Rice plant development: from zygote to spikelet.
ABSTRACT Rice is becoming a model plant in monocotyledons and a model cereal crop. For better understanding of the rice plant, it is essential to elucidate the developmental programs of the life cycle. To date, several attempts have been made in rice to categorize the developmental processes of some organs into substages. These studies are based exclusively on the morphological and anatomical viewpoints. Recent advancement in genetics and molecular biology has given us new aspects of developmental processes. In this review, we first describe the phasic development of the rice plant, and then describe in detail the developmental courses of major organs, leaf, root and spikelet, and specific organs/tissues. Also, for the facility of future studies, we propose a staging system for each organ.
Plant Cell Physiol. 46(1): 23–47 (2005)
doi:10.1093/pcp/pci501, available online at www.pcp.oupjournals.org
JSPP © 2005
Rice Plant Development: from Zygote to Spikelet
Jun-Ichi Itoh 1, Ken-Ichi Nonomura 2, Kyoko Ikeda 1, Shinichiro Yamaki 1, Yoshiaki Inukai 3, Hiroshi
Yamagishi 2, Hidemi Kitano 3 and Yasuo Nagato 1, 4
1 Graduate School of Agricultural and Life Sciences, University of Tokyo, Tokyo, 113-865 Japan
2 National Institute of Genetics, Mishima, Shizuoka, 411-8540 Japan
3 Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, 464-8601 Japan
Rice is becoming a model plant in monocotyledons and
a model cereal crop. For better understanding of the rice
plant, it is essential to elucidate the developmental pro-
grams of the life cycle. To date, several attempts have been
made in rice to categorize the developmental processes of
some organs into substages. These studies are based exclu-
sively on the morphological and anatomical viewpoints.
Recent advancement in genetics and molecular biology has
given us new aspects of developmental processes. In this
review, we first describe the phasic development of the rice
plant, and then describe in detail the developmental courses
of major organs, leaf, root and spikelet, and specific organs/
tissues. Also, for the facility of future studies, we propose a
staging system for each organ.
Keywords: Development — Rice — Staging.
Abbreviations: AC, archesporial cell; DAP, days after pollina-
tion; GMC, guard mother cell; LE, lateral element; PMC, pollen
mother cell; PPC, primary parietal cell; PSC, primary sporogenous
cells; SAM, shoot apical meristem; SC, synaptonemal complex; SEM,
scanning electron microscope; SMC, subsidiary mother cell.
Higher plants have many specialized organs, tissues and
cells. All these components are derived from a single cell (ferti-
lized egg, zygote) through a number of developmental events.
In animals, developmental and differentiation processes are
restricted mainly to a short period of embryogenesis. In con-
trast, development continues in plants until the end of the life
cycle. Apical meristems repeatedly differentiate lateral organs.
Meristematic tissues are generated in a variety of organs with
proper timing to achieve successive development. Therefore,
developmental studies of the entire plant life cycle are essen-
tial to elucidate the establishment of plant formation.
Importance of developmental study
Recent advancement of developmental studies has pro-
duced interesting results that show the way in which plant
development is regulated genetically. One fruitful result is the
establishment of the ABC model in flower development (Coen
and Meyerowitz 1991), which facilitates our better understand-
ing of flowers. Since the 1980s, numerous mutants have been
identified and characterized to study plant morphogenesis and
differentiation, and their causal genes have been cloned.
Detailed developmental analyses of mutant phenotypes and
spatiotemporal expression pattern of the causal genes contrib-
ute greatly to better understanding of plant architecture. In
addition, developmental studies are expected to answer the
identity of unknown organs, homology of similar organs and
other important subjects.
For a description of the developmental process to be uni-
versal, for the description to be applicable to the comparison
between wild type and mutant or between two species, catego-
rization of the process with landmark events is essential. One
categorization method is division into discrete stages. Stages
should be characterized by landmark events. Although such
landmark events are provided mainly by morphological and
anatomical studies, recent genetic and molecular studies would
make the staging system more available. For Arabidopsis, the
developmental course of the wild-type flower has been divided
into 12 stages using a series of events (Smyth et al. 1990). This
staging is now used widely for describing mutant phenotypes
and the site and timing of the cloned genes’ expression.
Concomitant with the progress of molecular and genetic
studies of plant development, a large amount of information,
such as the expression pattern of genes and molecular func-
tions of the proteins and their cellular localization, is being
accumulated. These numerous data are being integrated in sev-
eral website databases. To use information from various data
sets more effectively, it is extremely useful to establish devel-
opmental staging based on internationally accepted terminology.
Rice is a model plant for developmental study
At present, Arabidopsis is a major source of information
regarding plant development because it offers excellent advan-
tages as a model plant for developmental study (Meyerowitz
and Somerville 1994). Arabidopsis is a dicotyledonous spe-
cies. Therefore, another model plant in monocotyledons is
4Corresponding author: E-mail, firstname.lastname@example.org; Fax, +81-3-5841-5063.
Staging of rice development24
needed for a general understanding of angiosperms. Rice plant
(Fig. 1) offers various advantages as an experimental plant
compared with other monocot species, such as small genome
size, a known genome sequence (Sasaki and Burr 2000) and
self-fertilization. Moreover, it is an annual plant that is never-
theless able to survive for several years. It has a facility for
Agrobacterium-mediated transformation, easy cultivation and
accumulation of information on varieties and mutants. For
those reasons, rice is considered a model plant of monocots.
Rice belongs to the grass family (Poaceae) together with bar-
ley, wheat, maize and sorghum, which are important cereal
crops that support the global food supply. In addition, these
grasses are inferred to have a monophyletic origin (Clark et al.
1995), indicating that information obtained from rice is helpful
for studying other cereal crops. In fact, several reports have
elucidated genome synteny between rice and other grasses
(Bennetzen and Ma 2003). Accordingly, rice is not merely a
model plant for developmental study, it is a model crop for
evolutionary and agronomical studies of cereals.
In this review, we describe rice development from zygote
to spikelet, and attempt to categorize each developmental proc-
ess into several stages. We hope that the staging systems pro-
posed here are useful for describing mutant phenotypes and
spatiotemporal expression patterns of development-related
genes, and for comparative study with other species.
Categorization of Rice Plant Development
Plant development starts from egg cell fertilization with a
sperm nucleus to form a zygote (fertilized egg). From the first
zygotic division, plant development proceeds toward maturity
through a number of stages that are discernible according to
landmark events. Development finally culminates in the forma-
tion of male and female gametes that fertilize to form a zygote.
The plant developmental course is roughly divisible into three
phases: embryogenesis, vegetative and reproductive. Seed
dormancy and germination, and the onset of inflorescence
development typically delimit these three phases. In each
phase, numerous events occur sequentially. Consequently, each
phase can be divided into discrete stages. To date, develop-
mental studies on rice have been carried out from the stand-
point of morphological changes, and too detailed staging
systems for several processes have been proposed (Matsu-
shima and Manaka 1956, Kawata and Harada 1975). However,
recent advancement in molecular biology and developmental
genetics using useful mutants has revealed stage-specific gene
expression and stage-specific defects of mutants. It has lent
new insights into rice development. It would be useful to
review rice development from a viewpoint that incorporates
recent results of molecular and genetic studies.
Embryogenesis in most plant species is a period in which
the plant body plan (body axis) is established. That is, the
zygote undergoes cell division without morphogenetic events
to form a globular embryo. It then differentiates the shoot api-
cal meristem (SAM) and radicle in fixed positions that specify
the body axis. For that reason, embryogenesis is an extremely
important period for understanding the developmental nature of
After seed germination, the plant undergoes vegetative
development, usually the longest period, repeatedly forming a
number of leaves and branches as lateral organs. During this
phase, the stem also grows by the supply of cells from the rib
zone of the SAM. Recent studies have indicated that the vege-
tative phase can be divided into two phases—juvenile and adult
phases—which differ in many traits (Hackett 1985, Poethig
1990, Telfer et al. 1997, Asai et al. 2002).
The reproductive phase has been studied exhaustively
because anyone can recognize the remarkable event in this
phase: flowering. This phase marks a change of meristem iden-
tity from inflorescence meristem to floral meristem. In addi-
tion, flower formation is a complicated process because the
floral meristem changes the identities of lateral organs sequen-
tially: the sepal, petal, stamen and carpel (pistil). Therefore, the
Schematic representation of mature rice plant.
Staging of rice development 25
reproductive phase includes a large number of ingeniously reg-
The following sections explain phasic development of rice
plants with particular attention to the temporal landmark events
that characterize each phase, and we propose staging systems
that are useful for future study.
DESCRIPTION OF DEVELOPMENTAL COURSE
First, it should be mentioned that several foliage leaves
are present in mature grass embryos, including rice embryos
(Fig. 2A). This fact contrasts with the situation in other mono-
cot and dicot embryos, in which foliage leaves are not formed
before dormancy and germination. This phenotype means that
in grasses, early vegetative stages are incorporated into
embryos before dormancy takes place. The temporal reversion
of dormancy and early vegetative phase suggests that grasses
are heterochronic mutants (Freeling et al. 1992, Asai et al.
2002). In this report, we regard rice embryogenesis as a period
In addition to a heterochronic nature, grasses are unique in
that they have complex embryos (Fig. 2A). Several embryo-
specific organs are present aside from the shoot (plumule) and
radicle. The scutellum is the largest organ in the embryo corre-
sponding to the cotyledon, and differentiates independently of
the SAM, as indicated by Satoh et al. (1999). The epidermal
layer of the scutellum (scutellar epithelium) comprises pali-
sade-shaped cells. It is the site at which many maturation-
related genes are expressed (Sugimoto et al. 1998, Miyoshi et
al. 1999, Miyoshi et al. 2002). The coleoptile is the first recog-
nizable organ post-globular stage, which protrudes from the
ventral region just above the SAM. Although the identity of the
coleoptile has been discussed, it may be an appendage of the
scutellum. An epiblast, whose function is not clear, is formed
in the basal region on the ventral side. The epiblast is not
always observed in grass embryos; maize has no epiblast. Most
of the dorsal region is occupied by the scutellum. The radicle is
formed endogenously in the basal region. Thus, it is frequently
considered as a kind of adventitious root.
Pollination occurs in rice at the same time as flower open-
ing. It lasts for only about 15 min at around noon in summer.
Fertilization takes place several hours after pollination. Due to
the synchronous pollination (fertilization), it is possible to
mutagenize a large number of zygotes, engendering efficient
non-chimeric mutant production (Satoh and Omura 1979). The
zygote reiterates cell divisions to form a globular embryo with
no apparent morphological differentiation for 3 days after polli-
nation (3 DAP) (Fig. 2B–D, J). In contrast to the Arabidopsis
embryo, cell divisions are not regular, even at the very early
stage. Although the first cell division usually occurs horizon-
tally to form apical and basal cells, the directions of the second
and following cell divisions are not fixed. At 4 DAP, the first
morphogenetic event occurs: formation of SAM, coleoptile pri-
mordium and radicle primordium (Fig. 2E, K). Because the
SAM, radicle and coleoptile initiate at fixed positions, embry-
onic axis and regionalization are inferred to be established in
the globular embryo. The SAM at this stage is flat, not dome-
like. The first leaf primordium is recognized at 5 DAP (Fig. 2F,
L). Three foliage leaves are formed in the rice embryo. Soon
after the formation of the third leaf primordium, the rice
embryo is morphologically completed (Fig. 2G, M). Subse-
quently, maturation and dormancy processes take place.
For categorizing embryo development, it is reasonable to
discriminate stages by specific morphogenetic events such as
Embryo development in rice. (A) Longitudinal section of mature embryo. Arrow indicates the SAM. SC, scutellum; CO, coleoptile; EP,
epiblast; RA, radicle. (B) 1 DAP embryo. (C) 2 DAP embryo. (D) 3 DAP embryo. (E) 4 DAP embryo differentiating coleoptile primordium
(black arrowhead), SAM (arrow) and radicle primordium (white arrowhead). (F) 5 DAP embryo differentiating the first leaf primordium (arrow).
(G) Morphologically completed 10 DAP embryo. (H) OSH1 expression (arrowhead) in 3 DAP embryo. (I) RAmy1A expression (arrowhead) in
scutellar epithelium of 5 DAP embryo. (J–M) SEM images of 3, 4, 5 and 10 DAP embryos, respectively.
Staging of rice development 26
the initiation of new organs. However, during the globular
stage, no apparent morphogenetic events occur. Marker genes
that are expressed at a specific stage of globular embryo are not
known, except OSH1 (Sato et al. 1996). Considering the impor-
tance of the globular stage in embryo development, it is indis-
pensable to reveal the events that take place over the 3 d that
the globular embryo lasts. The number of cells in the embryo
increases rapidly at 1 DAP, but after about the 25-cell stage, the
rate of cell increase falls (Nagato 1978). That rate reduction
suggests that actively dividing cells are restricted in the
embryo. Detailed growth analysis reveals that another land-
mark event occurs at around the 150-cell stage. From this
stage, the embryo undergoes dual rhythmic growth and cell
division (Nagato 1976), and exponential growth (Nagato
Staging of embryogenesis in rice
a Days after pollination, b the stage at which respective gene is first expressed is shown; c Sato et al. (1996), d Miyoshi et al. (2002), e Miyoshi et al.
(1999), f Hong et al. (1995), g Satoh et al. (1999), h Itoh et al. (2000), i Miyoshi et al. (2000).
No. of cells EventsExpressed gene b
Em2 Early globular stage1 1–ca.25 First division of fertilized egg
Rapid cell division
Em3 Middle globular stage 2 ca.25–ca.150ca.25–ca.150-cell stagegle1 f, gle2 f, gle3 f
Relatively slow growth
Em4 Late globular stage3ca.150–ca.800 Oblong-shapedOSH1 c, OsVP1 d,
cle1 f, shl1 g, shl2 g,
shl4 g, sho1 h, sho2 h
Onset of exponential growth,
dual rhythmicity of growth and
Gradient of cell size along
Em5 SAM and radicle
4ca.800–Onset of coleoptile, SAM and
Em6 First leaf formation5–6Protrusion of 1st leaf
Enlargement of scutellum
Expression of RAmy1A in
Onset of juvenile vegetative
Em7 Second and third leaf
7–8Protrusion of 2nd and 3rd leaf
primordia in alternate
Protrusion of epiblast
Em8 Enlargement of
9–10Enlargement of organs and
OsVP1 d, OSEM d,
RAB16A e, REG2 e
riv1 i, riv2 i
Expression of maturation-
related genes such as OsEM,
Rab16A, REG2, etc.
Em9 Maturation11–20Expression of maturation-
related genes such as OsEM,
Em10 Dormancy 21–Dormancy
Staging of rice development27
1978). Furthermore, at about this stage, OSH1 expression starts
in a specific region where the SAM is later formed (Fig. 2H)
(Sato et al. 1996).
Based on these events, we can imagine a developmental
program in the globular embryo as follows. Initially, most
zygote-derived cells divide several times. Then, actively divid-
ing cells are restricted to a portion of the embryo (alterna-
tively, some embryo cells stop dividing), resulting in some
gradient of cell activity. The progress of this tendency may
cause apical–basal or dorsal–ventral axis formation of the
embryo. However, the embryonic axis does not immediately
induce SAM and radicle formation. In this respect, the 150-cell
stage seems to be a turning point for SAM and radicle differen-
tiation and other morphogenetic events because SAM and
radicle differentiation apparently start at this stage (dual rhyth-
micity of growth and recruitment of indeterminate cells, as
deduced from OSH1 expression). Correlation between OSH1
expression and SAM formation is confirmed by analyses of shl
mutants (Satoh et al. 1999, Satoh et al. 2003), strong shl alleles
lacking the SAM express OSH1 only in a small number of
cells, and weak shl alleles, forming incomplete SAMs, show an
intermediate number of OSH1-expressing cells between strong
alleles and the wild-type allele.
Morphological differentiation of the embryo takes place at
4 DAP (Fig. 2E). The coleoptile, SAM and radicle are recog-
nized almost simultaneously at 4 DAP, and the first leaf pri-
mordium at 5 DAP. Second and third leaf primordia
differentiate within 3 or 4 d. Most morphogenetic events are
completed by 10 DAP.
It is difficult to categorize late embryogenesis because
morphogenetic events do not occur after 10 DAP: only a slight
enlargement of organs occurs. In that case, maturation- related
and dormancy-related genes would be useful. One ABA-regu-
lated gene, Rab16A, is expressed after 10 DAP. Expression of
another ABA-regulated gene, REG2, is down-regulated from
the shoot and radicle after 10 DAP (Miyoshi et al. 1999).
Accordingly, embryo maturation is considered to progress rap-
idly after 10 DAP. Although viviparous mutants have been
reported (Miyoshi et al. 2000), late embryogenesis of rice has
not been understood well.
Staging of embryo development—Based on the landmark
events described above, we propose a staging system of
embryo development in rice (Table 1).
Stage Em1: Zygote. Most of the egg cells are fertilized
with self-pollen at around flower opening. On rainy or cool
days, pollination frequently occurs in closed flowers. This
stage lasts for several hours.
Stage Em2: Early globular stage. From first cell division
to about the 25-cell stage at 1 DAP (Fig. 2B). This stage is
characterized by rapid multiplication of cells. The cell division
pattern is not conserved. The first division is horizontal in most
cases, but oblique cell division is also observed. The second
and subsequent cell divisions are variably oriented.
Stage Em3: Middle globular stage. From the approxi-
mately 25-cell stage to the approximately 150-cell stage at
2 DAP (Fig. 2C). During this stage, we have not observed any
remarkable events. Cells on the dorsal side (endosperm side)
seem to be somewhat larger than those on the ventral side.
Interestingly, the maturation-related gene OSVP1 is expressed
at this stage (Miyoshi et al. 2002).
Stage Em4: Late globular stage. From about the 150-cell
stage to the onset of organ formation at 3 DAP (Fig. 2D, J).
During this stage, several events take place for differentiating
embryonic organs such as the SAM and radicle. The embryo
starts exponential growth that shows dual rhythmicity. The cell
size gradient becomes apparent along the dorsoventral axis:
ventral cells are smaller than dorsal cells. A rice kn1-type
homeobox gene OSH1 is first expressed in a small region on
the ventral side (Fig. 2H) (Sato et al. 1996), indicating that
indeterminate cells for constructing the SAM are recruited at
Stage Em5: Onset of coleoptile, SAM and radicle differen-
tiation. When the embryo comprises 800–900 cells and
becomes approximately 110 µm long at 4 DAP, the first mor-
phogenetic event is visible as a protrusion of the coleoptile on
the ventral side (Fig. 2E, K). Just below the coleoptile, the
SAM is found. Its shape is flat, not dome-like. In addition, the
scutellum is apparent in the apical region of the coleoptile. In
the internal basal region, the radicle primordium is recognized
as a population of densely stained small cells. OSH1 is
expressed in the SAM and the surrounding tissues.
Stage Em6: Formation of first leaf primordium. At 5 DAP,
the first leaf primordium is visible on the opposite side of the
coleoptile (Fig. 2F, L). Enlargement of embryonic organs, espe-
cially the scutellum, is remarkable. From this stage, the embry-
onic phase and juvenile vegetative phase coexist. A major α-
amylase gene, RAmy1A, is first expressed in the apical region
of scutellar epithelium (Fig. 2I), then its expression extends
toward the basal scutellar epithelium. One maturation-related
gene, OSEM, is expressed from this stage.
Stage Em7: Formation of second and third leaf promor-
dia. Second and third leaves are formed at 7 and 8 DAP,
respectively. By this stage, the SAM becomes dome-shaped. At
this stage, organs are enlarging. The epiblast becomes enlarged;
it is not present in maize embryos.
Stage Em8: Enlargement of organs. From 9 to 10 DAP, no
more morphological change is observed, but enlargement of
organs is marked (Fig. 2G, M). A maturation-related gene,
Rab16A, is expressed from this stage. Expression of another
maturation-related gene, REG2, is down-regulated from the
shoot, radicle and vascular bundle.
Stage Em9: Maturation of embryo: 11–20 DAP. Enlarge-
ment of embryonic organs continues, but to a small extent.
Maturation-related genes such as OSEM, Rab16A and REG2
are expressed strongly, but the expression becomes weak at