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Pollen Germination Methods
Conference Paper · December 2016
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Pollen Germination Methods
Pollen is anatomically simple organ compared to other highly differentiated tissues and
other plant organs. Pollen develops within the anther and at maturity contains the products of
sporophytic gene expression, arising from the tapetal layer of the anther wall, and gametophytic
gene expression from the vegetative and generative nuclei. When a pollen grain falls on a
receptive stigma, the stored RNA, protein, and bioactive small molecules allow rapid
germination and outgrowth of a tube that penetrates and grows within the style. Experiencing the
most rapid growth of any plant cell known, which is restricted exclusively to the tip of the tube,
the pollen tubes eventually deposit the two sperm cells in the embryo sac where they fuse with
the egg and central cell to form the zygote and endosperm respectively. Pollen quality must be
assessed to find plant fertility, to monitor pollen state during storage, ecological or taxonomic
studies, pollen biochemistry, stigma interactions, incompatibility systems and fertilization,
breeding, genetics and conservation ecology.
Pollen Viability
Pollen viability is one measure of male fertility. Viable pollen is responsible for high crop
yield. In hybridization programme pollen fertility and viability have a paramount importance. In
other contexts, pollen may be stored for germplasm conservation, to make hybrids between
plants that flower at different times or places, or for later use in hybridization programmes, and
the quality must be monitored (Kearns and Inouye 1993).
There are direct and indirect measures of pollen viability. Direct tests consist of
depositing the pollen on receptive stigmas and determining whether seeds are produced. Such
testing has the advantage of providing an unequivocal measure for the population of pollen
grains deposited on the stigma, but it has several disadvantages, such as time-consuming, labor
intensive and lager number of fresh samples. Pollen germination can also be scored in vitro.
Indirect methods rely on the correlation between ability to fertilize an ovule and some
physiological or physical characteristic that can be determined more rapidly. Indirect- methods
that correlate with pollen germination include (1) the fluorochromatic procedure (FCR), (2)
testing pollen for enzyme activity, and (3) testing stain ability of vegetative cells. The correlation
is greatest for FCR and lowest for stain ability (Heslop-Harrison et al. 1975, Heslop-Harrison
1992).
Germination Test
Pollen of most species will germinate and grow a tube when placed in a solution of
calcium, boron, and an osmoticant. Although it provides a control- led experimental system,
germination in-vitro does not completely mimic growth in-vivo such as temperature and
germination medium. In-vitro germination can be affected by time of pollen collection and
storage conditions as well as pollen density on the culture medium. A large numbers of pollens
have been successfully germinated under laboratory conditions on relatively simple media. The
composition of a germination medium to obtain optimal responses has to empirically formulate
for each species. Brewbaker and Kwack (1963) medium found to be suitable for most species.
Brewbaker- Roberts Hodgkin and
Ingredients
Kwack Medium Medium Lyon’s Medium
Sucrose (5 - 20%) Adjust according to species
Boric Acid 100 mg/l 10 mg/l 100 mg/l
Calcium Nitrate 300 mg/l 362 mg/l 400 mg/l
Potassium nitrate 100 mg/l 100 mg/l 100 mg/l
Tris - 60-130 mg/l -
Manganese sulfate - - 4.86 g/l
TAPS - - 200 mg/l
One must keep in mind that time and method of collection and storage age of pollen
affect viability (Shivanna and Johri 1989, Shivanna 1993). Pre germination relative humidity
probably affects the internal solute potential and wall properties of the pollen grain and has a
subsequent effect on pollen germination (Shi and Yang 2010). Any work requiring maximally
viable pollen should be conducted shortly after anther dehiscence. Germination is most
successful immediately after anthesis and viability deteriorates rapidly in most species. Several
methods of in vitro raising pollen cultures are available. The selection of a method depends on
requirement of the study and facility. The methods are as follows (Shivanna and Rangaswamy
1992).
Hanging Drop Culture
The hanging drop culture method has been most commonly used in the past. Essentially,
it involves suspending the pollen grains in a drop of nutrient medium (on a cover glass) hanging
over a shallow depression. To prevent evaporation of the culture medium, the hanging drop
culture is sealed suitably.
Sitting Drop Culture
The sitting drop culture method is simpler than the hanging drop method. It involves
culturing of pollen grains in a drop of culture medium placed on a microslide. The culture is then
maintained in a humid chamber to prevent evaporation.
Suspension Culture
In large samples of pollen grains in 2 to 10-ml culture medium in suitable vials incubated
on a shaker and observed for germination by placing drop in slide.
Pollen State
Pollen is like any living organism, its behavior and survival are influenced by both environment
and genotype. Therefore, pollen quality must be measured under defined conditions, and thought
about experimental and plant growth conditions is always required. In experimental treatments
possible variables must be carefully considered, and controls used. Pollen production and quality
vary from hour to hour, day to day and season to season. Many species have strong diurnal
rhythms: in the summer anther dehiscence may occur at 5 am and pollen collected 12 h later may
be in viable (Shivanna and Johri 1989, Heslop-Harrison 1992). The nutrition of the parent plant
can also have large effects on pollen viability may affect results from the in-vitro Germination
tests discussed below, more than growth on the stigma. Some pollen shows no viability in tests
unless it is correctly preconditioned by, for example, leaving in a humid atmosphere before
testing. For most species, the requirements for such preconditioning or post-maturation are not
established, so cytological methods to estimate viability may give an unrealistic estimate of
quality.
Microscopy
Light microscopy is profusely used for examining pollen quality, since it allows clear
observation of pollen morphology. It is useful to examine pollen before carrying out any of the
other tests described here to monitor its condition. Staining methods are discussed below, but
direct examination of anthers and pollen will confirm that grains are present in anthers, and show
undifferentiated or grossly shrunken grains. Most pollen samples will have a few such in viable
grains, but the presence of many aborted grains indicates substantial infertility. The causes may
include genetic sterility of hybrids, or severe environmental stress. Since microscopy is quick,
good pollen can be immediately used for pollinations, and field examination is possible. Pollen
can be observed as shed from the anther under a dissecting microscope dry at low power under a
transmitted light microscope, or at higher powers when mounted in a medium of suitable osmotic
strength (typically 10-20% sucrose to prevent bursting), or suspended in an organic solvent such
as acetone.
Pollen 'Staining' Tests
Stains are specific to pollen components can also be used to test for viability. Aniline
blue in lactophenol stains callose; acetocarmine stains chromosomes; phloxin-methyl green
stains cytoplasm and cellulose (Dafni et al. 2005). However, immature or non-viable pollen
sometimes contains enough of these elements to cause staining, and viable pollen of some
species does nor stain well (Heslop-Harrison and Heslop-Harrison 1985). Therefore, stains
provide (at best) only rough estimates of viability.
Dyes such as acetocarmine in glycerol jelly and aniline blue in lactophenol stain
nonabortive pollen but not abortive pollen. However, Alexander's stain differentially colors
abortive and nonabortive pollen; malachite green stains cellulose in pollen walls, and acid
fuchsin stains protoplasm. Thus abortive (or germinated) grains appear green, and grains with
protoplasm appear pink. Alexander's stain also be used to distinguish self versus outcross pollen
on stigmas in species where the incompatibility' mechanism acts at the stages of pollen
germination and self-pollen retains cytoplasm.
Pollen Quality
All the methods of testing discussed above depend on many variables and none is able to
confirm that a particular sample of pollen is in viable and will not be able to fertilize any dant:
they only give a likelihood estimate. Most are unsuitable for field use, and the variability
between different samples for the same species, even although fertile, can be high. Nevertheless,
cytological methods for testing pollen quality have proved valuable for research and in
agriculture, and give unique information about plant fertility.
Brewbaker, J.L. and Kwack, B.H., 1963. The essential role of calcium ion in pollen germination
and pollen tube growth. American Journal of Botany, 50, 859–865.
Dafni, A., Kevan, P.G., and Husband, B.C., 2005. Practical Pollination Biology. Environ quest
Ltd. Ontario.
Heslop-Harrison, J. and Heslop-Harrison, Y., 1985. Germination of stress-tolerant Eucalyptus
pollen. Journal of cell science, 73, 135–57.
Heslop-Harrison, J.S., 1992. Cytological techniques to assess pollen quality. Sexual plant
reproduction, 41–48.
Heslop-Harrison, R., Knox, B., Heslop-Herrison, J.Y., and Mattson, O., 1975. Pollen wall
proteins: emission and role in incompatibility response. In: J.G. Deckett and P.A. Racey,
eds. The Biology of Male Gamete. Biological Journal of Linnaean Society, 189–202.
Kearns, C.A. and Inouye, D.W., 1993. Techniques for pollination biologists. Colorado, USA.:
University Press of Colorado, Niwot,.
Shi, D.-Q. and Yang, W.-C., 2010. Pollen Germination and Tube Growth. In: E.C. Pua and M.R.
Davey, eds. Plant Developmental Biology - Biotechnological Perspectives: Volume 1.
Berlin, Heidelberg: Springer Berlin Heidelberg, 245–282.
Shivanna, K.R., 1993. Pollination biology: Contributions to fundamental and applied aspects.
Current Science, 65 (3), 226–233.
Shivanna, K.R. and Johri, B.M., 1989. The Angiosperm Pollen: Structure and Function. Wiley
Eastern Ltd., New Delhi.
Shivanna, K.R. and Rangaswamy, N.S., 1992. Pollen Biology - A laboratory Manual. Springer-
Verlag, Berlin.
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