全 文 ：Received 6 Aug. 2003 Accepted 30 Oct. 2003
Supported by the National Natural Science Foundation of China (30070454).
* Author for correspondence. E-mail: ; Fax: +86 (0)514 7349817; Tel: +86 (0)514 7979354.
http://www.chineseplantscience.com
Comparison of Caryopsis Development Between Two Rice Varieties
with Remarkable Difference in Grain Weights
WANG Zhong1*, GU Yun-Jie1, HIRASAWA Tadashi2 , OOKAWA Taiichiro2, YANAHARA Satogo2
(1. Department of Agronomy, Agriculture College, Yangzhou University, Yangzhou 225009, China;
2. Tokyo University of Agriculture and Technology, Tokyo 183-8509, Japan)
Abstract: Two rice (Oryza sativa L.) varieties, Ootikara with big grain (36 mg/grain) and Habataki with
small grain (18 mg/grain), were used to study the factors that control caryopsis development. We
compared the differences of caryopsis weight, the number of endosperm cell, the structures of pericarp
and endosperms, changes of physiological activities, and so forth during the caryopsis development. The
main results showed that the growth period of the cell of ovary wall and the caryopsis of Ootikawa was
longer compared with that of Habataki, resulting in the formation of more endosperm cells and heavier cell
weight. In Ootikawa, it has a longer period of maintaining a high level of dehydrogenase and catalase
activities, and higher level of respiratory rate of the ears, the degree of greening and photosynthetic rate
of the flag leaf, and a longer functional maintenance period of the dorsal carpellary bundles in the ovary.
And all these features indicated that the larger capacity and the longer active period of the caryopsis in
Ootikara would lead to the notable expansion of the caryopsis.
Key words: rice; caryopsis; endosperm; amyloplast; aleurone layer
In rice, the dry weight of the endosperm accounts for
more than 90 per cent of the caryopsis and its developing
determines the weight and quality of rice grains. Many in-
vestigations have been carried out on the development of
caryposis, especially the endosperm. For example,
Hoshikawa has successively and substantially studied the
formation of the endosperm of rice, the division and growth
of the endosperm cell, the differentiation of the aleurone
layer and the development of the amyloplast and the
proteosome, etc. (Hoshikawa, 1967a-g; 1968a-d). He
pointed out that the proliferation of the endosperm cells
were resulted from the division of cells around the
endosperm, and the endosperm cells in the center of
caryposis developed earlier with earlier starch accumulation.
The starch in the endosperm was compound starch. The
aleurone layer was transformed from the epidermal cells of
the endosperm after the ceasation of cell division. The de-
velopment of the endosperm varied with the environment
and varieties. The number of the endosperm cell and the
layers of the aleurone differ with varieties and grain shape.
Matsuda, with his ultrastructural observation, classified the
development of endosperm amyloplast into six phases, i.e.
(i) plasmid amplification; (ii) plasmid expansion; (iii) starch
accumulation; (iv) small-starch granule formation; (v) cap-
sula expansion; and (vi) large-starch granule formation
(Matsuda, 1984). And it was suggested by Tanaka that,
there were two types of formation of proteosome, PB1 and
PB2, in his study on the accumulation of the stored protein
in rice endosperm, and that the number of PB2 type had
much more effects on rice quality. Domestic investigators
(Qu et al., 1991; Yuan et al., 1994; Shen et al.,1997) analyzed
the shape of amyloplast in relation to rice quality, and sug-
gested that the rich-filled endosperm amyloplasts were
polyhedron, while the poor-filled amyloplasts were ball-like
with large interspace. Recently, investigations have been
started on the molecule structure of the stored protein of
endosperm (Muench and Okita, 1997), and genetic expres-
sion of the enzyme and stored protein associated with starch
formation (Yamagata et al., 1982; Muench et al., 1998), and
also on the expression of endosperm traits in transgenic rice
(Zheng et al., 1995). However there are few reports on the
discipline of the division, differentiation, filling of the en-
dosperm cells and the relationship of grain weight to grain
quality of rice, and the physical causes controlling the for-
mation of endosperm cells and the shape of caryopsis are
still unknown. Recently, we (Wang et al., 1995;1998; Gu et
al., 1999; 2002) have obtained many new results on the de-
velopment of the rice endosperm with optical and electron
microscopy. The division of the endosperm was character-
ized by amitosis followed immediately after the telophase of
mitosis (Wang et al., 1995), these accelerated the division
cycle of the free nuclei as well as the formation of endosperm
Acta Botanica Sinica
植 物 学 报 2004, 46 (6): 698-710
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WANG Zhong et al.: Comparison of Caryopsis Development Between Two Rice Varieties with Remarkable Difference in
Grain Weights
cells (Gu et al., 1999). It was found that the endosperm cell
wall was formed in the cytoplasm where phragmoplasts
were firstly formed in the cytoplasm, later transformed into
cell plate and finally to cell wall. Furthermore, the mecha-
nism of formation of the aleurone layer was also found to
be transformed from the endosperm epidermal cells that
accumulated minerals, fat, protein and other nutrients (Wang
et al., 1998). The effect of the development of epidermis to
the expansion of caryopsis has been elucidated (Gu et al.,
2002). Two rice varieties, Ootikara and Habataki, with re-
markably different dry weight, were used in this experiment
to further understanding the factors that controlling the
development of the rice caryopses by comparing their cary-
opsis weights, number of endosperm cells, structures of
the pericarp and endosperms, and the changes of their bio-
logical activities during the caryopsis development.
1 Materials and Methods
1.1 Plant materials
Two rice (Oryza sativa L.) varieties, cv. Ootikara (big
grain, average dry weight of caryopsis: 36 mg/grain) and
cv. Habataki (small grain, 18 mg/grain) were studied in the
experimental field of the Faculty of Agriculture, Tokyo Uni-
versity of Agriculture and Technology. The seeds were
sown every 10 to 15 d during the time from May 8 to July 6,
to prolong the periods of blooming and flower marking.
With two rice seedlings per hole, eight seedlings were cul-
tivated in a pot sized 0.05 m2 in area, which was filled with
equal paddy field soil and dry soil with a basic fertilizer of
1g N, P2O5, K2O respectively per pot. Additional fertilizer
of 1g (NH4)2SO4 per pot was properly applied at mid-tillering
and 3.4 g mixed fertilizer, with a ratio of N:P2O5: K2O = 14%:
14%:14%, per pot applied at the time of panicle initiation.
1.2 Marking dates of caryopsis development
During the period of rice flowering, either under natural
conditions (about 11 am) or being induced by CO2 (Wang
et al., 1989), the flowering dates were marked correctly by
dotting the glumes and hanging time tags on the rice plants.
The caryopsis of different days of development but
bloomed simultaneously was chosen for experimentation.
1.3 Measurement of fresh and dry weights of caryopsis
Every 5 d, 30 grains with high potence were collected to
obtain their fresh weight, and dry weight after being dried
at 90 ℃ to calculate the water content. After stripping off
the pericarp and embryos of the caryopsis, the endosperms
were dried and weighed.
1.4 Observation of the free nucleus and calculation of its
division cycle
The caryopsis, 48 h after flowering, was fixed with
Carnoy’s fixative solution for 3 h and then kept in a 70%
alcohol solution for use. Before observation, the ovule was
removed from the caryopsis with tweezers and hydrolyzed
with 1 mol/L HCl (60 ℃, 8 min). After being rinsed with
water, the ovule was treated with 4% iron vitriol and then
stained with 4% hematoxylin for 10 min respectively. The
stained ovule was placed on the glass slide, and the em-
bryo sac was picked out with tweezers or a pair of dissect-
ing needles under a stereomicroscope (Figs.1-3). The em-
bryo sac was lain flat and covered with cover glass for
morphological observation and counting of the free nuclei.
Because of the synchronized division of the endosperm
nuclei, about 0-2 d after flowering, the division cycle (Y) of
the free nuclei could be calculated according to the time (t)
after fertilization and the numbers (x) of the endosperm
nuclei from the following formula:
Y= ( lg 2/lg x) t
Where t refers to 45 h (48 h-3 h, 3 h indicates the time
for completion of the double fertilization in rice florets).
1.5 Separation and calculation of endosperm cells
The caryopsis collected from different days after
flowering, was fixed for 5-10 h with Carnoy’s fixative
solution, and then transferred to 70% alcohol, and stored
in the freezer for standby application. For cell separation,
three grains of caryopsis were taken, and their pericarp of
caryopsis, seed coats and embryos were stripped. After
being washed several times with water to clean the alcohol
in the endosperms, the endosperms were treated with 1
mol/L HCl for 1 h, and dyed with Schiff’s reagent for 2 h so
that the endosperm cell nuclei were stained red. Then the
endosperms were repeatedly washed until the water be-
came colorless. After which the endosperm was cut into
several pieces and decomposed with 1 mL of 0.5% cellu-
lase solution of pH 5.5 at 40 ℃ for 3-5 h. After the cells
were thoroughly separated, the cell suspension was di-
luted with water to 100 mL, and from which an alliquat of 5
mL was removed. The 5 mL alliquat, after some water being
added, was poured into an extractor holding a micropore
filtering membrane. The endosperm cells being extracted
were evenly distributed as a certain area (p 82 mm2) on the
membrane (Figs.6, 7). Finally, the filtering membrane was
taken out for calculating the number of cells (or nuclei) (x)
in a visual field (p 12 mm2) under a light microscope. The
number of endosperm cells (Y) in the caryopsis was calcu-
lated according to the following formula.
Numbers of endosperm cells = [the average cell number
in a visual field × (filtering membrane area ÷ visual field
area) × (general volume of cellulase solution ÷ volume of
cell suspension for extraction)] ÷ (the number of caryopsis
Acta Botanica Sinica植物学报 Vol.46 No.6 2004700
Figs.1-5. 1. Caryopsis development, the upper row, Habataki; the lower, Ootikara. Figure in the picture represents day after
flowering, ×2; 2. Habataki embryo sac, 2 d after flowering, ×40; 3. Ootikara embryo sac, 2 d after flowering, ×40; 4. Cross section
of caryopsis in Habataki, 6 d after flowering, ×40; 5. Cross section of caryopsis in Ootikara, 6 days after flowering, ×40. A, aleurone
layer; a, amyloplast; Dv, dorsal vascular bundle; E, embryo; ES, endosperm; n, nuclear; NE, nucellus epidermis; np, nucellus projection;
O, endosperm center spot; p, proteosome Pe, pericarp; s, sieve; SV, stigma vascular; t, trachea.
→
for enzyme decomposition)
Y = [x × (p 82 mm2 ÷ p 12 mm2)] × (100 mL ÷ 5 mL) ÷ 3 =
426.7x
Also the average dry weight of endosperm cells can be
calculated through the endosperm dry weight divided by
the number of endosperm cells.
1.6 Structural observation of caryopsis
The caryopsis, on the 2nd, 4th, 6th, 8th, 10th and 15th
day after flowering in both varieties were collected
respectively. The caryopsis was cut crosswide by a sharp
blade into pieces which were fixed in mixing solution of 2%
glutaraldehyde, 1% paraformaldehyde and 0.05 mol/L so-
dium cacodylate with pH 7.2 for 3 h. They were then post
fixed in 1% osmic acid for several hours, dehydrated with a
gradient concentration of alcohols which were replaced by
1, 2-epoxypropane, embedded with Spurr resin of low
viscosity. The specimens were cut into 1 µm semi-thin
sections, and dyed with toluidine blue (TBO) for light mi-
croscopic observation.
1.7 Determination of dehydrogenase activity and starch
in caryopsis
Dehydrogenase activity was determined by using the
tetrazolium (TTC) staining method. Briefly, the longitudi-
nal- or cross-sections of caryopsis were soaked in 0.5%
TTC for 1 h at 25 ℃. The red stains indicate the localization
of dehydrogenase activity where the respiration was most
vigorous. The starch in the caryopsis was determined by
using the I2-KI staining method. One or two drops of the
I2-KI solution were added to the longitudinal- or cross-
section of the caryopsis for 5-10 min before observing the
blue-black stained starch. Starch could also be localized by
observing the hand-cut slices of caryopsis stained with I2-
KI solution.
1.8 Measurement of physiological activities
1.8.1 Chlorophyll content of the flag leaf The relative
content of chlorophyll in each flag leaf estimated by the
chlorophyll detector was expressed as the level of greening.
1.8.2 Photosynthetic rate of the flag leaf This is mea-
sured by the LI-6400 photosynthesis system with initial
CO2 concentration of 350 µL/L at 25-30 ℃.
1.8.3 Respiratory rate of the panicle Measured by the
LI-6200 photosynthesis system, the panicles (containing
ear axle and branches) of various days of flowering were
kept in the assimilation room at 28 ℃ with the initial CO2
concentration of 350 µL/L, and the respiratory rate (µmol
CO2·g-1·s-1) was calculated through the amount of CO2-
released per second by the dry weight (g) of panicle.
1.8.4 Catalase activity in the caryopsis It was measured
by the oxygen electrode method (Wang et al., 1990).
Three grains of caryopsis, after their fresh weight was
recorded, were added to 2 mL of 50 mmol/L Hepes solution
with pH 7.5 for milling. The homegenate was diluted to 10
mL. Three mL of the crude catalase solution was added to
the reaction cup of the oxygen electrode, into which 20 µL
1% H2O2 solution was injected, and the oxygen-releasing
rate was recorded at constant temperature of 25 ℃. Cata-
lase activity was equivalent to the oxygen-releasing rate
divided by the fresh weights of caryopsis involved in the
reaction, which is shown in the following formula:
Catalase activity (µmol O2·g-1·min-1) = oxygen-releas-
ing rate (µmol O2·min-1)/caryopsis fresh weight (g)
2 Results
2.1 Growth of caryopsis and proliferation of endosperm
cells
In Habataki, it was found that the fresh weights, sizes
and the number of endosperm cell within 5 d after flowering,
the dry weights of the caryopsis and endosperms within 10
d after flowering, and the dry weight of a single endosperm
cell within 15 d after flowering, all exceeded those in Ootikara
(Figs.1, 28). Afterwards, the fresh and dry weights, size of
the caryopsis, the number of the endosperm cell and its dry
weight in Ootikara exceeded those in Habataki. That is,
compared with that of Ootikara, the caryopsis of Habataki
grew faster and accumulated more dry materials during the
primary stage of growth. However, in the later stage of
development, the opposite was true.
Also shown in Fig.28, the periods for the growth of
caryopsis, and the division and filling of endosperm cells
in Habataki, were shorter than those in Ootikara. For
example, the dry weights of the caryopsis in Habataki pri-
marily kept stable on the 15th day after flowering, while
they tended to increase in Ootikara on the 30th day after
flowering. The longer periods of caryopsis growth and the
more accumulation of the dry materials during the middle
and later stages in Ootikara resulted in the heavier dry
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Acta Botanica Sinica植物学报 Vol.46 No.6 2004702
Table 1 Numbers of endosperm free nucleus and their division cycles 2 d after flowering
Varieties
Interval between flowering and flower-picking
10 a.m., Aug. 16 to 10 a.m., Aug. 18 11 a.m., Aug. 26 to 11 a.m., Aug. 28
Number of free nucleus Division cycle Number of free nucleus Division cycle
(numbers/embryo sac) (h) (numbers/embryo sac) (h)
Ootikara 1 114 ± 275 4.47 ± 0.17 567 ± 144 4.95 ± 0.19
Habataki 1 816 ± 381 4.18 ± 0.16 1 094 ± 273 4.48 ± 0.15
Figs.6-15. Structure contrast of ovary wall between Habataki (left) and Ootikara (right), the shape of endosperm cell, structure of
ovary wall and the dorsal vascular bundle with different days after flowering, ×400; 6, 7. Endosperm cells 15 d after flowering,
×100 ; 8, 9. The ovary wall 4 d after flowering, ×400; 10, 11. Ovary wall 10 d after flowering, ×400; 12, 13. The dorsal carpellary
bundle and the aleurone layer 6 d after flowering, ×400; 14, 15. The dorsal bundle 15 d after flowering, ×400.
→
weights of both the caryopsis and single endosperm cell,
which were approximately as much as that in Habataki.
By measuring the number of free nuclei and calculating
their division cycles 2 d after flowering in both varieties
(Table 1), we found that the free nuclei in Habataki divided
faster than those in Ootikara, leading to remarkable increase
in the volume of the embryo sac (Figs.2, 3). Thus we can
conclude that the rapidity of caryopsis growth in Habataki
during the primary stage could be related to the high speedy
division of the free nuclei after flowering, and the large
basic numbers and fast proliferation of endosperm cells
after cell formation took place 3 d after flowering.
2.2 Changes of some physiological activities during cary-
opsis development
During the caryopsis development, remarkable differ-
ences in physiological activities of the caryopsis existed
between the two varieties, such as the colors of glume,
caryopsis and endosperm, activities of catalase and dehy-
drogenase in caryopsis, accumulation and declination of
starch in various parts of the caryopsis, respiratory rate of
the panicle, greening and photosynthetic rates of the flag
leaf (Table 2; Fig.29). Habataki has a shorter period of cary-
opsis development, higher rate of starch filling, and a shorter
duration of sustained greening in husk and caryopsis. There
were also shorter period of dehydrogenase activities in the
endosperm aleurone layer and in the dorsal vascular bundles
of the ovary and the vascular bundles in the rachillae of
H2O2 activity in the caryopsis, higher respiratory rate of
the panicle, greening level and photosynthetic rate of the
flag leaf as well as other physiological activities. In Ootikara,
however, all the afore-mentioned physiological activities
maintained longer periods of high level activity, that docu-
mented by a longer time of caryopsis development and a
delay in aging of the rice plant after flowering.
It was interesting to notice the abundance of starch
accumulated in the ovary wall during the primary period of
caryopsis in Ootikara, and gradually disappeared with full
extension of the ovary wall cells (Figs.8-11). This sug-
gests that starch accumulation in the ovary wall might pro-
vide the necessary material for the full growth of pericarp
cells, and plays a key role in the formation of the bulky sink
of caryopsis.
2.3 Changes of structure of endosperm cells and peri-
carp during caryopsis development
The structural changes of pericarp (Figs.8-11), en-
dosperm cells (Figs.16-27), dorsal vascular bundles and
aleurone layer (Figs.12-15) were analyzed. The main differ-
ences between two varieties are shown in Table 3.
In Habataki, the amyloplasts in the endosperm cells ap-
peared earlier and had a higher rate of starch filling. For
instance, on the 4th day after flowering amyloplasts con-
taining starch grains appeared in the cytoplasm of the en-
dosperm cells (Fig.18 ). And on the 10th day after flowering,
the endosperm cells in the middle part of the caryopsis
were filled with amyloplasts, and giving the shape of poly-
hedron resulted from the pressure between starch grains
and amyloplasts (tortoise shell-shape in cross section, Fig.
24). However in Ootikara the amyloplasts appeared a little
later in the endosperm cells. The amyloplasts containing
starch grains appeared approximately on the 5th day after
flowering and the time for the amyloplasts filling in en-
dosperm cells was longer than 15 d after flowering (Fig. 27).
Moreover, during the filling of cells, the average volume of
the amyloplasts and starch grains in the endosperm was
larger than that in Habataki.
Starch accumulated to its maximum quantity in the ovary
walls on the 4th day after flowering in Ootikara during the
primary period of caryopsis development. Starch accumu-
lation to it as most of the ovary wall cells filled with amylo-
plasts could be visualized (Fig.9). Moreover, the amylo-
plasts disappear gradually within 15 d after flowering. In
Habataki, however, although starch accumulation during
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Acta Botanica Sinica植物学报 Vol.46 No.6 2004704
Table 2 Development of caryopses and their dyed reaction to tetrazolium (TTC) and I2-KI in both varieties
Days after flowering (d) 0 5 10 15 20 25 30
Glume O Green Green Green Green Green-yellow Yellow Yellow
H Green Green Green Green Yellow Yellow Yellow
Caryopsis O Green Green Green Green back White White
White belly
H Green Green Green back White White White
Color White belly
Endosperm O White with latex White without latex Hard and transparent
H White with latex White without latex Hard and transparent
Endosperm O ++ ++ + + - -
H ++ + + - - -
Aleurone layer O ++ ++ + + -
H ++ + - - -
Dorsal vascular O ++ ++ ++ ++ + -
Reaction to bundle H ++ ++ + - - -
T T C
Rachilla vascular O ++ ++ ++ ++ ++ + -
bundle H ++ ++ ++ + - - -
Embryo O + ++ ++ ++ ++ ++
H + ++ ++ ++ ++ ++
Endosperm O + ++ ++ ++ ++ ++
H ++ ++ ++ ++ ++ ++
Embryo O - ++ + + + +
Reaction to H - + + + + +
I2-KI Pericarp O ++ + - - - -
H + - - - - -
Rachilla O + + + - - -
H + + - - - -
The deep TTC staining in the vascular bundle indicates high dehydrogenase activity and in turn a stronger ability in transporting milking
substance. The deep staining with I2-KI indicates a higher starch content. H, Habakati; O, Ootikara; +, slightly stained; ++, deeply stained; -,
barely stained.
Figs.16-27. Structure contrast of endosperm cells between Habataki (left) and Ootikara (right) with different days after flowering,
×400. 16, 17. Two days after flowering. 18, 19. Four days after flowering. 20, 21. Six days after flowering. 22, 23. Eight days after
flowering. 24, 25. Ten days after flowering. 26, 27. Fifteen days after flowering.
→
the primary periods of caryopsis development did occur,
yet amyloplasts were less and smaller. The ovary wall cells
were not so big as those in Ootikara, and exhibited prema-
ture senility. On the 10th day after flowering, the ovary
walls shrank and the cells disappeared on the 15th day
after flowering.
During the primary and middle periods of caryopsis
development, chloroplasts appeared in the internal layer of
the pericarp and the cells around the dorsal vascular bundles
of the ovary wall. The chloroplasts possessed thylakoids
of lamella structure with starch grain in their matrix. The
oxygen-releasing rate in photosynthesis of the green
pericarp, using HCO3- as substrate, was measured by the
oxygen electrode method (Tanaka, 1990). It was concluded
that the photosynthesis could be vigorously carried out in
the green cells of pericarp. Compared with the Habataki,
the pericarp of Ootikara developed more vigorously and its
cells were larger with longer life cycle, and the greening
time maintained in the pericarp especially the dorsal vascu-
lar bundle was 5-7 d longer in Ootikara, all of which fa-
vored the growth and filling of caryopsis.
As shown in Table 2, during the caryopsis development,
the dehydrogenase activities maintained in the aleurone
layer and dorsal vascular bundle were 5 to 10 d longer in
Ootikara. The same conclusion could also be obtained by
comparing the structural changes of aleurone layers and
dorsal vascular bundles during caryopsis development in
both varieties (Figs.12-15). In Habataki, the aleurone layer
was formed earlier, usually had three layers while that of
Ootikara usually had four or five layers, and its formation
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Grain Weights
Acta Botanica Sinica植物学报 Vol.46 No.6 2004706
was later and remained for a longer time. In terms of the
structure of the dorsal vascular bundles, whose sieve tubes
had approximately six bundles and 50 pieces in Habataki,
and shrank on the 15th day after flowering. However, in
Ootikara the structure of the dorsal vascular bundle, whose
sieve tubes had nine bundles and 80 pieces, was still in
perfect condition and a larger cross section could be visu-
alized on the 15th day of development.
3 Discussion
3.1 Physiological factors of caryopsis enlargement in
Ootikara
The physiological factors for the notable enlargement
of the caryopsis in Ootikara, whose dry weight is twice as
much as that of Habataki, can be attributed to the following
three aspects.
Sink strength, consisting of sink capacity and sink vigor,
means the ability of a plant organ to receive and transform
assimilates. In rice caryopsis, this means the volume of
endosperm and embryo was encircled by the pericarp, which
can be expressed by the fresh or dry weight of caryopsis,
or the products of the cell volume multiplied by the num-
bers of endosperm cells. Sink vigor refers to the metabolic
activity or the ability to absorb assimilates. In rice caryopsis,
it can be shown by the growth rate of the caryopsis, the
respiratory rate of the sink organ and the enzyme activities
associated with starch metabolism. In this study, the sink
vigor was expressed by the growth curve of the caryopsis,
the number and weight of the endosperm cells, the respira-
tory rate of the panicles, dehydrogenase and catalase
Fig.29. Changes of greening level and photosynthetic rates of the flag leaf, respiratory rate of the panicle and catalase activities in
caryopsis before and after flowering in both varieties.
Fig.28. Development of caryopses and the changes of numbers and weights of their endosperm cells. The reason that endosperm cell
number has a little decrease can be attributed to the disappearance of cells caused by the growth of embryos near them.
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Grain Weights
Table 3 Comparison of caryopses structures within 2-15 d after flowering in both varieties
Days after flowering Ootikara (big grain) Habataki (small grain)
2 d Smaller embryo sac, fewer free nuclei and Bigger embryo sac, more free nuclei and
bigger internuclear space smaller internuclear space
4 d Ovary wall filled with amyloplasts, less amyl- Fewer amyplasts in ovary wall, amyloplasts
oplasts in endosperm cell in endosperm cell easily observed
6 d Thickening of ovary wall, amyloplast in endosp- Thinning of ovary wall, amyloplasts in endosperm
erm cells occupies less space and their outer layer cells occupy larger space and their outer layer cells
cells have not developed into aleurone layer cells begin to develop into aleurone layer cells
8 d Continued thickening of ovary wall, outer Continued thinning of ovary wall, the cells in the
layer endosperm cells begins to convert into middle of the endosperm have been filled with
aleurone layer cells amyloplasts
10 d Endosperm cells continue to divide and to grow and Endosperm cells stop division and amyloplasts in the
amyloplasts become spherical shape middle appear a tortoise shell shape and polyhedron in shape
15 d The ovary wall is thick and the structure of the dorsal Ovary wall and dorsal vascular bundles are shrunken and all
vascular bundle appears normal. Dorsal aleurone layer the endosperm cells are filled with amyloplasts, tending to
increases and amyloplast is further filled and enlarged stop milking
activities of the caryopsis, etc. It was found that Ootikara
has stronger sink capacity and vigor, which played a key
role in the growth and filling of the caryopsis.
Source strength refers to the ability to generate and
output of assimilates from the sink organ. In rice, the pho-
tosynthetic rate of the flag leaf after heading is the most
direct indicator of source strength. As expressed in Fig.2,
compared with Habataki, although the photosynthetic rate
of the flag leaf in Ootikara was lower during the primary
stage of milking (within 10 d after flowering), it was higher
during the middle and later stages (10-30 d after flowering)
because of high level of greening. The strong source
strength was maintained by flag leaf in Ootikara during the
middle and later stage of milking provided a material supply
for the caryopsis filling.
The vascular bundles in the spikelets and dorsum of
ovaries are the channels for transporting the milking mate-
rials to the caryopsis. In contrast with Habaraki, these vas-
cular bundles in Ootikara dorsum not only were larger in
the transverse section and had larger number of sieve tubes,
but also maintained a longer period of integrated structure
and activity. The well developed vascular bundles in the
spikelets in Ootikara facilitated the transportation of pho-
tosynthetic materials to the caryopsis.
3.2 Effects of force on division and morphology of en-
dosperm cells
As it is well known, the glume is the limiting factor of
caryopsis growth. We have observed that the division of
the endosperm cell was affected by space and force. Com-
pared with the Hakiraka, the larger final numbers and vol-
ume of endosperm cells in the caryopsis of the Ootikara are
associated with the stronger sink capacity of its spikelets,
as well as the fact that the glume or pericarp had little effect
on the growth and division of endosperm cells during the
primary stage of caryopsis development.
The morphology of the endosperm cells was notably
affected by force. Taking the growth of the caryopsis in
Ootikara as example, within 6 d after flowering, when the
growth of caryopsis was not limited by glume and its trans-
verse section appeared circular (Fig.5). And most of the
endosperm cells dividing in the same periods were distrib-
uted around the circumference of the circle. However, when
the development of the caryopsis was restricted by glume,
the forces existed from different directions on the cary-
opsis were uneven and resulted in the change of the mor-
phology of the endosperm cells. If less force exerted along
the long axis of the transverse section of the caryopsis the
cells became longer in length (Fig.30), but if with force ex-
erted along the short axis, then the cells became shorter.
Fig.30. Cell shapes of caryopsis and endosperm in rice by
different forces. A. The development of the caryopsis has not
been restricted by glume. B. The development of caryopsis has
been restricted by glume. a, the external shape of the caryopsis; b,
the shape of the caryopsis in cross section; c, cell shape of the
cross section of middle part of the endosperm; arrow, the part by
the stronger force; *, core point in endosperm.
Acta Botanica Sinica植物学报 Vol.46 No.6 2004708
The caryopsis’ growth and its shape were first restricted
by the pericarp and then by the glume. The prerequisite for
the continuous development of endosperm tissue is the
pericarp, namely the ovary wall, fully extended in rice
production, particularly in cross breeding it is often seen
that the caryopses, which have no closure of the inner and
outer glume , all develop into abnormally smaller grains,
because their ovaries during the development were suf-
fered from the solar ultraviolet radiation and water stress.
Ultraviolet radiation restricts the division of the ovary cells
and water stress, i.e. water deficiency, inhibits the develop-
ment of the ovary epidermal cell, which means that the peri-
carp can not fully expand. The same phenomenon can be
documented by cutting glume during rice cross breeding.
The expansion of the pericarp plays a more important role
in the caryopsis development of larger grain. In some vari-
eties with larger grains, the reason why the caryopsis growth
could not occupy all the space formed by inner and outer
glume is related to the fact that the pericarp can not fully
extend. The Ootikara used in this experiment can fill the
space formed by both glumes and are associated with the
full extension of pericarp due to the smooth development
of the ovary cell.
3.3 Mechanism of aleurone layer formation
The aleurone layer belongs to the surface cells of the
endosperm, and the milking substances for the caryopsis
have to pass through the surface cells before entering the
endosperm. The reason why the endosperm cells can de-
velop into the aleurone layer cells is related to the accumu-
lation of minerals (Tanaka et al., 1977; Ogawa et al., 1979),
fats and other milking “wastes”, in addition to the storing
materials, i.e. starch and protein, for the inner endosperm
cells (Wang et al., 1998). Aside from the soluble sugars and
amino acids in the milking substances unloaded from the
dorsal vascular bundles to degenerated nucellus layer, there
are other materials such as P, K, Mg, Ca and other minerals,
and fatty acids. When absorbed by the surface endosperm
cells, most of the soluble sugars and amino acids are trans-
ported to the inner endosperm cells for the synthesis of
starch and protein, to be stored as amyloplasts and protein
bodies, respectively. Those materials such as minerals, fatty
acids and some amino acids surplus to the endosperm, are
left and stored as remnants in the surface layer cells of
endosperm. The mineral elements after entering the vacu-
ole of the surface layer cells of endosperm by self-metabo-
lizing and combining with other materials, such as some
proteins, form complex aleurone grains, and the fat synthe-
sized by fatty acids and phosphoglycerate accumulates in
a spherical body. These changes render the surface layer
cells of the endosperm evolving into an aleurone layer rich
in minerals, fat and protein.
Based on the view that the aleurone layer is the result of
the accumulation and conversion of “milking wastes” in
surface layer cells of the endosperm, it can be deducted
that the number of aleurone layers are associated with the
quantity of milking materials. Although the milking sub-
stances unloaded from the dorsal vascular bundles to the
degenerated nucellus layer are transported to the internal
endosperm by passing through the whole surface layer of
cells, but most of them go through the nearby dorsal epi-
dermal cells, in which more milking “wastes” are
accumulated. So more aleurone layers are formed along the
surface layers of the dorsal endosperm, while in the other
surface layer of the endosperm, only one aleurone layer
was formed because of less “waste” being accumulated.
As shown in the present study, the weight of the endosperm
in Ootikara is twice as that of Hakiraka, and in the former
more milking substances are transported from dorsal en-
dosperm surface to its inner part and it takes a longer time
for milking, thus more aleurone layers are formed and the
time for the complete formation of the inner aleurone layer
is prolonged. This further demonstrates the view that the
aleurone layer comes from the accumulation of “milking
wastes” in the endosperm surface layer cells.
During grain filling, “wastes” are also accumulated in
the cells of the milking channel, such as the parenchyma
cells of the vascular bundles and the surrounded thick-
walled cells in the spikelet, nucellus projection cells, cells
with color and so on. When these cells are fulfilled with the
“waste”, the milking rate may decrease. Therefore the deep
staining of the aleurone layer and the appearance of col-
ored cell layer may indicate a maturing caryopses.
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