Bull. Peony News 6: 2-9. (1918)
THE PRESERVATION OF POLLEN FOR HYBRIDIZING
A. P. Saunders

The hybridist does not as a rule make as much use as he might of the fact that pollen may be successfully kept for some time, and under favorable conditions maintains its fertilizing power to quite an amazing degree. For the benefit of those who are working on the cross-breeding of peonies or other flowers it has seemed worth while to gather together some of the results that have been obtained by experiment; all the more since the information has not found its way to any extent into general botanical literature. We may thus benefit by what has already been learned and perhaps be encouraged to devise still better means for carrying over the pollen beyond the blooming time of the plant from which it is taken.

Certain facts are fairly well known which show the great vitality of the pollen of a few species of plants. For instance, it is a common practice to gather pollen from tomato plants grown out of doors during the late summer and fall months, and hold it over for use on plants grown under glass in winter. Grape pollen also is said to retain its vitality for fully two months; and that of the date palm for a year or more. Carnation pollen may be kept in closely stoppered bottles for several weeks, and may thus be shipped from one part of the country to another. In preparing such pollen for preservation it is first to be thoroughly dried, and then sealed up tightly enough so that little or no moisture can get to it. For plants native to moist countries, the drying should be done in the shade, while for those native to arid regions it may be more quickly done in direct sunlight.

It is said that in the West Indies the pollen of certain plants is commonly preserved and distributed by laying it between sheets of dry blotting paper and enclosing these in cardboard boxes.

An important paper by M. Pfundt on the general subject of the influence of humidity on the vitality of pollen, appeared in Pringsheim's Jahrbücher der wissenschaftlichen Botanik, Vol. 47, 1910. From this paper most of the following material is taken.

It is of historical interest that in 1712, Kampfer noted the practice of the Orientals, who cut off the male blossoms of the date palm and hang them up in a dry place, by which means the pollen is preserved until the following year. Kölreuter in 1766 records his having successfully preserved the pollen of Chieranthus cheiri for 14 days; and Gartner in 1844 describes quite an elaborate set of experiments in which the pollen of various plants was studied with relation to the preservation of its vitality. In all of these experiments the test of vitality was the actual fertilization of blooms and the production of seed.

This method was tedious and involved a great amount of labor where anything like an elaborate series of experiments was to be carried out. Consider a moment the method of fertilization on the plant. The pollen grain having been deposited on the stigma, where it is held by the viscid fluid, sooner or later sends out a process called the pollen tube, which penetrates down to the ovum, and through the means of which a part of the contents of the pollen grain are ultimately conveyed to the ovum, effecting fertilization. Hence it is reasonable to suppose that the ability of pollen grains to send out a pollen tube is as good a test of their vitality as the actual fertilization and production of seed on the plant.

Now it has been found that by putting pollen grains into a sugar solution of appropriate strength, the formation of the pollen tube can be brought about on a glass slide which can be examined under the microscope. This is the method which has been used in the later researches on the subject. It is not at all difficult to observe the formation of the pollen tube in this way, the tube itself being a gut-like process extending to a surprising length in the liquid. If a solution of cane sugar in water is prepared of about 15 to 20 per cent. concentration—say one-half ounce (two level teaspoonfuls) of cane sugar in half a glass of water-the pollen of a good many species of garden plants will readily germinate in such a solution. Iris pollen tested during the preparation of these notes, showed very good pollen tube formation within three or four hours. A drop of the solution on a microscope slide answers very well if kept from evaporation; the drop may be covered with a thin cover glass, though there is danger of crushing the pollen grains unless this is very carefully done. The best arrangement, if it is available, is one of those slides with a little well in the centre; a drop of sugar solution is then placed on a cover glass with the pollen, and the cover glass inverted over the well. This forms a hanging drop which is protected from evaporation. By such observations, the vitality of pollen may be determined, but experiment has shown that pollen tube formation gets very much slower after the pollen has been preserved for some time; samples of pollen that had been kept several months took as much as three or four days before pollen tube formation began, and it may well be doubted whether such pollen would be of much use under field conditions.

In investigating the vitality of various pollens, Pfundt took samples of them and placed them either in the air of the room or in a desiccator (a glass vessel tightly closed from the air) containing a solution which would maintain a fixed degree of humidity within the vessel.

Ordinarily the air on a dry day contains about 40 per cent. of the maximum humidity. Immediately after rain this may rise to 90 or even 100 per cent. Sixty per cent, is said to be about right for our comfort. In the experiments Pfundt carried out, he records that the humidity of the air of the room in which the samples were kept ran from 30 to 50 per cent. in winter, and from 40 to 80 per cent, in summer. Hence the samples exposed to the air of the room were under a degree of humidity which varied between wide limits with the changes of the weather. In a desiccating vessel one may adjust the humidity to any desired degree by using mixtures of sulphuric acid and water of various concentrations.

Pure concentrated sulphuric acid gives a humidity very near zero, for it is a strong absorbent of water. A mixture of 54 per cent. sulphuric acid and 46 per cent, of water gives a humidity equal to about 30 per cent. of the maximum. That would be roughly dry weather conditions. Acid and water in the proportion 37 per cent. of acid to 63 per cent. of water gives 60 per cent. humidity; moist weather conditions. Whereas 15 per cent. acid and 85 per cent. water gives 90 per cent, humidity; muggy rainy weather conditions.

The experiments show that rainy weather conditions are very bad for the preservation of pollen, and that the best results in longevity are usually attained under dry weather conditions, and even complete dryness is very much more favorable to the life of the pollen grain than is too much moisture. The samples under observation were kept in a dark cupboard, and the temperature of the room was ordinarily about 65 Fahrenheit.

The following table gives the longevity of various pollens in days under the different conditions as specified, the figures 90, 60, 30 per cent. referring to the percentage humidity maintained by mixtures of sulphuric acid and water, as explained above.

ORDER SPECIES In air 90 p.c. 60 p.c. 30 p.c. Over strong acid,
i. e. zero humidity
Liliaceae
  Colchicum autumnale 92 16 32 229 121
  Hemerocallis fulva 11 2 6 20 26
  Tulipa Gesneriana 37 23 43 108 92
  Galanthus nivalis 42 27 42 76 56
  Iris graminea 20 3 16 48 57
Ranunculaceae
  Peonia albifiora 56 14 39 157 157
  Trollius europaeus 29 9 29 102 124
Glumiflorae
  Zea mays 1 1 1 1 1
  Poa compressa 1 1 1 1 1
Rosales
  Prunus avium 28 12 25 102 126
  Lupinus perennis 129 12 43 260 164
  Viola odorata 35 19 28 217 235
  Primula elatior 56 18 56 155 179

Several facts stand out strikingly from this table. One is the great variations in the different genera. The grasses are conspicuous for their short-livedness, whereas many of the others, especially some of the early spring and late autumn blooming plants,—Colchicum, Viola, Primula,—show an even more surprising longevity.

Wetting is very injurious to pollen, and diminishes its vitality a great deal. Pollen of Tropaeolum majus wetted for two minutes and then placed in artificially dried air, maintained its vitality for only two days, whereas the same pollen unwetted remained vital under similar conditions for 88 days. And the longer the wetting continues, the more disastrous the effect. The flowers—those that can do so-try their best to prevent the wetting of the pollen and stigma by furling up tightly when rain comes, and at night, when dew is falling.

The longevity of many of the pollens included in the above table, was certainly not to be expected, and one can hardly imagine what end in the economy of nature is met by endowing the pollen with a vitality of which it can hardly ever take advantage under natural conditions. Fertilization on the plant is generally completed within a few hours after the pollen is applied to the stigma, though there are a few exceptional cases on record, as for example Betula alba, which is reported as requiring about four weeks.

Most pollens will not form pollen tubes at low temperatures—anything approaching the freezing point—but in the case of a few of the spring flowers—Crocus, Snowdrop, Christmas Rose—it was found possible to get pollen tube formation at a temperature of only about 40 degrees Fahrenheit.

Pollen is particularly susceptible to the attack of mould, which is indeed an even worse enemy than moisture. Hence in exact experiments it is necessary to use precautions in making up solutions for use. After adding the required amount of cane sugar to make whatever concentration is desired, the solution should be sterilized, most easily by heating. Such a solution should be pretty free from mould spores; yet in the tests on very old samples of pollen, where pollen tube formation was slow, Pfundt states that it was impossible to keep the tests going for more than three or four days without mould appearing.

Pollen tube formation may sometimes be observed in pure water. If a little of the pollen be scattered on a microscope slide, a tiny drop of water added, and the whole gently covered with a cover glass, the pollen grains will be seen to swell, and within a few hours or even in less than one hour, pollen tube formation may begin. Too much must not be expected under these conditions, for at the best only about 20 or 30 per cent. of the pollen grains show vitality even when perfectly fresh.

For ordinary purposes of testing, it is probably best to use a sugar solution as I have described, though it is to be remembered that the same concentration does not suit all pollen equally well. However, if positive results are obtained showing the sample to have retained its vitality, nothing more is needed. If the results are negative, which will be more likely with pollen that has been kept over for some time, then it may be necessary to use sterilized solutions in order to be able to give the test a longer duration.

Later investigations on the germination and fertility of pollen, for instance those of Sandsten at the University of Wisconsin, have extended the conclusions of Pfundt to a larger range of species, and have thrown new light on some other aspects of the problem. For example, it is often supposed that frost is actually fatal to the vitality of pollen. This is not the case, for it is found that pollen subjected for several hours to temperatures two or three degrees below the freezing point, while it does not act as well nor as quickly as fresh unchilled pollen, nevertheless retains a fairly high fertilizing power and germinates much as usual in the warmth. On the other hand, the life of the pistil, at least in the case of tree fruits, is imperilled by frost; indeed the majority of the pistils after exposure to the same conditions as used in the test on the pollen, were found to be dead and quite incapable of being fertilized.

It is sometimes supposed that rain washes off the viscid juice from the stigma, and so makes fertilization impossible. The fact appears to be that the female part of the flower is not seriously affected by rain; but the pollen is so; and since the period of receptivity of the stigma is not very prolonged—a few days at the utmost—if rain is persistent it may prevent the dispersion of the pollen until the stigmas have passed the time when they could be fertilized.

CONCLUSIONS

The practical conclusions from all this for our purpose as peony hybridizers, are these:

1. Peony pollen under favorable conditions is very long lived and there should be no difficulty in carrying it through the entire season from the earliest blooming species to the falling of the last blooms of Chinese peonies, thus making it possible to cross P. Wittmanniana or other early species on those forms that come on later.

2. The safest conditions for preservation are where the fresh pollen is put into a desiccator provided with a mixture of sulphuric acid and water in the proportion by weight 54 of acid to 46 of water, which will be by volume 30 of acid to 46 of water, since concentrated sulphuric acid has a specific gravity of 1.8 where water is unity. But if the pollen is simply dried in the sun, folded in paper and kept in a dry room, it will probably retain its vitality for a couple of months, hence there should be no difficulty in hybridizers exchanging samples of pollen during the season.

3. Pollen that is to be kept must be carefully preserved from wetting; and as a corollary, it is probably hopeless to use in the field any pollen that has been wet and dried again.

4. Pollination should be carried out on days that are at least fairly warm in order to have the chances of its "taking" as good as possible.

Perhaps a few words may not be out of place here for the benefit of the beginner in cross-fertilization, who will probably have found this paper rather dry reading.

The fundamental necessities are, first, to take the bloom before it has been fertilized either by its own or by other pollen; second, to effect pollination with the desired pollen, and third, to prevent subsequent pollination.

To meet these aims we take for example a peony bloom that is just expanding, and before it is sufficiently opened to permit pollination either by wind or by bees. If it is a double, without stamens, there is no danger of self-fertilization; but if stamens are present they should be examined to determine whether or not they have already burst; if so, the flower is probably already fertilized; if not, they and all the petals should be stripped off with a pair of forceps. If the stigma is dry and not covered with viscid juice, the flower should be enclosed in a little paper bag and left until the stigma is in a receptive condition; when this time arrives an anther may be brought over from the bloom that is to be the male parent, and gently rubbed on the sticky surface of the stigma. It is very easy to see the yellow pollen adhering to the surface when this is done. The fertilized flower should then be enclosed in a little paper bag or in a bit of cloth tied over it; this may be left on for the rest of the summer, or it may be removed after a few days, when all danger of outside pollination has passed. A string tag should be attached to the stem, recording the two parents, and for precaution an entry should also be made in a note book, as the records on the plant may be washed off during the summer rains.