Rice Science, 2008, 15(2): 125–130

Copyright © 2008, China National Rice Research Institute.

Published by Elsevier BV. All rights reserved

Pollen Grain Germination and Pollen Tube Growth in Pistil of Rice

CHEN Shi-qiang, WANG Zhong, LIU Man-xi, XIE Zhao-wei, WANG Hui-hui
(Laboratory of Crop Inheritance, Cultivation & Physiology, Yangzhou University, Yangzhou 225009, China)

Abstract: The germination of pollen grain in vitro and the growth of pollen tube in the pistil of rice were observed with a
microscope. The stigma was removed at different time points after pollination to study its effect on seed setting rate. The rice
pollen grain started to germinate at 2 min after pollination and the pollen tube penetrated stigma into style in 5–10 min, 30 min
later the end of pollen tube reached the bottom of ovary, and only some pollen tubes arrived at embryo sac at 40 min after
pollination. Meanwhile, a small amount of callose began to deposit in the pollen tubes, a great deal of callose was observed
at 50 min after pollination, whereas the pollen grain began to shrink. The growing rates of pollen tube in the rice stigma, style
and ovary were 1500, 5000, and 5400 µm/h, respectively. The seed setting rate was quite low when the stigma was removed
at about 10–15 min after pollination, gradually increased when it removed at 20 min to 50 min after pollination, and over 60%
when it removed at 50 min after pollination and finally tended to be stable.
Key words: rice; pollen grain germination; pollen tube growth; seed setting rate; microscopic observation

Pollination occurs when pollen grains land on
stigma. If compatible, a pollen tube emerges through
one of the apertures of the grain and grows through
stigma, style, placenta, micropyle, and finally into
embryo sac. How does pollen tube grow? Which
factors are involved in its regulation? How can pollen
tube arrive at the embryo sac accurately? These are the
questions that researchers are concerned about all the
time. In 1979, Yang et al [1] studied the development
of pollen tube, and found that the pollen tube has two
different developmental pathways, sporophytic and
gametophytic developments. In the middle 1980s, Zee
and Xu [2] described the development of male organs
in rice sexual reproduction. After 1990, Tian and his
collaborators reported that there was obvious gradient
distribution of calcium in the stigma and style during
the pollen tube growth in tobacco [3-4]. Zheng et al [5]
thought that the requirement for calcium during the
development of anther might relate to the following
directional growth of the pollen tube. Zhao et al [6]
noted that G protein was involved in the regulation of
pollen tube growth. Huang et al [7] studied the formation
of pollen tube pathway in wheat, and divided the
developmental process of wheat pollen tube into three
stages: the germination of pollen tube, the formation
of pollen tube pathway, and the deposit of callose.
Wedzony and van Lammeren [8] studied the germination
of maize pollen grain and the pollen tube growth when
2,4-D was applied. The results showed that the pollen
tube grew slowly during 30 min after pollination, and
the tip of the pollen tube reached the micropyle in
5–24 h after pollination. Fan et al [9] studied the pollen
tube pathway in peony, and found that it was formed
at 1 h after pollination. Hu et al [10] observed the pollen
tube growth in the style of Arabidopsis by means of
aniline blue staining, and calculated the growing rate
of pollen tube. However, the growth dynamic of rice
pollen tube and its growth rate in the pistil of rice have
not been reported in detail.
In our study, the growth dynamic of rice pollen
tube – the growth rate and the pathway of pollen tube in
stigma and ovary – was investigated with a fluorescence
microscopy. We believed that our results would facilitate
the molecular biology research on rice pollen tube.
MATERIALS AND METHODS
Plant materials

Received: 16 January 2008; Accepted: 3 March 2008 Two rice (Oryza sativa subsp. indica) materials,
Corresponding author: WANG Zhong (wangzhong@yzu.edu.cn)
This is an English version of the paper published in Chinese in Chinese a variety Yangdao 6 and a cytoplasmic male sterile
Journal of Rice Science, Vol. 21, No. 5, 2007, Pages 513–517. (CMS) line YW-2S, were grown in pots at the
126
experimental farm of Yangzhou University, Yangzhou,
China. The YW-2S florets were pollinated with the
pollen of Yangdao 6 for studying the pollen grain
germination and tube elongation.
Methods
Observation of rice pollen grain germination in vitro
Melted potato culture medium, which was composed
of 15% sucrose, 5% potato starch, 0.0005% boric acid,
and 80% distilled water, was spread on a glass slide.
Rice pollen grains were put on the glass slide after the
hot culture medium cooled down. Then the slide was
observed under a microscope with a digital camera, and
the image of pollen grain germination was digitally
captured every 1 min.
Observation of pollen tube growth in pistil under a
fluorescence microscope
CO2 was applied to the spikelets of CMS line
YW-2S to facilitate flowering [11]. The flowering
spikelets of YW-2S were rapidly pollinated with the
pollen grains of Yangdao 6. The ovaries at 1, 2, 3, 5,
10, 20, 30, 40, 60, and 120 min after pollination were
put in FAA immediately and fixed for 24 h. Then they
were put in 70% alcohol solution. After rehydrated in
50%, 30% and 10% alcohol solutions, the ovaries
Fig. 1. Germination of pollen grains in the potato culture medium at 30°C.
A, Pollen grains at 0 min after cultured in the potato medium; B, Most pollen grains germinated 2 min later; C, The length of most pollen tubes
up to the diameter of pollen grain 4 min later; D, The length of pollen tubes exceeded twice of the diameter of pollen grain 6 min later; E, The tips of
pollen tubes had already enlarged 8 min later; F, The tips of pollen tubes had broken 10 min later.
Rice Science, Vol. 15, No. 2, 2008
were transferred to 2 mol/L NaOH solution for 12 h,
washed twice in distilled water, and then stained with
the solution of 0.05% aniline blue for 12 h. Softened
ovaries were put on the clean glass slides for
observation under a fluorescence microscope, and the
images were digitally captured [12].
The pollen tube growth rate was calculated by
measuring the length of pollen tube at different time
points after pollination [10].
Examination of seed setting rate when stigma was
removed at different time points after pollination
According to the method in studying wheat by
Huang et al [7], the stigma of pot-grown rice, which
was artificial pollinated, was removed at 5, 10, 15, 20,
30, 40, 50, 60, and 120 min after pollination, respectively,
and the seed setting rate was examined at maturity.
RESULTS
Germination of rice pollen grain and elongation of
pollen tube in the potato culture medium
When rice pollen was cultured on the potato
medium (Fig. 1-A), most pollen grains started to
germinate 2 min later (Fig. 1-B). Pollen tubes then
elongated, and 4 min later most pollen tubes were
CHEN Shi-qiang, et al. Pollen Grain Germination and Pollen Tube Growth in Pistil of Rice 127

approximately as long as the diameter of pollen grain
(Fig. 1-C). At 6 min after culture, the length of pollen
tubes exceeded twice of the diameter of pollen grain
(Fig. 1-D). At 8 min after culture, the pollen tubes
were three times as the diameter of pollen grain and
the tips of pollen tubes had already enlarged (Fig. 1-E).
At 10 min the tips of pollen tubes broke, meanwhile
the inclusions were released (Fig. 1-F), and the pollen
tubes stopped elongating. This was probably because
the potato medium could not simulate the real pistil of
rice, and it did not meet the requirement for the
normal elongation of pollen tube. In addition, the rate
of pollen tube growth was variable at different
temperatures, being 1400–1600 µm/h in the potato
culture medium at the optimum temperature of 30°C
for rice pollen grain germination.
Pollen grain germination and pollen tube growth
dynamics in the pistil of rice
Pollen grain germinated on the stigma at 2 min
after pollination (Fig. 2-A). At 5 min, the pollen tube
was longer than the diameter of pollen grain (Fig.
2-B), and the growth rate of pollen tube on the stigma
was about 1500 µm/h. At 10–15 min, most pollen

Fig. 2. Growth of rice pollen tubes observed with a fluorescence microscope.

A, Pollen grain germinated at 2 min after pollination; B, Pollen tube was longer than the diameter of pollen grain 5 min later; C, Most pollen
tubes grew into the style 10–15 min later; D, Pollen tubes growing in the ovary 25 min later; E, In ovary the tip of pollen tube enlarged 30 min later;
F, A pollen tube got through the micropyle 40 min later; G, Lots of pollen tubes abnormally enlarged and stopped growing 40 min later; H, Pollen
grains were shriveled and their color under the fluorescence microscope changed 50 min later; I, Pollen tubes growing in the whole pistil at 40 min
after pollination.
128
Table 1. Number of fertilized florets and seed setting rate of YW-2S with stigma removed at different time points.
Item
5 min 10 min
No. of treated florets 61 54
No. of fertilized florets 0 0
Seed setting rate (%) 0.0 0.0
tubes grew into the styles (Fig. 2-C), and the growth
rate reached about 5000 µm/h in the style. At 20 min,
the pollen tubes entered the ovary, then continued to
elongate in the ovary till 25 min after pollination (Fig.
2-D). At 30 min the tip of pollen tube enlarged in the
ovary (Fig. 2-E), and this may be resulted from the
rapid growth of pollen tubes. Moreover, many pollen
tubes were viewed at the bottom of the ovary at that
time, which suggested that the pollen tube pathway
from the stigma to the ovary was already formed at 30
min after pollination. The growth rate of pollen tube
in the ovary was about 5400 µm/h. At 40 min, a pollen
tube grew through the micropyle into the ovule (Fig.
2-F). Meanwhile a small amount of callose were
formed in the pollen tubes, which might prevent the
inclusions from flowing backward. There were several
pollen tubes growing into the ovule together, but most
pollen tubes developed abnormally, their tips enlarged
and finally stopped growing (Fig. 2-G, I). The
enlargement of the tip may involve some kinds of
mechanism that inhibit the elongation of the pollen
tubes. At 50 min, the pollen grains were shriveled (Fig.
2-H), and their inclusions were almost exhausted. The
pollen tube pathway from the stigma to the micropyle
was totally formed. At 60 min, the callose plug inside
the pollen tube was formed, thus the inclusions could
not flow to the ovule.
Seed setting rate when stigma was removed at
different time points after pollination
Fertilized floret number and seed setting rate of
rice CMS line YW-2S were recorded when the stigma
was removed at different time points after pollination.
As shown in Table 1, the seed setting rate was
regularly changed according to the time points when
the stigma was removed. No floret was fertilized when
the stigma was removed at 5 min after pollination,
which suggested that the pollen tubes had not grown
into the ovary at that time. When the stigma was
Rice Science, Vol. 15, No. 2, 2008
Time for removing stigma (Minutes after pollination)
15 min 20 min 30 min 40 min 50 min 60 min 120 min
46 57 56 65 72 66 80
4 8 16 23 44 44 54
8.7 14.0 28.6 35.4 61.1 66.7 67.5
removed at 10 min after pollination, only a few florets
were fertilized, and the seed setting rate was nearly
zero. Then the seed setting rate increased from 8.7%
to 61.1% during the time for removing the stigma at
15–50 min after pollination. The later the stigma was
removed, the higher the seed setting rate was. When
the time points for removing the stigma were delayed
from 50 min to 120 min after pollination, the seed
setting rate was increased quite slowly. The result was
consistent with the observations of pollen tube elongation
under a fluorescence microscope. Rice pollen tube
grew into the ovary at about 30 min after pollination,
meanwhile, the pollen tube pathway from the stigma
to the ovary was formed and the sperm cells in the
pollen tube were released to the embryo sac at that
time. Thus, the removal of stigma almost had no effect
on the seed set when the pollen tube had already
reached the ovary.
DISCUSSION
Pollen grain germination and pollen tube growth
dynamic in pistil of rice
The pollen tube growth in wheat had been
observed by Huang et al [7]. Our results in rice and the
results in wheat [7] are listed in Table 2. The
characteristics of pollen grain germination and pollen
tube growth in wheat and rice are as follows: 1) At
2–3 min after pollination, the pollen grains of rice and
wheat germinated. Whereas at 10 min after pollination,
the newly emerged pollen tubes did not reach the
ovaries, thus the removal of stigma at this period
would result in the failure of the fertilization of florets.
2) Only a few florets were fertilized and the seed
setting rate was quite low when the stigma was
removed within 20 min after pollination. When the
pollen tube elongated to some length, most inclusions
in the pollen grains moved to the front part of the
CHEN Shi-qiang, et al. Pollen Grain Germination and Pollen Tube Growth in Pistil of Rice 129

Table 2. Comparison on the pollen tube growth between wheat and rice.
Wheat Rice
Time for removing stigma
(Minutes after pollination) Seed setting rate Seed setting rate
Fluorescence observation Fluorescence observation
(%) (%)
2 min Pollen grains did not germinate 0.0 Pollen grains germinated 0.0
3 min Pollen grains germinated 0.0 Pollen tubes entered the style 0.0
5 min Pollen tubes grew in the style 0.0 Pollen tubes grew in the style 0.0
10 min Pollen tubes did not grow towards the ovary 0.0 Pollen tubes grew towards the ovary 0.0
20 min Pollen tubes grew towards the ovary 10.5 Some pollen tubes grew into the ovary 14.0
30 min Pollen tubes grew in the ovaries 31.4 Most pollen tubes grew into the ovary 28.6
40 min Pollen tube pathway was basically formed 35.1 Pollen tube entered the ovule 35.4
50 min Pollen tube pathway was formed 55.0 Pollen tube pathway was formed 61.1
60 min Callose plug was formed 67.4 Callose plug was formed 66.7

pollen tube to provide nutrition for the following

growth of the tube, thus the removal of stigma had a
few effect on its growth at that period. 3) During
30–40 min after pollination, lots of pollen tubes were
observed in the ovary of rice and wheat. Meanwhile,
some pollen tubes grew through the micropyle into the
ovule, and sometimes several pollen tubes grew into
the ovule together. The seed setting rate exceeded
35% as the stigma was removed at 40 min after
pollination, which suggested that pollen tube pathway
was basically formed at that time [7]. 4) During 40–50
min after pollination, the tips of some pollen tubes
abnormally enlarged, and the callose was deposited
inside. The pollen tube pathway was completely
formed at that time. The seed setting rate of rice and
wheat florets with the stigma removed was increased
largely, being more than 55%. The seed setting rate of
rice with the stigma removed during 20–50 min after
pollination was higher than that of wheat. This is
probably attributed to the different temperatures at
flowering. Wheat flowers in spring when the
temperature is relatively low, while rice flowers in hot
summer. Thus, rice pollen tubes grow faster and the
seed setting rate is higher compared with those of
wheat. 5) During 50–60 min after pollination, the
pollen grains shriveled, their inclusions were used up,
and a large amount of callose were formed, blocking
the pollen tubes. The seed setting rate was quite high
with the stigma removed at this period, and it was
55% in wheat, and reached 61.1% in rice when the
stigma removed at 50 min after pollination. The seed
setting rates of both wheat and rice were above 65%
when the stigma removed at 60 min after pollination,
closing to the rate under natural conditions.
Mechanisms probably involved in directional
growth of pollen tube
Tian and Yuan [4] had studied the growth of
tobacco pollen tube, and found that the growth of
pollen tube was polarized and the pollen tube elongation
resulted from the secretory vesicles produced by Golgi
apparatus, which might be concerned with the
directional growth of pollen tube into the embryo sac.
The germination of pollen grain and directionally
growth of pollen tube were thought to be closely
related to calcium. Zheng et al [5] pointed out that
calcium was found in the styles of tobacco. The study
on towel gourd by Chu et al [17] showed that low
calcium concentration of 0.01–1.00 mmol/L could
promote the pollen grain germination and the pollen
tube elongation, while the high concentration of 10
mmol/L inhibited. Moreover, Yang and Zhou [18]
suggested that the pollen tube growth was associated
with plant hormones.
It is common in plant that many pollen tubes get
into the ovary, but only few reached the bottom of the
ovary, and finally just one pollen tube gets through the
micropyle and undergoes double fertilization. In rice,
most pollen tubes, when they get to the ovary, stop
growing and their tips are abnormally enlarged.
Maybe it resulted from the lack of the ability to
elongate and enter the micropyle in pollen tubes, or
when one pollen tube gets through the micropyle, it
may then block the other pollen tubes into the
micropyle, thereby the other pollen tubes grow
abnormally and finally died out [2].
In our research, the stigma of CMS line YW-2S
was chosen for pollen tube germination. The number
130
of pollen grains in the stigma was controlled by
artificial pollination, thus the growing process of
pollen tubes could be better observed and photographed.
The pollination of fertile rice variety, however, could
not be controlled because of the influence of its own
pollen grains. When pollen tubes extended into the
ovule, neither their growth nor the release of the two
sperm cells into the embryo sac could be observed
under a fluorescence microscope because of its thick
integument. However, the following observation could
be done by means of paraffin sectioning [19] and resin
sectioning.
ACKNOWLEDGEMENTS
This work was supported by the National Natural
Science Foundation of China (Grants Nos. 30070454
and 30471045).
REFERENCES
1 Yang H Y, Zhou C. Two different developmental pathways of
pollen grains. Acta Bot Sin, 1979, 21(4): 345–351. (in
Chinese)
2 Zee S Y (Xu S X), Xu X B. Morphology and Anatomy of
Rice. Beijing: Agricultural Press, 1984. (in Chinese)
3 Xie C T, Qiu Y L, Ge L L, Chen S H, Tian H Q. The
distribution of calcium in the stigma and style of tobacco
during pollen germination and tube growth. Acta Physiol Sin,
2005, 31(1): 53-61. (in Chinese with English abstract)
4 Tian H Q, Yuan T. Calcium function in fertilization process
in angiosperms. Acta Physiol Sin, 2000, 26(5): 369–380. (in
Chinese with English abstract)
5 Zheng M Z, Yang Y H, Guo J, Qiu Y L, Xie C T, Tian H Q.
The primary observation of calcium distribution during the
anther development of tobacco. J Xiamen Univ: Nat Sci, 2004,
43: 126–132. (in Chinese with English abstract)
6 Zhao C P, Zhang S L. A review on regulatory effect of G protein
Rice Science, Vol. 15, No. 2, 2008
on pollen tube growth. Acta Bot Boreal Occident Sin, 2004,
24(11): 2177–2182.
7 Huang J H, Wang G J, Sun Y, Diao Y L. Studies on the formation
date of pollen tube way of wheat. Heilongjiang Agric Sci,
2004(2): 20–22. (in Chinese with English abstract)
8 Wedzony M, van Lammeren M A M. Pollen tube growth and
early embryogenesis in wheat × maize crosses influenced by
2,4-D. Ann Bot, 1996, 77: 639–647.
9 Fan B Y, Gao S P. Preliminary study on the form time of
pollen tube pathway of Peony (Paeonig suffruticosa Andr),
Henan Agric Sci, 2004(5): 51–52. (in Chinese)
10 Hu Y, Zhao J. Morphologic character of pollen tube growth
and embryo development during sexual reproductive process
of Arabidopsis thaliana. J Wuhan Univ, 2006, 52(2):
241–246.
11 Wang Z, Gu Y J, Gao Y Z. CO2 promoting flowering in rice.
Plant Physiol Comm, 1987(3): 29–31. (in Chinese)
12 Wang L, Liu X D, Lu Y G, Feng J H, Xu X B, Xu S X (Zee S
Y). Endosperm development in autotetraploid rice. Rice Sci,
2005, 12(2): 83–91.
13 Yang H Y. A century retrospect and prospect of the researches
on fertilization biology in plants. World Sci-tech R & D, 1998,
20(3): 9–13. (in Chinese)
14 Wang W H, Zhao J, Yang H Y. Embryological studies on in
vitro pollination in rice. Acta Bot Sin, 2001, 43(9): 905–909.
(in Chinese with English abstract)
15 Hu S Y. Embryology of Angiosperms. Beijing: People
Education Press, 1982. (in Chinese)
16 Hu S Y. Research History on Fertilization of Angiosperms and
Origin of Double Fertilization. Beijing: Science Press, 2002.
(in Chinese)
17 Chu L M, Jin A H, Xu D Q, Xu G D, Liu P. Effect of calcium
concentration on pollen germination and pollen tube growth
of Luffa Cylindrica Roem. J Henan Agric Sci, 2005(4): 56–58.
(in Chinese with English abstract)
18 Yang H Y, Zhou C. Review of Studies on Sexual Reproduction
in Plants. Wuhan: Wuhan University Press, 2001. (in Chinese)
19 Shen J H, Shen Y, Wang Y J. Pathway of pollen tube growth
and duration of different phases of fertilization process in
wheat. Acta Agron Sin, 2006, 32(4): 522–526. (in Chinese
with English abstract)