全 文 ：Received 5 Jan. 2004 Accepted 15 Feb. 2004
Supported by the National Natural Science Foundation of China (30170060).
* Author for correspondence. E-mail: .
http://www.chineseplantscience.com
Acta Botanica Sinica
植 物 学 报 2004, 46 (6): 719-723
Isolation of Two Populations of Sperm Cells
from the Pollen Tube of Tobacco
QIU Yi-Lan, YANG Yan-Hong, ZHANG Sai-Qun, TIAN Hui-Qiao*
(Key Laboratory of Ministry of Education for Cell Biology and Tumor Cell Engineering, Xiamen University, Xiamen 361005, China)
Abstract: Pollen of Nicotiana tabacum L. is bicellular type, a generative cell and a vegetative cell in a
mature pollen grain. To isolate sperm cells pollen tube should be induced in advance. Using an in vivo-in
vitro technique, the style was pollinated and kept in vivo growing for 37 h and then cut about 3.5-4.0 cm
long to be cultured in a medium containing 15% (W/V) sucrose, 0.01% (W/V) boric acid, 0.01% (W/V) CaCl2,
0.01% (W/V) KH2PO4, at pH 5.0. A numerous pollen tubes grew out of the cut end of the style after being
cultured for 3-5 h. When the pollen tubes were transferred to a broken solution just containing 9%
mannitol some tubes broke and released the tube content into the broken solution including two sperm
cells. Two sperm cells just released from the tube are connected with vegetative nucleus (VN) of pollen
tube consisting of a male germ unit (MGU). Two sperm cells of tobacco are dimorphism: one is big and the
other small. The small one (Svn) connects with vegetative nucleus and the big one only associates with
the small one. Two isomorphic sperm cells might be selective fusion with egg cell and central cell during in
vivo fertilization (preferential fertilization). When two associated sperm cells were transferred into a
solution containing 0.01% cellulase, 0.008% pectinase and 9% mannitol, the association between two brother
sperm cells disappeared, and both cells could be easily separated using a micromanipulator. To probe the
mechanism of preferential fertilization two sperm cells from one pollen tube have to be separated each
other to find the differences at the molecular level. Two sperm cells were respectively collected into two
individual populations, each containing over thousand big sperm cells or small one, using a micromanipulator.
The two sperm populations will offer a possibility to find the differences between two sperm cells in
genes and proteins by using molecular methods, which will help us to understand the mechanism of
preferential fertilization and gametic recognition of higher plants, especially in the species with bicellular
pollen.
Key words: sperm cell; sperm dimorphism; preferential fertilization; Nicotiana tabacum
Since the first isolation of sperm cells were reported by
Cass in 1973, the isolation of sperm cells has been con-
ducted in about 40 species of flowering plants. Among these
species, most are tricellular pollen grains in which two sperm
cells have formed and can be directly isolated from pollen
grains (Russell, 1991). To isolate sperm cells from bicellular
pollen grains, the pollen tubes have to be firstly induced, in
which the generative cell divides to form two sperm cells.
Many isolations of sperm cells from bicellular pollen grains
were conducted with an in vivo-in vitro technique (Shivanna
et al., 1988), in which the stigma was pollinated and the
pollen tubes grew in vivo for some time, and then the style
was excised and immersed into a culture solution. When
pollen tubes grew out of the cut end of the style, it could
release two sperm cells by bursting the pollen tubes.
The isolated sperm cell can be used in in vitro fertiliza-
tion to understand the mechanisms of fertilization, zygote
activation and embryo development (Kranz and Lörz, 1993).
Sperm cells for in vitro fertilization need to be viable and
high-quality but small number in quantity even just one.
The isolated sperm cells can also be used in molecular
biology researches to find the differences between two
brother sperm cells at molecular level and probe the mecha-
nism of gametic recognition. For this purpose, the number of
isolated sperm cells not only requires over thousand to make
a cDNA library of sperm cells or to detect surface proteins
on the cells, but also requires two individual populations of
two brother sperm cells to identify the differences between
the both from one pollen tube. In most of the former reports
two brother sperm cells mixed in one population. Using
these sperm cells, some special genes and proteins of
Brassica, Lilium and maize have been isolated and re-
searched (Southworth and Kwiatkowski, 1996; Xu and Tsao,
1997; Xu et al., 1999; Zhang et al., 1999; Singh et al., 2002;
Xu et al., 2002). In this paper, we report a protocol for the
individual collection of two populations of sperm cells from
pollen tubes of bicellular pollen of tobacco, which are es-
pecially adapted to intently molecular research.
Acta Botanica Sinica 植物学报 Vol.46 No.6 2004720
1 Materials and Methods
Plants of Nicotiana tabacum L. were grown in a con-
trolled growth room at 20 ℃ in the dark and at 27 ℃ in the
light for 16 h daylength at Xiamen University. Tobacco
pollen is of bicellular type, which contains a generative cell
and a vegetative cell during flower anthesis. Although pol-
len grains could germinate and pollen tubes grow very long
when pollen grains were cultured in a medium, the genera-
tive cell did not divide to form two sperm cells unless using
some special treatment (Read et al., 1993). The generative
cell division and two sperm cell formation only occur in a
pollen tube growing inside style about 6-8 h after pollina-
tion (Tian and Russell, 1997a).
Flowers were emasculated before anthesis and artifi-
cially pollinated at anthesis. The style of tobacco is about
3.5-4.0 cm in length and it will take 40-44 h for the pollen
tube to reach ovary (Tian and Russell, 1998). The whole
style was cut at the 38th hour after pollination and cultured
in a modified medium containing 15% (W/V) sucrose,
0.01% (W/V) boric acid, 0.01% (W/V) CaCl2, 0.01% (W/V)
KH2PO4, at pH 5.0. The osmolality of the medium was 455
mOsmol/kg H2O. About just 0.5 cm style was immersed into
the medium according to natural polarity. After incubated
in the medium about 3 h, a lot of pollen tubes grew out of
the cut end of the style. To release sperm cells, the cut end
of the style with many pollen tubes was immersed into a
bursting solution just containing 9% mannitol, pH 5.0, os-
molality 493 mOsmol /kg H2O. Some pollen tubes burst in
the solution by osmotic shock and pairs of sperm cell were
released.
Paired sperm cells released from one pollen tube could
be operated using a micromanipulator. Two brother sperm
cells of tobacco are different in size, and the small one con-
nects with the vegetative nucleus and the big one is only
associated with the small one. The small sperm cell was
named Svn and the big one Sua according to the study of
Russell’s (1985), in which Svn was connected with the veg-
etative nucleus and Sua was not associated with the veg-
etative nucleus. A few of paired sperm cells without con-
spicuous difference in size were omitted to keep the purity
of two groups.
2 Results
The quality of pollen tubes is very important for releas-
ing tube content including two sperm cells. If pollen tubes
grew very well, they were uniform in size and apparently
vigorous because cytoplasmic streaming with organelles
moving quickly. When the styles with the pollen tubes
were transferred to a bursting solution, the pollen tubes
quickly burst at their tips and the cytoplasm spurted out. In
this condition, two sperm cells were easily identified be-
cause the tube cytoplasm soon disappeared in the solution
and vegetative nucleus (VN) was associated with one sperm
cell (Svn). Generally, the small one (Svn) connected with
VN was an average diameter of 6.79 mm, the big one (Sua)
was just associated with the small one (Sua), was 7.76 mm.
If pollen tubes grown out of the cut end of a style appeared
badly, the tip of pollen tube swelled, and cytoplasm mov-
ing could not be observed. When these pollen tubes were
transferred into a bursting solution, only a few of pollen
tubes burst, and the cytoplasm was squashed out of the
pollen tubes. The two sperm cells could not be identified
because the tube cytoplasm surrounded the both. Normally,
the pollen tubes growing out of the cut end of style were
good quality within in vitro cultured 5 h. If the styles were
cultured over 9 h, the pollen tubes grew too long, only a
few pollen tubes could burst.
The newly released sperm cells from pollen tubes are
generally ellipsoid and elongated, and then quickly became
football-shaped in bursting solution with 9% mannitol. At
the beginning, some cytoplasm of pollen tube wrapped two
sperm cells and the vegetative nucleus (Fig.1), which con-
sisted of a male germ unit (MGU) (Fig.2). Of the two sperm
cells, the one initially connected with the vegetative nucleus
is almost always the small one. The big one is associated
with small one, but is not directly connected with the veg-
etative nucleus. The vegetative nucleus swelled and broke
down quickly. The association of the both sperm cells could
keep at least 30 min. There was a spindle-shaped envelop-
ing membrane around the two sperm cells, and the mem-
brane was divided into two rooms in which two sperm cells
located individually (Fig.2). This membrane could keep in-
tact about 20 min with time after isolation, the cytoplasmic
material disappeared from membrane which could not be
observed clearly under a microscope but still made two
sperm cells be associated with each other. When an enzy-
mic solution dripped into broken solution that made bro-
ken solution with a final concentration of 0.01% cellulase
(Onozuka R-10), and 0.008% pectinase (Serva), the asso-
ciation between the two sperm cells disappeared quickly.
Both the cells could be easily separated using microman-
ipulator (Figs.3, 4). Generally, it took 30 min to select a hun-
dred of sperm cells from two styles. After the sperm cells
were washed twice in bursting solution the organelles of
pollen tube around both could be essentially clean out (Figs.
5, 6). Bursting solution containing purified sperm cells was
then transferred into an Eppendorf microcentrifuge tube,
QIU Yi-Lan et al.: Isolation of Two Populations of Sperm Cells from the Pollen Tube of Tobacco 721
which was precooled with liquid nitrogen. Each tube could
store sperm cells over thousand. The sperm cells were stored
in liquid nitrogen to prepare for the isolation of mRNA or
proteins.
The sperm cells also have a developmental change from
the beginning of the sperm formation to the end of the
journey growing in a style. At early stage, the association
between two sperm cells is very weak, and many paired two
sperm cells automatically separate in bursting solution in
10 min. During sperm cell maturation, however, the
Figs. 1-6. 1. Newly released two sperm cells wrapped by some cytoplasm of pollen tube. Arrow indicates two sperm cells. 2. Twenty
minutes later, pollen tube cytoplasm disappears and the arrow indicates an enveloping membrane around two sperm cells. 3, 4. Two
sperm cells are being separated using a micromanipulator. The arrow indicates one sperm cell being drawn into flame-drawn capillaries.
5. A population of big sperm cells. 6. A population of small sperm cells. (All of the figures are enlarged 940 times)
Acta Botanica Sinica 植物学报 Vol.46 No.6 2004722
association between two sperm cells becomes stronger,
and it was difficult to separate two sperm cells even in the
solution over 30 min, which reflects a developmental
change.
3 Discussion
Among the isolation of sperm cells of higher plants,
most are tricellular pollen species in which two sperm cells
have formed when flower bloom. The sperm cells of these
species could be directly released from pollen grains by
osmotic shock or physical grinding, and en masse isolation
of sperm cells could be easily reached. The quantity of
sperm cells correlated with that of pollen, and more pollen
grains release more sperm cells. The sperm cells in these
species with bicellular pollen in which pollen grains con-
tain a precursor generative cell and a vegetative cell, are
formed in pollen tube, and it is necessary to develop a
pollen tube to produce two sperm cells before isolating
sperm cells. Therefore, the isolation of sperm cells from
bicellular pollen is more difficult than tricellular pollen be-
cause of limited pollen tubes growing in a style, and limited
pollen tubes burst in assay. By using the in vivo/in vitro
method to culture pollen tube containing sperm cells, Tian
and Russell (1997a) have isolated sperm cells of tobacco,
and in vitro fertilization of tobacco was also tried (Tian and
Russell, 1997b). The isolated sperm cells of tobacco were
also used in assay of molecular biology screening special
genes of male gamete (Xu et al., 2002). Now, two brother
sperm cells of tobacco are intently separated and collected
into two individual populations containing over thousand
purified sperm cells, which will prepare for using molecular
methods to find differences between two brother sperm
cells on special genes and proteins that may control double
fertilization of tobacco.
Since the preferential fertilization of both sperm cells
from one pollen tube was found in Plumbago zeylanica in
1985 (Russell, 1985), the phenomenon of sperm dimorphism
has been found in many plants (Russell, 1991; Mogenson,
1992; Hu and Tian, 2002). Some pairs of two sperm cells
with dimorphism are different in cellular size, and some in
organelles content. All of the sperm cells with dimorphism
may be the preferential fertilization: one sperm cell spe-
cially fuses with egg to produce an embryo and the other
fuses with central cell to produce the nutritive endosperm.
Unfortunately, so far the preferential fertilization has only
been confirmed in P. zeylanica because of the difficulty of
techniques. Besides the differences of size and the number
of organelles between two brother sperm cells, recently,
the difference of surface charge was also found between
two sperm cel ls of P. zeylanica by using a cel l
electrophoretic method (Zhang and Russell, 1999). Those
differences between both sperm cells occurring in other
angiosperms may also have an influence on the pattern of
double fertilization. The two sperm cells of tobacco have
been found dimorphic in pollen tube (Yu and Russell, 1994)
and the divergence becomes more evident with develop-
ment (Tian and Russell, 1998; Tian et al., 2001). The differ-
ence may reflect a gametic recognition during fertilization,
and needs to be intensively studied with molecular methods.
Preferential fertilization is a very interesting research
project in developmental biology of higher plants, but we
know nothing about its molecular mechanism. The prefer-
ential fertilization of higher plants may involve two
possibilities: egg cell and central cell select two sperm cells,
or two sperm cells select egg cell and central cell in vivo.
For the former, it is necessary to identify the molecular
difference between egg cell and central cell, which needs to
isolate egg cell and central cell. For the latter, it is neces-
sary to separate two sperm cells from one pollen tube and
to find the special genes that control gametic recognition
and preferential fertilization of higher plants. Only after in-
dividual type of two sperm cells is collected into two sepa-
rate populations and each over thousand sperm cells, can
the difference between two sperm cells be identified by
using general molecular methods. Two purified populations
of sperm cells can be used to improve the research of sperm
cell biology of higher plants from cellular level to molecular
level by sensitive techniques such as immunoblotting, elici-
tation of monoclonal antibodies or the preparation of PCR-
amplified cDNA libraries. The utility of these techniques in
sperm cell biology of higher plants will enhance our under-
standing of the unique properties that the male gametes of
angiosperms may possess and contribute to an understand-
ing of the nature of preferential fertilization (Zhang et al.,
1998). As a precondition of the use of these techniques, the
isolation of two individual populations containing over
thousand sperm cells is necessary for the research of mo-
lecular characteristics between two brother sperms.
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(Managing editor: WANG Wei)