Pollen Mixtures

When we think of pollination, we are inclined to think mainly of the ova and pollen tubes. The seed parent's influence is commonly thought of only as interference — blocking "illegitimate" pollen tubes from reaching the ovary. There is evidence, however, that the seed parent can "decide" to reject one fruit that has been self-pollinated, and redirect resources (nutrients) to another that has been cross-pollinated. In some cases, such as Amaryllis and its kin, viable seeds may be produced from self-pollination if the scape is removed from the bulb. This prevents the plant from withdrawing nutrients from the developing seeds.

The use of pollen mixtures offended the Mendelianists, who insisted that nothing would be produced in the mixture that would not also be found from single pollinations of the same pollen parents. Evidence is against this opinion. I recently learned of a working hypothesis: that the seed parent "decides" which fruit to support by the number and diversity of pollen tubes entering the style — rather than the "genotypes" of the growing seeds.

If each pollen tube presents certain "recognition substances" for the seed parent to detect, the greater diversity of pollen tube types should provoke the seed parent to provide more and better nutrition to the seeds in that ovary — or portion of the inflorescence. This leads to the surprising possibility that part of "inbred decline" may be the result of the seed parent being less inclined to lavish attention on the seeds. It is entirely possible that a pollen tube can reach the ovary and fuse with an ovum, but produce no seed because the seed parent does not recognize that particular pollen tube as legitimate. But if another, legitimate pollen tube is also present, the seed parent could be "tricked" into feeding the seed. A clear example of this is shown in the paper by Mangelsdorf and Reeves, noted below.

In my own very limited experiments, Brunsvigia rosea (Lam.) Hannibal, refused to set seed on its own pollen, but seeded well when pollinated by other specimens and other genera. The offspring all resembled the seed parent entirely. In another, I applied a mixture of pollen from several cultivars to the triploid Amaryllis (Hippeastrum) 'Lemon Lime'. This parent rarely yields more than a handful of viable seeds from a pod; I raised 18 seedlings from one pod.

A possible explanation for some of the results may be RNA interference. One pollen tube may fuse with the ovum, but siRNAs and miRNAs from other pollen tubes, living or dead, may alter gene expression in the embryo. If small RNAs from plants can alter gene expression in animals that ingest them, it should be just as likely that they could influence gene expression in other plants.

Zdanov, L. A. (1963) Supplementary pollination of sunflower with foreign pollen. Genetics for Agriculture pp. 387-98.
Compared with sunflower inbreds produced in the usual way, those pollinated with self pollen + pollen of maize or Ricinus set more seed, were taller and slower in development and had a higher oil content and a higher yield. Maize pollen was more beneficial to some genotypes and Ricinus pollen to others. The effects of pollination with self pollen + foreign pollen for two or more years were additive.

Šimanskij, N. K. (1966) Mentor capacity of foreign pollen in supplementary pollination of sunflower. Problems of breeding, genetics and seed production. 7. pp. 56-58.
The sunflower variety Armavir 3497 obtained by repeated self pollination, supplemented with thistle and pumpkin pollen, formed 8.6-40.6% of normal seeds. In the second and third generations the vegetative period of the plants was found to have been shortened by 3-5 days and oil content raised by 0.6-2.1%. Foreign pollination of sunflower affects the development of progeny characters such as plant height, pollen colour, and shape and size of achenes.

Stepanov, K. I. (1960) A study of self pollinated lines of maize obtained by supplementary pollination with foreign pollen. Trud. 1. nauc. Konf. molod. Ucen. Moldav. (Proc. 1st sci. Conf. jun. Sci. Moldav.) pp. 199-205
The depressive effects of inbreeding in two maize varieties were reduced at Kišinev by adding pollen of Atriplex, Hibiscus, pumpkin, sorghum, African millet, rye, wheat or sunflower to obtain the first-generation inbreds. The inbreds produced in this way acquired morphological stability more quickly than inbred lines from pollination with self pollen only.

Ohsawa, R., Y. Hirata, and N. Yagishita (1989) Pollen-mixture effect on germination and tube growth of pollen in Cyclamen and Primula. Bull. General Educ. Tokyo Univ. Agr. & Techn. 26:183-189. [In Japanese]

The New Phytologist 138(3): 481 (March 1998)
Pollen–pollen interactions in Betula pendula in vitro
H. L. PASONEN & M. KÄPYLÄ
Direct pollen interactions, as well as interactions mediated by a recipient, can have a remarkable influence on pollen fertilization ability. Under conditions of pollen competition it could be advantageous if pollen grains interfered with the germination of other pollen. The aim of this study was to find out if there are direct negative or positive pollen–pollen interactions between pollen grains from genetically slightly different donors. The in vitro germinability of the pollen from several Betula pendula Roth clones was investigated. The pollen interactions between the clones were examined pairwise by using equal pollen mixtures. In three of the eight cases the germination percentage of the pollen mixture was significantly higher than the average germination percentage of the separate clones that formed the mixture, which indicates some type of interaction between the pollen populations. We found only positive interactions between the pollen of clones. This study also documented density-dependent germination of pollen grains in vitro (= pollen population effect). Adding an aqueous pollen extract to the incubation medium increased the germination percentages of poorly germinating pollen and small pollen populations. Germination-stimulating effects were found to exist both with fresh and dead pollen. Such direct pollen–pollen interactions could be explained by specific water-soluble substances diffusing from pollen grains.

Arnold: Immediate effects of pollination (1873)
As stated above, both the ears of corn grew upon the same stalk, and from the seed of a dark purple corn like that upon the large ear. This ear was allowed to be fructified by pollen grown upon its own stalk. The pollen of this purple variety was then all removed and destroyed. And as the silken pistils of the smaller ear began to show themselves, pollen of a yellow variety of corn was supplied, by suspending small bottles filled with water and the stalk bearing the pollen plunged therein, then after a short time this yellow pollen was removed, and pollen of a white variety of corn was furnished in the same way. By examining the individual grains upon the small ear you will observe that they are yellow at the base and white upon the top. You will then please remember the purple seed from which the stalk and the two entirely different ears grew, and after a thorough examination, I feel confident that every unbiased intelligent person will agree with me that in corn at any rate, two different varieties of pollen can be made to influence one seed, and that the pollen will have an immediate effect upon the color of corn, if upon nothing else.

Collins & Kempton (1913)
In the resulting ear the two kinds of seed were easily distinguished. The pure seeds resulting from the first pollination were pure white, while the hybrid seed resulting from the second pollination were yellow. Unlike the ears where mixed pollen was used, the two kinds of seed were not indiscriminately distributed. All the white seeds were on the lower portion of the ear; all the colored were on the upper portion. This segregation of the two kinds of seed must have deprived the hybrid seed of any advantage that might be secured by crowding weak neighbors, while the time which elapsed between the two applications of pollen precluded the possibility of the hybrid seed appropriating material in advance of the pure seed.
    The ear produced 212 white, or pure, seeds and 161 that were yellow, or hybrid. The average weight of the pure seed was 283 grams per 1,000. The average weight of the hybrid seed was 292.5 grams per 1,000, a difference of 9.5±1.06 grams, or 3.4 per cent. The seeds in the lower portion of the ear are usually somewhat larger than those on the upper portion, a fact that should be considered in connection with the observed increase.

Mangelsdorf & Reeves: Maize-Tripsacum hybrids (1931)
Figure 4
Some ears set seed almost 100 per cent. The hybrid seeds, however, are very small compared to maize seeds of the same age. A few maize seed near the middle resulted from pollinating with maize 24 hours after pollinating with Tripsacum. Note that some of the hybrid seeds adjacent to the maize seeds are larger than their sibs.

Grieve: Raising New Pelargoniums (1875)
About the middle of last September I repeated an experiment which I had previously tried, with the view of ascertaining whether or not superfetation could be induced in the case of the Pelargonium, and, if so, it might of course be inferred that the same might take place amongst other hermaphrodite plants; or, at all events to ascertain for the second time whether or not the application of foreign pollen to the stigmas would have any perceptible effect upon the progeny in cases in which such stigmas had been previously fertilised by their own pollen; accordingly two plants of Pelargonium peltatum were again selected and placed in a structure where it was hardly possible that pollen from kindred species could by any means reach them, and, as soon as the stigmas were found to be in proper condition, a few blooms upon each plant were fertilised with their own pollen, and all other blooms were removed. On the following day the stigmas of one of the plants were covered with pollen taken from a zonal variety. The blooms upon the other plant were left untouched after the application of their own pollen. Both plants duly ripened seed, and the produce of each was sown in a separate pot, and germinated simultaneously; but, as was expected, the produce of the plant whose blooms had the second application of pollen were exceedingly diversified in aspect, although they all appeared to be of the peltatum type, the leaves of some of them were large, others exceedingly small; some had zoned foliage, the leaves of others were quite zoneless; while the plants raised from the seeds produced by the blooms which had received only one application of pollen were quite uniform in appearance. All the plants, however, were kept growing slowly throughout the winter months, and were planted in the open air about the end of May. All the seedlings raised from the plant whose blooms received only one application of pollen have now flowered, and their blossoms differ in no respect from those of the parent plant; while, as regards the produce of the twice fertilised plant, none of the seedlings have yet flowered, and—what is more remarkable—the plants are rapidly losing that diversity of appearance which distinguished them during the earlier stages of their development, although they are still very distinct from each other, and the appearance of flowers is looked forward to with interest. Should they approximate to the zonal type, this circumstance may perhaps be considered as favourable to the theory of superfetation; while, should the flowers be in no degree different from those of P. peltatum, it will be difficult to account for the diversity of appearance among the seedlings apparently occasioned by the second application of pollen, otherwise than by supposing that, even after fertilisation has been effected, the ovules may still, to some extent, be nourished or fed by the application of pollen to the stigma.

Dubious Dahlia Crosses (1897)
M. MARTIN CAHUZAC has sent to the editor of the Semaine Horticole six flowers said to be the result of a cross between a Chrysanthemum and a Dahlia. The evidence of hybridisation does not appear to have been very marked. A week or two ago, Mr. E. J. LOWE sent us flowers of a cross between a Sunflower and a Dahlia; the flowers were those of a Dahlia, but the central disc was proportionately larger and deeper. Improbable as such crosses appear, it would be rash to assert that they are not possible. Very often the application of the foreign pollen seems to induce enhanced growth of the seedling without actual change of form having taken place.

Morse: Pollen Selection (1902)
Mixed pollens from differently colored flowers produced all flaked flowers in Gladiolus.

Wichura on Hybrids (1866) p. 73
Wichura confirms Gaertner in the assertion that where hybrid pollen is used for the impregnation of simple or complicated hybrids, as also in pure species, there is a great predominance of individual forms, while hybrid ovules impregnated by the pollen of pure species, even in the most complicated combinations, give very uniform products.

Soviet Plant Physiology pp. 190-193 (1958)
Sedov: The effect of the variety of the pollinators and of pollen mixtures on various physiological processes in apple seedlings
The higher degree of hydration in apple seedlings obtained from seeds resulting from pollination with mixtures of pollen, and also the increased rate of photosynthesis in these seedlings indicates their high vitality. Proceeding from this we should also note that it is necessary to have 2-3 varieties of pollinators in the maternal seed plantings of apple together with pairs of the main variety suitable for that place; this guarantees pollination with a mixture of pollens under natural conditions in each instance, and significantly increases not only the percentage of fruits set, but also guarantees getting vital pairs of seedlings. It is also necessary to expand the use of mixtures of pollen in selective breeding since more vital hybrids are produced as the result.

Cell Res. 2011 Oct 25;: 22025251
Ingested plant miRNAs regulate gene expression in animals.
Hervé Vaucheret, Yves Chupeau
Institut Jean-Pierre Bourgin, INRA, 78000 Versailles, France.
The incidence of genetic material or epigenetic information transferred from one organism to another is an important biological question. A recent study demonstrated that plant small RNAs acquired orally through food intake directly influence gene expression in animals after migration through the plasma and delivery to specific organs.

Cook: Hybridizing (1907)
I have several [rose] seedlings, where the pollen was taken from three and four different varieties mixed together, and they are the richest color in red of any I have ever raised.

CybeRose note: Regarding the next two items, I am not sure whether Knight claimed that two pollen parents could influence a single offspring, or that two seeds in the same pod might have different fathers.

Herbert: On the Production of Hybrid Vegetables (1822)
I suspect that the germen is very speedily fertilized when the dust has touched the stigma; but I doubt whether, after being fecundated, it is closed against any further impression. Mr. Knight has assured me that by touching the stigma of a smooth cabbage with the dusts of a curled and of a red cabbage, he had given both the curl and the red colour to the seedlings; but I am uncertain whether both dusts were applied at the same time.

Hovey: Cross-breeding Flowering Plants (1837)
Mr. Knight has stated that he dusted the stigma of a smooth cabbage with the pollen of a Savoy and of a red cabbage, and obtained seedlings which were both curled and of a red color. This shows that a plant may be impregnated, and with effect, with two, and perhaps more, different sorts.

Principles and Practices in Plant Ecology: Allelochemical Interactions (1999)
8.4.2.2.2 Pollen-pistil interaction
The experiments on pollination included fluorescence measurements 2 to 5 min following germination and 1 month following ovary development. The fluorescence spectra of the Hippeastrum stigma was analyzed after pollination by foreign and own pollen (Figure 8.8). When pollen of wind-pollinated species such as Dactylis glomerata L. (Figure 8.8), Populus balsamifera L., and Betula verrucosa were added, the weak own intrinsic fluorescence of the Hippeastrum pistil responded to the pollen addition on the stigma. The character of spectra was changed by both cross-pollination and self-pollination, where new peaks arose (Figures 8.8, i-iii), whereas pollination by pollen from insect-pollinated Hemerocallis fulva L. only increased the whole influorescence intensity (Figure 8.10, ii). After 1 month of observation, the ovary formation was at variance with that following cross- or self-pollination. In other cases, fruit maturation was not seen.
    Simultaneously with the measurements on the pistil, the fluorescence of the same species pollen added on the Hippeastrum stigma was also recorded (Figure 8.8). The pollen grains of foreign pollen weakly or practically did not change the autofluorescence spectra. However, own-pollen fluorescence intensity decreased 2 to 3 min after its addition on the pistil. Its maximum at 480 nm shifted to the long wavelength region (Figure 8.8). This phenomenon appeared to be associated with the chemosensory features of the surfaces of both the stigma and the pollen grains.

Principles and Practices in Plant Ecology: Allelochemical Interactions (1999)
8.4.2.3 Pollen-Pollen Interaction in Mixtures
One of the suitable modes of estimating pollen-pollen interactions is observation of fast changes in their autofluorescence. The responses are obviously dependent on both the composition of the excretion of donor pollen and the chemosensory peculiarities of the surface component of the plant-acceptor pollen. In this section we will consider the chemical interactions between dry pollen grains, for example, through a communication by volatile excretion, unlike our experiments with moistened pollen and leachates from pollen (Roschina and Melnikova, 1996).
    The present data involve pollen of meadow wind-pollinated species; forest-living wind-pollinated species; meadow wind-pollinated and insect-pollenates species, grown in flower gardens; and weeds.
    The fluorescence spectra of pollen mixtures may reflect initial fast responses in pollen allelopathy. One example is shown in Figure 8.13, where the fluorescence spectra of mixtures from wind-pollinated herbaceous meadow plants such as medow foxtail (Alopecurus pratensis L.) and orchardgrass (Dactylis glomerata L., Poaceae), and cultural flower garden species poppy (Papaver orientale L., Papaveraceae) and day lily (Hemerocallis fulva L., Liliaceae) are given. The changes are mainly in the fluorescence intensity. The value is 1.5-fold higher in Alopecurus in a mixture of Alopecurus and Papaver (var. i, ii), whereas poppy has no visible shifts. In conrast, meadow foxglove decreases its autofluorescence intensity and shifts the main maximum to the long wavelength region in a mixture with day lily pollen (var. iii, iv). The latter shows no changes in its autofluorescence. When pollen grains of Hemerocallis and Dactylis (var. v, vi) are mixed, orchardgrass undergoes significant changes and a new maximum at 480 nm arises, with major maximum at 525 nm becomes more flat, and red fluorescence with maximum at 650 nm is more obvious, while the intensity of fluorescence in maxima decreases more than twofold. The autofluorescence of day lily pollen also decreases. If pollen of orchardgrass and poppy are mixed, the fluorescence of both remains unchanged (var. vii, viii).
<snip>
    Pollen-pollen antagonism, estimated by the ability to germinate, has been known since the 1930s for cultural plants of parterres (Bransheidt, 1930; Zanoni, 1930), whereas a mutual stimulation was first found for natural field plants by Golubinskii in 1946. Unlike the experiments needed for 1 to 24 h for the pollen tube growth, our experiments permitted testing the chemosensitivity of different pollen species in mixtures for 1 to 2 min, and can indicate compatible or incompatible species in phytocenosis. The ability to depress or stimulate fertilization by foreign pollen could be one of the determinants of the mutual existence of the species. Certainly, pollen of Papaver orientale and Hemerocallis fulva, as parterre species, were either insensitive or weakly sensitive to pollen of field-grown, wind-pollinated Alopecurus pratensis L. and Dactylis glomerata. Only the fluorescence of Hemerocallis pollen was stimulated by pollen of weeds Artemisia vulgaris and Urtica dioica. In contrast, the pollen grains of wind-pollinated weeds and woody species were extremely sensitive to each other, decreasing their autofluorescence. The sensitivity between pollen of wind-pollinated meadow plants Alopecurus and Dactylis was marked, but without a strong correlation with their germination.

Beaton: The Process of Fertilisation (1862)
The lesson to learn from the fact that pods and seeds do swell and ripen sometimes without the germ having been fertilised is this—that you should apply the pollen in such doubtful cases four or five times the same day; for there never was such a thing as a stigma getting fit a second time, or after a day's interval. But as we know to a certainty that there are three if not four degrees in the quickening process, we may be excused for supposing that more pollen and most pollen will push the degrees farther and farthest if we apply it in time— that is, ere the first and final moisture on the stigma is dried up; and those which never show the moisture are more safe, one would think, if all the pollen it needs has been collected on the same day, or at the farthest on the morrow.

14 'Beitrage zur Kenntniss,' &c., 1844, s. 345.
15 'Nouvelles Archives du Museum,' tom. i. p. 27. 1865

Darwin: Variation of Animals and Plants vol. 2
With respect to plants, nearly the same results were obtained by Kölreuter and Gärtner. This last careful observer, after making successive trials on a Malva with more and more pollen-grains, found,14 that even thirty grains did not fertilise a single seed; but when forty grains were applied to the stigma, a few seeds of small size were formed. In the case of Mirabilis the pollen grains are extraordinarily large, and the ovarium contains only a single ovule; and these circumstances led Naudin15 to make the following experiments: a flower was fertilised by three grains and succeeded perfectly; twelve flowers were fertilised by two grains, and seventeen flowers by a single grain, and of these one flower alone in each lot perfected its seed: and it deserves especial notice that the plants produced by these two seeds never attained their proper dimensions, and bore flowers of remarkably small size. From these facts we clearly see that the quantity of the peculiar formative matter which is contained within the spermatozoa and pollen-grains is an all-important element in the act of fertilisation, not only for the full development of the seed, but for the vigour of the plant produced from such seed.

Darwin: Gardeners' Chronicle 11 Aug 1866: 756
Gärtner crossed 36 flowers of different varieties of the common Pea, and he did not get a single pod perfectly developed and with the full complement of seed; he crossed 10 flowers of Kidney Beans, and did not get a single pod. Some years ago I crossed the varieties of the Sweet Pea, and many more flowers dropped off unimpregnated than were fertilised. The difficulty arises from the anthers opening at so early an age that they must be removed long before the flower expands. After the operation the immature stigma is liable to exposure to the air; and it is difficult to judge when to apply the pollen. Moreover there is some reason to suspect that the stigma requires successive applications of pollen.

The Development and Properties of Raw Cotton By William Lawrence Balls pp. 63-64 (1915)
    Of the many pollen-tubes which germinate on the style, only some twenty or so can find an ovule. Those which were too late in starting, or too slow in growing down the style, also perish, and their remains may partly be traced in the walls of the young fruits; while the rest are thrown off when the style breaks away from the point of the young boll.
    Some remarkable features of this race between the pollen-tubes require further study. The style of some kinds of cotton is either non-nutritious, or more probably poisonous, to the pollen of other kinds. Thus, crosses between the Indian group of cottons and the Upland or Peruvian groups do not appear to be possible. Uplands and Peruvians can, on the other hand, be artificially intercrossed with ease. Even in this case, however, if equal amounts of self and foreign pollen are placed on the style simultaneously, so that both have an equal chance, 97 per cent. of the victors will be self-tubes; so that, although Egyptian pollen can grow down an Upland style quite satisfactorily, it cannot grow so fast as the Upland's own pollen can do, and vice versa. If, lastly, the pollen mixture is made with pollen from the first cross between Upland and Egyptian, the percentage of wins credited to the home team falls to about 60 per cent.; the hybrid pollen is said to be more "prepotent." These facts have considerable economic bearing on the possibility of keeping cotton strains pure with fewer precautions, but it will take a great deal of tedious research to find whether they have any utility.

Physiology and Biochemistry of Plant Cell Walls p. 198 (1996)
Brett and Waldron
The diffusion of materials from pollen wall to stigma surface is clearly involved in cell-cell recognition in these systems. This is clear from experiments in which mixtures of pollen are applied to stigmas. Normally incompatible pollen can be induced to fertilize flowers by application as a mixure with compatible pollen, the compatible pollen having been first sterilized by gamma irradiation to avoid competition. An even more effective method of overcoming incompatibility is to mix the incompatible pollen with diffusates from compatible pollen. Neither method, hower, permits fertilization of a flower by all types of incompatible pollen, so clearly other mechanisms are also involved in incompatibility.

Currants, Gooseberries, and Jostaberries p. 206 (2005)
Danny L. Barney, Kim E. Hummer
In some cases of wide crosses, such as 'Crandall' (R. aureum) x gooseberries, pollen mixtures are more likely to result in fertilization and seed set than pollen from a single donor (Tolmacev, 1940). Pollen mixtures have also been popular with Russian breeders when crossing American and European gooseberries to produce mildew-resistant offspring (Keep, 1975). Whenever possible, use the freshest pollen available.

Jour. Roy. Hort. Soc. London (1847)
Herbert: On Hybridization amongst Vegetables pt. 1
I stated (Amaryllidaceae, p. 371) that I had found flowers of every cross-bred kind of Hippeastrum, after its stigma had been touched with the pollen of another bred by a different cross, produce seed abundantly; while those on the same stem, which were touched with their own pollen only, either failed to produce seed, or produced few, and those in a capsule very deficient in size and vigour. The observation of several years' enables me now to say that this remarkable fact is almost invariable, and that, although the hybrids in this genus are capable of bearing seed by their own pollen, the admission of the pollen of another cross-bred plant of the same genus (however complicated the cross) to any one flower of the umbel, is almost sure to check the fructification of the others, so that the excision of the anthers in such case is quite superfluous, the difficulty being to get the individuals to fertilize their own germens. This remarkable fact led me to try a further experiment, and the result has brought to light a startling fact, that in the same genus the pollen of a cross-bred plant can even overpower the natural fertilization of a wild bulb of an uumixed species. For this experiment I chose a bulb lately dug up by Mr. Gardner on the Organ Mountains in Brazil, and sent to me by the kindness of G. Wailes, Esq., of Newcastle; closely allied to H. aulicum, of which it may be called var. Organense, or, if it be separated as a species, H. Organense, having the scape usually two-flowered, the red not intense or shining as it is in Aulicum, and the screen in the throat ragged and half-bearded. The very bulb lately dug up in Brazil was used. It produced two two-flowered scapes; the first pair of flowers were touched with their own dust, and the germens swelled; of the second scape, which was several days later, one flower was touched with its own, and the other by the dust of a fine triple cross from H. bulbulosum, var. pulverulentum by reginae-vittatum, otherwise called Johnsoni. The ovaries of the three flowers impregnated by the natural pollen for a few days after the decay of the last flowers appeared to have the advantage, and the fourth continued smaller, and seemed likely to fail, when it unexpectedly made a rapid advance, and immediately the three others ceased to grow, and after a few days perished entirely; while the progress of the pod impregnated by the mule made vigorous and rapid progress to maturity, and bore good seed, which vegetated freely. The impregnation by the cross-bred pollen was therefore slower in taking effect, but had the same decided superiority over the pollen of the natural species as over that of any other cross-bred variety. The anthers had been taken out of the flowers before their expansion. This is a strange truth, and the more remarkable from the difficulty of obtaining cross-bred seed at all in the genera which are most nearly related to Hippeastrum, namely, Habranthus and Zephyranthes. Where no access from the dust of another individual is admitted, the hybrid Hippeastrum Johnsoni is capable, as I have stated, of reproducing itself by seed. A bulb of Solandrifloro-Johnsoni, of which all the flowers were set by their own pollen, produced seed vigorously from all of them.

Genetics Today: Proceedings of the XI International Congress of Genetics (1963) p. 235
13.77. New Methods in Cotton Hybridization.
D. V. Ter-Avanesyan (U.S.S.R.)
    Achievements in biological science reached in the last decade are well known. It should be noticed, however, that as profound is our knowledge with regard to a living cell, so poor it is in the field of biology of pollination and fertilization. The experimental data remain unexplained so far, and they are strictly empirical. Here are some interesting results of a long period of our work at the Central Asia Experimental Station of the All-Union Research Institute of Plant Breeding. There was a task to work out the method of cotton hybridization using the pollen of heterochromosomic species and even of genera of Malvaceae family.
    As it is known, in the case of hybridization of G. hirsutum L. cotton species with G. herbaceum L. and G. arboreum L. (African-Asiatic species) having twice less chromosomes in their cells, pollen tubes of the latter, as a rule, do not fertilize even if they reach embryo sac of diploid form. At the same time the pollen of the Malvaceae family (Hibiscus esculentus, H. rosa sinensis, H. cannabinus and Malva neglecta) does not germinate on the cotton stigma at all.
    But in spite of the evident genetical incompatibility of chromosomic gametes we succeeded in using them when working out two breeding methods. The first method is as follows: at the growing period of maternal cotton there were 2-3 buds left on the plant, the rest being removed. This method stimulated the accumulation of nutrients in flowers. 10-20 pollen grains were placed on the stigma of emasculated flower followed by unlimited pollination with the pollen of a paternal form (in this case of an alien species) for 3 hours.
    The second method with similar results was the following: in that very evening emasculated flowers were pollinated with unlimited quantity of paternal pollen. Next day unlimited quantity of parental pollen was again placed on the stigma of the same flower, and at noon (about 20 hours after emasculation) there was pollination with the mixture of paternal and maternal pollen in different amounts.
    The first and second generations from seeds developed by the described method, gave some interesting plants. The progeny considerably differed from maternal plants. It got the characters of non-crossing cotton species and changed completely its qualitative and quantitative characteristics being affected by the pollen of Malvaceae plants. These characters became hereditarily fixed, and they were transmitted to progenies. The suggested methods can be used for cotton hybridization. They will improve our possibilities of using diversified Gossypium genus in cotton hybridization.

Genetics Today: Proceedings of the XI International Congress of Genetics (1963) p. 213
13.13. Pollination with Pollen Mixture to Produce Interspecific Hybrids of Plum and Cherry (Prunus),
K. K. Enikeyev (Moscow, U.S.S.R.).
    Soviet horticulturists use the method of pollination with pollen mixture in interspecific crosses.
    This method was worked out by I. V. Michurin in order to overcome incompatibility of different fruit species. I. V. Michurin based this method on the regularities of selective fertilization and complex physiological interaction between the components of pollen mixture and the pistil of a maternal form.
    The author has used two variations of the pollination with pollen mixture and got the following results:
    The first variation consists of the pollination of one species with the pollen mixture of several varieties of another species which is almost or entirely unable to cross with the former, it secures the selective fertilization by means of the most biologically adapted pollen grains of one of the varieties of a paternal form.
    As a result of pollinating Cerasus Besseyi Bail. with pollen mixture of several European varieties of apricot (Armeniaca vulgaris L.) quite viable and fertile hybrids have been produced.
    The hybridization of several Prunus species with different chromosome numbers in F1 results in the development of low-fertile hybrids of an intermediate type. Such are P. spinosa L. x P. domestica L. hybrids (2n = 40) and P. cerasus L. x P. avium L. hybrids (2n = 24).
    When these low-fertile hybrids (F1) were pollinated with the pollen mixture of several varieties of one of parental species, F2 consisted of quite fertile and commercially valuable plants with the characteristics of both species.
    The second variation consists of the addition of small amounts of own or alien pollen related to maternal form to the pollen of an uncrossable species. This variation is based on the fact that in the process of fertilization own or alien pollen favourably affects the pistil, creating conditions which stimulate the growth of pollen tubes and the fertilization with the pollen of an uncrossable or hardly crossable species.
    The analysis of morphological characteristics and chromosome numbers in F1 hybrids shows that preferable fertilization with own or closely related pollen takes place in these crosses, though in some cases this method helped to cross two hardly crossable species, for example, Cerasus Besseyi Bail. x Prunus cerasifera Ehrh, (2n = 16); C. Besseyi x P. spinosa. (2n = 24.)

Genetics Today: Proceedings of the XI International Congress of Genetics (1963) pp. 234-235
13.76. Multiple Effect of Fertilization in Floral Plants.
I. M. Polyakov (U.S.S.R.).
    The widespread conception of fertilization considers this process only as syngamy (double fertilization in floral plants). Gamete combination is considered to be of random character (with some exceptions).
    Physiological and biochemical aspect of fertilization is regarded as one that has no direct effect on formation and modification of hereditary disposition of the progeny. According to this conception fertilization is nothing but a "bridge" that helps the germinal plasma to pass from one progeny to another.
    There is a considerable number of facts now showing that physiology of fertilization has a direct effect on heredity, variability and vitability of progeny. The main facts are connected with finding the effect of polyfatherhood. This effect is expressed both in general stimulating and sometimes specific modifying action on behalf of that part of the pollen, which physiologically "participates" in the process without participating in double fertilization (in some cases the pollen may belong to other species). This fact also manifests itself in changing the character and range of offspring variability when pollination changing conditions are changed (especially under limited pollination). In connection with the aforesaid, well-known facts of metaxenis are becoming more important.
    Our trials with 15 species of floral plants (especially with Nicotiana and Zea mays species) together with the investigations carried out by other authors showed the double role of the effect of polyfatherhood.
    On one side physiological "combined action" of pollen tubes maintains typical hereditary disposition, on the other side the effect of polyfatherhood through changing conditions of pollination and fertilization (change of qualitative and quantitative composition of pollen, its physiological state and so on) may lead to the change of normal heredity.
    It may be also considered that syngamy is not only a simple combination of both parents' heredity, it can lead to the development of new formations as a result of metabolic interactions of two gametes.
    In trials with corn it was shown that the effect of polyfatherhood concerns not only the first progeny, in some trials this effect reaching even the fourth generation. Thus, the constant interrelation of fertilization processes and genetic phenomena was deeper than it had been supposed.
    In our trials we tried also to find out physiological and biochemical foundation of the effect of polyfatherhood. In 1953-1962 we recommended and used the new method for studying fertilization processes with the help of the pollen labelled with radioactive isotopes S35 and P32. For the first time these tests were direct physiological evidence that pollen without taking direct part in double fertilization is active in metabolic processes of growing seeds. We have partly studied these processes and tried to give their quantitative characteristics.
    The further development of metabolic theory of fertilization and the study of two most important aspects of fertilization—polyfatherhood and selectiveness will be of great significance for genetics.

Darwin: The Effects of Cross and Self Fertilisation in the Vegetable Kingdom
The stigmas on two lately expanded flowers on a variety of cabbage called Ragged Jack, were well covered with pollen from the same plant. After an interval of twenty-three hours pollen from the Early Barnes cabbage growing at a distance was placed on both stigmas, and as the plant was left uncovered pollen from other flowers on the Ragged Jack would certainly have been left by the bees during the next two or three days on the same two stigmas. Under these circumstances, it seemed very unlikely that the pollen of the Barnes cabbage would produce any effect; but three out of the fifteen plants raised from the capsules thus produced were plainly mongrelized, and I have no doubt that the twelve other plants were affected, for they grew much more vigorously than the self-fertilized seedlings from the Ragged Jack planted at the same time and under the same conditions.

The Journal of Cell Biology 66: 556-567 (1975)
Large Electrical Currents Traverse Growing Pollen Tubes
Weisenseel, Nuccitelli and Jaffe
Using a newly developed vibrating electrode, we have explored the electric fields around lily pollen germinating in vitro. From these field measurements, we infer that each wetted pollen drives a steady current of a few hundred picoamperes through itself. Considered as a flow of positive ions, this current enters an ungerminated grain's prospective growth site and leaves its opposite end. After a grain germinates and forms a tube, this current enters most of the growing tube and leaves the whole grain. The current densities over both of these extended surface regions are relatively uniform, and the boundary zone, near the tube's base, is relatively narrow. This current continues as long as the tube grows, and even continues when elongation, as well as cytoplasmic streaming, are blocked by 1 μg/ml of cytochalasin B.
    After an otherwise indistinguishable minority of tubes have grown to lengths of a millimeter or more, their current comes to include an endless train of discrete and characteristic current pulses as well as a steady component. These pulses are about 30 s long, never overlap, recur every 60-100 s, and seem to enter a region more restricted to the growing tip than the steady current's sink.
    CybeRose: I don't know if this is relevant to pollen mixtures, but it's something to be considered.

Collins & Kempton: Effect of cross-pollination on seed size in maize (1913)
In order to distinguish between pure-bred and hybrid seed on the same ear only those hybrids with colored aleurone could be utilized. It might be urged that there was a tendency for colored seeds to be heavier than white and that we have been measuring differences between colored and white seed rather than differences between pure and hybrid seed. Fortunately, there was material at hand to test this point. In 1911 a cross had been made between Variegated and Mexico Black, two of the varieties used in these experiments. This hybrid was grown and a number of self-pollinated ears secured in 1912. These ears all had both white and colored seeds. A comparison of the weight of the white and colored seed from each of these ears showed only the ordinary fluctuating differences between the two classes.

Glushchenko: The Phenomenon of Polyfertilization in Plants (1957)
V. J. Bazavluk made experiments on polyfatherhood in Mirabilis jalapa. Let us examine the results of the experiment with pollination by two father forms: white x (crimson + yellow), and with three father pollinators: white x (crimson + yellow + white). It is interesting that besides the white, yellow and crimson parent forms, plants with orange-crimson corolla, i.e., bearing the characters of their two father forms have been produced in F1. Such results have not been observed in control pair-crosses.

American Journal of Botany. 1999; 86:261-268.
The effects of pollen load size and donor diversity on pollen performance, selective abortion, and progeny vigor in Mirabilis jalapa (Nyctaginaceae).
Richard A. Niesenbaum
The influence of pollen competitive environment on pollen performance (pollen germination, stigmatic penetration, and pollen tube growth rate), the maturation or abortion of initiated fruit, seed size, and seedling vigor was explored by manipulating the size and diversity of stigmatic pollen loads on Mirabilis jalapa. All aspects of pollen performance significantly increased with the number of pollen grains on a stigma or pollen tubes in a style, but was not influenced by the diversity of pollen donors. Plants tended to mature single-ovulate fruits that came from flowers where pollen load size and diversity were greatest and aborted those where these were lowest. No plants from seeds resulting from pollinations with a single pollen grain survived, but other fitness measures were mostly determined by maternal plant.

Laterrot: Pollen mixtures for tomato hybrids (1983)
With this technique, 1 to 5 hybrids per 100 pollinated flowers are obtained. It is probably possible to improve this technique by using a male sterile line to eliminate emasculation of the female parent. In this case the tomato pollen comes from a male-fertile line bearing the same marker gene as the female line. Efficiency of the method might also be enhanced by double pollination; the first by L. peruvianum and the second a short time later, with tomato pollen.

Yenikeyev: Pollination with a pollen mixture (1965)
Hybridization of the blackthorn (Prunus spinosa L.) with the domestic plum (P. domestica L.) was also facilitated by using a pollen mixture of several plum varieties (cf. also YENIKEYEV, 1960, 1962). It can be seen from Table I that the percentage fruit set resulting from pollination of the blackthorn with a pollen mixture of three plum varieties, was much higher than that obtained after pollination with each variety separately.

Vysotskii: Wide Hybridization in the Malvaceae (1960)
When starting work in this direction, we encountered an apparently serious obstacle: the high incompatibility between different genera and tribes of the Malvaceae, in particular, between the different genera and cotton. To overcome this obstacle, we resorted to a number of new methods; firstly, multiple saturating pollination. A pollen mixture from different genera of the Malvaceae was applied three times and, subsequently, pollen from the maternal plant was used. Sometimes these operations were effected on the same pair of parental forms over three, four and even seven years.

Babajanyan: Inbreeding Rye (1948)
Rye, as is known, is a strict cross-pollinator; isolation leads to utter sterility, and inbreeding to depression in the progeny. In one of the experiments that we have been conducting at our Institute for the past three years and more, self-fertilizing rye plants in isolators were given the pollen of spring wheat. In a number of cases this led to a heightened grain formation. In 1948 one plant by this method showed 20% of grain development compared with only 1% in the case of pure inbreeding; another plant showed 22% of grain development in the presence of foreign pollen and 0 under pure inbreeding; a third plant showed 24% and 0; other cases showed 33% and 2%, 39% and 0, 48% and 0, and 54% and 0. This is the highest figure we have obtained. Thus, such strict cross-pollinators like rye, which produce no seeds with ordinary inbreeding, produce them under the influence of foreign pollen.
   But this is not the most important, although this in itself is an indication of the diminution of the depression of self-pollination. The important thing is that in plants produced in this way the depression of inbreeding is diminished, and in many cases it disappears in the progeny.

Lysenko: Overcoming cross-incompatibility in wheat hybrids (1954)
In experiments conducted by Avakian at Gorki Leninskiye, castrated plants of Hostianum 0237 wheat were pollinated with the pollen of 1160 (as we have already said, this combination usually results in nonviable progeny) mixed with the pollen of the maternal form Hostianum 0237. Some of the plants grown from the resulting seeds were obviously hybrids. These plants proved to be viable, they did not perish. Thus, the presence of the pollen from Hostianum 0237 influenced the process and result of fertilization with the pollen from 1160, as a consequence of which a viable instead of a lethal, nonviable, progeny was obtained.