American Journal of Botany 100(11): 2121–2131. 2013.

ORCHID PROTOCORM-LIKE BODIES ARE SOMATIC EMBRYOS1

YUNG-I LEE2,5, SHAN-TE HSU3, AND EDWARD C. YEUNG4
2Botany Department, National Museum of Natural Science, 404 Taichung, Taiwan, ROC; 3Department of Horticulture,

National Chiayi University, Chiayi, Taiwan, ROC; and 4Department of Biological Sciences, University of Calgary, Calgary,
AB, T2N 1N4, Canada

• Premise of the study: Protocorm-like bodies (PLBs) of orchids are important in orchid micropropagation and outwardly re-
semble somatic embryos in form and development. To determine whether PLBs are truly embryogenetic, we compared PLBs
with somatic embryos and zygotic embryos to determine whether they had similar surface molecules and whether hydroxypro-
line-rich glycoprotein (HRGP) inhibitors similarly alter their growth.
• Methods: Embryogenic calluses (ECs), zygotic embryos, and protocorms were collected for histological and histochemical
studies with light microscopy. The presence of JIM11 and JIM20 epitopes was determined using immunodot blots and immu-
nolocalization procedures. The importance of wall proteins in the formation of PLBs was investigated using 3,4-dehydro-L-
proline (3,4-DHP), an inhibitor of HRGP biosynthesis.
• Key results: At the early stages of PLB formation, the cytoplasm of the globular cell clusters and meristemoids took on a vacu-
olated appearance. Starch granules and protein bodies appeared, albeit transitory in nature. Positive localizations of JIM11 and
JIM20 were noted in the embryogenic culture and developing PLBs similar to zygotic embryos. The inclusion of an inhibitor
to HRGPs inhibited PLB formation.
• Conclusions: This study demonstrates that during the early stages of PLB formation, the cells show cytological characteristics
and cell wall markers similar to zygotic embryo development, justifying the statement that PLBs are indeed somatic embryos
of orchids. Thus, these results suggest that PLBs could be used as an experimental embryological system for physiological or
molecular characterization.

Key words: hydroxyproline-rich glycoproteins; JIM11; JIM20; meristemoids; Orchidaceae; Phalaenopsis; protocorm-like
bodies; somatic embryo.

Phalaenopsis orchids are one of the most important flower
crops in the world. For orchids, an efficient plant regeneration
system is beneficial for the micropropagation of selected elites
and genetic transformation studies. The generation of proto-
corm-like bodies (PLBs) is an important technique for micro-
propagating orchids (Arditti and Ernst, 1993). PLBs were first
noted in the shoot-tip culture of Cymbidium orchid by Morel
(1960). Since the general characteristics of growth and struc-
ture are similar to those of protocorms, the regenerated struc-
tures are termed PLBs.
Protocorm-like bodies can be induced directly from various
explants, such as shoot tips (Tokuhara and Mii, 1993), flower
stalk buds (Ichihashi, 1992), root tips (Tanaka et al., 1976), and
leaf segments (Park et al., 2002). The indirect regeneration of
PLBs from embryogenic callus culture has also been documented
using solid (Ishii et al., 1998; Tokuhara and Mii, 2001) and liq-
uid suspension cultures (Tokuhara and Mii, 2003). Although
the morphology and aspects of structural development are
known, detailed ontogenetic studies for PLBs are absent. After
1 Manuscript received 1 June 2013; revision accepted 8 July 2013.
The authors thank Dr. Mei-Chu Chung at the Institute of Plant and
Microbial Biology, Academia Sinica, Taipei, Taiwan (IPMB), and Dr.
Wann-Neng Jane (Plant Cell Biology Core laboratory, IPMB) for the
use of the transmission electron microscope. This research was supported
by a Discovery grant from the Natural Sciences and Engineering Research
Council of Canada to ECY, by grants from National Chiayi University
to S.T.H., and by grants from the National Museum of Natural Science
to Y.I.L.
5 Author for correspondence (e-mail: leeyungi@mail.nmns.edu.tw)
doi:10.3732/ajb.1300193
American Journal of Botany 100(11): 2121–2131, 2013; http://www.amjbot.org/ © 2013 Botanical Society of America
2121
plating liquid suspension cells on solid medium, Tokuhara and
Mii (2003) demonstrated that PLBs from Phalaenopsis orchids
have a single cell origin. A similar observation was reported by
Jheng et al. (2006) for Oncidium. PLBs can also form directly
from the surface of the explants via differentiation of epidermal
cells (Vij et al., 1984; Chen et al., 1999). In this case, it is likely
that PLBs also have a single cell origin. Subepidermal cells
(Park et al., 2002, 2003) can also give rise to PLBs. Judging
from histological evidence presented, this type of PLB has a
multicellular origin. Individual cells give rise first to meriste-
moids; meristemoids divide further, merge, and give rise to
PLBs (Park et al., 2003). These examples indicate that there are
varied patterns of PLB formation.
In the literature, it is often suggested that PLBs are so-
matic embryos simply based on morphology (e.g., Ishii et al.,
1998; Huan et al., 2004) or that somatic embryogenesis is an
early step in PLB formation (see Zhao et al., 2008). Somatic
embryogenesis is the development of somatic cells into dif-
ferentiated plants through embryo stages under appropriate
conditions (Zimmerman, 1993). Are PLBs somatic embryos?
Do they take on characteristics similar to zygotic embryos
prior to developing into PLBs? To answer these questions
and to determine whether PLBs are indeed somatic embryos,
we carried out a detailed ontogenetic study of PLBs from a
friable embryogenic callus of Phalaenopsis using histologi-
cal and histochemical methods. We compared their ontogeny
to normal in situ zygotic embryo development and subse-
quent protocorm formation. Additionally, we used zygotic
embryo cell wall markers specific to JIM11 and JIM20 anti-
bodies to probe whether similar wall proteins are present
during the course of PLB formation to further document
similarities between them.
2122 AMERICAN JOURNAL OF BOTANY
Our initial survey identified through the use of JIM antibod-
ies that JIM 11 and JIM20 epitopes are abundant in the cell
walls of Phalaenopsis zygotic embryos. The JIM11 and JIM20
antibodies recognize the arabinosylation motifs of HRGPs such
as extensins (Xu et al., 2011). During tobacco zygotic embryo
development, JIM11 and JIM20 epitopes play an important role
in cotyledon primordia formation and influence shoot apical
meristem activities (Zhang et al., 2008). Besides using JIM11
and JIM20 as zygotic embryo cell wall markers, we used an
inhibitor, 3,4-DHP, to alter HRGPs in cell walls of the explants
to identify any important consequences of altering HRGPs on
PLB formation.
MATERIALS AND METHODS
The establishment of a callus culture system—The callus was generated
from in vitro grown PLBs of the breeding line Phalaenopsis Taisuco Yellow.
These initial PLBs were derived from flower bud stalk culture as described by
Ichihashi (1992). The PLBs (~5–6 mm in diameter) were cut in half trans-
versely and cultured on 1/4-strength macroelements and full-strength microele-
ments of Murashige and Skoog (MS) medium (Murashige and Skoog, 1962),
supplemented with 100 mg·L−1 myo-inositol, 0.5 mg·L−1 niacin, 0.5 mg·L−1
pyridoxine HCl, 0.1 mg·L−1 thiamine HCl, 2 mg·L−1 glycine, 100 mg·L−1 glu-
tamine, 0.1 mg·L−1 biotin, 10 g·L−1 sucrose, 3 g·L−1 Gelrite, 1 mg·L−1 2,4-di-
chlorophenoxyacetic acid (2,4-D), and 1 mg·L−1 thidiazuron (TDZ). The pH of
the media was adjusted to 5.7 with 1 N KOH prior to autoclaving at 121°C for
20 min. The explants were incubated in darkness at 25 ± 2°C. The formation of
callus was first observed from the necrotic tissue of PLBs after 5 mo of culture.
The callus with a friable nature was selected and subcultured on the same me-
dium every 4 wk in darkness. After 12 mo of continual subculture, the embryo-
genic callus (ECs) and nonembryogenic calluses (NECs) were selected for
further analysis.
PLB regeneration from friable embryogenic calluses—Selected friable
ECs were transferred to a maltose-containing medium to induce PLBs. The
PLB induction medium contained 1/4 strength MS basal medium supplemented
with 100 mg·L−1 myo-inositol, 0.5 mg·L−1 niacin, 0.5 mg·L−1 pyridoxine HCl,
0.1 mg·L−1 thiamine HCl, 2 mg·L−1 glycine, 100 mg·L−1 glutamine, 1 mg·L−1
biotin, 0.1 mg·L−1 2,4-D, 1 mg·L−1 TDZ, 10 g·L−1 maltose, and 3 g·L−1 Gelrite.
The cultures were maintained at 25 ± 2°C with a 12 h light/12 h dark photope-
riod and a photon flux of 25–30 µmol·m–2·s−1 (daylight fluorescent tube FL-
20D/18; 20 W). The friable cultures were fixed at regular intervals for
histological and histochemical studies until mature PLBs were clearly visible.
To confirm that single cells can give rise to PLBs, we placed one piece of
friable callus mass (50 mg fresh mass) in each 100 mL Erlenmeyer flask con-
taining 10 mL liquid medium (similar to the medium for callus culture). The
flasks were shaken for 30 s in a vortex mixer (3200 rpm, Vortex Genie 2, Sci-
entific Industries, Bohemia, New York, USA). After the vortex treatment, 2 mL
of each cell suspension was removed and spread on a 9 cm Petri dish each con-
taining 20 mL solid medium (the same as the liquid medium with the addition
of 3 g·L−1 Gelrite). The cultures were examined regularly for PLB formation.
To examine details of development, some cell masses were removed and exam-
ined using interference contrast microscopy at regular intervals.
Light microscopy—The ECs, NECs, and developing PLBs were fixed in a
solution of 2.5% glutaraldehyde and 1.6% paraformaldehyde in 0.05 mol·L−1
phosphate buffer (pH 6.8) overnight at 4°C. After fixation, the samples were
dehydrated using an ethanol series, and embedded in Technovit 7100 resin
(Kulzer & Co., Wehrheim, Germany) as described by Yeung (1999). Serial, 3
µm thick sections were cut with glass knives using a Reichert-Jung (Vienna,
Austria) 2040 Autocut rotary microtome. These sections were stained with pe-
riodic acid–Schiff’s reagent for total insoluble carbohydrates and counter-
stained with amido black 10B for proteins (Yeung, 1984). All bright field
images were captured using a Leica 480 digital camera on a Leica Aristoplan
light microscope and the contrast of images was adjusted using Photoshop CS3
software (Adobe, Seattle, Washington, USA).
Histological observation of zygotic embryo development and the formation
of protocorms from mature seeds—To investigate zygotic embryo development,
[Vol. 100
the flowers of Phalaenopsis breeding line K678 were hand-pollinated at the
time of anthesis to ensure good fruit and seed set. Developing seeds were col-
lected and processed for histological studies as detailed by Lee et al. (2008). For
the study of protocorm development, 1/4 strength MS basal medium supple-
mented with 20 g·L−1 sucrose, 30 g·L−1 potato homogenate and 3 g·L−1 Gelrite
was used for seed germination. After sowing, the cultures were maintained at
25 ± 2°C with 12 h light/12 h dark and photon flux of 2530 µmol·m–2·s−1 (day-
light fluorescent tube FL-20D/18; 20 W). Developing protocorms were col-
lected and fixed for histological and histochemical studies until the first leaf
became clearly visible.
Transmission electron microscopy—Structural changes during the early
stages of PLB formation were also investigated using electron microscopy. Se-
lected callus masses were fixed with 2% paraformaldehyde and 2.5% glutaral-
dehyde in 0.1 mol·L−1 sodium phosphate buffer, pH 6.8, at 4°C overnight. After
three 15-min buffer rinses, the materials were postfixed with 1% OsO4 in the
same buffer for 4 h at room temperature and then rinsed in three 15-min changes
of buffer. Following fixation, the materials were dehydrated in an acetone se-
ries, and embedded in Spurr’s resin (Electron Microscope Sciences, Washing-
ton, Pennsylvania, USA). Ultrathin sections of 60–70 nm were cut using a
diamond knife on an ultramicrotome (Richert-Jung Ultracut E). These sections
were stained with uranyl acetate and lead citrate (Hayat, 2000). The sections
were examined and photographed using a Philips CM 100 transmission electron
microscope at 80 kV.
The detection of HRGPs using JIM11 and JIM20 antibody—A set of JIM
antibodies (JIM 8, JIM 11, JIM 12, JIM13, JIM14, JIM15, JIM16 and JIM20)
was purchased from PlantProbes (Leeds, UK). The most abundant HRGPs in
the zygotic embryo were determined with a preliminary survey using an im-
munodot blot assay (detailed next). JIM11 and JIM20 gave the best response
(data not shown); therefore, these two antibodies were used as cell wall markers
for zygotic embryos.
Immunodot blot assay—ECs, NECs, developing PLBs, and zygotic em-
bryos (0.3–0.4 g) were collected and ground into fine powders in liquid nitro-
gen. Proteins were extracted using 0.7 mL extraction buffer [100 mmol·L−1
Tris, 900 mmol·L−1 sucrose, 10 mmol·L−1 EDTA, 100 mmol·L−1 KCl and
0.4% (v/v) mercaptoethanol, pH 8.8] and 0.7 mL of Tris-saturated phenol (pH
8.8). After centrifugation at 8000 rpm for 5 min at 4°C, the supernatant was
collected for protein precipitation. The proteins were precipitated by the ad-
dition of five volumes of 0.1 mol·L−1 ammonium acetate (in 100% methanol)
to the phenol phase, and left at −20°C overnight. The precipitate was centri-
fuged at 16 000 × g for 20 min at 4°C. The pellet was dissolved in rehydration
buffer (8 mol·L−1 urea, 2% CHAPS, 2% Triton X-100, 50 mmol·L−1 1,4-di-
thiothreitol [DTT]). Samples were boiled at 96°C for 5 min and spotted on a
nitrocellulose membrane by a micropipette (as 5 µL drops). The membrane
was air-dried at room temperature for 1 h and blocked in phosphate-buffered
saline (PBS) buffer containing 5% (w/v) milk powder and 0.5% bovine serum
albumin (BSA) for 1 h, followed by labeling with primary monoclonal anti-
bodies, JIM11 and JIM20. The primary antibodies were diluted 1 : 2000 in
PBS containing 1% BSA. After three washes with PBST (PBS buffer contain-
ing 0.2% Tween 20) for 10 min, the membrane was probed with a 1 : 2500
dilution of the secondary antibody, horseradish peroxidase (HRP)-conjugated
anti-rat IgG at room temperature for 1 h. After the final wash with PBST, the
detection of signal was performed by adding chemiluminescent HRP sub-
strate (WBKL S0500, Millipore, Billerica, Massachusetts, USA) and captured
by a luminescent image analyzer, LAS-4000 (FUJIFILM, Tokyo, Japan).
Immunofluorescence labeling of JIM11 and JIM20—For immunofluo-
rescence labeling of HRGPs, ECs, NECs, PLBs and zygotic embryos were
collected and fixed in 4% (v/v) paraformaldehyde in the microtubule stabi-
lizing buffer (MTSB) [50 mmol·L−1 piperazine-N,N′-bis(2-ethanesulfonic
acid) (PIPES), 5 mmol·L−1 MgSO4.7H2O, 5 mmol·L−1 ethylene glycol-bis
(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA), pH 6.9] and left over-
night at 4°C. Samples were dehydrated in an ethanol series (30%, 50%, 70%,
90% and 100%) and placed in pure acetone. The samples were infiltrated grad-
ually (3 : 1, 1 : 1, and 1 : 3 100% acetone to Technovit 8100; Kulzer & Co., Weh-
rheim, Germany), followed by two changes of pure Technovit 8100. The resin
was polymerized at 4°C, and 3 µm thick sections were cut and placed in a drop
of water on a slide and dried at room temperature.
Sectioned samples were rehydrated in PBS, pH 7.3, for 5 min followed by
blocking in a 2% BSA in PBS for 5 min. The sections were labeled with primary
November 2013] LEE ET AL.—ORCHID PROTOCORM-LIKE BODIES ARE SOMATIC EMBRYOS 2123

monoclonal antibodies, JIM11 and JIM20, and left overnight at 4°C. The pri-
mary antibodies were diluted 1 : 20 in PBS containing 1% BSA. After washing
in PBS three times (5 min each time), sections were incubated with a 1 : 20 dilu-
tion of the secondary antibody (anti-rat IgG-FITC, F6258, Sigma, St. Louis,
Missouri, USA) in PBS containing 1% BSA for 1 h in the dark, followed by
three washes in PBS (10 min each). For quenching tissue autofluorescence, sec-
tions were stained with 0.01% (w/v) toluidine blue O (TBO) in PBS for 10 min.
After washing in PBS three times (5 min each), sections were mounted in me-
dium containing an antifade mounting reagent (VECTASHIELD Mounting
Medium, Vector Laboratories, Burlingame, California, USA) before observa-
tion. Controls were prepared by incubating with the blocking solution instead
of the primary antibodies. The sections were examined and the images were
captured using a digital camera attached to an epifluorescence microscope
(Axioskop 2, Carl Zeiss AG).

Treatment with 3,4-DHP—The importance of HRGPs in PLB develop-

ment was studied using 3,4-DHP (200 µmol·L−1) added to the PLB regenera-
tion medium. Calluses without 3,4-DHP treatment were used as controls. One
piece of friable ECs (500 mg fresh mass) was divided into 10 portions and
placed on a 9 cm Petri dish containing 20 mL solid medium. Each Petri dish
was regarded as a replicate, and every treatment was repeated five times. After
8 wk of culture, explants were evaluated in terms of percentage of PLB for-
mation. At the same time, the expression of JIM11 and JIM20 antigens in
3,4-DHP-treated cultures was examined by immunodot blot assay as de-
scribed already. Experiments were performed in a randomized design. The
data were statistically analyzed using ANOVA followed by Fisher’s protected
least significant difference test.

RESULTS

Friable callus morphology— Continuous subculturing of the

initial callus derived from PLB sections results in the formation
of the friable yellowish-white ECs (Fig. 1A). The callus masses
are composed of compact and loose aggregates of cells; histo-
logical sections revealed that callus cells at the surface are more
compact, while cells near the center are loosely packed (Fig. 1B).
A majority of cells within the friable callus have dense cyto-
plasm with a few starch grains and small vacuoles. Each nu-
cleus is prominent with a densely stained nucleolus. After
transfer to the PLB induction medium, the callus gives rise to
PLBs of different sizes (Fig. 1C).

PLB formation from loosely packed cells— To determine

whether single cells from the friable callus are capable of giv-
ing rise directly to PLBs, we first focused on the structural
changes to the loosely packed callus cells. In histological sec-
tions, a number of isodiametric single cells can be seen embed-
ded within the friable callus (Fig. 1B). On the basis of the cell
division pattern, these single cells can give rise to PLBs. Figure 2
of plastic-embedded specimens documents the formation and
cytological changes of a PLB from a single cell, and corre-
sponding morphological changes are shown in Appendix S1
(see Supplemental Data with the online version of this article)
using interference contrast optics.
A brief vortex treatment in liquid medium separated the
friable cell masses into small clumps and individual cells. A
majority of single cells take on a spherical shape with a cen-
trally located nucleus surrounded by prominent starch grains. Fig. 1. Regeneration of protocorm-like bodies (PLBs) from friable
Distinct vacuoles are present within the cytoplasm of the embryogenic callus. (A) Macroscopic view of friable embryogenic cal-
cells (Fig. 2A). The first mitotic division gives rise to two lus. Scale bar = 2 mm. (B) Light micrograph of section, stained with pe-
cells of unequal size (Fig. 2 B). The smaller apical cell di- riodic acid–Schiff’s reagent and amido black 10B, of embryogenic callus
vides to give rise to the “protocorm proper”, which subse- showing cells in the more tightly packed region (arrow) and the loosely
quently develops into a PLB (Fig. 2C–H). The larger basal packed cells (arrowhead). Scale bar = 40 µm. (C) PLBs that formed from
cell has fewer mitotic divisions, and its daughter cells re- the embryogenic callus after brief vortexing. Scale bar = 4 mm.
main vacuolated (Fig. 2D–F).
2124 AMERICAN JOURNAL OF BOTANY
Fig. 2. Light micrographs of stained sections showing formation of
protocorm-like bodies (PLBs) from loosely packed embryogenic callus af-
ter a brief vortex. (A) Isolated single cell from friable callus. The cell has a
prominent nucleus with large starch grains (red dots) around it. Scale bar =
30 µm. (B) Two-celled PLB with a smaller terminal cell (arrow) and a
larger basal cell (arrowhead). Starch grains are still present. Scale bar = 30 µm.
(C) PLB at the three-celled stage. Large vacuoles are replaced by small
ones, causing cytoplasm to look vesiculated. Starch granules are scarce.
Scale bar = 30 µm. (D) Derivatives of the terminal cell continue to divide,
whereas the basal cell becomes vacuolated. Cytoplasm of cells at the apical
end still looks vesiculated. Scale bar = 10 µm. (E) PLB continues to expand
by anticlinal and periclinal divisions. Vesiculated appearance of cytoplasm
is maintained in the derivatives of the terminal cell. Scale bar = 25 µm. (F)
As the PLB continues to increase in size, the cytoplasm becomes vacuo-
lated. At this stage, the cells within the PLB are of similar sizes. Scale
[Vol. 100
Distinct cytological changes occur within the globular cell
clusters at the beginning stages of PLB formation. The starch
grains become smaller (Fig. 2B), and the large vacuoles are
gradually replaced by many tiny vacuoles, imparting a highly
vesiculated appearance to the cytoplasm (Figs. 2C, 3A). Con-
comitant with the formation of the tiny vacuoles, a small
amount of protein deposit, as determined by amido black 10B
stain, can be found within them (Fig. 3A). The protein deposit
is further confirmed by the electron-dense deposits within
small vacuoles (Fig. 3B). The vesicular appearance of the cy-
toplasm and the presence of protein deposits are transient
events because large vacuoles reappear and the protein depos-
its disappear from the cytoplasm as development progresses.
The morphology of a PLB begins to be visible. The cells of
the future shoot pole continue to divide and become smaller,
while the remaining cells enlarge (Fig. 2G). After 12 wk of
culture, PLBs are readily seen. A distinct gradient of cell size
can be found within each PLB, with the shoot pole comprising
small cytoplasmic cells (Fig. 2H).
PLB formed from the compact region of the callus— Histo-
logical studies were also conducted to determine the origin of
PLBs from the more compact regions of the friable callus. In
the compact region, the callus is composed of aggregates of
meristemoids. On the basis of the arrangement of the cell walls,
each meristemoid appears to have derived from a single cell
(Fig. 4A). The compactly arranged aggregates subsequently
merge and organize into growth centers (Figs. 4B–D). Polar-
ized development occurs with the surface cells of each growth
center, giving rise to the future shoot pole of a PLB (Fig. 4D).
Continual cell divisions occur within the small protuberances,
resulting in PLB formation (Fig. 4E). The PLBs have a distinct
gradient of cell size, with the smaller cytoplasmic cells occupy-
ing the future shoot pole and larger, highly vacuolated cells oc-
cupying the basal end (Fig. 4F).
Cytological changes similar to that described for PLBs
from single cells are also present within the meristemoids
during the formation of the PLBs. The cells of the meriste-
moids are initially vacuolated (Fig. 4A). As development
progresses, the cytoplasm of the cell aggregates begins to
take on a vesiculated appearance (Fig. 4B, C). Similar to
PLB formation from single cells, the small vacuoles are soon
replaced by larger ones.
Zygotic embryo development— Figure 5 illustrates the de-
velopment of the zygotic embryo of Phalaenopsis emphasiz-
ing changes in the storage deposition pattern. A more complete
description of zygotic embryo development can be found in
Lee et al. (2008) for P. amabilis. At the early globular stage,
the embryo cells are vacuolated, and a few tiny starch grains
are visible (Fig. 5A). By the globular stage, a distinct proto-
derm and a marked gradient of cell size can be seen in the
embryo proper (Fig. 5B). At this time, the embryo cells re-
main vacuolated, and more starch grains are found to surround
the nuclei. As cells cease to divide within the globular embryo,
bar = 60 µm. (G) As development progresses, a distinct protoderm is formed.
The protoderm cells are smaller than the internal cells. Scale bar = 100 µm.
(H) As it develops further, the PLB becomes elongated. An obvious gradi-
ent of cell size can be found at this stage. The future shoot pole (arrow) is
marked by small cytoplasmic cells, which eventually give rise to a shoot
apical meristem. B = basal cell; T = terminal cell. Scale bar = 150 µm.
November 2013] LEE ET AL.—ORCHID PROTOCORM-LIKE BODIES ARE SOMATIC EMBRYOS
Fig. 3. Cytological changes during early development of protocorm-like body (PLB). (A) Light micrograph of protein deposits (blue dots) in the
highly vesiculated cytoplasm of a developing PLB similar to Fig. 2D. Scale bar = 10 µm. (B) Transmission electron micrograph showing the vesiculated
cytoplasm of a developing PLB. A small amount of protein deposits are present within small vacuoles. N = nucleus; P = plastid; PD = protein deposit; V =
vacuole. Scale bar = 2 µm.
storage products begin to appear (Fig. 5C). Starch grains re-
main abundant during the early stage of embryo maturation.
The large vacuoles are replaced by smaller ones, and protein
deposits begin to appear within these small vacuoles (Fig. 5C).
As the seed approaches maturity, the cytoplasm of the embryo
cells takes on a dense appearance with abundant storage prod-
uct deposits (Fig. 5D).
Early stages of protocorm development— Two weeks af-
ter sowing, the zygotic embryos begin to increase in size by
the process of vacuolation (Appendix S2A, see online Sup-
plemental Data). Protein deposits are no longer visible in the
cytoplasm; instead, many starch grains appear. Mitotic ac-
tivities begin to occur in the future shoot pole. After the pro-
tocorm emerges from the seed coat, a layer of meristematic
cells is clearly visible (Appendix S2B). These cells are
smaller than the rest of the cells of the protocorm. Soon, a
small depression forms at the shoot pole (Appendix S2C),
quickly followed by the appearance of a small dome-shaped
shoot apical meristem and, surrounding it, the first true pro-
tocorm leaf (Appendix S2D).
Immunodot blots and immunofluorescence localization of
JIM11 and JIM20 HRPG epitopes in zygotic embryos— Both
JIM11 and JIM20 antibodies gave similar staining intensity,
and the signals intensified as the embryos matured (Fig. 6A).
Hence, these two antibodies were used as zygotic embryo mark-
ers for developing PLBs.
Positive immunofluorescence staining of JIM11 and JIM20
epitopes were also found, and the results concurred with the
immunodot blot assays. The JIM11 and JIM20 epitopes gave a
similar immunostaining pattern. At the proembryo stage, the
fluorescence signal of JIM11 and JIM20 epitopes were detected
in the walls of the embryo proper (online Appendix S3). As the
embryo reached the early globular stage, obvious fluorescence
signals of JIM11 and JIM 20 epitopes were observed and were
stronger in the basal regions of the embryo (Appendix S3C, G).
A similar pattern was present as the embryo reached maturity,
with a stronger signal in the larger cells near the micropylar end
2125
and weaker fluorescence signals at the chalazal end of the em-
bryo (Appendix S3D, H).
Immunodot blots and Immunofluorescence localization
of JIM11 and JIM20 HRPG epitopes in NECs, ECs, and
PLBs— For immunodot blot assays, strong signals of JIM11
and JIM20 staining were found in ECs, while only weak sig-
nals were detected in NECs. When compared with ECs, the
signals were weaker in PLBs (Fig. 6B). Results from immuno-
fluorescence staining concurred with the immunodot blot as-
says. In the NECs, little to no fluorescence signals of JIM11
and JIM20 epitopes was detected (Fig. 6A, B). In contrast,
much stronger fluorescence signals of JIM11 and JIM20 epitopes
were found in ECs. It is important to note that JIM11 epitope
was localized to some substance present between cells in
addition to the cell walls (Fig. 7C). An obvious fluorescence
signal of JIM20 epitope was only detected in cell walls of
ECs, while no signal was detected in the intercellular region
(Fig. 7D). During the formation of meristemoids, the signal
for JIM11 was stronger in the intercellular region, while the
signal for JIM20 was more uniform in the walls of the meris-
temoids (Fig. 7E, F). In PLBs, a stronger fluorescence signal
was localized at the epidermis for both epitopes, and only
weak fluorescence was present at the walls of the inner cells
(Fig. 7G, H). In the negative controls, no labeling can be found
in the ECs after incubation without primary antibodies of JIM11
and JIM20 (photos not shown). The evaluation of immuno-
fluorescence staining intensity of JIM11 and JIM20 epitopes
in NECs, ECs, meristemoids, and PLBs is summarized in Table 1.
Overall, signals from the binding of JIM11 epitope was stron-
ger than from the JIM20 epitope.
Effects of 3,4-DHP treatment on PLBs formation and devel-
opment from callus culture—To evaluate the effects of 3,4-DHP
on the expression of HRGPs and PLB regeneration, immunodot
blot assays were carried out using JIM11 and JIM20 antibodies
with careful morphological examination of PLB development.
When compared with untreated control cultures (ECs and PLBs),
2126 AMERICAN JOURNAL OF BOTANY [Vol. 100

Fig. 4. Light micrographs of the formation of protocorm-like bodies (PLBs) from the tightly packed region of the embryogenic callus. (A) In the more
tightly packed region of the callus, single cells divide and develop into meristemoids (arrows). Each meristemoid appears as a spherical cell aggregate.
Scale bar = 20 µm. (B) The meristemoids (arrows) merge with neighboring meristemoids. The outline of individual meristemoids becomes less distinct.
Cytoplasm of most cells within the meristemoid is dense. Scale bar = 20 µm. (C) The merged meristemoids subsequently organize into growth centers (*).
The cytoplasm of the cells also has a vesiculated appearance. Scale bar = 20 µm. (D) Cells of the growth center become polarized, with smaller cells form-
ing at the future shoot pole (*) of a PLB. Scale bar = 50 µm. (E) Continual cell divisions within the small protuberance result in the formation of a PLB.
Scale bar = 100 µm. (F) Well-developed PLB with a distinct protoderm and a gradient of cell size. The larger, highly vacuolated cells occupy the basal end,
while the smaller cytoplasmic cells occupy the future shoot pole (arrow). Scale bar = 200 µm.

very low expression of the JIM11 and the JIM20 epitopes was
detected in ECs treated with 3,4-DHP (Fig. 6B). About 4 wk after
transfer of the ECs to the PLB induction medium with 3,4-DHP,
small cell aggregates became necrotic, unlike the fully viable cell
aggregates in the control. After 8 wk of culture, both the fresh
mass and PLBs regeneration capacity were significantly reduced
in treated cultures as compared to controls (Table 2).
DISCUSSION
In a majority of flowering plants, zygotic embryo develop-
ment first establishes a body plan with the formation of tissues,
apical meristems, and cotyledon(s). The other key events are
the synthesis and accumulation of storage products and the
preparation for developmental arrest (Goldberg et al., 1994).
Subsequent seed germination elaborates the body plan as estab-
lished during the early stages of embryo development. An inter-
vening structure such as the orchid protocorm is absent during
seed germination.
It is generally known that orchid embryo has no obvious
structural histodifferentiation within the embryo proper (Yam
et al., 2002); apical meristems are distinctly absent from or-
chid embryos. At best, a gradient of cell size is present within
a mature embryo with smaller cells located at the apical end in
the future shoot pole. Germination of orchid embryos first gives
November 2013] LEE ET AL.—ORCHID PROTOCORM-LIKE BODIES ARE SOMATIC EMBRYOS 2127

Fig. 5. Light micrographs of development of a zygotic embryo showing changes in storage deposition pattern. (A) Early globular embryo with well-
developed suspensor cells (S). The embryo cells are initially vacuolated, with several tiny starch grains (arrow) in the cytoplasm. V = vacuole. (B) As de-
velopment progresses, the embryo enlarges. A distinct protoderm and a marked gradient of cell size can be seen in the embryo proper, which has highly
vacuolated cells and abundant starch grains (arrow). V = vacuole. (C) As the cells cease to divide within the globular embryo, storage products begin to
appear. The large vacuoles (V) are replaced by smaller ones, and protein bodies (arrows) begin to appear within these small vacuoles. (D) As seeds ap-
proached maturity, numerous protein bodies (arrows) can be found within the embryo cells. Scale bars = 20 µm.
2128 AMERICAN JOURNAL OF BOTANY
Fig. 6. Semiquantitative analysis by immunodot blots of the relative
abundance of JIM11 and JIM20 epitopes in extracts prepared from devel-
oping zygotic embryos (A) and protocorm-like bodies (PLBs) of Phalaen-
opsis orchids. PE: proembryo stage; EG: early globular embryo stage; GE:
globular embryo stage; E: embryogenic calluses (with meristemoids); N:
nonembryogenic calluses; P: protocorm-like bodies; DHP: embryogenic
calluses treated with 200 µmol·L 3,4-DHP.
rise to an ephemeral tubercle structure called a protocorm
from which a shoot and a root subsequently differentiate (Cribb,
1999). The term protocorm was first coined by Treub to de-
scribe a stage in lycopod development (Arditti and Krikorian,
1996). Bernard applied this term to describe the corm-like
stage of orchid seed germination (see Arditti and Krikorian,
1996). The strategy of delaying histodifferentiation is impor-
tant in maximizing massive seed production upon a single suc-
cessful pollination event. One event can lead to the formation
of a huge number of seeds, e.g., in Anguloa ruckeri, a single
capsule contains 3.9 million seeds (Arditti, 1992). In order that
a proper seedling can develop from each embryo, the shoot apical
meristem must form for vegetative development to continue.
As Sussex (1989, p. 225) wrote in his review, “meristems make
a plant”; without the presence of a shoot apical meristem, con-
tinuous vegetative development cannot take place. The forma-
tion of a protocorm is a solution to vegetative establishment in
orchids, i.e., the formation of a functional shoot meristem for
postembryonic growth. Chances are the developmental pro-
grams for protocorm development are established during
embryo maturation. Even though the protocorm is a postem-
bryonic structure, it has been considered as a continuation of
embryogeny, an integral part of embryogenesis (Curtis and
Nichol, 1948).
Orchid protocorm-like bodies are somatic embryos— From
the evidence presented, PLBs should be regarded as somatic
embryos because they show features similar to that of their zy-
gotic counterparts. We demonstrated that (1) the early division
pattern of embryogenic cells from EC is similar to that in zy-
gotic embryos, (2) soon after PLB formation, the globular-
shaped PLBs have the ability to synthesize storage product,
albeit transient in nature, and (3) wall proteins similar to those
in the zygotic embryos are present.
As seen in Fig. 2 and Appendix S1, the early stages of PLB
formation closely recapitulate zygotic embryo development.
[Vol. 100
The first division of an embryogenic cell is unequal, giving rise
to two cells of unequal size. The smaller terminal cell continues
to divide and gives rise to the “protocorm proper”, and the basal
cell is arrested after a few mitotic divisions. In P. amabilis, its
zygotic counterpart, the first division of the zygote, is unequal,
with the smaller terminal cell giving rise to the embryo proper;
the basal cell has a limited number of divisions, and the daugh-
ter cells differentiate into suspensor cells (Lee et al., 2008). The
lack of suspensor cell formation in the developing PLB can be
attributed to the culture environment, which is not inductive of
suspensor formation.
During the early stages of PLB formation, the cells show
transient cytological characteristics similar to zygotic embryo
development (Figs. 3, 4), i.e., having a vesiculated appearance
of the cytoplasm with protein deposits. In zygotic embryos of
flowering plants, after histodifferentiation, storage products
begin to appear in the embryo cells. In general, starch deposi-
tion begins first, followed by the formation of storage proteins
and lipids. Storage proteins are localized within small vacu-
oles. In zygotic embryos of orchids, the pattern of storage
product deposition appears to be similar to that in other flow-
ering plants (Fig. 3; Yeung and Law, 1992; Yeung et al., 1996).
In this study, the early accumulation of starch and the tran-
sient appearance of proteins within the tiny vacuoles of devel-
oping PLBs recapitulate early events of storage product
biosynthesis of zygotic embryos. The failure to continue to
accumulate storage products within PLBs is most likely a re-
sult of the in vitro culture environment. In zygotic embryos,
once the body plan is established, the zygotic embryo can be
easily cultured; precocious germination soon follows, bypass-
ing the storage product synthesis phase of embryo develop-
ment (Yeung and Sussex, 1979; Haslem and Yeung, 2011). It
is no surprise to see that once PLB development is initiated, in
the absence of developmental arrest, the small globular cell
clusters will develop rapidly into PLBs bypassing storage pro-
duction biosynthesis.
The formation of PLB requires the synthesis of similar zy-
gotic embryo cell wall components to ensure proper devel-
opment. In the NECs, its inability to synthesize proper wall
components such as JIM11 and JIM20 epitopes leads to a
loss of morphogenetic potential of the callus. A properly de-
veloped cell wall is required to coordinate cell division and
expansion leading to organized development (Dupuy et al.,
2010). When the inhibitor 3,4-DHP is used to interfere with
HRGP biosynthesis in the EC, PLB formation is reduced,
indicating that HRGPs are integral parts of the wall structure
in PLBs.
In the more tightly packed region of the callus (Fig. 1),
single cells divide and develop into meristemoids, and mer-
istemoids subsequently “merge” to form single PLBs (Fig. 5).
This pattern of development has been observed in shoot or-
ganogenesis in a variety of species, e.g., the cotyledons of
radiata pine (Yeung et al., 1981). On the basis of the cell profile,
each individual meristemoid is similar to a small globular
cell cluster from a single cell of EC. The fact that individual
meristemoids fail to develop into separate PLBs is likely due
to the close physical contact between them. Mechanical
stresses, the nature of the extracellular matrix, and intrinsic
factors can mediate growth and development (Boudaoud,
2010; Peters et al., 2000). The resulting physical stresses
generated by mitotic activities among the meristemoids may
have altered their morphogenetic potential resulting in the
formation of a single PLB.
November 2013] LEE ET AL.—ORCHID PROTOCORM-LIKE BODIES ARE SOMATIC EMBRYOS 2129

Fig. 7. Immunofluorescence localization of JIM11 and JIM20 epitopes during regeneration of protocorm-like bodies (PLBs) of Phalaenopsis. (A)
Nonembryogenic calluses with very weak signals of JIM11 epitopes (arrow) located primarily at surface of cell aggregates. Scale bar = 40 µm. (B) NECs
showing indistinct signals of JIM20 epitopes (arrow). Scale bar = 40 µm. (C) Embryogenic calluses with strong signals of JIM11 epitopes (arrow). The
signals are present mainly in the wall and the intercellular substance. Scale bar = 40 µm. (D) ECs with strong signals of JIM20 epitopes (arrow), mainly
localized in the cell wall. Scale bar = 40 µm. (E) Meristemoids showing prominent signals of JIM11 epitopes (arrow) in the intercellular substance and
moderate signals on the surface walls of meristemoids. Scale bar = 40 µm. (F) Meristemoids with moderate signals of JIM20 epitopes (arrow) in the cell
wall and the intercellular substance. Scale bar = 40 µm. (G) PLBs showing the epidermis with more surface signals of JIM11 epitopes (arrow) than the inner
tiers. Scale bar = 200 µm. (H) PLBs showing moderate signals of JIM20 epitopes (arrow) mainly at the epidermis. Scale bar = 200 µm.
2130 AMERICAN JOURNAL OF BOTANY [Vol. 100

TABLE 1. Intensity of immunofluorescence labeling with JIM11 and rapid progression in genome sequencing, future molecular ge-
JIM20 antibodies of developmental stages during the formation of netics studies will provide new insight into the regulatory pro-
protocorm-like bodies (PLBs) from callus culture of Phalaenopsis. cess for protocorm development.
Antibody signal
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