Nezv Phytol.

(1991), 118, 527-533

Development of the ovular structures in

peach [Prunus persica (L.) Batsch]

BY A R A N C H A A R B E L O A ^ AND MARIA HERRERO^

1 U.F.I. Pomologia, E.E. Aula Dei, Apartado 202, ^ U. Fruticultura, SJ.A.-D.G.A.
(INIA), Apartado 727 Campus de Aula Dei, 50080 Zaragoza, Spain
(Received 19 July 1990; accepted 22 April 1991)

SUMMARY

Changes in the ovular tissues of peach (Prunus persica (L.) Batsch) were studied from anthesis to fertilization or
degeneration in pollinated and unpolHnated flowers. During this time the ovule undergoes a continuous
developmental process. At anthesis the megagametophyte is immature and is not fully developed until 12 days
later. Durmg the first three weeks following anthesis the ovule increases in length from 400 to 1200/fm, and the
nucellus and integuments undergo various changes particularly in the distribution of starch and cutin. These
appear to be connected with early embryo sac nutrition and protection. As the ovule matures, starch accumulates
mainly in the embryo sac, integuments and at the nucellar tip. This starch disappears during early embryo sac
elongation. Concomitant with starch depletion, the cutin that separated the integuments from the nucellus
vanishes. Other cutin accumulates between cells in the nucellar tip, thus protecting and isolating the embryo sac.
Most of these changes appear to be a fixed feature of ovule development since they were also observed in a few
long lived ovules in unpollinated flowers. However, in unpolHnated fiowers development of most of the ovules is
soon interrupted: callose accumulates at the chaiaza, starch disappears and the ovules degenerate.

Key words: Prunus persica, peach, ovule, fertilization.

ovular tissues during embryo sac development may

INTRODIiCTIOK shed new light on embryo sac nutrition and pro-
While much work has been done on megagame- tection.
tophyte development in angiosperms (Willemse & In peach (Prunuspersica (L.) Batsch), while details
van Went, 1984), information is scarce on the of megagametophyte and emhryo or endosperm
function of the ovular tissues that envelop the development have been described (Ragland, 1934;
embryo sac. Work on the ovule itself has mainly been Lilien-Kipnis & Lavee, 1971), no information is
from a purely structural or taxonomic standpoint available on how ovular structures develop during
(Bouman, 1984) and tbe developmental changes that the course of flowering. In this paper we follow the
occur in these tissues during the reproductive changes in peach ovular structures from anthesis to
process have been largely neglected. The fact that in fertilization or degeneration paying special attention
most angiosperm species the embryo sac is enclosed to nutritional and isolation processes.
in a well differentiated ovular structure, suggests
that these tissues may have a role in development. MATERIALS AND METHODS
It has been tacitly assumed that these tissues have The plant material
a protective role since the embryo sac is buried in
Peach flowers cv ' Sudanell' were used in this
them and a cuticular layer covers most ovules (Esau,
experiment. To make the flowers unattractive to
1977). On the other hand, Bueli (1952) suggested
insects and to avoid self-pollination flowers w-ere
that ovular tissues might act as a source of nutrients.
emasculated and their petals removed one day before
In many species the nucellus is considered to be a
anthesis (Free, 1964). At anthesis a group of flowers
food storage reservoir, thus being a remote source of
was left unpollinated and a different group was
photoassimilates (Panchaksharappa & Rananaware,
pollinated with poUen from cv 'Baby Gold 9'.
1976; Prasad, 1976; Bhandari, Bhargava & Sach-
deva, 1985). Likewise, the integuments also have
Microscope preparations
been considered a source of reserves in many species
(Mogensen, 1973; Sehgal & Gifford, 1979; Hawker Pistils from both treatments were fixed every
& Buttrose, 1980). A study of the changes in the alternate day from anthesis until 21 days later and
528 A. Arbeloa and M. Herrero

Figure 1. Longitudinai section of an ovule at anthesis. The outer (oi) and inner (ii) integuments envelop the
nucellus (n). JB4 embedded. PAS + Toluidine blue stain. X 170. Figure 2. Mature embryo sac 12 days after
anthesis showing the synergids; cytoplasm and nucleus are at the micropylar pole, and a large vacuole (v) is
located in the opposite poW. Paraffin embedded. Gerlach stain, x 1250, Figure 3. Same embryo sac showing
the egg and central cells. The egg cell (ec) contains a large vacuole towards the micropyle. The polar nuclei
(arrow) within the central cell (cc) have already fused. Paraffin embedded, Gerlach stain, x 1175. Figure 4,
Column of cells (arrow) degenerate at the nucellar tip extending from the micropyle (m) to embr>'o sac (es).
Paraffin embedded. Gerlach stain, x 530. Figure 5. Pollen tube (arrow) growing through the micropyle and
reaching the nuceWar tip (nt) 19 days after pollination. Paraffin embedded, Calcofiuor stain, x 490. Figure 6.
Embryo sac 19 days after pollination. The egg ceil (ec) and central cell (cc) contain starch reserves (arrows) in
their cytoplasm, the synergid (s) has degenerated. JB4 embedded. PAS + Toluidie blue stain, x 560,
 Ovule development in peach 529

12'

Figure 7. Nucellar tip stained with auramine. External cells of nuceilus and the adjacent cells of the inner
integuments have culinized walls (arrows) at anthesis. JB4 embedded, x 550. Figure 8. Nucellar tissue stained
for starch, 12 days after anthesis. Starch reserves are mainly present at the nucellar tip (nt), in central areas of
the nuceilus and inside the embryo sac (es). Paraffin embedded. IKI stain, x 220. Figure 9. Nucetlar tip of a
fertilized ovule, 28 days after pollination, stained with auramine. Cutin is present not only in the external walls
surrounding the nuceMus but also between cells, A young embryo (em) can be seen inside the embryo sac.
Paraffin embedded, x 630. Figure 10. Nucellar tip (nt) showing the external cells with a thick wall (arrow)
towards the micropyle. Inner integumentary- cells (ii) are degenerate and deeply stained with safranin. Paraffin
embedded. Gerlach stain, x 550, Figure 11. Unstained ovule seen with fluorescent light. Inner integumentary
cells surrounding the micropyle (m) are degenerate and show autofluorescence, Paraffin embedded, x 205.
Figure 12. Outer integumentary cells (oi) projecting towards the obturator (ob). These cells show PAS-positive
staining (arrows) when a pollen tube lies in close proximity. JB4 etnbedded. xS30.
530 A. Arbeloa and M. Herrero
then on days 28 and 36 after anthesis. Five pistils per
treatment were fixed in formalin - acetic acid-
alcohol (FAA), dehydrated in a tertiary butyl alcohol
series and then embedded in paraffin wax. In
pollinated flowers, five extra pistils were similarly
fixed on the same days to observe pollen tube
growth. Additionally five pistils per treatment were
fixed at weekly inter\-als in 2-5 % glutaraidehyde in
003 M phosphate bufTer (Sabatini, Bensch & Barr-
nett, 1963), dehydrated in an ethanol series and
embedded in JB4 plastic resin (Polysciences Inc).
Resin-embedded specimens were sectioned at
2/im and stained with periodic acid-SchifPs reagent
(PAS) for insoluble carbohydrates (Feder & O'Brien,
1968), with calcof^our (Hughes & McCuliy, 1975) for
cellulose, and with PAS counterstained with tol-
uidine blue (Feder & O'Brien, 1968) for general
histological observations.
Paraffin-embedded material was sectioned at
10/.im and stained with safranin, crystal violet and
light green according to Gerlach (1969), with IKI
for starch reserves (Johansen, 1940) and with
auramine O (Heslop-Harrison, 1977) for cutin.
Autofiuorescence was monitored in these sections
with epifiuorescence and with a filter combination
violet-ultraviolet (exciting filter BP3 55-425 and
barrier filter LP460).
Pollen tube growth was observed in squash
preparations until the time of arrival of the tube at
the base of the style. Pistils previously autoclaved in
5 per cent sodium sulphite to soften the tissues
(Jefferies & Belcher, 1974), were stained with 0-1 per
cent aniline blue in 01 N K3PO4 for the presence of
callose (Linskens & Esser, 1957). Penetration of the
pollen tube into the ovule was observed on paraffin
sections stained in a similar manner.
RESULTS
Embryo sac
The ovule of peach is crassinucelate and bitegmic
(Fig. 1). The fully developed nucellus encloses the
emhryo sac and is covered by two integuments that
are distinguishable only at the tnicropylar end where
the outer integument overtops the inner. At anthesis
the embryo sac is immature and the megagame-
tophyte undergoes a similar developmental process
in both pollinated and unpollinated flowers. Embryo-
genesis is of the Po/v^oKum type (Maheshwari, 1950).
During the 12 days after anthesis, the megaspore
gives rise to an eight nucleate embryo sac. Synergids
are well developed with a vacuole at the chalazai end
(Fig. 2); the egg cell sits near them and contains a
vacuole at the micropylar end (Fig- 3). Antipodals
are ephemeral and degenerate three days after
formation. One day later the polar nuclei fuse (Fig.
3) and the synergids begin to degenerate. The pollen
tube reaches the nucellus, penetrates through a
previously degenerate column of cells (Eigs 4, 5) and
enters the embryo sac via the degenerate synergids.
Eertilization occurs approximately 19 days after
pollination. Following fertilization, the fused polar
nuclei quickly divide forming free nuclear endo-
sperm. The embryo attains a globular shape of about
eight cells one week after fertilization and seven
weeks later it contains two incipient cotyledons.
By the time of fertilization the embryo sac
elongates and reaches the chalazal end of the
nucellus. As the embryo sac matures starch accum-
ulates both in the central and in the egg cells (Eig. 6).
Eollowing fertilization, starch disappears progress-
ively from the embryo and the endosperm.
Nucellus
The embryo sac is inside a conspicuous nucellus
comprising several layers of cells. The nucellus is
surrounded by a thick layer that contains cutin since
it stains with auramine O (Eig. 7). This layer is
continuous around the nucellus except at the chalazal
end. At the nucellar tip, the cells are rich in starch
reserves (Eig. 8) and maintain their structure
throughout ovule development. In growing ovules
starch is also present in a central area between the
embryo sac and the chalaza. This area follows the
path through which the embryo sac will elongate
(Eig. 8).
Eollowing fertilization, the starch in the nucellar
tip cells gradually disappears. At the same time the
cutin layer that separated the nucellus from the
integuments becomes restricted to the micropylar
area. The cutin that surrounds the nucellar tip
remains in place and it accumulates further between
the cell walls of this area and towards the micropyle
(Fig. 9).
In the first three weeks following anthesis, both in
unpollinated and pollinated flowers, the ovule in-
creases in length from 400 to 1200/im.
Integuments
In peach ovules the inner and outer integuments are
clearly differentiated in the vicinity of the micropyle
(Eig. 1). The micropyle is simple at anthesis and
then it twists and elongates. At anthesis cutin is also
present between both integuments and along the
inner surface of the micropyle (Eig. 7). The in-
tegumentary cells lining the micropylar canal contain
abundant starch at anthesis. These reserves soon
disappear. As these cells subsequently degenerate
they stain deeply with safranin (Eig. 10) and display
autofiuorescence (Eig. 11). In the micropylar region
of the outer integument after anthesis large vacuo-
lated cells grow outwards towards the obturator
forming the exostome. These cells are rich in starch
reserves. In the ovules approached by a pollen tube,
at the same time that the starch disappears, PAS
stained material (Eig. 12) that also stains with
calcofiuor appears in these cells.
 Ovule development in peach S31

V 14

Figure 13. Unpollinated ovule 36 days after anthesis. An elongated embryo sac (es) is still visible. Paraffin
embedded. Gerlach stain. x216. Figure 14. Nucellar tip (nt) of unpotlinated ovule 28 days after anthesis
stained with auramine O. Cutin is present not only in the external walls surrounding the nuceilus but also in
the intercellular spaces. The egg cell (ec) is still apparent. Paraffin embedded. x295.

Starch accumulates in both inner and outer
integumentary cells from anthesis and throughout
ovule development up until fertilization. After
fertilization starch progressively disappears and both
integuments degenerate.
Ovule degeneration
Peach pistils contain two ovules although only one
seed is normally produced. There is a delay of three
days between tbe maturation of the two ovules in the
same Bower. One of the ovules stops growing and
degenerates within two weeks of anthesis while the
other one continues to grow until fertilization or
degeneration. Both ovules follow a similar pattern of
degeneration. The first clear symptom is the ap-
pearance of callose, identified with aniline blue stain,
in a localized region at the chalazal end of the ovule.
It then spreads to the whole ovule. Starch disappears
from the ovular tissues, the nuceilus shrinks and
separates from the integuments and the integuments
collapse. In unpollinated flowers, pistil degeneration
manifested by the collapse of both ovules starts 19
days after anthesis, and degeneration is observed in
most of the pistils 28 days after anthesis. However, a
proportion of the ovules were still viable 36 days
after anthesis (Fig. 13). Although lacking embryos or
endosperm, they showed the same changes in starch
and cutin distribution (Fig. 14) as well as embryo sac
elongation as in pollinated fiowers.
DISCUSSION
Development of ovule
It has been tacitly assumed that the tissues of the
ovule play a passive role enveloping the ennbryo sac.
The present work clearly shows that the ovule
continues to develop throughout the lifespan of the
flower. Apart from the formation of a mature
megagametophyte, ovule development includes a
tripling in the size of the nuceilus and integutnents,
from anthesis until fertilization. Changes in the
different tissues enveloping the embryo sac may play
important roles in fertilization and early postfertili-
zation development.
Embryo sac development and fertilisation
At anthesis the embryo sac is immature and
maturation is not synchronous between the two
ovules of a flower. While early reports (Bradbury,
1929; Tukey, 1933) assumed that in Prunus mega-
gametophyte maturation coincided with anthesis,
later work has also recorded a delay m tnega-
gametophyte maturation in other Prunus species
(Jefferies, 1975; Pimienta & Polito, 1983). However,
despite this delay, by the time the pollen tubes reach
the embryo sac (Herrero & Arbeloa, 1989) develop-
ment is complete. Once the pollen tubes enter the
ovary, by tracking along the obturator (Arbeloa &
Herrero, 1987), they meet the exostome since
during ovule development the outer integuments
project outwards towards the obturator. This integu-
mentary growth may facilitate pollen tube penetra-
tion into the micropyle as it brings both structures
into closer proximity. However, physical proximity
probably is not the only requirement for pollen tube
entry into the ovule; some kind of chemotropic
stimulus may also be required (Herrero, Arbeloa &
Gascon, 1988). Further research is now needed to
understand the presence of PAS positive material in
the external cells of the exostome associated with the
pollen tube.
After the pollen tube enters the exostome, it grows
in the micropylar inner canal and enters the nuceilus.
The column of cells that degenerates in the nucellar
532 A. Arbeloa and M. Herrero
tip may be related to pollen tube growth since this
would facilitate pollen tube passage from the micro-
pyle to the embryo sac. A similar column of
degenerate cells was first observed by Jensen (1969)
in cotton and later in other species (Wilms, 1981;
Tilton & Lersten, 1981; Pimienta & Polito, 1983).
Embryo sac protection
The thick layer of material surrounding the nucellus
is almost certainly cutin since it stains with auramine
O (Considine & Knox, 1979; Heslop-Harrison &
Heslop.-Harrison 1981; Boland & Sedgley, 1986)
and because of the epidermal origin of these ovular
tissues (Esau, 1977). While the presence of an
external cuticular layer may act as a protective and
transpiration barrier, the possible functional signi-
ficance of an inner cuticle in ovules (Ryser et al.,
1988) is far from clear. A cuticular layer has been
described in the ovules of various species sur-
rounding either the micropyle (Tilton, 1980; Tilton
& Lersten, 1981), nucellus (Wilms, 1981; Ryser
et al., 1988; Bruun & Olesen, 1989), or the embryo sac
(iVlogensen, 1981). In peach it surrounds the nuc-
ellus leaving the chalazal end free. During ovule
development it disappears from lateral regions of the
nucellus becoming relegated to the micropylar half
of the nucellus and accumulates in the intercellular
spaces at the nucellar tip. This accunnulation at the
nucellar tip could be related to embryo sac isolation.
Cutin accuinulation would play a protective role
avoiding later pathogen infection. Embryo sac iso-
lation has also been pointed out in Ornithogalum
(Tilton, 1980) although mediated by a different
mechanism since in this genus a plug occludes the
micropyle during embryogenesis.
Early embryo sac nutrition
Embryo sac nutrition appears to be via the chalazal
end of the ovule (Mogensen, 1973). However, the
way the ennbryo sac derives nutrition during early
development, when it elongates until it reaches the
chalazal end, is still unclear. Results herein indicate
that the ovule's own reserves could account for this
early phase of development. The disappearance of
starch at the nucellar tip concomitant with early
embryo sac elongation may be associated with early
nutrition of the embryo sac and with providing the
reserves required for the elongation process. Alter-
natively, Buell (1952) pointed out that the reserves
located at the nucellar tip might be used during
pollen tube growth along the nucellus. This idea
gathers further support from the suggestion by
Herrero and Dickinson (1979) that the pollen tube
grows in an heterotrophic manner through the style
and along the obturator (Arbeloa & Herrero, 1987).
However, it is unlikely that this is the case in the
peach nucellus since there were no clear changes in
starch distribution accompanying pollen tube pas-
sage throughout the nucellus. Likewise, early
embryo sac nutrition probably relies on starch
reserves that accumulate in the integuments and
embryo sac of mature ovules; these reserves are also
depleted following fertilization.
In this respect, cutin modifications may be
significant in allowing communication between, or
isolating, particular structures. A cuticle around the
nucellus, except at the chalazal end, would act as an
impermeable barrier to the passage of nutrient
reserves, supporting the idea of a nutrient pathway
through the chalaza (Zhang & Zheng, 1988; Olesen
& Bruun, 1990). In peach it would facilitate starch
consumption from the nucellar tip and the embryo
sac itself. Afterwards., the disappearance of the cutin
that separates the nucellus from the integuments
could be related to the passage of carbohydrate
reserves from the integuments to the nucellus and
embryo sac. This idea is further supported by the
fact that, following the disappearance of this cutin
barrier, starch also disappears from the integuments.
The significance of ovule development
Results herein indicate that the ovular tissues in
peach, far from being passive structures that envelop
the embryo sac, undergo a number of changes during
the lifespan of the flower that may play an important
part in the reproductive process. It is clear that
pollination triggers a number of biochemical and
developmental changes in the ovary (Fuller &
Leopold, 1975; Deuremberg, 1976) and even in the
final stages of megagametophyte development (Pimi-
enta & Polito, 1983; Herrero and Gascon, 1987).
Likewise, in other species the entire ovule matu-
ration process depends on pollination (Law & Yeung,
1989; Yeung & Law, 1989). However, in peach, the
fact that most of the changes reported here take place
in a similar way in unpoJlinated flowers indicates that
they are not triggered by pollination or fertilization
but rather they are intrinsic to ovule development
and play subsequent roles in fertilization and early
embryo sac development.
ACKNOWLEDGEMENTS
Thanks are due to CICYT, to IN IA and to the U.S.-Spain
Joint Committee for Scientific and Technological Co-
operation for financial support.
REFERENCES
ARBELOA, A. & HEHREBO, M . (1987). The significance of the
obturator in the control of the pollen tube entry into the ovary
in peach [Prunus persica). Annals of Botany 60, 681-685.
BHANDARI, N , N . , BHABGAVA, M . & SACHDEVA, A. (1985), The
mature ovule of Papaver somniferum. A histochemical study.
Phytomorphology 35, 111-119.
BOLAND, D . J. & SEDGLEY, M . (1986). Stigma and style mor-
phology in relation to taxonomy and breeding systems in
 Ovule development in peach
Eucalyptus and Angophora (Mytaceae) Australian Journal of
Botany 34, 569-584.
BOUMAN, F, (1984), The ovule. In: Embryology of Angtosperms
(Ed. by B, M, Johri), pp, 123-153, Springer-Verlag, Berlin,
BRADBURY, A, D, (1929V A comparative study of the developing
and aborting fruits of Prunus cerasus. American Journal of
Botany 16, 525-542.
BRUUN, L . & OLESEN, P , (1989), A structural investigation of the
ovule in sugar beet Beta vulgaris: The micropylar nuceilus,
Nordic Journal of Botany 9, 81-87,
BuEiX, K. M, (1952). Developmentai morphology in Dianthus.
II. Starch accumulation in ovule and seed, American Journal
of Botany 39, 458-1-67,
CoNSIDiNE, J, A. & KNOX, R . B, (1979), Development and
histochemistry of the ceils, cell walls and cuticle of the dermal
system of fruit of the grape, Vitis vinifera L, Protoplasma 99,
347-^65,
DEI'REMBERG, J, J, M , (1976), Activation of protein synthesis in
ovaries from Petunia hybrida after compatible and incompatible
pollination, Acta Botanica Neerlandica 25, 221-226,
EsAi:, K, (1977). Anatomy of Seed Plants. J, Wiley & Sons I n c ,
New York.
FEDEK, N , & O'BRIEN, T . P. (i968). Plant microtechnique: some
principles and new methods. American Journal of Botany 55,
123-142.
FREE, J, B, (1964), Comparison of the importance of insect and
wind pollination of apple trees. Nature 201, lld-lH.
FL'LLER, G , L . & LEOPOLD, A, C, (1975). Pollination and the
timing of fruit set in cucumber. Hortscience 10, 617-619,
GERLACH, D , (1969), A rapid aafranine-crystal violet-light green
staining sequence for paraffin sections of plant materials. Stain
Technology 44, 210-211,
HAWKER, J. S, & BUTTROSE, M , S, (1980). Development of the
almond nut [Prunus dulcis (Mill.) D, A, Webb], Anatomy and
chemical composition of fruit parts from anthesis to maturity.
Annals of Botany 46, 313-321,
HERRERO, M . & .ARBELOA, A, (1989). Influence of the pistil on
pollen tube kinetics in peach {Prunus persica). American Journal
of Botany 76, 1441-1447,
HERRERO, M , , ARBELO,^, A, & GASCON, M , (1988). Pollen-pistil
interaction in the ovary in fruit trees. In: Sexual Reproduction
in Higher Plants (Ed, by M, Cresti, P, Gori & F. Pacinj), pp,
297-302, Springer-Verlag, Berlin,
HERRERO, M , & DICKLNSON, H , G , (1979), Pollen-pistil incom-
patibility in Petunia hybrida: changes in the pistil following
compatible and incompatible intraspecific crosses. Journal of
Cell Science 36. 1-18,
HERREKO, M , & GASCON, M , (1987). Prolongation of enibryo sac
viability in pear (Pyrus commums) following pollination or
treatment with gibberellic acid. Annals of Botany 60, 287-293.
HESLOP-HARRISON, V, (1977), The pollen stigma interaction:
pollen tube penetration in Crocus. Annals of Bol any 4\, 9 \i-922.
HESLOP-HARRISO.N, Y , & HESLOP-HARRISON, J, (1981), The
digestive glands of Pinguicula: structure and cytochemistry.
Annah of Botany 47, 293-319.
HUGHES, J, & MCCUI.LV, M . E . (1975), The use of an optical
brightener in the study of plant structure. Stain Technology 50,
319.
JEFFERIE.S, C , J, (1975), Floral biology and fruit development in the
European plum. PhD thesis, University of Bristol, Research
Station of Long Asthon,
JEFFERIES, C , J, & BELCHER, A. R. (1974), A fluorescent bnghtener
used for pollen tube identification in vivo. Stain Technology 49,
199-^202,
JENSEN', W , A, (1969), Cotton embryogenesis: pollen tube de-
velopment in the nuceilus, Canadian Journal of Botany 47,
383-^385.
JOH,^NSEN, D, A. (1940). Plant Mierotechnique. McGraw-Hill,
New York.
533
LAW, S, K, & YEL^NG, E . C , (1989), Embryology of Calypso
bulbosa. I, Ovule development. American Journal of Botany 76,
1668-1674,
LiLiEN-KiPNis, H, & LAVEE, S, (1971), Anatomical changes
during the development oi" 'Ventura' peach fruits. Journal of
Horticultural Science 46, 103-110.
LiNSKENS, H, F, & EssER, K. (1957), Uber eine spezifische
Anfarbung der Pollenschlauche im Griffel und die Zahl
Kallosepfropfen nacb Selbstung und Fremdung. Naturwissen-
schaften 44, 16.
MAHESHWARI, P . (1950). An Introduction to the Embryology of the
Angiosperms. McGraw-Hill I n c , New York,
MoGENSEN, H. L, (1973), Some histochemical, ultrastructural
and nutritional aspects of the ovule of Quercus gambeiii.
American Journal of Botany 60, 48-54,
MoGENSEN, H, L, (1981). Ultrastructural localization of aden-
osine triphosphatase in the ovules of Saintpaulia ionantha
(Gesneriaceae) and its relation to synergid function and embryo
sac nutrition. American Journal of Botany 68, 183-194,
OLESEN, P . & BRUUN, L , (1990), A structural investigation in
sugar beet {Beta vulgaris): integuments and micropyle, Nordic
Journal of Botany 9, 499-506,
PANCHAKSHARAPPA, M , G , & RANANAWARE, M , B , (1976). Con-
tribution to the physiology of ovule and seed development in
Dioscorea bulbifera L . A histochemical s t u d y . I n : Physiology of
Sexual Reproduction in Flowering Plants, p p , 2 3 7 - 2 4 8 . Kalyani
Publications, New Delhi.
PIMIENTA, E , & POLITO, V. S, (1983), Embryo sac development in
almond [Prunus dulcis (Mill) D, A, Webb] as affected by cross-
self- and non-pollination, Annah of Botany 51, 469-479,
PRASAD, K , (1976), Histochemistry of nuceilus and endosperm in
certain species of Cruciferae, In: Physiology of Sexual Re-
production in Flowering Plant, pp, 225-230, Kalyani Publi-
cations, New Delhi,
RAGLAND, C , H , (1934), The development of tbe peach fruit with
special reference to split pit and gumming. Proceedings of the
American Societv for Horticultural Science 31, 1-21,
RYSER, V., ScHORDERET, M,, JAUCH, V, & MEIER, H . (1988),
Ultrastructure of the 'fringer-layer': the innermost epidermis
of cotton seed coats, Protoplasma 147, 81-90.
SABATINI, D , D , , BENSCH, K , & B.ARRNETT, R . J. (1963). Cyto-
chemistry and electron microscopy. The preservation of cellular
ultrastructure and enzimatic activity by aldehyde fixation.
Journal of Cell Biology 17, 19-58,
SEHGAL, C, B , & GiFFORD, E. M, JR (1979), Developmental and
histochemical studies of the ovules of Nicotiana rustica L.
Botanical Gazette 140, 180-188,
TiLTON, V, R, (1980), The nucellar epidermis and micropyle of
Ornithogalum caudatum (Liliaceae) with a review of these
structures in other taxa. Canadian Journal of Botany 58,
1872-1884.
TiLTON, V, R. & LERSTEN, N . R, (1981), Ovule development in
Ornithogalum caudatum (Liliaceae) with a review of selected
papers on Angiosperms reproduction. Ill Nuceilus and mega-
gametophyte. New Phytologist 88, 477-504,
TUKEY, H , B , (1933), Embryo abortion in early ripening varieties
of Prunus avium. Botanical Gazette 94, 433-468,
WiLMS, H, J, (1981). Pollen tube growth tbrough nuceilus into
embryo sac of Spinacia. An ultrastructural investigation, Acta
Societas Botanicorum Poloniae 50, 191-194.
WiLLEMSE, M. T, M. & VAN WENT, J , L , (1984), The female
gametophyte. In: Embryology of Angiosperms, (Ed. by B, M.
Johri) pp, 159-191, Springer-Verlag, Berlin,
YEUNG, E , C . & LAW, S, K . (1989), Embryology of Epidendrum
ibaguense. I, Ovule development, Canadian Journal of Botany
67, 2219-2226.
ZHANG, 2 . J, & ZHENG, G , J, (1988). Translocation of uranin
within the living ovules of Vanilla. Acta Botanica Sinica 30,
490-493.
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