A HISTOCHEMICAL STUDY OF
IN VANDA (ORCHIDACEAE)
MARVIN RAY ALVAREZ
A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF
THE UNIVERSITY OF FLORIDA
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE
DEGREE OF DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
The author wishes to acknowledge with gratitude the
guidance and encouragement given by his advisory committee
chairman, Dr. Yoneo Sagawa, without whose assistance and
teaching this investigation could not have been conducted.
The author also wishes to thank the members of his
advisory committee, Drs. R. H. Biggs, A. D. Conger, R. R.
Cowden, J. R. Edwardson, and G. R. Noggle, for their advice
and criticism of the manuscript. Special thanks are due to
Dr. Cowden for his invaluable aid with histochemical tech-
Grateful acknowledgement is herewith given for finan-
cial support provided by the U. S. Atomic Energy Commission,
Contract No. AT-(40-1)-3088, and by the Graduate School of
the University of Florida in the form of a Graduate School
The writer is thankful to Jones and Scully, Inc.,
Miami, and Thornton Orchids, West Palm Beach, for the plants
used in this study.
Finally, the author wishes to express his sincere
thanks to his wife for her unending support and patience
throughout this work and for typing the manuscript.
TABLE OF CONTENTS
ACKNOWLEDGEMENTS . . . . . . . . . .
LIST OF FIGURES . . . . . . . .
LIST OF TABLES . . . . . . . . ... .. vii
INTRODUCTION . . . . . . . .
Botanical Histochemistry . . . .
The Embryo as a Tissue Source . . .
MATERIALS AND METHODS . . . . . .
Culture Techniaue . . . . . .
Categorization of Developmental Stages
Histochemical Methods and Technioue .
Method of Cell Volume Analysis . . .
OBSERVATIONS . . . . . . . . . . .
Background . . . . . . . . . . .
Size Relationships During Embryo Development . .
Megaspore Stage . . . . . . . . .
Ganetophyte Stage . . . . . . . . .
Embryo Stage . . . . . . . . . .
Protocorm Stage . . . . . . . . .
DISCUSSION OF OBSERVATIONS . . . . . . . .
The Cell Wall . . . .
The Cell W'all in the Megaspore
The Cell Wall in the Protocorm
The Synergids . . . .
Starch Distribution . . .
Proteins and RNA . . . .
The I egasporangiun . . .
The Gametophyte . . . .
The Embryo . . . . .
The Protocorm . . . .
The Histones . . . . .
SUMMARY . . . . . . .
and Embryo Stages
Stage . . .
LITERATURE CITED . . . . . . . . .
. . 6
. . 6
. . 7
. . 7
. . 9
I I I I I I
BIOGRAPHICAL SKETCH . . . . . . . . .. 70
LIST OF FIGURES
1. Sequence of Embryo Development . . . . .. 21
2. Comparison of Meristematic and Parenchymatous
Cell Volumes During Development . . . . 23
3. Comparison of Cell and Nuclear Volumes
in the Protocorm . . . . . . ... .24
4. PAS Stained Cross Section through the
Megasporangium. . . . . . . .. .28
5. Protein Concentration Gradient in the
Megasporangium. . . . . . . .. .28
6. PAS Stained Longitudinal Section of the Embryo Sac 28
7. Protein Localization in a Megasporangium with
a Pre-mitotic Megaspore . . . . . ... 28
8. A Longitudinal Section of an Embryo Sac
Stained for Protein . . . ... . . .30
9. A Longitudinal Section of an Embryo Sac
Stained for Ribonucleic Acid . . . ... 30
10. A Longitudinal Section of a Three-cell Embryo
Stained with PAS . . . . . . ... 30
11. A Longitudinal Section of a Multicellular Embryo
Stained with PAS . . . . . . ... 30
12. A Longitudinal Section of a Three-cell Embryo
Stained for Total Protein . . . . ... 32
13. Total Protein Distribution in Early
Ilulticellular Embryo . . . . . ... 32
14. Total Protein Distribution in Early
Multicellular Embryo . . . . . ... 32
15. Total Protein Distribution in Early
Multicellular Embryo . . . . . ... 32
16. A Longitudinal Section of an Embryo Sac
Showing a Post Fertilization Total Protein
Increase at the Chalazal End . . . .
17. A Longitudinal Section of a Two-cell Embryo
Stained for Ribonucleic Acid . . . .
18. A Longitudinal Section of an Early
Stained for Ribonucleic Acid . . . .
19. A Longitudinal Section of an Early Protocorm
Stained with PAS . . . . . . .
20. A Longitudinal Section of a Late Protocorn
Stained with PAS . . . . . . .
21. A Longitudinal Section of an Early Protocorm
Stained for Total Protein . . . .
22. A Longitudinal Section of an Early Protocorm
Stained for Ribonucleic Acid . . . .
23. A Longitudinal Section of an Ammonium
Oxalate Extracted Early Protocorm
Showing Pectin Distribution .
24. Longitudinal Section of Early
and Late Protocorm Showing
Changes in Histone Concentration
25. Longitudinal Section of Early
and Late Protocorm Showing
Changes in Histone Concentration
. . . 36
. . . 38
. . . 38
26. A Longitudinal Section of a Protocorm
Stained for Tyrosine-containing Proteins . 38
27. A Longitudinal Section of a Protocorm
Stained for Sulfhydryl-containing Proteins 38
LIST OF TABLES
1. Histochemical Methods . . . . . . . 8
2. Comparison of Mean Cell Volume (3)
of Meristematic and Parenchymatous
Cells throughout Development . . . ... 22
3. Changes in Mean Cell and Nuclear Volume (/3)
in 4 Quarters of Protocorms from the
Apical through the Parenchymatous Regions 22
4. Summary of Results in
Megaspore and Gametophyte Stages ...... 25
5. Summary of Results in
Embryo and Protocorm Stages . . . ... 26
Lost higher plants and animals originate from a sin-
gle cell, the zygote. From this common source arise all of
the cell types which ultimately comprise the complex,
mature organism. What are the factors which govern the
transformation of this general and unspecialized cell into
heterogeneous, specialized groups of cells? The purpose of
this investigation is to characterize the biochemical
changes which occur in the major cellular constituents of
the developing plant embryo with reference to the foregoing
Cellular differentiation is reflected in both morpho-
logic and physiologic characteristics and consequently the
problem has been investigated from both viewpoints resulting
in an impressive assemblage of facts and theories relating
to the problem. In the last 15 years the mechanisms of
differentiation and the acquisition of tissue specificity
have been subjected to extensive investigation by animal
embryologists (Brachet, 1957) and this has resulted in the
basic idea that differentiation is the result of the synthe-
sis of specific proteins. The investigations of Avery et al.
(1944) and Boivin et al. (1948) led to the recognition of
the genetic role of deoxyribonucleic acid (DNA) which
through ribonucleic acid (RNA) controls protein synthesis.
As a result of the work of Watson and Crick, Kornberg,
Nirenberg, Ochoa, and others (Taylor, 1963), the facts con-
cerning the replication of the genetic material and the
theory of the genetic code have been established. Yet, we
still lack a completely coordinated explanation backed by
experimental facts as to how cells of supposedly identical
genetic constitution synthesize different proteins at dif-
ferent times thus resulting in differences between cells.
The results of biochemical and physiological studies
traditionally have been expressed in terms of fresh or dry
weight of the tissue concerned. While such an approach has
provided the basis for our understanding of fundamental
biochemical reactions, it cannot yield information about
the basic unit of life, the cell (Avery and Engel, 1954).
As a result two other methods of approach have recently
been utilized in the investigation of the problem of differ-
entiation. The first method, quantitative histochemistry,
involves the use of scaled down biochemical techniques
which are applied to small samples of tissue obtained by
sectioning. Quantitative histochemistry has received impe-
tus from the efforts of Linderstrim-Lang et al. in the
Carlsberg Laboratory. The second method, microscopic histo-
chemistry, utilizes the specificity of certain dyes for
specific chemical substances thus achieving intracellular
localization of substances. These latter methods have been
applied extensively by zoologists in the study of differen-
tiation of vertebrate and invertebrate embryos and have
resulted in many interesting facts.
Botanists, on the other hand, have been slow to apply
histochemical methods to botanical problems (Jensen, 1962).
Within the last decade, however, a number of botanical
investigators have applied histochemical methods to problems
of cellular differentiation. Developmental studies concern-
ing the correlation of respiration and total proteins utiliz-
ing root tip cells were conducted by Brovn and Broadbent
(1950). Whaley et al. (1952) investigated histochemically
the cellulose patterns in root tip cells. Studies on the
content and variation of nucleic acids in the nuclei of root
tip cells have been numerous (Patau and Swift, 1953; Deeley
et al., 1957; Holmes et al., 1955; Jensen, 1956, 1958;
McLeish, 1959; Woodard et al., 1961; Das and Alfert, 1961).
RNA, protein, and enzyme distribution have been investigated
in the meristematic regions of both root and shoot (Jensen,
1955, 1956; Avers, 1958; Avers and King, 1960; Avers, 1961;
Gifford and Tepper, 1962a, 1962b).
The Embryo as a Tissue Source
Most of the above mentioned investigations have been
carried out on either shoot or root meristems which provide
a convenient source of permanently embryonic tissue in
plants. The chief disadvantage of meristems for the study
of cellular differentiation is that it is often difficult to
trace the lineage of a given cell in a tissue section. The
embryo, on the other hand, provides us with a simple, dif-
ferentiating tissue in which one can, by sampling all stages
of development, relate each cell to the zygote. The disad-
vantages of using the embryo for studies of differentiation
are that it is usually quite small and is often masked by
nutritive and integumentary tissue in the seed.
The Orchidaceae is particularly well suited for
embryological studies since the embryo in most of the genera
consists of a relatively undifferentiated group of cells
contained in a thin, transparent seed coat and the endosperm
is completely absent. This undifferentiated condition is
also found in the parasitic and saprophytic members of the
Balanophoraceae, Rafflesiaceae, Gentianaceae, Pyrolaceae,
Orobanchaceae, and the Burmanniaceae (Maheshwari, 1950).
In this study the genus Vanda was chosen because the
morphology of embryogenesis was well known (Swamy, 1942;
Alvarez, 1962) and the embryo is relatively large. In addi-
tion each ovulary contains close to one million (Correll,
1950) developing ovules and synchronization among the ovules
is high (Duncan, 1959).
Because of these advantages, the relative concentra-
tion of the insoluble polysaccharide fraction of the cell
wall, total proteins, ribonucleic acid, histone, and sulf-
hydryl and tyrosine-containing proteins over the entire
embryonic sequence of events from the Megaspore Stage
through the Protocorm Stage were investigated. It is hoped
that the construction of he profile of the distribution of
these major biochemical constituents during embryogeny will
serve as a contribution to the ever growing body of infor-
mation relating to cellular differentiation.
MATERIALS AND METHODS
The following cultivars of Vanda were used in this
study: Vanda xHelen Paoa (Univ. of Fla.--UF #1109), Vanda
xli. Foster X Vanda xE. Noa (UF #1115), Vanda xBurgeffii
(UF #1116), and Vanda xHawaiian Blue (UF #1531). Since no
differences were noted in the embryogeny of these hybrids,
they will be given a common description here.
Several flowers on each of the plants were pollina-
ted with fresh pollen and the plants were kept in the green-
house. One pod on each plant was periodically sampled to
determine the degree of embryo development which had occurred.
A plug of tissue containing a portion of the placental ridge
was removed from the wall of the ovulary and fixed in CRAF
(Johansen, 1940). The remainder of the plug was reinserted
and the pod was tightly wrapped with Saran Wrap. A pod
from each plant could thus be sampled repeatedly. Small
fragments of the excised placental ridge were smeared in
aceto-orcein and the stage of development noted.
About five days after fertilization occurred, the
ovularies were excised and washed externally with a warm
detergent solution. The placental ridges were aseptically
removed and each was cultured in 125 ml. Erlenmeyer flasks
on the surface of a solidified culture solution (Vacin and
'ent, 1949) modified by the addition of 8 grams of agar
and 250 ml. of coconut milk per liter of solution. The
ovule cultures were kept at approximately 300C at 120 foot
candles of illumination from two cold fluorescent strip
Categorization of Develoomental Stages
The developmental stages investigated were:
1. Megaspore Stage. Includes development from the
completion of meiosis to the initiation of embryo sac mito-
2. Gametophyte Stage. Development from mitosis to
3. Embryo Stage. Includes embryo development occur-
ring within the ovule.
4. Protocorm Stage. 3abryo development from germi-
nation up to primordial leaf and primary root formation.
Histochemical !Methods and Technioue
When the desired stage of development was available,
fragments of the placenta with attached ovules were fixed,
dehydrated in a tertiary butyl alcohol series, embedded in
Parawax at 5600 for 8 to 12 hours, and sectioned to appro-
priate thicknesses (Johansen, 1940). The histochemical
methods used for the identification and localization of par-
ticular constituents are summarized in Table 1.
Table 1--Histochemical Methods
Method or Reagent
Hot ammonium oxalate
4% sodium hydroxide
by pectin removal
and followed by PAS
Cellulose 17% sodium hydroxide
by pectin and hemi-
followed by PAS
FAA Yasuma and
(4 hrs.) Ichikawa (1953)
Iazia et al.
Azure B, pH 4, fol-
Fast green, pH 8.0
Flax and Himes
Table 1, continued
Chemical Method or Reagent Fixation Reference
Sulfhydryl Azo-aryl mercaptide FAA Bennett and
(-SH)-contain- coupling (4 hrs.) Watts (1958)
Tyrosine Morel-Sisley reac- FAA Lillie (1957)
tion (4 hrs.)
Method of Cell Volume Analysis
Measurements of cell size for the purpose of demon-
strating the differential in cell enlargement evident between
the meristematic and parenchymatous regions through all
stages of development were made with a calibrated eyepiece
micrometer in a Bausch and Lomb research microscope. Only
the largest diameter of each cell was measured in approxi-
mately median longitudinal sections. All distinct cells in
each tissue section were measured. Measurements were made
in the two-cell proembryo phase of the Embryo Stage and in
both meristematic and parenchymatous regions in early and
late phases of the Protocorm Stage. Standard deviations for
all samples were calculated and found in all cases to be
small. Cell volumes were calculated using the formula
In the Monandrae (taxa having one fertile anther),
the ovulary at anthesis contains three parietal placental
ridges which, upon pollination or auxin stimulus (Hubert
and Maton, 1930; Magli, 1958), initiate archesporial tissue
(Wirth and Withner, 1959). Further placental development
has been shown in Phalaenoosis (Niimoto and Sagawa, 1962)
and Dendrobium (Sagawa and Israel, in press) to consist of
the formation of a system of dichotomously branched protru-
berences, or megasporangia, which are organized into a
central column of nucellar cells surrounded by a single
epidermal layer with an enlarged terminal archesoorial cell
which functions directly as a megaspore mother cell. Sub-
sequent meiotic divisions of this cell give rise to four
megaspores, the one nearest the chalazal region forming the
eight-nucleate embryo sac surrounded by the integuments.
In Vanda, fertilization occurs between 60 and 70 days
after pollination and shortly thereafter the outer and end
walls of the outer integumentary cells become dense and the
nuclei disappear. The inner integument persists until the
embryo is in a multicellular stage.
The sequential steps in embryo development are illus-
trated in Fig. 1. The zygote divides transversely forming
two cells. Subsequently, the basal (micropylar) cell divides
in the same manner thus forming a two-cell proembryo and a
suspensor initial. Subsequent vertical divisions of the sus-
pensor initial result in an eight-cell suspensor apparatus.
Meanwhile the two cells of the proembryo divide irregularly
producing a cluster of cells. Continued cell divisions in
the embryo result in the formation of an oval mass of cells
surrounded by finger-like projections of the suspensor appa-
Growth of the embryo results in the differentiation
of two intergrading regions. The cells proximal to the sus-
pensor apparatus enlarge and become parenchymatous while
those distal to the suspensor remain meristematic. Contin-
ued growth of the embryo results in the splitting of the
seed coat and the protocorm either continues to adhere to
the placental ridge or falls off onto the culture medium.
Soon after the embryo is free of the seed coat, rapid
division of the cells of the meristematic region ensues
forming an apical structure. Trichomes arise from the epi-
dermal cells of the parenchymatous region. By this time the
cells of the suspensor have become necrotic.
At this stage in the developmental sequence a pri-
mordial leaf sheath forms around the apex. The necrosis of
the cells of the parenchymatous region is concomitant with
the initiation of subsequent primordial leaves and is complete
when the seedling is photosynthetic.
Size Relationshios During Embryo Develooment
Differentiation of the embryo of Vanda is character-
ized by the formation of two regions differing primarily in
cell size. Calculations of the volumes of the cells of the
two regions were made in the two-cell proembryo and also in
protocorms of 185 to 200 microns and 700 to 800 microns
(Table 1). The cells in the two-cell proembryo are of
approximately the same volume. As the embryo increases in
total size, however, the difference in cell volume in the
two regions increases and is maximal in the Protocorm Stage
prior to the necrosis of the parenchymatous region (Fig. 2).
Measurements of cell and nuclear diameters were also
made on several longitudinal sections of protocorms (Table 2).
As can be seen from Fig. 3, the increase in cell and nuclear
volume from the meristematic to the parenchymatous region is
The megasporangium consists of an extension of the
placental ridge in which the cells are organized into a
nucellar row surrounded by a single epidermal layer with an
enlarged terminal, tenuinucellate megaspore. Observation
of this structure stained with morphological stains reveals
only a difference in size and position between the mega-
spore and the attending tissue. Treatment of cross sections
of the megasporangium with the periodic acid-Schiff's reac-
tion reveals, however, that the inner periclinal and the
anticlinal walls of the nucellar epidermis (integumentary
initials) immediately surrounding the megaspore are more
highly PAS positive than the outer walls of these cells.
The cell walls of the megaspore itself are also highly PAS
positive indicating a high concentration of insoluble poly-
saccharides (Fig. 4). Efforts to determine the nature of
these polysaccharides by the differential extraction of the
cell wall were unsuccessful due to the extremely small size
and delicate nature of the tissue.
Staining of the megasporangium with either mercuric
bromphenol blue or by the ninhydrin-Schiff's reaction shows
that the highest concentration of total protein in the
megasporangium occurs in the megaspore and in the nucellar
cells immediately surrounding the megaspore (Fig. 5). All
cells of the megasporangium appear to have a higher concen-
tration of total proteins than the cells of the placental
ridge and total protein in all of the megasporangial cells
is most highly concentrated in the nuclei. The nucleoli
of all these cells give a very positive reaction to total
protein stains and in the megaspore the chromatin is parti-
PAS staining of the embryo sac shows a marked con-
trast in the concentration of insoluble polysaccharides
between the components of the megagametophyte (Fig. 6).
The cell walls and cytoplasm of the egg are faintly PAS
positive. Particles are often present at the base of the
egg and appear to be small starch grains. These particles
were never observed in the distal end of the egg which
appears to be somewhat vacuolated, although the vacuole is
not clearly distinct from the cytoplasm.
Markedly contrasting with the egg are the two syner-
gids which exhibit vivid cytoplasmic staining with the PAS
reaction. The stain is ubiquitously distributed in these
cells and only a few PAS positive bodies are detectable in
the cytoplasm. The nature of this carbohydrate is unknown
and attempts at differential extraction were unsuccessful.
The cell wall at the micropylar end of the embryo
sac appears greatly thickened in contrast with the rest of
the wall and exhibits a high concentration of insoluble
Prior to megaspore mitosis,the concentration of total
protein in the cytoplasm undergoes a marked rise. The elon-
gation and subsequent vacuolation of the megaspore prior to
the first mitotic division results in the formation of a
strand of cytoplasm approximately two-thirds the width of
the embryo sac and extending.the length of the cell (Fig. 7).
The observed increase in total protein concentration in this
portion of the cell probably results from a drastic decrease
in total cytoplasmic volume with a concomitant uptake of
water into the vacuolated portion. Nuclear total protein
concentration appears to remain constant until the mitotic
division ensues. During the increase in premitotic total
cytoplasmic protein concentration, the nucleolus increases
to two or three times its original volume and exhibits
intense staining with mercuric bromphenol blue.
At the completion of the mitotic divisions of the
megaspore, the thin cytoplasmic strands which remain evident
in the embryo sac show a very low concentration of total
protein (Fig. 8). The polar nuclei before degeneration and
the antipodal nuclei exhibit a slightly higher protein con-
centration than the cytoplasmic strands of the embryo sac
but still appear relatively low.
The suspensor and embryo initials differ in total
protein content in the two-cell phase (Fig. 11). Repeated
observations indicate a somewhat higher total protein con-
centration in the embryo initial than in the suspensor ini-
tial. In later multicellular stages of the Eboryo Stage,
the difference in total protein concentration between the
suspensor apparatus and the embryo proper becomes cuite
marked (Figs. 13, 14, 15). The cells of the suspensor
appear highly vacuolated and show a very low total protein
concentration. No differences with respect to this consti-
tuent, however, appear between the individual cells of the
An interesting phenomenon encountered in the embryo
sac shortly after fertilization is the formation of an ill-
defined region of very high protein concentration at the
chalazal limits of the embryo sac which gradually disappears
as the embryo increases in size (Fig. 16).
During the zygote and two-cell proembryo phases of
the Embryo Stage, the ovule contains RNA only in the nucle-
oli. Repeated observations of similarly treated sections
containing two-cell phases of the proembryo occasionally
show the presence of a "shell" of RNA in the cytoplasm imme-
diately surrounding the nucleus of the proembryo initial
and suspensor initial. The RNA "shell" in the suspensor
initial, however, was not as distinct as in the embryo ini-
tial and disappears during enlargement of the suspensors
The egg apparatus exhibits a distinct differential
staining for total protein (Fig. 8) which is low in the egg
cytoplasm but high in the nucleus. The egg nucleolus is
also distinct, having stained darker than the nucleoplasm.
In contrast, the cytoplasm of the two synergids is very high
in total protein concentration and the nucleus in these
cells is indistinguishable from the cytoplasm.
Treatment of longitudinal sections of the embryo sac
with pH 4.0 Azure B reveals a low concentration of ribonu-
cleic acid in the entire embryo sac. The staining was very
light and limited to a small area in the cytoplasmic strands
surrounding the polar nuclei (Fig. 9). Observation of early
phases of the embryo sac often shows a high concentration of
RNA in the polar nuclei (Fig. 9).
No difference in the amount of insoluble polysaccha-
rides was detectable at the two and three-cell stages (Fig. 10).
'Then the suspensor apparatus becomes morphologically dis-
tinct, however, the cell walls of these cells become thicker
and more highly PAS positive than those of the embryo pro-
per (Fig. 11). This difference does not appear, however,
until the suspensor apparatus consists of at least two cells.
In the multicellular Embryo Stage, cytoplasmic iNA
concentration is low in all cells but appears to be somewhat
higher in the cells of the embryo proper (Fig. 18).
One of the characteristic features of the Protocorm
Stage is the presence and distribution of starch grains
throughout the tissues during the developmental sequence.
These inclusions are found in large numbers in both differ-
entiated regions but are much larger in the parenchymatous
region (Fig. 19). In the later phases of the Protocorm
Stage, starch grains are found only in the parenchymatous
region which is becoming necrotic (Fig. 20).
These inclusions stain vividly by the PAS method,
showing layering and a distinct hylum. They give a slightly
positive reaction with mercuric bromphenol blue and the nin-
hydrin-Schiff's reaction and also with sudan III. Polariz-
ing optics, however, show them to be birefringent.
In the early Protocorm Stage, total protein distribu-
tion is uniform in the meristematic cells and the parenchy-
matous cells. This result was obtained by staining with
mercuric bromphenol blue and the ninhydrin-Schiff's reaction
RNA also appears to be uniformly distributed in this
stage. Repeated observations, however, show what appear to
be regions of high RNA concentration in the cytoplasm of
the parenchymatous region. These areas are morphologically
indistinguishable from the surrounding cells which exhibit
lower staining intensity (Fig. 22). At this stage, two
nucleoli are distinguishable in some of the nuclei of the
parenchymatous cells. These appear frequently in those
cells exhibiting high concentrations of cytoplasmic RNA. In
the very latest phases of the Protocorm Stage, RNA concentra-
tion is distinctly higher in the meristematic apical cells,
particularly in those at the base of the leaf primordium.
Hot ammonium oxalate extraction of the pectic sub-
stances from the cell walls in the Protocorm Stage with sub-
sequent PAS staining shows that the cell walls of the meri-
stematic cells contain greater amounts of pectic substances
than those of the parenchymatous cells (Fig. 23). Further
extraction of the hemicelluloses and cellulose with sodium
hydroxide indicates that these constituents are in relatively
higher concentrations in the cell walls of the parenchyma-
tous cells than in the meristenatic cells and that hemicel-
lulose comprises the greatest part of the non-pectic fraction
of the cell wall.
Treatment of similar sections with hydroxylamine and
ferric chloride after the method of Reeve (1959) indicates
that the esterification of pectin is very low, if not
indeed absent, in the cell walls of all the cells during the
Protocorm Stage. Certain sections exhibited a very weak
positive reaction in the walls of some parenchymatous cells,
indicating slight pectin esterification, but the results
are not considered conclusive.
Staining of the early protocorm with fast green at
pH 8.0 results in an equally intense stain in all nuclei
indicating a proportionately higher amount of histones in
the nuclei of the parenchymatous cells than in the meristem-
atic cells (Fig. 24). In the late phases of the Protocorm
Stage, however, the nuclei of the parenchymatous region
stained considerably more intensely (Fig. 25) indicating a
very high histone concentration. This observation is con-
current with the morphologically detectable initiation of
necrosis in this region. Synchronous with this event is the
appearance of slight cytoplasmic staining with fast green
at pH 8.0 suggesting the presence of basic proteins in the
Feulgen staining of the late protocorm shows a marked
contrast between the staining intensity of the meristematic
and parenchymatous nuclei, the latter being considerably
Tyrosine and sulfhydryl-containing proteins are
localized primarily in the nuclei of all cells and staining
intensity is greatest in the nuclei of the parenchymatous
cells. This relative distribution does not appear to change
extensively during development (Figs. 26, 27).
Results are summarized in Tables 3 and 4.
Fig. 1--Sequence of Embryo Development
The first division of the zygote (a) is transverse
producing a terminal and a basal cell (b) which also divides
transversely (c). The first division of the terminal cell
is vertical producing a four-cell embryo (d). A second ver-
tical division occurs in the terminal cell at right angles
to the first (e). Several successive divisions of the basal
cell give rise to the globular embryo proper while continued
vertical division of the terminal cells give rise to the
suspensor apparatus which forms finger-like projections over
the globular embryo (f).
The cells of the embryo distal to the suspensor
remain meristematic while those proximal to the suspensor
enlarge and become vacuolated (g). This results in the
formation of intergrading meristematic and parenchymatous
regions (h, i).
iSEOi 4 E BORYO DEVL OPEN T
o or C- H m
E 0 H '--
o o a11) t
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OCd 0 ) -i 4 z to H r)
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Fig. 2--Comparison of 1'eristematic
and Parenchymatous Cell Volumes During Development
ICL~-C-~` I -111~.~~ 1 ----~--~I I _
H / a )o
100" [ 10
lst 1/4 2nd 1/4 3rd 1/4 4th 1/4
Apical Region Parenchymatous Region
Fig. 3--Comparison of Cell
and Nuclear Volumes in the Protocorm
a co rd 9-:
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) o4 C) +- C) 44 o4 (1) +D-1 0 p m sr -D (a q o ar E 'r, (L
c0 3n -4 e c 3 H C D 0 s H C ( 1) (1) Z r-l j a co Z
0) C L) a r1 63 0 H O a rc; -q r 3I Cd
Fig. 4--PAS Stained Cross Section
through the Megasporangium
The anticlinal and inner cell walls of the nucellar
epidermis surrounding the megaspore as well as the walls
of the megaspore are thicker and more highly PAS positive
than the other cell walls of the megasporangium.
Fig. 5--Protein Concentration Gradient
in the Megasporangium
The negaspore and surrounding nucellar cells exhibit
a higher total protein concentration than the basal nucel-
lar epidermis and axial row.
Fig. 6--PAS Stained Longitudinal
Section of the Embryo Sac
The synergid cells show a high, uniform concentra-
tion of insoluble polysaccharides. All other embryo sac
components are low in insoluble carbohydrates.
Fig. 7--Protein Localization in a L.egasporangium
with a Pre-mitotic Megaspore
Immediately prior to mitosis the megaspore elongates
and becomes laterally vacuolated while protein concentra-
tion in the cytoplasm increases.
FIe. FI. 5
I I. :I ^I,*.....
i ;: .
\- -' '/
^a^ ~ rs Y.-' -;l^S
Fig. 8--A Longitudinal Section of
an Embryo Sac Stained for Protein
Total protein concentration is high and uniform in
the synergid cells. Total protein concentration in the
polar nuclei diminishes as the embryo sac matures.
Fig. 9--A Longitudinal Section of
an Embryo Sac Stained for Ribonucleic Acid
RNA is low in all embryo sac components except the
Fig. 10--A Longitudinal Section of
a Three-cell Embryo Stained with PAS
No differences in insoluble polysaccharides are evi-
dent between the cells at this stage. Note the thick,
highly PAS positive cell wall at the micropylar end of the
Fig. 11--A Longitudinal Section of
a l:ulticellular Ebryo Stained with PAS
At this stage the cell walls of the suspensor appa-
ratus are thicker and more highly PAS positive than the
cell walls of the embryo proper.
FIG. 8 FIG.9
Fig. 12--A Longitudinal Section of
a Three-cell Embryo Stained for Total Protein
As early as the two-cell stage,the embryo initial
exhibits a higher total protein concentration than the sus-
Figs. 13, 14, 15--Total Protein Distribution
in Early Multicellular embryos
As development proceeds, the higher total protein
concentration in the embryo proper versus the suspensor
apparatus becomes more marked.
FI G. 12
FI G. 1 -
Fig. 16--A Longitudinal Section of an Embryo Sac
Showing a Post Fertilization
Total Protein Increase at the Chalazal End
Shortly after fertilization the concentration of
total protein at the chalazal end of the embryo sac
Fig. 17--A Longitudinal Section of
a Two-cell Embryo Stained for Ribonucleic Acid
A marked basophilia is exhibited at the periphery of
the nuclei at the two-cell stage. This reaction disappears
from the cells of the suspensor apparatus during subsequent
Fig. 18--A Longitudinal Section of
an Early Multicellular Embryo
Stained for Ribonucleic Acid
RNA distribution in the multicellular embryo paral-
lels closely that of protein.
Fig. 19--A Longitudinal Section of
an Early Protocorm Stained with PAS
Note that starch grains are present in both meri-
stematic and parenchymatous cells.
FI G. 19
Fig. 20--A Longitudinal Section of
a Late Protocorm Stained with PAS
During late phases of the Protocorm Stage, starch
grains become limited to the parenchymatous region.
Fig. 21--A Longitudinal Section of
an Early Protocorm Stained for Total Protein
Note that the total protein concentration is the
same in the meristematic and parenchymatous cells at stage
Fig. 22--A Longitudinal Section of
an Early Protocorm Stained for Ribonucleic Acid
Certain cells in the inner parenchymatous region
often exhibit high basophilia. These cells appear to be
involved in the formation of vascular tissue.
Fig. 23--A Longitudinal Section of
an Ammonium Oxalate Extracted Early Protocorm
Showing Pectin Distribution
The cell walls of the meristematic cells contain
proportionately greater amounts of pectic substances than
the parenchymatous cells.
:, " "-1'
S. *} -;* ,' ,
, -; '- i '" '. (
' ; ,
, ...-'* .. -* ':' ,, * ;
'-. _' .^'.". '.,
Figs. 24, 25--Longitudinal Sections
of Early and Late Protocorms Showing
Changes in Histone Concentration
In the early protocorm stage (Fig. 24), histone
concentration in the nuclei of the meristematic and paren-
chymatous cells is the same. In the late protocorm (Fig. 25),
however, histone concentration rises markedly in the
Fig. 26--A Longitudinal Section of a Protocorm
Stained for Tyrosine-containing Proteins
Tyrosine-containing protein concentration is higher
in the nuclei of the parenchymatous cells.
Fig. 27--A Longitudinal Section of a Protocorm
Stained for Sulfhydryl-containing Proteins
Sulfhydryl-containing protein concentration is
higher in the nuclei of the parenchymatous cells.
FIG. 2z 4
DISCUSSION OF OBSERVATIONS
The Cell Wall
Early studies of the cell wall were limited primarily
to the definition of certain gross properties such as swell-
ing (Naegli, 1864), birefringence (von Mohl, 1859), and
cytoplasmic proximity (Fitting, 1900). From these studies
evolved the inquiries resulting in the extensive assays of
the chemical nature of the cell wall during the early to mid
nineteen hundreds (Katz, 1924; Akbronn and Frey, 1926;
Frey-Wyssling, 1935, 1936). Following the advent of the
electron microscope, emphasis was placed upon the correla-
tion of chemical and physical characteristics, obtained with
polarizing optics and x-ray defraction techniques, with the
observable ultrastructure as revealed by ultrathin section-
ing ('mhaley et al., 1952; Scott et al., 1956). In recent
years, emphasis in cell wall studies has centered around the
elucidation of the mechanisms involved in cell expansion
(Green, 1958) and on the distribution and biosynthesis of
cell wall constituents during cellular differentiation
(Bishop et al., 1958; Jensen, 1960; Flemion, 1961). As a
result of the dominant role played by the cell wall in hor-
monal regulation of plant cell growth, the effect of auxin
treatment on wall biosynthesis has also received extensive
investigation (Ordin et al., 1955; Bonner et al., 1955;
Lamport, 1963). For recent reviews on this aspect see Audus
(1959), Thimann (1960), Galston and Purves (1960), and Ray
(1961). For general reviews on the cell wall, Preston (1959),
KGhlethaler (1961), and Northcote (1963) may be consulted.
The investigation described herein reveals that the
cell wall and other insoluble polysaccharide structures
undergo ordered changes in distribution and concentration
during embryogenesis. These shifts in distribution appear to
be closely associated with cellular growth and differentia-
The Cell Wall in the Megasoore and Embryo Stages
During the Megaspore Stage the cell walls of the mega-
spore and the anticlinal walls of the surrounding nucellar
e-idermis appear considerably thicker than those of the
other megasporangial cells. Similar changes also occur in
the cell walls of the suspensor apparatus during the marked
enlargement of these cells in the Embryo Stage. Evidence of
the loss of plasmadesmatic connections between the negaspore
mother cell and the surrounding nucellar cells has been
obtained with the electron microscope in Dendrobium (Israel,
1962). Thus, while thickened cell walls do not necessarily
indicate few plasmadesmata, they do serve to emphasize the
fact that the megaspore is embarked on a course of develop-
ment different from the cells around it and that the cell
wall is at least involved in such development.
The Cell Wall in the Protocorm Stage
An interesting correlation exists between the
observed cell wall thickening of the negaspore and the sus-
pensor apparatus and the cell walls of the meristenatic cells
of the protocorm. Differential extraction of the cell walls
of the protocorm reveal that the pectic substances are con-
tained in higher amounts in the growing cells of the meri-
stematic region than in the cells of the parenchymatous
region. Jensen (1960) reports similar results in the differ-
entiating protodermal cells of the onion root tip. Methyl
esterification of the pectins, however, is practically absent
in all of the cell walls of the protocorm. This finding con-
flicts with the proposition of Ordin et al. (1955) that cell
wall plasticity is the result of the formation of methyl
bridges between carboxyl groups which prevent or reduce cross
linkage of the molecules of galacturonic acid by ionic bind-
ing of calcium ions and that this biosynthesis is increased
by indole-3-acetic acid. Jansen et al. (1960), however,
report that while the hot-water soluble pectin is almost
fully esterified, the residual insoluble "protopectin," which
comprises 80 percent of the pectic substance, is only approx-
imately 30 percent esterified. Since little reaction for
methyl esterification was obtained in the protocorn, it is
possible that the esterified hot-water soluble fraction was
not present since the tissue was not in contact with hot
water at any time during the localization procedure. It is
considered possible, however, that hot paraffin immersion
may have caused the removal of the hot-water soluble frac-
tion. But regardless of this, the pectin fraction detected
after ammonium oxalate extraction--probably protopectin--is
proportionately higher in the expanding cells which concurs
with the idea proposed by Albersheim and Bonner (1959) that
the increased wall plasticity resulting from auxin stimulus
is due to an over-all softening of the cell wall by the
addition of pectic material.
Further extraction of the cell walls of the protocorm
indicates that the major non-cellulosic component of the
walls of all the cells is hemicellulose. This finding is in
agreement with the suggestion of Jensen (1960) that the non-
cellulosic polysaccharides may play a major role in deter-
mining the characteristics of the cell wall. It appears
that cell wall thickening in the megaspore and suspensor
apparatus may also be due to the same phenomenon observed in
the expanding cells of the protocorm. This increase in cell
wall polysaccharide content without secondary wall formation
seems to be characteristic of expanding cells.
Another hypothesis regarding cell wall extensibility
that merits discussion is that proposed by Lamport (1963).
Until recently there was little support for the view that
protein might be the wall component which controls its plas-
ticity since the amount of protein in the cell wall had been
shown to be ouite small; 4 to 12 percent by weight. Lamport,
however, has implicated the amino acid hydroxyproline in the
structure of a cell wall protein which he refers to as
extension. He speculates that this protein is directly
involved in controlling cell wall plasticity by providing a
network of labile cross-linkages between the cellulose
microfibrils. The existence of hydroxyproline in peptide
linkage in the cell wall has been demonstrated by Lamport.
This protein is soluble when first synthesized but later
becomes firmly attached to the cellulose, perhaps through a
covalent protein-cellulose link. Lamport has further shown
that the oxygen of the hydroxyproline is derived from atmos-
pheric oxygen and he proposes experiments in which the
oxygen content of a tissue is varied--but never allowed to
become limiting to the function of the cytochrome system--
to determine what, if any, effect the presence of oxygen has
on cell expansion. Such an experiment might be conducted
utilizing the protocorm of Vanda, since in this tissue cell
expansion is a normal function of embryo growth and no other
complicating paths of differentiation are present nor is
auxin stimulus necessary.
The synergid cells are usually ephemeral structures
in the angiosperm gametophyte which disappear soon after or
even before fertilization (Maheshwari, 1950). In some
cases, however, as in Vanda, one or both of these cells may
persist and show considerable activity. A similar condi-
tion has been observed in the cases of Allium unifolium and
A. rotundum (Weber, 1929). While the synergids are usually
considered to be morphological remnants of the archegonium
(Fuller and Tippo, 1954), the unusually high concentration
of insoluble carbohydrates and total protein, as well as
the ubiquitous distribution of these classes of compounds
in the cytoplasm, suggests the possibility of a nutritional
role for these cells in support of the egg and early embryo.
Similar results and conclusions were obtained by Pritchard
(1962) in his histochemical study of the embryogeny of
Unlike Stellaria, the synergids in Vanda also exhibit
a high concentration of total protein, the distribution of
which is similar to the polysaccharide distribution observed
in these cells. Since the embryo sac and, indeed, the ovule
at this stage, is very small, differential extraction of
tissue sections was not successful. While discrete PAS pos-
itive particles are also found in these cells as well as in
the basal portion of the egg, and while the presence of
starch has also been reported in other angiosperm embryo
sacs (Caheshwari, 1950), and because of its uniform distri-
bution, it seems likely that the PAS positive material in
the case of Vanda is more in the nature of a mixture of hemi-
celluloses such as the mannans found in the seed of the date
palm or the hexosans in leguminous seeds which have been
shown to function as reserve foodstuffs (IuGhlethaler, 1961).
In the above cited cases, however, the hemicellulosic com-
pounds are contained as a part of the cell wall. The possi-
bility of a mucopolysaccharide nature of this substance is
alluded to by the similarity of the distribution of the
protein and the PAS positive material although no analagous
cases can be found in the literature.
The early Protocorm Stage is characterized by the
presence of starch grains in the cells of both the meri-
stematic and parenchymatous cells. In the later phases of
protocorm development, however, starch grains become confined
to the parenchymatoas portion where they become progressively
smaller and ultimately disappear subsequent to the initiation
of necrosis of the parenchymatous region. In this phase of
development, the remaining starch grains appear in greater
number in the cells in the central portion of the protocorm
which appear as vascular initials. Since this occurs prior
to the estaJlishment of full photosynthetic activity, as evi-
denced by the external greenness of the plant, it appears
that the entire parenchymatous region serves as a store of
nutrients for the actively dividing cells of the meristem.
Proteins and RNA
The correlation of the activity of RNA and protein
metabolism has been the subject of intensive investigation in
recent years and the literature is far too extensive to
review here. For general reviews consult Brachet (1957) and
Within recent years, the role of RNA and protein in
plant morphogenesis has emerged as a leading factor and
numerous studies concerning the synthesis, activity, and
distribution of these compounds have been forthcoming. Out-
standing among these studies is the work of Sunderland et al.
(1957) in which they correlated protein concentration and
respiration in the various tissues of the apical meristem of
Luoinus alba. Also noteworthy are the studies of Taylor
(1958, 1959) on the synthesis and distribution of nucleic
acids during microsporogenesis in Lilium and Tulbawhia.
The metabolism of the nucleic acids and proteins was
recently investigated in seedlings of barley by Ledoux et al.
(1962) in which they followed changes in the content of
these substances betv.een the various organs of the seedling
and the endosperm during early development. The distribution
of RNA and proteins in the apical meristem has also been
studied by Gifford and Tepper (1962a, 1962b) and these
authors were able to correlate fluctuations in these classes
of compounds in the various histologically differentiated
regions of the shoot apex during floral induction.
All of the above cited investigations, together with
the impressive number of studies of the interrelation of the
metabolism of these compounds in animal tissue, point to the
conclusion that the control of protein synthesis resides in
the nucleus and that this control is mediated to the cyto-
plasm via RNA. The strongest support for this view is sum-
marized in the following facts: 1. RNA is required for pro-
tein synthesis to occur in the cytoplasm. 2. The RNA frac-
tion of the Tobacco Mosaic Virus determines what type of
protein is made in cases of virus infection. 3. The rate of
incorporation of RNA precursors is much higher in the nucleus
than in the cytoplasm. 4. Transplantation experiments have
shown that labelled RNA can pass from the nucleus to the
cytoplasm (Iirsky and Allfrey, 1958).
In the investigation described herein,all of the find-
ings relating to RNA and protein distribution are in agree-
ment with this fundamental idea.
Observation of the megasporangium stained for total
protein shows that the concentration is considerably higher
in the cells of the distal end, particularly in the megaspore
and in the nucellar cells immediately surrounding it. Obser-
vation of the megaspore itself between the time of its forma-
tion until the beginning of the mitotic divisions reveals no
striking increases in the protein concentration of the cell.
In fact, after the enlargement of the embryo sac following
the mitotic divisions of the nucleus, protein concentration
in this structure appears to be considerably reduced.
Ultrastructure studies of megasporogenesis in
Dendrobium (Israel, 1962) have shown that the megaspore mother
cell actively absorbs the surrounding nucellus and this may
be a mechanism for the rapid increase of protein in a rapidly
enlarging cell. The occurrence of this phenomenon was alluded
to by Esau (1953) and the active uptake of intact protein has
been demonstrated in vertebrates (Ebert, 1954), in tissue
culture (Francis and Winnick, 1953), and in plant cells
(Jensen and McLaren, 1960). In view of the above, it appears
that the elevated protein concentration observed in the
nucellus of the distal tip is a remnant of the condition of
active intact protein absorption, which existed during mega-
sporogenesis, but which is not functioning in the Megaspore
Observation of the embryo sac before fertilization
indicates that the concentration of total proteins and RNA is
low in all components except the synergids which have already
been discussed. The polar nuclei and the antipodal cells
appear to have a higher concentration of these classes of
compounds during the early phases of the Gametophyte Stage.
The endosperm nucleus disappears, however, during later
embryo sac development.
In the case of the polar nuclei, RNA appears to be
confined entirely to the nucleus and its distribution therein
is uniform except for a deeper stain in the nucleolus. RNA
was never observed in the thin cytoplasmic strand surround-
ing the secondary nucleus. Any explanations given for the
observed decrease in the protein and RNA content of the
polar nuclei would be purely speculative since comparable
studies are nonexistent. However, Ledoux et al. (1962) report
that in the endosperm of barley,RNA'ase activity increases
as RNA in this tissue decreases. Thus one might speculate a
similar occurrence in Vanda but at an earlier phase. This
condition might be related to the increase in ploidy in the
secondary nucleus resulting from the fusion of the polar
nuclei in which a factor, or factors, for RNA'ase production
may be contained in double dose. In most angiosperms, the
polar nuclei lie just above the egg and are connected to it
by a conspicuous cytoplasmic strand (Maheshwari, 1950). In
the embryo sac of Vanda, however, the polar nuclei are
located in the center of the embryo sac and appear to be
suspended between two large vacuoles by thin cytoplasmic
strands emanating from the long sides of the embryo sac. Pos-
sibly this position makes access to the precursor ribose
nucleotides difficult (Ambellan, 1955).
Certainly a cytochemical comparison of the embryo sac
of Vanda with a species in which the endosperm is well devel-
oped would be enlightening.
Shortly after fertilization, the chalazal end of the
embryo sac shows a small region of intense protein staining.
As the embryo enlarges, this region increases in area and in
staining intensity. When the embryo nearly fills the embryo
sac, the region begins to disappear. This activity appears
to be associated with the antipodal cells.
The role of the antipodals in angiosperm embryology
is not clear. Phylogenetically, they have been regarded as
vestiges of the prothalial tissue (Fuller and Tippo, 1954).
In the Gramineae, Gentianaceae, and Compositae, the
antipodals frequently show a considerable increase in size
or number (Maheshwari, 1950). This event is sometimes
accompanied by an increase in ploidy in these cells and is
indicative of their high metabolic activity. The increase
in protein staining observed in Vanda suggests the possibi-
lity of an increase in the number of these cells. Feulgen
staining of the embryo sac does not indicate, however, any
increase in DNA in this region. In cases of an increase in
cell number, the cells maintain their individuality. In
Vanda, on the other hand, the region giving the intense pro-
tein reaction does not appear to be composed of individual
cells. One may speculate that the antipodal cells in Vanda
do increase in number, but that the cellular membranes break
down liberating their cellular content into the cytoplasm of
the embryo sac, thus making added protein available to the
growing embryo. The possibility also exists that this
region is nothing more than the cytoplasm of the embryo sac
which has been pushed up against the chalazal limits by the
growing embryo resulting in an increase in total protein
concentration due to a reduction in volume.
The first division of the zygote results in a two-cell
proembryo. At this early stage the morphological difference
between the two cells is slight. As development progresses,
however, the micropylar cell gives rise to the suspensor
apparatus and the chalazal cell to the embryo. These two
portions of the embryo undergo very different courses of
cytodifferentiation. The cells of the suspensor apparatus
enlarge considerably and become highly vacuolated while those
of the embryo proper remain small, non-vacuolated, and typi-
cally meristematic. The cells of the suspensor begin to
disappear soon after germination of the embryo.
If one examines the two-cell proembryo for its dis-
tribution of total protein and RNA, it becomes evident that
the two cells differ chemically from each other, if not
structurally, even at this early stage. The embryo initial
shows a markedly higher concentration of total protein in its
nucleus and cytoplasm and a "shell" of RNA appears around
the nuclear membrane. While this accumulation of basophilic
material is also present in the suspensor initial, it soon
disappears. Thus, in the cell forming the dividing tissue,
RNA and total protein is high in both nucleus and cytoplasm,
while in the cell destined to form cells enlarging primarily
as a result of water uptake, these constituents are low.
Such a condition is what one would expect to find if,
indeed, protein synthesis is under nuclear control via RNA,
and correlates well with the findings of Jensen (1958) in the
root tip. Differentials in RNA and protein activity in early
embryo development are well known in amphibian embryos where
it has been shown that the incorporation of RNA and protein
precursors occurs primarily in the nucleus during segmenta-
tion but increases in the cytoplasm following gastrulation
resulting in the formation of dorso-ventral gradients of acti-
vity (Fico, 1954; Sirlin, 1955; Brachet and Ledoux, 1955).
In the sea urchin egg, RNA synthesis is detectable in the
blastula stage (Hultin, 1950) but increases markedly during
gastrulation (Elson, Gustafson, and Chargaff, 1954).
The fact that the content of RNA in an initial
determines the subsequent growth of a cell was illustrated
recently by Woodstock and Skoog (1962) in Zea. Nuclear
modification during early differentiation in specialized
cells, such as in secretary cells of the pancreas, has been
amply demonstrated in animals (Zalokar, 1961). All of the
above provide further evidence for the direct relationship
existing between RNA, protein synthesis, and morphogenesis.
What, then, is the controlling mechanism which ena-
bles one cell in a two-cell system to synthesize and ela-
borate RNA into the cytoplasm and prevents a morphologically
and, indeed, genetically identical cell from doing the same?
Does this control reside in the genetic material of these
cells, or is it mediated by virtue of the environment of the
The only readily detectable differences between the
suspensor initial and embryo, aside from their protein and
RNA content, is the position that they occupy in the embryo
sac. The suspensor initial lies in intimate contact with
the synergids while the embryo initial protrudes into the
cytoplasm or possibly into the vacuole of the embryo sac.
If one considers the hypothesis proposed by Markert (1958)
that the activation of a gene or gene complex is dependent
on the condition of the cytoplasm, it follows that possibly
RNA synthesis in the cells of the embryo occurs only in the
absence of certain substrates in the cytoplasm. In the
suspensor initial, such substrates might be furnished by the
synergids, whereas in the case of the embryo initial, these
substrates may be lacking due to the slow rate of movements
of macromolecules through the cytoplasm of the suspensor
initial. Thus certain gene groups may be activated in this
cell resulting in the synthesis of specific proteins. In
view of the increased thickness of the cell wall of the sus-
pensor apparatus, which is apparent when it consists of at
least two cells, one might further speculate that transfer
of macromolecules between the embryo and suspensor becomes
increasingly difficult, ultimately resulting in the necrosis
of the*suspensor following the demise of the synergids.
Such a proposal is, of course, purely speculative but is in
agreement with the observations and with current hypotheses
of the mechanisms involved in cellular differentiation.
The Protocorm Stage is characterized morphologically
by the establishment of two intergrading regions of cell
types; the meristematio and parenchymatous regions. During
the Embryo Stage, all of the cells of the embryo proper are
similar morphologically and in protein, RNA, and insoluble
polysaccharide distribution. Shortly after emergence of
the embryo from the seed, however, the cells proximal to
the suspensor apparatus begin to enlarge and become dis-
tinctly vacuolated, while those distal to the suspensor
remain meristematic. Growth of the protocorm results from
the continued enlargement of the subapical meristematic cells.
During this enlargement, the protein concentration
in the parenchymatous cells appears to remain nearly con-
stant. Thus while these cells are enlarging ultimately to
roughly four times their original diameter, the relative
concentration of total proteins remains very nearly the same
as in the initials from which they derived. This observation
indicates that while the major cause of enlargement in these
cells, as well as in the premitotic megaspore, is due pri-
marily to the vacuolation of the cytoplasm resulting from
increased water uptake, protein synthesis is occurring at a
rate sufficient to maintain the concentration of total pro-
tein roughly equal to that of the actively dividing meri-
These findings are partially in conflict with the
generalization pertaining to plant cell growth voiced
recently by Ray (1961) in which he states: "Unlike typical
growth in animals or microorganisms, plant cell growth does
not consist primarily of protein synthesis. While cell
enlargement in the intact plant is normally accompanied by
vigorous synthesis of proteins, this synthesis commonly is
far from sufficient to keep pace with the volume increase of
the cell." Jensen (1955),in his morphological and biochemi-
cal analysis of root tip growth in Vicia faba, also has
shown that while during the stage of radial enlargement
protein synthesis does occur, elongation of these cells is
marked by a cessation of protein synthesis accompanied by
an increase in water uptake. Avery and Engel (1954), on the
other hand, found that in the Avena coleoptile total nitrogen
continues to increase when section growth is dependent solely
upon enlargement of the existing cells. These authors found
that this increase is roughly proportional to the increase
in,cell volume. They conclude that: "Cell enlargement thus
is not solely a matter of water uptake, but is accompanied by
the synthesis of additional protoplasm." This condition
certainly appears to be the case in the protocorm of Vanda.
Further evidence-for the occurrence of protein syn-
thesis in the enlarging parenchymatous cells of the proto-
corm is provided by the fact that cytoplasmic RNA is evident
and the relative concentration of this compound, like protein,
is roughly equal in the two regions of the protocorm. The
cytoplasmic RNA disappears from the parenchymatous region
after the completion of cytodifferentiation of the component
cells. This latter observation is in accord with the find-
ings of Brachet (1950) on early developing amphibian embryos
in which he reports that RNA increases in every organ just
before differentiation but decreases after differentiation
is complete unless the organ in question is active in protein
Observation of longitudinal protocorm sections reveals
the presence of groups of-cells in the parenchymatous region
in which the concentration of cytoplasmic RNA is consider-
ably higher than in the surrounding cells. These groups of
cells appear to occur at random in the parenchynatous region
but are usually located toward the center of the parenchymatous
mass and usually encompass those cells which form the vas-
cular strands. Similar results have been obtained in embryos
of Capsella bursa-pastoris by Pritchard (personal communi-
cation). The formation of vascular tissue from parenchyma
involves cell elongation. Thus it appears that elongation
of these cells requires protein synthesis at least during
the initial stages of differentiation. The possibility
exists, however, that the observed increase in concentration
of cytoplasmic RNA results from a reduction in cytoplasmic
volume due to plasmolysis induced by fixation. However, in
view of the fact that cells surrounding the region of high
cytoplasmic RNA concentration exhibit similar degrees of
vacuolation without noticeable change in RNA distribution,
the likelihood of fixation artifact seems remote.
Longitudinal sections of protocorms show that during
early phases of protocorm development the relative concen-
tration of histones in the nuclei of the two regions is
approximately equal. In later phases, however, the relative
concentration of histones in the large nuclei of the paren-
chymatous region is seen to increase. This increase in his-
tone staining occurs immediately prior to the initiation of
visible signs of the necrosis of this region. After the
increase in histone concentration, cytoplasmic RNA and total
protein decrease until the withering of the entire region is
Stedman and Stedman (1950) first proposed that his-
tones might act as regulators of genetic activity. Huang
and Bonner (1962) presented evidence that DNA bound to
histones is inactive in the support of DNA-dependent RNA
synthesis and these authors speculated that histones might
act as gene regulators. Allfrey et al. (1963) have further
shown that histones, protamines, and polylysine inhibit the
rate of incorporation of C14-adenosine into RNA and of
C14-labelled amino acids into protein in isolated thymus
nuclei. These authors conclude that histones inhibit a
large number of nuclear biosynthetic activities, probably
by interference with nuclear ATP synthesis. Thus the
mechanism of repression by histones, if it indeed exists,
is probably ouite complex. As pointed out by Barr and
Butler (1963), any model for the mode of action of gene
repression by histones would have to include a mechanism
which would insure the "presence and removal of particular
histone molecules at appropriate times."
The possibility exists that the increased histone
staining observed in the nuclei of the protocorm is due to
the depolymerization of the nucleoproteins during pycnotic
nuclear degeneration as reported by Leuchtenberger (1950).
Three basic changes in nuclear morphology may be observed
during the death of a cell: 1. pycnosis, which involves a
decrease in the size of the nucleus and a concentration of
the chromatin; 2. karyorrhexis, in which fragmentation of
the chromatin occurs; and 3. karyolysis, in which chromatin
basophilia decreases markedly. Examination of the cells of
the parenchymatous region during the developmental phase of
the protocorm in which an increase in histones is apparent
does not reveal any of the above listed symptoms of a dying
cell; in fact, the nuclei are large and exhibit a high
degree of basophilia with the Feulgen stain. Thus it
appears that the increase in nuclear histones observed in
this region accompanies an increase in the amount of DNA in
these nuclei, probably as a result of polyploidy.
The purpose of this investigation was to examine and
correlate the changes in relative concentration and dis-
tribution of total proteins, ribonucleic acid, insoluble
polysaccharides, histones, and sulfhydryl and tyrosine-con-
taining proteins throughout the development of the embryo
sac and embryo of Vanda. In this investigation, emphasis
was placed on the elucidation of the relationship between
the biochemical composition of a structure and its form.
Below are listed the major results and interpretations
derived from this study.
1. The inner and anticlinal primary walls of the
integumentary cells surrounding the megaspore and the walls
of the megaspore proper are thicker and richer in insoluble
polysaccharides than those of the other megasporangial cells.
The loss of plasmadesmatic connection between the megaspore
mother cell and the surrounding nucellar epidermis in
Dendrobium, and the unusually thick primary wall observed,
suggest that this condition might be peculiar to cells
embarked on a course of development quite unlike those of
its immediate neighbors.
2. There is a concentration gradient of total pro-
teins in the megasporangium, higher at the distal than
proximal end. It appears that this is a remnant of the
condition of active protein absorption through rhopheocy-
tosis previously described in Dendrobium.
1. Only the synergid cells have a high concentration
of insoluble polysaccharides. They also exhibit a high
concentration of total protein. Both of these classes of
compounds are ubiquitously distributed in the cells. It was
proposed that the synergids serve a nutritional role in
support of the egg, since no endosperm is present and the
egg is low in these compounds.
2. RNA concentration is low in all parts of the
embryo sac. The highest concentration appears in the polar
nuclei. After fertilization, the concentration of RNA in
the endosperm nucleus becomes progressively lower until it
is no longer detectable.
3. During the initiation of megaspore mitosis, the
cytoplasm becomes confined to a central strip bordered by
vacuoles, the concentration of total protein in the cytoplasm
rising markedly to a peak and then decreasing again during
cell enlargement. The process of embryo sac enlargement
appears to consist primarily of an increased water uptake
by the megaspore during mitotic division, protein synthesis
4. Around the time of fertilization, the chalazal
region of the embryo sac shows a high concentration of total
protein, which diminishes after the enlargement of the
embryo. This localized increase in total protein is postu-
lated to be due to an increase in the number of antipodal
cells followed by their breakdown. This process liberates
protein into the embryo sac which may then be taken up by
1. There is no difference in insoluble polysaccha-
rides between the suspensor initial and embryo initial in
the two-cell stage. When the suspensor apparatus consists
of two or more cells, however, the cell walls of the suspen-
sor become thicker than those of the embryo.
2. There is a difference in total protein and RNA
concentration between the suspensor initial and embryo ini-
tial at the two-cell stage. The embryo proper exhibits a
higher concentration of both these classes of compounds and
this difference persists until the demise of the suspensor
1. The Protocorm Stage is characterized by the pres-
ence of starch grains in the parenchymatous region, which
diminish in size as development progresses. It was proposed
that the parenchymatous region serves as a storage tissue for
the active meristematic portion in lieu of an endosperm tis-
2. The differentiation of a meristematic cell into
a parenchymatous cell involves an increase in over-all size.
This increase is due partially to vacuolation. Thus this
growth involves water uptake accompanied by protein synthe-
sis at a rate sufficient to maintain a constant protein
3. RNA distribution in the protocorm parallels that
of protein. However, in the parenchymatous region, groups
of cells associated with the formation of vascular tissue
exhibit inordinately high concentrations of cytoplasmic RNA.
4. Differential extraction of longitudinal sections
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A. Methyl esterification of the pectins is low in all cells.
B. The walls of the meristematic cells contain proportion-
ately higher amounts of pectic material than the parenchy-
matous cells. C. The major non-pectic components of the
cell wall of all cells in the protocorm are the hemicellu-
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Marvin Ray Alvarez was born April 3, 1936, at Tampa,
Florida. In June, 1954, he was graduated with honors from
Thomas Jefferson High School. He received the degree of
Bachelor of Science with a major in biology and a minor in
chemistry from the University of Tampa in June, 1958. From
1958 to 1961,Mr. Alvarez taught biology and chemistry at
Hillsborough High School in Tampa. In 1960, he was awarded
a National Science Foundation summer institute fellowship
to pursue graduate study at the University of Florida.
In September, 1961, he enrolled at the University of
Florida as a graduate student. He received the degree of
Master of Science and has since been pursuing his work
toward the degree of Doctor of Philosophy. During this
time, Mr. Alvarez has held a Graduate Teaching Assistant-
ship, a Research Assistantship, and was awarded a Graduate
Marvin Ray Alvarez is married to the former Delma
Barbara Flores and is the father of two children. He is a
member of the Phi Sigma Biological Society and the Botanical
Society of America.
This dissertation was prepared under the direction of
the chairman of the candidate's supervisory committee and
has been approved by all members of that committee. It was
submitted to the Dean of the College of Agriculture and to
the Graduate Council, and was approved as partial fulfill-
ment of the requirements for the degree of Doctor of
,/Dean, College of Agriculture
Dean, Graduate School
1' G.R ^//
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