Anther, ovule, seed, and nucellar embryo
development in Citrus sinensis cv. Valencia
Anna M. Koltunow, Kathleen Soltys, Nobumasa Nito,
and Stuart McClure
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Abstract: 'Valencia' orange, a commercially important cultivar of Citrus, forms polyembryonic seeds
by an apomictic process called nucellar embryony in which many embryos initiate directly from nucellar
cells surrounding the sexual embryo sac. We observed anther, ovule, seed, and fruit development in
relation to nucellar embryo development in seeds and unfertilized ovules of 'Valencia'. Pollination and
fertilization are required to set fruit in 'Valencia', and low seed set was found to be related to defects in
both male and female gametogenesis. Nucellar embryo initial cells were evident histologically in ovules
of flowers just prior to anthesis. However, in vitro culture of ovules from flowers at different
prepollination stages showed that embryos could develop from ovules cultured as early as the binucleate
stage of megagametogenesis in which nucellar initial cells were absent histologically. During fruit
development, the timing and sequence of the early events of nucellar embryo formation were
synchronous in seeds and unfertilized ovules, indicating a co-ordinated control of embryo development
in spatially and developmentally distinct structures. In both developing seeds and unfertilized ovules,
embryo initial cells first formed thick walls, which isolated them from surrounding maternal tissue. In
later stages, the cell walls thinned in some initial cells and embryogenesis became asynchronous.
Cleavage of embryogenic cells coincided with degenerative processes linked to embryo sac expansion in
seeds and to a previously unreported, localized degeneration in the central portion of the nucellus in
unfertilized ovules. Some initial cells never divided. Nucellar embryo development was restricted to the
central portion of unfertilized ovules and to the micropylar region of seeds. Only fertilized ovules had
the capacity to form mature polyembryonic seeds. In unfertilized ovules a specialized vascular structure
formed linking developing embryos to the chalaza1 vasculature of the ovule. Embryo development
arrested at the globular stage in unfertilized ovules and the integuments differentiated to form a seed
coat. The timing of reproductive events described was linked to floral and fruit morphological
characteristics to facilitate molecular characterization of nucellar embryogenesis and seed formation in
this cultivar.
Key words: Citrus, nucellar embryony, seed, ovule, apomixis.
R6sum6 : L'orange de 'Valencia', un important cultivar commercial de Citrus, forme des graines
polyembryoniques par un processus apomictique appelt embryonie nucellaire, dans lequel la formation
de plusieurs embryons dtbute directement a partir de cellules nucellaires entourant le sac embryonnaire.
Les auteurs ont observt le dtveloppement de l'anthere, de l'ovule, de la graine et du fruit en relation
avec le dtveloppement d'embryons nucellaires dans les graines et les ovules non fertilistes du
C.V. Valencia. La pollinisation et la fertilisation sont necessaires pour la mise a fruit chez ce cultivar et
les petits nombres de graines formtes sont relits 2 des manquements de la gamttogtnbse mile aussi
bien que femelle. L'observation histologique dtmontre la prtsence de cellules initiales d'embryons
nucellaires dans les ovules des fleurs juste avant l'anthbse. Cependant, des cultures in vitro d'owles
provenant de fleurs a difftrents stades de pollinisation, montrent que les embryons peuvent se
dtvelopper aussi t6t que le stade binuclte de la mtgagamCtogCnbse, oh les cellules initiales nucellaires
sont histologiquement absentes. Au cours du dCveloppement du fruit, le moment et la stquence des
premiers Cvbnements de la formation des embryons nucellaires est synchrone chez les graines et chez les
ovules non fertilisees, indiquant un contr6le co-ordonne du dtveloppement embryonnaire dans des
structures distinctes par leur localisation et leur dtveloppement. Chez les graines en developpement aussi
bien que chez les ovules non fertilistes, les cellules initiales des embryons forment d'abord une paroi
I Received November 2, 1994.
I1
A.M. Koltunowl and K. Soltys. C.S.I.R.O., Division of Horticulture, G.P.O. Box 350, Adelaide, South Australia 5001, Australia.
N. Nito. Department of Agriculture, University of Saga, Saga, 840 Japan.
S. McClure. C.S.I.R.O., Division of Soils, Private Bag No. 2, Glen Osmond, South Australia 5064, Australia.
Author to whom all correspondence should be addressed
Can. J. Bot. 73: 1567- 1582 (1995). Printed in Canada 1 ImprimC au Canada
Can. J. Bot. Vol. 73, 1995
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tpaisse, laquelle les isole des tissus maternels ambiants. Aux stades plus avancCs, les parois cellulaires
s'amincissent chez certaines cellules initiales et 1'embryogCnkse devient asynchrone. Le clivage des
cellules embryogknes coi'ncide avec des processus de dCgCnCrescence lies B l'expansion du sac
embryonnaire dans les graines, et i une dCgCnCrescence jamais mentionnke, 1ocalisCe dans la partie
centrale du nucelle des ovules non fertilisies. Certaines cellules initiales ne se divisent jamais. Le
dCveloppement des embryons nucellaires est restreint i la partie centrale des ovules non fertilisCes et i
la region micropylaire des graines. Seules les ovules fertilisCes ont la capacitC de former des graines
polyembryoniques matures. Chez les ovules non fertilisCes, une structure vasculaire spCcialisCe se forme
et relie l'embryon en dCveloppement au systkme vasculaire chalazale de I'ovule. Le dCveloppement de
l'embryon s'arrete au stade globulaire chez les ovules non fertiliskes et les tCguments internes se
dCveloppent pour former une enveloppe sCminale. La chronologie des Cvknements de la reproduction
dCcrits a CtC reliCe aux caracteristiques morphologiques de la fleur et du fruit, afin de faciliter la
caractkrisation molCculaire de 1'embryogCnkse nucellaire et la formation de la graine chez ce cultivar.
Mots cle's : Citrus, embryonie nucellaire, graine, ovule, apomixie.
[Traduit par la redaction]
Introduction
Developmental studies of floral initiation, floral organ formation, and fruit and seed development in Citrus have been
made with varying degree of detail. Given the commercial
interest in Citrus fruit and juice, many of the earlier studies
focussed on the development of the fruit itself. For example,
the study by Bain (1958) provides a detailed account correlating changes in the morphology, anatomy, and composition of
'Valencia' fruit during its development on the tree. Studies
on floral and inflorescence ontogeny in sweet orange have
been made recently (Lord and Eckhard 1985; Plummer
1987) and these complement the more general (Davenport
1990) and in some cases, limited, histological studies on
floral development (Schneider 1968) and formation of the
male and female reproductive organs and gametophytes
(Osawa 1912; Frost and Soost 1968). Descriptions of seed
development in vivo in Citrus at the histological level have
not been extensive and have concentrated only in part on the
development of nucellar embry 0s.
In Citrus, nucellar embryos develop from the maternal
nucellar tissue of the ovule surrounding the sexual embryo
sac and have the same genetic constitution as the female
plant. Many nucellar embryos can develop alongside the
zygotic embryo in an individual seed in certain Citrus species
that are classified as polyembryonic (Bruck and Walker
1985; Cameron and Frost 1968; Kobayashi et al. 1979,
1981; Wakana and Uemoto 1987, 1988). Nucellar seedlings
have been ex~loitedas sources of material for rootstocks in
Citrus propagation because of their uniform genetic composition but are a nuisance in Citrus breeding programs because
it is often difficult to identify zygotic seedlings (Frost and
Soost 1968).
Nucellar embryony is one of many apomictic reproductive
pathways. In recent reviews, apomixis has been described in
detail as a widely variant phenomenon in angiosperms
(Asker and Jerling 1992) and has been considered from a
developmental and molecular viewpoint in comparison with
sexual reproduction (Koltunow 1993). Other apomictic pathways are more complex than nucellar embryony and they
involve the formation of an embryo sac without meiosis. In
all apomictic pathways, however, the male gametophyte
makes no contribution to the genetic constitution of the
embryo. Apomictic processes eliminate the need for events
considered essential for the formation of a seed because
meiosis is uncoupled from female gametophyte development
and double fertilization is uncoupled from embryo and endosperm development (Koltunow 1993).
The existence of nucellar embryony raises questions about
how reproductive developmental events are regulated spatially and temporally in the ovule and how certain cells in the
nucellus, a structure destined to be degraded during seed
development, are directed onto an embryogenic developmental pathway in the absence of fertilization. Understanding the sequence of developmental events during nucellar
embryogenesis forms a foundation for addressing such questions at the molecular level.
Our present interest in understanding the molecular basis
for the formation of nucellar embryos and our aim of controlling seed formation in Citrus by gene introduction via
plant transformation warranted a fresh look at the morphological events of Citrus floral organ formation and seed
development. In this study, we observed these processes in
'Valencia', a commercially important, polyembryonic cultivar. We divided flower formation and fruit development in
'Valencia' into distinct stages on the basis of size and
morphological criteria and observed the processes of anther,
ovule, and zygotic and nucellar seed development with scanning electron and light microscopy. Nucellar embryo development was also described histologically in fertilized and
unfertilized ovules at sequential stages of 'Valencia' fruit
development in a detailed study of the developmental
sequence and synchrony of these events in a single cultivar.
Materials and methods
Histology
Plant material was harvested from eight different orchard
grown Citrus sinensis cv. Valencia trees grafted onto Troyer
citrange rootstock. Trees were from 8 to 16 years old. Collected material was measured and in the case of fruit, also
weighed. Floral tissue and seeds extracted from fruits were
either fixed whole or dissected prior to fixation in 4 % formaldehyde, 0.25 % glutaraldehyde in 50 mM PIPES pH 7,
and then dehydrated in ethanol as described by McFadden
et al. (1988). For scanning electron microscopy the tissue
was dehydrated to 100% ethanol, dissected if necessary to
expose floral organs, critical point dried, sputter-coated with
gold, and observed with a Cambridge stereoscan 250 MK3
scanning electron microscope (SEM) operated at 20 kV. For
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Fig. 1. Photographic representation of vegetative and inflorescence shoot types A-E of 'Valencia' as described
by Sauer (1954). Scale bar = 50 mm separated into 10-mm lengths.
sectioning, the tissue was dehydrated to 70% ethanol and
embedded in LR Gold resin as described by McFadden et al.
(1988), serially sectioned at 2 pm using a Reichert Jung
microtome, stained in 0.1 % alkaline toluidine blue (O'Brien
and McCully 1981), and photographed under bright field or
Nomarski optics using a Zeiss Axioplan microscope. Developing seeds and unfertilized ovules extracted from various
fruit stages were measured for length and width once they
were fixed and infiltrated in 100% LR Gold resin using a
Zeiss dissecting microscope fitted with an eyepiece graticule.
Observations and data reported are based on the information
collected over two growing seasons (1991 and 1992).
Culture of Citrus ovules of various developmental stages
Ovules were excised aseptically from the pistils of various
'Valencia' floral bud stages. Pistils were surface sterilized in
70% ethanol for 5 min, 100% White King (5% available
sodium hypochlorite) for 5 min, and washed in five changes
of sterile water. Excised ovules were then cultured on the
medium described by Moore (1985). A total of 100 ovules
were cultured per floral stage. In addition, 200 unfertilized
ovules were extracted from sterilized mature 'Valencia'
fruits and cultured as described by Moore (1985). As a
control, 200 unfertilized ovules were extracted from mature
f ~ ioft the monoembryonic cultivar Ellendale tangor (C. sinensis X Citrus reticulata).
Results
Floral development
Citrus is a day-neutral, woody perennial genus. In contrast
with herbaceous annuals, flowering in Citrus plants derived
from seed only occurs after a very prolonged juvenile period
(5 - 10 years). Once a plant reaches maturity, only some of
the apices flower (Figs. 1A- ID); other apices are committed to a vegetative mode of growth that ailows continued
growth of the tree (Fig. 1E). It is difficult to predict, therefore, which bud-bearing shoots will produce flowers. The
morphology of floral shoots is not constant and Sauer (1954)
described four distinct floral shoot types in 'Valencia' that
are shown in Figs. 1A- ID. Floral development is not even
uniform down a floral shoot. Lord and Eckard (1985)
showed that in 'Valencia' floral shoots of the type shown in
Fig. 1B (left), development of the subapical bud (arrow) is
delayed relative to the tertiary bud in the shoot. For these
reasons, preliminary experiments in which flower buds were
tagged did not always yield a homogeneous sample representing a particular stage of reproductive organ development. The variable time of development of individual
flowers, coupled with seasonal variations in length of time
taken for flowers to reach anthesis, made it necessary to
choose a more reliable marker for floral development. We
observed that bud size, irrespective of position on the floral
shoot and the time of tree flowering, was a suitable reproducible marker for a particular developmental stage of male and
female reproductive organ development. Our general observations of 'Valencia' reproductive organs and seed structure
were collated with those of others and are summarized in
Table 1. The four floral bud developmental stages, A to D,
that we chose for histological analysis are described in detail
in Table 2.
Citrus floral organs, i.e., sepals, petals, stamens, and the
ovary locules that give rise to the fruit segments, are initiated
acropetally. Floral organ development has quite advanced
Can. J. Bot. Vol. 73, 1995
Table 1. Diagnostic characteristics of reproductive organs and seeds of 'Valencia'.
Structure
Calyx-corolla
Stamen-pollen
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Ovary-ovules
Stigma-style*
Embryo-seed
Features
Five distinct white petals alternate with five sepals; occasionally, four petal-sepal
combinations are found
Twenty stamens with white filaments and yellow, four-lobed anthers; anther walls
have persistent epidermis, fibrous endothecium, and two to three ephemeral
middle layers; tapetum glandular and multinucleate; meiotic divisions in
microspore mother cells are simultaneous with tetrads predominantly tetrahedral;
irregular-sized grains indicative of microspore DNA content irregularities; pollen
shed at two-cell stage; exine is reticulate and apertures colporate
Ovary contains on average 10 fused carpels or locules, progenitors of fruit
segments; ovules number four to five per locule and are anatropous, suspended,
bitegmic, and crassinucellate; micropyle formed by both integuments; outer
integument three to six cells thick, and inner integument two to five cells thick;
nucellus massive; embryo sac of polygonum type; high degree of sterility with
collapsed or absent embryo sacs at anthesis
Stigma spherical at top of style; stylar canals open on stigma surface; epidermal
stigma cells give rise to papillae that secrete exudate; style cylindrical, has about
10 slit-shaped stylar canals that open into locules; stigma receptive to pollen from
1.to 3 days prior to anthesis and 6 to 8 days postanthesis
Seed endospermic, embryogeny a result of random unpredictable divisions;t
endosperm development of nuclear-type, centripetal wall formation begins at
micropylar end; nucellar polyembryony occurs; seed has a double seed coat; outer
coat derived from outer integument and comprises an exotestal palisade composed
of fibrous cells with a mucilaginous outer wall and mesotestal cell layers; inner
seed coat very compact, consisting of the remnants of inner integument, nucellus,
and endosperm
Note: Data are taken from observations in this study and comparison with descriptors used by Tisserat et al.
(1990) and Johri et al. (1992).
*Data from Frost and Soost (1968).
t
~ and ~Walker
~ (1985).
k
by stage A (Fig. 2) where the 20-30 anthers were well developed (Fig. 3) and the microspore mother cells were undergoing meiosis in the four locules of the anther (Figs. 4, 5).
The ovules were primordia at this stage (Figs. 6 , 7). At
stage B (Figs. 8, 9), the microspores had separated in the
anther (Figs. 10, 11) and a megaspore mother cell had differentiated in some of the ovules (Figs. 12, 13). Stage C was
characterized by bud length (Figs. 14, 15) and by the fact
that tapetal degeneration and some abnormal pollen were
evident (Figs. 16, 17). In ovules at stage C, the outer integument had completed development (Fig. 18) and a small proportion of the ovules were undergoing megagametogenesis
with the developing embryo sacs containing two nuclei
(Fig. 19). At stage D, the five petals opened easily with
externally applied pressure, exposing the anthers, some of
which had begun to dehisce (Fig. 20), and the pistil
(Figs. 20, 21). Pollen maturation was complete (Figs. 22,
23) and a small proportion of the ovules (Fig. 24) contained
embryo sacs with four nuclei (Fig. 25).
In the majority of ovules observed, megagametogenesis
was defective, and most ovules were sterile and lacked an
embryo sac. Furthermore, a high percentage of pollen in
'Valencia' also had characteristics indicative of developmental abnormalities. For example, developing pollen grains
were observed to vary in size, with very small to giant grains
evident (Figs. 17, 23). Some grains were empty (Fig. 17)
and crushed (Fig. 23). Figure 26 shows the elongated structure of a field of normal, mature, dry 'Valencia' pollen that
rounds up on hydration as shown in Fig. 27, while the
aberrant pollen grains remain shrunken and crushed in
appearance. These observations suggest that defects in
gametogenesis are likely to contribute to the low levels of
seed set commonly observed in this Citrus cultivar.
Nucellar embryo initiation during 'Valencia' flower
development
Nucellar embryos of Citrus have been shown to initiate from
individual cells in the nucellus of the ovule, which is the tissue that surrounds the developing embryo sac. Nucellar cells
initiating an embryogenic developmental pathway have been
termed adventive or nucellar embryo initial cells (Kobayashi
et al. 1979, 1981; Wilms et al. 1983) and are first morphologically distinguishable by their large, deeply staining nucleus
and granular, deeply staining cytoplasm (Kobayashi et al.
1979; Naumova 1993). Using these histological criteria, we
first observed nucellar embryo initial cells histologically in
the nucellus of ovules obtained from 'Valencia' flowers at
around anthesis (stage D, Figs. 20-25). The nucellar initial
cells (Fig. 28) were not plentiful and averaged at most two
to three per ovule.
Biochemical changes probably precede histological morphogenesis; therefore, we wanted to assess more carefully
at what stage of ovule development nucellar cells may
become committed to embryogenic development. We cdltured ovules excised from floral bud stages B (Fig. 121, C
(Fig. 18), and D (Fig. 24) in vitro on simple hormone-free
Koltunow et al.
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Table 2. Morphological characteristics of floral stages A-D.
Stage
Bud length
(mm)*
A
2.5
B
5
C
10
D
13- 15
Bud morphology
Stamen development
Ovule development
Ovule primordia present (Figs. 6, 7)
Sepals parted slightly and expose dome Anther tips level with stigma tip
(Fig. 3), but filament is very
of petals; sepal tips level with petal
and average 4 - 5 per locule
short; all cell layers of anther
dome (Fig. 2)
formed (Fig. 4); microspore mother
cells in meiosis (Fig. 5)
Anther tips slightly shorter than
Inner and outer ovule integuments well
Petals emerged from sepals; tips of
initiated and these enclose half of the
stigma (Fig. 9); microspores
sepals midway between petal base
separated (Fig. 10); tapetum intact
nucellus (Fig. 12); megaspore
and tip (Fig. 8)
and some pollen grains appear empty
mother cell evident in some ovules
(Fig. 11)
(Fig. 13)
Filament elongation very evident
Outer integuments fully enclose
Petals almost fully extended;
nucellus and the inner integument
(Fig. 15); tapetum of anther almost
separation between petals not
that has not fully joined (Fig. 18);
fully degraded (Figs. 16, 17); pollen
apparent (Fig. 14)
grains binucleate; signs of abnormal
normal megagametophy te
pollen development with a number
development difficult to observe;
of empty and variable sized grains
in embryo sacs first mitotic division
(Figs. 16, 17)
completed and two nuclei evident
(Fig. 19)
Flower just prior to opening;
Some anthers have dehisced, others
Inner integument formation complete;
not yet opened (Fig. 20); pollen
separation between petals obvious;
many ovules sterile; others have
mature, high percentage of
embryo sacs with four nuclei
petals spring open if bud gently
nonviable grains (Figs. 16, 17)
(completed second mitotic division
touched (Fig. 20)
(Fig. 25))
*Measured from rounded base of sepal to petal tip.
medium (Moore 1985) to assess the capacity for embryo
development of these ovule developmental stages. A total of
100 ovules were cultured per stage and after a period of 4
months, three of the stage C ovules developed a mass of
green embryos while 16 of the stage D ovules formed similar
masses of embryos. Embryos were not formed from the stage
B ovules that were at a very early stage of ovule development
in which integument formation was incomplete (Fig. 12),
and all of these ovules either underwent necrosis or desiccation. These results show that in 'Valencia', ovular cells have
the capacity to differentiate embryos as early as the binucleate stage of megagametogenesis, in which histodifferentiation of nucellar embryo initial cells is not yet apparent.
Seed development in 'Valencia'
'Valencia' is a weakly parthenocarpic cultivar and needs
pollination and fertilization to set fruit or the flowers abscise
(Sauer 1954). Although 'Valencia' pistils contain around 50
or so ovules (Table 1): not all of them were observed to form
mature seeds, and a high proportion of ovules persisted and
remained very small compared with developing seeds. We
termed these ovules unfertilized because they often did not
contain an embryo sac at anthesis and (or) embryo sac
expansion never occurred. We extracted all the seeds and
unfertilized ovules found in fruit from the stages of fruit
development labelled E-R shown in Fig. 29. The dimensions of fruit, seeds, and ovules collected are shown in
Fig. 30, graphs A-C, respectively. Physiological changes
in 'Valencia' fruit development were studied previously and
divided by Bain (1958) into three distinct phases of development. Our fruit stages E -J span phase I, the tissue differentiation phase (Fig. 30; Bain 1958). Stages K-Q span
phase 11, the cell expansion phase (Fig. 30; Bain 1958),
while stage R is the only representative of phase 111, the fruit
ripening phase and is a ripe, mature 'Valencia' fruit at the
end of fruit development (Fig. 30; Bain 1958).
Changes in seed and unfertilized ovule dimensions from
the different fruit stages are compared in Figs. 30B and 30C,
respectively. Both fertilized and unfertilized ovules increased
in size during fruit development. During seed development,
the ovule increased in size approximately 13-fold (Fig. 30B),
while an unfertilized ovule was observed to almost double in
size (Fig. 30C).
Morphological characteristics of seed and unfertilized
ovule development during the course of fruit maturation
were studied by scanning electron microscopy. Unfertilized
ovules maintained a constant size and general appearance
from anthesis to stage L of fruit development. At stage M,
a change in unfertilized ovule morphology was observed.
The surface cells changed from a smooth, regular pattern
observed in stage G fruit ovules (Fig. 3 1) to a more crushed,
roughened appearance at stage M (Fig. 32). An aril-like
structure also began to form at this stage and there was a
noticeable swelling in the midregion of many ovules from
M-stage fruits (Fig. 32). These characteristics became more
exaggerated in many of the unfertilized ovules as fruit development proceeded (Fig. 33). Ovules from stage R fruit were
desiccated and were of variable size as a result of variation
in aril length (compare Figs. 34, 35) and in the width of the
swelling in the ovule midregion (Fig. 34) containing the
undeveloped embryos (Fig. 35).
By contrast, fertilized seeds were distinguishable by eye
from the unfertilized ovules by their comparative size at fruit
stage G (Fig. 30). Although they had increased in size, they
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ABBREVIATIONS:
A, anther; C, chalaza1 end; CO, connective; E, embryo; EN, endosperm; ES, embryo sac; EO, endothecium;
EP, exotestal palisade; F, filament; 11, inner integument; L, locule; M, micropyle; MT, mesotesta; MY, meiocytes (pollen);
MMC, megaspore mother cell; N, nucellus; NE, nucellar embryo; 0 , ovary, 01, outer integument; OV, ovule; OVP, ovule
primordium; P, petal; PO, pollen grain; PW, placental wall; S, stigma; SE sepal; SEM, scanning electron micrograph; ST, style;
T, tapetum; V, vascular network; ZE, zygotic embryo.
Figs. 2-25. SEMs and light micrographs showing development of floral organs and gametophytes in Valencia. Fig. 2. SEM of
stage A floral bud. Scale bar = 1 mm. Fig. 3. SEM of dissected stage A bud. Scale bar = 1 mm. Fig. 4. Cross section of anther
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from stage A bud. Scale bar = 100 pm. Fig. 5. Section through anther locule from stage A bud showing pollen meiocytes.
Nomarski optics. Scale bar = 10 pm. Fig. 6. SEM of ovule primordia from stage A bud. Scale bar = 40 pm. Fig. 7. Longitudinal
section through ovule primordium from stage A bud. Scale bar = 20 pm. Fig. 8. SEM of stage B floral bud. Scale bar = 1 mrn.
Fig. 9. SEM of dissected stage B bud. Scale bar = I mm. Fig. 10. Cross section of anther from stage B bud. Scale bar = 100 pm.
Fig. 11. Cross section through anther locule from stage B bud showing intact tapetum and pollen grains. Scale bar = 10 pm.
Fig. 12. SEM of developing ovule from stage B bud. Scale bar = 40 pm. Fig. 13. Longitudinal section through ovule from stage B
bud showing megaspore mother cell (outlined) in nucellus and incomplete integument formation. Nomarski optics. Scale bar =
20 pm. Fig. 14. SEM of stage C floral bud. Scale bar = 1 mm. Fig. 15. SEM of dissected stage C bud. Scale bar = 1 mm.
Fig. 16. Cross section through anther from stage C bud. Scale bar = 40 pm. Fig. 17. Cross section through locule of anther from
stage C bud showing degenerating tapeturn and irregular-sized pollen grains. Scale bar = 10 prn. Fig. 18. SEM of ovule from stage
C bud. Scale bar = 40 pm. Fig. 19. Longitudinal section through ovule from stage C bud showing two-nucleate embryo sac
(outlined) embedded in nucellar tissue. Nomarski optics. Scale bar = 20 pm. Fig. 20. SEM of top third of open floral bud at
stage D. Scale bar = 1 mm. Fig. 21. SEM of dissected pistil from stage D bud. Scale bar = I mrn. Fig. 22. Cross section of
anther from bud at stage D. Scale bar = 100 pm. Fig. 23. Cross section through locule of anther at stage D showing mature pollen
grains and compacted nonviable grains. Scale bar = 10 pm. Fig. 24. SEM of ovules at floral stage D. Scale bar = 40 pm. Fig. 25.
Longitudinal section of ovule from stage D bud showing four-nucleate embryo sac. Scale bar = 20 pm.
Fig. 26. SEM of normal-sized dry 'Valencia' pollen. Scale bar = 20 pm. Fig. 27. SEM of hydrated 'Valencia' pollen. Arrows
indicate nonviable grains. Scale bar = 20 pm. Fig. 28. Histological section stained with toluidine blue (see Methods) of a nucellar
embryo initial cell (arrow) from an ovule taken from floral stage D. Scale bar = 20 pm. Fig. 29. 'Valencia' fruit stages E-R from
which seeds and unfertilized ovules were extracted for analysis. Stages E - 0 are green fruit, stage P has a touch of colour, and
stage R is a ripe fruit. Scale bar = 50 mm.
Can. J. Bot. Vol. 73, 1995
Fig. 30. Dimensions of collected 'Valencia' fruit, seeds, and
ovules. (A) Fruit length (a), diameter (m), and weight (-.-).
(B) Seed length (a) and diameter (m) during fruit
development. The diagrams show embryo stages observed in
the micropylar third of the seed. Globular, heart, and late
torpedo stages are shown. (C) Unfertilized ovule length (@)
and diameter (m) during fruit development. The diagrams
indicate significant morphological events of early nucellar
embryo development. Lines at the bottom show stages over
which the nucellus (n) and inner integuments (ii) persist and
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Fruit development
phase
F
H
J
L
N
Fruit stage
P
R
also when a specific portion of the nucellus in the central to
chalazal region degrades and a vascular network forms and
subsequently degenerates (v). Standard deviations are shown
for samples that are of minimum size of n = 10.
maintained their ovule-like shape until stage J (Fig. 36).
Numbers of such seeds averaged about four per fruit. By
stage K (Fig. 37) curvature in the micropylar end of the seed
was evident and expansion in the chalazal end gave this
region a more rounded appearance. This shape was also
common at stage N (Fig. 38) when the chalazal end had
expanded further. Abortion of seed development was common in subsequent stages of fruit development (Figs. 39, 40,
41); however, some seeds were also observed to abort development at the earlier stages. A typical, mature 'Valencia'
seed is shown in Fig. 42. Mature, fully developed seeds
numbered one to four per fruit at stage R. Therefore, only
2-8 % of ovules developed into mature seeds. Although seed
abortion was a factor in decreasing mature seed numbers,
especially during stages 0 to R, the low numbers of seed in
'Valencia' are probably primarily a result of malfunctions in
male and female gametogenesis, as less than 10% of ovules
appear to initiate seed development.
Nucellar embryo development in unfertilized ovules of
'Valencia'
At stage E, the first fruit stage examined after style abscission, the numbers of nucellar initial cells had increased to at
least 50 per ovule based on estimates from serial sectioning
of ovules. Not all ovules contained such embryo initial cells,
however, and nucellar embryogenesis was evident in 50% of
unfertilized ovules sectioned. The rectangular, nucellar embryo
initial cells in unfertilized ovules were situated in the position
normally taken up by the embryo sac or surrounded the
crushed remnants of an embryo sac. Therefore, the initial
cells had a central location in the nucellus of the unfertilized
ovule and formed an elliptoid-like mass (Fig. 43). By stage
G (Fig. 44), most of these nucellar initial cells had developed
very thick cell walls.
A localized degeneration of nucellar tissue was first
observed at fruit stage H, in a region of the nucellus slightly
off centre and towards the chalazal end of unfertilized
ovules. This degeneration became quite pronounced by stage
J (Figs. 45 -47) and had obliterated a number of cells in the
central portion of the ovule, in some cases creating a cavity.
Whether the thick-walled nucellar embryo initial cells were
resistant to degradation is not certain. Two other developmental features were associated with this degeneration of
nucellar tissue: (i) a change in the morphology of the nucellar
embryo initial cells and (ii) the initiation of a new vascular network connecting this region to the chalazal end of the ovule.
During stages H to J, the nucellar embryo initial cells in the
vicinity of the area of nucellar degeneration became thinner
walled, rounder, larger, and more vacuolate (Figs. 45 -47).
In these cells the nucleus was very prominent. By stage J,
some of these large vacuolate cells had begun to divide
(Fig. 47). A heterogeneity of developmental morphologies
of nucellar embryogenic cells was evident within each ovule.
By stage K (Fig. 48,49), nucellar embryos were present, yet
some nucellar embryo initial cells were still thick walled and
angular (Fig. 48).
Koltunow et al.
1575
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Figs. 31-42. SEMs of unfertilized ovules and seeds during fruit development. Fig. 31. Unfertilized ovule, stage I. Scale bar =
100 pm. Fig. 32. Unfertilized ovule, stage M. Scale bar = 100 pm. Fig. 33. Unfertilized ovule, stage 0. Scale bar = 100 pnl.
Fig. 34. Unfertilized ovule, stage R. Scale bar = 200 pm. Fig. 35. Unfertilized ovule, stage R with portion of outer seed coat
removed to show sac of nucellar embryos. Scale bar = 200 pm. Fig. 36. Seed from Stage H. Scale bar = 200 pm. Fig. 37.
Seed from stage K. Scale bar = 400 pm. Fig. 38. Seed from stage N. Scale bar = 400 pm. Fig. 39. Aborted seed from
stage 0. Scale bar = 400 pm. Fig. 40. Aborted seed from stage P. Scale bar = 1 mm. Fig. 41. Aborted seed from stage Q.
Scale bar = 1 mm. Fig. 42. Mature polyembryonic 'Valencia' seed from stage R. Scale bar = 1 mm.
Nucellar embryogenesis was always more advanced in the
central to chalaza1 region of the ovule adjacent to the localized degeneration of the nucellus. Embryogenic development
was not observed to be initiated from the angular thick-
walled initial cells in the micropylar region of unfertilized
ovules at these stages. Furthermore, embryogenic cells that
were not fully surrounded by degenerating nucellar cells and
retained an association with intact nucellar tissue appeared to
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1576
Can. J. Bot. Vol. 73. 1995
Figs. 43-57. Nucellar embryo development in unfertilized ovules. All sections are stained in toluidine blue as described in
Methods. Fig. 43. Longitudinal section of a female sterile ovule from stage E fruit. Arrows indicate nucellar embryo initial cells.
Scale bar = 100 pnl. Fig. 44. Ovule from stage G fruit. Arrows indicate nucellar initial cells with thickened cell walls. Normarski
optics. Scale bar = 10 pm. Fig. 45. Section through ovule from fruit stage J. Arrowheads indicate rounded embryogenic nucellar
cells. Some of these are surrounded by degenerating non embryogenic nucellar cells in the central to chalazal portion of ovule. Scale
bar = 10 pm. Fig. 46. Another ovule from fruit stage J. Arrowheads indicate different morphologies of embryogenic nucellar cells.
Scale bar = 10 pm. Fig. 47. Ovule from fruit at stage J with large arrowhead indicating rounded embryogenic cell undergoing
division. Small arrow indicates grains that may be starch. Scale bar = 10 pm. Fig. 48. Ovule from stage K of fruit developnlent
showing embryogenic cells. Large arrowhead indicates a thick-walled cell delayed in development relative to the embryo (small
arrow). Scale bar = 10 pm. Fig. 49. Another ovule from stage K. Arrow shows enlarged embryo. Scale bar = 10 pm. Fig. 50.
Oblique longitudinal section of ovule from a fruit at stage M of development. A well-developed vascular network is visible
connected to the embryogenic mass in the central to chalazal portion of the nucellus. Scale bar = 100 pm. Fig. 51. A closer view
of the embryo mass shown in Fig. 50. Five distinct embryos can be seen. Nomarski optics. Scale bar = 10 pm. Fig. 52.
Longitudinal section of another ovule from stage M fruit. The vascular network has degenerated and atrophy of nucellar cells is
evident. Scale bar = 100 pm. Fig. 53. A magnified view of the embryo mass shown in Fig. 52. Arrowhead indicates undivided
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initial cell. Scale bar = 10 pm. Fig. 54. Cross section of ovule from fruit at stage N. Outer integument has differentiated to
exotestal palisade and underlying mesotesta. Two embryogenic clumps are visible, each connected to the vascular network. Scale
bar = 100 pm. Fig. 55. Longitudinal section of ovule from stage Q. Embryogenic masses have increased in size with a
corresponding decrease in nucellar tissue. Inner integument has compressed to almost a single layer. Exotestal layers differentiated.
Scale bar = 100 pm. Fig. 56. Enlarged view of embryo tissue from Fig. 55, with arrowheads indicating embryo initial cells that did
not develop further. Nomarski optics. Scale bar = 10 pm. Fig. 57. An ovule from fruit at stage R that appears as a miniseed. The
nucellus is absent and replaced with a mass of globular stage embryos. Scale bar = 100 pm.
be in more advanced stages of embryogenesis during stages
H to K (Figs. 45-49).
By stage M of fruit development, small clumps of
embryos had formed in the region that was the site of nucellar degeneration (Figs. 50, 51). Sometimes several of these
clumps surrounded central cavity (not shown). A vascular
network had formed that linked the groups of developing
embryos to the chalazal vasculature of the ovule (Fig. 50).
a large portion of the nucellus remained intact and
apart from cellular expansion appeared morphologically
unchanged (Fig. 50). In ovules in which the vascular network had failed to form between the embryo and the chalazal
end, such as in the ovule shown in Figs. 52 and 53, the nucellar tissue surrounding the embryogenic clumps atrophied and
was probably being utilized as a nutrient source for the developing embryos. Embryogenesis did not proceed further in
such ovules. Even at this stage of development, some
embryogenic initial cells remained undivided. For example,
the thick-walled, undivided nucellar embryo initial cell
shown in Fig. 53 was again surrounded by degenerated
nucellar cells.
At stage N of fruit development, most of the unfertilized
ovules contained multiple clumps of globular nucellar embryos
connected to the chalazal end by a vascular network. The
funicle had begun to form an aril structure and the outer
integument had differentiated to form what appeared to be
an exotestal palisade and tegumen structures (Fig. 54) as
described during seed coat maturation in Citrus seeds
(Boesewinkel 1978). In ovules from Stage Q fruit (Fig. 5 3 ,
the outer seed coat structure was fully developed with long
integument palisade cells and a distinguishable mesotestal
layer. This was separated from the remaining structures for
most of the ovule length. The inner integument had compacted and decreased in size (Fig. 55). The nucellar embryos
had increased in size and the nucellus had begun to decrease
in size. The connection of the embryo mass to the vascular
network appeared severed (Fig. 55). Initial cells that had not
developed past the single cell stage could be seen embedded
in the globular embryogenic mass (Fig. 56). At the end of
stage R, only a vestige of nucellus remained (Fig. 57). The
exotesta and mesotesta were fully differentiated and detached
from the single layer of inner integument (tegumen) and
remnants of nucellar cells surrounding the embryo mass
(Fig. 57). The globular embryos of various sizes were
arrested at this stage and did not develop further. The morphological events in nucellar embryo development in unfertilized ovules are illustrated in Fig. 30C.
The capacity of the arrested globular embryos to develop
further in unfertilized ovules was determined by culturing the
undeveloped ovules extracted from mature stage R 'Valencia' fruits on a simple hormone-free culture medium (Moore
1985). Embryos were produced by 67% of the 200 ovules
a
ow ever,
u
cultured and these developed into normal seedlings. The
capacity to form seedlings from undeveloped ovules in culture was restricted to the polyembryonic 'Valencia'. Embryos
did not emerge from undeveloped ovules extracted from
mature fruit of the monoembryonic tangor 'Ellendale'.
Taken together, our observations show that in unfertilized
'Valencia' ovules, division of the nucellar initial cells first
occurs in the central to chalazal portion of the ovule adjacent
to a zone of degenerated nucellar cells. Embryos develop
within the confines of the nucellar tissue, digest it, and
become arrested at a late globular stage. The unfertilized
ovule differentiates to form a miniseed with a differentiated
seed coat. Only 67 % of unfertilized ovules in 'Valencia' had
the capacity to undergo nucellar embryogenesis and complete embryo development when cultured in vitro.
Nucellar embryo development in 'Valencia' seeds
Nucellar embryo development in fertilized seeds was carefully studied in other Citrus cultivars by Wakana and Uemoto
(1988) and Wilms et al. (1983). As we were primarily interested in determining the timing of events of nucellar embryo
development in seeds relative to unfertilized ovules, particularly in the early stages of fruit development, this study in
'Valencia' was limited to characterization of that phenomenon.
In an ovule extracted from fruit stage G containing an
intact fertilized embryo sac, nucellar initial cells with thick
walls completely surrounded the embryo sac from chalazal
end to micropyle (Figs. 58, 59). By fruit stage I, embryo sac
expansion had occurred and this expansion was predominantly in the chalazal end of the seed. There was a concomitant division and expansion of nucellar cells at the
chalazal end of the seed that contributed to the initial increase
in seed size. As a result, the initial cells appeared to be concentrated in distinct clusters. Little ovule expansion occurred
at the micropylar end so the initial cells were easy to locate
(Fig. 60). From serial sections, nucellar embryo initial cells
were predominantly clustered in the micropylar third of the
seed and our remaining observations at subsequent stages of
develoument focused on this Dart of the seed;
Nucellar embryogenesis proceeded in the same manner as
that observed for unfertilized ovules. By stage
- J , the angular
thick-walled cells became larger, thinner walled, rounded,
and vacuolate and were most clearly evident at the edges of
the embryo sac (Figs. 61, 62). At this stage, some of the
rounded initial cells were surrounded by degraded nucellar
cells (Fig. 62). By stage K, some of the rounded initial cells
had divided (Fig. 63). Embryo development appeared to be
more advanced the closer the embryo was situated to the
embryo sac and where some contact was maintained with
adjoining nucellar cells. Zygotic embryo development was
also underway by stage L (Figs. 64, 65). At stage M, a few
small, globular embryos could just be observed by SEM in
~
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Figs. 58-65. Embryo development in seeds. All sections were stained with toluidine blue as described in Methods. Fig. 58. Cross
section through a developing seed from stage G of fruit development. Thick-walled initial cells (arrows) are present surrounding the
expanding embryo sac. Scale bar = 50 pm. Fig. 59. Micrograph using Nomarski optics of the embryo sac region in Fig. 58. Note
the concentration of thick-walled cells (arrowheads) immediately surrounding the embryo sac. Scale bar = 10 pm. Fig. 60. Section
through micropylar end of developing seed from stage I fruit. Thick-walled initial cells are shown by arrows. Size of nucellar cells
in the micropylar region of the seed are smaller than elsewhere in the nucellus. Scale bar = 50 pm. Fig. 61. Section through
embryo sac of a seed from stage J. The nucellar embryogenic cells have become rounded (arrowheads). Scale bar = 10 pm.
Fig. 62. Nucellar embryo cell in seed from stage J fruit. Arrowhead points to cell coated with degenerated nucellar cells. Nomarski
optics. Scale bar = 10 pm. Fig. 63. Nucellar embryo with two cells (arrowhead) in a seed from stage K fruit. Nomarski optics.
Scale bar = 10 pm. Fig. 64. Section through seed from stage L fruit, showing position of the zygotic embryo relative to a
developing nucellar embryo. Scale bars = 100 pm. Fig. 65. Magnified view of nucellar and zygotic embryo from Fig. 64.
Unmarked arrowhead shows an embryo initial cell. Scale bar = 50 pm.
the micropylar region of the seed. By stage N, the number
of globular embryos had increased (Figs. 66, 67). Occasionally, globular embryos could also be seen developing more
towards the chalazal end of stage N seeds (Figs. 66, 68). The
process of embryo development became obviously asynchronous after this stage, as it appeared that embryos at the
surface of the micropylar mass developed more rapidly. By
stage 0 , a high proportion of embryos in the micropylar third
were at the heart stage (Fig. 69) and by stage P, at the
torpedo stage of development (Fig. 70). Figure 30B summarizes the developmental stages where globular, heart, and
torpedo stage embryos are typically first located in the
micropylar portion of the seed.
Mature seeds from stage R fruit contained a variety of
developmental embryo stages crowded together (Fig. 71).
Typically, the largest embryo occupied the chalazal third of
the seed, and the remaining embryos decreased in size
towards the micropylar end of the seed. Very small globular
embryos, not shown in Fig. 71, were also present in the
micropylar region of mature seeds.
These observations show that nucellar embryogenesis is
initiated at the same time in both the developing seeds and
unfertilized ovules during 'Valencia' fruit development. In
both cases the process follows the same sequence of events,
i.e., the initial cells differentiate from the nucellus and then
become isolated from surrounding nucellar cells by a thick
wall that thins as the cells enlarge and become rounded. Division in both cases is associated with nucellar cell degenera-
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Figs. 66-71. Micrographs of advanced stages of embryo development in seeds of 'Valencia'. In all
of these figures except Fig. 71, the micrographs show embryos developing in the micropylar end of
the seed. It was not possible to distinguish which was the zygotic embryo in each case. Fig. 66.
Schematic diagram, not drawn to scale, of a seed extracted from fruit at stage N showing relative
locations of embryos photographed in Figs. 67 and 68. Fig. 67. SEM of clusters of globular
embryos (arrowheads) covered in residual endosperm. Scale bar = 100 pm. Fig. 68. SEM of a
smaller globular embryo located more towards the chalazal end of the seed. Scale bar = 40 pm.
Fig. 69. SEM of late globular and heart stage embryos in a seed from stage 0 fruit. Scale bar =
100 pm. Fig. 70. SEM of torpedo embryos in a seed from stage P fruit. Scale bar = 200 pm.
Fig. 71. Multiple embryos dissected from a single mature seed taken from stage R fruit. All
embryos stained with 0.05% toluidine blue for contrast. Scale bar = 8 mm.
tion events and a re-establishment of contact with maternal
nucellar cells. The site of nucellar embryo initiation, however, differs for developing seeds and unfertilized ovules,
occurring in the micropylar end and central to the chalazal
region, respectively.
Discussion
Fruit development is usually initiated by two independent
stimuli. Following pollination and during pollen tube growth,
auxin and gibberellins act as the primary stimulus that initi-
ates fruit development causing ovary expansion (Roth 1977;
Lee 1987). A second stimulus is often required to maintain
fruit growth and is thought to emanate from the seed that produces high levels of auxin (Luckwill 1959; Lee 1987). In
some Citrus cultivars, seedless fruit can be set parthenocarpically in the absence of fertilization and even pollination
(Sykes and Possingham 1992). 'Valencia', however, requires
both pollination and fertilization for fruit set or all of the
small fruitlets will abscise soon after style fall (Wakana and
Uemoto 1987; Sauer 1954).
Seed set in 'Valencia' is low (one to four seeds per fruit)
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Can. J . Bot. Vol. 73, 1995
and our observations suggest this could be due to defects
during gametogenesis. We did not study aberrations in
'Valencia' embryo sac development extensively; however
Naumova (1993) observed several cases in which members
of the genus Citrus occasionally abort megagametophyte
development at the tetrad stage, which usually leads to the
collapse of the ovule. Generally, female sterility in Citrus is
attributable to embryo sac degeneration later in megagametogenesis, prior to flowering. In some species, embryo sacs
also form without meiosis, and if these are fertilized, a seed
develops containing a triploid embryo with pentaploid endosperm ("small" seeds; Naumova 1993). Defects in microspore development in sweet oranges were documented by
Iwamasa (1966). The variation in pollen grain size and the
occurrence of empty grains that we also observed in 'Valencia' (Figs. 11, 17, and 23) was attributed by Iwamasa (1966)
to irregular chromosomal segregations during meiosis, with
giant grains containing a larger number of chromosomes than
the normal. 'Valencia' clearly shows defects in both male
and female gametogenesis. In addition, however, 'Valencia'
has the ability to form nucellar embryos, and this paper
focuses on the description and discussion of this phenomenon.
Recent studies of nucellar embryony in unfertilized,
undeveloped Citrus ovules (Wakana and Uemoto 1987) and
seeds (Wakana and Uemoto 1988) provide significant descriptive information about this process. These studies established that nucellar embryos are initiated autonomously and
develop with or without pollination in both unfertilized
ovules and developing seeds in a number of polyembryonic
Citrus cultivars. Furthermore, as endosperm is not necessary
for the early development of nucellar embryos, they must
therefore be dependent upon the nucellus as a direct nutritional source (Wakana and Uemoto 1987, 1988). Those
studies have related nucellar embryo development in seeds to
days postfertilization, a criterion that varies depending on the
climatic conditions and the cultivar under study. Our study
has added to those of Wakana and Uemoto (1987, 1988) by
addressing the question of the timing of nucellar embryo
initial cell specification in ovules. We also studied histologically the early development of nucellar embryos in unfertilized ovules of a weakly parthenocarpic cultivar.
To determine when the nucellar embryo initial cells are
s~ecifiedin 'Valencia'. we cultured ovules of 'Valencia' at
early stages of female gametophyte formation. This functional assay showed that cells capable of initiating embryogenesis are specified as early as the binucleate stage of
megagametogenesis in 'Valencia'. At this stage, nucellar
embryo initial cells could not be identified histologically.
This extends the findings of Kobayashi et al. (1981) who sectioned ovules just priorto anthesis in a number of Citrus cultivars to identify the presence of nucellar embryo initial cells
and also cultured ovules extracted from flowers just before
anthesis and observed embryo formation. ~ o b a y a s h iet al.
(1981) observed that embryos formed only from the nucellar
region of the ovule and not from other structures. Given the
limitations of our ovule culture experiments, it may be that
under suitable conditions, nucellar embryony could be initiated from nucellar tissue at stages preceding early megagametogenesis.
The events of nucellar embryogenesis in unfertilized ovules
and also in developing seeds were not studied previously in
the course of fruit development in a single cultivar. We
observed histologically that in 'Valencia', nucellar embryo
initial cells are first evident by their large nucleus and deeply
staining cytoplasm and occur in an elliptical zone surrounding a sexual embryo sac. If the embryo sac has degraded, as
often occurs in unfertilized ovules, initial cells occur in an
elliptical zone in the vicinity of the normal position of the
embryo sac. Their cell walls increase in thickness compared
with the surrounding nucellar cells. The embryogenic initial
cells then become more rounded with thinner walls, the cells
enlarge in size, become vacuolate, and have prominent
nuclei. In unfertilized ovules, these spherical cells are mainly
confined towards the chalaza1 end of the central region
of the
"
ovule and their development is accompanied by a degeneration of the nucellar tissue in that region of the ovule.
This localized degeneration was not reported in previous
studies of nucellar embryo formation in unfertilized ovules
(Kobayashi et al. 1979, 1981; Wakana and Uemoto 1987,
1988; Tadeo and Primo-Millo 1988). After further expansion, division of these cells occurs, but additional major
localized degeneration of the nucellus is not observed. In fertilized seeds during the early stages of embryo sac expansion, several layers of nucellar cells gradually degenerate
and the angular, thick-walled, initial cells that then abut the
embryo sac become rounded, enlarge, and subsequently
divide. Wilms et al. (1983) observed changes in cell wall
thickness during nucellar embryogenesis in seeds by electron
microscopy. The thickened cell walls are probably due to
callose deposition as Wakana and Uemoto (1988) observed
a strong aniline blue-induced callosic fluorescence in adventive embryo initial cells in developing Citrus seeds. Wilms
et al. (1983) demonstrated that formation of this thickened
cell wall blocked plasmodesmata, isolating the embryogenic
initial cells from the surrounding tissue. This isolation may
allow a switch to an embryogenic developmental pathway to
occur by preventing receipt of contrary signals from surrounding cells. Wilms et al. (1983) postulated that in developing seeds, division of the nucellar embryo initial cell
depended on appropriate development of the initial cell and
also the degeneration and breakdown of the surrounding
nucellar tissue.
Our observations of nucellar embryo development in
seeds and unfertilized ovules of 'Valencia' provide more
direct evidence for the hypothesis of Wilms et al. (1983). In
both cases, division of nucellar embryo initial cells coincides
with a controlled breakdown of nucellar tissue. Furthermore.
our observations of embryo formation in unfertilized ovules
showed that a higher frequency of dividing embryogenic
cells was always found when these cells retained some contact with surrounding nucellar cells. If the rounded embryo
initial cells were isolated and surrounded by degenerating
nucellar cells, then division of those cells was delayed. Some
maintenance of contact to maternal nucellar tissue amears to
be important for progression through embryogenesis. Therefore; although severing of contacts from maternal tissue may
be required to allow reprogramming towards an embryogenic pathway, re-establishment of contact with maternal
tissue is also essential for rapid embryogenic development.
This apposition is analogous to development of a zygotic
embryo in the sense that the zygotic embryo does not initially
float freely in the embryo sac but is linked to maternal tissue
..
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Koltunow et al.
in the micropylar region by a suspensor.
The localized degeneration of nucellus in unfertilized
ovules also is intriguing because it can create a cavity
reminiscent of an embryo sac in a developing seed. Selective
degeneration of particular nucellar cells may be serving as a
nutrient source akin to endosperm in the seed, and the
majority of embryo development that subsequently occurs in
the central region of the unfertilized ovule may be the result
of a competitive advantage to those embryo cells closest to
available nutrients.
The establishment of a vascular network linking developing nucellar embryos to the chalazal vasculature in the ovule
is another significant developmental event that begins during
nucellar degeneration in unfertilized ovules of 'Valencia'.
The nature of the progenitor cells that give rise to this structure and the signal(s) initiating its formation remain unclear.
The structure is transient in nature and appears essential for
growth of the globular embryos, or the nucellus is rapidly
degraded and embryos arrest very early in development.
Our observations of the differing location of embryo
development in unfertilized ovules and developing seeds of
'Valencia' are consistent with the results obtained by Wakana
and Uemoto (1987, 1988) in other Citrus cultivars. In unfertilized ovules of 'Valencia' in our study and the unfertilized
ovules in cultivars observed by Wakana and Uemoto (1987),
embryo development occurs in the central region of the
ovule, while in seeds, zygotic and nucellar embryo formation
is restricted to the micropylar region of the seed. Wakana
and Uemoto (1988) observed that in seeds, nucellar embryo
initial cells in the chalazal region rarely divided at all. An
obvious difference between fertilized seeds and unfertilized
ovules is the presence of endosperm in all but the micropylar
end of the sexual embryo sac where the developing zygotic
embryo is situated. In seeds, the inhibition of nucellar
embryo development in the chalazal region means that all
embryo development, sexual and nucellar, is restricted to the
micropylar end. The nucellar embryo initials in the chalazal
end of the embryo sac may be inhibited by as yet unknown
mechanisms that normally act to polarize zygotic embryo
development to the micropylar end of an embryo sac, such
as gradients of morphogens or even nutrients. Alternatively,
Wakana and Uemoto (1988) suggested that a component of
the endosperm in the chalazal end of the embryo sac may
specifically act to inhibit nucellar embryo initial cell division
in that region.
Seed coat development continues to occur in unfertilized
'Valencia' ovules even though the nucellar embryos remain
at a globular stage. This indicates that differentiation of the
seed coat is independent of the later events of embryo
maturation. Why do nucellar embryos arrest at the globular
stage in unfertilized ovules? These ovules lack endosperm
and it is possible that exhaustion of nutrient supply prohibits
further embryo development. Alternatively, the arrest at the
globular stage could be a combination of lack of nutrients and
of physical space restrictions. Unfertilized ovules undergoing nucellar embryo development only double in size compared with seeds where the ovule increases 13-fold in size.
Therefore, there is very little space to accommodate the
developing embryos in unfertilized ovules. These globular
nucellar embryos are not dead, just arrested in development
because they can complete development and form plants if
cultured on a simple medium containing basal salts and
sucrose. Both unfertilized and fertilized ovules have the
capacity to produce embryos in 'Valencia', yet only seeds
significantly increase in size. Seed expansion is evident well
before the developing embryos are at the globular stage.
Therefore, the endosperm or processes related to fertilization
must provide important signals that trigger the ovule expansion necessary to accommodate embryo growth.
The similarity and the coordination of the early events of
nucellar embryogenesis in seeds and unfertilized ovules of an
individual fruit is striking given the spatial separation of
these structures in the fruit and that only seeds are fertilized.
The coordination of these events is also surprising given that
not all ovules had the capacity to produce embryos in 'Valencia' as determined by in vitro culture. It is not known
whether this temporal coordination of nucellar embryogenesis within a fruit is universal for polyembryonic Citrus cultivars. It may be specific to 'Valencia', in which pollination
and fertilization are absolutely necessary for fruit set.
In Citrus, therefore, embryogenesis is initiated within the
environment of an embryo sac following fertilization and
also from maternal nucellar cells external to an embryo sac.
During nucellar embryogenesis in unfertilized ovules, many
nucellar cells appear to have the potential to embark on an
embryogenic pathway. However, whether they divide to initiate embryogenesis is dependent on their initial isolation
from maternal tissue, a localized degeneration in the nucellus,
a re-establishment of connections to maternal tissues, their
position in the nucellus, and their early access to nutrient via
a specialized vascular structure. A viable seed is not produced in unfertilized ovules because the nucellar embryos
arrest after the globular stage of development, yet a seed coat
differentiates. By contrast, nucellar embryos initiated in a
fertilized seed are able to develop past the globular stage
through to embryo maturity. What molecular signal provides
some nucellar cells with an egglike developmental potential
capable of embryo formation without fertilization and restricts
others from embarking on an embryogenic pathway? Nucellar embryogenesis may reflect an alteration in the spatial and
temporal expression of a gene(s) normally involved in sexual
embryo development, but this speculation remains to be
tested by molecular investigation.
Acknowledgements
We thank Margaret Minter for typing and Dr. Nigel Scott,
Dr. Susan Barker, and Dr. Bob Fischer for very helpful criticisms during the preparation of this manuscript.
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