteen days before the normal development of the pistil). I cut the corolla and the pistil as far as the ovary, dried the incision with blotting-paper and fertilised... The fertilisation succeeded perfectly.
And I noticed that with the Clivia, in all fertilisation (after operation) before the normal period, the rapid development of the seeds in formation forces the outer covering of the ovary to stretch prematurely. The result is a change in the general outer aspect of the latter, all the more noticeable the farther it takes place from the time of natural fertilisation. When fertilisation takes place after operation, as in the case quoted above, ten to twelve days before the possible opening of the flower, the following facts are established:
The colouring of the outer envelope of the ovary changes rapidly in a few week from a shiny dark green (natural aspect) to a glaucous greyish, dull green, crossed by brownish, longitudinal stripes, starting from the top. This covering stretches tightly over the ovary, which assumes a round form with a smooth surface, very different from that of an ovary fertilised at the normal period. I noted the fact three times. The photo (fig. 5) clearly shows the phenomenon (fertilisation in the middle). The plant represented in the photograph still bears a mature fertilisation of the preceding year and another of about the same as that showing the phenomena already mentioned. The comparison between these fertilisations is interesting.
I am curious to see what my series of Clivia will give, bred from seeds coming from ovaries fertilised after operation, two, four, one of them even ten to twelve days before the possible opening of the flower. Up to now (the plants are more than two years old), there is nothing abnormal in the leaves. I am awaiting the appearance of the flowers. It is not out of the question that it may be necessary to repeat the same influence for several consecutive generations before seeing the appearance of any new phenomenon.
I also fertilised some orchids, some Cypripedium, in this way with equal success. The seeds of this Cypripedium come up with difficulty. Still, I got a seedling, but unfortunately it died. All this allows me to state that the seeds were generally good and that the experiment with the Cypripedium has fully succeeded (especially with Cypr. insigne sanderae). I tried on Cattleya. I diluted the pollen by fertilising any Cattleya available; the next day I cut the corolla of the individual upon which I wanted to try the experiment as far as the ovary, I dried the incision with blotting-paper and covered it over with the diluted pollen taken from the Cattleya fertilised the day before. I made three, four similar trials without success.
The fertilisation after operation right into the ovary of a full-blown flower, that is to say, of an ovary normally developed might sometimes be of real interest; also in cases in which the object would not be precisely to provoke the appearance of fresh phenomena, but simply to attempt crosses between allied species (for example between tomatoes and potatoes or tobacco, etc. etc.) in which the conformation of the pistil might (?) form an obstacle to the success.
|(1) I must, however, point out that in fertilising after
operation direct in the ovary, even a fortnight before the possible
opening of the flower, one is not so certain of being able to produce
premature fertilisation. It is not impossible that the pollen scattered on
the ovary remains alive without acting for several days, until the ovules
have attained their normal development and that, then only, fertilisation takes place. In this case it would not be premature. This is
an important question which might be solved, I think, by our means of
I must, however, remark that besides the abnormal individuals obtained with Impatiens, I have just observed (May 1926) the appearance of abnormal flowers in a seedling of Clivia, bred from seeds obtained from an ovary fertilised 10 to 12 days before the possible opening of the flower (see p. 15 on this subject). This would tend to confirm my thesis of the possibility of premature fertilisation (after operation direct in the ovary).
A vast field of experiments opens out for the man of science as well as for the practical breeder. It would be interesting to see where fertilisation would lead us after direction operation in the ovary, practised on two, three, several consecutive generations, by making use each time of individuals showing the most interesting results.
I might enlarge more fully on this, and enter into considerations logically permissible. The subject is too new and too vast, and I hand it over to researchers, leaving all scientific discussion to experts. I take the liberty, however, of adding that there will probably be optimum points to be established for the utmost limit of possibility of fertilisation of the ovules and for each species of plant.
The possibility of fertilising the ovules after operation direct in the ovary at a time when it is not effected by nature suggested the following thought to my mind: We note the posibility of premature fertilisation. How do the seeds themselves act? Does their apparent maturity correspond with the optimum point possible of germination? I immediately made experiments and again it was the Impatiens sultani of which I made use.
I fertilised the specimens normally. In a greenhouse the seeds take (June-July) twenty-three to twenty-four days to mature. From the seventeenth day, I open the ovaries and sow the seed, still green, on very wet Sphagnum. After one or two days, the green colour changes, the seeds assume the brown tone of ripe seeds, then they germinate and yield plants more puny than the normal type.
I then take still younger ovaries, some even perhaps too young, and sow all the seeds pell-mell. The germination takes place very slowly and produces very puny specimens. They revive, however, after a time and become perfect plant, among these, there are some the flowers of which seem to have a colour that I believe to be new; but this I cannot, however, vouch for. Several other plants, on the other hand, especially two, produced flowers of a clearly different shape, not opening so well, of a rounder form and with petals showing curly edges (see photo.) Here, also, I observed physiological disturbances, but only with the flower, the plants being vigorous and the leaves normal. I kept these two individuals: they produced, by self-fertilisation, offspring showing the same phenomena. Here, once more, the morphological changes were definitely established in the offspring (fig. 6). (The two flowers below are normal).
|The three abnormal flowers (at the top) come from seeds which have germinated before their time (the anomaly is hereditary). The two flowers at the bottom are normal.|
After this success, I immediately resolved to repeat these experiments on rye. This was in 1923. The fertilisation having taken place at the end of May, the maturity could be expected from the 8th to the 15th of July. On the 23rd of June I began the experiment. I chose a well-formed ear, of course still quite green at this date. I removed a grain at the top, another in the middle, and a third at the bottom of the ear. I had previously prepared some very damp turf, superficially divided into twenty-four compartments. I sowed the three grains together in one compartment. On the twenty-fourth of June, I repeated the same operation, and likewise on the following days. Thus I had theoretically (for in reality it is not so certain) subjects of different ages, differing by one day. I saw the green grain assuming a colour something like that of ripe grain, that is to say, brownish; then germination took place, but only slowly. It went on very irregularly. I believe the experiment was badly arranged. Many of the subjects rotted. At last I kept a few of them and planted them in the open ground. The following year, in 1924, I had some very fine specimens with a good crop. In June 1924, I decided to make the experiment again, using plants resulting from the trials of 1923. I did not observe anything abnormal in these plants bred from grains which had prematurely germinated. The fertilisation took place quite well. In short, except that the specimens seemed to me to be very strong, which might proceed from causes absolutely foreign to my intervention, there was nothing special to note. I decided to make use of the strongest individual, having fifty-four ears, saying to myself that if it owes its strength to the premature germination, my mind will be settled on this point at the second influenced generation.
On the 22nd of June, I once more take my pan of turf, divided into twenty-four compartments, and every day I deposit in one compartment three grains of green rye, as in the preceding year. The same phenomena were reproduced as in 1923. Besides, I am led to a new discovery: on the 1st of July, I notice that one of the three grains sown the previous day has a tiny white point at its tip. I look at it through a magnifying glass and I see that in tearing the grain too roughly from the ear, I have injured and laid bare the germ. (The little white point was the germ). I was interested, especially as I found that the exposure of the germ was caused by the tearing away of two thick, outer coverings. The following day I only notice a slight change in the white point. But on the third day of July, to my great amazement, I see that the germ has grown, though not one of the seeds sown since the 22nd of June gives any appearance of life. I immediately place beside this green germ «operated on» by chance, a grain of normal rye «witness», coming from one or other of the crops of 1923; this grain, germinating after twenty-four hours, allows me to make observations by comparison. Here is the copy of the notes taken concerning this green grain, the germ of which has been accidentally liberated: Germination supposed to have begun on the third of July. Germination clearly ascertained on the 4th of July. A little shoot of from 4 to 5 millimeters showed itself on the 5th of July. The grain put in a pot on the 6th of July (with the «witness»), when the shoot is at least 8 millimeters.
|Various phases of the premature germination with rye after the liberation of the germ by operation.|
|(1) The liberation of the germ cannot be effected in cereals without a certain amount of skill. (Moreover, this will be the case in every trial attempted with other sorts of seeds.) Consequently, it will be interesting to state how I proceed. Fig. A shows the side of the grain bearing the germ; fig. B, the other side. I make a horizontal incision round the germ, then a longitudinal incision on the side opposite to the germ C. With my nail or with fine pincers, I open the coverings in C, then I try, by pulling quickly, to remove them. It is unnecessary to say that deep incisions must not be made, as a general they ought simply to cut the coverings as far as the cotyledons. Perhaps practice may teach simpler methods.|
On the following days, only the little green stick continues to grow, the part that has become opaque not moving any more. The growth of this individual is slow. The rye «witness» grows three times as quickly at least (fig. 7). In order to control the experiment, I took, on July 6th, from any available field, some green grains of rye not older than from eighteen to nineteen days since their fertilisation. Before planting them, I liberated the germs by operating. The same phenomenon of the glassy shoot manifested itself. Thus it was not a phenomenon peculiar to subjects coming from a plant bred from grain which has prematurely sprouted, it is a phenomenon to all grains of green rye that are made to germinate prematurely after the liberation of the germ by operation. Green grains of wheat act in the same way, but in the two cases the complete success depends on the age of the grain in formation for cereals, the younger giving the greater success. This causes curious variations of the phenomenon and the research for the optimum point of germination possible after liberation of the germ will be very interesting (1).
By comparison with the natural germination of rye takes place by the appearance of an opaque shoot of an ivory colour which grows rapidly. In our experiment repeated dozens of times, we have always had the growth in three stages: a) the appearance of the glassy shoot which attains the maximum growth, then stops (8 to 10 millimeters); b) the appearance of the little green stick which grows up the central canal of the glassy shoot; c) the rapid change in the glassy shoot which, losing its transparency, takes an opaque, greenish colour. On the whole, we observe, besides the appearance of absolutely fresh phenomena, a modification in the rhythm of growth. The «seedlings» that I kept, grew very slowly, they remained puny for a long time. The photo already mentiond (fig. 7) shows the comparative sizes between the normal, individual witness and the grain of green rye of which the germ had been laid bare. But this puny plant soon recovered and became a very strong specimen. Here I expected, considering the result obtained with the Impatiens under conditions far less abnormal, extraordinary results; for I had established for the individual in formation conditions of growth entirely deviating from the way of nature. Well! it was not so at all; at least, I did not observe anything special visible; I have obtained an ample crop of grains which I sowed in October 1925.
It will not be without interest to describe the result of the following experiment. I had noticed that it was not very easy to lay bare the germ in the grain still green. The latter often becomes detached, and this accident suggested to me the idea of seeing if the germ alone would be satisfied with heat and moisture for its development.
A) Germ taken from
corn still green.
B) Development of this germ after remaining 3 days on the damp sphagnum.
I have experimented with several germs; they do not all grow equally quickly, but they all tend to take the shape of fig. B. By comparing them with germs laid bare by operation, but attached to the cotyledons remaining during the same time on the damp turf, pose that the faculty of easily absorbing the dampness is the cause of the rapid development of these isolated germs. At a certain moment they rotted. Perhaps by placing them on a material resembling in chemical composition that of the cotyledons, one might hope for a better result. It would, in any case, be worth while to control the degree and time of vitality of these isolated germs.
These experiments made a great impression on me. I do not wish to draw many conclusions from them; however, I can affirm, after all the preceding experiments, that it is indisputably shown that in the plant-world the interaction of vital forces is not necessarily manifested in the rhythm established by heredity, but that it can manifest itself by fresh phenomena and by unknown morphological variations. Moreover, this is what I had learnt from the excellent experiment of Gerassimow, related by H. De Vries in «Oorsprong en bevruchting der bloemen». Gerassimow, by operating on the filamentous Seaweeds of the Spirogira family (which propagate by division of the cells) had noted that, if, at a certain moment during the phenomenon, the temperature is suddenly lowered, the two halves of the nucleus of the divided cell place themselves on one side of the division, so that one half of the cell contains two nuclei and the other none at all. The former multiplies itself by cells with two nuclei, whilst the latter is destined to die (1).
I have always been so enthusiastic about this experiment that I am glad to be able to quote it here and to give it the priority of principle (2).
|Fig. 8. Long-bearded ear of corn has undergone an influence caused by temperature before fertilisation.
The other ear from the same stalk is normal.
In June 1923, I made some experiments by the process which I have called (see theoretical part) the cultivation in a different environment, by means of temperature, on ears of rye.
The flowering of the rye, begun about 6-8th of June, was terminated about the 15-16th of June. The experiments began on the 17th of June, immediately after the fertilisation. They consisted in submitting the individuals in formation (the ear) to a temperature impossible to be realised by nature, the stalk and root remaining in the natural environment. Among others, I made the following experiments:
a) An ear (immediately after fertilisation) was placed in a temp of +2° centigr. (= about 35 Fahrenheit) from the 15th of June till the 7th of July. After this date it was put back in its natural environment.
b) Another ear was placed from the 15th of June till the 3rd of July, at +32° to 37° centigr. (90 to 100 F.). After this date it was also put back into its natural environment.
In both cases the roots and stem remained intact. I should like to point out that in both cases the grains in formation were influenced by different extremes of temperature with great exactness. I repeat, this represents a collection of conditions which could not be realised in the natural environment. I made several other experiments, but I will not dwell on them here. I shall only quote this one which is an experiment of collective influence. On the 15th of June, I placed a plant of rye in a pot inside a pan of which the sides were double, the space between being filled with ice. I thus obtained a temperature of +2° to +3° to +4° centigr. (35 to 38 to 40 Fahr.). The whole: roots, stem and plant, were thus forced for ten days in these abnormal temperatures. I have only a few short notes on this subject. I can, however, say that, in spite of the exposure to such a low temperature and in the dark, the ear and stalk remained perfectly green.
In August the crop of all these ears had a surprise in store for me. I obtained grains different in shape and colour from the normal for each kind of influence. There were, especially, grains with quite a pink tip and of a very curious tapering shape. On comparing the grains one with the other, one would have said that they came from different species. In October I sowed the grains of each ear in a square of special soil, and neither the ears nor the crop of the following year gave me anything visibly abnormal.
In 1924, I began my experiments once more. But now I influenced before the fertilisation. I set an ear from the 23rd of May to the 3rd of June at +2° centigr. (35° Fahr.); after the 3rd of June it was put back in the natural environment. The fertilisation took place much later than with the ears that had not been influenced.
I obtained several very fine grain, which yielded, in 1925, some ears of exceptional strength. I dare not attribute this result to the influence they had undergone, my observations not having been made with sufficient accuracy. I had a plentiful crop from these ears. I shall sow them in October 1925. Besides, I did this. On the 23rd of May, I placed an ear at 2° centigr. (35° Fahr.), the stalk remaining in the natural environment, but the roots at +30° centigr. (55° Fahr.) and this during ten days. Then the whole was put back into the normal environment. The ear developed very curiously, the photo (fig. 8) shows it compared with a normal ear, the latter with little awns. It flowered and yielded several forming grains of a large size and of a curious appearance, that I could not bring to maturity, once again for want of skill in manipulating.
I must add that for all the preceding experiments, the parts of the plant influenced (ear or stalk) remained in darkness.
Thus there were, in reality, influences working in conjunction with obscurity. If necessary, this latter influence might be eliminated.
In the communication of no. 7 of Floralia, February 15th 1929, concerning new views in the domain of horticulture, especially of orchids, it was casually mentioned how one of Reychler's methods to produce mutation artificially consists in cutting away the stigma before fecundation and applying pollen on the wound. In spite of the absence of the stigma seeds form, but the plants grown from them show deviations, that experimentally prove to be hereditary. According to a written communication of Reychler's, mutation was originated in this way not only with Impatiens Sultani but also with Clivia.
Not long ago I repeated these experiments of Reychler's provisionally in order to ascertain whether effectively in the said circumstances germination of the pollen on the wounded surface took place and the pollen tubes penetrated into the cavity of the ovary, as far as the ovules.
I wrongly stated in the said communication of no. 7 of Floralia, that in Reychler's experiments pollen was brought on the surface of the wound some days after the cutting away of the stigma. On the contrary, Reychler impressively states that pollination must occur immediately after the operation, but that first the surface of the wound must be carefully dried with blotting paper and afterwards during some days protected against the sun and humidity. According to these rules I continued the preliminary experiments in the laboratory with cut flowers, scarcely or not yet opened, of Narcissus poeticus and of the Darwin tulip, of which the stamens had not yet opened and no pollen showed on the pistils.
With the narciss the corolla with the style enclosed in its tube was cut, sometimes at the level of the ovary, at other times at a quarter, a half, three quarters of the height of the tube of the corolla.
With the tulip I clipped off corolla and stamens and cut the pistils quite away. After the operation I dried carefully with filtering paper and immediately pollinated abundantly.
Three resp. six days later the ovaries were put into alcohol and examined in the usual way under the microscope.
With both species the result was favourable; with the narciss it was an extraordinary success. Fig. 1 shows, powerfully magnified, the pollen grains of Narcissus poeticus germinating, three days after pollination, on the surface of the wound caused by cutting away the tube of the corolla and the style just above the ovary. The cut cells and those immediately below them died off. Already the living, deeper lying cells here and there are forming new partitions parallel with the surface of the wound, a first indication of a layer of tissue that will isolate the wound.
Fig. 2 summarily sketches, very feebly magnified, a longitudinal section section of the upper part of the ovary of Narcissus poeticus, with the lower end of the tube of the corolla and style cut away just above the ovary (six days after the operation and pollination took place). On the surface of the wound the pollen grains can be seen. Through the middle of the style the tube continues into the cavity of the ovary. In the ovary at the left one sees a partition, at the right the cavity of one of the three compartments with some ovules. Above in the cavity of the ovary a clew of pollen tubes can be detected.
Fig. 3 gives a strong enlargement of a longitudinal section through the style, with the rootlike tubes of pollen grains, growing from the wound towards the cavity of the ovary, three days after pollination.
Finally we see on fig. 4, feebly magnified, a preparation of a free ovule from the ovary of narciss, six days after pollination occurred on the surface of the wound. The pollen tube that penetrated through the micropyle still adheres to it.
With the Darwin-tulip as well germination of the pollen on the wound was ascertained as also growing of the pollen into the cavity of the ovary.
It can therefore not be doubted that in this way effective pollination can take place on the surface of the wound, after removal of the stigma. Whether it will succeed with all plants, is as yet an open question, but considering the fact that up to now it succeeded in all the five cases in which it was tried, by Reychler with Impatiens Sultani, Clivia and Cypripedium Sanderae, I deem it probable that the possibility will hold generally.
There is some reason to suppose that pollen grain and germ cell, the germ itself and the young germing plant, are in a sensitive period, in which they can react on all kinds of disturbing influences with hereditary deviations, with mutation. It is a well known phenomenon that after crossing different forms there sometimes appear new properties, not founded on a mere combination of the qualities of the parents. This too can be considered as mutation resulting from the abnormal character of the combination of a pollen grain and a germ cell, that are not well matched.
In the posterity of crosses we must indeed distinguish between normal products of crosses in which we have exclusively all sorts of combinations of the properties of the parents, and mutants, in which quite new properties appear.
The method of Reychler, pollination on the surface of the wound after removal of the stigma, helps the practical breeder to an easy way of artificially provoking the appearance of new forms.
In order to make the experiments successful, it is of capital importance to cut away the corolla before it unfolded and before the stamens opened, to dry carefully the surface of the wound with blotting-paper or filtering-paper, richly pollinating it immediately afterwards and finally protecting it during some days against direct heat of the sun and wetting either by rain or by dew.