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Volume 49, 2001
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 Aust. J. Bot., 2001, 49, 199–207

Floral morphology and embryology of two Australian species

of Citrus (Rutaceae)

Kerri Clarke and Nallamilli Prakash

Botany Building, School of Rural Science and Natural Resources,

University of New England, Armidale, NSW 2351, Australia.
Email: Clarkekerri@hotmail.com

Abstract. The floral morphology and embryology of two species of Australian Citrus L. occurring in the most
southerly range of the genus, C. australasica F.Muell. and C. australis (Mudie) Planchon, have been studied.
Cytokinesis in the microsporocytes was simultaneous resulting in tetrahedral tetrads. Tapetal cells were bi- to
multinucleate and unevenly one- or two-layered. Microspore development was frequently asynchronous. Anther
wall consisted of a layer of endothecium, three to five middle layers and one or two layers of Secretory tapetum.
The ovules were anatropous, bitegmic and crassinucellate. Although multiple sporogenous cells that grew into
multiple megasporocytes were common, occurrence of twin or multiple gametophytes was rare. Development of the
female gametophyte was of the Polygonum type, with antipodal cells frequently persisting until after fertilisation.
Endosperm was of the Nuclear type while embryogeny was of the Onagrad type. Both integuments contributed to
the seed coat. Cells of the outer layer of the testa developed fibrous thickenings and secreted mucilage. Seeds were
monoembryonate and seed germination was hypogeal. The recent move incorporating Australian native citrus
species in to the genus Citrus was supported on the grounds of close embryological similarities.
BT99045
K.FlKeCloriClarlmorkearandphkeN.olandgPryNaakndl ashembril yoPralogkayofshCitrus

Introduction Prakash and Lim (1996) reviewed the embryology of the

Rutaceae providing a comprehensive literature survey that
Eremocitrus Swingle and Microcitrus Swingle were among highlighted the patchy nature of our knowledge about the
the 40 genera of Australian Rutaceae (Harden 1991). family and the need for more research even on such a popular
Mabberley (1998) presented a case for reuniting these two genus as Citrus. The current embryological study is
genera with Citrus L. and he recognised six Australian significant in that there have been (1) few such investigations
species—C. glauca (Lindley) Burkill (= Eremocitrus), of the Australian Rutaceae and (2) no investigations for any
C. australasica F.Muell., C. australis (Mudie) Planchon, of the native Citrus species that are among the very few wild
C. garrawaye F.M.Bailey, C. gracilis Mabb. sp. nov., and species of the genus known in the world, as all previous
C. inodora F.M.Bailey (= all Microcitrus). investigations have been on cultivated Citrus species.
Australian aborigines (Lazarides and Hince 1993) and Citrus australasica (= Microcitrus australasica (F.Muell.)
early European settlers (Anon. 1927) were known to have Swingle), commonly called finger lime, is found in northern
used native citrus fruits as a food source. There has been New South Wales (from Lismore extending northwards) and
renewed interest in Australian native food and medicinal south-eastern Queensland. Citrus australis (= Microcitrus
plants. The wide variation of fruit shape, size and colour of australis (Mudie) Swingle) known as Australian lime or
Citrus australasica (finger lime) have provided added dooja, occurs around Brisbane and further north in
potential for marketing native Citrus (Swingle 1915; Taylor Queensland.
1996).
Breeding experiments indicated that commercial citrus Materials and methods
species were closely related genetically to the native Floral and fruit materials of various developmental stages of Citrus
Australian species. The Australian species along with australasica F.Muell. var. australasica Swingle, C. australasica
Clymenia Swingle, Fortunella Swingle, and Poncirus Raf. F.Muell. var. sanguinea Bailey, and C. australis (Mudie) Planchon,
were able to be interbred (Mullins et al. 1989). There were were collected periodically from March 1995 until March 1997 from
two sites. Material was collected from disjunct populations of
problems, however, with intergeneric breeding, such as
C. australasica growing in naturally regenerated forests on private
pollen tube retardation, and ovule and seed sterility (Frost property at Mount Burrell, New South Wales. Viola orchard situated at
and Soost 1968; Soost and Cameron 1975; Barrett 1977; Bangalow, New South Wales, provided nursery grown pot plants of
Sedgley and Griffin 1989). C. australasica var. sanguinea and C. australis that had been budded

© CSIRO 2001 10.1071/BT99054 0067-1924/01/02199

200
onto ‘Troyer’ citrange rootstock two years prior to collection. Plants
often had floral and fruit material at various stages of development,
with unopened buds through to immature fruits on the same branch.
The incidence of floral and fruit abortion was high in the cultivated
plants although flowering was prolific. Flowering in the wild plants was
less predictable. Voucher specimens were collected for all the plants
sampled and deposited in the N. C. W. Beadle Herbarium (NE), Botany,
University of New England.
Microtechnique
Flower buds and fruits were fixed on site in formalin–propiono–alcohol
(FPA—5 mL formalin, 5 mL propionic acid and 90 mL 70% ethanol),
then stored in 70% ethanol.
Fixed buds, fruits, and seeds were prepared for sectioning by
following conventional methods (Bhandari 1997). Dehydrated
specimens were infiltrated with and embedded in Paraplast for
sectioning. Longitudinal and transverse sections of small buds, fruitlets
and seeds were stained with either safranin and fast green, or with
Heidenhains haematoxylin, safranin and fast green—the latter
providing superior results for pollen cytology. All prepared slides were
deposited in the Botany Building.
Scanning electron microscopy
Scanning electron microscopy was used to observe the external
structure of pollen and pollen germination on the stigmatic surface of
both Citrus australasica and C. australis. Stigmas and mature anthers
that were previously fixed and stored in 70% ethanol, were subjected to
critical point drying and sputter-coated with gold. Scanning electron
microscopy was performed with a JEOL JSM-5800LV Scanning
Microscope at 20 kV.
Seed germination
Ripe seeds of both species were placed on germination pads moistened
with tap water in Petri dishes under a temperature regime of 30°C day/
20°C night.
Results
Floral morphology
The morphology of the flower in Citrus australasica was
broadly similar to C. australis. Flowers were axillary and
occurred singly or in pairs. When in bud, the imbricate petals
were white with a purple tinge at the base. They were regular
and hypogynous. Floral parts were free with stamens that
were partly fused at the point of attachment below the floral
disc. Flowers of C. australis were tetra- or pentamerous, with
petals that were dotted with oil glands and reflexed when
open. In C. australasica, flowers were tri- or tetramerous,
with petals reflexed or assurgent when open.
Each flower had about 25 stamens in C. australis and
16–20 in C. australasica. Anthers were tetrasporangiate
with introrse and longitudinal dehiscence and had a
prominent apical oil gland in the connective tissue.
The pistil was attached to a floral disc and was syncarpous
and multilocular. Many lysigenous oil glands were found in
the ovary wall. The ovary of C. australis had six or seven
chambers while C. australasica had three to five. The ovary
of C. australis was subglobose with a cylindrical terminal
style and a hairy capitate stigma. In C. australasica, the
cylindrical ovary supported a terminal style that was itself
K. Clarke and N. Prakash
cylindrical or geniculate with a capitate or slightly clavate
stigma. Stylar canals were present in both species, arising
from the locules and terminating at or near the surface of the
stigma. Each locule contained two rows of ovules attached
along the axis of the placenta.
It was common to find pistils with suppressed growth in
flowers that had already dehisced anthers. Longitudinal
sections of such pistils revealed that they were in early stages
of embryo sac development. Often there were pistils with
aborted ovules; in some cases the ovules had aborted even
before the integuments were formed.
Initiation of juice vesicles was not consistent. While some
ovaries had juice vesicles initiated during early embryo sac
development, in others they were initiated about the time of
fertilisation. Carpellary outgrowths, initiated at the time of
fertilisation, occurred between juice vesicles.
Fruits of C. australasica had a thin leathery skin and
varied in size, shape and skin colour, and colour of internal
juice vesicles. Citrus australasica var. sanguinea was
red-fruited, differing from the green fruited C. australasica
var. australasica. Fruits of C. australis were thick-skinned,
small, round and yellow when ripe. The fruits were usually
many seeded, but in some samples of C. australasica there
were few or no seeds.
As few differences were observed in the embryology of
C. australis, C. australasica var. australasica and
C. australasica var. sanguinea, the observations below apply
to all three unless otherwise stated.
Anther and pollen development
By the time petals became visible in the floral bud the
stamens showed a definite filament and a tetrasporangiate
anther with an apical oil gland. Once microsporocytes
formed, the tapetal cells became bi- or multinucleate. The
anther wall had a well-defined epidermal layer,
undifferentiated endothecium, three to five middle layers,
and an uneven, 2-layered Secretory type of tapetum with bi-
or multinucleate cells. Degeneration of middle layers
commenced at the microsporocyte and early tetrad stages.
Anther development was asynchronous within the same
floral bud, particularly in the early stages of
microsporogenesis. The development of microsporangia in
the same anther was also asynchronous, as the two inner
microsporangia lagged behind the outer. Even in the same
microsporangium, there was variation in the stages of
microsporogenesis between basal and apical portions.
During anther maturation, the middle layers became
obliterated leaving only remnant layers against the
endothecial wall. The endothecium developed fibrous
thickenings, and epidermal cells remained intact persisting
in the mature anther. Developing pollen grains accumulated
cytoplasm and stained deeply with safranin, preventing
observation of pollen mitosis, however, staining with
haematoxylin allowed identification of 2-celled pollen. The
Floral morphology and embryology of Citrus
endothecium was one cell thick at the line of dehiscence but
two or 3-layered nearer the connective tissue. There was a
single longitudinal line of dehiscence and dehisced anthers
contained two empty pollen sacs. Pollen sacs of fresh mature
anthers were filled with a yellow oily substance that
surrounded the pollen.
The spherical pollen of Citrus australasica was 20 µm in
diameter and pentacolporate. Citrus australis pollen had a
diameter of 17 µm across and was tetracolporate. Exine
patterning for both species was reticulate.
Megasporogenesis
Ovule initials were identified before the locules fused in
the pistil and the stigma differentiated. In pistils with
suppressed growth, ovule initiation did not occur until
the pollen was almost mature. The primary parietal and
sporogenous cells resulted directly from the division of
the archesporial cell. Repeated divisions of the parietal
cell produced the many parietal layers that constituted
the nucellar cap of the mature ovule in Citrus
australasica and C. australis. In both species, five to
seven sporogenous cells were frequently observed.
Directly below the parietal tissue the sporogenous cells
enlarged to become multiple megaspore mother cells
(Fig. 1A). In most ovules, only one megaspore mother
cell persisted to undergo meiosis, producing a linear
tetrad.
Development of female gametophyte
Only the chalazal megaspore of the tetrad was functional
(Fig. 1B). Following mitosis, a large vacuole separated the
nuclei that had migrated to opposite poles of the embryo sac
(Fig. 1C). Successive mitotic divisions resulted in an
8-nucleate gametophyte forming the 7-celled Polygonum
type of embryo sac (Fig. 1D). The mature female
gametophyte was located deep within the nucellus, under
eight or nine (to 13) parietal layers. Folds in the aging
synergid gave the synergid a hooked appearance. The polar
nuclei remained separated until the time of fertilisation. The
three antipodal cells became shrunken and deeply stained,
but often persisted until after fertilisation.
The occurrence of multiple embryo sacs was rare, as only
one instance of two binucleated embryo sacs was observed in
a C. australis ovule, when all other ovules in the ovary were
already at the mature stage.
Ovule anatomy
Both inner and outer integuments of the bitegmic ovule in
Citrus australasica and C. australis formed a micropyle that
is either zigzag (Fig. 2A), or straight, depending on the
evenness of integument growth. At the sporogenous cell
stage, the inner integument could be seen as a rudimentary
protuberance from the ovular epidermis. The outer
integument was initiated from subepidermal layers as the
201
multiple sporogenous cells began to elongate. By the
functional megaspore stage, the inner and outer integuments
reached the apex of the nucellar cap that was now six to eight
parietal layers deep. At the 4-nucleate embryo sac stage the
outer integument extended beyond the inner integument. The
mature ovule was anatropous and showed a well-defined
hypostase with deeply stained cells in the chalaza. The
nucellar cap could be up to 13 layers thick with starch grains
prominent, particularly in the micropylar region. The single
vascular bundle contained in the funicle terminated at the
chalaza and joined the hypostase. Elongated epidermal hairs
formed an obturator that developed from the placenta, the
funicle, or both. The obturator hairs in C. australis were
composed of a single elongated cell (Fig. 2B) whereas those
of C. australasica were multicellular (Fig. 2C).
Citrus australis occasionally showed twin nucelli; only
the larger nucellus contained an embryo sac. Normally two
rows of ovules developed from the placenta, but three rows
were present in one sample of this species.
Pollination and fertilisation
Pollen was attached to the mucilaginous papillae on the
stigma and pollen tubes grew through the stylar canal to
enter the ovary and into the micropyle. Pollen tube entry
into the embryo sac was through a synergid (Fig. 3A).
Triple fusion occurred before syngamy. The zygote lay
dormant while the endosperm formed. Starch grains
appeared around the primary endosperm nucleus and the
zygote at the time of fertilisation.
Development of endosperm and embryo
Endosperm was of the Nuclear type. In many ovules of
Citrus australasica, endosperm formed an inverted
horseshoe shaped structure in the centre of the embryo sac.
The zygote did not show polarity early in endosperm
development. Despite active growth of the endosperm, some
ovules degenerated. Divisions in the endosperm were
synchronous in the early stages of development.
Seed development
The few stages in early embryo development observed
indicated Onagrad embryogeny. The many starch grains in
the nucellar cap seemed to enlarge at the time of fertilisation.
Juice vesicles and intercarpellary growths intruded into the
locule and surrounded the young seeds. Tannins were
deposited on the inside layer of cells of the inner integument
and the chalazal region. The nucellar tissue surrounding the
embryo sac broke down as the endosperm grew while the
cells of the hypostase accumulated tannin and developed
thickened walls.
As the seed enlarged, the endosperm expanded at the
expense of central nucellar cells. However, the nucellus
continued to produce new cells around its periphery. The
globular embryo in the young seed of C. australis, as seen in
202 K. Clarke and N. Prakash

Fig. 1. (A) A developing ovule in longitudinal section of Citrus australasica var. australasica. One
of the multiple megaspore mother cells (arrow) at early prophase I of meiosis (scale bar = 20 µm).
(B) The functional megaspore (arrow) of a linear tetrad of megaspores was situated at the chalazal
end of the developing ovule of C. australasica var. sanquinea. (C) A developing embryo sac seen in
longitudinal section of a C. australis ovule at the 4-nucleate stage of development. The two nuclei in
view were situated at the chalazal end of the embryo sac. (D) A mature embryo sac of C. australis in
longitudinal section was of the Polygonum type (scale bars = 10 µm for B–D).

transverse section, was situated centrally and surrounded by The outer integument became unevenly convoluted as the
the endosperm that had now become cellular near the epidermal cells differentiated into the outer seed coat. The
micropyle. The nucellar tissue, bounded by the inner young seed had a single vascular bundle in the raphe. The
integument, still constituted a significant part of the seed. globular embryo broke away from the small suspensor and
Floral morphology and embryology of Citrus 203

Fig. 2. (A) Uneven growth of the integuments formed a zigzag micropyle (m) in a mature ovule
of Citrus australis (scale bar = 40 µm). (B) Obturator in C. australis was formed by single-celled
hairs. (C) In C. australasica obturator was composed of multicellular hair (scale bars = 20 µm for
B and C).
204
moved down into the embryo sac, still surrounded by the
cellular endosperm (Fig. 3B). Endosperm in the chalazal
region, however, remained nuclear at this stage of
development.
Mature seed and germination
The mature seed in Citrus australasica was small, round on
top and flat underneath, about 6 × 4 mm, and was
cream-coloured with a patterned testa. The cotyledons were
thick and fleshy, and were surrounded by three remnant
layers of cellular endosperm (Fig. 3C). About four cell
layers of nucellus also persisted around the endosperm.
Both integuments contributed to the seed coat although
only a single layer of tannin-filled cells represented the
inner integument. In the testa, the epidermal cells became
elongated, thickened and covered by a layer of mucilage
(Fig. 3D). Although the cotyledons are green, germination
was hypogeal in both C. australasica and C. australis and
resulted in single seedlings.
Discussion
Corner (1976) and Vaughan (1970), based on their work on
seeds, demonstrated that genera and even species in
Rutaceae, could be differentiated based on embryological
characters. Sometimes, although the characters were broadly
similar, the size and shape of various embryological
structures allowed demarcation.
Anther development
Microsporogenesis and pollen development in the Australian
Citrus species was similar to that in other species of Citrus
and in other Rutaceae (Davis 1966; Johri et al. 1992), in
showing simultaneous cytokinesis and tetrahedral
microspore tetrads.
The non-synchronous microspore development among
anthers of the same flower and among microsporangia of the
same anther, seen in the present materials, also occurred in
Severinia buxifolia Ten. (Lim and Tinggie 1991). Citrus in
general was reported to have relatively synchronous anther
development, although Horner and Lersten (1971) noted a
difference in microspore stages between distal and proximal
ends of the microsporangia in Citrus limon (L.) Burm.f.
Tapetal cells in Citrus species (Osawa 1912; Banerji
1954; Horner and Lersten 1971) and Severinia buxifolia
(Lim and Tinggie 1991) became binucleate before meiosis
commenced in the microsporocytes. In the species studied by
us, some of the tapetal cells became binucleate even before
the microsporocytes had rounded-off, and by early prophase
binucleate and frequently multinucleate tapetal cells formed
two layers around the microsporocytes. Horner and Lersten’s
(1971) report of the dissolution of tapetal cell walls in Citrus
limon during the early microspore tetrad stage, with their
contents moving into the microsporangial cavity, was
interpreted by Sedgley and Griffin (1989) as indicative of
K. Clarke and N. Prakash
periplasmodial type of development. Secretory tapetum was,
however, the common mode of tapetal development in the
Rutaceae (Davis 1966; Lim and Tinggie 1991; Johri et al.
1992; Prakash and Lim 1996). In both species studied here
tapetal cells remained intact until pollen development was
almost complete and at no stage did the tapetal nuclei or
protoplasts move into the microsporangium.
Pollen grains of Citrus australis and C. australasica were
smaller than in other species of the genus where the pollen
size ranged from 21 to 39 µm (Sedgley and Griffin 1989).
Mature pollen of Citrus contained two cells (Sedgley and
Griffin 1989), excepting Citrus grandis (Burm.) Merrill,
which had three-celled pollen (Banerji 1954). The reticulate
pattern of the exine in our study was similar to that in Citrus,
Fortunella (Scora 1988), and other members of the Rutaceae
(Davis 1966; Boyd 1992; Johri et al. 1992; Prakash and Lim
1996).
Obturator
Obturator hairs were multicelled in Citrus australasica but
unicelled in C. australis. Unicellular obturator hairs occur in
Citrus grandis (Banerji 1954) and Poncirus trifoliata (L.)
Raf. (Boeswinkel 1978) and possibly in Severinia buxifolia
(Lim and Tinggie 1991). A two-celled condition was seen in
Citropsis aff. tanakae (Boeswinkel 1978).
Ovule development
Megasporogenesis and embryo sac development in Citrus
appeared to be generally uniform (Osawa 1912; Bacchi
1943; Banerji 1954; Soost and Cameron 1975) as with the
family Rutaceae as a whole (Johri and Ahuja 1957; Ghosh
1974; Lim and Tinggie 1991).
Multiple sporogenous cells, known to occur in Citrus
(Bacchi 1943), were also present in both species we have
studied. Sporogenous cells formed multiple megaspore
mother cells and could potentially develop multiple embryo
sacs. In some angiosperms multiple archesporial and
megaspore mother cells were known to lead to
polyembryony and apomixis (Bouman 1984). Multiple
embryo sacs, known in Citrus paradisi Macf. (Bacchi 1943)
and Ravenia spectabilis Engl. (Ghosh 1974), were very rare
in our materials.
The antipodal cells of Citrus australasica and C. australis
frequently persisted until after fertilisation, in contrast to
C. paradisi, C. aurantium L. (Bacchi 1943), C. grandis
(Banerji 1954) and Severinia buxifolia (Lim and Tinggie
1991), in which they degenerated early. The polar nuclei did
not fuse prior to fertilisation, as was also the situation in
Citrus aurantium and C. nobilis Lour. (Osawa 1912). On the
other hand, in C. grandis (Banerji 1954) and Severinia
buxifolia (Lim and Tinggie 1991), the polar nuclei fused to
form the secondary nucleus before fertilisation.
At times, in C. australasica and C. australis flowers could
effectively be staminate due to ovule abortion and retarded
Floral morphology and embryology of Citrus 205

Fig. 3. (A) Longitudinal section from a Citrus australis embryo sac, at post-fertilisation, with
primary endosperm nucleus (pen) in view. Pollen tube entry via a synergid (s) (scale bar = 10 µm).
(B) Globular embryo from a developing seed of C. australis in longitudinal section surrounded by
endosperm (ce) that has become cellular (scale bar = 20 µm). (C) Longitudinal section from a
mature seed of C. australasica showing the embryo consisting of massive cotyledons (c) and small
triangular area of the radicle and hypocotyls (white arrow) (scale bar = 500 µm). (D) Section of the
seed wall of Fig. 3C at increased magnification. The epidermis (e) of the testa had elongated
thickened cells and a well-developed mucilage layer. The single layer of tegmen (double white
arrowhead) was filled with tannins (scale bar = 40 µm).
206
ovule development, with embryo sacs that were 4-nucleate in
open flowers containing already dehisced anthers. Such
retarded or suppressed growth has earlier been recorded in
other species of Citrus (Osawa 1912; Bacchi 1943; Soost and
Cameron 1975). In experiments involving Microcitrus
papuana H.Winters (= Citrus wintersii, Mabberley 1998),
Barrett (1990) found that 75% of pistils in the flowers had
suppressed growth.
Pollination and seed development
In Citrus australasica and C. australis triple fusion occurred
before syngamy and the zygote had a prolonged resting stage
before the onset of embryogenesis, as in Poncirus trifoliata
(Osawa 1912). Embryogeny in the two Australian Citrus was
of the Onagrad type while embryogeny of the few Citrus
species previously studied showed either Solanad or
Onagrad types (Davis 1966).
Structure of the mature seed in C. australasica and
C. australis was similar to that in Poncirus trifoliata,
Fortunella margarita (Lour.) Swingle, and Citrus as a whole
(Schneider 1968; Corner 1976; Boeswinkel 1978).
Seedlessness, seen as a desirable attribute in commercial
species (Sykes 1993), occasionally occurred in
C. australasica.
Citrus australasica and C. australis showed hypogeal
type of germination as also in C. sinensis (Schneider 1968),
C. grandis and C. reticulata Blanco (Frost and Soost 1968).
However, the two Australian species lacked polyembryony,
so common in many commercial species.
Relationships
Various aspects of embryological development in
Citrus australasica and C. australis were so similar to other
species of the genus that there is little justification, at least
on these grounds, to maintain a separate genus, Microcitrus,
to accommodate these species. We, therefore, support
Mabberley’s (1998) treatment of reuniting the Australian
species with Citrus.
Acknowledgments
Grateful thanks go to Jim and Barbara O’Brien from Mount
Burrell, NSW, and Judy and Ashley Viola of Bangalow,
NSW, for allowing access to plants on their properties.
Thanks also go to Dr Jeremy Bruhl for advice. Financial
support from N. C. W. Beadle Grants in Aid and Friends of
Botany Foundation to Kerri Clarke is gratefully
acknowledged.
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Manuscript received 15 July 1999, accepted 14 September 2000