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Lebensm.-Wiss. u.-Technol. 37 (2004) 317–322
The microstructure of almond (Prunus dulcis (Mill.)
D.A.Webb cv. ‘Nonpareil’) cotyledon$
Clyde T. Younga,{, William E. Schadela, Harold E. Patteeb,*, Timothy H. Sandersc
a
Department of Food Science, North Carolina State University, Box 7624, Raleigh, NC 27695, USA
Department of Botany, USDA-ARS-SAA-MQRHU, North Carolina State University, Box 7625, Raleigh, NC 27695, USA
c
Department of Food Science, USDA-ARS-SAA-MQRHU, North Carolina State University, Box 7624, Raleigh, NC 27695, USA
b
Received 3 September 2003; accepted 4 September 2003
Abstract
Microstructure of almond (Prunus dulcis (Mill.) D.A. Webb cv. ‘Nonpareil’) cotyledon was observed with light, scanning and
transmission electron microscopy. The objective of this study was to characterise almond cotyledon surfaces as well as to describe
internal and subcellular organisation. The testa has an outer epidermis, which consists of relatively large thin-walled cells, which
range from 100 to 300 mm in width. The major portion of the testa consists of approximately 14–20 layers of flattened parenchymal
cells with the total thickness of the layers ranging from 80 to 120 mm. The remainder of the testa was comprised of a small amount of
vascular tissue. The embryo consisted primarily of parenchymal tissue with relatively thin cell walls (1–3 mm in thickness) and a small
amount of provascular tissue. Protein bodies up to 12 mm in width and spaces once occupied by lipid bodies up to 3 mm in width were
present in all cells of the embryo.
r 2003 Swiss Society of Food Science and Technology. Published by Elsevier Ltd. All rights reserved.
Keywords: Protein body; Lipid body; Light microscopy; Scanning electron microscopy; Transmission electron microscopy
1. Introduction
Almond (Prunus dulcis (Mill.) D.A. Webb) is one of
the most versatile of all tree nuts. Several superior
almond cultivars have been developed in California, the
most important being the ‘‘Nonpareil’’’ which accounts
for over 50% of the total California production
(Rosengarten, 1984).
The vast majority of almonds are sold shelled; shelled
almonds may be sold as whole natural almonds or may
be processed into numerous forms (Rosengarten, 1984).
Because almonds are further processed (e.g. cooked), it
is helpful to evaluate how such processes affect almond
cotyledon microstructure. Undesirable changes in mi$
The research reported in this publication was a co-operative effort
of the Agricultural Research Service of the United States Department
of Agriculture and the North Carolina Agricultural Research Service,
Raleigh, NC 27695-7643. The use of trade names in this publication
does not imply endorsement by the United States Department of
Agriculture or the North Carolina Agricultural Research Service of the
products named, nor criticism of similar ones not mentioned.
*Corresponding author.
E-mail address: harold [email protected] (H.E. Pattee).
{
Deceased.
crostructure may lead to undesirable alterations in
almond shelf-life, flavor and texture.
It is the purpose of this paper, therefore, to document
the microstructure of almond cotyledons as observed
with light, scanning and transmission electron microscopy (TEM). This study will serve as a reference for
future evaluation of the microstructural changes, which
occur as almonds are cooked or processed into other
forms such as paste or butter. Earlier work on the
microstructure of almond cotyledons has been limited to
light microscopy (LM) (Vaughan, 1970; Decke, 1982)
and to TEM of almond protein bodies (Lott & Buttrose,
1978).
2. Materials and methods
Almond cotyledons (Prunus dulcis (Mill.) D.A. Webb
cv. ‘Nonpareil’) were obtained from the California
Almond Growers Exchange. Tissue blocks (1 mm3) of
outer surface testa and cotyledon, mid-region cotyledon
and inner surface cotyledon were cut from the almonds
and fixed in Karnovsky’s fixative (Karnovsky, 1965) as
0023-6438/$30.00 r 2003 Swiss Society of Food Science and Technology. Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.lwt.2003.09.007
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modified by Young and Schadel (1989). The modified
fixative was prepared by mixing 25 ml of 8 g/100 ml
formaldehyde, 3.6 ml of 70 g/100 ml glutaraldehyde and
28.6 ml of 0.1 mol/l sodium phosphate buffer. The tissue
blocks were fixed under vacuum for 30 min at 23 C and
then fixed at atmospheric pressure for 48 h at 4 C.
Following six changes of 0.1 mol/l sodium phosphate
buffer (4 C, pH 7.0), the material was post-fixed for 1 h
in 1% osmium tetroxide in 0.1 mol/l buffer (4 C, pH
7.0) and dehydrated at room temperature at 15 min
intervals in a graded series of aqueous ethanol (10%,
25%, 50%, 75% and 95%) and then finally at two
30 min intervals in absolute ethanol.
2.1. Preparation for scanning electron microscopy
(SEM)
Dehydrated almond tissue was critical point dried in a
Tousimis unit (Ladd) using liquid carbon dioxide. Dried
sections were mounted on aluminum specimen stubs with
double-sided tape and silver conducting paint. Sections
of almond tissue on stubs were coated with 30 nm of
gold–pallidium alloy with a Hummer V sputter coated
fitted with a Technics digital thickness monitor. Specimens were viewed with a Phillips 505T SEM at working
distance of 15 mm and an accelerating voltage of 15 kV.
Fig. 1. Intact almond fruit (A) almond seed enclosed within testa (B)
and separated into its two cotyledons (C). Bar=7.5 mm.
2.2. Preparation for light microscopy (LM)
Dehydration almond tissue was embedded using the
methodology of Spurr (1969) for long pot-life resin.
Sections were cut to a thickness of 7 mm using a Reichert
ultramicrotome and glass knives. After mounting on
glass slides, the sections were stained with 1% acid
fuchsin and 1% toluidine blue using the methods of
Feder and O’Brien (1968). Stained sections were
observed using a Wild light microscope equipped for
brightfield and phase contrast microscopy and photographed with 35 mm black and white film (Kodak Tri-X
pan, 400).
2.3. Preparation for transmission electron microscopy
(TEM)
Dehydrated walnut tissue was also embedded in
Spurr’s resin for TEM. Ultrathin sections cut with a
Reichert ultramicrotome were stained with 4.0% uranyl
acetate for 45 min, followed by 0.4% lead citrate for
4 min. Sections were examined with a JEOL 100S
transmission electron microscope.
3. Results
The edible almond seed has a brown testa enclosing
the two white cotyledons (Fig. 1). The testa has an outer
Fig. 2. Scanning electron micrograph of: outer surface of the relatively
large, thin-walled cells of testa. Bar =50 mm.
epidermis which consists of relatively large thin-walled
cells which range from 100 to 300 mm in width (Fig. 2).
The major portion of the testa consists of approximately
14–20 layers of flattened parenchymal cells with the
total thickness of the layers ranging from 80 to 120 mm
(Fig. 3). The testa also contains occasional vascular
bundles which have spiraled secondary wall thickenings
in the protoxylem cells (Fig. 4).
Examination of the outer surface of the cotyledon
beneath the testa reveals cells that are irregular in
outline and about 15–20 mm in width (Fig. 5). When
viewed in cross section, the epidermis of the cotyledon
consists of a single layer of cells (Fig. 6), which is
subtended by the parenchymal cells of the cotyledon
(Figs. 6 and 7). The flat inner surface of each cotyledon
also consists of cells that are irregular in shape and
likewise are about 15–20 mm in width (Figs. 8 and 9).
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Fig. 5. Scanning electron micrograph of: outer surface of cotyledon
beneath testa. Bar=10 mm.
Fig. 3. Scanning electron micrograph of: cross section of testa with
flattened parenchymal cells (large arrow) closely adpressed on the
outer surface of the cotyledon (small arrow). Bar=10 mm.
Fig. 6. Scanning electron micrograph of: cross section of flattened
parenchymal tissue of testa (A) is subtended by single layer of cells
comprising cotyledon’s outer epidermis (B). Parenchymal cells of
cotyledon (C) subtend the epidermis. Bar=10 mm.
Fig. 4. Scanning electron micrograph of: cross section of testa with
vascular bundle (vb). Note the spiraled secondary wall thickenings in
the protoxylem cells (arrow). Bar=25 mm.
Each cotyledon contains a relatively small amount of
provascular tissue that extends throughout the parenchymal tissue. The provascular tissue is characterised by
narrow cells approximately 5 mm thick and 20–40 mm
long arranged in bundles (Fig. 10).
Each cotyledon consists primarily of thin-walled
(1–3 mm in thickness) parenchymal cells which range
from 15 to 40 mm in width (Figs. 10–12). The parenchymal cells possess a cytoplasmic network, which
surrounds the angular protein bodies which range from
2 to 12 mm in diameter and the spaces once occupied by
lipid bodies which can be up to 3 mm in width.
The protein bodies contain calcium oxalate crystals,
globoid crystals, and numerous spaces once occupied by
protein crystalloids (Fig. 12). The thin cytoplasmic
network that surrounds the spaces once occupied by
lipid bodies demonstrates a limited amount of cytoplasm in mature almond parenchymal cells (Fig. 13). It
can also be observed that plasmodesmata provide
cytoplasmic connections between adjacent parenchymal
cells.
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Fig. 7. Scanning electron micrograph of: cross section of mid-region
parenchymal cells of cotyledon with cell wall (w), protein bodies (p)
and spaces once occupied by lipid bodies which are demarcated by
cytoplasmic network (cn). Bar=5 mm.
Fig. 9. Scanning electron micrograph of: cross section of flat, inner
surface of cotyledon. Bar=10 mm.
Fig. 8. Scanning electron micrograph of: surface view of flat, inner
surface of cotyledon. Bar=10 mm.
4. Discussion
The present study of almond cotyledon microstructure will serve as a means of comparison for future
observations on microstructural changes that occur as
almonds are cooked or otherwise processed. Earlier
works by Vaughan (1970) and Decke (1982) provide
only drawings and light micrographs of almond microstructure. Work by Lott and Buttrose (1978) provided
transmission electron micrographs of almond protein
bodies. The current study therefore provides more
thorough characterisations of the edible portion of the
shelled almond as evaluated by light, scanning and
TEM. Previous work on peanuts by Young and Schadel
(1993) has demonstrated the importance of documenting and evaluating the native state of a nut’s micro-
Fig. 10. Light micrograph of a cross section of mid-region tissue of
cotyledon with a provascular bundle (arrow) among parenchymal
tissue. Bar=20 mm.
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Fig. 11. Transmission electron micrographs of: cross section of midregion parenchymal cells of cotyledon depicting cell walls (w), angular
protein bodies (p) and space once occupied by calcium oxalate crystal
(co) within protein body. Bar=10 mm.
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Fig. 13. Transmission electron micrographs of: cross section of midregion parenchymal cell of cotyledon depicting protein bodies (p) and
spaces once occupied by lipid bodies which are demarcated by
cytoplasmic network (cn). Note the limited amount of cytoplasm in
mature almond parenchymal cells. Bar=3 mm.
structure before observing the processed microstructure
of the form.
Acknowledgements
We are grateful to Ms. Valerie M. Knowlton of the
North Carolina State University Electron Microscopy
Center for assistance with transmission electron microscopy. Dr. Young passed away during the final
preparation of this manuscript.
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Fig. 12. Transmission electron micrographs of: cross section of midregion parenchymal cell of cotyledon depicting angular protein bodies
with spaces once occupied by protein crystalloids (pc) and globoid
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