Pollen Grain Size and Surface Morphology for a Perennial
Rye Hybrid
Stacey Lacoste
Department of Biological Sciences
University of Lethbridge
Lethbridge, Alberta
Abstract
To determine whether pollen grain size and surface morphology impacts fertility
in a perennial rye hybrid, pollen and stigmas were collected from hybrids experiencing
fertility problems. All samples were placed in Karnovsky's fixative then
dehydrated in an ethanol series. Pollen was then further processed through
HMDS drying, while stigmas were dried using Critical Point Drying. The Scanning
Electron Microscope was used to view pollen and stigma samples to help determine
possible differences in size and surface morphology. Image capture and measurements
were accomplished using the Quartz PCI program. There were no significant
differences in length or width between pollen grains of the three plants sampled.
The majority of pollen grains for all three samples were elliptical in shape,
with a single pore present at one end. The surface of the grains was rough and
pebbly in texture, most grains had a relatively flat exine. No difference
in stigma morphology was apparent. Stigma samples from plants 121 and 256
contained pollen at attachment sites. In this preliminary investigation
there appeared to be no difference in pollen size and surface morphology for the
three hybrids sampled.
Introduction
Rye is a widely adapted forage crop because of its winter hardiness and ability
to grow on marginal soils. Its extensive root system also helps to provide
for drought tolerance. Both perennial and annual varieties of rye also have
the advantage of providing high quality forage for livestock, they are highly
digestible and thus are suitable for all classes of ruminants (Fehr and Hadley,
1980). There is interest in the development of hybrid varieties of perennial
rye to provide higher quality, self-seeding forage crops for livestock.
However, there are often pre- and post-zygotic barriers to fertility in hybrids
that must be overcome (DeJeu and Caldere, 1997; Sarmah and Sarla, 1998).
Temporal and mechanical barriers can be easily overcome for example by harvesting
and storing pollen or by mechanically manipulating the recipient plant, but
delivery of pollen to stigmas does not ensure reproductive success. Rye
is cross-pollinated so several mechanisms have developed to screen out unwanted
pollen and stimulate growth of desired pollen (Fehr and Hadley, 1980). The recognition
event is most likely genetically-mediated as in Solanaceae (Kao and McCubbin,
1996). It is believed that the incompatibility reaction in rye is controlled
by a multiple allelic series in one or two loci. The pollen grain will
be inhibited if both incompatibility loci in the stigma have alleles common
to the two loci present in the pollen (Fehr and Hadley, 1980). The trigger
for recognition is often a protein coating the pollen surface and/or the surface
of the stigma (Fehr and Hadley, 1980). The recipient styles may
impose incompatibility barriers that prevent germination of the pollen on the
stigma or prevent combination of the gamete with the egg. It is believed
that pollen grains may receive nutrition from the stylar tissue to aid pollen
tube growth (Cruzan, 1990), so the recipient plant may withhold those nutrients
to prevent tube growth.
The pollen grain itself may have a role in compatibility/incompatibility.
Pollen grain surface morphology may impact mating success if recognition
works like a lock-and-key model, although it has been difficult to prove
this. In addition, differences in pollen size may represent functional
mating barriers, if the volume of the grain is proportional to its resource
content, implying that larger grains contain more nutrients and thus have
an increased likelihood of generating longer pollen tubes that have an
increased chance of reaching the ovum for a fertilization event. Cruzan
(1990) found that in Erythronium grandiflorum smaller grains showed less
stylar attrition than larger ones and thus had reduced fertility in comparison
to larger grains.
The purpose of this study was to compare pollen grains and stigmas from a number
of plants of a perennial rye hybrid, using scanning electron microscopy to evaluate
differences in size or surface features of the grains. The implication
is that these possible differences in size and surface features may be influencing
the low fertility rates in the hybrid.
Materials and Methods
Pollen and spikelets were collected from plants of this perennial rye hybrid.
Stigmas were dissected out of the spikelets and both stigmas and pollen
were placed into Karnovsky's fixative, a combination of 2.5% to 4% glutaraldehyde
and 4% formaldehyde. All samples were left for 48 hours then put
through an ethanol dehydration series consisting of 50%, 70%, 85%, 95%
for two changes each at 15 minutes per change and 100% for two changes
at 30 minutes each.
The stigma specimens were further processed using Critical Point Drying
(CPD), then mounted using silver paste and sputter-coated (Polaron Division
Bio-Rad). The pollen was run through an Hexamethyldiethylsulfoxide
(HMDS) series consisting of 1 run at 50:50 HMDS and Ethanol for 15 minutes
each, 1 run of <100% HMDS for 15 minutes each and 2 runs of 100%
HMDS for 15 minutes each. The pollen was then mounted on stubs using
double-sided sticky tape and sputter-coated. The Scanning Electron
Microscope (Hitachi S-450 and Hitachi S-500) was used to view pollen and
stigma samples to help determine possible differences in size and surface
morphology. Images were captured, processed and labeled with the
Quartz PCI© program. Sizes were determined through measurements
with Quartz PCI and a t-test was used to determine whether the differences
were significant.
Results
| Hybrid Number |
90 |
121 |
256 |
Significance |
| length (µm) |
48.4±6.77 |
51.6±4.64 |
47.1±3.82 |
Not Significant |
| width (µm) |
31.8±5.59 |
32.7±3.04 |
35.3±3.03 |
Not Significant |
Table 1. Significance of differences in mean length and width values
for hybrid pollen grain samples. Significance was determined via
the t-test.
Discussion and Conclusions
The pollen grains appeared to be relatively uniform in shape (Figures 1-3) although
some were occasionally crescent-shaped or spherical. They were elliptical
and generally contain a pore nearer to the larger end, which concurs with descriptions
of pollen grains of other species of rye (Figure 3a) (Erdtman, 1971). Two
grains in sample 256 featured double pores on the exine (Figure 4). The surfaces
of the grains had a pebbly or rough texture, although the exine was generally
quite flat which is indicative of the wind method of dispersal utilized by this
type plant (Figure 5) (Wodehouse, 1959). A number of specimens had
very wrinkled surfaces, which may be due to the HMDS technique used to prepare
them. However, this deformed type of grain appeared to be much more prevalent
in sample 90 (Figures 1 and 6) than in either of the other two samples, although
no statistical analysis was performed. The prevalence of this feature in
sample 90 in comparison with the other samples may indicate that this feature
is not due to the drying technique and is a natural characteristic of the pollen
in this sample of hybrid plant and thus may play a role in recognition by the
stigma. Mean lengths (t-test; P>7.05E-05, df=69) and widths (t-test; P>2.5E-03,
df=69) of the pollen grains were determined to be not significant via t-tests
(Table 1). Thus, incompatibility is not related to pollen grain size.
Stigmas were also sampled from each of the three plants. They appeared
to be very uniform in structure and surface features among all three specimens
with the ends appearing slightly wrinkled or variegated (7-11). Pollen
grains were found attached to the stigmas in samples 121 and 256. Two
situations were apparent; in some cases there was a web-like substance present
between the pollen grains and the stigma surface (Figure 12-14). Rejection
mechanisms have been shown to be biochemical in other angiosperms (Fehr and
Hadley, 1980), this substance was probably secreted by the stigma as some sort
of rejection mechanism, possibly to prevent pollen tube growth, which has been
postulated to be a mechanism of self-rejection in rye plants (Landes,
1939). In other cases the pollen appeared to be attached to the stigma
and the aforementioned substance was not present, which may indicate acceptance
by the plant of the pollen grain (Figures 12, 15-17).
Due to the reduced fertility experienced by these hybrids, there is obviously
some sort of rejection mechanism occurring. However, this study found
no apparent differences in pollen size and surface morphology between these
three plants. Therefore, it is most likely that compatibility in this
species is not due to variation in pollen size or pollen and stigma surface
features. It is probably due recognition of the protein coat present on
the surface of the pollen or stigma. However, the data presented here
is a preliminary study, and a number of experimental factors should be improved
upon before pollen size and surface morphology can be conclusively ruled out
as mechanisms of rejection due to two factors. The surface wrinkling experienced
by pollen of sample 90 may be due to the HMDS drying. Thus, this technique
should be compared to the CPD technique which has been used in the past for
pollen and has been shown to be less damaging for other plant tissues (Robards,
1985). Larger pollen sample sizes from a larger population of plants are
also necessary to provide more conclusive evidence. It was also difficult
to accurately measure the sizes and determine the shapes of the pollen grains,
as due to their small size the glue used obscured parts of them from view, this
will have to be controlled for in future studies.
Works Cited
Cruzan, M.B. Variation in pollen size, fertilization ability,
and post-fertilization siring ability in Erythronium grandiflorum.
Evolution. 1990 (44):843-856.
DeJeu, M.J., and F.G. Caldere. Retarded embryo growth and early
degeneration of sporophytic tissue are associated with embryo abortion
in the interspecific cross Alstroemeria pelegrina x Alstroemeria aurea.
Can. J. Bot. 1997 (75):916-924.
Erdtman, G. Pollen Morphology and Plant Taxonomy. New York:
Hafner Publishing Company, 1971.
Fehr, W.R., and H.H. Jones, eds. Hybridization of Crop Plants.
Wisconsin: American Society of Agronomy and Crop Science Society of America,
1980.
Kao, T.H. and A.G. McCubbin. How flowering plants discriminate
between self and non-self pollen to prevent inbreeding. PNAS.
1996 (22):12059-12065.
Landes, M. The cause of self-sterility in rye. Am. J. Bot.
1939 (26):567-571.
Robards, A.W., ed. Botanical Microscopy 1985. New York:
Oxford University Press, 1985.
Sarmah, B.K., N. Sarla. Erucastrum abyssinicum x Brassica oleracea
hybrids
obtained by ovary and ovule culture. Euphytica.1998 (102):37-45.
Wodehouse, R.P. Pollen Grains. New York: Hafner Publishing
Co., 1959.
Acknowledgments
I would like to thank Doug Bray and Roy Pescador for all of their advice
and help with the SEM, and especially for all their patience with my endless
questions and crises. I would also like to thank Dr. Jim Thomas for
his help with sample collection and questions.
