Advances in Botanical Research, 2: 223-227 (1965)
Embryology in Relation to Physiology and Genetics
P. Maheshwari and N. S. Raangaswamy

B. Germination of Pollen

Whereas the life span of the pollen of many species can be extended by artificial devices, this should not be taken to imply that stored pollen would always retain its capacity for germination and subsequent potency for fertilization to the same degree as fresh pollen. Some probable explanations for this are: loss of moisture, depletion of food reserves, and a decrease and /or inactivation of the enzymes or other key substances.

The logical corollary is that stored pollen may require for its germination a higher relative humidity and a higher level of nutrient material than fresh pollen. This has been confirmed by some investigators. Pfeiffer (1944) observed that on providing a favourable humidity and temperature the pollen of Chinchona showed some recovery of its capacity for germination. The pollen of Pinus freshly collected in June yielded optimum germination in 2% sucrose, while a sample stored until December required as much as 20% sucrose (Kühlwein and Anhaeusser, 1951). Similarly, the corresponding requirements of Arachis hypogaea and of the varieties T.55 I.C. 1472 and T.25 of Pennisetum typhoideum wore 10, 12.5 and 25% sucrose for fresh pollen; and 12.5, 15 and 27.5% for stored pollen (Vasil, 1982). For tomato Gorobec (1958) observed that 4-day-old pollen stored in the laboratory yielded fruits of normal size while older pollen gave only small fruits. At the other extreme it is recorded that the pollen of apple gave some germination even 9 years after storage (Ushirozawa and Shibukawa, 1951).

Nielsen (1956) observed that the content of pantothenic acid (chief constituent of coenzyme A) decreases substantially in the pollens of Pinus and Alnus after storage for one year. This imbalance in coenzyme A upsets the general metabolism which in turn shortens the life of the pollen.

It is of interest to note that stored pollen which fails to germinate in vitro may nevertheless cause a satisfactory seed set. The investigations of Olmo (1942) on grape, of Stone et al. (1943) on pistachio, of Hagiaya (1949) on tobacco and of Visser (1955) on tomato serve as examples. The stored pollen of these plants showed only 5% germination in vitro but proved quite effective for field use. Similarly, the pollen of potato stored at -34.4°C for 7-13 months gave almost no germination in culture but effected a good fruit set when used on the stigma (King, 1955). The pollen of cacao stored over calcium chloride at -20 to -30°C for 1-4 weeks showed only a low germination on an agar-dextrose medium, but field pollinations gave 40-50% fruit set (Soria and Denys, 1961). This is readily understandable because the stigmatic and stylar tissues help to make up some of the deficiencies imminent to storage and provide a natural environment for the germination of the pollen and the growth of the pollen tube.

Thus, the failure of germination of stored pollen in cultures does not necessarily mean that the pollen is dead or useless. Similarly, the mere germination of stored pollen in vitro is no assurance that it will effect fertilization. For example, the pollen of wheat showed 5-10% germination on an agar medium but proved unsatisfactory for pollination in nature (Kováčik and Holienka, 1962). This implies that there are other factors which also control the germination of pollen.

The pollen requires an adequate supply of moisture, inorganic elements, and a source of energy—usually a sugar. Some of these requirements may be met from the reserves of the pollen itself, but very often one or more of these act as limiting factors.

The ambient humidity is critical for the germination of pollen. As substrates the lower surface of fresh leaves of aquatic plants and sometimes even moist parchment paper have proved adequate. In some instances good germination has also been obtained by merely placing the pollen near a hanging drop of water in a microchamber. Excessive moisture is deleterious to the pollen, and it is a common experience of plant breeders that pollinations carried out soon after rain or dew are frequently infructuous.

Schmucker (1933) reported that the pollen of Nymphaea germinated in a solution of glucose only when mixed with the stigmatic extract of the plant. Later, he detected boron in the extract and, therefore, replaced it by traces of boric acid. This proved successful and quantitative estimations revealed that the pollen required almost the same concentration of boron as was present in the stigma. Following Schmucker, many others have identified boron in the stigma and style and have confirmed its favourable effect on the germination of pollen.

It was believed that boron is related, in some unknown way, to the incorporation of carbohydrates in the pollen tube membrane. However, some studies indicate that it is essential to the synthesis of peotins in the middle lamella of newly formed cells (Spurr, 1957) and in the pollen tubes (Raghavan and Baruah, 1959). Stanley (1964) and Stanley and Loewus (1964) suggested that myo-inositol is probably an intermediate in the conversion of hexose sugar to pectin. On using tritiated myo-inositol with two concentrations of boron (0.75 and 7.5 µg/ml) in cultures of the pollen of Pyrus communis they found that (a) boron combined with a specific enzyme enabling it to bind and react with inositol, (b) myo-inositol was readily converted into pectin, and (c) pectin synthesis increased at higher levels of boron. These observations suggest that boron plays a significant role in pectin synthesis in germinating pollen.

Like boron, calcium has been shown to enhance the germination of pollen as well as the growth of the pollen tubes, and especially the latter. Its role is discussed under Section II C.

Low concentrations (0.001-0.0001 M) of dicarboxylic acids, such as succinic, fumaric and adipic, have also been reported to stimulate the germination of pollen (Petrochenko, 1962). The pollens of many members of the family Malvaceae germinate only with some difficulty, but Bronckers (1961) obtained 76-82% germination of the pollen of cotton in artificial media in the presence of acenaphthene.

It is well known that during germination the pollen grains exercise a mass effect (Brink, 1924). The ancient Arabs did not discard their stocks of old pollen of date palm but mixed it with the fresh lot and used the mixture quite liberally. It is possible that the old pollen, although inviable, contributed some chemical substances to the mixture. Savelli and Caruso (1940) also mentioned a "mutual stimulation effect" in Nicotiana. When a large sample of pollen of one species was added to a small population of pollen of another species both pollens were stimulated and showed better growth. Several investigators have also reported that in cultures a denser sowing of the pollen grains results in better germination than when only a few grains are used (Fig. 1). Tišin (1962) found that the addition of pollen of Hibiscus, Malva neglecta and Gossypium arboreum to that of cotton 108F, promoted the germination of pollen and the development of bolls as compared to selfing and pollination with limited pollen. Similarly, Ščedrina (1982) observed that supplementary pollinations as well as mixtures of pollen from more than one variety of maize increased the frequency of fertilization from 71.8-97.4% and fertilization was accomplished in 24-28 hours. On the contrary, selfing and pollinations with a restricted number of pollen grains caused a delay in fertilization and a decrease in the amount of food reserves in the grain. Likewise, Vožda (1962) has reported that mixed pollinations in maize resulted in an increased weight of the ears but the other characters remained unaltered. According to Bardier (1960) in wheat the grain set was considerably improved if rye pollen was used either before or a few hours after pollination by wheat pollen. Such a "mentor" effect of foreign pollen has also been reported for sugar beet (Kovarskij and Guzan, 1960), rye (Sulima, 1960) and sorghum (Zajceva, 1961), but all these observations need confirmation on the basis of more critical data.

The receptivity of the stigma, is another link in the chain of events leading to fertilization, although it is often rather difficult to control. Generally the receptivity of the stigma is determined by the age of the flower, and the ambient humidity and temperature. Jones and Newell (1948) have reported that under favourable conditions the stigma remained receptive for 19 days in Buchloe dactyloides and for 24 days in Zea mays. According to Eghiazarjan (1962) stigmas of Nicotiana, less than a day old after anthesis, possessed greater receptivity than those of unopened or older flowers, and mature pistils preferred self pollen to foreign pollen.

The effect of the stigma is well known in certain plants in which the pollen fails to germinate after self-pollinations (see Brewbaker, 1957). Jost (1907) attributed this self-sterility to a retarded growth of the pollen tubes. On the basis of his work on Corydalis cava, Lilium bulbiferum, Secale cereale and other plants, he propounded the concept of "Individualstoffe", meaning that many plants have a characteristic substance in the pistil which inhibits the growth of their own pollen tubes. Correns (1912) noted, however, that while individuals of the same clone or pure line were cross-sterile, they could hybridize freely with individuals of other clones or pure lines. He therefore postulated the presence of "Linienstoffe" as the underlying cause of self-sterility. In other words, each pure line is characterized by a substance which is specific to all of its individuals and is inhibitory to their pollen. This theory opened the genetical approach to self-sterility, and Compton (1913) attempted to explain it in Mendelian terms. He assumed that substances are formed in the pistil which stimulate or retard the growth of the pollen tube. He also drew an analogy between self-sterility and the growth of fungous hyphae into the host tissue and compared the mechanism of self-sterility with that of immunity against a pathogen.

In some self-sterile plants successful self-pollinations can be made in the bud stage of the flower. Brassica oleracea (Attia, 1950), Trifolium hybridum (Williams, 1951) and Nicotiana alata (Pandey, 1988) are some examples. In Musa the frequency of fertilization is often discouragingly low, even if abundant pollen is supplied. However, several clones show an increased fertility if bud pollinations are made (Shepherd, 1954, 1960). No conclusive explanation is available for this phenomenon but it is postulated that some factors, which would inhibit the germination of pollen soon after natural self-pollination, might be absent or ineffective in the immature stigma. At the same time it may be noted that bud pollination is not always satisfactory and may lead to the formation of weaklings, heteroploids and sterile individuals (Iizuka, 1960). Like bud pollination, bee pollination has also been reported to be beneficial. In certain intravarietal crosses of cotton, bee pollination resulted in an increased number of bolls, seed set, and the quality and yield of the fibre (Arutjunova and Skrebcov, 1962).

In the Cruciferae the incompatibility reaction is localized in the stigma and after self-pollinations the percentage of germination of pollen is negligible. Sometimes, this can be improved by increasing the ambient humidity. El Murabaa (1957) has reported that high temperatures (26°C) proved more favourable in overcoming self-incompatibility in Raphanus sativus than lower temperatures (17°C) which promoted cross-pollination.

Sexual incompatibility is common in interspecific and intergeneric crosses. Nevertheless, several examples can be mentioned of successful interspecific and intergeneric crosses, especially among the orchids, where the stigma has little or no deleterious effect on the germination of foreign pollen.

Pollen Mixtures