POLLEN GRAIN, POLLEN MORPHOLOGY, POLLEN GERMINATION, AND POLLEN VIABILITY

Pollen grain

Pollenis a fine to coarse powder containing micro gametophytes produce malegametes(sperm cells).Pollen grains have a hard coat that protects the sperm cells during the process of their movement between thestamensto thepistilof flowering plants or from the maleconeto the female cone ofconiferous plants. When pollen lands on a compatible pistil of flowering plants, itgerminatesand produces a pollen tube that transfers thespermto the ovule of a receptive ovary. The individual pollen grains are small enough to require magnification to see detail.

Structure of pollen

Pollen grains come in a wide variety of shapes (most often spherical), sizes, and surface markings characteristic of the species

Pollen grains ofpines,firs, andsprucesare winged. The smallest pollen grain, that of theForget-me-not(Myosotisspp.), is around 6m(0.006 mm) in diameter. Wind-borne pollen grains can be as large as about 90-100m. The study of pollen is calledpalynology

The pollen wall protects the sperm nucleus while the pollen grain is moving from the anther to the stigma, it protects the vital genetic material from drying out and solar radiation. The pollen grain surface is covered with waxes and proteins, which are held in place by structures called sculpture elements on the surface of the grain. The outer pollen wall prevents the pollen grain from shrinking and crushing the genetic material during desiccation and it is composed of two layers. These two layers are the tectum and the foot layer, which is just above the intine. The tectum and foot layer are separated by a region called the columella, which is composed of strengthening rods. The outer wall is constructed with a resistant biopolymer called sporopollenin. The pollen tube passes through the wall by way of structures called apertures.

Pollenaperturesare any modification of the wall of the pollen grain. These modifications include thinning, ridges and pores, they serve as an exit for the pollen contents and allow shrinking and swelling of the grain caused by changes in moisture content. The elongated apertues/ furrows in the pollen grain are called colpi (s. colpus) which along with pores, are a chief criteria for the identifying pollen classes.Pollen grains may have furrows, the orientation of which (relative to the original tetrad of microspores) classify the pollen ascolpateorsulcate. The number of furrows or pores helps classify the flowering plants, witheudicotshaving three colpi (tricolpate), and other groups having one sulcus.

Except in the case of some submerged aquatic plants, the mature pollen-grain has a double wall, a thin delicate wall of unaltered cellulose (the endospore orintine) and a tough outer cuticularized exospore orexine. The exine often bears spines or warts, or is variously sculptured, and the character of the markings is often of value for identifying genus, species, or even cultivar or individual. In some flowering plants,germinationof the pollen grain often begins before it leaves the microsporangium, with the generative cell forming the two sperm cells.

Pollen germination

Another germination event during the life cycle ofgymnospermsandflowering plantsis the germination of a pollen grain afterpollination. Like seeds,pollengrains are severely dehydrated before being released to facilitate their dispersal from one plant to another. They consist of a protective coat containing several cells (up to 8 in gymnosperms, 2-3 in flowering plants). One of these cells is atube cell. Once the pollen grain lands on the stigma of a receptiveflower(or a femaleconein gymnosperms), it takes up water and germinates. Pollen germination is facilitated byhydrationon the stigma, as well as by the structure andphysiologyof the stigma and style. Pollen can also be induced to germinatein vitro(in aPetrior test tube).

During germination, the tube cell elongates into apollen tube. In the flower, the pollen tube then grows towards theovulewhere it discharges thespermproduced in the pollen grain for fer The germinated pollen grain with its two sperm cells is the mature male microgametophyteof these plants

Pollen tube growth and guidance

Once the pollen interacts with the silk/stigma and begins to hydrate, a pollen tube is extruded through the pollen tube.

At a growth rate of some 0.5 cm/h in maize, the pollen tube grows rapidly through the transmitting tissue aiming to reach the female gametophyte via the micropyle region of the ovary.

We are interested in genes regulating pollen tube growth and have applied a functional genomics approach to identify oligopeptides involved in guiding the tube to the female reproductive cells. Our recent experiments have shown, that peptides secreted by the female gametophyte are involved in the final stage of pollen tube attraction.

when pollen reaches the stigma, it grows a pollen tube to extend down the style. Two sperm nuclei enter the ovule: one fuses with the egg to produce the zygote, the other fuses with two polar nuclei to make the 3nendosperm. The endosperm is part of the seed and provides nutrition for the sporophyte embryo. The fusion of two sperm nuclei with megagametophyte nuclei is calleddouble fertilization.

Pollen development in flowering plantPollen grains develop in the anthers of the staminae. In the anthers mostly four, but sometimes only two loculi are present. In the loculi sporogenic tissue (from the Greek spora = seed and the Latin generare = produce) can be found from which pollen develop. At the inner side of each loculus a layer of large, rectangular cells, the tapetum (from the Greek tapes = carpet; t) can be found. The tapetum serves for the nutrition of the developing pollen, the deposition of cell wall material in the outer part of the pollen grain and other compounds in and over the wall.

First, free pollenmothercells (PMC) are formed, which become spores by a meiotic divisionThemeiosisinvolves two divisions, which lead to the formation of four daughter cells, the spores. Those four cells are originally still interconnected and are called tetrads (Greek Tetra = four; figure C). Later they come apart and the tapetum deposits the outer wall or exine (more on the pollen wall inpollen morphology). The exine protects the spore against dessication, mechanical pressure and ultraviolet radiation.

Sometimes the exine layer is covered by sticky substances which are also produced by the tapetum. This adhesive material facilitates the attachment of pollen grains to insects, and in this way also zoophilic pollination. It also plays an important role in the adhesion of pollen grains to the female stigma and in the recognition between pollen and pistil. Also substances responsible forpollen allergyare often products originating from the tapetum

A. Cross-section through an anther of Lilie (Lilium)sp.) with on the left and the right side two loculi each. In the loculi sporemothercells (SMCs) can be seen from which the four spores develop through meiosis I and II. In between the loculi of each pair a thin layer of cells (arrow) is visible along which the loculus can burst open at maturity and release the pollen grains. In the middle the cross-sectioned filament (Fi) to which the anther is attached is indicated. In the upper part the vascular bundle (v) of the loculus can be distinguished.B. Loculus. The lumen contains developing pollen. On the inner wall (w) of the loculus a layer constitued of block-shaped single cells is present, the tapetum (t). The tapetum feeds the developing spore and -later- pollen.

C. Tetrad stage during pollen development. After the two meiotic divisions the four daughter cells are still interconnected and form a tetrad. They are still surrounded by the wall (arrow) of the original cell, the microspore mother cell (MMC). D. Mitotic division in the spore leading to the formation of a microgametophyte or pollen. Only the metaphase is shown here. The chromosomes lay in the equatorial plane of the cell. E. Nearly ripe pollen grain: visible are a vegetative cell with nucleus (VN), which later will form the pollen tube, and a generative cell with its own nucleus (GN), which later will divide into two sperm cells.

Pollen viability

Viability is defined as the ability to live, develop, or in the case of plants, to germinate when conditions favorable to the plant exist.Measuring Viability One method scientists use is to stain collected anthers, the male reproductive organ that holds the pollen, in aniline blue dye. The dye will be absorbed by the viable pollen grains. A slide is prepared and the dyed grains are counted under a microscope.

Pollen StorageThough pollen has the best chance of being viable when it is fresh, some horticulturalists cultivate pollen and store it. Pollen intended for use outside of a one- or two-week window when the grains are considered viable are refrigerated

Assessing pollen viability

frequently used to assess pollen viability. methods, a more extensive overview has recently been provided by Dafni and Firmage The fastest way of analyzing pollen viability is using vital stains that react with pollen enzymes, thereby indicating the presence of intact cellular contents. The most commonly used vital stain is the fluorochromatic reaction (FCR) which reveals esterase activity in pollen with an intact plasma membrane This test can be performed very quickly in the laboratory, and often the pollen viability results correlate with seed set.

Another frequently used method to assess pollen viability is in vitro germination. Because pollen grains of many species will easily germinate in a medium that contains boric acid and an osmoticum, this method is widely used (Taylor and Hepler, 1997). Furthermore, in recent years protocols have been developed that allow high germination frequencies of pollen of the more recalcitrant pollen. However, despite the simple basic requirements of pollen tube growth media, the optimal composition may vary from species to species and the use of suboptimal media may underestimate pollen viability.

Still, pollen germination rates usually provide more reliable data on pollen viability than vital stains. Finally, pollen viability may be measured after pollination, by analyzing germination on the stigma or seed set derived from that pollination. Both methods are time consuming and may lead to an overestimation of pollen viability if the pistil is overpollinated. Although all methods are valuable with respect to predicting seed set, they are prone to errors and results should be interpreted carefully. Furthermore, obtaining accurate pollen viability rates may depend strongly on storage conditions, protocols used and other factors. Because of these reasons, it is difficult to compare the results obtained in individual experiments.

Factor affecting pollen viability

Relative humidity Temperature UV-B radiation Transport

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