Group Title: histochemical study of embryo development in Vanda (Orchidaceae)
Title: A Histochemical study of embryo development in Vanda (Orchidaceae)
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Title: A Histochemical study of embryo development in Vanda (Orchidaceae)
Physical Description: vii, 70 leaves : ill. ; 28 cm.
Language: English
Creator: Alvarez, Marvin Ray, 1936-
Publication Date: 1964
Copyright Date: 1964
 Subjects
Subject: Orchids   ( lcsh )
Plant physiology   ( lcsh )
Botany thesis Ph. D
Dissertations, Academic -- Botany -- UF
Genre: bibliography   ( marcgt )
non-fiction   ( marcgt )
 Notes
Statement of Responsibility: by Marvin Ray Alvarez.
Thesis: Thesis (Ph. D.)--University of Florida, 1964.
Bibliography: Includes bibliographical references (leaves 63-69).
Additional Physical Form: Also available on World Wide Web
General Note: Typescript.
General Note: Vita.
 Record Information
Bibliographic ID: UF00097919
Volume ID: VID00001
Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
Resource Identifier: alephbibnum - 000415194
oclc - 37441447
notis - ACG2433

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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)

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Ho St 0 ) tO DH 0
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0 0

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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

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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.












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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 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)- __




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