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, email@example.com; Fax, +81-3-5841-5063.
Staging of rice development 24
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 development25
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 development26
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 Events Expressed gene b
Em2 Early globular stage1 1–ca.25First division of fertilized egg
Rapid cell division
Em3 Middle globular stage 2ca.25–ca.150ca.25–ca.150-cell stagegle1 f, gle2 f, gle3 f
Relatively slow growth
Em4 Late globular stage3 ca.150–ca.800 Oblong-shaped OSH1 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
4 ca.800–Onset of coleoptile, SAM and
Em6 First leaf formation 5–6Protrusion of 1st leaf
Enlargement of scutellum
Expression of RAmy1A in
Onset of juvenile vegetative
Em7 Second and third leaf
7–8 Protrusion of 2nd and 3rd leaf
primordia in alternate
Protrusion of epiblast
Em8 Enlargement of
9–10 Enlargement 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,
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
Staging of rice development28
around 20 DAP. Expression of these genes is also detected in
the aleurone layers of the endosperm.
Stage Em10: Dormancy. After 20 DAP, the rice embryo
would be dormant.
Rice plants produce 10 or more foliage leaves before
entering the reproductive phase. Because leaf length differs
with its position, the longest leaf being three or four leaves
below the last one (flag leaf), the vegetative stage can be char-
acterized by each leaf position. However, such a categorization
would be complicated and not useful because the total number
of leaves differs largely among cultivars and almost no differ-
ence is detected between two adjacent leaves except for the
first and second leaves. Accordingly, in this review, we divide
the vegetative phase into two phases: juvenile and adult. As in
other species, many traits differ in juvenile and adult phases
(Hackett 1985, Poethig 1990, Telfer et al. 1997). It is marked
that the differences between juvenile and adult phases are not
discrete, but continuous. For that reason, it is difficult to spec-
ify the boundary of the two phases explicitly. We only recog-
nize the difference when two remote stages (e.g. 2nd leaf and
10th leaf) are compared. An intermediate period exists between
juvenile and adult phases; the juvenile phase is converted grad-
ually into the adult phase. For rice, Asai et al. (2002) first
reported the differences between juvenile and adult phases in
rice in analysis of a heterochronic mutant mori1. Those differ-
ences are observed in all the traits they examined. mori1 is a
heterochronic mutant that reiterates the second-leaf stage and
suppresses the induction of adult phase. Their data suggest that
the juvenile phase is the first and second leaf stages; third to
fifth leaf stages are intermediate. The later stage is the adult
phase. The following section describes how juvenile and adult
phases differ in many traits, as summarized in Fig. 3 and Table 2.
The SAM becomes enlarged gradually with development.
For example, the SAM at the 10th leaf stage is nearly two-fold
larger than that at the second leaf stage (Fig. 3A). Expression
of histone H4 gene indicates that the cell division frequency in
the SAM is higher at the second-leaf-primordium stage than at
the stages after the fourth leaf primordium stage (Fig. 3B).
Various leaf traits vary largely during development. Plas-
tochrons (the rate of leaf production) of the second and third
leaves are approximately 1.6 d; after the fifth leaf, it is approxi-
mately 4 d. Leaf size and shape also vary with development.
The first leaf is quite small and lacks a blade. The second leaf
has both a blade and sheath, but it is very small (Fig. 3C). Sub-
sequently, leaf size increases gradually, achieving its maximal
size in the three or four leaves below the last leaf (flag leaf).
The leaf blade shape is rather rounded in earlier leaves. Ana-
tomical differences are also observed. Adult leaves are
strengthened mechanically by the presence of a large midrib,
whereas the midrib is not prominent in the second leaf; it is
small in the third leaf (Fig. 3D). Regarding physiological traits,
photosynthesis has been examined. The apparent photosyn-
thetic rate per unit area is very low in the second leaf blade. It
increases gradually in subsequently developed leaf blades. Low
photosynthesis at the early stages engenders heterotrophism.
Stem structure also differs between juvenile and adult
phases. In the adult stem, node and internode are differentiated
clearly, but in the stem where first through third leaves are
inserted, node and internode are not clearly distinguished (Fig.
3E). Vascular bundles are regularly oriented in the adult stem,
but they are rather randomly oriented in the basal stem where
the first through third leaves are inserted (Fig. 3E). Many
Comparison of juvenile and adult traits in rice. (A) SAM at
second (left) and 10th (right) leaf stages. (B) Histone H4 expression in
SAM at second (left) and 10th (right) leaf stages. (C) Second (left) and
sixth (right) leaf blade. (D) Cross-sections of midrib region of second
(left) and 10th (right) leaf blades. Arrowhead indicates midrib (E)
Longitudinal sections of basal stem region where second and third
leaves are inserted (left) and apical stem region where seventh and
eighth leaves are inserted (right). Arrowheads indicate nodes.
Staging of rice development29
crown roots (adventitious roots) are formed from basal stem
nodes, but almost no crown roots emerge from the adult stem
The traits described above indicate that juvenile and adult
phases show not only quantitative differences, but also qualita-
tive changes that are regulated by a developmental switch. The
phase change is a simultaneous alteration in the expressionof
many genes. Although MORI1 is the only gene reported so far
to be involved in the phase change of the whole plant, it is
inferred that specific genes are responsible for the phase
change of each organ and tissue.
Reproductive Phase: Inflorescence
DESCRIPTION OF DEVELOPMENTAL COURSE
When environmental conditions and internal factors
become favorable for floral induction, a SAM that has regu-
larly formed foliage leaves and tillers (branches) is converted
into an inflorescence meristem. The inflorescence meristem is
discernible from the SAM by the identity of lateral organs. The
inflorescence meristem forms bracts (small degenerate leaves)
and inflorescence branches that bear flowers (spikelets) later.
The following sections describe inflorescence development and
its staging only briefly because Ikeda et al. (2004) have already
proposed a detailed staging system.
A mature rice inflorescence is shown in Fig. 4A. The rice
inflorescence is categorized as a raceme together with those of
Sorghum, Panicum and Avena, in which spikelets are not
attached directly to the main axis, but are formed on the lateral
branches. The rice inflorescence is also called a panicle
because of its conical shape. In grasses, the main axis of the
inflorescence is called the rachis (Bell 1991). Lateral branches
that are attached directly to the rachis are called primary
(rachis) branches. Those on the primary branch are termed sec-
ondary (rachis) branches. The mature inflorescence of rice has
10 or more primary branches that bear approximately 150
spikelets. Two types of inflorescence meristem are recognized:
rachis and branch meristems. The rachis meristem forms bracts
and branches as lateral organs, and finally aborts. In contrast,
the branch meristem differentiates spikelets and branches; it is
ultimately converted into a terminal spikelet. Moreover, the
phyllotaxy of lateral organs differs for rachis and primary
branch meristems. Primary branches are produced in spiral
phyllotaxy, but spikelets on the primary branches are arranged
in a biased distichous phyllotaxy. The length of primary inflo-
rescence branches is reduced acropetally.
When the rachis meristem forms a bract 1 primordium, it
is larger than the final SAM (Fig. 4B). The rachis meristem
forms bracts and primary branches sequentially in 2/5 spiral
phyllotaxy (Fig. 4C, E). The first primary branch primordium
is visible at the axile of bract 1 when bract 2 primordium is
formed. The rachis meristem increases its size, reaching its
maximum size at the bract 3 primordium stage. Subsequently, it
shrinks gradually. The growth of bracts is severely suppressed:
the distal cells of the third and subsequent bracts become hairs
(Fig. 4D, F). When all branch primordia are formed, the rachis
meristem loses its activity and aborts. The first branch primor-
dium does not elongate first: all branch primordia elongate
simultaneously (Fig. 4F). When primary branches elongate to
some extent, secondary branches are formed in the basal
regions (Fig. 4G). Then the branch meristem is converted into a
spikelet meristem to form glumes. Then it converts into a
flower (floret) meristem to form floral organs (Fig. 4H). The
inflorescence remains short (<4 cm) at this stage. It commences
rapid elongation of rachis and branches after floral organ pri-
mordia are differentiated. Maturation of anthers and ovules
takes place during rapid branch elongation.
Staging of inflorescence development—Development of
rice inflorescence is categorized into nine stages (Table 3),
according to Ikeda et al. (2004).
Stage In1: Establishment of rachis meristem. This stage is
the period from the conversion of the SAM to rachis meristem
until bract 1 primordium formation (Fig. 4B). The rachis meris-
Comparison of juvenile and adult characters in rice
Cell division ActiveInactive
Orientation of VBIrregularRegular
Adventitious rootManyFew or none
Disease resistanceWeak Strong
Staging of rice development30
tem is larger than the last vegetative meristem. The –3 and –4
stem internodes have already started their elongation and the
–2 internode starts elongation at this stage. Herein, the second,
third and fourth stem internodes counted from the uppermost
one are designated as –1, –2 and –3 internodes, respectively.
Stage In2: Formation of primary rachis branches I. Dur-
ing this stage, primordia of bract 2, bract 3 and the first primary
branch are formed (Fig. 4C). At the end of this stage, the rachis
meristem attains its maximum size. Most bract positions are
determined at this stage, as inferred from the bract-specific
expression of the PLA1 gene (Miyoshi et al. 2004).
Stage In3: Formation of primary rachis branches II. This
stage corresponds to the period of primary branch formation
after the In2 stage (Fig. 4D). Primary branches are produced in
spiral phyllotaxy (Fig. 4E). The number of primary branches
depends on the time when the rachis meristem loses its activity
(loss of OSH1 expression). Stages In1—In3 are completed
within 3 or 4 d. Elongation of the –1 stem internode starts at
The lax and pap1 mutants would be related to this stage
(Komatsu et al. 2001, Takahashi et al. 1998).
Stage In4: Elongation of primary branches. Although
basal primary branch primordia are formed earlier than the api-
cal ones, all primordia commence elongation simultaneously
(Fig. 4F). Therefore, In4 is the stage from the onset of primary
branch elongation to the formation of lateral organs. The epi-
dermal cells of bracts elongate like hairs.
Stage In5: Differentiation of higher-order branches. Soon
after the onset of elongation, primary branches (especially
basal ones) produce secondary branches as lateral organs. For
that reason, the stage In5 is the period when the primary branch
meristem produces secondary branches, but not spikelets (Fig.
4G). Basal secondary branches frequently produce tertiary
branches. Seven to nine days elapse from the conversion of the
meristem until the end of this stage. The uppermost stem inter-
node starts elongation at this stage. By this time, –3 and –4
internode elongation has been completed.
Stage In6: Differentiation of glumes. Primary and second-
ary branch meristems are converted into terminal spikelet mer-
istems and form rudimentary glumes (Fig. 4H). Subsequently,
lateral meristems become lateral spikelet meristems. Lateral
organs of primary branches are arranged in a biased distichous
phyllotaxy with a divergence angle of about 110° (Fig. 4H).
Two empty glumes, lemma and palea, are formed after two
Stage In7: Differentiation of floral organs (inflorescence
length 1.5–40 mm). After glume formation, floral organs (two
lodicules, six stamens and one carpel) are formed. At this
stage, rachis elongation is not yet remarkable. Even when car-
pel primordia are observed in all flowers, the inflorescence is
only 10 mm long. The –2 stem internode elongates rapidly.
Stage In8: Rapid elongation of rachis and maturation of
reproductive organs. When the inflorescence becomes approxi-
mately 40 mm long, it starts rapid elongation. Each organ
increases in size. Maturation of reproductive organs, the sta-
men and pistil, takes place to form pollen grains and the
embryo sac. At this stage, the uppermost and –1 stem inter-
nodes elongate rapidly.
Stage In9: Heading and flowering. A few days after com-
pletion of flowers, the inflorescence emerges from the sheath
of the flag leaf; flowering occurs at around noon.
Inflorescence development in rice. (A) Mature rice inflorescence. (B) Formation of first bract (arrowhead). (C) Second bract formation.
(D) Early stage of primary branch formation. (E) Cross-section of rachis apex differentiating four primary branches in spiral phyllotaxy. Numer-
als indicate first, second, third and fourth branches, respectively. (F) Onset of primary rachis branch elongation. (G) Formation of secondary
branches (*). (H) Formation of spikelets. ra, rachis; prb, primary rachis branches; srb, secondary rachis branches; sp, spikelet; B1, first bract; B2,
Staging of rice development31
Development of Major Organs
DESCRIPTION OF DEVELOPMENTAL COURSE
Leaves are produced repetitively as lateral organs of the
SAM. Analyses of a large number of mutants in several model
plants (for reviews, see Scanlon 2000, Byrne et al. 2001, Bow-
man et al. 2002) suggest that leaf development is a complex
process comprising cell division and expansion, axis determi-
nation, tissue differentiation and specification.
Grass leaves have a different architecture from those of
dicots. For maize, developmental analyses of leaves have been
made (Freeling 1992, Sylvester et al. 1996). However, for rice,
only a few studies have been attempted from histological and
morphological viewpoints (Kaufman 1959a, Hoshikawa 1989).
This section presents a description of rice leaf development
that incorporates recent genetic and molecular results.
The mature rice leaf is strap-like; it is usually divided into
three distinct regions along the proximal–distal axis. The leaf
blade (lamina) is the distal region of a leaf and a major site of
photosynthesis (Fig. 5A). The leaf sheath is the proximal
region and surrounds the shoot apex and younger leaves to pro-
tect them from physical damage. The boundary of the blade
and sheath consists of three distinct parts: the lamina joint (col-
lar), the ligule and the auricle (Fig. 5A). The lamina joint is a
whitish region in the base of the blade; it functions in bending
the leaf blade toward the abaxial side (Fig. 5A). The ligule is
membranous and acuminate; it usually splits into two seg-
ments in adult leaves. The auricles are a pair of small append-
ages with long hairs that are positioned at the leaf margins (Fig.
The rice leaf is also polarized along the adaxial–abaxial
axis (Fig. 5B, E). Numerous papilla and two kinds of tri-
chomes are found over the entire leaf surface, except for the
Staging of inflorescence development in rice
a Values are for cv. Taichung 65.
b Itoh et al. (1998), c Komatsu et al. (2001), d Iwata and Omura (1971b) e Nagao and Takahashi (1963), f Jones (1952), g Futsuhara et al. (1979),
h Takahashi et al. (1998).
length (mm) a
0.05–0.1Conversion of vegetative meristem
to rachis meristem. Enlargement of
rachis meristem. Formation of bract
1 primordium. Onset of –2 stem
In2 Formation of
primary branches I
Rachis meristem Branch
0.1–0.2 Rachis meristem reaches maximum
size. Formation of first two primary
branch primordia and bract 2 and 3
pla1 b, laxc, sp d
Dn1 e, Dn2 f, Dn3 g
primary branches III
Rachis meristem Branch
0.2–0.4Formation of primary branch
primordia in spiral arrangement.
Abortion of rachis meristem at the
end of this period. Onset of –1 stem
lax c, pap1 h
In4Elongation of primary
Branch meristem0.4–0.6 Simultaneous elongation of primary
In5Formation of higher-
secondary and tertiary
0.6–0.9Formation of secondary and tertiary
branches. Onset of uppermost stem
lax c, Dn1 f, Dn2 g,
Spikelet meristem Floret
0.9–1.5Differentiation of two rudimentary
glumes, two empty glumes, lemma
and palea in 1/2 alternate
(see Table 6)
1.5–40Differentiation of floral organs, two
lodicules, six stamens and a carpel
(see Table 6)
In8Rapid elongation of
rachis and branches
Loss of meristem40–220Rapid elongation of rachis and
branches. Completion of anther and
ri e (see Table 6)
ca.220 Inflorescence emergence out of the
sheath of flag leaf. Flowering at
Staging of rice development 32
adaxial surface of the sheath. In the adaxial epidermis of the
blade, bulliform cells are arranged in vertical rows between
vascular bundles (Fig. 5C). Two types of vascular bundles—
large and small ones—are observed (Fig. 5B). The xylem is
located on the adaxial side of the vascular bundles, and the
phloem on the abaxial side (Fig. 5C). Bundle sheath cells (Fig.
5C) enclose the vascular bundles. Sclerenchymatous fiber cells
are observed on the adaxial and abaxial sides of the vascular
bundles of leaf blade (Fig. 5C) and on the abaxial side of the
sheath. The shapes of the blade and sheath margins are differ-
ent. The margin of the sheath is pointed and membranous (Fig.
Leaf primordium is first recognized as a small bulge on
the flank of the SAM (Fig. 5J). After the protrusion, the bulge
grows toward the apex and toward the opposite side of the
SAM, forming a crescent-shaped primordium (Fig. 5H). The
primordium becomes hood-shaped by rapid cell division and
elongation in the apical and marginal regions (Fig. 5J). At this
stage, initiation of the procambial strand is visible in the leaf
center (Fig. 5K). After the two margins of the leaf primordium
overlap and enclose the SAM, the shape of the leaf primor-
dium becomes cone-like, and the blade (lamina)–sheath bound-
ary is established (Fig. 5L). A protrusion of ligule primordium
appears at the blade–sheath boundary of the adaxial surface.
That protrusion originates from periclinal divisions of epider-
mal cells (Fig. 5M, N). Furthermore, the onset of various inter-
nal tissues occurs at this stage. The large vascular bundles
cover the total width of the leaf, and xylem and phloem are rec-
ognized in the mid-vein. In addition, small vascular bundles are
found between the large vascular bundles, and macrohairs dif-
ferentiate on the leaf-tip epidermis. Stomata formation in the
blade proceeds basipetally from the distal region, but the proxi-
mal region of the epidermis remains immature.
After differentiation of the ligule primordium, the leaf
blade elongates rapidly. Although the leaf blade reaches its
maximum length around this stage, leaf sheath elongation
remains suppressed (Fig. 5O). Following the completion of leaf
blade elongation, the leaf sheath commences rapid elongation.
Epidermis-specific cell types such as bulliform cells, silica
cells and stomata cells become apparent from the apex. Vascu-
lar bundles become mature, and sclerenchymatous cells differ-
entiate just outside the vascular bundles (Fig. 5P). When the tip
of the leaf blade emerges out of the sheath of the preceding
leaf, the internal and epidermal structures of the leaf are almost
complete except those in the most proximal region. Lacuna,
which are air spaces interrupted by septa, are formed basipe-
tally in the inner tissue of the midrib and the leaf sheath (Fig.
5E). The leaf blade bends at the lamina joint as a result of une-
Leaf development in rice. (A) Mature leaf. (B) Cross-section of mature leaf blade. Upper side is adaxial. (C) Close-up of the midvein in
(B). (D) Longitudinal section of shoot apex. (E) Cross-section around shoot apex. Lacuna formation is observed in the leaf sheath of P5 (asterisk).
Margin of the leaf sheath is pointed (arrowhead). (F) Expression of OSH1 in the shoot apex. Down-regulation of OSH1 is observed in the P0
(arrow). (G) Expression of OsPNH1 in the shoot apex. The arrowhead indicates the expression in the central region of the P0. (H) SEM image of
SAM and late P1 primordium. (I) Expression of OsSCR around shoot apex. OsSCR expression is observed in the P1 but not in the SAM. (J) SEM
image of early P1 and P2 primordium. (K) Cross-section of the center of the P2 differentiating procambial strand. (L) SEM image of the P3. The
arrow indicates the blade–sheath boundary. (M, N) Early development of ligule. Periclinal division is shown (arrow). (O) SEM image of the P4.
Elongation of the sheath (below the arrow) does not yet start. (P) Cross-section of large vascular bundle of P4 leaf sheath. LB, leaf blade; LG,
ligule; AU, auricle; LJ, lamina joint; LS, leaf sheath; LV, large vascular bundle; SV, small vascular bundle; SC, sclerenchymatous cell; PL,
phloem; XY, xylem; BS, bundle sheath cell; BC, bulliform cell; SA, shoot apical meristem; PS, procambial strand; LP, ligule primordium.
Staging of rice development33
qual elongation that occurs between the adaxial and abaxial
cells (Maeda 1961).
Staging of leaf development—Although the word plasto-
chron is originally defined as a time interval between the initia-
tion of two successive leaves, plastochron number (Pi) is
usually used to classify stages of leaf development (Sharman
1942, Hill and Load 1990). P1 represents the youngest leaf pri-
mordium just after the protrusion on the flank of the SAM. P2
is the next youngest primordium, and P3 represents the third
youngest primordium (Fig. 5D, E). In addition, the term P0 is
frequently used (Jackson et al. 1992, Sylvester et al. 1996). P0
represents a state of cells determined as a new leaf primordium
in the SAM. Similarly, P–1 can be defined as a stage of cells
that will become P0 cells in the next plastochron. It is reasona-
ble to use the plastochron number to represent leaf stages
because two successive stages (Pi and Pi+1) are easily distin-
guished. Moreover, it can be used ubiquitously in other model
plants such as Arabidopsis and maize (Jackson et al. 1992,
Lynn et al. 1999).
The developmental course of a rice leaf sometimes differs
with position: the first and second leaves are quite different
from adult leaves. However, adult leaves follow a similar
developmental course. Here, we propose a staging system for
adult leaves (Table 4).
Stage P0: Formation of leaf founder cells. Cells at the P0
stage, called leaf founder cells, are those whose fates are deter-
mined to be leaf primordium but are not distinguishable mor-
phologically from other cells in the SAM. They are detected by
down-regulation of OSH1 expression (Fig. 5F). The leaf
founder cells are distributed in a half-ring domain in the cir-
cumference of the SAM, opposite the youngest leaf primor-
dium. OsPNH1, which is preferentially expressed in the
developing vascular bundle, is first expressed in the central
region of P0, where the midvein will develop later (Fig. 5G)
(Nishimura et al. 2002).
Stage P1: Formation of leaf primordium. The P1 leaf pri-
mordium is a small protrusion on the flank of the SAM or a
crescent-shaped primordium (Fig. 5H). Cell division activity in
P1 is much higher than that in the SAM, as confirmed by his-
tone H4 expression (Itoh et al. 2000). Several molecular mark-
ers distinguish P1 from P0. The OsSCR expression starts in the
P1 epidermal layer (Fig. 5I); it is then restricted gradually to
the specific cell files of epidermis (Kamiya et al. 2003a). The
DROOPING LEAF (DL) gene, a regulator of midrib formation
and carpel specification, is first expressed in the central region
of the P1 primordium (Yamaguchi et al. 2004).
Stage P2: Hood-shaped primordium. The P2 leaf primor-
dium is hood-shaped (Fig. 5J). Molecular markers distinguish-
ing P1 and P2 primordia have not been reported so far.
Stage P3: Formation of ligule primordium. The margins
of the P3 leaf overlap and completely enclose the SAM. The P3
leaf is long and conical (Fig. 5L). The main morphological
change at this stage is the establishment of the blade–sheath
boundary. The ligule primordium is observed (Fig. 5M, N). In
the leaf tip, epidermal cells are morphologically distinguished
from the internal cells, indicating the cessation of apical meris-
tematic activity (Kaufman 1959a). Epidermal differentiation
and small vascular bundle formation proceed basipetally. Cell-
file-specific expression of OsSCR in the epidermis is observed
at the P3 stage (Kamiya et al. 2003a).
Staging of leaf development in rice
a Nishimura et al. (2002), b Kamiya et al. (2003a), c Yamaguchi et al. (2004), d Itoh et al. (2000), e Obara et al. (2004), f Maekawa (1988).
Events Gene expressionRelated mutant
P0 Formation of leaf founder cellsRecruitment of leaf founder cells Down-regulation of OSH1
sho1 d, sho2 d,
P1 Formation of leaf primordiumProtrusion of primordium. Elongation of
leaf margin around SAM
OsPNH1 a OsSCR b DL c
P2 Hood-shaped primordiumHood-like shape. Overlapping of two
margins. Differentiation of vascular bundle
OsPNH1 a OsSCR b DL c
P3Formation of ligule primordium Formation of ligule primordia. Formation
of leaf blade–sheath boundary.
Differentiation of sclerenchymatous cells.
Initiation of epidermal specific cells
OsPNH1 a OsSCR b DL c
P4Rapid elongation of leaf blade Differentiation of epidermal specific cells
(bulliform cells, silica cells, cork cells and
stomata). Elongation of leaf blade
OsPNH1, OsSCR and DL
P5 Rapid elongation of leaf sheathElongation of leaf sheath. Emergence of
leaf blade from the sheath of preceding
leaf. Formation of lacunae. Maturation of
leaf epidermal cells
P6MaturationBending of leaf blade at the lamina joint
Staging of rice development34
Stage P4: Rapid elongation of leaf blade. A salient fea-
ture of the P4 leaf is its rapid leaf blade elongation (Fig. 5O).
This elongation is believed to be attributable to the activity of
the intercalary meristem located in the basal region of the leaf
blade (Kaufman 1959a). Expression of genes that are associ-
ated with tissue differentiation, such as OsPNH1, OsSCR and
DL, becomes down-regulated (Nishimura et al. 2002, Kamiya
et al. 2003a, Yamaguchi et al. 2004).
Stage P5: Rapid elongation of leaf sheath. Following the
completion of leaf blade elongation, rapid elongation of the
leaf sheath occurs. The leaf blade tip emerges from the sheath
of the P6 leaf. The lacuna are formed basipetally in the midrib
and the sheath (Fig. 5E).
Stage P6: Mature leaf. The leaf becomes mature and
growth is completed. Leaf blade bending occurs at the lamina
Root (Crown Root)
DESCRIPTION OF DEVELOPMENTAL COURSE
The rice root system consists of seminal, crown and lateral
roots, which correspond to pole-borne, stem-borne and root-
borne roots, respectively (Barlow 1994). Generally, the root
system of most dicot plants develops from the radicle formed
in the embryo, whereas monocot plants have a so-called fibrous
root system that is characterized by numerous crown roots
(Klepper 1992). For example, a field-grown rice plant usually
has several hundreds (sometimes over 1,000) crown roots
(Kawata et al. 1978, Kawashima 1988). To date, organization
and cell differentiation processes in lateral root development
have been well characterized in a model dicot, Arabidopsis
(Malamy and Benfey 1997). In contrast, only a few studies
have explored root development in monocots. Recently, sev-
eral molecular markers expressed in specific tissues of the rice
root have been reported. The QHB gene is expressed specifi-
cally in the central cells of the quiescent center in the root api-
cal meristem, and the OsSCR gene in the endodermis (Kamiya
et al. 2003a, Kamiya et al. 2003b).
Based on morphological observations, Kaufman (1959b)
and Kawata and Harada (1975) described root development in
rice. They also proposed a staging system of crown root devel-
opment. Here, we describe rice root development and propose
a staging system with a small modification to that by Kawata
and Harada (1975). The proposed system incorporates recent
results of genetics and molecular biology.
The crown root primordium of rice originates from the
innermost ground meristem cells, which are adjacent to the
peripheral cylinder of vascular bundles in the stem (Fig. 6A).
Those initial cells gradually differentiate various tissues in suc-
cession, such as the epidermis, endodermis, cortex, stele (cen-
tral cylinder) and root cap, to form the complete organization
of the apical meristem of the crown root. At the next stage,
each tissue increases the number of cells through a regulated
division pattern in the following manner. Initial cells of crown
root primordium are formed in a few layers by one or two peri-
clinal divisions of the innermost ground meristem cells (Fig.
6A). These initial cells and the nuclei of these cells become
enlarged and their protoplasm density gradually increases. Sub-
sequently, cells in the inner layers of the initial divide anticli-
nally and periclinally to form an epidermis–endodermis initial
and a central cylinder initial. Then, the cells in the outer layer
of the initial begin to divide mainly anticlinally and form the
root cap initial (Fig. 6B). The epidermis–endodermis initial dif-
ferentiates into epidermis and endodermis by periclinal divi-
sions in a specific cell layer (Fig. 6C). The root cap initial cells
and central cylinder initial cells undergo anticlinal and pericli-
nal divisions and consequently increase their own size (Fig.
The endodermal cells undergo a number of periclinal, but
asymmetrical divisions to produce several cortical cell layers
(Fig. 6D) (Kawata and Lai 1965). Then root cap initial cells
Crown root development
in rice. (A) Establishment of ini-
tial cells. (B) Establishment of
epidermis–endodermis and root
cap initials. (C) Differentiation of
epidermis–endodermis initial into
epidermis and endodermis. (D)
Cortex differentiation. (E) Estab-
lishment of fundamental organi-
zation of root primordium. (F)
Onset of cell vacuolation (arrow-
head) in cortex and elongation
(arrow) in stele. (G) Crown root
emergence. IC, initial cells; PV,
peripheral cylinder of vascular
bundle; C, root cap or its initial;
EE, epidermis–endodermis ini-
tials; S, stele (central cylinder);
EP, epidermis; EN, endodermis;
CO, cortex; COL, columella;
MXII, late meta-xylem vessel.
Staging of rice development 35
form columella through periclinal divisions. The cells in the
central cylinder initial (stelar initial) continue anticlinal and
periclinal divisions to become dome-shaped (Fig. 6E). At the
same stage, cells that constitute various tissues of the vascular
bundle are gradually differentiated (Fig. 6E). Then, in the basal
region of the primordium, cells of all tissues become vacu-
olated and elongated concurrently with the emergence of the
crown root from the stem (Fig. 6F, G). It is noted that the stage
when the vascular bundle system connects the stem and crown
root coincides with the time of root emergence (Kawata and
Staging of crown root development—Crown root develop-
ment is divided into seven stages (Table 5).
Stage Cr1: Establishment of initial cells. Initial cells of
crown root primordium are formed in a few layers by one or
two periclinal divisions of the innermost ground meristem cells
(Fig. 6A). The QHB and OsSCR genes are first expressed in the
outer layer cells derived from the first periclinal division.
Thereafter they maintain their specific expression: QHB in the
quiescent center and OsSCR in endodermis (Kamiya et al.
2003a, Kamiya et al. 2003b). The first periclinal division is
suppressed in the crl1 mutant (Inukai et al. 2001).
Stage Cr2: Establishment of epidermis–endodermis, cen-
tral cylinder and root cap initials. The initial cells of crown
root primordium begin to divide anticlinally and periclinally to
form epidermis–endodermis initial, central cylinder initial and
root cap initial (Fig. 6B).
Stage Cr3: Differentiation of epidermis and endodermis.
The epidermis–endodermis initial differentiates into epidermis
and endodermis by periclinal divisions in a specific cell layer
(Fig. 6C). The root cap initial cells and central cylinder initial
cells undergo anticlinal and periclinal divisions (Fig. 6C).
Stage Cr4: Differentiation of cortex. Endodermal cells
begin to form cortical cells by peliclinal divisions (Fig. 6D).
When endodermal cells divide periclinally to produce cortex,
the expression of OsSCR is down-regulated in the daughter
cortex cells (Kamiya et al. 2003a).
Stage Cr5: Establishment of fundamental organization.
Root cap initial cells form columella by periclinal divisions
(Fig. 6E). In the central region of the stele, a large meta-xylem
vessel is observed (Fig. 6E). At this stage, fundamental organi-
zation of the root is established.
Stage Cr6: Onset of cell elongation and vacuolation. At
this stage, cells in the basal region of the stele show com-
mencement of cell elongation and vacuolation, and those of the
cortex show vacuolation (Fig. 6F). In the crl2 mutant, the
growth of these cells is suppressed (Inukai et al. 2001). Around
this stage, the root apex reaches the stem epidermis.
Stage Cr7: Emergence of crown root. In the basal region
of the root primordium, cells of all tissues elongate concur-
rently with the emergence of the crown roots (Fig. 6G).
DESCRIPTION OF DEVELOPMENTAL COURSE
Spikelet development and its staging have already been
published in detail (Ikeda et al. 2004). Thus in this paper,
spikelet development is reviewed only briefly.
The rice spikelet consists of a single floret because the
spikelet meristem is converted into a floret meristem after pro-
ducing two pairs of sterile glumes (rudimentary glumes and
empty glumes) (Fig. 7A). Rice florets comprise lemma, palea
and three kinds of organs: two lodicules (petals), six stamens
and one pistil constituted by a single carpel (Fig. 7B)
After producing lateral branches, branch meristems (inflo-
rescence meristems) are converted into spikelet meristems (Fig.
7C). The spikelet meristem first differentiates a pair of sterile
glumes in a 1/2 alternate arrangement. They are rudimentary in
their shape and bear no axillary buds (Fig. 7D). They are called
Staging of crown root development in rice
a Kamiya et al. (2003b), b Inukai et al. (2001).
Cr1Establishment of initial cellsEstablishment of initial cells of crown root primordium by
periclinal divisions of ground meristem
Cr2Establishment of epidermis–
endodermis, central cylinder and
root cap initials
Formation of epidermal–endodermal and central cylinder
initials from the inner layer of the initial. Formation of root
cap initial from the outer layer of the initial
Cr3 Differentiation of epidermis and
Differentiation of epidermis and endodermis from
Cr4Differentiation of cortexFormation of cortex by periclinal divisions of endodermal
Cr5Establishment of fundamental
Establishment of fundamental organization of root. Root cap
reaches the stem epidermis
Cr6Onset of cell elongation and
Cells in the most basal region of stele start cell elongationcrl2 b
Cr7Emergence of crown rootElongation of basal cells of all tissues. Emergence of crown
root out of stem
Staging of rice development36
rudimentary glumes in rice or merely ‘glumes’ in other grasses.
Grass species such as barley and wheat produce several florets
in one spikelet after the pair of (rudimentary) glumes. In rice,
however, one more pair of sterile glumes, called empty glumes,
is formed (Fig. 7E); then the spikelet meristem is transformed
to a floret meristem to produce two kinds of glumes (one
lemma and one palea) and floral organs (Fig. 7F, G). The
empty glumes are much larger than rudimentary glumes, but
much smaller than lemma. The six glumes: two rudimentary
glumes, two empty glumes, lemma and palea, are arranged in
1/2 alternate phyllotaxy. After the palea, floral organs are
formed. The meristem at this stage is called a floral meristem.
Rice has only three kinds of floral organs: two lodicules, six
stamens and one pistil. The lodicules that are positioned on the
lemma side are small and whitish. They correspond to petals.
Sepals have been lost during the evolution of grasses. Six sta-
mens are formed in a whorl. Finally, one carpel primordium is
differentiated from the lemma side of floral meristem, and then
encloses the floral meristem. The floral meristem is trans-
formed to an ovule primordium; then an embryo sac is formed
(Fig. 7H, I).
The rice (grass) flower structure differs from that of dicots
and other monocots in that organs that correspond to sepals are
lacking. In addition, only two lodicules (petal) exist and are
biased to the lemma side, although the basic number of floral
organs in monocots is three. Thereby, one lodicule to be
formed on the palea side would have been lost in the past.
Recent studies have revealed that the ABC model is par-
tially modified in rice (Nagasawa et al. 2003) even if we do not
consider the loss of organs corresponding to sepals. Rice
homologs of class A, B and C genes are expressed as expected
(Kyozuka et al. 2000, Nagasawa et al. 2003). However, in con-
trast to Arabidopsis, carpel identity is predominantly regulated
by the DL gene (Nagasawa et al. 2003).
Staging of spikelet development—Spikelet development is
divided into eight stages (Table 6) according to Ikeda et al.
Stage Sp1: Formation of a pair of rudimentary glume pri-
mordia. The spikelet meristem first produces a pair of rudimen-
tary glumes in a 1/2 alternate arrangement (Fig. 7C). The outer
one is positioned on the adaxial side of the spikelet meristem.
Two rudimentary glumes are vestigial.
Stage Sp2: Formation of a pair of empty glume primor-
dia. Following two rudimentary glumes, a pair of empty
glumes is formed in a 1/2 alternate arrangement (Fig. 7D). The
empty glumes grow to some extent, but they are sterile. The
FZP gene is expressed at this stage and is considered to be nec-
essary for floret meristem formation (Komatsu et al. 2003).
Stage Sp3: Formation of lemma primordium. The lemma
primordium is formed at a position that is 180° apart from the
second empty glume (Fig. 7E). The two empty glumes are
growing at this stage. The LHS gene regulates the identity of
the lemma and palea (Jeon et al. 2000); RAP1 is expressed in
lemma (Kyozuka et al. 2000).
Stage Sp4: Formation of palea primordium. The palea pri-
mordium is formed at a position that is 180° apart from the
lemma (Fig. 7F). The two empty glumes and lemma are grow-
ing. RAP1 is expressed in the palea (Kyozuka et al. 2000).
Many mutants are known to affect lemma and palea develop-
ment (Iwata and Omura 1971a, Iwata and Omura 1971b).
Stage Sp5: Formation of lodicule primordia. Two lodi-
cule primordia are formed on the lemma sides. The two empty
glumes, lemma and palea are growing. The RAP1, OsMADS45,
OsMADS2 and SPW1 genes are expressed in lodicules (Kyo-
Spikelet development in rice. (A) Mature spikelet. (B) Mature flower. (C) Early spikelet meristem producing rudimentary primordium.
(D) Formation of empty glume primordia. (E) Formation of lemma primordium. (F) Formation of palea primordium. (G) Formation of stamen pri-
mordia. (H) Formation of carpel and ovule primordia. (I) Formation of embryo sac. PA, palea; LE, lemma; EG, empty glume; RG, rudimentary
glume; ST, stamen; PI, pistil; LO, lodicule; CA, carpel; OV, ovule.
Staging of rice development37
zuka et al. 2000, Nagasawa et al. 2003). In spw1, lodicules are
transformed to glumes (Nagasawa et al. 2003). The number of
lodicules is increased in fon1 and fon2 mutants (Nagasawa et
Stage Sp6: Formation of stamen primordia. Six stamen
primordia are formed in the whorl (Fig. 7G). The OsMADS45,
SPW1 and RAG are expressed in stamens (Kyozuka et al. 2000,
Nagasawa et al. 2003). The stamens are transformed to carpels
in the spw1 mutant (Nagasawa et al. 2003). The number of sta-
mens is increased in fon1 and fon2 mutants (Nagasawa et al.
Stage Sp7: Formation of carpel primordium. A carpel pri-
mordium is formed on the lemma side of the floral meristem
(Fig. 7H). At this stage, stamen primordia differentiate into an
anther and filament.
OsMADS45, RAG and DL are expressed in the carpel pri-
mordium (Kyozuka et al. 2000, Yamaguchi et al. 2004). In the
dl mutant, the carpel is transformed to stamens (Nagasawa et
al. 2003). fon1 and fon2 increase the number of pistils
(Nagasawa et al. 1996).
Stage Sp8: Formation of ovule and pollen. When the car-
pel primordium extends to the palea side, OSH1 expression is
down-regulated from the floral meristem. In addition, it is con-
verted to ovule primordium (Fig. 7H, I). The carpel encloses
the ovule at this early stage (Fig. 7H). The ovule undergoes
female gamete formation. Simultaneously, pollen grains are
formed in the anther. For detailed descriptions of ovule and
pollen development, see below.
The OsMADS45 and OsMADS13 genes are expressed in
the ovule (Lopez-Dee et al. 1999). The MSP1 gene is expressed
in the surrounding tissues of male and female sporocytes (Non-
omura et al. 2003).
Development of Specific Organs/Tissues of Interest
DESCRIPTION OF DEVELOPMENTAL COURSE
The ovule is a female reproductive organ where megaspo-
rogenesis, megagametogenesis, fertilization and embryogene-
sis take place. The ovule comprises the nucellus, chalaza and
funiculus along the proximal–distal axis. In the radial direc-
tion, inner and outer integuments differentiate from the
chalaza. To date, most studies on ovules have used dicot spe-
cies. They have revealed a number of genes that are associated
with placental formation, ovule primordium differentiation,
ovule identity, differentiation of each ovule component and
integument development (for review, see Gasser et al. 1998,
Grossniklaus and Schneitz 1998, Skinner et al. 2004). Analysis
of petunia MADS-box genes is a pioneer in the field of ovule
identity establishment (Angenent et al. 1995, Colombo et al.
1995). By contrast, only a few studies have examined ovule
development in monocots. In rice, the OsMADS13 gene is con-
sidered to regulate ovule identity (Lopez-Dee et al. 1999).
OsMADS24 and OsMADS45 are also considered to assist the
function of OsMADS13 (Favaro et al. 2002).
Staging of spikelet development in rice
a Komatsu et al. (2003), b Iwata et al. (1983), c Jeon et al. (2000), d Kyozuka et al. (2000), e Misro (1981), f Nagao and Takahashi (1963), g Iwata and
Omura (1971a), h Iwata and Omura (1971b), i Nagasawa et al. (2003), j Greco et al. (1997), k Yamaguchi et al. (2004), l Nonomura et al. (2003),
m Lopez-Dee et al. (1999).
Sp1 Formation of a pair of rudimentary
Formation of two rudimentary glumes in 1/2
Sp2Formation of a pair of empty
Formation of two empty glumes in 1/2 alternate
Sp3Formation of lemma primordium Formation of lemma in 1/2 alternate phyllotaxy.
Elongation of empty glumes
LHS c, RAP1 d
lhs c, bd1 e,
bd2 e, An1 f,
An1 f, An2 f
Sp4Formation of palea primordiumFormation of palea in 1/2 alternate phyllotaxy.
Elongation of empty glumes and lemma
LHS c, RAP1 d
dp1 g, dp2 h
Sp5Formation of lodicule primordia Formation of two lodicules on the lemma side.
Elongation of empty glumes lemma and palea
RAP1 d, SPW1 i,
Sp6Formation of stamen primordiaFormation of six stamen primordia in whorl SPW1 i, OsMADS2 d,
RAG d, OsMADS45 j
Sp7Formation of carpel primordium Formation of carpel. Differentiation of stamen
into filament and anther
RAG d, OsMADS45 j,
Sp8Formation of ovule and pollenDifferentiation of integuments and embryo sac.
MSP1 ll, OsMADS13 m,
Staging of rice development38
Ovule development in rice was described correctly by
Lopez-Dee et al. (1999). They also proposed a useful staging
system for the ovule. Here, we describe rice ovule develop-
ment and staging system with a small modification to that pro-
posed by Lopez-Dee et al. (1999).
Rice has a hemianatropous ovule in the monocarpellary
pistil. The ovule attaches its basal part to the ovary wall on the
palea side; its apical part inclines toward the basal lemma side
(Fig. 8A). The micropyle and egg apparatus are positioned in
the receptacle end on the lemma side. In rice, the funiculus is
vestigial; the chalaza seems to be attached directly to the ovary
wall (Fig. 8A). The inner integument covers most of the nucel-
lus leaving a small pit, a micropyle (Fig. 8B, C). The outer
integument, however, covers only a quarter of the inner integu-
ment on the style side (Fig. 8B).
The floral meristem is converted into an ovule primor-
dium when the carpel primordium formed on the lemma side of
the floral meristem elongates to some extent (Fig. 8F). This
conversion is confirmed by the down-regulation of OSH1
expression and the complementary onset of OsMADS13
expression (Fig. 8D, E), although the ovule primordium is first
identified morphologically when the carpel primordium
becomes ring-shaped (Fig. 8G). Just before the carpel primor-
dium encloses the ovule primordium, a ring-shaped integu-
ment primordium is formed from the base (charaza) of the
ovule (Fig. 8H). At this stage, the archesporial cell (AC) in the
nucellus tip becomes enlarged. Soon after integument primor-
dium differentiation, the carpel completely encloses the ovule
primordium to form an ovary locule. Then, the single integu-
ment primordium divides into inner and outer integuments
(Fig. 8I). The AC elongates and differentiates directly into the
megaspore mother cell.
Although both inner and outer integuments elongate, elon-
gation of the inner integument is remarkable. It envelops most
of the nucellus except for the micropyle area near the
megaspore mother cell (Fig. 8C). The outer integument elon-
gates as long as the inner integument on the receptacle side, but
that elongation is restricted to a quarter of the inner integument
on the style side (Fig. 8B, K). The inner integument is stained
more intensely than the outer integument by hematoxylin or
Ovule development in rice. (A) Longitudinal section of mature semianatropous ovule. (B) SEM image of mature integument. The black
line indicates the boundary between the inner and outer integuments. (C) Close-up image of micropyle region. (D) Down-regulation of OSH1
expression from the floral meristem/ovule primordium when the carpel primordium is formed. (E) Expression of OsMADS13 in the floral meris-
tem/ovule primordium where OSH1 expression is down-regulated. (F) The early stage of the ovule primordium. (G) Ovule primordium just before
integument protrusion. (H) Formation of integument primordium. (I) Differentiation of integument primordium into inner and outer integuments.
(J) Formation of linearly arranged four megaspores (arrowheads). (K) Degeneration of three megaspores and formation of micropyle. The arrow
indicates a functional megaspore and the arrowheads three degenerating megaspores. (L) Formation of the eight-nucleate embryo sac. Four nuclei
exist in each region indicated by an arrowhead. (M) Formation of cellularized embryo sac. NU, nucellus; AN, antipodals; EG, egg cell; II, inner
integument; OI, outer integument; MP, micropyle; CA, carpel; OV, ovule primordium; IN, integuement primordium; MMC, megaspore mother
cell; CC, central cell; PN, polar nucleus; SY, synergid.
Staging of rice development39
toluidine blue. Then, the ovule starts to incline toward the
Concomitant with integument elongation, the polygonum-
type embryo sac is formed in the nucellus. The megaspore
mother cell undergoes meiotic division and the four
megaspores are arranged linearly in the micropyle–chalaza
direction (Fig. 8J). Only the chalazal megaspore remains func-
tional; the other three micropylar spores degenerate (Fig. 8K).
Following meiosis, the functional megaspore undergoes
mitotic nuclear division to become a two-nucleate cell. The
nucellar cells degenerate gradually and the megagametophyte
becomes enlarged. In time, the megagametophyte forms a large
vacuole and each of the two nuclei undergoes a second mitotic
division. Successively, the megagametophyte becomes eight-
nucleated through the third mitotic division.
After mitotic division, the polarization of egg cell, syn-
ergid cells, polar nuclei and antipodal cells occurs (Fig. 8L, M).
The antipodal cells of rice continue to divide and form an
antipodal cell cluster at the chalazal end, as in maize (Huang
and Sheridan 1994). The ovule inclines to form a hemianatro-
pous one. The pistil is enlarged along the style–receptacle axis.
Staging of ovule development—Based on the events de-
scribed above, we propose a staging system of ovule develop-
ment (Table 7). This system is a small modification of that by
Lopez-Dee et al. (1999).
Stage Ov1: Ovule primordium formation. Soon after the
carpel primordium is differentiated morphologically, the
OsMADS13 gene begins to be expressed in the cells where
OSH1 expression becomes down-regulated in the floral meris-
tem (Fig. 8D–F). That expression is the first sign of ovule for-
mation. The ovule primordium becomes obvious: the proximal
end is attached to the palea-side carpel (Fig. 8G).
Stage Ov2: Integument primordium formation. The integ-
ument primordium protrudes from the base of the ovule pri-
mordium (Fig. 8H). The carpel encloses the ovule to form an
ovary locule. The AC becomes enlarged. The rice homolog of
the AINTEGUMENTA gene starts to be expressed in the integu-
ment primordium (Yamaki, S., Ito, M. and Nagato, Y. unpub-
Stage Ov3: Division of integument primordium. The sin-
gle integument primordium divides into inner and outer integu-
ments (Fig. 8I). The AC differentiates into a megaspore mother
cell (Fig. 8I). SUPERWOMAN1/OsMADS16 (Nagasawa et al.
2003) begins to be expressed in the abaxial side of the outer
integument. MULTIPLE SPOROCYTE1 (MSP1) is expressed
Staging of ovule development in rice
a Values are for Taichung 65.
b Lopez-Dee et al. (1999), c Yamaki, S., Ito, M. and Nagato, Y. (unpublished data), d Nagasawa et al. (2003), e Nonomura et al. (2003).
length (cm) a
Ov1Ov 1Ovule primordium differentiation0.3–1 OsMADS13 a
Ov2Ov 2–Ov 3 Integument primordium
differentiation. Ovary locule
formation. AC enlargement
of ANT b
Ov3Ov 2–Ov 3Division of integument primordiumDivision of integument primordium
into inner and outer integuments.
3–5 SPW1 c
Ov4Ov 4Meiosis of MMCIntegument elongation. Micropyle
formation. Meiosis of MMC
Ov5Ov 4Degeneration of three micropylar
Degeneration of three micropylar
Ov6Ov 5First mitotic nuclear division First mitotic nuclear division of
chalazal spore. Onset of ovule
Ov7 Ov 6Second mitotic nuclear division Second mitotic nuclear division.
Vacuole formation in
Ov8Ov 7 Third mitotic nuclear division Third mitotic nuclear division
resulting in eight-nucleate
Ov9Ov 8 Polarization of nucleiPolarization of nuclei. Completion of
Ov10Ov 9MaturationMaturation 18–25
Staging of rice development40
in the whole carpel and ovule (Nonomura et al. 2003). The
msp1 mutant has a large number of female gametocytes.
Stage Ov4: Meiosis of megaspore mother cell. Inner and
outer integuments elongate. Elongation of the outer integu-
ment on the style side is limited. The megaspore mother cell
undergoes meiosis to form four linearly arranged megaspores
Stage Ov5: Degeneration of three micropylar spores. The
integuments complete their elongation and form a micropyle
(Fig. 8K). At this stage, the ovule is anatropous. Three of the
four megaspores degenerate: only the chalazal one develops to
a functional megagametophyte (Fig. 8K).
Stage Ov6: First mitotic nuclear division. The nucellar
cells gradually degrade. The enlarged megagametophyte under-
goes the first nuclear division. The ovule starts inclining
toward the receptacle side.
Stage Ov7: Second mitotic nuclear division. The two
nuclei of the megagametophyte undergo a second nuclear divi-
sion. Degeneration of nucellar cells continues. Ovule inclina-
Stage Ov8: Third mitotic nuclear division. A large central
vacuole is formed in the megagametophyte and the third
nuclear division occurs. Consequently, an eight-nucleated meg-
agametophyte is formed (Fig. 8L). Ovule inclination continues.
Stage Ov9: Migration of nuclei and cellularization. The
eight nuclei migrate to their respective positions, and cellulari-
zation occurs (Fig. 8M).
Stage Ov10: Maturation. This stage marks the comple-
tion of the hemianatropous ovule with embryo sac, comprising
one egg cell, two synergids, one central cell (fusion of two
polar nuclei) and antipodal cells (Fig. 8A).
Anther: Sporogenesis and Gametogenesis
DESCRIPTION OF DEVELOPMENTAL COURSE
In Arabidopsis, many mutants and genes have been
reported as associated with anther development, sporogenesis
and gametogenesis (Scott et al. 2004, McCormick 2004, Yade-
gari and Drews 2004). On the other hand, genetic regulation of
these reproductive pathways remains unclear in rice, despite
the fact that the staging and dissecting reproductive pathway of
rice helps to surmount the reproductive barrier for introgression
of agronomic traits into cultivars from distantly related species.
The stamen initiates in the third whorl of the rice flower.
The anther primordium comprises three layers, designated L1,
L2 and L3 (Satina et al. 1940, Goldberg et al. 1993). In rice the
L1 layer gives rise to the epidermis and the stomium, which
play important roles in anther dehiscence; the L2 layer devel-
ops into the archesporial cells, which are the primordial germ
cells from which pollen and microsporangium differentiate;
and the L3 layer gives rise to the connective cells, vascular
bundles and circular cell cluster adjacent to the stomium
(Raghavan 1988, Nonomura et al. 2003). The connective cells
are positioned between microsporangia and vascular bundles,
and degenerate during gametogenesis
In rice, a few cylindrical rows of hypodermal anther cells
differentiate into ACs in the four corners of a transverse section
(Fig. 9A). The ACs can be distinguished from other cells by a
slightly enlarged nucleus and cytoplasm. After differentiation,
they continue cell divisions to form primary sporogenous cells
(PSCs) and primary parietal cells (PPCs) (Fig. 9B). The PSCs
undergo several mitotic divisions and differentiate into pollen
mother cells (PMCs). The PPCs repeat periclinal divisions and
generate endothecium, a middle layer and a tapetum layer (Fig.
9C). Each of the four-walled layers expands through anticlinal
divisions. The tapetal cells are binucleated and serve as a nurse
tissue to provide nutrition and pollen wall materials for game-
tophytes (reviewed by Scott et al. 2004).
After completion of anther wall formation, the PMCs
undergo pre-meiotic DNA synthesis (pre-meiotic S) and enter
meiosis (Fig. 9D) (Nonomura et al. 2004b). Meiotic events are
described in the following section of this review.
Spores develop into haploid gametophytes (Fig. 9E),
which produce haploid gametes. Angiosperm sporophytes pro-
duce two types of spores—microspores and megaspores—
Sporogenesis and gametogenesis
in rice anther. (A) The ACs differentiate
into the hypodermis of anthers. (B) The
ACs continue periclinal division (arrow)
and develop PSCs and PPCs. (C) Forma-
tion of four-layered anther wall through
periclinal divisions of PSCs. (D) Forma-
tion of PMCs. PMCs enter into meiosis.
(E) Formation of tetrad spores. (F) The
uninucleated microgametophyte becomes
spherical and enlarged. (G) A first pollen
mitosis produces a vegetative nucleus
(arrow) and generative nuclei (arrow-
heads). (H) Mature pollen grain contains
two sperm nuclei (arrowheads), and an
amorphous vegetative nucleus (arrow). EP,
epidermis; EN, endocethium; Ml, middle
layer; TA, tapetum.
Staging of rice development41
which give rise to pollen grain and embryo sac, respectively.
After release from the tetrads, the free microspores become
spherical and enlarged without mitotic cell division (Fig. 9F).
In maize, intine and exine layers, which are components of the
pollen wall, are formed at this stage (Chang and Neuffer 1989).
In most flowers, this uninucleate status of male gametophytes
is retained until the inflorescence heading. Although anther
length is correlated roughly with development of microspor-
angia, most anthers reach their maximum length at the end of
the uninucleate stage (Table 8). The first pollen mitosis pro-
duces a generative nucleus and a vegetative nucleus. The gen-
erative cell subsequently divides into two sperm cells (Fig.
9G). We have observed rice gametogenesis by the chromatin
staining method. Therefore, we use the terms ‘sperm nuclei’
and ‘binucleate stage’ in this study in place of ‘sperm cells’ and
‘bicellular stage’, respectively.
Staging of anther development—Based on the develop-
mental processes described above, we propose a staging sys-
tem for anther development (Table 8).
Stage An1: Formation of hypodermal ACs. In transverse
section, the anther initial is ovoidal; it subsequently becomes
four-cornered in shape. At the four corners, the ACs initiate at
the L2 layer (Fig. 9A).
Stage An2: Formation of anther wall layer. The ACs dif-
ferentiate into PSCs and PPCs; the PPCs continue to divide
periclinally (Fig. 9B). Transcripts of the MSP1 gene, suppress-
ing the AC derivatives entering into sporogenesis, start to accu-
mulate in PPCs and are retained in the developed tapetum, but
not in PSCs and PMCs (Nonomura et al. 2003).
Stage An3: Completion of anther wall layer. Formation of
the four-layered anther wall is completed (Fig. 9C). PSCs
mature into PMCs and undergo pre-meiotic S at this stage
(Nonomura et al. 2004b) (Fig. 9C).
Stage An4: Meiosis. PMCs and a megaspore mother cell
(MMC) enter into meiosis. The PMCs gradually become spher-
ical as a result of callose accumulation in anther locules (Fig.
Stage An5: Formation of tetrad spores. The PMC pro-
duces four haploid spores (Fig. 9E). The tapetal cells are binu-
cleated; the endothecium and middle layer begin to collapse
(Fig. 9E). MSP1 mRNAs in both anthers and ovules has disap-
peared by this stage (Nonomura et al. 2003).
Staging of anther development in rice
a AL: anther length in cv. Nipponbare, b FL: floret length in cv. Nipponbare, c ND: not determined; d Nonomura et al. (2003), e Tsuchiya et al.
(1994), f Zhu et al. (2004), g Lee et al. (2004), h Kazama and Toriyama (2003), i Komori et al. (2004).
* Osc4 e, Osc6 e, AID1 f, OsCP1 g, Rf1 h,i. ** aid1 f oscp1 g, rf1 h,i
ACs differentiation at four
corners of hypodermis of
An2Formation of anther
Periclinal division of
hypodermis to form layered
structure of anther wall
0.15–0.3 0.7–2.0Ov1–Ov2MSP1 d
anther wall layer
Establishment of PMCs and
four-layered anther wall. Pre-
meiotic DNA synthesis in
An4Meiosis(see Table 9)0.45–0.951.6–4.8 Ov5(see Table 10) (see Table 10)
An5Formation of tetrad
Formation of four haploid
spores. Onset of endothecium
and middle layer degradation
Formation of spherical and
Formation of exin, intine and a
pore for pollen tube
1.1–2.25.0–7.0 Ov6–Ov8* **
Formation of vegetative
nucleus and a generative
nucleus by pollen mitosis I
2.2 7.0 Ov9* **
Formation of two sperm nuclei
by pollen mitosis of generative
2.2 7.0 Ov9***
Staging of rice development 42
Stage An6: Formation of uninucleate gametophyte. The
microspores become spherical and enlarged (Fig. 9F), and a
pore for pollen tube germination differentiates. In the 1.6 mm
long anthers, tapetal cells have almost degenerated (Fig. 9F).
At this stage, the expression of Osc4 and Osc6 mRNA is
detected in tapetal cells, although the function of both genes
remains unknown (Tsuchiya et al. 1994). Promoter activity of a
cysteine protease gene OsCP1, whose T-DNA-tagged mutation
affects pollen development, is also found (Lee et al. 2004). The
AID1 (Zhu et al. 2004) and Rf1 (Kazama and Toriyama 2003,
Komori et al. 2004) genes function at this stage, as inferred
from their mutant phenotypes, although they have not yet been
Stage An7: Formation of binucleate gametophyte. First
pollen mitosis produces two nuclei, a vegetative nucleus and a
generative nucleus (Fig. 9G).
Stage An8: Formation of mature pollen. The generative
nucleus successively undergoes a second pollen mitosis. The
mature pollen contains two smaller sperm nuclei, and a larger
but amorphous vegetative nucleus (Fig. 9H).
COURSE OF MEIOSIS
Meiosis is a crucial event for the sexual reproduction of
eukaryotes to form haploid spores and gametes. This event is
characterized by a single round of pre-meiotic S followed by
two rounds of chromosome segregation. The pre-meiotic S is
known to affect meiotic events such as homologous chromo-
some pairing, reductional chromosome segregation and homol-
ogous recombination in wheat (Martinez-Perez et al. 1999),
fission yeast (Watanabe et al. 2001) and Arabidopsis (Mercier
et al. 2003).
Homologous pairing in early meiosis, which plays impor-
tant roles in faithful cell division and homologous recombina-
tion, is divided into several substages based on the state of
synapsis and condensation. Homologous chromosomes begin
to condense at leptotene. They become partially synapsed at
zygotene, become fully synapsed at pachytene, subsequently
becoming desynapsed but held together by chiasmata at diplo-
tene and diakinesis. The synapsis guarantees a strong and close
connection of the homologous pairs, facilitated by a network of
longitudinal and transversal protein fibers called the synaptone-
mal complex (SC) (Moses 1969, Westergaard and Wettstein
1972, Gillies 1975). The function of the SC in the early
prophase remains to be elucidated.
Homologous recombination is also an important event for
generating genetic diversity in offspring. In yeast, recombina-
tion initiation depends on DNA double-strand breaks by the
SPO11 machinery (Bergerat et al. 1997, Keeney et al. 1997),
and on the RAD51 and DMC1 complex, which catalyses
homologous recombination (Masson and West 2001). Many
genes and mutations such as AtSPO11, AtRAD51 and AtDMC1
have been reported in Arabidopsis (reviewed by Caryl et al.
2003). Two DMC1 homologs, OsDMC1A and OsDMC1B, have
been identified in rice, although their functions in meiosis
remain unclear (Kathiresan et al. 2002).
A number of spontaneous and induced synapsis mutations
have also been reported in rice (Kitada et al. 1983, Kitada and
Omura 1983, Nonomura et al. 2004a, Nonomura et al. 2004b),
but the genetic regulation of rice meiosis is poorly understood.
The genus Oryza consists of more than 20 wild and two culti-
vated species. The interspecific F1 hybrids between cultivars
and wild species frequently show high sterility, which is partly
attributable to the low ability of homologous pairing at meiosis
(Katayama 1963, Brar and Khush 1997). Dissecting the meiotic
process of Oryza will contribute to reducing reproductive barri-
ers between different genome species.
Staging of meiosis—The staging of meiosis described here
is based on events in anthers. Zhang and Zhu (1987) have esti-
mated the absolute time course of male meiosis through statis-
tical analysis in rice. In this review, anther length is used
mainly as a parameter to represent the approximate time course
in addition to the floret length because the longitudinal lengths
of anthers and floret are correlated roughly with meiotic stages.
Optical sectioning shows that the progression of female meio-
sis is synchronized loosely with that of male meiosis (Table 9).
Stage Mei1: Pre-meiotic S/G2. Decondensed chromatins
fill up the nucleoplasm (Fig. 10A). Pre-meiotic S occurs syn-
chronously among the PMCs in anthers of 0.3–0.4 mm in
length (Nonomura et al. 2004b). However, it is difficult to dis-
tinguish pre-meiotic S from G2 in meiocytes. Localization of
the PAIR2 protein into the PMC nuclei takes place subsequent
to pre-meiotic S (Nakano et al. unpublished data). Results of
RT–PCR and mutant analysis suggest that expression of PAIR1
mRNA is linked to early meiosis or pre-meiosis (Nonomura et
Stage Mei2: Leptotene. Meiosis-specific chromosome
condensation starts (Fig. 10B). The lateral element (LE) of the
SC is associated with chromosomal axes (Nonomura, K.I.,
Eiguchi, M., Nakano, M., Suzuki, T. and Kurata, N. unpub-
Stage Mei3: Zygotene. Synapsis of homologous chromo-
somes progresses to form central components of the SC
between LEs of homologous chromosome axes (Fig. 10C).
Chromosomes transiently become a compact sphere and adhere
to the nucleolus at early zygotene to form a so-called synizetic
knot (Nonomura et al. 2004b). The importance of the synizetic
knot, however, is poorly understood.
Stage Mei4: Pachytene. Homologous chromosome synap-
sis is completed, and the paired chromosomes are thickened
(Fig. 10D). Homologous recombination is inferred to occur at
around this stage.
Stage Mei5: Diplotene. Because of the rapid removal of
SCs from most chromosomal regions, homologous chromo-
somes are separated from each other, while pairing is main-
tained at the chiasmata (Fig. 10E).
Staging of rice development43
Stage Mei6: Diakinesis. Chromosome pairs are highly
condensed (Fig. 10F) and attached closely to the nuclear enve-
lope. The nucleolus begins to disappear.
Stage Mei7: Metaphase I. The nuclear envelope is broken
down. Bipolar kinetochores of homologous chromosome pairs
are captured by microtubule bundles of bipolar spindles; the
captured pairs align on the equatorial plate (Fig. 10G). The
nucleolus disappears completely.
Stage Mei8: Anaphase I/telophase I. Homologous chro-
mosomes separate and move to opposite poles (reductional
division) (Fig. 10H). At the end of this stage, a phragmoplast, a
cytoskeletal structure held by two arrays of microtubule bun-
dles, determines the position of the cytokinetic plate (Non-
omura et al. 2004a).
Stage Mei9: Interkinesis (prophase II). Two daughter cells
and nuclei are reconstructed. Small nucleoli appear with con-
densed chromosomes (Fig. 10I).
Stage Mei10: Metaphase II. Chromosomes align on the
equatorial plate of each daughter cell (Fig. 10J). The nucleoli
Stage Mei11: Anaphase II/telophase II. Sister chromatids
of chromosomes separate and move to opposite poles (equa-
tional division) (Fig. 10K).
Stage Mei12: Tetrad. Meiotic cell division is completed,
producing four haploid spores (Fig. 10L).
DESCRIPTION OF DEVELOPMENTAL COURSE
Stomata are cell complexes that are specialized for gas
exchange between the leaf and its environment. Stomata in rice
are distributed in vertical rows on the leaf surface (Fig. 11A),
but those on the adaxial surface of the leaf sheath are rudimen-
tary. Cell rows in which stomata are to be formed later are
called stomatal cell rows. The distribution of stomatal cell rows
in the epidermis is not random. In rice, the stomatal cell rows
are located on the flanks of vascular bundles. The rows are usu-
ally separated, but are sometimes formed in two adjacent files.
Stomata in rice consist of two guard cells that are narrow and
have thickened walls and two subsidiary cells flanking the
Development of stomata in rice has been well described
by several authors (Kaufman 1959a, Hoshikawa 1989). There-
fore, we briefly summarize the course while incorporating
recent results. At the P3 stage of leaf development, the stomatal
cell row is determined basipetally on the leaf epidermis (Fig.
11B). Symmetric transverse cell divisions in the stomatal cell
Staging of meiosis in rice anther
a AL: anther length in cv. Nipponbare, b FL: floret length in cv. Nipponbare, c ND: not determined.
d Nonomura et al. (2004a), e Nonomura et al. (2004b), f Katayama (1963), g Chao and Hu (1960), h Kitada and Omura (1983).
Events in PMC AL a (mm)
Mei1 Pre-meiotic S/G2Pre-meiosis specific DNA synthesis0.02–0.450.9–1.6 ND c
Mei2LeptoteneInitiation of meiotic chromosome
condensation. Association of LEs to
Mei3 Zygotene Establishment of central components
between homologous chromosome axes
0.45–0.65 1.9–2.4NDpair1 d,
pair2 e, as f
Mei4PachyteneCompletion of SC formation. Homologous
0.60–0.80 2.3–3.4Zygotene to
ds2~d, s11 h
Mei5Diplotene Degradation of SC 0.75–0.85 2.8–3.4ND
Mei6 Diakinesis Breakdown of nuclear envelope.
Disappearance of a nucleolus
0.70–0.90 2.9–3.4Pachytene to
Mei7Metaphase I Alignment of homologous chromosome
pairs at equatorial plate
Reductional division of each homolog 0.80–0.90 4.0–4.8ND
Formation of daughter cells and nuclei 0.80–0.90 4.0–4.8ND
Mei10Metaphase II Alignment of sister-chromatid pairs at
Equational division of each sister
Mei12 TetradFormation of four haploid spores 0.80–1.10 4.0–5.0Interkinesis
Staging of rice development44
row produce numerous cells smaller than those on either side
of the row. The first asymmetric divisions produce guard
mother cells (GMCs) and non-specialized epidermal cells (Fig.
11C). The GMC is a small cell that is stained strongly; the non-
specialized epidermal cell is a large and weakly stained cell.
The second asymmetric divisions occur in the two lateral epi-
dermal cells (subsidiary mother cells, SMCs) adjacent to the
GMC to form subsidiary cells, resulting in a three-cell com-
plex (GMC + two subsidiary cells) (Fig. 11D). Finally, trans-
verse symmetric divisions in GMC produce a guard cell pair
(Fig. 11E). At maturity, the guard cells elongate and become
dumb bell-shaped, while the subsidiary cells become ellipsoi-
dal (Fig. 11F).
Staging of stomata development—Staging of stomata for-
mation in rice was first proposed by Stebbins and Shah (1960).
Kamiya et al. (2003a) subsequently described the staging in
detail. According to Kamiya et al. (2003a), stomata formation
is divided into six stages. Their staging system is based on the
cell division pattern and the expression pattern of the OsSCR
gene. The SCR gene was first reported in Arabidopsis as regu-
lating asymmetric division of the cortex/endodermis in root.
SCR expression is restricted to the quiescent center and the
endodermal cell layer in root and some kinds of cell layers in
above-ground tissue (Di Laurenzio et al. 1996, Wysocka-Diller
et al. 2000). OsSCR is thought to be an orthologous gene of
SCR. The expression pattern in the root is conserved (Kamiya
et al. 2003a). Notwithstanding, OsSCR expression in rice
leaves is different from that in Arabidopsis. The expression of
OsSCR in leaves is correlated with stomatal development
(Kamiya et al. 2003a). For that reason, the profile of OsSCR
expression is a useful marker for stomata development and epi-
The staging suggested by Kamiya et al. (2003a) is reason-
able in most respects, but their division of the subsidiary cell
formation process into two stages (stage 2 and stage 3) is not
explicit. Asymmetric division of the SMC and the formation of
the three-cell complex are events in a single continuous proc-
ess. Therefore, we unified their stage 2 and stage 3 as a single
stage. In this review, we divide stomatal development into five
stages (Table 10).
Stage Sto0: Determination of stomatal cell row. Cell rows
(stomatal cell row) where stomata are to be formed later are
determined (Fig. 11B), but stomata-specific cells are not yet
Meiosis in rice anther. (A) Pre-meiotic stage. Chro-
mosomes are stained with 4,6-diamidino-2-phenylindole
(magenta). (B) Leptotene stage. (C) Zygotene stage. (D)
Pachytene stage. (E) Diplotene stage. (F) Diakinesis stage. (G)
Metaphase I. Spindle fibers (green) capture and align homolo-
gous chromosome pairs (magenta) at metaphase I plate. (H)
Anaphase/telophase I. (I) Interkinesis. (J) Metaphase II. (K)
Anaphase II/telophase II. (L) Tetrad stage.
Staging of rice development 45
specified. OsSCR is specifically and uniformly expressed in the
stomatal cell rows (Fig. 11G).
Stage Sto1: Formation of GMC. Asymmetric cell divi-
sions in the potential stomatal cell row produce GMCs (Fig.
11C). OsSCR expression is maintained in the GMC but is
down-regulated in other epidermal cells of the stomatal cell
row (Fig. 11H). Polarized expression of OsSCR is observed on
the GMC side of SMCs at the later stage of Sto1 (Fig. 11I).
Stage Sto2: Formation of three-cell complex. A pair of
subsidiary cells is formed by asymmetric divisions of the two
SMCs (Fig. 11D). As a result, a three-cell complex comprising
a GMC and two subsidiary cells is formed. OsSCR is expressed
in both the GMC and the two subsidiary cells (Fig. 11J).
Stage Sto3: Formation of guard cell pair. Transverse sym-
metric divisions of GMC produce a guard cell pair (Fig. 11E).
At this stage, the level of OsSCR expression decreases rapidly
Stage Sto4: Completion of stomatal complex. The forma-
tion of the stomatal complex, comprising two narrow guard
cells and two large subsidiary cells is completed (Fig. 11F).
Staging of stomatal development in rice
Events OsSCR expression
Sto0Determination of stomatal cell
Determination of stomatal cell rowsUniform expression in the stomatal cell row
Sto1Formation of GMC Formation of GMC by asymmetric
division in the stomatal cell row
Down-regulation in non-stomata-forming
cells in the stomatal cell row
Sto2 Formation of three-cell
Asymmetric division of SMC. Formation
of three-cell complex comprising GMC
and two subsidiary cells
Localized expression in the SMC adjacent to
GMC. Cell specific expression in GMC and
Sto3 Formation of guard cell pairFormation of a pair of guard cells by
transverse symmetric divisions in GMC
Sto4 Completion of stomatal
Completion of stomatal complex
Development of stomatal complex in rice. (A) Mature stomata in leaf blade. Arrows indicate guard cells and arrowheads subsidiary
cells. (B–F) Longitudinal sections of developing stomatal complex stained with Toluidine Blue-O. (B) Stomatal cell row (arrow). (C) Production
of GMC (arrow) and non-specialized cell (arrowhead) by asymmetric division. (D) Asymmetric division of the SMC (arrowheads) and the forma-
tion of a three-cell complex consisting of two subsidiary cells (arrow) and the GMC. (E) Formation of guard cell pair by transverse symmetric
division (arrows). (F) Mature stomatal complex (arrow). (G–K) Expression pattern of OsSCR. (G) Uniform expression in the stomatal cell row
(arrow). (H) Down-regulation in the non-specialized epidermal cell (arrow), with strong expression in the GMC (arrowhead). (I) Polarized expres-
sion on the GMC side of the SMC (arrowheads). (J) Expression in both the GMC and two subsidiary cells (arrows). (K) No expression in mature
stomatal complex (arrowhead).
Staging of rice development46
This review has explicitly described a number of develop-
mental processes that drive the life cycle of rice. The staging
systems proposed here can be used in the description of mutant
phenotypes and interpretation of gene expression and other bio-
logical aspects, as well as in establishing a conceptual frame-
work of rice development. The rice plant is becoming an
important model monocot plant and model cereal crop. For
those reasons, elucidation of rice plant development contributes
to the advancement of comparative biological studies regard-
ing grasses (cereals) and between monocots and dicots. Many
other processes that are not mentioned in this review also exist:
endosperm development, vascular formation, stem develop-
ment, air space formation in roots and leaf sheath (adaptation
for anaerobic conditions) and others. These processes have not
been examined sufficiently, but are expected to be understood
soon. In addition, genes associated with rice development are
being revealed rapidly. Thus, we expect that the staging sys-
tems described here will be refined in the future using new
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