Citation
A Histochemical study of embryo development in Vanda (Orchidaceae)

Material Information

Title:
A Histochemical study of embryo development in Vanda (Orchidaceae)
Creator:
Alvarez, Marvin Ray, 1936- ( Dissertant )
Sagawa, Yoneo ( Thesis advisor )
Biggs, Robert H. ( Reviewer )
Conger, A. D. ( Reviewer )
Cowden, R. R. ( Reviewer )
Edwardson, J. R. ( Reviewer )
Noggle, G. R. ( Reviewer )
Place of Publication:
Gainesville, Fla.
Publisher:
University of Florida
Publication Date:
Copyright Date:
1964
Language:
English
Physical Description:
vii, 70 leaves : ill. ; 28 cm.

Subjects

Subjects / Keywords:
Cell walls ( jstor )
Cells ( jstor )
Cytoplasm ( jstor )
Embryo sac ( jstor )
Embryos ( jstor )
Histones ( jstor )
Internet search systems ( jstor )
Megaspores ( jstor )
Protocorms ( jstor )
RNA ( jstor )
Botany thesis Ph. D
Dissertations, Academic -- Botany -- UF
Orchids ( lcsh )
Plant physiology ( lcsh )
City of Tampa ( local )
Genre:
bibliography ( marcgt )
non-fiction ( marcgt )

Notes

Abstract:
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 differentiation. 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 impetus from the efforts of Linderstrom-Lang et al. in the Carlsberg Laboratory. The second method, microscopic histochemistry, 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 differentiation 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 concerning the correlation of respiration and total proteins utilizing root tip cells were conducted by Brown 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....Lfost 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, differentiating tissue In which one can, by sampling all stages of development, relate each cell to the zygote. The disadvantages 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 addition 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 concentration of the insoluble polysaccharide fraction of the cell wall, total proteins, ribonucleic acid, histone, and sulfhydryl and tyro sine-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 information relating to cellular differentiation.
Thesis:
Thesis (Ph. D.)--University of Florida, 1964.
Bibliography:
Includes bibliographical references (leaves 63-69).
General Note:
Typescript.
General Note:
Vita.
Statement of Responsibility:
by Marvin Ray Alvarez.

Record Information

Source Institution:
University of Florida
Holding Location:
University of Florida
Rights Management:
Copyright [name of dissertation author]. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
Resource Identifier:
029995952 ( AlephBibNum )
37441447 ( OCLC )
ACG2433 ( NOTIS )

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A HISTOCHEMICAL STUDY OF
EMBRYO DEVELOPMENT

IN VANDA (ORCHIDACEAE)











By
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
June, 1964












ACKNOWLEDGEMENTS


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-

niaues.

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

Fellowship.

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 . . . . . . . . .


Page

Sii


S v


6



9
. . 6
. . 6
. . 7
. . 7
. . 9


10
10
12
12
13
16
17

39


S. 39
S. 40
S. 41
S 43
S. 45
S 45
S. 47
S 48
S. 50
S. 53
S. 56

S. 59

. .63


I I I I I I









Page

BIOGRAPHICAL SKETCH . . . . . . . . .. 70












LIST OF FIGURES


Figure Page

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








Figure


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
Lulticellular Embryo
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


. 34


. 34


. 36


Page











LIST OF TABLES


Table Page

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













INTRODUCTION


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

problem.

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.


Botanical Histochemistry

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





5


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.

Culture Technicue

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

lamps.

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-

sis.

2. Gametophyte Stage. Development from mitosis to

fertilization.

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


Chemical
Conrstl tnenta


Method or Reagent


Fixation Reference


Total insolu-
ble polysac-
charides




Starch


Pectins


Hemicellu-
loses


Periodic acid-
Schiff's reaction
(PAS)




1% 12KI-polarizing
optics

Hot ammonium oxalate
extraction

4% sodium hydroxide
extraction preceded
by pectin removal
and followed by PAS
reaction


Formalin-
acetic
acid-
alcohol
(FAA)
(4 hrs.)

None


FAA
(4 hrs.)

FAA
(4 hrs.)


Hotchkiss
(1948)




Johansen (1940)


Jensen (1962)


Jensen (1962)


Cellulose 17% sodium hydroxide
extraction preceded
by pectin and hemi-
cellulose removal
followed by PAS
reaction

Total Ninhydrin-Schiff's
proteins reaction

Mercuric bromphenol
blue


FAA
(4 hrs.)


Jensen (1962)


FAA Yasuma and
(4 hrs.) Ichikawa (1953)

Iazia et al.
(1953)-


Ribonucleic
acid (RNA)





Deoxyribonu-
cleic acid
(DNA)

Histones


Azure B, pH 4, fol-
lowing deoxyribonu-
clease treatment
(12 hrs.)




Feulgen reaction



Fast green, pH 8.0


Carnoy (3
parts al-
cohol: 1
part gla-
cial ace-
tic acid
(24 hrs.)

Carnoy
(3:1)
(24 hrs.)

FAA
(4 hrs.)


Flax and Himes
(1950)





Jensen (1962)



Alfert and
Geschwind
(1953)


Cnnctitunnta~~








Table 1, continued

Chemical Method or Reagent Fixation Reference
Constituents

Sulfhydryl Azo-aryl mercaptide FAA Bennett and
(-SH)-contain- coupling (4 hrs.) Watts (1958)
ing proteins

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

1/6 td3.











OBSERVATIONS


Background

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-

ratus.

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

approximately linear.

Megaspore Stage

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-

cularly evident.

C-ametophyte Stage

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

polysaccharides.

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

embryo proper.

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

(Fig. 17).

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).


Embryo Stare

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).


Protocorm Stage

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

(Fig. 21).








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

cytoplasm.

Feulgen staining of the late protocorm shows a marked

contrast between the staining intensity of the meristematic

and parenchymatous nuclei, the latter being considerably

darker.

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


C,


'I


C













h


1.1
f


'I

a.









4r






22








co

o or C- H m
E 0 H '--
o o a11) t

0 ;m 01 0



c) C 0o ),
OCd 0 ) -i 4 z to H r)
+ O 0) H I' tO

0 oH -d




1H Q' a)

O ,0 Z Io 0
H ) C) H LO C'2 (0 0 C
Ho St 0 ) tO DH 0
'ci 0 H C) -- ---) 0) 0 V) CO
0z 0 Hu 0 z 9 __
H O 0 Q) a)I
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0 0

4- 0 a 0 )

04-' 4- 4- 0 0 a

C 0 H C C6 c) o

C)O) 00 C) -4 a
0 0) COD o CL C) cY rd C



to 1 o P0
1 H H o

4- zi aH )~ PH





0)











Apical

A Parenchymatous


Early Protocorm
(185-200.t )


Late Protocorm
(700-800')


Fig. 2--Comparison of 1'eristematic
and Parenchymatous Cell Volumes During Development


1000-



500-





200-


i -
Two-Cell
Proembryo


ICL~-C-~` I -111~.~~ 1 ----~--~I I _




24



100,000- -10,000

SCell volume

Nuclear volume








10,000- -1,000





-*
H / a )o




I I



0
1,000- /100












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
















0

OQ





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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. 7


FI&. (o











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
polar nuclei.







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
embryo sac.







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. 11


FIG. 10
















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-
pensor initial.





















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.





























FIG. 13


FIG. 15


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
increases markedly.





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
development.






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.




























FIG. 16


FI G. 19


FIG. 18










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
of development.







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.































FIG. 20


'' '-
.,7- :.;,
A^:--,,
:, " "-1'





;Ii
S. *} -;* ,' ,






, -; '- i '" '. (


' ; ,
, ...-'* .. -* ':' ,, * ;
'-. _' .^'.". '.,


FIG. 23


FIG. 21


FIG.22















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
parenchymatous nuclei.









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.





.38


FIG. 2z 4


FIG. 25


FIG. 26


FIG. 27












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-

tion.

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 Synergids

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

Stellaria media.

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.

Starch Distribution

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

Zalokar (1961).

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.


The Megasoorangium

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

Stage.

The Gametoohyte

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 Embryo

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

cell?

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

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-

stematic cells.

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

synthesis.

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.


The Histones

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

complete.








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




58


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.












SUMMARY


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.


Megaspore Stage

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.

Gametophyte Stage

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

being minimal.








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

the embryo.

Embryo Stage

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

apparatus.

Protocorm Stage

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-

sue.








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

concentration.

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

of the protocorm for cell wall constituents reveal that:

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-

loses.

5. Concentration of histones in the nuclei of the

meristematic and parenchymatous cells appears approximately

equal in the early phases of the Protocorm Stage. Immediately

prior to the morphologically detectable initiation of necro-

sis of the parenchymatous region, the concentration of his-

tones in the nuclei of these cells increases markedly. The

question of the role of histones as gene repressors in con-

nection with this phenomenon was discussed.












LITERATURE CITED


Albersheim, P. and J. Bonner. 1959. Metabolism and hormo-
nal control of pectic substances. Jour. Biol. Chem.
234:3105-3108.

Alfert, M. and I. I. Geschwind. 1953. A selective stain-
ing method for the basic proteins of cell nuclei.
Proc. Nat. Acad. Sci. (U.S.) 39:991-999.

Allfrey, V. G., V. C. Littau, and A. E. Mirsky. 1963. On
the role of histones in regulating ribonucleic acid
synthesis in the cell nucleus. Proc. Nat.Acad. Sci.
(U.S.) 49:414-421.

Alvarez, M. R. 1962. Studies on the ontogeny of Vanda.
Master's Thesis, University of Florida.

Ambellan, E. 1955. Effect of adenine mononucleotides on
neural tube formation of frog embryo. Proc. Nat. Acad.
Sci. (U.S.) 41:428-432.

Ambronn, H. and A Frey. 1926. Cited after Mihlethaler.
1961.

Audus, L. J. 1959. Plant Growth Substances. Interscience
Publishers, Inc., New York. Pp. 553.

Avers, C. J. 1958. Histochemical localization of enzyme
activity in the root epidermis of Phleum oratense.
Amer. Jour. Bot. 45:609-612.

__. 1961. Histochemical localization of enzyme
activities in root meristematic cells. Amer. Jour. Bot.
48:137-142.

Avers, C. J. and E. E. King. 1960. Histochemical study of
intracellular enzymatic heterogeneity of plant mito-
chondria. Amer. Jour. Bot. 47:220-225.

Avery, G. S. Jr. and F. Engel. 1954. Total nitrogen in
relation to age and position of cells in Avena coleop-
tiles. Amer. Jour. Bot. 41:310-315.








Avery, 0. T., C. M. McLeod, and M. McCarty. 1944. Studies
on the chemical nature of the substance inducing trans-
formation of pneumococcal types. J. Exptl. Med.
79:137-158.

Barr, G. C. and J. A. V. Butler. 1963. Histones and gene
function. Nature 199(4899):1170-1172.

Bennett, H. S. and R. H. Watts. 1958. The cytochemical
demonstration and measurement of sulphydryl groups by
azo-aryl mercaptide coupling, with special reference to
Mercury Orange. Gen. Cytochem. Methods 1:318-374.

Bishop, C. T., S. T. Bayley, and G. Setterfield. 1958.
Chemical constitution of the primary walls of Avena
coleoptiles. Plant Physiol. 30:283-288.

Boivin, A., R. Vendrely, and C. Vendrely. 1948. L'acide
desoxyribonuclique du noyau cellulaire, de'positaire
des caracteres here'ditaires; arguments d'ordre analy-
tique. Compt. Rend. 226:1061-1063.

Bonner, J., L. Ordin, and R. Cleland. 1955. Auxin-induced
water uptake. In The Chemistry and Iode of Action of
Plant Growth Substances. Acad. Press, New York.
pp. 260-270.

Brachet, J. 1950. Queloues observations sur le mode
d'action de l'organisateur chez les amphibiens.
Experimentia 6:56-57.

S1957. Biochemical Cytology. Acad. Press, New
York. Pp. 535.

Brachet, J. and L. Ledoux. 1955. L'action de la ribonu-
clease sur la division des ouefs d'amphibiens. Exptl.
Cell Research suppl. 3, 27-39.

Brown, R. and D. Broadbent. 1950. The development of cells
in the growing zones of the root. Exptl. Botany
1:249-263.

Correll, D. S. 1950. Native Orchids of North America.
Chronic Botanica Company, Waltham, Mass. Pp. 329.

Das, N. K. and M. Alfert. 1961. Accelerated DNA synthesis
in onion root meristem during x-radiation. Proc. Nat.
Acad. Sci. (U.S.) 47:1-6.

Deeley, E. M., H. G. Davis, and J. Chayen. 1957. The DNA
content of cells in the root of Vicia faba. Exptl.
Cell Research 12:582-591.








Duncan, R. E. 1959. Orchids and cytology. In The Orchids.
Ed. Carl L. Withner. Ronald Press Co., New York.
pp. 189-260.

Ebert, J. D. 1954. The effects of chorio-allantoic trans-
plants of adult chicken tissues and homologous tissues
of the host chick embryo. Proc. Nat. Acad. Sci. (U.S.)
40:337-347.

Elson, D., T. Gustafson, and E. Chargaff. 1954. The
nucleic acids of the sea urchin during embryonic devel-
opment. J. Biol. Chem. 209:285-293.

Esau, K. 1953. Plant Anatomy. John Wiley and Sons, New
York. Pp. 376.

Fico, A. 1954. Analyse de l'induction neurale chez les
amphibiens au moyen d'organistatems marquis. J. Embryl.
Exptl. Morphol. 2:194-203.

Fitting, A. 1900. Cited after MUhlethaler. 1961.

Flax, M. H. and M. H. Himes. 1950. A differential stain
for ribonucleic acid and desoxyribonucleic acid. Anat.
Record 108:529.

Flemion, F. 1961. Cytochemical studies of the developing
primary cell wall in the apical shoots of normal and
physiologically dwarf peach seedlings. Plant Physiol.
36(suppl. XXVII):51.

Francis, '. D. and T. Winnick. 1953. Studies on the path-
way of protein synthesis in tissue culture. J. Biol.
Chem. 202:273-289.

Frey-Wyseling, A. 1935. Cited after Mihlethaler. 1961.

1936. Cited after iMlhlethaler. 1961.

Fuller, H. J. and 0. Tippo. 1954. College Botany. Henry
Holt and Co., New York. Pp. 993.

Galston, A. W. and W. K. Purves. 1960. The mechanism of
action of auxin. Ann. Rev. of Plant Physiol. 11:239-276.

Gifford, E. M. Jr. and H. B. Tepper. 1962a. Histochemical
and autoradiographic studies of floral induction in
Chenopodium album. Amer. Jour. Bot. 49:706-714.

and 1962b. Ontogenetic and histochemical
changes in the vegetative shoot tip of Chenopodium
album. Amer. Jour. Bot. 49:902-911.







Green, P. B. 1958. Concerning the site of the addition of
new wall substances to the elongating Nitella cell wall.
Amer. Jour. Bot. 45:111-116.

Holmes, B. E., L. K. Mee, S. Hornsey, and L. H. Gray. 1955.
The nucleic acid content of cells in the meristematic
elongating and fully elongated segments of roots of
Vicia faba. Exptl. Cell Research 8:101-113.

Hotchkiss, R. D. 1948. A microchemical reaction resulting
in the staining of polysaccharide structures in fixed
tissue preparations. Arch. Biochem. 16:131-141.

Huang, R. C. and J. Bonner. 1962. Histone, a supressor of
chromosomal RNA synthesis. Proc. Nat. Acad. Sci. (U.S.)
48:1216-1221.

Hubert, B. and J. Maton. 1939. The influence of synthetic
growth-controlling substances and other chemicals on
postfloral phenomenon in tropical orchids. Biol. Jaarb.
6:244-285.

Hultin, T. 1950. The protein metabolism of sea urchin
eggs during early development studied by means of N15-
labelled ammonia. Exptl. Cell Research 1:599-602.

Israel, H. W. 1962. An electron microscope study of mega-
spore development in Dendrobium orchids. Doctoral
Dissertation, University of Florida.

Jansen, E. F., R. Jang, P. Albersheim, and J. Bonner. 1960.
Pectic metabolism of growing cell walls. Plant Physiol.
35:87-97.

Jensen, W. A. 1955. A morphological and biochemical analy-
sis of the early phases of cellular growth in the root
tip of Vicia faba. Exptl. Cell Research 8:506-522.

1956. The cytochemical localization of acid
phosphatase in root tip cells. Amer. Jour. Bot.
43:50-54.

1956. On the distribution of nucleic acids
in the root tip of Vicia faba. Exptl. Cell Research
10:222-226.

S1958. The nucleic acid and protein content
of root tip cells of Vicia faba and Allium cepa. Exptl.
Cell Research 14:575-583.

1960. The composition of the developing pri-
mary wall in onion root tip cells. II. Cytochemical
localization. Amer. Jour. Bot. 47:287-295.








Jensen, W. A. 1962. Botanical Histochemistry. McGraw-Hill,
New York. Pp. 408.

Jensen, W. A. and A. McLaren. 1960. Uptake of proteins by
plant cells--the possibility of pinocytosis in plants.
Exptl. Cell Research 19:414-416.

Johansen, D. A. 1940. Plant Microtechniaue. McGraw-Hill,
New York. Pp. 523.

Katz, J. R. 1924. Cited after MGhlethaler. 1961.

Lamport, D. T. A. 1963. Oxygen fixation of plant cell wall
protein. Jour. Biol. Chem. 238:1438-1440.

Ledoux, L., P. Galand, and R. Huart. 1962. Nucleic acids
and protein metabolism in barley seedlings. II. Inter-
relations of the different organs. Exptl. Cell Research
27:132-136.

Leuchtenberger, C. 1950. A cytochemical study of pycnotic
nuclear degeneration. Chromosoma 3:449-473.

Lillie, R. D. 1957. Adaption of the Morel Sisley protein
diazotization procedure to the histochemical demonstra-
tion of protein bound tyrosine. Jour. Hist. and Cytochem.
5:528-532.

Magli, G. 1958. The possibility of substitution with auxin
for the action of pollen on the development of the
ovules of the orchid. Nuovo Jiorn. Bot. Ital.
65(3):401-416.

Maheshwari, P. 1950. An Introduction to the Embryology of
Angiosperms. McGraw-Hill, New York. Pp. 453.

Mazia, D., P. A. Brewer, and M. Alfert. 1953. The cyto-
chemical staining and measurement of protein with mer-
curic bromphenol blue. Biol. Bull. 104:57-67.

Markert, C. L. 1958. Chemical concepts of cellular differ-
entiation. In The Chemical Basis of Development. Ed.
W. D. McElroy and B. Glass. Johns Hopkins Press,
Baltimore. pp. 3-16.

McLeish, J. 1959. Comparative microphotometric studies of
DNA and arginine in plant nuclei. Chromosoma
10:686-710.

Mirsky, A. E. and V. Allfrey. 1958. The role of the cell
nucleus in development. In The Chemical Basis of
Development. Ed. W. D. McElroy and B. Glass. Johns
Hopkins Press, Baltimore. pp. 94-99.








KGhlethaler, K. 1961. Plant cell walls. In The Cell.
Acad. Press, New York. pp. 85-135.

Naegli, C. 1864. Cited after MGhlethaler. 1961.

Niimoto, D. H. and Y. Sagawa. -1962. Ovule development in
Phalaenopsis. Caryologia 15(1):89-97.

Northcote, D. H. 1963. The biology and chemistry of the
cell walls of higher plants, algae, and fungi. Inter-
nat. Rev. Cyt. 14:223-265.

Ordin, L., R. Cleland, and J. Bonner. 1955. Influence of
auxin on cell wall metabolism. Proc. Nat. Acad. Sci.
(U.S.) 41:1023-1029.

Patau, K. and H. Swift. 1953. The DNA content (Feulgen)
of nuclei during mitosis in a root tip of onion.
Chromosoma 6:149-160.

Preston, R. D. 1959. Wall organization in plant cells.
Internat. Rev. Cyt. 8:33-58.

Pritchard, H. 1962. Cytochemical studies on the megasporo-
genesis and embryogenesis in Stellaria media (L) Cyrill.
Doctoral Dissertation, Lehigh University.

Ray, P. M. 1961. Hormonal regulation of plant cell growth.
In Control Mechanisms in Cellular Processes. Ronald
Press Co., New York. pp. 185-212.

Reeve, R. M. 1959. Histological and histochemical changes
in developing and ripening peaches. II. The cell walls
and pectins. Amer. Jour. Bot. 46:241-247.

Sagawa, Y. and H. W. Israel. Post-pollination ovule devel-
opment in Dendrobium orchids. I. Introduction. In
press. Caryologia.

Scott, F. M., K. C. Hammer, E. Baker, and E. Bowler. 1956.
Electron microscope studies of cell wall growth in the
onion root. Amer. Jour. Bot. 43:313-324.

Sirlin, J. L. 1955. Nuclear uptake of methionine-S35 in
the newt embryo. Experimentia 11:112-113.

Stedman, E. and E. Stedman. 1950. Cell specificity of his-
tones. Nature 166(4227):780-781.

Sunderland, N., J. K. Heyes, and R. Brown. 1957. Protein
and respiration in the apical region of the shoot of
Lupinus alba. J. Exptl. Bot. 8:55-70.

Swamy, B. G. L. 1942. Morphological studies in three spe-
cies of Vanda. Current Science 11:285-286.








Taylor, J. H. 1958. Incorporation of P-32 into nucleic
acids and proteins during microgametogenesis of Tulbaghia.
Amer. Jour. Bot. 45:123-131.

1959. Autoradiographic studies of nucleic
acids and proteins during meiosis in Lilium longiflorum.
Amer. Jour. Bot. 46:477-484.

1963. Molecular Genetics. Acad. Press, New
York. Pp. 544.

Thimann, K. V. 1960. Plant growth. In Fundamental Aspects
of Normal and Malignant Growth. Elsevier Publ. Co.,
Amsterdam. Pp. 1025.

Vacin, E. and F. Went. 1949. Culture solution for orchid
seedlings. Bot. Gaz. 110:605-613.

von Mohl, H. 1859. Cited after MGhlethaler. 1961.

Weber, E. 1929. Entwichlungs geschichtliche Untersuchungen
iber die Gattung Allium. Bot. Arch. 25:1-44.

"Whaley, W. G., L. W. Mericle, and C. Heimsch. 1952. The
wall of the meristematic cell. Amer. Jour. Bot.
39:20-26.

Wirth, M. and C. L. Withner. 1959. Embryology and develop-
ment in the Orchidaceae. In The Orchids. Ed. Carl L.
Withner. Ronald Press Co., New York. pp. 155-158.

Woodard, J., E. Rasch, and H. Swift. 1961. Nucleic acid
and protein metabolism during the mitotic cycle in Vicia
faba. J. Biophys. Biochem. Cytol. 9:445-462.

Woodstock, L. W. and F. Skoog. 1962. Distributions of
growth, nucleic acids, and nucleic acid synthesis in
seedling roots of Zea mays. Amer. Jour. Bot.
49:623-633.

Yasuma, A. and T. Ichikawa. 1953. Ninhydrin-Schiff and
alloxan-Schiff staining. A new histochemical staining
method for proteins. J. Lab. and Clin. Med. 41:623-633.

Zalokar, E. 1961. Ribonucleic acid and the control of
cellular processes. In Control Mechanisms of Cellular
Processes. Ronald Press Co., New York. pp. 87-140.










BIOGRAPHICAL SKETCH


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

School Fellowship.

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
Philosophy.


Ap~eb, 1964


,/Dean, College of Agriculture



Dean, Graduate School
Supervisory Committee:


Chairman


-J






1' G.R ^//
,2^"i ^m)- __




Full Text

PAGE 1

A HISTOCHEMICAL STUDY OF EMBRYO DEVELOPMENT IN VANDA (ORCHIDACEAE) By 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 June, 1964

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ACKNOWLEDGEMENTS 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 histochemlcal techniques. Grateful acknowledgement is herewith given for financial 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 Fellowship. 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. 11

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TABLE OF CONTENTS Page ACKNOWLEDGEMENTS ii LIST OF FIGURES v LIST OF TABLES vii INTRODUCTION 1 Botanical Histochemistry 2 The Embryo as a Tissue Source 3 MATERIALS AND METHODS 6 Culture Technique 6 Categorization of Developmental Stages 7 Histochemical Methods and Technique 7 Method of Cell Volume Analysis 9 OBSERVATIONS 10 Background 10 Size Relationships During Embryo Development .... 12 Megaspore Stage 12 Gametophyte Stage 13 Embryo^ Stage 16 Protocorm Stage 17 DISCUSSION OF OBSERVATIONS 39 The Cell Wall 39 The Cell Wall in the Megaspore and Embryo Stages . . 40 The Cell Wall in the Protocorm Stage 41 The Synergids 43 Starch Distribution 45 Proteins and RNA 45 The Mega sporangium 47 The Gametoohyte 48 The Embryo 50 The Protocorm 53 The Histones 55 SUMMARY 59 LITERATURE CITED 63 ill

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Page BIOC-RAPHICAL SKETCH 70 lv

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LIST OF FIGURES Figure Page 1. Sequence of Embryo Development 21 2. Comparison of Merl sterna tic 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-mltotic 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 Multicellular Embryo 32 14. Total Protein Distribution in Early Multicellular Embryo 32 15. Total Protein Distribution in Early Multicellular Embryo 32

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Figure Page 16. A Longitudinal Section of an Embryo Sac Showing a Post Fertilization Total Protein Increase at the Chalazal End 34 17. A Longitudinal Section of a Two-cell Embryo Stained for Ribonucleic Acid 34 18. A Longitudinal Section of an Early Multicellular Embryo Stained for Ribonucleic Acid 34 19. A Longitudinal Section of an Early Protocorm Stained with PAS 34 20. A Longitudinal Section of a Late Protocorm Stained with PAS 36 21. A Longitudinal Section of an Early Protocorm Stained for Total Protein 36 22. A Longitudinal Section of an Early Protocorm Stained for Ribonucleic Acid 36 23. A Longitudinal Section of an Ammonium Oxalate Extracted Early Protocorm Showing Pectin Distribution 36 24. Longitudinal Section of Early and Late Protocorm Showing Changes in Hi stone Concentration 38 25. Longitudinal Section of Early and Late Protocorm Showing Changes in Histone Concentration 38 26. A Longitudinal Section of a Protocorm Stained for Tyrosine-contalning Proteins ... 38 27. A Longitudinal Section of a Protocorm Stained for Sulfhydryl-containing Proteins . . 38 vi

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LIST OF TABLES Table Page 1. Hlstochemical Methods 8 2. Comparison of Mean Cell Volume {/*$) of Merlstematlc 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 vil

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INTRODUCTION Most higher plants and animals originate from a single 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 problem. Cellular differentiation is reflected in both morphologic 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 synthesis 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

PAGE 9

through ribonucleic acid.(RNA) controls protein synthesis. As a result of the work of Watson and Crick, Kornberg, Nlrenberg, Ochoa, and others (Taylor, 1963), the facts concerning 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 different times thus resulting in differences between cells. Botanical Histochemistry 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 differentiation. The first method, a_uantitative histochemistry, involves the use of scaled down biochemical techniques which are applied to small samples of tissue obtained by sectioning. Quantitative histochemistry has received impetus from the efforts of Linderstrom-Lang e_t al. in the Carlsberg Laboratory. The second method, microscopic histochemistry, utilizes the specificity of certain dyes for specific chemical substances thus achieving Intracellular

PAGE 10

localization of substances. These latter methods have been applied extensively by zoologists in the study of differentiation 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 concerning the correlation of respiration and total proteins utilizing root tip cells were conducted by Brown and Broadbent (1950). Whaley et al. (1952) investigated hi stochemically 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; Glfford and Tepper, 1962a, 1962b). The Embryo as a Tissue Source Lfost 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

PAGE 11

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, differentiating tissue In which one can, by sampling all stages of development, relate each cell to the zygote. The disadvantages 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 addition 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 concentration of the insoluble polysaccharide fraction of the cell wall, total proteins, ribonucleic acid, histone, and sulfhydryl and tyro sine-containing proteins over the entire embryonic sequence of events from the Megaspore Stage

PAGE 12

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 information relating to cellular differentiation.

PAGE 13

MATERIALS AND METHODS The following cultlvars of Vanda were used in this study: Vanda xHelen Paoa (Univ. of Pla.— UP #1109), Vanda xM. Foster X Vanda xE. Noa (UF #1115), Vanda xBurgeffii (UF #1116), and Vanda xHawailan Blue (UF #1531). Since no differences were noted in the embryogeny of these hybrids, they will be given a common description here. Culture Technique Several flowers on each of the plants were pollinated with fresh pollen and the plants were kept in the greenhouse. 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 6

PAGE 14

removed and each was cultured In 125 ml. Erlenmeyer flasks on the surface of a solidified culture solution (Vacin and Went, 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 30°C at 120 foot candles of illumination from two cold fluorescent strip lamps. Categorization of Developmental Stages The developmental stages investigated were: 1. Megaspore Stage. Includes development from the completion of melosis to the initiation of embryo sac mitosis. 2. G-ametophyte Stage. Development from mitosis to fertilization. 3. Embryo Stage. Includes embryo development occurring within the ovule. 4. Protocorm Stage. Embryo development from germination up to primordial leaf and primary root formation. Hlstochemlcal Methods and Technique 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 56°C for 8 to 12 hours, and sectioned to appropriate thicknesses (Johansen, 1940). The hlstochemlcal methods used for the Identification and localization of particular constituents are summarized in Table 1.

PAGE 15

Table 1 — His to chemical Methods Chemical Constituents

PAGE 16

Table 1, continued Chemical Constituents Method or Reagent Fixation Reference Sulfhydryl Azo-aryl mercaptide FAA (-SH) -containcoupling (4 hrs.) ing proteins Tyrosine MorelSi sley reacFAA tion (4 hrs.) Bennett and tfatts (1958) Lillie (195?) Method of Cell Volume Analysis Measurements of cell size for the purpose of demonstrating the differential in cell enlargement evident between the meristematlc 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 approximately 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 meristematlc 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 1/6 7Td 5 .

PAGE 17

OBSERVATIONS Background 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 Phalaenopsls (Niimoto and Sagawa, 1952) and Dendroblum (Sagawa and Israel, in press) to consist of the formation of a system of dichotomously branched protruberences, or megasporangia, which are organized into a central column of nucellar cells surrounded by a single epidermal layer with an enlarged terminal archesporial cell which functions directly as a megaspore mother cell. Subsequent 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. 10

PAGE 18

11 The sequential steps in embryo development are illustrated in Fig. 1. The zygote divides transversely forming two cells. Subsequently, the basal (mlcropylar) cell divides in the same manner thus forming a two-cell proembryo and a suspensor initial. Subsequent vertical divisions of the suspensor initial result in an eight-cell suspensor apparatus, lleanwhile 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 fingerlike projections of the suspensor apparatus. Growth of the embryo results in the differentiation of two intergrading regions. The cells proximal to the suspensor apparatus enlarge and become parenchymatous while those distal to the suspensor remain meristematic. Continued 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 epidermal cells of the parenchymatous region. By this time the cells of the suspensor have become necrotic. At this stage in the developmental sequence a primordial 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 photo synthetic.

PAGE 19

12 Size Relationships During Embryo Development Differentiation of the embryo of Vanda is characterized 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 pro embryo 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 approximately linear. Kegaspore Stage 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 megaspore and the attending tissue. Treatment of cross sections of the megasporangium with the periodic acid-Schif f ' s reaction reveals, hov/ever, that the inner periclinal and the

PAGE 20

13 anticlinal walls of the nucellar epidermis (integumentaryInitials) 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 polysaccharides (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 megasporanglum with either mercuric bromphenol blue or by the ninhydrin-Schif f • s reaction shows that the highest concentration of total protein in the megasporanglum occurs in the megaspore and in the nucellar cells immediately surrounding the megaspore (Fig. 5). All cells of the megasporanglum appear to have a higher concentration 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 particularly evident. G-ametophyte Stage PAS staining of the embryo sac shows a marked contrast in the concentration of insoluble polysaccharides between the components of the raegagametophyte (Fig. 6). The cell walls and cytoplasm of the egg are faintly PAS

PAGE 21

14 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 synergids 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 polysaccharides. Prior to megaspore mitosis, the concentration of total protein in the cytoplasm undergoes a marked rise. The elongation 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

PAGE 22

15 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 concentration 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 concentration in the embryo initial than in the suspensor initial. In later multicellular stages of the Embryo Stage, the difference in total protein concentration between the suspensor apparatus and the embryo proper becomes quite 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 constituent, however, appear between the individual cells of the embryo proper. An interesting phenomenon encountered in the embryo sac shortly after fertilization is the formation of an illdefined region of very high protein concentration at the chalazal limits of the embryo sac which gradually disappears as the embryo increases in size (Fig. 15).

PAGE 23

16 During the zygote and two-cell proembryo phases of the Embryo Stage, the ovule contains RNA only in the nucleoli. 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 immediately 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 initial and disappears during enlargement of the suspensor s (Fig. 17). 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 ribonucleic 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). Embryo Stage No difference in the amount of insoluble polysaccharides was detectable at the two and three-cell stages (Fig. 10)

PAGE 24

17 V/hen the suspensor apparatus becomes morphologically distinct, ho?/ever, the cell walls of these cells become thicker and more highly PAS positive than those of the embryo proper (Fig. 11). This difference does not appear, however, until the suspensor apparatus consists of at least two cells. In the multicellular Embryo Stage, cytoplasmic RNA concentration is low in all cells but appears to be somewhat higher in the cells of the embryo proper (Fig. 18). Protocorm Stage 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 differentiated 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 hylura. They give a slightly positive reaction with mercuric bromphenol blue and the ninhydrin-Schiff • s reaction and also with sudan III. Polarizing optics, however, show them to be birefrlngent. In the early Protocorm Stage, total protein distribution is uniform in the meri sterna tic cells and the parenchymatous cells. This result was obtained by staining with mercuric bromphenol blue and the ninhydrln-Schiff ' s reaction (Fig. 21).

PAGE 25

18 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 concentration is distinctly higher in the meristematlc apical cells, particularly in those at the base of the leaf primordium. Hot ammonium oxalate extraction of the pectic substances from the cell walls in the Protocorm Stage with subsequent PAS staining shows that the cell walls of the meristematlc 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 parenchymatous cells than in the meristematlc cells and that hemlcellulose 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 esteriflcation of pectin is very low, if not indeed absent, in the cell walls of all the cells during the

PAGE 26

19 Protocorm Stage. Certain sections exhibited a very weak positive reaction In the walls of some parenchymatous cells, Indicating slight pectin esterlflcation, 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 hlstones in the nuclei of the parenchymatous cells than In the meristematic 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 concurrent 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 cytoplasm. Feulgen staining of the late protocorm shows a marked contrast between the staining intensity of the meristematic and parenchymatous nuclei, the latter being considerably darker. Tyrosine and sulfhydryl-contalning 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.

PAGE 27

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 vertical 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 meristematlc while those proximal to the suspensor enlarge and become vacuolated (g). This results in the formation of intergrading meristematlc and parenchymatous regions (h, 1).

PAGE 28

21 MNCE °f EMBRYO OEl/EL OPM ENT ->

PAGE 29

22 o •H +3 e CD +3+3 CQ C •H CD F* S CD (X ~z o > CD to i += s 2 O CD.C B bD rH O O Fh >^ +3 H H
PAGE 30

23 1000. CO 4^ •H C •H 5 O > H H CU O c cd cu r 50020010080604020104H 2© Apical & Parenchymatous O & A TwoCell Proembryo Early Protocorm (185-200/*) v Late Protocorm (700-800^) Fig. 2 — Comparison of Meristematic and Parenchymatous Cell Volumes During Development

PAGE 31

24 100,000• Cell volume © Nuclear volume 10,0003, H o > o 1,000-10,000 -1,000 100-=" 1-100 1st 1/4 2nd 1/4 3rd 1/4 4th 1/4 Apical Region Parenchymatous Region L,io 3, o > u
PAGE 32

25 o xi a c a E o o *H o o o O 03 C) f-c o c o H p cd ^ p c CD O c o o 22 •H en o Q W E< 03 hi E* O a s o cd s 03 CO cd O i-l >» a k o ,a -p £ >» W O &0 bO CSV • H M cd «J cd co Q > 0) CO O CO cd "^ jj CD Pi t3 E •H c -d cd C Jh CD o P.rri co cd a3 p bD co CD H 3 *d o 3 cd e X O *d JVi C A< CD O H O 03 Cd E CD CO CO 03 cd O OHP >> P*-H ^ o e .a p E >-> o Wop i a CD Hffl'dO ca o cd a, c P c H H o cd h cd 5h o Sh .c su p -^p p p co ^SS c p H rH E rH CD P O bD bO w c a w iH cd -d o P. co H rH P iH C CD < o tjs CO M H a E O U H O P s CD CQ O 03 d CD H H JU ,a cd 3.C rH O O O 03 C3 C co M >> iH O A. CD fafl cd p CQ O H I H H cd I CD B HOP CD P ? CD p c cd o cd ^h P. rift 03 •• Cd CD 03 Cd H C rri bDH Hf-ri cd cd H cd cd S £ O rH £ o p. CQ id bD o J •d i C H I CD P CD >> SU P o o cd an to CO rH ^ E cd cd E co tD o u a CD 3 CD H SCdft k e H O • H • c CO H CD o -d •H bfl ID c P c CO o ! -H H 'd p OP^to J^ C, ^ • bD CD CD • H MO • CO^H c c 3 CD O bO bO W o cd E CO 03 Cd O iH >J p, P o XP CD P x: a o p CD o Pi rH cd -d o a co HH P H So 1

PAGE 33

26

PAGE 34

Fig. 4 — PAS Stained Cross Section through the Liega sporangium 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 megasoorangium. Fig. 5 — Protein Concentration Gradient in the Megasoorangium The megaspore and surrounding nucellar cells exhibit a higher total protein concentration than the basal nucellar eoidermis and axial row. Fig. 6 — PAS Stained Longitudinal Section of the Embryo Sac The synergic cells show a high, uniform concentration of insoluble polysaccharides. All other embryo sac components are low in insoluble carbohydrates. Fig. 7 — Protein Localization in a I.'egasporanglum with a Pre-mitotic Megaspore Immediately prior to mitosis the megaspore elongates and becomes laterally vacuolated while protein concentration in the cytoplasm Increases.

PAGE 35

28 FIO. H FIG. 5 FIG. a> FIG-. 7

PAGE 36

Fig. 8 — A Longitudinal Section or 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 polar nuclei. Fig. 10 — A Longitudinal Section of a Threecell Embryo Stained with PAS No differences in insoluble polysaccharides are evident between the cells at this stage. Note the thick, highly PAS positive cell wall at the mlcropylar end of the embryo sac. Fig. 11 — A Longitudinal Section of a Multicellular Embryo Stained with PAS At this stage the cell walls of the suspensor apparatus are thicker and more highly PAS positive" than the cell walls of the embryo proper.

PAGE 37

30 FIG. 8 FIG. 9 FIG. 10 FIG. 11

PAGE 38

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 susoensor initial. 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 aooaratus becomes more marked.

PAGE 39

32 F I G. l^fFIG. 15

PAGE 40

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 increases markedly. 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 develooment. Fig. 18 — A Longitudinal Section of an Early Multicellular Embryo Stained for Ribonucleic Acid RNA distribution in the multicellular embryo parallels closely that of protein. Fig. 19 — A Longitudinal Section of an Early Protocorm Stained with PAS Note that starch grains are present in both merlstematlc and parenchymatous cells.

PAGE 41

34 FIG. 18 F I G. 19

PAGE 42

Pig. 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 merlstematic and parenchymatous cells at stage of develODment. 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 merlstematic cells contain proportionately greater amounts of pectic substances than the parenchymatous cells.

PAGE 43

36 FIG. 20 FIG. 2 FIG.22 FIG. 23

PAGE 44

Figs. 24, 25 — Longitudinal Sections of Early and Late Protocorms Showing Changes in Hlstone Concentration In the early protocorm stage (Fig. 24), histone concentration in the nuclei of the merlstematic and parenchymatous cells is the sane. In the late protocorm (Fig. 25), however, histone concentration rises markedly in the parenchymatous nuclei. 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-contalning Proteins Sulfhydryl-contalnlng protein concentration is higher in the nuclei of the parenchymatous cells.

PAGE 45

38 FIG. 2H FIG. 26 FIG. 27

PAGE 46

DISCUSSION OF OBSERVATIONS The Cell '.Tall Early studies of the cell wall were limited primarily to the definition of certain gross properties such as swelling (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; Ambronn and Frey, 1926; Frey-7/yssling, 1935, 1936). Following the advent of the electron microscope, emphasis was placed upon the correlation of chemical and physical characteristics, obtained with polarizing optics and x-ray defraction techniques, with the observable ultrastructure as revealed by ultrathin sectioning (7/haley et al. , 1952; Scott et al. , 1955). 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. i 1958; Jensen, 1960; Flemion, 1961). As a result of the dominant role played by the cell wall in hormonal regulation of plant cell growth, the effect of auxin treatment on wall biosynthesis has also received extensive 39

PAGE 47

40 Investigation (Ordin et al. , 1955; Bonner et al. , 1955; Lamport, 1963). For recent reviews on this aspect see Audus (19 59), Thimann (1960), Gals ton and Purves (1960), and Ray (1961). For general reviews on the cell wall, Preston (1959), Kiihle thaler (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 embryogenesls. These shifts in distribution appear to be closely associated with cellular growth and differentiation. The Cell '.Vail in the .Megaspore and Smbryo Stages During the Megaspore Stage the cell walls of the megaspore and the anticlinal walls of the surrounding nucellar ei dermis 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 Smbryo Stage. Evidence of the loss of plasmadesmatic connections between the megaspore mother cell and the surrounding nucellar cells has been obtained with the electron microscope in Dendroblum (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 development different from the cells around it and that the cell wall is at least Involved in such development.

PAGE 48

41 The Cell Wall in the Protocorm Stage An Interesting correlation exists between the observed cell wall thickening of the megaspore and the suspensor apparatus and the cell walls of the meristematic cells of the protocorm. Differential extraction of the cell walls of the protocorm reveal that the pectic substances are contained in higher amounts in the growing cells of the merlstematlc region than in the cells of the parenchymatous region. Jensen (1960) reports similar results in the differentiating 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 conflicts 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 binding of calcium ions and that this biosynthesis is increased by indole3-acetic acid. Jan sen et al. (1960), however, report that while the hot-water soluble pectin is almost fully esterified, the residual insoluble "protopectln, " which comprises 80 percent of the pectic substance, is only approximately 50 percent esterified. Since little reaction for methyl esterification was obtained in the protocorm, 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

PAGE 49

42 may have caused the removal of the hot-water soluble fraction. But regardless of this, the pectin fraction detected after ammonium oxalate extraction— probably orotopectin— 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 overall 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 hemicelliilcse. This finding is in agreement with the suggestion of Jensen (1960) that the noncellulosic polysaccharides may play a major role in determining the characteristics of the cell wall. It appears that cell wall thickening in the megasoore 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 plasticity since the amount of protein in the cell wall had been shown to be a.uite 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

PAGE 50

43 " extensin." He speculates that this protein is directly involved in controlling cell wall plasticity by providing a network of labile crosslinkages 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 atmospheric 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 Syne reel ds The synergid cells are usually ephemeral structures in the angiosperm garaetophyte which disappear soon after or even before fertilization (Llaheshwari, 1950). In some cases, however, as in Vanda , one or both of these cells may persist and show considerable activity. A similar condition has been observed in the cases of Allium unl folium and A. ro tundum (Weber, 1929). V/hile the synergids are usually considered to be morphological remnants of the archegonium

PAGE 51

44 (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 Stellaria media . 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 positive 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 angio sperm embryo sacs (Maheshwari, 1950), and because of its uniform distribution, it seems likely that the PAS positive material in the case of Vanda is more in the nature of a mixture of hemicelluloses 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 (Mtthle thaler, 1951). In the above cited cases, however, the hemicellulosic compounds are contained as a part of the cell wall. The possibility of a mucopolysaccharide nature of this substance is alluded to by the similarity of the distribution of the

PAGE 52

45 protein and the PAS positive material although no analagous cases can be found in the literature. Starch Distribution The early Protocorm Stage is characterized by the presence of starch grains in the cells of both the merlstematic and parenchymatous cells. In the later phases of protocorm development, however, starch grains become confined to the parenchymatous 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 establishment of full photosynthetic activity j as evidenced 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 RITA 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 Zalokar (1961). 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

PAGE 53

46 distribution of these compounds have been forthcoming. Outstanding among these studies is the work of Sunderland et al . (195?) in which they correlated protein concentration and respiration in the various tissues of the apical meristera of Luolnus alba . Also noteworthy are the studies of Taylor (1958, 1959) on the synthesis and distribution of nucleic acids during microsporogenesis in Lllium and Tulbaghla . 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 between the various organs of the seedling and the endosperm during early development. The distribution of RNA and proteins in the apical merlstem has also been studied by Qifford and Tapper (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 cytoplasm via RNA. The strongest support for this view is summarized in the following facts: 1. RNA is required for protein synthesis to occur in the cytoplasm. 2. The RNA fraction of the Tobacco Mosaic Virus determines what type of protein is made in cases of virus infection. 3. The rate of

PAGE 54

47 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 (Mirsky and Allfrey, 1958). In the investigation described herein, all of the findings relating to RNA and protein distribution are in agreement with this fundamental idea. The Hegasoorangium 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. Observation of the megaspore itself between the time of its formation 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. Ultras true ture studies of megasporogenesis in Dendroblum (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

PAGE 55

48 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 megasporogenesls, but which is not functioning in the Megaspore Stage. The G-ametoohyte 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 surrounding 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 ploldy in the

PAGE 56

49 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 (Llaheshwari, 1950). In the embryo sac of Van da , 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. Possibly 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 developed 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 anglosperm embryology is not clear. Phylogenetlcally, they have been regarded as vestiges of the prothalial tissue (Fuller and Tippo, 1954). In the G-ramineae, G-entianaceae, and Compositae, the antipodals frequently show a considerable increase in size or number (Maheshwari, 1950). This event is sometimes accompanied by an Increase in ploldy in these cells and is

PAGE 57

50 indicative of their high metabolic activity. The Increase in protein staining observed in Vanda suggests the possibility 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 protein 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 Embryo 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 cytodifferentlatlon. The cells of the suspensor apparatus enlarge considerably and become highly vacuolated while those

PAGE 58

51 of the embryo proper remain small, non-vacuolated, and typically merlstematlc. The cells of the suspensor begin to disappear soon after germination of the embryo. If one examines the two-cell proembryo for its distribution 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 segmentation but increases in the cytoplasm following gastrulatlon resulting in the formation of dorso-ventral gradients of activity (Flcq, 1954; Slrlin, 1955; Brachet and Ledoux, 1955). In the sea urchin egg, RNA synthesis is detectable in the blastula stage (Hultln, 1950) but Increases markedly during gastrulatlon (Elson, G-ustafson, and Chargaff, 1954).

PAGE 59

52 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 secretory 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 enables one cell in a two-cell system to synthesize and elaborate 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 cell? 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 Marker t (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

PAGE 60

53 synerglds, 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 suspensor 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 synerglds. 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 The Protocorm Stage is characterized morphologically by the establishment of two lntergrading regions of cell types; the merlstematic 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 distinctly vacuolated, while those distal to the suspensor remain merlstematic. Growth of the protocorm results from the continued enlargement of the subapical merlstematic cells.

PAGE 61

54 During this enlargement, the protein concentration In the parenchymatous cells appears to remain nearly constant. 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 primarily 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 protein roughly equal to that of the actively dividing meristematic cells. 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 biochemical analysis of root tip growth In Vicla 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

PAGE 62

55 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 synthesis in the enlarging parenchymatous cells of the protocorm 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 findings 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 synthesis. Observation of longitudinal protocorm sections reveals the presence of groups of cells In the parenchymatous region in which the concentration of cytoplasmic RNA is considerably higher than in the surrounding cells. These groups of cells appear to occur at random in the parenchymatous region but are usually located toward the center of the parenchymatous

PAGE 63

56 mass and usually encompass those cells which form the vascular strands. Similar results have been obtained in embryos of Capsella bur sa pas tori a by Prltchard (personal communication). 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 vacuo la tion without noticeable change in RNA distribution, the likelihood of fixation artifact seems remote. The Hi stones Longitudinal sections of protocorms show that during early phases of protocorm development the relative concentration 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 parenchymatous region is seen to increase. This increase In hlstone staining occurs immediately prior to the initiation of visible signs of the necrosis of this region. After the Increase in hi stone concentration, cytoplasmic RNA and total protein decrease until the withering of the entire region is complete.

PAGE 64

57 Stedman and Stedman (1950) first proposed that histories 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 C 14 adenosine into RNA and of C^ 4 labelled amino acids into protein in Isolated thymus nuclei. These authors conclude that histones inhibit a large number of nuclear bio synthetic activities, probably by interference with nuclear ATP synthesis. Thus the mechanism of repression by histones, If it Indeed exists, is probably quite 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 hlstone molecules at appropriate times." The possibility exists that the Increased hi stone staining observed in the nuclei of the protocorm is due to the depolymerization of the nucleoprotelns 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. pycnosls , which involves a decrease in the size of the nucleus and a concentration of the chromatin; 2. karyorrhexls , in which fragmentation of the chromatin occurs; and 3. karyolysls , in which chromatin

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58 basophilia decreases markedly. Examination of the cells of the parenchymatous region during the developmental phase of the protocorm in which an Increase in hi stones 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 hi stones observed in this region accompanies an Increase in the amount of DNA in these nuclei, probably as a result of polyploidy.

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SUMMARY The purpose of this investigation was to examine and correlate the changes in relative concentration and distribution of total proteins, ribonucleic acid, insoluble polysaccharides, hlstones, and sulfhydryl and tyroslne-contalning 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. Megaspore Stage 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 Dendroblum , 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. 59

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60 2. There is a concentration gradient of total proteins in the mega sporangium, higher at the distal than proximal end. It appears that this is a remnant of the condition of active protein absorption through rhopheocytosls previously described In Dendroblum . Gametophyte Stage 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 synerglds 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 inoreased water uptake by the megaspore during mitotic division, protein synthesis being minimal.

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61 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 postulated 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 the embryo. Embryo Stage 1. There is no difference in insoluble polysaccharides 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 suspensor become thicker than those of the embryo. 2. There is a difference in total protein and RNA concentration between the suspensor initial and embryo initial 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 apparatus. Protocorm Stage 1. The Protocorm Stage is characterized by the presence 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 merlstematic portion in lieu of an endosperm tissue.

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62 2. The differentiation of a merlstematlc cell into a parenchymatous cell involves an increase in over-all size. This increase is due partially to vacuo lation. Thus this growth involves water uptake accompanied by protein synthesis at a rate sufficient to maintain a constant protein concentration. 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 of the protocorm for cell wall constituents reveal that: A. Methyl esterificatlon of the pectins is low in all cells. B. The walls of the merlstematlc cells contain proportionately higher amounts of pectic material than the parenchymatous cells. C. The major non-pectlc components of the cell wall of all cells in the protocorm are the hemicelluloses. 5. Concentration of hlstones in the nuclei of the merlstematlc and parenchymatous cells appears approximately equal in the early phases of the Protocorm Stage. Immediately prior to the morphologically detectable initiation of necrosis of the parenchymatous region, the concentration of hlstones in the nuclei of these cells increases markedly. The question of the role of hlstones as gene repressors in connection with this phenomenon was discussed.

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LITERATURE CITED Albersheira, P. and J. Bonner. 1959. Metabolism and hormonal control of pectlc substances. Jour. Biol. Chem. 234:3105-3108. Alfert, Iff* and I. I. Geschwlnd. 1953. A selective staining method for the basic proteins of cell nuclei. Proc. Nat. Acad. Sci. (U.S.) 39:991-999. Allfrey, V. G. , V. C. Littau, and A. E. Mirsky. 1963. On the role of hlstones in regulating ribonucleic acid synthesis in the cell nucleus. Proc. Nat. Acad. Sci. (U.S.) 49:414-421. Alvarez, M. R. 1962. Studies on the ontogeny of Vanda . Master's Thesis, University of Florida. Ambellan, E. 1955. Effect of adenine mononucleotides on neural tube formation of frog embryo. Proc. Nat. Acad. Sci. (U.S.) 41:428-432. Ambronn, H. and A Frey. 1926. Cited after Muhlethaler. 1961. Audus, L. J. 1959. Plant Growth Substances . Interscience Publishers, Inc., New York. Pp. 553. Avers, C. J. 1958. Hlstochemical localization of enzyme activity in the root epidermis of Phleum pratense . Amer. Jour. Bot. 45:609-612. . 1961. Hlstochemical localization of enzyme activities in root meristematic cells. Amer. Jour. Bot. 48:137-142. Avers, C J. and E. E. King. 1960. Hlstochemical study of intracellular enzymatic heterogeneity of plant mitochondria. Amer. Jour. Bot. 47:220-225. Avery, G. S. Jr. and F. Engel. 1954. Total nitrogen in relation to age and position of cells in Avena coleoptiles. Amer. Jour. Bot. 41:310-315. 63

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64 Avery, 0. T. , C. M. McLeod, and M. McCarty. 1944. Studies on the chemical nature of the substance inducing transformation of pneumococcal types. J. Sxptl. Med. 79:137-158. Barr, 0. C. and J. A. V. Butler. 1963. Histones and gene function. Nature 199(4899) : 1170-1172. Bennett, H. 3. and R. H. Watts. 1958. The cytochemical demonstration and measurement of sulphydryl groups by azo-aryl mercaptlde coupling, with special reference to Mercury Orange. G-en. Cytochem. Methods 1:318-374. Bishop, C. T. , 3. T. Bayley, and G. Setterfleld. 1958. Chemical constitution of the primary walls of Avena coleoptiles. Plant Physiol. 30:283-288. Boivln, A., R. Vendrely, and C. Vendrely. 1948.. L'aclde desoxyribonucleique du noyau cellulaire, deposltaire des caracte'res hereditaires; arguments d'ordre analytlque. Compt. Rend. 226:1061-1063. Bonner, J., L. Ordln, and R. Cleland. 1955. Auxin-induced water uptake. In The Chemistry and Mode of Action of Plant Growth Substances . Acad. Press, New York, pp. 260-270. Brachet, J. 1950. Quelques observations sur le mode d' action de l'organisateur chez les amphibiens. Experimentia 6:56-57. . 1957. Biochemical Cytology . Acad. Press, New York. Pp. 535. Brachet, J. and L. Ledoux. 1955. L' action de la rlbonucle'ase sur la division des ouefs d' amphibiens. Sxptl. Cell Research suppl. 3, 27-39. Brown, R. and D. Broadbent. 1950. The development of cells in the growing zones of the root. Exptl. Botany 1:249-263. Correll, D. S. 1950. Native Orchids of North America. Chronica Botanlca Company, Waltham, Mass. Pp. 329. Das, N. K. and M. Alfert. 1961. Accelerated DNA synthesis in onion root merlstem during x-radiatlon. Proc. Nat. Acad. Sci. (U.S.) 47:1-6. Deeley, E. M. , H. G. Davis, and J. Chayen. 1957. The DNA content of cells in the root of Viola faba. Exptl. Cell Research 12:582-591.

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65 Duncan, R. E. 1959. Orchids and cytology. In The Orchids . Ed. Carl L. Withner. Ronald Press Co. , New York, pp. 189-260. Ebert, J. D. 1954. The effects of chorlo-allantolc transplants of adult chicken tissues and homologous tissues of the host chick embryo. Proc. Nat. Acad. Scl. (U.S.) 40:337-347. Elson, D. , T. Gustafson, and E. Chargaff. 1954. The nucleic acids of the sea urchin during embryonic development. J. Biol. Chem. 209:285-293. Esau, K. 1953. Plant Anatomy . John Wiley and Sons, New York. Pp. 376. Flcq, A. 1954. Analyse de l 1 Induction neurale chez les "araohiblens au moyen d' organ! statems marauds. J. Embryl. Exptl. Morphol. 2:194-203. Fitting, A. 1900. Cited after Muhlethaler. 1961. Flax, IE. H. and M. H. Hlmes. 1950. A differential stain for ribonucleic acid and desoxyrlbonuclelc acid. Anat. Record 108:529. Flemion, F. 1961. Cytochemical studies of the developing primary cell wall in the apical shoots of normal and physiologically dwarf peach seedlings. Plant Physiol. 36(suppl. XXVII): 51. Francis, ff. D. and T. Winnick. 1953. Studies on the pathway of oroteln synthesis in tissue culture. J. Biol. Chem. 202:273-289. Frey-Wyssllng, A. 1935. Cited after Muhlethaler. 1961. . 1936. Cited after Muhlethaler. 1961. Fuller, H. J. and 0. Tiooo. 1954. College Botany . Henry Holt and Co., New York. Pp. 993. G-alston, A. ff. and W. K. Purves. 1960. The mechanism of action of auxin. Ann. Rev. of Plant Physiol. 11:239-276, G-ifford, E. M. Jr. and H. B. Tepper. 1962a. Histocheraical and autoradiographic studies of floral induction in Chenopodlum album . Amer. Jour. Bot. 49:706-714. and . 1962b. Ontogenetic and histochemical changes in the vegetative shoot tip of Chenopodlum album. Amer. Jour. Bot. 49:902-911.

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66 Green, P. B. 1958. Concerning the site of the addition of new wall substances to the elongating Nitella cell wall. Amer. Jour. Bot. 45:111-116. Holmes, B. E. , L. K. Mee, S. Hornsey, and L. H. Gray. 1955. The nucleic acid content of cells in the merlstematlc elongating and fully elongated segments of roots of Vicia faba . Sxptl. Cell Research 8:101-113. Hotchklss, R. D. 1948. A microchemlcal reaction resulting in the staining of polysaccharide structures In fixed tissue preparations. Arch. Biochem. 16:131-141. Huang, R. C and J. Bonner. 1962. Hlstone, a supressor of chromosomal RNA synthesis. Proc. Nat. Acad. Sci. (U.S.) 48:1216-1221. Hubert, B. and J. Ma ton. 1939. The Influence of synthetic growth-controlling substances and other chemicals on oostfloral phenomenon in tropical orchids. Biol. Jaarb. 6:244-285. Hultln, T. 1950. The protein metabolism of sea urchin eggs during early development studied by means of Mislabelled ammonia. Exptl. Cell Research 1:599-602. Israel, H. W. 1962. An electron microscope study of megaspore development in Dendroblum orchids. Doctoral Dissertation, University of Florida. Jansen, E. F. , R. Jang, P. Albersheim, and J. Bonner. 1960. Pectic metabolism of growing cell walls. Plant Physiol. 35:87-97. Jensen, W. A. 1955. A morphological and biochemical analysis of the early phases of cellular growth in the root tip of Vlcia faba . Exptl. Cell Research 8:506-522. 1956. The cytochemlcal localization of acid phosphatase in root tip cells. Amer. Jour. Bot. 43:50-54. . 1956. On the distribution of nucleic acids In the root tip of Vlcia faba . Exptl. Cell Research 10:222-226. 1958. The nucleic acid and protein content of root tip cells of Vlcia faba and Allium cepa . Exptl. Cell Research 14:575-583. 1960. The composition of the developing primary wall in onion root tip cells. II. Cytochemlcal localization. Amer. Jour. Bot. 47:287-295.

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67 Jensen, ff. A. 1962. Botanical Histochemistry . McGraw-Hill, New York. Pp. 408. Jensen, W. A. and A. McLaren. 1960. Uptake of proteins by plant cells — the possibility of pinocytosls in plants. Exptl. Cell Research 19:414-416. Johansen, D. A. 1940. Plant Microtechnique . McGraw-Hill, New York. Pp. 523. Katz, J. R. 1924. Cited after Muhlethaler. 1961. Lamport, D. T. A. 1963. Oxygen fixation of plant cell wall protein. Jour. Biol. Chem. 238:1438-1440. Ledoux, L. , P. Galand, and R. Huart. 1962. Nucleic acids and protein metabolism in barley seedlings. II. Interrelations of the different organs. Exptl. Cell Research 27:132-136. Leuchtenberger, C. 1950. A cytochemical study of pycnotic nuclear degeneration. Chromosoma 3:449-473. Lillie, R. D. 1957. Adaption of the Morel Sisley protein diazotlzation procedure to the hlstochemical demonstration of protein bound tyrosine. Jour. Hist, and Cytochem. 5:528-532. Magll, G. 1958. The possibility of substitution with auxin for the action of pollen on the development of the ovules of the orchid. Nuovo Jiorn. Bot. Ital. 65(3)1401-416. Maheshwari, P. 1950. An Introduction to the Embryology of Anglosperms . McGraw-Hill, New York. Pp. 453. Mazia, D. , P. A. Brewer, and M. Alfert. 1953. The cytochemical staining and measurement of protein with mercuric bromphenol blue. Biol. Bull. 104:57-67. Markert, C. L. 1958. Chemical concepts of cellular differentiation. In The Chemical Basis of Development . Ed. W. D. McElroy and B. Glass. Johns Hopkins Press, Baltimore, pp. 3-16. McLeish, J. 1959. Comparative ralcropho tome trie studies of DNA and arginlne in plant nuclei. Chromosoma 10:686-710. Mlrsky, A. E. and V. Allfrey. 1958. The role of the cell nucleus In development. In The Chemical Basis of Development . Ed. W. D. McElroy and B. Glass. Johns Hopkins Press, Baltimore, pp. 94-99.

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68 Muhle thaler, K. 1961. Plant cell walls. In The Cell. Acad. Press, New York. pp. 85-135. Naegll, C 1864. Cited after Muhle thaler. 1961. Nllmoto, D. H. and Y. Sagawa. 1962. Ovule development In Phalaenoosls . Caryologla 15(1) :89-97. Northcote, D. H. 1963. The biology and chemistry of the cell walls of higher plants, algae, and fungi. Internet. Rev. Cyt. 14:223-265. Ordln, L. , R. Cleland, and J. Bonner. 1955. Influence of auxin on cell wall metabolism. Proc. Nat. Acad. Sci. (U.S.) 41:1023-1029. Patau, K. and H. Swift. 1953. The DNA content (Feulgen) of nuclei during mitosis in a root tip of onion. Chromosoma 6:149-160. Preston, R. D. 1959. Wall organization in plant cells. Internat. Rev. Cyt. 8:33-58. Prltchard, H. 1962. Cytochemical studies on the megasporogenesis and embryogenesis in Stellarla media (L) Cyrlll. Doctoral Dissertation, Lehigh University. Ray, P. M. 1961. Hormonal regulation of plant cell growth. In Control Mechanisms in Cellular Processes . Ronald Press Co., New York. pp. 185-212. Reeve, R. M. 1959. Histological and histochemlcal changes in developing and ripening peaches. II. The cell walls and pectins. Amer. Jour. Bot. 46:241-247. Sagawa, Y. and H. W. Israel. Post-pollination ovule development in Dendroblum orchids. I. Introduction. In press. Caryologla. Scott, F. M., K. C. Hammer, E. Baker, and E. Bowler. 1956. Electron microscope studies of cell wall growth in the onion root. Amer. Jour. Bot. 43:313-324. Slrlin, J. L. 1955. Nuclear uptake of methlonlne-S 35 in the newt embryo. Experimentia 11:112-113. Stedman, E. and E. Stedman. 1950. Cell specificity of histones. Nature 166(4227) : 780-781. Sunderland, N. , J. K. Heyes, and R. Brown. 1957. Protein and resolration in the apical region of the shoot of Luplnus* alba. J. Exptl. Bot. 8:55-70. Swamy, B. G. L. 1942. Morphological studies in three species of Vanda. Current Science 11:285-286.

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69 Taylor, J. H. 1958. Incorporation of P-32 Into nucleic acids and Droteins during mlcrogametogenesis of Tulbaghia. Araer. Jour". Bot. 45:123-131. 1959. Autoradiographic studies of nucleic acids and proteins during melosls in Li Hum longlflorum . Aroer. Jour. Bot. 46:477-484. . 1963. Molecular Genetics. Acad. Press, New York. Pp. 544. Thlmann, K. V. 1960. Plant growth. In Fundamental Aspects of Normal and Malignant Growth. Elsevier Publ. Co., Amsterdam. Pp. 1025. Vacin, S. and F. Went. 1949. Culture solution for orchid seedlings. Bot. Gaz. 110:605-613. von Mohl, H. 1859. Cited after liuhle thaler. 1961. Weber, E. 1929. Entwichlungs geschichtllche Untersuchungen uber die Gattung Allium. Bot. Arch. 25:1-44. 7/haley, W. G. , L. W. Mericle, and C. Heimsch. 1952. The wall of the meristematlc cell. Amer. Jour. Bot. 39:20-26. Wlrth, M. and C. L. Withner. 1959. Embryology and development in the Orchldaceae. In The Orchids . Ed. Carl L. Withner. Ronald Press Co., New York. pp. 155-158. Woodard, J. , E. Rasch, and H. Swift. 1961. Nucleic acid and oroteln metabolism during the mitotic cycle in Vlcla faba'. J. Biophys. Biochem. Cytol. 9:445-462. Woodstock, L. W. and F. Skoog. 1962. Distributions of growth, nucleic acids, and nucleic acid synthesis in seedling roots of Zea mays . Amer. Jour. Bot. 49:623-633. Yasuma, A. and T. Ichikawa. 1953. Ninhydrln-Schif f and alloxanSchlff staining. A new hlstochemical staining method for proteins. J. Lab. and Clin. Med. 41:623-633. Zalokar, !.:. 1961. Ribonucleic acid and the control of cellular processes. In Control Mechanisms of Cellular Processes . Ronald Press Co., New York. pp. 87-140.

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BIOGRAPHICAL SKETCH 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 Assistantship, a Research Asslstantshlp, and was awarded a Graduate School Fellowship. Marvin Ray Alvarez is married to the former Delma Barbara Flore s and is the father of two children. He is a member of the Phi Sigma Biological Society and the Botanical Society of America. 70

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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 fulfillment of the requirements for the degree of Doctor of Philosophy. Aui ' il , 1964 gy^L^^vCV ^/Dean, College of Agriculture Supervisory Committee Chairman aV ^ rDean, Graduate School


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