~s
in the Phloem of Salix
ughout, intermingled with othe
iceably in the circled area. An r
back~rohund m.aterial. In this view
asm 1~ s own m Plate 2B. x 23
:ed With tubular and non-tubular
ton-tubular material in a clump
:spectively. x 9000.
at
f tubular type only a small part of
8 86<)-72, 1974
,.t/111· B0 t . 3 '
occurrence and Structure of Epicuticular Wax on
the Juice Vesicles of Citrus Fruits
A. FAHN, I. SHOMER, and I. BEN-GERA 1
Department of Botany, The Hebrew University of Jerusalem, Jerusalem, Israel
Received: 19 November 1973
ABSTRACT
1b surface of the juice vesicles of some citrus fruits was studied with the aid of the transmission and
e ing electron microscopes. The epidermis of the vesicles is covered with a thin cuticle on top of which
:found wax secretions .of various str?~turall?attez:ns. It is as~umed that the secreted epicuticular wax
serves as an adhesive which holds the JUICe vesicles m each fruit segment together.
INTRODUCTION
The citrus fruit develops from a syncarpous gynoecium with axile placentation. The fruit
Jocules are filled with stalked spindle-shaped juice vesicles. These vesicles develop from
cells of the inner epidermis and subepidermal layers of the pericarp, i.e. from the endoc;arp. Each juice vesicle consists of an external layer of elongated epidermal cells which
enclose large thin-walled juice cells (Fahn, 1974). The juice vesicles of each locule adhere
to each other and form, together with the endocarps of each carpel, the fruit segments.
One of the problems of citrus fruits to the canning industry is the loss of adherence of
the juice vesicles to each other as a result of the commonly-used methods of preservation.
Experiments in applying relatively high temperatures (50-70 oq or organic solvents and
shaking the fruit segments have shown that the juice vesicles readily separate from each
other (Plate IA, B). To learn how to prevent this separation it was first necessary to find
out what agent keeps the vesicles together.
In this paper we present the results of an anatomical study of the surface of the
vesicles.
MATERIALS AND METHODS
For examination, fruits of the following Citrus species growing in the coastal plain of
Israel were used: C. paradisi Macf. cv. Marsh Seedless (seedless grapefruit), C. sinensis
Osbeck cv. Shamouti (Shamouti orange), and C. sinensis Osbeck cv. Valencia (Valencia
orange).
Fruits of various stages of development were studied: ripe-stored, young, still growing
grapefruits collected on 14 and 21 August and I I September 1973; developing Shamouti
oranges collected on 14 August and 11 September 1973; developing Valencia oranges
collected on 21 August 1973. For observations using the transmission electron microscope portions of fruit segment, consisting of parts of juice vesicles, were fixed in 3·5 per
cent glutaraldehyde in o·r M buffer cacodylate (pH 6·9) for 2 h, washed in the buffer and
kept ~n it overnight, post fixed in buffered 2 per cent Os0 4 for 2 h, washed in buffer for
15 mm and dehydrated in ethanol. All the above treatments were carried out at room
~mperature. The mate_rial was embedded in Spurr's low-viscosity embedding medium
tJurr, 1969). The sections were prepared with a LKB ultramicrotome, using glass
IVes, and viewed and photographed with a Philips 300 electron microscope.
1
Agricultural Research Organization, Division of Food Technology, Bet Dagan, Israel.
Fahn, Shomer, and Ben-Gera
For examination with a scanning electron microscope, portions of fruit segllle
taining several incised juice vesicles were used. These portions were fixed and deh~ ~
using the above-mentioned method. Before examination, several juice vesicles ~
0
dehydrated material were separated so as to expose their surfaces, especially t ~
facing the free intervesicular spaces. The vesicles were then attached to standard ~
dried and coated with gold in a vacuum evaporator, and examined with a Cam~~
Stereoscan S4 microscope.
·"'F
RESULTS
I
Hand-cut sections of grapefruit juice vesicles, stained with Sudan IV, revealed that !be
epidermis of the vesicles was covered with a thin cuticle.
Studies using the transmission electron microscope confirmed the presence of a cu ·
and revealed that at least part of the cuticle, in .the sp~ce bet":een neighbouring vesi~ \
was covered by a layer of electron-dense matenal. This matenal appeared in two follll, :
homogeneous and granular, and was thicker in the corners where more than two vesicb
meet and where free space was available (Plate IC, D).
Chromatographic analysis of this material, which was washed away from the petlc4
fruit segments by organic solvents, showed that the material between the vesicles was
waxy and similar in composition to the wax covering the fruit surface and, in great pan,
to the wax of the leaves of other plants, e.g. cabbage. Results of this investigation will ~
discussed in more detail in a separate paper.
Assuming the material covering the juice vesicles to be an epicuticular wax we hall
studied its structure in the grapefruit and in two varieties of orange.
The patterns of the wax structure as seen on the edges of juice vesicles exposed to rne
intervesicular spaces, were found to vary. Different structures occurred even in one and
the same species.
a
'
il
l
f
d
n
I\
a
(1
(1
o:
gt
Grapefruit
In the grapefruit three types of wax structure were observed: (I) continuous laym
with papillae-like protrusions (Plate 2A) which, as seen in cross section, were local ir!llations of the wax layer (Plate IE) and large bizarre-shaped protrusions (Plate 2B). In some
cases the papillae-like protrusions formed aggregate structures (Plate 2c). ~ometimestlat
sheaths of wax occurred on top of the papillae-like protrusions (Plate 2D); (2) dispersed
groups of rod-like structures (Plate 3A, B). These structures sometimes appear to be
melted and fused to form bizarre masses (Plate 3c) ; (3) aggregate coating which consisted I
of superimposed fibrillar or thread-shaped structures forming a complicated three dimensional meshwork (Plates 3D, 4A).
bl
Sl
(I
lc1
I
Oranges
On the juice vesicles of oranges wax secretion in the form of a three dimensional mesh·
work of thread-shaped structures was observed (Plate 4B, c). The meshwork pattern may
be continuous with flat homogeneous layers (Plate SA, B). It may also occur both in continuation and in alternation with such layers, forming a complicated storied structure
of several strata (Plates 4D, SB). Several layers of platelets with irregular borders, so:
times giving the impression of being a result of breaking of larger sheaths, were observ
especially in the Shamouti oranges (Plate sc, D).
DISCUSSION
With the aid of the transmission electron microscope a distinct cuticle, with an addit~
layer of electron dense material on top of it, was shown to occur on the epidermal ce
juice vesicles of citrus fruits.
I
th
ti~
be
fo:
of
by
Slr
ho:
Sb:
]
I
ext
clu
tax
Th
tee
zd Ben-Gera
icroscope, portions of fruit
fhes.e p~rtions were fixed and
mmmatw~ .several juice vesicles
expose t err surfaces, especial!
es were then attached to stand y
)rator, and examined with a arct
'S
taine~ with Sudan IV, revealed
n cutiCle.
cope confirmed the presence of
he sp~ce between neighbouring a
:!. Thrs material appeared in two
le corners where more than tw 0
C, D).
bich was washed away from th
e
.the material between the
rmg the fruit surface and, in great
tge. Results of this investigation
:les .to. be an epicuticular wax we
vanetles of orange.
~ edges of juice vesicles exposed
1t structures occurred even in
were observed: (I) continuous
seen in cross section, were local
.h aped protrusions (Plate 2B). In
·
e structures (Plate 2c).
! protrusions (Plate 2D); ( 2)
: structures sometimes appear
(3) aggregate coating which
s forming a complicated three
be form of a three dimensional
te 4B, c). The meshwork .-~··-·~·.,
iA, B). It may also occur both in
aing a complicated storied
~telets with irregular borders,
mg of larger sheaths, were
a distinct cuticle, with an
rn to occur on the epidermal
Occurrence and Structure of Epicuticular Wax on Juice Vesicles
871
'd r c 68) mentioned previously that the epidermal cells of the juice vesicles
schiJel e 19
h .
'd '
waxy cuticle on t err outer sr e .
.
.
.
·have a ·cal and physical methods used by the present authors m an~ther re~earch mve~tl~heCnot yet published) suggest~d that the electr~n dense mater~a~ outsr~e the cuticle
gauon After applying an orgamc solvent to a frurt segment the JUICe vesrcles separate
was w~f; (Plate 1A, B). It can thus be assumed that the wax represents the adhesive by
pr~m: the juice vesicles of each segment keep together.
whrc hologically, many types of epicuticular wax structure ha~e been described ~y
0
Ma?'authors (DeBary, 1877; Amelunxen, Morgenroth, and Prcksak, 1967; Martm
severJ ·per 197o). One of the structural patterns found by us in the grapefruit (Plate
and )u.m Unilar to that of the wax layer with papillae of the Papaver orientale capsule as
ZA-D IS S
"
dm
. t he grapef rmt
.
'b d by Amelunxen eta!. (1967). Another structura1pattern 1oun
~es~nt ~f dispersed groups of rod-like structures (Plate JA, B). This pattern resembles
IS~~ ~f the wax structure of some Eucalyptus leaves (e.g. E. perriniana) described by
~allam and Chambers (1970). The elements of the above Eucalyptus wax, h~wever, were
described by Hallam and Chambers as tubes, and not as rods as seen by us m the grapefruit.
· consrstmg
· · o f s~pe!r~pose
·
d th rea d - or ro d -s h ape d st rucThe aggregate wax coa~mg
tures of the citrus juice vesrc~es (Plates JD, 4A) rs srmrlar to the wax of Eucalyptus globulus
and Atriplex hortensis descnbed by Amelunxen eta/. (1967).
Wax in the form of plates arranged in one layer or in several superimposed layers
(Plate 5c, D) as well as in continuation of aggregate meshwo:ks of thread-shape~ ~ax
(Plate SA, B), are common. Of SJ?ecial interest is the several-stoned wax pattern consrstmg
of intermingled layers of complicated meshworks of thread-shaped structures and homogeneous flat wax layers (Plates 40, SB).
Platelets which according to their shape and arrangement appear to have formed by
breaking up of larger wax sheaths were observed especially on the juice vesicles of the
Shamouti orange.
Wax deposition in the fonn of platelets was also observed by Albrigo and Brown
(1970) on the fruit surface of the Valencia orange. Albrigo (1972) assumed that the platelets are a result of cracking of continuous wax layers during the growth of the fruit.
The above structural patterns of wax secretions of citrus juice vesicles were seen in
the regions facing free intervesicular spaces. In the regions where the vesicles pressed
tightly on each other one thin wax layer occurred, apparently formed by the two neighbouring vesicles.
As to the relation between taxonomic status and the structural patterns of the wax the
following can be concluded:
I. Both in the grapefruit and in the oranges, the pattern of the complicated meshwork
of thread-shaped or rod-shaped structures occurs.
2. The pattern of a continuous layer of wax with papillae-like protrusions was found
by us only in the grapefruit.
J. Homogeneous layers of wax, in combination with meshworks of thread-shaped
structures or without them, were observed in the two orange varieties examined. The
homogeneous wax layers were often in the form of superimposed platelets, as seen in the
Shamouti orange.
It should be mentioned, however, that as the patterns of wax structure vary to a great
extent even within the same species and variety, it will only be possible to draw final conclusions as to taxonomic differences after examination of many more specimens of the
taxa involved.
ACKNOWLEDGEMENT
Thanks are due to the staff of the SEM Unit of the Hebrew University of Jerusalem for
technical assistance.
Fahn, Shomer, and Ben-Gera
'""~~
LITERATURE CITED
Aco>uco,
L. G., '97'.
Uitrutructw-c
of'"'''"'"
the 'Valencia'
orange.
J. Am. Soc.
hort. Sci. 97, 761-5.'"d >tomata of dcwlopfug
''"~ ""
--and
470-2. G. E., 1970. Orange peel topography as affected by a preharvest plastic
Sci. 5,BROWN,
AMRDNX>N, F., MORG<NRorn, K., >nd P>CK.<AK, T., >967. Uol<m>ch<mgeo '" d" Epidc,""
Stereoscan-Elektronenmikroskop. Z. Pflanzenphysiol. 57, 79--95.
DEBARY, A., 1877. Vergleichende Anatomie der Vegetationsorgane. W. Engelmann, Leipzig.
FAHN, A., 1974. Plant Anatomy, 2nd edn. Pergamon Press, Oxford.
Bot. 18,
335-86.
HAU.AM,
N.D.,
Md
T. C., >970. Tire loaf""'"' of the gcn"' E"roiYJ>t"' L'Ffidtk,.
c.._,.,,
MARTIN, J. T., and JlTNIPER, B. E., 1970. The Cuticles of Plants. E. Arnold, London.
ScHNEIDER, H., 1968. The anatomy of Citrus. In The Citrus Industry, voi. ii, pp. I-85. Eds. W.
L. D. Batchelor, and H . J. Webber. University of California.
SmR,
A. R.,
A
cpozy re.<in embedding medium fot clcctson mioto"<>PY.'
struct.
Res.t9ii9.
26, 31-43.
low-,i~o,ity
EXPLANATIONS OF PLATES
PLATE I
An untreated fruit segment of a seedless grapefruit from which the endocarp was removed.
B.
As in A, but after immersion in CHC13 for a few seconds. X o·8.
C-E. Electron micrographs of portions of cross sections of juice vesicles, showing outer cell walls
the epidermis,
cuticle
and epicuticular wax secretion (wa) in the space between the
c. Shamouti
orange.
X 28(c)
ooo
D.
Valencia orange. x 16 ooo
E. Marsh seedless grapefruit. x IO ooo
A.
A
. x 65ooelectron
Scanning
B. X58oo
c.
D.
PLATE 2
micrographs of the surface of juice vesicles of the Marsh seedless grapefruit.
X 58oo
X4400
PLATE 3
A.
X2IOOelectron micrographs of the surface of juice vesicles of the Marsh seedless grapefruit.
Scanning
B. X5300
c . X2200
D.
X5500
PLATE 4
Scanning
electron
micrographs
of
the
surface
of
juice vesicles of Marsh seedless grapefruit (A),
Drange (B) and Shamouti orange (c, D).
A.
B.
c.
D.
x 6zoo
X5ooo
X 5500
X 2400
PLATE 5
Drange
(c,electron
D).
Scanning
micrographs of the surface of juice vesicles of Valencia orange (A, B) and
A. X IIOO
B. X2400
<:. X2400
D. X2500
1en-Gera
!TED
md stomata of developing leaves and
affected by a preharvest plastic spray.
Untersuchungen an der Epidermis lllit
7, 79--95.
Jrgane. W. Engelmann, Leipzig.
Oxford.
of the genus Eucalyptus L'Heritier.
'.mts. E. Arnold, London.
Industry, vol. ii, pp. 1-85. Eds. W.
ornia.
medium for electron microscopy. J.
PLATES
1
which the endocarp was removed.
s. X o·B.
;e vesicles, showing outer cell walls
(wa) in the space between the
'\-
of the Marsh seedless grapefruit.
of the Marsh seedless grapefruit.
I[
Marsh seedless grapefruit (A),
Jf Valencia orange (A, B) and
.
~-.
r
' '
·,~')
·, .,:_... ·~·.· ;~
fl'~ sH OM ER , AND BEN-GERA-Occurrence and Structure of Epicuticular Wax on the Juice
FA ·'
Vesicles of Citrus Fruits
PLATE 1
Ann. Bot. 38, 869-72, 1974
Thq
ultr.i(
largcj
trcatd
lit.H!
nppa~
n1ract
In th
(Lind
sing!
a pSC'
spora
pscud
situat
ocdu1
as ad
cxhibi
apex,
angioJ
struct1
Cu!tut
Youn~
The su
commi
outer, ,
The
minute
roughe
cerned.
fibrils <
Ann. Bot. 38, 869-72 1974