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A cytochemical study of the effects of growth regulators on the shoot apical meristem of certain seed plants

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Title:
A cytochemical study of the effects of growth regulators on the shoot apical meristem of certain seed plants
Creator:
Varnell, R. J
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English
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viii, 95 leaves : ill. ; 28 cm.

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Subjects / Keywords:
Apical meristems ( jstor )
Internet search systems ( jstor )
Lipids ( jstor )
Meristems ( jstor )
Metabolism ( jstor )
Mother cells ( jstor )
Nucleic acids ( jstor )
Plants ( jstor )
RNA ( jstor )
Seedlings ( jstor )
Agronomy thesis Ph. D
Dissertations, Academic -- Agronomy -- UF
Growth (Plants) ( lcsh )
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bibliography ( marcgt )
theses ( marcgt )
non-fiction ( marcgt )

Notes

Thesis:
Thesis (Ph. D.)--University of Florida, 1974.
Bibliography:
Includes bibliographical references (leaves 86-93).
General Note:
Typescript.
General Note:
Vita.
Statement of Responsibility:
by Ray Judson Varnell.

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University of Florida
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University of Florida
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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.
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37876287 ( OCLC )

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Full Text













A CYTOCHEMICAL STUDY OF THE EFFECTS
OF GROWTH REGULATORS ON THE SHOOT
APICAL MERISTEM OF CERTAIN SEED PLANTS














By

RAY JUDSON VARNELL


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


1974




A CYTOCHEMICAL STUDY OF THE EFFECTS
OF GROWTH REGULATORS ON THE SHOOT
APICAL MERISTEM OF CERTAIN SEED PLANTS
By
RAY JUDSON VARNELL
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
1974


To
Dr. Earnest Ball,
viko&d
whcr+s advice, encouragement, and example
many years ago became the sustaining inspiration
which led me to this point in my career.


ACKNOWLEDGMENTS
I wish to acknowledge the guidance, encouragement,
and helpful criticism received from the members of my
Supervisory Committee, and especially that from the late
Dr. R.G. Stanley, and from Dr. I.K. Vasil who supervised
and provided facilities for this study. Acknowledgment
is also made of the help received from Dr. J.W. Brookbank
with the microspectrophotometric work. I gratefully
acknowledge the patience, encouragement, and assistance
provided me by my wife, Emily, who typed the manuscript
and prepared the graphs. Support for this study was
provided by an N.D.E.A., Title IV, fellowship.
in


TABLE OF CONTENTS
ACKNOWLEDGMENTS iii
ABSTRACT vi
INTRODUCTION 1
LITERATURE REVIEW 4
Concepts of Shoot Morphogenesis 4
Metabolism in the Apical Meristem 12
Effects of Chemicals Applied to the Apical Meristem. 16
MATERIALS AND METHODS 18
Plants 18
Treatments 19
Harvesting 22
Staining 24
Microspectrophotometry 28
Analyses 29
RESULTS 37
Lupinus 37
Morphological Analyses of Treatments 37
Cytochemical Analyses of IAA Effects 42
Pinus 57
Morphological Analyses of Treatments 57
Cytochemical Analyses of Kinetin Effects 50
IV


Coleus
71
DISCUSSION 72
Lupinus 72
Pinus 78
APPENDIX 84
LITERATURE CITED 86
BIOGRAPHICAL SKETCH 94
V


Abstract of Dissertation Presented to the Graduate
Council of the University of Florida
in Partial Fulfillment of the Requirements
for the Degree of Doctor of Philosophy
A CYTOCHEMICAL STUDY OF THE EFFECTS
OF GROWTH REGULATORS ON THE SHOOT
APICAL MERISTEM OF CERTAIN SEED PLANTS
By
Ray Judson Varnell
August, 1974
Chairman: Robert G. Stanley
Cochairman: Sherlie H. West
Major Department: Agronomy
Previous studies of seed plants have described whether
auxins applied directly to the shoot apical meristem altered
development. The present study repeated these observations
and extended them to include the effects of a cytokinin and
a gibberellin. Attempts were also made to quantitate some
of the effects of these substances by measuring cytophoto-
metrically changes in ribonucliec acid (RNA), total protein,
and unsaturated lipids in the cytoplasm, and histones in the
nucleus.
Results of this study show that application of 1.5 yg
indolacetic acid (IAA) in a lanolin droplet to the exposed
apical meristem of Lupinus albus seedlings caused: (1)
vi


axillary buds to form closer to the apex than normal, (2)
displacement of primordia formed during the first two plasto
chrons following treatment, and (3) significant increases in
concentration of RNA, protein, unsaturated lipids, and
histones in the meristems. Primordial displacement tended
to be random relative to the site of treatment, which may be
a feature common to dicotyledonous plants exhibiting spiral
phyllotaxis. That IAA conferred initiation site capabilities
to all of the peripheral zone for a short time was indicated
by (2) and (3) above and by decreases in concentrations of
the observed compounds toward control levels after the
second plastochron following treatment. Effects of IAA
on RNA and histones suggest that nucleic acid metabolism,
and possibly gene expression, was involved in the response.
Kinetin and gibberellic acid (GA) had no apparent morpho
genetic effect on Lupinus meristems.
Treating shoot apical meristems of Pinus elliottii
seedlings with kinetin resulted in: (1) bud scale formation
and the onset of dormancy, and (2) changes in concentrations
of the cytochemically determined compounds listed above.
IAA and GA produced no morphogenetic effects. Interactions
of kinetin with nuclear-based events and with membranes in
the cytoplasm were suggested by levels of histones and RNA
in treated meristems exceeding control levels through the
sixth day following treatment, during which time unsaturated
lipids sharply fell below control values. Rapid declines


in RNA, protein, and histones by day eight coincided with the
first appearance of bud scales, signaling the onset of
dormancy in kinetin-treated meristems.
It was observed that the central mother cell zone is
not metabolically quiescent in vegetative shoot meristems
of Lupinus and Pinus seedlings.
viii


INTRODUCTION
Because most of the growth and development of the
shoot is initiated at or regulated by the shoot apex,
the importance of the shoot apex to the plant, and to the
plant scientist, can hardly be over-emphasized. The shoot
apex consists of an apical meristem, a subapical meristem,
and a region of maturation. It is within the apical meristem
that lateral organs and, to a limited extent, shoot form
arise. The initial orderly disposition of organs around
the periphery of the shoot, and of tissues within the shoot,
is due to localized centers of growth and differentiation
within the apical meristem. The apical meristem, in addition,
contains groups or zones of cells which differ from one
another in the plane of cell division, affinity for various
stains and dyes, mitotic rate, extent of vacuolization, and
nuclear as well as cell sizes and volume ratio. The factors
maintaining the developmental and cytological heterogeneity
within the apical meristem are unknown.
Information about these unknown factors may be
obtained, however, by studying the effects of treatments
1


2
which disrupt the normal course of development in the
apical roeristem. One kind of treatment for disrupting
normal development involves applying substances directly
on the surface of the meristem. The rationale is that
some of the applied substance penetrates the apical
meristem and disrupts the orderly disposition of organs
and/or tissues, and inferences are drawn concerning the
internal regulatory factors from the nature of the applied
substance and its effects.
Effects of substances applied directly to the intact,
growing apical meristem have been studied relatively
little. Lack of such experimental information is undoubt
edly due to the small size of the meristem, the difficulty
of exposing and working with the intact meristem without
injuring it, and the complex interactions between the
apical meristem and the rest of the plant. But the
advantages of experimenting with the intact apical meristem
should not be ignored. Unlike excised apical meristems,
intact ones have not suffered the trauma of being cut
off from their natural life support systems and of wound
and healing reactions. Physiological parameters, such
as chemical contents, gradients, and fluxes, that are
characteristic of the normally developing apical meristem,
probably are accurately represented only in the intact
meristem. Moreover, although it may be easier in some


3
respects to experiment with excised shoot tips, the
results must eventually be verified with intact meristems.
Previous studies have described whether applied
auxins altered meristem development. The present study
repeated these observations and extended them to include
the effects of a cytokinin and a gibberellin. Attempts
were also made to quantitate some of the effects of these
substances by measuring changes in ribonucleic acid,
total protein, and unsaturated lipids in the cytoplasm,
and histones in the nucleus. These data provide information
about the internal factors regulating growth and cytological
heterogeneity within the apical meristem in control and
experimentally treated plants.


LITERATURE REVIEW
Concepts of Shoot Morphogenesis
The two most widely accepted concepts pertaining
to morphogenesis at the shoot apex are Wardlaw's (1957,
1960) and Plantefol's (1946, 1947a, 1947b, 1948) interpreta
tions of apical organization. According to Wardlaw (1965a,
1965b), the apex is a dynamic geometrical reaction system,
"...of integrated and interrelated ..." regions. The
regions, forming a morphologically distinguishable vertical
series based partially on anatomical studies by Schoute
(1936), are: (1) the distal region, (2) the subdistal
region, (3) the organogenic region, (4) the subapical
region, and (5) the maturation region (Fig. 1A). The first
three regions comprise the apical meristem in which
cytological and/or histological zonation may be evident.
The cytological zones, which were first described
by Foster (1938) for Ginkgo biloba, but which are generally
applicable to all seed-bearing plants, are:(1) the apical
initial cell zone, (2) the central mother cell zone,
(3) the peripheral zone, and (4) the rib meristem (Fig. IB).
4


Fig. 1
A. Morphological regions in longitudinal section of
the shoot apex of Podocarpus macrophyllus,
stained with safranin and fast green. X300.
B. Cytological zonation in longitudinal section of
the shoot apex of Lupinus albus, stained with
Azure B. X200.
C. Histological zonation in longitudinal section of
the shoot apex of Quercus spp., stained with
safranin and fast green. X550.
D = distal region; SD = subdistal region; 0 =
organogenic region; SA = subapical region;
M = maturation region; I = initial cell zone;
Pz = peripheral zone; R = rib meristem; T =
tunica layer; C = corpus; P = primordium ;
L = young leaf; Cz = central mother cell zone.


6


7
A transition zone, described by Foster (1938) and located
at the lateral and basal margins of the central mother
cell zone, is not evident in many apices and is often
omitted from discussion of cytological zonation (Romberger
1963) .
Apical meristems of angiosperms (Clowes 1961), and
certain gymnosperms (Johnson 1951; Griffith 1952; Fagerlind
1954), typically exhibit histological zonation in the
form of (1) one or more outer layers of anticlinally
dividing cells, and (2) an inner mass of cells with randomly
oriented mitoses (Fig. lc). The former is called the
tunica if it is composed exclusively of anticlinally
dividing cells (Popham 1951; Clowes 1961), or the mantle if
an occasional periclinal division occurs (Popham and Chan
1950). The inner mass of cells underlying the tunica/
mantle is called the corpus. This nomenclature of histo
logical zonation is based on Schmidt's (1924) interpretation
of growth in the apical meristem. Although Wardlaw
(1965b) recognized the existence of a tunica/mantle
layer(s), his concept of growth in the apical meristem
differed radically from Schmidt's (1924).
Wardlaw (1965a) proposed: (1) that differential growth
rates throughout the apical meristem determine the shape
and size of the apex, and (2) that localized active growth
in the p^i :ieral zone determines the position of new
primordia. The growth rate declines steadily from the
subapical region to the summit of the distal region and to


8
the center of the shoot axis the rate of decline being faster
for low or flat apices, and slower for conical or cylindrical
apices.
Formation of a primordium, in Wardlaw's (1965a)
view, results from the comparatively active growth of a
small number of superficial and underlying cells at a
localized locus or site, the growth center, in the peripheral
zone in the subdistal region. This growth center, according
to Schoute (1913), inhibits the formation of other similar
growth centers in the immediate vicinity, and the apical
cells inhibit the formation of growth centers in their
immediate vicinity. Hence, primordia occur (1) between
older ones where sufficient space is available, and (2)
at some characteristic distance from the summit of the
apex. Support for this concept of primordial inception
comes from exhaustive studies of phyllotaxis (Snow and Snow
1931, 1933, 1935; Richards 1951, 1956), microsurgical experi
ments (Wardlaw 1949, 1950, 1955, 1956; Wetmore and Wardlaw
1951; Cutter 1954, 1956; Wardlaw and Cutter 1956), and theo
retical analyses of endogenous circadian rhythms (Biinning
1952) and of diffusion-reaction systems (Turing 1952).
Wardlaw's (1965a, 1965b) concept is founded on the
supposition that distribution of growth and morphological
pattern in the apical meristem are expressions of inter
related physiological-genetical processes. He further
supposes that each region and zone in the shoot apex has


9
its own characteristic metabolism and concentration gradients
of incoming nutrients and outgoing metabolic products, and
that the same considerations apply to the growth centers
and nascent primordia.
In contrast, Plantefolls (1946, 1947a, 1947b, 1948)
concept of apical organization and morphogenesis differs
from Wardlaw's (1957, 1960) in (1) the function of the
cytological zones in the apical meristem, and (2) the
regulation of primordium inception. According to Plantefol
(1946, 1947a, 1947b, 1948), during vegetative growth the
most distal region of the apical meristem as well as the
central mother cell zone, together defined as the "mristme
d'ttente" (Bersillon 1951), are more or less completely
inactive and contribute little or nothing to histogenesis
or organogenesis. Accordingly, it is the peripheral zone,
the so-called "anneau initial" (Plantefol 1947b), where
initial vegetative growth and development take place.
Growth and histogenesis also occur in the "mristme
mdullaire," which is the same as the rib meristem.
The concept of an inactive "mristme d'attentc was
based primarily on the absence or infrequent evidence of
cell divisions in this zone. Additional criteria are low
concentrations of cytoplasmic ribonucleic acid and protein,
low nucleocytoplasmic ratio, comparatively large vacuoles,
and few mitochondria; all characteristics of a more differ
entiated and less active state than that observed in the


10
highly meristematic peripheral zone. That these cytological
characteristics exist in the apical meristem of many plants
has been well documented (Lance 1957; Gifford and Tepper
1962a, 1962b; Nougarede et ad. 1965; Steeves et al. 1969)
and has been conceded by Wardlaw (1965b). What these cyto
logical characteristics mean in the morphogenetic, physio
logical, or metabolic sense is not known.
Regulation of primordial inception, in Plantefol's
(1950) view, is achieved by a small number of foliar helices
which spiral around the shoot axis. Each helix has a gener
ative center located in the peripheral zone (the "anneau
initial"), and the activities of the several generative
centers are regulated by an organizer. What instigates
the leaf generating center, their helical course around
the shoot axis, and the apical organizer and how it exerts
its effects have never been satisfactorily explained.
The salient points on which these two concepts of
apical organization agree regarding morphogenesis at the
shoot apex are: (1) the cytological and histological zones
of the apical meristem evidently have different functions,
and (2) the development and growth in the normal vegetative
apex rigidly follows a pattern characteristic for the species
The peripheral zone is the site of primordial initiation
and, in some rapidly growing apices (Wetmore 1943; Wardlaw
1956; Lance 1957), the site of inception of provascular
tissue. Thus, the peripheral zone is an organogenic region,


11
and in some instances a histogenic region also. The rib
meristem characteristically gives rise to the pith and is,
therefore, exclusively histogenic. In contrast, the initial
cell zone and central mother cell zone form neither organs
nor tissues as such, but apparently are comparatively active
metabolically. The latter point will be discussed below.
As Wardlaw (1965b) pointed out, the patternized
development of regularly spaced primordia in a characteristic
phyllotactic sequence is the primary organogenic activity
of the apical meristem. Furthermore, apical meristems
of different species may be large or small in absolute
size, or in relative size to primordia, or to the rest of
the shoot apex, and they may be typically conical, parabolic,
cylindrical, low and flat, or concave in shape. Whatever
its size, shape, or sequence of primordial initiation,
the apical meristem develops along a characteristic pattern
determined by the distribution of growth. Although no one
has questioned this interpretation of apical morphogenesis,
the physiological-genetical events which determine and
evidently regulate the morphogenic pattern at the shoot
apex are unknown.


12
Metabolism in the Apical Meristem
Metabolism within the apical meristem has not been
studied directly because of the small size and inaccessabil-
ity of the structure. It has been only in the last few
years that data have become available from which inferences
could be drawn. These data consist of cytochemical
localization, and quantification in some cases, of metaboli-
cally active substances such as nucleic acids, proteins,
specific enzymes, carbohydrates, and lipids. The distri
bution of nucleic acids and nucleohistone in the apical
meristem, for example, has been studied extensively recently
because of their presumptive role in gene activated
control of development. The initial discoveries that
the nucleic acids and nucleohistones were more concentrated
in the peripheral zone than in the initial cell and
central mother cell zones (Wardlaw 1965a, 1965b; Nougarde
1967) have stood the test of time. Seasonal changes
in the absolute amounts and in relative ratios of these
substances within the apical meristem have been tentatively
correlated with bud break, shoot elongation, bud development,
and dormancy (Cecich et_ al. 1972; Varnell and Vasil 1972).
Although cells in the central mother cell zone and initial
cell zone may not divide often in many species, they are
not arrested in any particular part of the cell cycle but
rather cycle comparatively slowly (Steeves et al. 1969).


13
The data of Steeves et_ a^. (1969) suggest a longer
phase than G2 phase for cells in these zones, but what this
means metabolically is not known.
The greater amounts of ribonucleic acid (RNA) in the
peripheral zone than in the other zones is the basis for the
differential basophilia within the apical meristem that
led Koch as early as 1891 to visualize cytological differ
ences within the shoot apex. The greater concentration of
RNA in the peripheral zone is associated with the greater
mitotic activity of this zone. The highest concentrations
of RNA are in the rapidly dividing cells of the growth
centers and of the newly initiated primordia (Lance 19 54;
Gifford and Tepper 1962a, 1962b). Hence, the RNA content
and the physiological activity of a cell appear to be
associated.
Nougarde (1967) suggested that the high concentration
of RNA in the peripheral zone is probably connected with a
high requirement for protein synthesis to support the
catalytic activities of rapidly dividing cells. While
Nougarde's (1967) suggestion seems reasonable, cytochemical
determinations of total protein in the apical meristem
do not support her. That total protein is rather evenly
distributed throughout the apical meristem has been
documented by Cecich et al. (1972), Gifford and Tepper
(1962a, 1962b), and Riding and Gifford (1973). If
V


14
Nougarde's (1967) suggestion is modified to correlate
RNA concentration with certain proteins, especially
particular enzymes, rather than total protein, then
substantiating evidence is present. Peroxidase activity
and nucleohistone content, for example, closely parallel
RNA concentration in the peripheral zone (Vanden Born 1963;
Van Fleet 1959; Riding and Gifford 1973; Cecich et al.
1972). Nonspecific esterase activity and sulfhydryl-
containing proteins, while present throughout the apical
meristem, are most evident in the peripheral zone
(Vanden Born 1963; Fosket and Miksche 1966).
Negative correlations involving RNA distribution
in the apical meristem are also known. Succinate dehydro
genase, for instance, either shows uniform activity through
out the apex (Vanden Born 1963) or is more evident in the
initial cell and central mother cell zones than in the
peripheral zone (Fosket and Miksche 1966; Evans and Berg
1972; Riding and Gifford 1973). Similarly, when storage
products, i.e., lipid bodies and starch grains, occur in
the apical meristem, they are almost always found only
in the initial cell and central mother cell zones and the
rib meristem (Gifford and Tepper 1962a, 1962b; West and
Gunckel 1968; Riding and Gifford 1973; Varnell and Vasil
1972) and not in the peripheral zone.
What these correlations mean in physiological or
metabolic terms relative to morphogenetic events has not


15
been determined, but some tentative conclusions can be
formulated. The presence of succinate dehydrogenase and
cytochrome oxidase (Thielke 1965) in the central mother
cell and initial cell zones, associated with a low mitotic
rate and occasionally with storage products, suggests
that these zones have high rates of respiration and
interconversion of metabolites, and limited deposition
of metabolic products. Thus, it follows that the central
mother cell zone and the initial cell zone must export
many substances to sites of deposition. The site of
deposition nearest the central mother cell and initial
cell zones is the peripheral zone, and especially the
growth centers and nascent primordia where cell and
organelle multiplication proceed rapidly. Moreover,
localization of peroxidase, esterase, and phosphatase
activity in the peripheral zone and particularly at sites
of primordial initiation (Vanden Born 1963; Evans and
Berg 1972; Fosket and Miksche 1966) indicates manifold
utilization of substrates in biosynthesis of cellular
material. The foregoing postulate is an extension of a
hypothesis advanced by Sunderland et al. (1956) before the
distribution of enzymes in the apical meristem was known.
The distribution of growth regulators within the apical
meristem is unknownk


16
Effects of Chemicals Applied to the Apical Meristem
To date, application of chemicals directly to the
shoot apex of seed plants has been carried out on only
two occasions. First, Snow and Snow (1937) applied
0.05% heteroauxin, presumably indole-3-acetic acid, in
lanolin to the exposed apices of Lupinus and Epilobium.
Examination of treated meristems approximately two to
three weeks later revealed enlargement and connation of
primordial bases, and alteration of normal phyllotaxis.
Phyllotaxis on Epilobium changed from the normally decussate
type to spiral. Changes in phyllotaxis were attributed
to displacement of growth centers and of existing primordia,
presumably by asymmetric growth. Displacement usually
was in the direction toward the site of application.
Second, Ball (1944) applied a variety of auxins,
including indole-3-acetic acid, to the shoot apex of
Tropaeolum. All of the compounds tested produced essentially
similar responses. A 1% concentration of indole-3-acetic
acid produced the greatest alteration of morphogenesis
at the apex without causing mortality. As with lupine
and Epilobium (Snow and Snow 1937), affected primordia
of Tropaeolum exhibited enlarged and connate bases.
Phyllotaxis of Tropaeolum was altered erratically for a
time but eventually returned to normal in surviving
plants. In addition, the more detailed observations by


17
Ball (1944) revealed (1) a reduction in the number of
tunica layers from two or three for normal meristems to
one for treated apices, and (2) abnormal formation of axil
lary buds and tissue hypertrophy in the organogenic region
of the Tropaeolum apex. Neither Snow and Snow (1937) nor
Ball (1944), however, included cytochemical analyses in
their studies. Thus, the question of how growth substances
affect metabolism and hence morphogenesis in the apical
meristem remains unanswered.
Experiments described in the following sections
were designed to answer some of these questions.


MATERIALS AND METHODS
Plants
Seedlings of lupine (Lupinus albus L.) and pine (Pinus
elliottii Engelm. var. elliottii) were grown in vermiculite
in a Percival reach-in incubator, model 1-35 LVL, maintained
at 26C day- and 18C night-temperature, and 16-hour
photoperiod with 3500 foot-candles (37,800 lux) of
fluorescent illumination. Cuttings of coleus (Coleus
blumei Benth.) were rooted in water, without benefit of
rooting hormone, and subsequently grown in vermiculite
on the laboratory bench with supplemental lighting of 16-
hour photoperiod and 4000 foot-candles (43,200 lux) of
fluorescent illumination augmented by 1000 foot-candles
(10,800 lux) of incandescent illumination.
Experiments were conducted with lupine seedlings be
tween the ages of 14 and 40 days, with pine seedlings be
tween the ages of four and ten weeks, and with coleus plants
after they formed strong root systems. Coleus remained suit
able for use for about 75 days, although the plastochron
lengthened considerably during this time. A plastochron is
the interval between the appearance of successive primordia
on a meristem.
18


19
Treatments
Three growth regulators, indole-3-acetic acid (IAA),
6-furfurylaminopurine (kinetin), and gibberellin-3 (GA),
were applied separately to apical meristems in the form of
paste-like emulsions. Pastes were prepared by emulsify
ing 1% (w/w) solubilized growth regulator in anhydrous
lanolin in a stainless steel micro cup attachment of a
Virtis "23" hi-speed homogenizer. Pure ethanol was the
solvent for IAA and GA; kinetin was dissolved in acidified
water. A minimum of solvent was used in each case. Pastes
were stored at 4C. Selection of the 1% concentration
of each growth regulator was based on the concentration
of a variety of auxin pastes which caused optimal disruption
of normal morphogenesis in Tropaeolum meristems (Ball
1944). Using each growth regulator at the same concentration,
-2
approximately 5 x 10 M, permitted direct comparisons
of their effects.
Pastes were applied to meristem surfaces as small drops,
one drop per meristem, with an eyelash cemented to a wooden
applicator. An eyelash applicator was made for each kind of
paste and labelled to avoid using it in the other pastes.
Drops were applied manually to meristems magnified 32X
in a Leitz dissecting microscope with head and stage
reversed on the base. Plants were held securely in the
microscope field by adjustable forceps taped to a stick
clamped to a ringstand. Each meristem was exposed by
pushing the overlying tips of primordia out of the way


20
with a dissecting needle. Drop size, controlled by visual
observation at about 25X, ranged from approximately 0.1 to
0.2 X". Thus, each treated meristem was confronted with
about 1.5 yg of hormone.
Growth regulator was applied to lupine meristems at
the site where the next primordium was expected to arise,
the so-called 1^ site. This site was determined by visually
projecting the phyllotactic spiral around the apical meristem
through an arc of 136 beyond the last-formed primordium.
The arc of 136 was based on the average angle of divergence
between successive primordia in lupine, as determined by
Snow and Snow (1931). Complexities of primordial initiation
in the pine meristem discouraged attempts to place growth
regulator on preassigned sites, and so paste was simply
placed on the summit of the meristem. Placement of growth
regulator in coleus was at either of the two 1^ sites or on
the summit of the meristem. The 1^ sites in coleus, a decus
sate species, are located 90 around the apical meristem from
each of the last-formed primordia.
A group of five to seven plants of the same species,
treated with the same growth regulator, and designated
for the same analysis constituted an experiment. Most
experiments were conducted over a 10-day period, with har
vesting every second day. Other experiments lasted 12 to 14
days, with harvesting every third or every fourth day. Each


21
experiment was replicated three times.
Untreated meristems were included in the initial exper
iments, in which RNA and nucleohistone were determined.
Untreated meristems were not included in subsequent experi
ments, in which protein and unsaturated lipids were deter
mined, because the untreated controls added little informa
tion as the paramount comparison was between lanolin and
growth regulator treatments.


22
Harvesting
Harvesting consisted of severing the shoot apex from
the plant, dissecting away all unnecessary tissue, mounting
the terminal millimeter of apex on a thin, 3.0 mm diameter,
aluminum disc, and freezing the apex and disc instantly
in Freon-22 chilled in liquid nitrogen. The aluminum
disc greatly facilitated handling of the frozen apex and
alignment of the apex for sectioning.
Frozen apices were mounted in chilled Cryoform
(Damon/IEC) or Tissue-tek O.C.T. Compound (Ames) and trans
versely sectioned at 10 u in an International Equipment
Company Microtome-Cryostat, model CTF. Serial sections
were picked up, one at a time, on 0.75-mm-thick glass
microscope slides kept at above-freezing temperature.
All the sections for each apex were mounted on the same
slide and subsequently treated alike. Sectioning of each
apex proceeded well into the region of maturation.
Immediately after sectioning, tissues were killed
and fixed to minimize degradation and movement of key
compounds. Based on Swift's (1966) recommendation,
ribonucleic acid (RNA) was fixed in Carnoy's ethanol-
acetic acid fluid (Jensen 1962). Proteins were also
fixed in Carnoy's fluid, because of the rapid penetration
and action of this fluid on plant proteins. Unsaturated
lipids were immobilized, as recommended by Baker (1946),


23
by treating the sections with formalin-calcium (Jensen
1962) for 30 minutes. After fixation, sections were
washed briefly in distilled water and air dried.


24
Staining
Sections designated for RNA determinations were acet-
ylated, then stained with 0.1% Azure B in citric acid
buffer at pH 4.0 at 55C for two hours (Jensen 1962).
After staining, sections were thoroughly washed in distilled
water, destained in tertiary butyl alcohol (TBA) for
30 minutes, transferred to fresh TBA overnight, passed
through two changes of xylol, and mounted in Permount.
Number one cover slips were used throughout the study.
Acetylation of lupine and pine sections significantly
reduced tissue affinity for Azure B. Acetylation of
Carnoy-fixed lupine sections with 100% acetic anhydride
at 55C for four hours reduced Azure B staining by 38%.
Staining of pine sections was reduced 23% by acetylation
with 10% acetic anhydride in pyridine at room temperature
for four hours, and 28% by heated 100% acetic anhydride.
Acetylation did not alter the wavelength at which maximum
optical density was obtained, namely 590 nm, for the
Azure B-RNA complex in either lupine or pine tissues.
Acetylation blocks carboxyl activity of amino acids,
and since free amino acids are not retained, the only
candidates left in section for acetylation to affect are
proteins. Thus, despite Flax and Himes' (1950, 1952)
contention, echoed by Jensen (1962), that Azure B staining
of proteins at pH 4.0 is negligible, lupine and pine


25
tissues evidently contain proteinaceous carboxyl groups
which retain their activity even at pH 4.0.
Specificity of Azure B for RNA in the material
studied was confirmed by extraction procedures. Complete
extraction of lupine RNA was accomplished in 0.5 M per
chloric acid at 70C for 30 minutes. Optical density
of the Azure B-RNA complex was reduced 21% in pine sections
following extraction with 0.1% aqueous RNAase, at pH 6.8,
at 35C, for 90 minutes. Longer incubation in enzyme
would be expected to further reduce stain intensity.
Sections designated for protein determinations were
stained in 1% naphthol yellow S (NYS) dissolved in 1%
acetic acid for 15 minutes, rinsed in distilled water,
and destained overnight in fresh 1% acetic acid, in accord
ance with the procedure developed by Deitch (1966b).
After destaining, sections were passed through two changes
of TBA, two changes of xylol, and mounted in Permount.
Specificity of NYS for protein was determined by
blocking and enzymic extraction procedures. Blocking
was partially accomplished in pine tissues by acetylation
in heated 100% acetic anhydride, as described above;
optical density of NYS-stained sections was reduced 27%.
Less vigorous acetylation, in 10% acetic anhydride in
pyridine at room temterature, did not reduce affinity of
pine tissue for NYS.
Enzymic extraction of protein reduced NYS staining


26
of both pine and lupine tissues. Protease, at 1% concen
tration, in 0.2 M cacodylate buffer, at pH 7.2, at 35C,
for 90 minutes, reduced protein staining by 50% in pine
tissues and 70% in lupine tissues.
Unsaturated lipids were colored black by exposure
to osmium tetroxide fumes for one hour, followed by washing
for 15 minutes in distilled water, and mounting in 80%
Karo brand corn syrup, in accordance with Cain (1950).
Specificity of osmium for unsaturated lipids in pine
sections was verified by extraction with pyridine at 27C
for 30 minutes, followed by fresh pyridine at 60C for 24
hours. Solvent extraction reduced tissue affinity for
osmium by 75% in pine sections. Pyridine extraction of
lupine tissues did not reduce their affinity for osmium.
Why pyridine occasionally fails to extract lipids is
unknown, but in such cases extraction with an alcoholic
solution containing ether or chloroform is usually successful
(Pearse 1968). Extracting lupine tissues with alcohol-
ether (Jensen 1962) reduced affinity for osmium by 62% in the
peripheral zone and 58% in the central mother cell zone.
Sections selected for histone determinations were
chosen from those in which RNA had been determined. The
procedure used was Deitch's (1966a) modification of Alfert
and Geschwind (1953). Cover slips were removed in xylol,
and Azure B was dissolved in 70% ethanol containing 1%
trichloroacetic acid (TCA). Nucleic acids were removed


27
in 5% TCA at 90C for 20 minutes. Sections were rinsed
in cold 5% TCA, followed by distilled water, and stained
in 0.1% aqueous fast green FCF in 0.005 M phosphate
buffer at pH 8.0, at room temperature, for 30 minutes.
After staining, sections were thoroughly rinsed in distilled
water, dehydrated in an ethanol series, passed into
xylol, and mounted in Permount.


28
Microspectrophotometry
Microspectrophotometric determinations were made
with a Reichert Zetopan microscope equipped with a Binolux
II light source, an achromatic, aplanatic 6-lens condensor
with 1.35 N.A., an achromatic 25X objective, and a complete
microphotometer attachment including a 12.5X, coated,
plane eyepiece and sliding interference wedge filter
with 11 to 14 nm half-width transmission band. Except
for lipids, determinations were made at maximum optical
densities; thus RNA at 590 nm, total protein at 430 nm,
and histone at 635 nm. Since osmium absorbs visible
light rather uniformly, regardless of wave length, un
saturated lipids were determined from optical densities
measured at the midpoint (550nm) of the range of maximum
light transmission of the interference wedge filter.
Relative amounts of cytoplasmic RNA, total protein, un
saturated lipid, and histone were determined from extinction
values displayed on a deflection meter.


29
Analyses
Treatment effects were evaluated by examining each
treated meristem as it was harvested, as well as its tissues
in section, for any visible, qualitative change from
normal development. Expected changes in morphogenesis
included those listed by Wardlaw (1965b), i.e., death
of the apex, bud initiation on the flank of the apex,
(usually preceded by death of the cells at the summit of
the apex), and altered differentiation of primordia.
Existence of nascent axillary buds was determined by
examination of transversely sectioned shoot tips.
Plastochron length in lupine and coleus was determined
by the difference in number of foliar primordia plus an
assessment of the stage of intra-plastochronic development
observed at the beginning and end of a given interval.
A reference primordiumwas marked with india ink at the
beginning of the interval. Intra-plastochronic development
was divided into early, middle, and late stages of presumably
equal duration, as judged by the extent of development
of the last-formed primordium.
Assessment of treatment effects on primordium
displacement was based on the assumption that only certain
angles of divergence would be affected. Snow and Snow
(1937) reported (1) that only the angles formed by
primordia adjacent to the treatment site were affected,
and (2) that affected primordia tended to be displaced


30
toward the treatment site. The latter finding would result
in affected angles being smaller than average. It follows
from (1) above that the angles of divergence expected to be
affected by treatment of a preassigned site on the apical
meristem would be a function of the number of elapsed plasto-
chrons since treatment. A list was accordingly prepared of
the angles expected to be affected according to the number
of plastochrons since treatment at 1^ (Table 1) .
In the case of one elapsed plastochron, 1^ would have
become P^, and would have become P2 (Table 1). It follows
from (2) above that, if treatment at 1^ was effective, after
one elapsed plastochron, P2 would be displaced towards P^,
resulting in a smaller-than-average angle of divergence
P^-P2* Displacement of P2 towards P^ would further result
in P2 being further away from P^ and a larger-than-average
angle P2~P2.
A similar rationale applied to the situation of more
than one elapsed plastochron. The only added factor was
that I2 may have been displaced if treatment at 1^ was
effective. Then, after two or more elapsed plastochrons
and after I2 had become an observable primordium, the angle
formed by that primordium with the next older one would be
smaller than average. According to Snow and Snow (1937),
treatment at 1^ would not affect I^, and 1^ would be located
136 of arc around the meristem from I2, regardless of
whether I2 was displaced.


TABLE 1
Angles of divergence expected to be affected according
to the number of elapsed plastochrons since treatment
at l1
Number of
elapsed
plastochrons
Stage of
development of
initiation sites
Angles of divergence affected
and expected direction of change*
Angle Direction of change
1
x**
< X
2
x
< X
< X
OJ


Table 1
continued
Number of
elapsed
plastochrons
Stage of Angles of divergence affected
development of and expected direction of change *
initiation sites
Angle Direction of change
3
Z1
P3
P4
-P5
X**
Z2
P2
P3
"P4
<
X
Z3
P1
P2
"P3
<
X
Pn
-P
=
X
1
2
4
Z1
P4
P5
-P6
>
X
Z2
->
P3
P4
"P5
<
X
Z3
P2
P3
-P4
<
X
P2
-P3
X
* < means less than; > means greater than or equal.
** x is the average angle of divergence for untreated shoot tips.


33
Thus, angles of divergence between primordia adjacent
to the treatment site were expected to be smaller than
average. The angle formed by the older adjacent primordium
and its older neighbor, angle in the case of one
elapsed plastochron (Table 1), was expected to be greater
than or equal to the average. All other angles of
divergence on treated meristems should approximate the
average. Angles of divergence expected to be affected
by treatment compared with those expected not to be
affected on treated meristems and with angles from untreated
meristems, using statistical methods,were the basis
for assessing treatment effects on primordium displacement.
Angles of divergence were measured with a protractor
after connecting with straight lines the mid-point of the shoot
axis with the mid-point of each primordium in transverse
section. Mid-points were plotted with approximately
100X enlargement of the image through a camera lucida
attachment on a Wild compound microscope.
Cytophotometric determinations were taken from
specific sample points in certain lupine and pine sections.
Only the sections immediately above the point of insertion
of the last-formed primordium and the immediately subjacent
section were sampled. Sample points in each lupine section
were the three presumptive initiation sites, referred
to as 1^, I2, and 1^, the three areas of intervening
peripheral zone between the initiation sites, and three


34
randomly chosen points at the center of the central
mother cell zone, for a total of nine from each section
(Fig. 2). Uniformity in locating sample points of initiation
sites in section, since initiation sites might be displaced
by treatment effects, was accomplished by visually project
ing the phyllotactic spiral through an arc of approximately
136 beyond the last-formed primordium to the presumptive
site of I thence continuing through another arc of 136
to I2, and similarly on to I3.
Initiation sites were numbered in accordance with the
procedure established in the literature (Snow and Snow 1931).
The numerical order corresponds to the order in which the
sites were created; i.e., 1^ arose first, I2 second, and I3
third. Existing primordia were numbered in reverse order of
formation; i.e., the youngest as P^, the next as P2, the
next as P3, etc.
Sample points in each pine section consisted of five
points distributed as evenly as possible throughout the
peripheral zone and five points clustered in the central
mother cell zone.
Quantitative data, such as changes in phyllotactic
angles and the cytophotometric data, were analyzed by
conventional statistical methods for comparisons among two
or more means (Snedecor and Cochran 1967) (ref. Appendix.)
All stated differences were significant at the 95% level of
confidence.


Fig. 2 Location of cytophotometric sample points in
transverse section of apical meristems of
Lupinus albus, stained with Azure B. X 200.
1= sample points in initiation sites; Fz =
sample points in intervening peripheral zone
C= sample points in central mother cell zone
P'l' P2' P3' leaf primordia, numbered
in order from youngest to oldest.


36


RESULTS
Lupinus
Morphological Analyses of Treatments
Morphogenetic effects of growth regulators applied
to the apical meristem of lupine shoots were evaluated
by determining whether changes occurred in (1) the distance
from the shoot tip to the last-formed axillary bud,
and (2) the displacement of primordia. Analysis of
treatment effect on plastochron length was attempted.
Axillary buds appeared closer to the shoot tip only
in IAA-treated meristems (Table 2). The distance, expressed
in numbers of the intervening nodes, averaged 5.2 for IAA-
treated meristems, as compared to 8.4 for untreated
meristems and 9.0 for lanolin-treated ones. Kinetin and
GA treatments did not cause axillary bud formation to
occur significantly closer to the shoot tip than untreated
controls.
Length of the plastochron was variable. The plastochron
lengthened in successive crops as the seed from which
the crops were grown aged. The plastochron lengthened
within crops as the plants aged. Average plastochron
length by experiments ranged from 1.9 days to 5.9 days.


38
TABLE 2
Summary of treatment effects on morphology
of the shoot tip of Lupinus
Number of nodes from
apex to last-formed Angle (degrees)
axillary bud of divergence*
Treatments
Average**
Sx
Average**
Sx
Untreated
8.4
0.9
134.5
2.0
Lanolin only
9.0
0.3
134.3
3.0
IAA
5.2
0.4
139.0
7.2
Kinetin
7.6
0.3
138.6
2.3
GA
7.4
0.4
137.6
3.0
* Computed
only for
those
angles expected
to be affected
by treatment (ref. Table 1).
** Averaged over harvest dates and replications.


39
Treatment effects, when superimposed on the variation
caused by progressive ageing of seeds and plants, could
not be realistically evaluated. Average plastochron
length for each experiment was taken into account in the
analysis of treatment effects on displacement of primordia.
Primordia were displaced only by treatment with IAA.
The average angle of divergence for affected angles in the
IAA treatment did not differ significantly from that for
untreated meristems or lanolin controls, but variance,
indicated by the sample mean standard error, was greater
in the IAA treatment than in the other treatments and
controls (Table 2). The average and the variance for
angles not expected to be affected in IAA-treated meristems
did not differ from control values.
In the case of one meristem harvested after the first
elapsed plastochron following treatment with IAA, the angle
of divergence which had formed between and P2 was 90
(Fig. 3), as compared to an average of 134.5 for untreated
meristems (Table 2) and 139 for unaffected angles in
treated meristems. The angle P^-P2 a^ter one elapsed
plastochron was expected to be smaller than average (Table 1).
However, in this meristem, contrary to Snow and Snow's
(1937) hypothesis that pirmordia adjacent to the treatment
site are displaced, the initiation site 1^ evidently was
displaced instead. P^ must not have been displaced
significantly, because when harvested the angle P2~P^


Fig. 3 Displacement of towards P2 following treatment
with 1.5 yg IAA in transection of apical meristem
of Lupinus albus, stained with alkaline fast
green. X 300.
A= apical meristem; P^, P2, P^, .... = leaf
primordia, numbered in sequence from youngest
to oldest.


41


42
was only 151 which was less than two standard deviations
from the average. If had been displaced, the angle
P2-P3 woul^ have been significantly greater than the average
(Table 1).
Another meristem, harvested eight days after treatment
with IAA, showed displaced 282 from P2 (Fig. 4). In this
case, enough time elapsed since treatment for ^ to develop
into P^, and 1^ into P2 (plastochron 2, Table 1). Hence,
I2 was displaced around the meristem away from the point
of treatment and away from 1^. 1^ was not displaced,
because the resulting angle of divergence between P2 and
P^ of 121.5 was less than a standard deviation from the
average. In this meristem, treatment with IAA caused reversal
of the phyllotactic spiral.
In view of the fact that morphological responses
in lupine were elicited only by IAA, cytochemical analyses
were restricted to the effects of this treatment.
Cytochemical Analyses of IAA Effects
Cytochemical analyses involved microphotometric
determinations of changes with time in concentration of
RNA, total protein, and unsaturated lipids in the cytoplasm
and histones in the nuclues.^ Comparisons were made between
^Results of experiments justifying the use of certain
cytochemical procedures and confirming the specificity of
the stains are listed in MATERIALS AND METHODS under the
sections headed Staining


Fig. 4 Displacement of P away from P2 following
treatment with 1.5 yg IAA in transection of apical
meristem of Lupinus albus, stained with alkaline
fast green. X300.
A = apical meristem; P^, P2, P^/ .... = leaf
primordia numbered in sequence from youngest to
oldest.


44


45
the initiation sites, the intervening peripheral zone,
and the central mother cell zone in lupine meristems.
RNA concentration in the initiation sites or in the
central mother cell zone of lupine apices was not affected
by treatment. However, IAA treatment caused RNA concen
tration of the intervening peripheral zone to increase by
29.5% on day two (Fig. 5). RNA concentration in the
intervening peripheral zone following the increase on day
two was comparable to that in the initiation sites.
Elevated RNA concentration in the intervening peripheral
zone tended to persist for the remainder of the study
period.
Evaluation of the possible effects of different
plastochron stages on RNA concentration in untreated
lupine meristems revealed that RNA in the three cytological
sites did not vary among plastochron stages (Table 3).
Protein concentrations in initiation sites and in the
intervening peripheral zone of IAA-treated lupine meristems
were increased relative to lanolin controls (Fig. 6). The
data indicate a tendency for protein concentration to
increase from day two through day eight, but differences
from control values were statistically significant only
for day eight. IAA did not significantly affect protein
concentration in the central mother cell zone. Uniformity
of protein concentration throughout the untreated apical
meristem, as previously reported for other species (Cecich


Fig. 5 Changes with time in RNA concentrations in the
intervening peripheral zone (), expressed as a
percentage of RNA in the initiation sites ( ),
in IAA-treated meristems of Lupinus albus. Each
point is the average of three replications.


RNA CONCENTRATION (%)


TABLE 3
Average relative concentrations of RNA
during the plastochron in shoot apical meristems
in Lupinus*
-
Plastochron
stage
Initiation
sites
Intervening
peripheral
zone
Central
mother cell
zone
Means
Early
1.52
1.35
1.12
1.33
Middle
1.78
1.64
1.17
1.53
Late
1.65
1.48
1.11
1.41
Means
1.64
1.48
1.13
*
Averaged over replications and expressed in arbitrary units.


Fig. 6 Changes with time in protein concentration in
the initiation sites (A), and the intervening
peripheral zone (B) of IAA-treated meristems
() expressed as percentage of lanolin controls
( ) of Lupinus albus. Each point is the
average of three replications.


PROTEIN CONCENTRATION (%)
O ro od o o ro a> o
o ooo ooooooooo
U1
o


51
st al. 1927; Gifford and Tepper 1962a, 1962b;Riding and
Gifford 1973), was observed in lupine apices treated with
lanolin only. Apices treated with IAA, however, had
higher concentrations of protein in the initiation sites
and intervening peripheral zone than in the central mother
cell zone.
Concentrations of unsaturated lipids in the intervening
peripheral zone and central mother cell zone were affected
by IAA treatment (Fig. 7). Based on the assumption that
the optical density of the lipid-bound osmium oxidation
product is a linear function of the concentration of
unsaturated lipids, concentration of unsaturated lipid in
IAA-treated apices increased nearly twofold in the interven
ing peripheral zone by the sixth day and approximately
threefold in both zones by the eighth day.
Nucleohistone concentration throughout IAA-treated
lupine meristems was affected by treatment (Fig. 8).
Significant differences in nucleohistone concentration
between IAA- and lanolin-treated meristems were observed
on day two and day six. The differences on day two,
however, were due largely to unusually low control values
and probably do not represent real treatment effects. The
peak on day six apparently was a genuine treatment effect.
After day six, nucleohistone concentrations in treated
meristems declined sharply. Relative to lanolin controls,
fluctuations in nucleohistone concentrations following


Fig. 7
Changes with time in unsaturated lipid concentration
in the intervening peripheral zone (A), and
the central mother cell zone (B) of IAA-treated
meristems (), expressed as percentage of
lanolin controls ( ) of Lupinus albus.
Each point is the average of three replications.


LIPID CONCENTRATION (%)

Ln
CO


Fig. 8 Changes with time in nucleohistone concentration
in the initiation sites (A), the intervening
peripheral zone (B), and the central mother cell
zone (C) of IAA-treated meristems (), expressed
as percentage of lanolin controls ( ), of
Lupinus albus. Each point is the average of three
replications. Because of an unusually low average
for lanolin controls, the percentage for day two
was based on the overall average for control.


55


56
treatment with IAA were similar for initiation sites,
the intervening peripheral zone, and the central mother
cell zone. The dramatic rise and fall of nucleohistone
concentration on the sixth day following treatment with
IAA coincided with the time of displacement of primordia
during the second plastochron.
Although IAA appeared to increase concentrations of RNA,
proteins, unsaturated lipids, and nucleohistones in lupine
meristems, the effect tended to diminish by the end of the
observation period. Diminution of effects should be
expected as the IAA that penetrated into the meristems
was diluted by growth and metabolic activities, including
translocation, and as the lanolin-IAA droplet was displaced
continually further away from the meristem by growth of
the shoot tip. Concentrations were rapidly approaching
control values after the eighth day for proteins (Fig. 6)
and for unsaturated lipids (Fig. 7) in affected cytological
zones. Diminishing effects of IAA on RNA concentration
in the intervening peripheral zone towards the end of the
experiment were perhaps equivocal (Fig 5). Nucleo
histone concentration decreased sharply after day six
(Fig. 8) and may have been approaching control values
towards the end of the experiment.


57
Pinus
Morphological Analyses of Treatments
Morphogenic effects of growth regulators applied to the
shoot apical meristerns of pine, as determined in this study,
were confined to the differentiation of primordia. Primordia
developed normally into foliar needles in all treatments,
except kinetin. Kinetin-treated meristems showed no response
to treatment until the eighth or tenth day at which time
differentiation of primordia into bud scales became evident
on some meristems (Fig. 9a). Out of 18 meristems harvested
on the eighth or tenth day following treatment with kinetin,
five produced bud scales. Six other kinetin-treated seed
lings which were not harvested all produced bud scales by day
14 and appeared at that time to be dormant. Immature bud
scales developing on kinetin-treated meristems appeared normal
in size, shape, coloration, and position on the meristem
(Figs. 9a and c). Mature scales on kinetin-treated meristems
were more brightly russet in color than scales subsequently
produced by unused plants (Fig. 9b) some weeks after the
study had been completed.
An attempt was made to determine whether the growth
regulators tested affected axillary bud formation.
Appearance of nascent axillary buds in the sectioned
portion of pine shoot tips, however, was too infrequent
to permit determination of treatment effects, although


Fig. 9 A. Dormant terminal bud following treatment
with 1.5 yg kinetin applied to the exposed
apical meristem of seedling of Pinus
elliottii. X14.
B. Dormant terminal bud of untreated seedling
of Pinus elliottii some weeks after the
study was completed. X14.
C. Excised bud scale from kinetin-treated shoot
tip of Pinus elliottii seedling. X23.
S = bud scale; L = young primary needle; N =
mature primary needle; B = axillary short-
shoot bud; SN = axillary short shoot with
secondary needles.


59


60
sectioning of the shoot tips extended 150 to 200 y below
the base of the apical meristem. Examination of sections
from approximately 50 meristems revealed only seven
meristems with axillary buds, and the bud-containing
meristems were distributed among almost all treatments.
No attempt was made to determine angles of divergence
or plastochron length in pine meristems because of the
difficulty in identifying the last-formed primordium.
Because kinetin was the only growth regulator tested
which elicited a morphogenetic response in pine meristems,
this treatment was the only one analyzed cytochemically.
Cytochemical Analyses of Kinetin Effects
Cytochemical analyses of pine meristems, as in the case
of lupine meristems, involved microspectrophotometric
determinations of changes with time in concentration of RNA,
total protein, unsaturated lipids, and nucleohistones.
Comparisons within pine meristems, however, were confined to
the peripheral and central mother cell zones.
Treating shoot apices of pine seedlings with kinetin
increased the concentration of cytoplasmic RNA only in the
central mother cell zone (Fig. 10). The increase was
significant only on day six, at which time RNA in treated
meristems exceeded controls by almost 300%. RNA concentra
tion in treated meristems had returned to control values
by day eight. The sharp rise and fall of RNA in treated


Fig. 10 Change with time in RNA concentration in the central
mother cell zone of kinetin-treated meristerns (),
expressed as percentage of lanolin controls (*) ,
of Pinus elliottii. Each point is the average of
three replications.


62


63
meristems preceded appearance of bud scales by two to four
days.
Kinetin affected protein concentration throughout the
pine meristem (Fig. 11). Protein was 20% to 30% more
concentrated in the peripheral zone of treated plants
than in controls on days two and six, respectively
(Fig. 11A). Concentrations then fell to approximately
20% less than control values on day eight. Protein in
treated meristems was still significantly less concentrated
than in controls on day ten.
In the central mother cell zone, protein concentration
in treated meristems differed from control only on day
eight (Fig. 11B). On this day, treated meristems had about
30% less protein than controls. Decline in protein
concentration in the central mother cell zone on day
eight corresponded to the decline in protein in the
peripheral zone on the same day.
Reduced concentrations of proteins in treated
meristems tended to correspond with the appearance of
bud scales and the onset of dormancy.
Unsaturated lipids were 25% less concentrated in the
central mother cell zone of kinetin-treated pine meristems
than in the lanolin controls (Fig. 12). Kinetin had no
apparent effect on unsaturated lipids in the peripheral
zone. Decreased lipid concentration in the central mother
cell zone was evident on the fourth day following treatment


Fig. 11 Change with time in protein concentration in the
peripheral zone (A) and the central mother cell zone
(B) of kinetin-treated meristems (t), expressed
as percentage of lanolin controls ( ) of Pinus
elliottii. Each point is the average of three
replications.


PROTEIN
CONCENTRATION (%)
o o
00
o
ro
o
*
o
o>
o
00
o
o
o
ro
o
A
o
Ln


Fig. 12
Change with time in unsaturated lipid concentration
in the central mother cell zone of kinetin-
treated meristems (i), expressed as percentage
of lanolin controls ( ) of Pinus elliottii
Each point is the average of three replications.


LIPID CONCENTRATION (%)
O) 00 5 N o
ooo ooooo
o>


68
(Fig. 12), but was gradually returning towards control
levels afterwards.
Concentrations of nucleohistones increased in both
the peripheral zone and the central mother cell zone
following treatment of pine meristems with kinetin
(Fig. 13). Kinetin-increased nucleohistone concentrations
were significantly higher in both zones on day six and
rapidly decreased toward control values thereafter. Peak
nucleohistone concentration in kinetin-treated meristems
corresponded with peak RNA concentration in the central
mother cell zone (Fig. 10) and preceded appearance of bud
scales by two days.


Fig. 13
Change with time in nucleohistone concentrations
in the peripheral zone (A) and the central
mother cell zone (B) of kinetin-treated meristems
(), expressed as percentage of lanolin controls
( ) of Pinus elliottii.
Each point is the average of three replications.


HISTONE CONCENTRATION (%)
70
TIME (DAYS)


Coleus
Growth regulators, as tested in this study, did
not affect morphogenesis at the shoot tip of coleus.
Lateral buds were formed in the second or third leaf
axils in all treatments and controls. Primordia were
not displaced by any treatment, and primordia an all
treatments appeared to develop normally, except that
development was considerably retarded during the course
of the study as the plastochron increased substantially
during this time. The plastochron at the beginning of
the study appeared to be two to three days in length.
When the plants had been grown under the conditions
reported above for about 50 days, the plastochron was
estimated to be 40 days in length in untreated meristems.
Treatments did not appear to shorten the plastochron.
Cytochemical analyses originally planned for the coleus
meristems were abandoned when the treatments failed to
elicit a morphogenetic response.


DISCUSSION
Lupinus
Of the three growth regulators tested, only IAA
affected shoot meristems in lupine. Treatment with IAA
resulted in: (1) axillary buds forming closer to the apex
than normal, (2) displacement of primordia, and (3)
increases in concentrations of RNA, total protein, and
unsaturated lipids in the cytoplasm, and histones in the
nucleus, of cells in the subdistal and organogenic regions
of the apical meristem.
Initiation of axillary buds closer to the apex than
normal and displacement of primordia in IAA-treated lupines
in this study agree with the observations of other workers
(Ball 1944; Snow and Snow 1937). Ball (1944), working with
Tropaeolum, observed lateral buds on the flanks of auxin-
treated apices, and primordia were displaced as much as 40
to 50 of arc from their normal positions on the meristem.
Snow and Snow (1937) observed primordial displacement on
lupine and Epilobium meristems treated with heteroauxin.
The three studies taken together, therefore, indicate that
IAA applied to the apical meristem of dicots disrupts the
normal physiological patterns within the apex and affects
72


73
physiological interactions between the apex and the rest of
the plant.
The only point of disagreement among these three studies
concerns the direction of primordial displacement. Snow and
Snow (1937) claimed that the direction of displacement tended
to be towards the treatment site. This claim was based pri
marily on observations in Epilobium, a decussate species, in
which the first pair of primordia to arise after treatment
were, without exception, displaced toward the site of treat
ment. Snow and Snow (1937) conceded that their data for
lupine, a species exhibiting spiral phyllotaxis, did not
provide much support for their claim.
Ball's (1944) observations of Tropaeolum, which also
features spiral phyllotaxis, as well as the present study of
lupine, does not support Snow and Snow's (1937) interpreta
tion. Greater variation in angles of divergence, indicated
by larger sample mean standard errors, was observed in auxin-
treated meristems than in controls for Tropaeolum by Ball
(1944), and for lupine in the present study. Average angles
of divergence for treated meristems in each of the three
studies did not differ significantly from that for untreated
meristems in the same species, except for Epilobium (Snow and
Snow 1937). Affected angles of divergence for treated
Epilobium meristems were always less than the normal 180.
Greater variation around an average angle of divergence which
approximately equals that for untreated meristems means that


74
affected angles tended to be greater or smaller than the
normal, and there are about as many large angles as small
ones. Developmentally, these results support Ball's (1944)
conclusion that primordial displacement in auxin-treated
apices is random relative to the site of treatment. This
conclusion may apply to all dicotyledonous species with
spiral phyllotaxis.
Application of IAA to the apical meristem of lupine
appeared to confer initiation site capabilities to the
intervening peripheral zone for a short time. This was
demonstrated not only by displacement of initiation sites
to what must have formerly been the intervening peripheral
zone, but also by metabolic activity in the intervening
peripheral zone being increased to the same level as
observed in the initiation sites. Increased metabolic
activity in the intervening peripheral zone of IAA-treated
meristems was shown by increased concentrations of all of the
cytochemical substances observed relative to control
values (Figs. 5, 6, 7, and 8). Concentration of RNA and
unsaturated lipids increased in the intervening peripheral
zone, as compared to the initiation sites, in IAA-treated
meristems. Enhanced metabolism in the intervening
peripheral zone was additionally indicated by increased
concentration of protein, relative to the central mother
cell zone, in IAA-treated meristems. Increased histone
concentration in the intervening peripheral zone indicated


75
that not only the cytoplasm but also the nuclei in this
zone were affected by IAA treatment. Responses in RNA and
unsaturated lipids within two to four days after treatment
correspond weU with the duration of the first plastochron,
during which time some primordia were displaced. Responses
of all observed substances, except nucleohistone, through
day eight would extend IAA effects into the second and
third plastochron when other primordia were displaced. An
enlarged zone of initiation potential would help explain
not only the displacement of primordia observed in this
and other studies but also the enlarged leaf bases and
connation of primordia observed in Tropaeolum by Ball
(1944) and in lupine and Epilobium by Snow and Snow (1937).
Diminishing concentrations of nucleohistones after
the sixth day (Fig. 8) and of proteins and unsaturated
lipids after the eighth day (Figs. 6 and 7) indicate that
a single application of a minute droplet of IAA produces
a dramatic but transient effect. If treatment effects
had largely disappeared by the tenth day, only three or
four plastochrons could be affected. Primordia produced
thereafter in subsequent plastochrons would be expected
to arise in normal positions around the apex (Table 1),
and phyllotaxis would once again be characteristic for
the species. Although the present study did not monitor
treated plants beyond the tenth or twelveth day, Ball
(1944) reported that treated plants, exhibiting altered


76
phyllotaxis, soon reverted back to normal phyllotaxis.
One possible mechanism for the cytological effects
of IAA observed in this study involves interactions between
the growth regulator and nucleic acid metabolism, and,
conceivably, gene expression. Involvement of auxin in
nucleic acid metabolism was suggested 20 years ago (Skoog
1954), and support for the idea has been forthcoming
(Key and Shannon 1964; Key 1964). RNA concentrations in
the present study were affected relatively soon (two days)
after treatment (Fig. 5), while unsaturated lipid concentra
tions were not affected until day four or later (Fig. 7).
The change in RNA concentration in IAA-treated apices
was well within the normal response time for auxin-
mediated changes in nucleic acid metabolism (Kamisaka 1972;
Key and Ingle 1968). Changes in protein and unsaturated
lipid concentration at about the same time or after the
change in RNA would be expected if existing cytoplasmic RNA
had to be augmented by more of the same kind in order to
meet requirements for enhanced synthesis of existing
proteins and unsaturated lipids, and if different genetic
information had to be translated by new messenger RNA
species for the synthesis of novel compounds.
According to Swift (1966), cytoplasmic RNA determined
by the Azure B technique, which was used in this study,
consists largely of ribosomal RNA. Total cytoplasmic
protein, determined by the NYS technique, consists of


77
soluble and structural protein (Deitch 1966b). Much of the
structural protein in the cytoplasm is contained in the
ribosomes. Thus, close correspondence in fluctuations with
time between cytoplasmic RNA and proteins in the intervening
peripheral zone of IAA-treated lupines (Figs. 5 and 6B) would
be expected through their common association in ribosomes.
Changes in protein concentration unaccompanied by
obvious changes in RNA in the initiation sites of IAA-
treated meristems (Fig. 6A) might be explained by changes
in soluble proteins and structural, non-ribosomal
proteins. If ribosomal protein was involved, corresponding
changes in ribosomal RNA in these zones must have been
obscured by compensatory changes in other kinds of
cytoplasmic RNA.
Because unsaturated lipids in the cytoplasm are
largely contained in membranes, changes in unsaturated
lipid concentration would probably reflect changes in
membrane-related phenomena. Membrane changes regulated
by differential gene expression could have widespread
implications on the physiology of cells, tissues, and
organs, as well as on morphogenetic events.
Changes in nucleohistone concentrations (Fig. 8),
accompanied by changes in RNA, further suggest growth
regulator involvement within the nucleus. That auxin
binds nucleohistone, causing conformational changes in the
protein, has been suggested (Venis 1968). Thus, the


78
possibility exists that auxins, interacting with histones
may modulate the latter's gene-repressor functions.
Results of the present study indicate involvement of
the central mother cell zone in the functions of the apex.
The central mother cell zone responded to IAA treatment
with increased concentrations of unsaturated lipids and
nucleohistones (Figs. 7 and 8). Fluctuations in concentra
tions of these substances in the central mother cell zone
were identical to those in the intervening peripheral
zone for treated meristems. RNA and protein in the central
mother cell zone apparently were unaffected by IAA.
Pinus
Application of IAA, kinetin, or GA to shoot apical
meristems of pine seedlings in this study resulted in a
morphogenetic response, the appearance of bud scales and the
onset of dormancy only to the kinetin treatment. Bud
formation, as observed here, is a common response to
kinetin treatment (Paulet and Nitsch 1959; Schraudolf and
Reinert 1959; Sinnott and Miller 1957) in those plants which
have a natural tendency to form buds (Miller 1961). Bud


79
formation is regarded as one of the two primary activities
of kinetin (Cutter 1965). The mechanism by which kinetin
stimulates bud formation is not known, but results of the
cytochemical analyses from the present study shed some
light on the problem.
Cytochemical effects of kinetin were first apparent
the second day after treatment. At that time, protein
concentrations exceeded controls by about 18% in the
peripheral zone (Fig. 11A). This was followed by an
abrupt decrease in concentrations of unsaturated lipids
(Fig. 12) in the central mother cell zone on day four.
RNA in the central mother cell zone and nucleohistones
in both zones exceeded control levels on day six (Figs. 10
and 13), two days before bud scales were first observed.
The effect of kinetin on RNA and nucleohistones suggests
involvement of the growth regulator in nucleic acid
metabolism and/or gene expression, which might explain
the sharp decrease in protein concentration in treated
meristems on the eighth day following treatment (Fig. 11).
Kinetin has been shown to interact with events surrounding
transcription (Datta and Sen 1965; Matthysee 1969)+ transla
tion (Datta and Sen 1965), and enzyme activity (Mann et al.
1963; Steinhart et: al. 1964; Boothby and Wright 1962).
Decreased concentrations of unsaturated lipids in the
cytoplasm probably reflected reduced amounts of membrane
material. Reduction in unsaturated lipids and membranes


80
could be achieved if the enzymes catalyzing their synthesis
were adversely affected by kinetin. Kinetin-related changes
in enzymes can occur by any one of at least three
mechanisms. One, kinetin can act directly on specific
enzymes by directing their synthesis (Boothby and Wright
1962; Steinhart et ad. 1964) or by altering their activity
(Bergman and Kwienty 1958; Henderson et al. 1962). Two,
kinetin can act indirectly on specific enzymes by affecting
nucleic acid metabolism (Fox and Chen 1968) or gene
expression (Boothby and Wright 1962; Clum 1967). Three,
kinetin can affect general enzyme activity by altering
physical properties of the cell, conceivably via (1) or (2)
above, and thereby changing the availability of substrate
and the utilization of product (Mothes and Engelbrecht
1961). Thus, there is ample evidence, albeit circumstantial
that kinetin could influence lipid metabolism and membrane
structure in treated pine meristems. Direct evidence of
kinetin influencing lipid metabolism was not discovered
in the literature.
Examination of cytoplasmic membranes from intact,
kinetin-treated plants apparently has not been done. Nitsch
(1968) observed no effect of kinetin in the medium on ultra
structure of i_n vitro tissues, but kinetin can affect mem
brane morphology in excised roots (Vasil 1973). Whether
altered membrane morphology involved changes in lipid metab
olism was not determined (Vasil (1973). However, this infor


81
mation may not apply to intact plants, because kinetin effects
often differ between intact and excised tissues from the
same plant (Pilet 1968; Fletcher and Adedipe 1972).
Kinetin-activated fluctuations in RNA in the central
mother cell zone on day six (Fig. 10) probably reflected
ribosomal-related events, despite the apparent absence of
treatment effects on proteins in this zone. Effects on
ribosomal protein might have been obscured by compensatory
changes in other structural proteins or in soluble proteins
in the cytoplasm. That compensatory changes in concentrations
of different proteins could result in significant biological
effects without altering the total concentration of
cytoplasmic protein is an inherent problem in the study of
total protein and was discussed above (ref. p. 13) regarding
Nougarde's (1967) hypothesis. A less likely explanation
for the fluctuations of RNA in the central mother cell zone
would be that the fluctuations resulted from massive
changes in concentration of messenger or transfer RNA.
In kinetin-treated apices, changes in RNA and nucleo-
histones on day six probably reflected activity within the
nuclei. Perhaps it was about this time that the physiological
state of treated meristems shifted from one favoring
growth to one favoring dormancy, and, as growth processes
slowed down and dormancy began to set in, novel enzymes
were needed in newly activated metabolic pathways. Genetic
information from previously unused segments of the


82
chromosomes would then be transcribed, and other segments
of the chromosomes would be repressed, perhaps by some of
the additional nucleohistones. Transcription of newly
derepressed genetic information could, conceivably, lead
to differentiation of primordia into bud scales and to
other events which, similarly, had never before occurred in
the brief lives of the pine seedlings used in this study.
A similar suggestion has been advanced to rationalize
the episodic annual growth habit of shoots in trees
(Romberger 1966).
Manifestations of the onset of dormancy in kinetin-
treated meristems included the rapid decline of RNA,
proteins, and nucleohistones (Figs. 10, 11, and 13) after
day six, the appearance of bud scales on day eight and ten,
and buds completely formed on all treated meristems on day
14. The rapid decrease in nucleohistones after day six
(Fig. 13) could correspond with declining metabolic activities
in the nuclei and elsewhere as dormancy was imposed. An
abrupt decrease in nucleohistone content in shoot meristems
has been correlated with cessation of elongation growth and
winter bud formation in white spruce (Cecich el: al. 1972).
Indirect evidence indicates that as dormancy sets in
decreases would be expected in total RNA (Chen and Osborne
1970; Pilet 1970; Poulsen and Beevers 1970; Bex 1972) and
proteins (Varner and Johri 1968; Chen and Osborne 1970;
Pollard 1970; Poulsen and Beevers 1970; Paranjothy and
Wareing 1971; Rijven and Parkash 1971). Reduced levels of


83
RNA and protein could be at least partially accounted for by
reduced ribosomal activity (Chen and Osborne 1970), by
fewer polysomes (Poulsen and Beevers 1970; Paranjothy and
Wareing 1971; Bex 1972; Evins and Varner 1972), and by a
lower rate of polysome formation (Evins and Varner 1972) as
dormancy is imposed.
The central mother cell zone was involved in metabolic
functions of the pine seedling shoot meristem. Changes in
all the observed compounds occurred in the central mother
cell zone in response to kinetin treatment (Figs. 10, 11B,
12, and 13B). Fluctuations with time in RNA (Fig. 10) and
nucleohistones (Fig. 13) were the same in the central
mother cell zone. Responses of RNA (Fig. 10) and unsaturated
lipids (Fig. 12) to treatment appeared to be restricted
to the central mother cell zone. Involvement of the
central mother cell zone in metabolic functions of the
vegetative apex were also observed in lupine in this
study and in shoots of mature spruce trees (Cecich et al.
1972), despite Plantefol's (1946, 1947a, 1947b, 1948)
hypothesis to the contrary.
This study demonstrated that topical treatment of the
apical meristem can be successfully combined with
cytochemical analyses. The results add to our knowledge
of how developmental and cytological heterogeneity is
maintained within the apical meristem. Perhaps this study
will stimulate additional analytical research on development
within the vegetative shoot apical meristem.


APPENDIX
The average for angles of divergence expected to be
affected was compared with that for angles expected not
to be affected as paired samples from each plant in a
given treatment. A two-tailed "Student's" t-test was
used to evaluate mean differences.
The analysis of variance used to evaluate growth
regulator effects versus controls over five harvest dates
with three replications in each of the cytochemical
experiments was as follows:
Source of variation Degrees of freedom
Total 29
Treatment 1
Dates 4
Interaction 4
Error 20
The unit of variation was the mean per plant. If the F-
ratio for interaction was significant at the 5% level,
separate analyses were made comparing dates within each
treatment and comparing treatments within each harvest
date. The analysis for harvest dates within a treatment
was as follows:
84


85
Source of variation Degrees of freedom
Total
14
Between dates
4
Within dates
10
The analysis of treatments within a harvest date was a
"Student's" t-test, using the error mean square from the
two-way analysis above as an estimate of the standard
error.
If interaction was negligible, the error mean
square was used for testing date and treatment main effects.


LITERATURE CITED
Alfert, M. and I.I. Geschwind. 1953. A selective staining
method for the basic proteins of cell nuclei. Proc.
Nat. Acad. Sci. (U.S.) 39:991-999.
Baker, J.R. 1946. The histochemical recognition of lipine.
Quart. J. Microscopical Sci. 87:441-470.
Ball, E. 1944. The effects of synthetic growth substances
on the shoot apex of Tropaeolum majus L. Araer. J. Bot.
31:316-327.
Bergman, F. and H. Kwietny. 1958. Oxidation of kinetin by
mammalian xanthine oxidase. Biochem. Biophys. Acta
28:100-103.
Bersillon, G. 1951. Sur le point vgtatif de Papaver
somniferum L.; structure et fonctionnement. Acad, des
Sci. Compt. Rend. 232:2470-2472.
Bex, J.H.M. 1972. Effects of abscisic acid on nucleic acid
metabolism in maize coleoptiles. Planta 103:1-10.
Boothby, D. and S.T.C. Wright. 1962. Effects of kinetin and
other plant growth regulators on starch degradation.
Nature 196:389-390.
Biinning, E. 1952. Morphogenesis in plants. Survey of Biol.
Prog. 2:105-140.
Cain, A.J. 1950. The histochemistry of lipoids in animals.
Biol. Rev. Trans. Camb. Phil. Soc. 25:73-112.
Cecich, R.A., N.R. Lersten, and J.P. Miksche. 1972. A
cytophotometric study of nucleic acids and proteins in
the shoot apex of white spruce. Amer. J. Bot. 59:442-
449.
Chen, D. and D.J. Osborne. 1970. Hormones in the trans
lational control of early germination in wheat embryos.
Nature 226:1157-1160.
Clowes, F.A.L. 1961. Apical meristems. Bot. Monog. 2.
Oxford, Blackwell.
Clum, H.H. 1967. Formation of amylase in disks of bean
hypocotyl. Plant Physiol. 42:568-572.
86


87-
Cutter, E.G. 1954. Experimental induction of buds from fern
leaf primordia. Nature 173:440-441.
1956. Experimental and analytical studies of
pteridophytes. XXXIII. The experimental induction of
buds from leaf primordia in Dryopteris aristata Druce.
Ann. Bot. N.S. 20:143-165.
1965. Recent experimental studies of the shoot
apex and shoot morphogenesis. Bot. Rev. 31:7-113.
Datta, A. and S.P. Sen. 1965. The mechanism of action of
plant growth substances: Growth substance stimulation
of amino acid incorporation into nuclear protein.
Biochem. Biophys. Acta 107:352-357.
Deitch, A.D. 1966a. Cytophotometry of nucleic acids. In:
Intro, to Quant. Cytochem.. G.L. Weid, ed. Academic
Press, New York. pp:327-354.
1966b. Cytophotometry of proteins. In: Intro.
to Quant. Cytochem. G.L. Weid, ed. Academic Press,
New York. pp:451-468.
Evans, L.S. and A.R. Berg. 1972. Qualitative histochemr.
istry of the shoot apex of Triticum. Can. J. Bot.
50:241-244.
Evins, W.H. and J.E. Varner. 1972. Hormonal control of
polyribosome formation in barley aleurone layers.
Plant Physiol. 49:348-352.
Fagerlind, F. 1954. The apical embryo- and shoot-meristem
in Gnetum, Ephedra and other gymnosperms. Svensk. Bot.
Tidskr. 48:449-470.
Flax, M.H. and M.H. Himes. 1950. A differential stain for
ribonucleic and desoxyribonucleic acids. Anat.
Record. 108:529.
1952. Microspectrophotometric analysis of meta-
chromatic staining of nucleic acids. Physiol. Zoo.
25:297-311.
Fletcher, R.A. and N.O. Adedipe. 1972. Hormonal regulation
of leaf senescence in intact plants. In: Plant Growth
Substances 1970. D.J. Carr, ed. Springer-Verlag,
Berlin. pp:571-580.
Fosket, D.E. and J.P. Miksche. 1966. A histochemical study
of the seedling shoot apical meristem of Pinus
lambertiana. Amer. J. Bot. 53:694-702.


88
Foster, A.S. 1938. Structure and growth of the shoot apex
in Ginkgo biloba. Torrey Bot. Club Bull. 65:531-556.
Fox, J.E. and C.M. Chen. 1968. Cytokinin incorporation in
to RNA and its possible role in plant growth. In:
Biochem. and Physiol, of Plant Growth Substances.
F. Wightman and G. Setterfield, ed. Runge Press,
Ottawa. pp:777-789.
Gifford, E.M., Jr., and H.B. Tepper. 1962a. Histochemical
and autoradiographic studies of floral induction in
Chenopodium album. Amer. J. Bot. 49:706-714.
1962b. Ontogenetic and histochemical changes in
the vegetative shoot of Chenopodium album. Amer. J.
Bot. 49:902-911.
Griffith, M.M. 1952. The structure and growth of the shoot
apex in Araucaria. Amer. J. Bot. 39:253-263.
Henderson, T.R., C.G. Skinner, and R.E. Eakin. 1962.
Kinetin and kinetin analogues as substrates and
inhibitors of xanthine oxidase. Plant Physiol. 37:
552-555.
Jensen, W.A. 1962. Botanical Histochemistry. Freeman &
Co., San Francisco.
Johnson, M.A. 1951. The shoot apex in gymnosperms.
Phytomorph. 1:188-204.
Kamisaka, S. 1972. Auxin-induced growth of tuber tissue of
Jerusalem artichoke. VII. Effect of cyclic 3',
5'-adenosine monophosphate on the auxin-induced cell
expansion growth. In: Plant Growth Substances 1970.
D.J. Carr, ed. Springer-Verlag, Berlin. pp:654-660.
Key, J.L. 1964. Ribonucleic acid and protein synthesis as
essential processes for cell elongation. Plant
Physiol. 39:365-370.
Key, J.L. and J. Ingle. 1968. RNA metabolism in response
to auxin. In: Biochem. and Physiol, of Plant Growth
Substances. F. Wightman and G. Setterfield, ed.
Runge Press, Ottawa. pp:711-722.
Key, J.L. and J.C. Shannon. 1964. Enhancement by auxin of
ribonucleic acid synthesis in excised soybean hypocotyl
tissue. Plant Physiol. 39:360-364.
Koch, L. 1891. Ueber Bau und Wachsthum der Sprosspitze der
Phanerogamen. I. Die Gymnospermen. Jahrb. f. Wiss.
Bot. 22:491-680.


Lance, A. 1954. Repartition de 1'acide ribonuclique dans
les mristemes apicaux de deux composes. Acad. des
Sci. Compt. Rend. 239:1238-1239.
1957. Recherches cytologeques sur 1'evolution de
quelques mristemes apicaux et sur ses variations
provoques par des traitements photopriodiques. Ann.
Sci. nat. Bot. Ser. XI. 18:91-422.
Mann, J.D., C.E. Steinhart, and S.H. Mudd. 1963. Alkaloids
and plant metabolism. V. The distribution and form
ation of tyramine methylpherase during germination of
barley. J. Biol. Chem. 238:676-681.
Matthysee, A.G. 1969. Interaction of effector substances
with chromosomal receptors. Abst. Int. Bot. Cong.
11th., Seattle. pp:143.
Miller, C.O. 1961. Kinetin and related compounds in plant
growth. Ann. Rev. Plant Physiol. 12:395-408.
Mothes, K. and L. Engelbrecht. 1961. Kinetin-induced
directed transport of substances in excised leaves in
the dark. Phytochem. 1:58-62.
Nitsch, J.P. 1968. Studies on the mode of action of
auxins, cytokinins and gibberellin at the subcellular
level. In: Biochem. and Physiol, of Plant Growth
Substances. F. Wightman and G. Setterfield, ed.
Runge Press, Ottawa. pp:563-580.
Nougarde, A. 1967. Experimental cytology of the shoot
apical cells during vegetative growth and flowering.
Int. Rev. Cyto. 21:203-351.
Nougarde, A., E.M. Gifford, Jr., and P. Rondet. 1965.
Cytohistological studies of the apical meristem of
Amaranthus retroflexus under various photoperiodic
regimes. Bot. Gaz. 126:281-298.
Paranjothy, K. and P.F. Wareing. 1971. The effects of
abscisic acid, kinetin and 5-fluorouracil on ribonucleic
acid and protein synthesis in senescing radish leaf
discs. Planta 99:112-119.
Paulet, P. and J.P. Nitsch. 1959. Stimulation chimique du
bourgeonnement chez Cardamine pratensis L. Bull. Soc.
Bot. France 106:425-441.
Pearse, A.G.E. 1968. Histochemistry: Theoretical and
Applied. Vol. 1., 3rd ed. Little, Brown & Co.,
Boston.


90
Pilet, P.E. 1968. Ln vitro and in vivo auxin and cytokinin
translocation. In: Biochem. and Physiol, of Plant
Growth Substances. F. Wightman and G. Setterfield, ed.
Runge Press, Ottawa. pp:993-1004.
1970. The effect of auxin and abscisic acid on
the catabolism of RNA. J. Exp. Bot. 21:446-451.
Plantefol, L. 1946. Fondements d'une theorie phyllotaxique
nouvelle. I. Historique et criteque. II. La.
phyllotaxie des monocotyledons. Ann. Sci. nat. Bot.
Ser. XI. 7:153-229.
1947a. La phyllotaxie des dikotyldons. IV.
Generalizations et conclusions. Ann. Sci. nat. Bot.
Ser. XI. 8:1-66.
1947b. Helices foliare, point vgtatif et
stele chez les dicotyldones. La notion d'anneau
initial. Rev. gen. Bot. 54:49-80.
1948. La theorie des helices multiples foliaires.
Masson, Pairs.
1950. La phyllotaxie. Anne Biol. 54(Ser. III.
26) : 447-460.
Pollard, C.J. 1970. Initiation of responses in aleurone
layers by gibberellic acid. Biochem. Biophys. Acta
222:501-507.
Popham, R.A. 1951. Principal types of vegetative shoot
apex organization in vascular plants. Ohio J. Sci.
51:249-270.
Popham, R.A. and A.P. Chan. 1950. Zonation in the vegeta
tive stem tip of Chrysanthemum morifolium Bailey.
Amer. J. Bot. 37:476-484.
Poulsen, R. and L. Beevers. 1970. Effects of growth regu
lators on ribonucleic acid metabolism of barley leaf
segments. Plant Physiol. 46:782-785.
Richards, F.J. 1951. Phyllotaxis: Its quantitative
expression and relation to growth in the apex. Phil.
Trans. Roy. Soc. B. 235:509-563.
1956. Spatial and temporal correlations involved
in leaf pattern production in the apex. In: Growth
of Leaves. F.L. Milthorpe, ed. Butterworths Sci.
Publ., London. pp:66-75.


Riding, R.T. and E.M. Gifford, Jr. 1973. Histochemical
changes occurring at the seedling shoot apex of Pinus
radiata. Can. J. Bot. 51:501-512.
Rijven, A.H.G.C. and V. Parkash. 1971. Action of kinetin
on cotyledons of fenugreek. Plant Physiol. 47:59-64.
Romberger, J.A. 1963. Meristems, growth, and development
in woody plants. U.S.D.A. tech Bull. No. 1293.
1966. Developmental biology and the spruce tree.
J. Washington Acad. Sci. 56:69-81.
Schmidt, A. 1924. Histologische Studien an phanerogamen
Vegetationspunkten. Bot. Arch. 8:345-404.
Schoute, J.C. 1913. Beitrage zur Blattstellungslehre.
Rec. Trav. Bot. Neerl. 10:153-339.
1936. Fasciation and dichotomy. Rec. Trav. Bot.
Neerl. 33:649-669.
Schraudolf, H. and J. Reinert. 1959. Interaction of plant
growth regulators in regeneration processes. Nature
184:465-466.
Sinnott, E.W. and C.O. Miller. 1957. Chemical regulation
of growth and organ formation in plant tissues. Symp.
Soc. Exp. Biol. 11:118-131.
Skoog, F. 1954. Substances involved in normal growth and
differentiation of plants. Brookhaven Symp. Biol.
(BNL 258):1-21.
Snedecor, G.W. and W.G. Cochran. 1967. Statistical Methods.
6th. ed. Iowa State Univer. Press, Ames.
Snow, M. and R. Snow. 1931. Experiments on phyllotaxis. I.
The effects of isolating a primordium. Phil. Trans.
Roy. Soc. B. 221:1-43.
1933. Experiments on phyllotaxis. II. The
effect of displacing a primordium. Phil Trans. Roy.
Soc. B. 221:354-400.
1935. Experiments on phyllotaxis. III. Diagonal
splits through decussate apices. Phil. Trans. Roy.
Soc. B. 225:63-94.
1937. Auxin and leaf formation. New Phytol.
36:1-18.


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A CYTOCHEMICAL STUDY OF THE EFFECTS
OF GROWTH REGULATORS ON THE SHOOT
APICAL MERISTEM OF CERTAIN SEED PLANTS
By
RAY JUDSON VARNELL
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
1974

To
Dr. Earnest Ball,
wjkos.«
whcr+s advice, encouragement, and example
many years ago became the sustaining inspiration
which led me to this point in my career.

ACKNOWLEDGMENTS
I wish to acknowledge the guidance, encouragement,
and helpful criticism received from the members of my
Supervisory Committee, and especially that from the late
Dr. R.G. Stanley, and from Dr. I.K. Vasil who supervised
and provided facilities for this study. Acknowledgment
is also made of the help received from Dr. J.W. Brookbank
with the microspectrophotometric work. I gratefully
acknowledge the patience, encouragement, and assistance
provided me by my wife, Emily, who typed the manuscript
and prepared the graphs. Support for this study was
provided by an N.D.E.A., Title IV, fellowship.
in

TABLE OF CONTENTS
ACKNOWLEDGMENTS iii
ABSTRACT vi
INTRODUCTION 1
LITERATURE REVIEW 4
Concepts of Shoot Morphogenesis 4
Metabolism in the Apical Meristem 12
Effects of Chemicals Applied to the Apical Meristem. 16
MATERIALS AND METHODS 18
Plants 18
Treatments 19
Harvesting 22
Staining 24
Microspectrophotometry 28
Analyses 29
RESULTS 37
Lupinus 37
Morphological Analyses of Treatments 37
Cytochemical Analyses of IAA Effects 42
Pinus 57
Morphological Analyses of Treatments 57
Cytochemical Analyses of Kinetin Effects 50
IV

Coleus
71
DISCUSSION 72
Lupinus 72
Pinus 78
APPENDIX 84
LITERATURE CITED 86
BIOGRAPHICAL SKETCH 94
V

Abstract of Dissertation Presented to the Graduate
Council of the University of Florida
in Partial Fulfillment of the Requirements
for the Degree of Doctor of Philosophy
A CYTOCHEMICAL STUDY OF THE EFFECTS
OF GROWTH REGULATORS ON THE SHOOT
APICAL MERISTEM OF CERTAIN SEED PLANTS
By
Ray Judson Varnell
August, 1974
Chairman: Robert G. Stanley
Cochairman: Sherlie H. West
Major Department: Agronomy
Previous studies of seed plants have described whether
auxins applied directly to the shoot apical meristem altered
development. The present study repeated these observations
and extended them to include the effects of a cytokinin and
a gibberellin. Attempts were also made to quantitate some
of the effects of these substances by measuring cytophoto-
metrically changes in ribonucliec acid (RNA), total protein,
and unsaturated lipids in the cytoplasm, and histones in the
nucleus.
Results of this study show that application of 1.5 yg
indolacetic acid (IAA) in a lanolin droplet to the exposed
apical meristem of Lupinus albus seedlings caused: (1)
vi

axillary buds to form closer to the apex than normal, (2)
displacement of primordia formed during the first two plasto
chrons following treatment, and (3) significant increases in
concentration of RNA, protein, unsaturated lipids, and
histones in the meristems. Primordial displacement tended
to be random relative to the site of treatment, which may be
a feature common to dicotyledonous plants exhibiting spiral
phyllotaxis. That IAA conferred initiation site capabilities
to all of the peripheral zone for a short time was indicated
by (2) and (3) above and by decreases in concentrations of
the observed compounds toward control levels after the
second plastochron following treatment. Effects of IAA
on RNA and histones suggest that nucleic acid metabolism,
and possibly gene expression, was involved in the response.
Kinetin and gibberellic acid (GA) had no apparent morpho¬
genetic effect on Lupinus meristems.
Treating shoot apical meristems of Pinus elliottii
seedlings with kinetin resulted in: (1) bud scale formation
and the onset of dormancy, and (2) changes in concentrations
of the cytochemically determined compounds listed above.
IAA and GA produced no morphogenetic effects. Interactions
of kinetin with nuclear-based events and with membranes in
the cytoplasm were suggested by levels of histones and RNA
in treated meristems exceeding control levels through the
sixth day following treatment, during which time unsaturated
lipids sharply fell below control values. Rapid declines

in RNA, protein, and histones by day eight coincided with the
first appearance of bud scales, signaling the onset of
dormancy in kinetin-treated meristems.
It was observed that the central mother cell zone is
not metabolically quiescent in vegetative shoot meristems
of Lupinus and Pinus seedlings.
viii

INTRODUCTION
Because most of the growth and development of the
shoot is initiated at or regulated by the shoot apex,
the importance of the shoot apex to the plant, and to the
plant scientist, can hardly be over-emphasized. The shoot
apex consists of an apical meristem, a subapical meristem,
and a region of maturation. It is within the apical meristem
that lateral organs and, to a limited extent, shoot form
arise. The initial orderly disposition of organs around
the periphery of the shoot, and of tissues within the shoot,
is due to localized centers of growth and differentiation
within the apical meristem. The apical meristem, in addition,
contains groups or zones of cells which differ from one
another in the plane of cell division, affinity for various
stains and dyes, mitotic rate, extent of vacuolization, and
nuclear as well as cell sizes and volume ratio. The factors
maintaining the developmental and cytological heterogeneity
within the apical meristem are unknown.
Information about these unknown factors may be
obtained, however, by studying the effects of treatments
1

2
which disrupt the normal course of development in the
apical roeristem. One kind of treatment for disrupting
normal development involves applying substances directly
on the surface of the meristem. The rationale is that
some of the applied substance penetrates the apical
meristem and disrupts the orderly disposition of organs
and/or tissues, and inferences are drawn concerning the
internal regulatory factors from the nature of the applied
substance and its effects.
Effects of substances applied directly to the intact,
growing apical meristem have been studied relatively
little. Lack of such experimental information is undoubt¬
edly due to the small size of the meristem, the difficulty
of exposing and working with the intact meristem without
injuring it, and the complex interactions between the
apical meristem and the rest of the plant. But the
advantages of experimenting with the intact apical meristem
should not be ignored. Unlike excised apical meristems,
intact ones have not suffered the trauma of being cut
off from their natural life support systems and of wound
and healing reactions. Physiological parameters, such
as chemical contents, gradients, and fluxes, that are
characteristic of the normally developing apical meristem,
probably are accurately represented only in the intact
meristem. Moreover, although it may be easier in some

3
respects to experiment with excised shoot tips, the
results must eventually be verified with intact meristems.
Previous studies have described whether applied
auxins altered meristem development. The present study
repeated these observations and extended them to include
the effects of a cytokinin and a gibberellin. Attempts
were also made to quantitate some of the effects of these
substances by measuring changes in ribonucleic acid,
total protein, and unsaturated lipids in the cytoplasm,
and histones in the nucleus. These data provide information
about the internal factors regulating growth and cytological
heterogeneity within the apical meristem in control and
experimentally treated plants.

LITERATURE REVIEW
Concepts of Shoot Morphogenesis
The two most widely accepted concepts pertaining
to morphogenesis at the shoot apex are Wardlaw's (1957,
1960) and Plantefol's (1946, 1947a, 1947b, 1948) interpreta¬
tions of apical organization. According to Wardlaw (1965a,
1965b), the apex is a dynamic geometrical reaction system,
"...of integrated and interrelated ..." regions. The
regions, forming a morphologically distinguishable vertical
series based partially on anatomical studies by Schoute
(1936), are: (1) the distal region, (2) the subdistal
region, (3) the organogenic region, (4) the subapical
region, and (5) the maturation region (Fig. 1A). The first
three regions comprise the apical meristem in which
cytological and/or histological zonation may be evident.
The cytological zones, which were first described
by Foster (1938) for Ginkgo biloba, but which are generally
applicable to all seed-bearing plants, are:(1) the apical
initial cell zone, (2) the central mother cell zone,
(3) the peripheral zone, and (4) the rib meristem (Fig. IB).
4

Fig. 1
A. Morphological regions in longitudinal section of
the shoot apex of Podocarpus macrophyllus,
stained with safranin and fast green. X300.
B. Cytological zonation in longitudinal section of
the shoot apex of Lupinus albus, stained with
Azure B. X200.
C. Histological zonation in longitudinal section of
the shoot apex of Quercus spp., stained with
safranin and fast green. X550.
D = distal region; SD = subdistal region; 0 =
organogenic region; SA = subapical region;
M = maturation region; I = initial cell zone;
Pz = peripheral zone; R = rib meristem; T =
tunica layer; C = corpus; P = primordium ;
L = young leaf; Cz = central mother cell zone.

6

7
A transition zone, described by Foster (1938) and located
at the lateral and basal margins of the central mother
cell zone, is not evident in many apices and is often
omitted from discussion of cytological zonation (Romberger
1963) .
Apical meristems of angiosperms (Clowes 1961), and
certain gymnosperms (Johnson 1951; Griffith 1952; Fagerlind
1954), typically exhibit histological zonation in the
form of (1) one or more outer layers of anticlinally
dividing cells, and (2) an inner mass of cells with randomly
oriented mitoses (Fig. lc). The former is called the
tunica if it is composed exclusively of anticlinally
dividing cells (Popham 1951; Clowes 1961), or the mantle if
an occasional periclinal division occurs (Popham and Chan
1950). The inner mass of cells underlying the tunica/
mantle is called the corpus. This nomenclature of histo¬
logical zonation is based on Schmidt's (1924) interpretation
of growth in the apical meristem. Although Wardlaw
(1965b) recognized the existence of a tunica/mantle
layer(s), his concept of growth in the apical meristem
differed radically from Schmidt's (1924).
Wardlaw (1965a) proposed: (1) that differential growth
rates throughout the apical meristem determine the shape
and size of the apex, and (2) that localized active growth
in the p^i neral zone determines the position of new
primordia. The growth rate declines steadily from the
subapical region to the summit of the distal region and to

8
the center of the shoot axis , the rate of decline being faster
for low or flat apices, and slower for conical or cylindrical
apices.
Formation of a primordium, in Wardlaw's (1965a)
view, results from the comparatively active growth of a
small number of superficial and underlying cells at a
localized locus or site, the growth center, in the peripheral
zone in the subdistal region. This growth center, according
to Schoute (1913), inhibits the formation of other similar
growth centers in the immediate vicinity, and the apical
cells inhibit the formation of growth centers in their
immediate vicinity. Hence, primordia occur (1) between
older ones where sufficient space is available, and (2)
at some characteristic distance from the summit of the
apex. Support for this concept of primordial inception
comes from exhaustive studies of phyllotaxis (Snow and Snow
1931, 1933, 1935; Richards 1951, 1956), microsurgical experi¬
ments (Wardlaw 1949, 1950, 1955, 1956; Wetmore and Wardlaw
1951; Cutter 1954, 1956; Wardlaw and Cutter 1956), and theo¬
retical analyses of endogenous circadian rhythms (Biinning
1952) and of diffusion-reaction systems (Turing 1952).
Wardlaw's (1965a, 1965b) concept is founded on the
supposition that distribution of growth and morphological
pattern in the apical meristem are expressions of inter¬
related physiological-genetical processes. He further
supposes that each region and zone in the shoot apex has

9
its own characteristic metabolism and concentration gradients
of incoming nutrients and outgoing metabolic products, and
that the same considerations apply to the growth centers
and nascent primordia.
In contrast, Plantefolls (1946, 1947a, 1947b, 1948)
concept of apical organization and morphogenesis differs
from Wardlaw's (1957, 1960) in (1) the function of the
cytological zones in the apical meristem, and (2) the
regulation of primordium inception. According to Plantefol
(1946, 1947a, 1947b, 1948), during vegetative growth the
most distal region of the apical meristem as well as the
central mother cell zone, together defined as the "méristéme
d'áttente" (Bersillon 1951), are more or less completely
inactive and contribute little or nothing to histogenesis
or organogenesis. Accordingly, it is the peripheral zone,
the so-called "anneau initial" (Plantefol 1947b), where
initial vegetative growth and development take place.
Growth and histogenesis also occur in the "méristéme
médullaire," which is the same as the rib meristem.
The concept of an inactive "méristéme d'attente1 was
based primarily on the absence or infrequent evidence of
cell divisions in this zone. Additional criteria are low
concentrations of cytoplasmic ribonucleic acid and protein,
low nucleocytoplasmic ratio, comparatively large vacuoles,
and few mitochondria; all characteristics of a more differ¬
entiated and less active state than that observed in the

10
highly meristematic peripheral zone. That these cytological
characteristics exist in the apical meristem of many plants
has been well documented (Lance 1957; Gifford and Tepper
1962a, 1962b; Nougarede et ad. 1965; Steeves et al. 1969)
and has been conceded by Wardlaw (1965b). What these cyto¬
logical characteristics mean in the morphogenetic, physio¬
logical, or metabolic sense is not known.
Regulation of primordial inception, in Plantefol's
(1950) view, is achieved by a small number of foliar helices
which spiral around the shoot axis. Each helix has a gener¬
ative center located in the peripheral zone (the "anneau
initial"), and the activities of the several generative
centers are regulated by an organizer. What instigates
the leaf generating center, their helical course around
the shoot axis, and the apical organizer and how it exerts
its effects have never been satisfactorily explained.
The salient points on which these two concepts of
apical organization agree regarding morphogenesis at the
shoot apex are: (1) the cytological and histological zones
of the apical meristem evidently have different functions,
and (2) the development and growth in the normal vegetative
apex rigidly follows a pattern characteristic for the species
The peripheral zone is the site of primordial initiation
and, in some rapidly growing apices (Wetmore 1943; Wardlaw
1956; Lance 1957), the site of inception of provascular
tissue. Thus, the peripheral zone is an organogenic region,

11
and in some instances a histogenic region also. The rib
meristem characteristically gives rise to the pith and is,
therefore, exclusively histogenic. In contrast, the initial
cell zone and central mother cell zone form neither organs
nor tissues as such, but apparently are comparatively active
metabolically. The latter point will be discussed below.
As Wardlaw (1965b) pointed out, the patternized
development of regularly spaced primordia in a characteristic
phyllotactic sequence is the primary organogenic activity
of the apical meristem. Furthermore, apical meristems
of different species may be large or small in absolute
size, or in relative size to primordia, or to the rest of
the shoot apex, and they may be typically conical, parabolic,
cylindrical, low and flat, or concave in shape. Whatever
its size, shape, or sequence of primordial initiation,
the apical meristem develops along a characteristic pattern
determined by the distribution of growth. Although no one
has questioned this interpretation of apical morphogenesis,
the physiological-genetical events which determine and
evidently regulate the morphogenic pattern at the shoot
apex are unknown.

12
Metabolism in the Apical Meristem
Metabolism within the apical meristem has not been
studied directly because of the small size and inaccessabil-
ity of the structure. It has been only in the last few
years that data have become available from which inferences
could be drawn. These data consist of cytochemical
localization, and quantification in some cases, of metaboli-
cally active substances such as nucleic acids, proteins,
specific enzymes, carbohydrates, and lipids. The distri¬
bution of nucleic acids and nucleohistone in the apical
meristem, for example, has been studied extensively recently
because of their presumptive role in gene activated
control of development. The initial discoveries that
the nucleic acids and nucleohistones were more concentrated
in the peripheral zone than in the initial cell and
central mother cell zones (Wardlaw 1965a, 1965b; Nougaréde
1967) have stood the test of time. Seasonal changes
in the absolute amounts and in relative ratios of these
substances within the apical meristem have been tentatively
correlated with bud break, shoot elongation, bud development,
and dormancy (Cecich et_ al. 1972; Varnell and Vasil 1972).
Although cells in the central mother cell zone and initial
cell zone may not divide often in many species, they are
not arrested in any particular part of the cell cycle but
rather cycle comparatively slowly (Steeves et al. 1969).

13
The data of Steeves et_ a^. (1969) suggest a longer
phase than phase for cells in these zones, but what this
means metabolically is not known.
The greater amounts of ribonucleic acid (RNA) in the
peripheral zone than in the other zones is the basis for the
differential basophilia within the apical meristem that
led Koch as early as 1891 to visualize cytological differ¬
ences within the shoot apex. The greater concentration of
RNA in the peripheral zone is associated with the greater
mitotic activity of this zone. The highest concentrations
of RNA are in the rapidly dividing cells of the growth
centers and of the newly initiated primordia (Lance 19 54;
Gifford and Tepper 1962a, 1962b). Hence, the RNA content
and the physiological activity of a cell appear to be
associated.
Nougaréde (1967) suggested that the high concentration
of RNA in the peripheral zone is probably connected with a
high requirement for protein synthesis to support the
catalytic activities of rapidly dividing cells. While
Nougaréde's (1967) suggestion seems reasonable, cytochemical
determinations of total protein in the apical meristem
do not support her. That total protein is rather evenly
distributed throughout the apical meristem has been
documented by Cecich et al. (1972), Gifford and Tepper
(1962a, 1962b), and Riding and Gifford (1973). If
V

14
Nougaréde's (1967) suggestion is modified to correlate
RNA concentration with certain proteins, especially
particular enzymes, rather than total protein, then
substantiating evidence is present. Peroxidase activity
and nucleohistone content, for example, closely parallel
RNA concentration in the peripheral zone (Vanden Born 1963;
Van Fleet 1959; Riding and Gifford 1973; Cecich et al.
1972). Nonspecific esterase activity and sulfhydryl-
containing proteins, while present throughout the apical
meristem, are most evident in the peripheral zone
(Vanden Born 1963; Fosket and Miksche 1966).
Negative correlations involving RNA distribution
in the apical meristem are also known. Succinate dehydro¬
genase, for instance, either shows uniform activity through¬
out the apex (Vanden Born 1963) , or is more evident in the
initial cell and central mother cell zones than in the
peripheral zone (Fosket and Miksche 1966; Evans and Berg
1972; Riding and Gifford 1973). Similarly, when storage
products, i.e., lipid bodies and starch grains, occur in
the apical meristem, they are almost always found only
in the initial cell and central mother cell zones and the
rib meristem (Gifford and Tepper 1962a, 1962b; West and
Gunckel 1968; Riding and Gifford 1973; Varnell and Vasil
1972) and not in the peripheral zone.
What these correlations mean in physiological or
metabolic terms relative to morphogenetic events has not

15
been determined, but some tentative conclusions can be
formulated. The presence of succinate dehydrogenase and
cytochrome oxidase (Thielke 1965) in the central mother
cell and initial cell zones, associated with a low mitotic
rate and occasionally with storage products, suggests
that these zones have high rates of respiration and
interconversion of metabolites, and limited deposition
of metabolic products. Thus, it follows that the central
mother cell zone and the initial cell zone must export
many substances to sites of deposition. The site of
deposition nearest the central mother cell and initial
cell zones is the peripheral zone, and especially the
growth centers and nascent primordia where cell and
organelle multiplication proceed rapidly. Moreover,
localization of peroxidase, esterase, and phosphatase
activity in the peripheral zone and particularly at sites
of primordial initiation (Vanden Born 1963; Evans and
Berg 1972; Fosket and Miksche 1966) indicates manifold
utilization of substrates in biosynthesis of cellular
material. The foregoing postulate is an extension of a
hypothesis advanced by Sunderland et al. (1956) before the
distribution of enzymes in the apical meristem was known.
The distribution of growth regulators within the apical
meristem is unknown

16
Effects of Chemicals Applied to the Apical Meristem
To date, application of chemicals directly to the
shoot apex of seed plants has been carried out on only
two occasions. First, Snow and Snow (1937) applied
0.05% heteroauxin, presumably indole-3-acetic acid, in
lanolin to the exposed apices of Lupinus and Epilobium.
Examination of treated meristems approximately two to
three weeks later revealed enlargement and connation of
primordial bases, and alteration of normal phyllotaxis.
Phyllotaxis on Epilobium changed from the normally decussate
type to spiral. Changes in phyllotaxis were attributed
to displacement of growth centers and of existing primordia,
presumably by asymmetric growth. Displacement usually
was in the direction toward the site of application.
Second, Ball (1944) applied a variety of auxins,
including indole-3-acetic acid, to the shoot apex of
Tropaeolum. All of the compounds tested produced essentially
similar responses. A 1% concentration of indole-3-acetic
acid produced the greatest alteration of morphogenesis
at the apex without causing mortality. As with lupine
and Epilobium (Snow and Snow 1937), affected primordia
of Tropaeolum exhibited enlarged and connate bases.
Phyllotaxis of Tropaeolum was altered erratically for a
time but eventually returned to normal in surviving
plants. In addition, the more detailed observations by

17
Ball (1944) revealed (1) a reduction in the number of
tunica layers from two or three for normal meristems to
one for treated apices, and (2) abnormal formation of axil¬
lary buds and tissue hypertrophy in the organogenic region
of the Tropaeolum apex. Neither Snow and Snow (1937) nor
Ball (1944), however, included cytochemical analyses in
their studies. Thus, the question of how growth substances
affect metabolism and hence morphogenesis in the apical
meristem remains unanswered.
Experiments described in the following sections
were designed to answer some of these questions.

MATERIALS AND METHODS
Plants
Seedlings of lupine (Lupinus albus L.) and pine (Pinus
elliottii Engelm. var. elliottii) were grown in vermiculite
in a Percival reach-in incubator, model 1-35 LVL, maintained
at 26°C day- and 18°C night-temperature, and 16-hour
photoperiod with 3500 foot-candles (37,800 lux) of
fluorescent illumination. Cuttings of coleus (Coleus
blumei Benth.) were rooted in water, without benefit of
rooting hormone, and subsequently grown in vermiculite
on the laboratory bench with supplemental lighting of 16-
hour photoperiod and 4000 foot-candles (43,200 lux) of
fluorescent illumination augmented by 1000 foot-candles
(10,800 lux) of incandescent illumination.
Experiments were conducted with lupine seedlings be¬
tween the ages of 14 and 40 days, with pine seedlings be¬
tween the ages of four and ten weeks, and with coleus plants
after they formed strong root systems. Coleus remained suit¬
able for use for about 75 days, although the plastochron
lengthened considerably during this time. A plastochron is
the interval between the appearance of successive primordia
on a meristem.
18

19
Treatments
Three growth regulators, indole-3-acetic acid (IAA),
6-furfurylaminopurine (kinetin), and gibberellin-3 (GA),
were applied separately to apical meristems in the form of
paste-like emulsions. Pastes were prepared by emulsify¬
ing 1% (w/w) solubilized growth regulator in anhydrous
lanolin in a stainless steel micro cup attachment of a
Virtis "23" hi-speed homogenizer. Pure ethanol was the
solvent for IAA and GA; kinetin was dissolved in acidified
water. A minimum of solvent was used in each case. Pastes
were stored at 4°C. Selection of the 1% concentration
of each growth regulator was based on the concentration
of a variety of auxin pastes which caused optimal disruption
of normal morphogenesis in Tropaeolum meristems (Ball
1944). Using each growth regulator at the same concentration,
-2
approximately 5 x 10 M, permitted direct comparisons
of their effects.
Pastes were applied to meristem surfaces as small drops,
one drop per meristem, with an eyelash cemented to a wooden
applicator. An eyelash applicator was made for each kind of
paste and labelled to avoid using it in the other pastes.
Drops were applied manually to meristems magnified 32X
in a Leitz dissecting microscope with head and stage
reversed on the base. Plants were held securely in the
microscope field by adjustable forceps taped to a stick
clamped to a ringstand. Each meristem was exposed by
pushing the overlying tips of primordia out of the way

20
with a dissecting needle. Drop size, controlled by visual
observation at about 25X, ranged from approximately 0.1 to
0.2 X". Thus, each treated meristem was confronted with
about 1.5 yg of hormone.
Growth regulator was applied to lupine meristems at
the site where the next primordium was expected to arise,
the so-called 1^ site. This site was determined by visually
projecting the phyllotactic spiral around the apical meristem
through an arc of 136° beyond the last-formed primordium.
The arc of 136° was based on the average angle of divergence
between successive primordia in lupine, as determined by
Snow and Snow (1931). Complexities of primordial initiation
in the pine meristem discouraged attempts to place growth
regulator on preassigned sites, and so paste was simply
placed on the summit of the meristem. Placement of growth
regulator in coleus was at either of the two 1^ sites or on
the summit of the meristem. The 1^ sites in coleus, a decus¬
sate species, are located 90° around the apical meristem from
each of the last-formed primordia.
A group of five to seven plants of the same species,
treated with the same growth regulator, and designated
for the same analysis constituted an experiment. Most
experiments were conducted over a 10-day period, with har¬
vesting every second day. Other experiments lasted 12 to 14
days, with harvesting every third or every fourth day. Each

21
experiment was replicated three times.
Untreated meristems were included in the initial exper¬
iments, in which RNA and nucleohistone were determined.
Untreated meristems were not included in subsequent experi¬
ments, in which protein and unsaturated lipids were deter¬
mined, because the untreated controls added little informa¬
tion as the paramount comparison was between lanolin and
growth regulator treatments.

22
Harvesting
Harvesting consisted of severing the shoot apex from
the plant, dissecting away all unnecessary tissue, mounting
the terminal millimeter of apex on a thin, 3.0 mm diameter,
aluminum disc, and freezing the apex and disc instantly
in Freon-22 chilled in liquid nitrogen. The aluminum
disc greatly facilitated handling of the frozen apex and
alignment of the apex for sectioning.
Frozen apices were mounted in chilled Cryoform
(Damon/IEC) or Tissue-tek O.C.T. Compound (Ames) and trans¬
versely sectioned at 10 u in an International Equipment
Company Microtome-Cryostat, model CTF. Serial sections
were picked up, one at a time, on 0.75-mm-thick glass
microscope slides kept at above-freezing temperature.
All the sections for each apex were mounted on the same
slide and subsequently treated alike. Sectioning of each
apex proceeded well into the region of maturation.
Immediately after sectioning, tissues were killed
and fixed to minimize degradation and movement of key
compounds. Based on Swift's (1966) recommendation,
ribonucleic acid (RNA) was fixed in Carnoy's ethanol-
acetic acid fluid (Jensen 1962). Proteins were also
fixed in Carnoy's fluid, because of the rapid penetration
and action of this fluid on plant proteins. Unsaturated
lipids were immobilized, as recommended by Baker (1946),

23
by treating the sections with formalin-calcium (Jensen
1962) for 30 minutes. After fixation, sections were
washed briefly in distilled water and air dried.

24
Staining
Sections designated for RNA determinations were acet-
ylated, then stained with 0.1% Azure B in citric acid
buffer at pH 4.0 at 55°C for two hours (Jensen 1962).
After staining, sections were thoroughly washed in distilled
water, destained in tertiary butyl alcohol (TBA) for
30 minutes, transferred to fresh TBA overnight, passed
through two changes of xylol, and mounted in Permount.
Number one cover slips were used throughout the study.
Acetylation of lupine and pine sections significantly
reduced tissue affinity for Azure B. Acetylation of
Carnoy-fixed lupine sections with 100% acetic anhydride
at 55°C for four hours reduced Azure B staining by 38%.
Staining of pine sections was reduced 23% by acetylation
with 10% acetic anhydride in pyridine at room temperature
for four hours, and 28% by heated 100% acetic anhydride.
Acetylation did not alter the wavelength at which maximum
optical density was obtained, namely 590 nm, for the
Azure B-RNA complex in either lupine or pine tissues.
Acetylation blocks carboxyl activity of amino acids,
and since free amino acids are not retained, the only
candidates left in section for acetylation to affect are
proteins. Thus, despite Flax and Himes' (1950, 1952)
contention, echoed by Jensen (1962), that Azure B staining
of proteins at pH 4.0 is negligible, lupine and pine

25
tissues evidently contain proteinaceous carboxyl groups
which retain their activity even at pH 4.0.
Specificity of Azure B for RNA in the material
studied was confirmed by extraction procedures. Complete
extraction of lupine RNA was accomplished in 0.5 M per¬
chloric acid at 70°C for 30 minutes. Optical density
of the Azure B-RNA complex was reduced 21% in pine sections
following extraction with 0.1% aqueous RNAase, at pH 6.8,
at 35°C, for 90 minutes. Longer incubation in enzyme
would be expected to further reduce stain intensity.
Sections designated for protein determinations were
stained in 1% naphthol yellow S (NYS) dissolved in 1%
acetic acid for 15 minutes, rinsed in distilled water,
and destained overnight in fresh 1% acetic acid, in accord¬
ance with the procedure developed by Deitch (1966b).
After destaining, sections were passed through two changes
of TBA, two changes of xylol, and mounted in Permount.
Specificity of NYS for protein was determined by
blocking and enzymic extraction procedures. Blocking
was partially accomplished in pine tissues by acetylation
in heated 100% acetic anhydride, as described above;
optical density of NYS-stained sections was reduced 27%.
Less vigorous acetylation, in 10% acetic anhydride in
pyridine at room temterature, did not reduce affinity of
pine tissue for NYS.
Enzymic extraction of protein reduced NYS staining

26
of both pine and lupine tissues. Protease, at 1% concen¬
tration, in 0.2 M cacodylate buffer, at pH 7.2, at 35°C,
for 90 minutes, reduced protein staining by 50% in pine
tissues and 70% in lupine tissues.
Unsaturated lipids were colored black by exposure
to osmium tetroxide fumes for one hour, followed by washing
for 15 minutes in distilled water, and mounting in 80%
Karo brand corn syrup, in accordance with Cain (1950).
Specificity of osmium for unsaturated lipids in pine
sections was verified by extraction with pyridine at 27°C
for 30 minutes, followed by fresh pyridine at 60°C for 24
hours. Solvent extraction reduced tissue affinity for
osmium by 75% in pine sections. Pyridine extraction of
lupine tissues did not reduce their affinity for osmium.
Why pyridine occasionally fails to extract lipids is
unknown, but in such cases extraction with an alcoholic
solution containing ether or chloroform is usually successful
(Pearse 1968). Extracting lupine tissues with alcohol-
ether (Jensen 1962) reduced affinity for osmium by 62% in the
peripheral zone and 58% in the central mother cell zone.
Sections selected for histone determinations were
chosen from those in which RNA had been determined. The
procedure used was Deitch's (1966a) modification of Alfert
and Geschwind (1953). Cover slips were removed in xylol,
and Azure B was dissolved in 70% ethanol containing 1%
trichloroacetic acid (TCA). Nucleic acids were removed

27
in 5% TCA at 90°C for 20 minutes. Sections were rinsed
in cold 5% TCA, followed by distilled water, and stained
in 0.1% aqueous fast green FCF in 0.005 M phosphate
buffer at pH 8.0, at room temperature, for 30 minutes.
After staining, sections were thoroughly rinsed in distilled
water, dehydrated in an ethanol series, passed into
xylol, and mounted in Permount.

28
Microspectrophotometry
Microspectrophotometric determinations were made
with a Reichert Zetopan microscope equipped with a Binolux
II light source, an achromatic, aplanatic 6-lens condensor
with 1.35 N.A., an achromatic 25X objective, and a complete
microphotometer attachment including a 12.5X, coated,
plane eyepiece and sliding interference wedge filter
with 11 to 14 nm half-width transmission band. Except
for lipids, determinations were made at maximum optical
densities; thus RNA at 590 nm, total protein at 430 nm,
and histone at 635 nm. Since osmium absorbs visible
light rather uniformly, regardless of wave length, un¬
saturated lipids were determined from optical densities
measured at the midpoint (550nm) of the range of maximum
light transmission of the interference wedge filter.
Relative amounts of cytoplasmic RNA, total protein, un¬
saturated lipid, and histone were determined from extinction
values displayed on a deflection meter.

29
Analyses
Treatment effects were evaluated by examining each
treated meristem as it was harvested, as well as its tissues
in section, for any visible, qualitative change from
normal development. Expected changes in morphogenesis
included those listed by Wardlaw (1965b), i.e., death
of the apex, bud initiation on the flank of the apex,
(usually preceded by death of the cells at the summit of
the apex), and altered differentiation of primordia.
Existence of nascent axillary buds was determined by
examination of transversely sectioned shoot tips.
Plastochron length in lupine and coleus was determined
by the difference in number of foliar primordia plus an
assessment of the stage of intra-plastochronic development
observed at the beginning and end of a given interval.
A reference primordiumwas marked with india ink at the
beginning of the interval. Intra-plastochronic development
was divided into early, middle, and late stages of presumably
equal duration, as judged by the extent of development
of the last-formed primordium.
Assessment of treatment effects on primordium
displacement was based on the assumption that only certain
angles of divergence would be affected. Snow and Snow
(1937) reported (1) that only the angles formed by
primordia adjacent to the treatment site were affected,
and (2) that affected primordia tended to be displaced

30
toward the treatment site. The latter finding would result
in affected angles being smaller than average. It follows
from (1) above that the angles of divergence expected to be
affected by treatment of a preassigned site on the apical
meristem would be a function of the number of elapsed plasto-
chrons since treatment. A list was accordingly prepared of
the angles expected to be affected according to the number
of plastochrons since treatment at 1^ (Table 1) .
In the case of one elapsed plastochron, 1^ would have
become P^, and would have become P2 (Table 1). It follows
from (2) above that, if treatment at 1^ was effective, after
one elapsed plastochron, P2 would be displaced towards P^,
resulting in a smaller-than-average angle of divergence
P^-P2* Displacement of P2 towards P^ would further result
in P2 being further away from P^ and a larger-than-average
angle P2~P2.
A similar rationale applied to the situation of more
than one elapsed plastochron. The only added factor was
that I2 may have been displaced if treatment at 1^ was
effective. Then, after two or more elapsed plastochrons
and after I2 had become an observable primordium, the angle
formed by that primordium with the next older one would be
smaller than average. According to Snow and Snow (1937),
treatment at 1^ would not affect I^, and 1^ would be located
136° of arc around the meristem from I2, regardless of
whether I2 was displaced.

TABLE 1
Angles of divergence expected to be affected according
to the number of elapsed plastochrons since treatment
at I1
Number of
elapsed
plastochrons
Stage of
development of
initiation sites
Angles of divergence affected
and expected direction of change*
Angle Direction of change
1
¿ x**
< x
2
¿ x
< X
< X
OJ

Table 1
continued
Number of
elapsed
plastochrons
Stage of Angles of divergence affected
development of and expected direction of change *
initiation sites
Angle Direction of change
3
Z1
P3
P4
-P5
X**
*2
-*â– 
P2
P3
"P4
<
X
*3
P1
P2
"P3
<
X
Pn
-P„
=
X
1
2
4
Z1
P4
P5
-P6
>
X
Z2
->
P3
P4
"P5
<
X
Z3
P2
P3
-P4
<
X
P2
-P3
X
* < means less than; > means greater than or equal.
** x is the average angle of divergence for untreated shoot tips.

33
Thus, angles of divergence between primordia adjacent
to the treatment site were expected to be smaller than
average. The angle formed by the older adjacent primordium
and its older neighbor, angle in the case of one
elapsed plastochron (Table 1), was expected to be greater
than or equal to the average. All other angles of
divergence on treated meristems should approximate the
average. Angles of divergence expected to be affected
by treatment compared with those expected not to be
affected on treated meristems and with angles from untreated
meristems, using statistical methods,were the basis
for assessing treatment effects on primordium displacement.
Angles of divergence were measured with a protractor
after connecting with straight lines the mid-point of the shoot
axis with the mid-point of each primordium in transverse
section. Mid-points were plotted with approximately
100X enlargement of the image through a camera lucida
attachment on a Wild compound microscope.
Cytophotometric determinations were taken from
specific sample points in certain lupine and pine sections.
Only the sections immediately above the point of insertion
of the last-formed primordium and the immediately subjacent
section were sampled. Sample points in each lupine section
were the three presumptive initiation sites, referred
to as 1^, I2, and I^/ the three areas of intervening
peripheral zone between the initiation sites, and three

34
randomly chosen points at the center of the central
mother cell zone, for a total of nine from each section
(Fig. 2). Uniformity in locating sample points of initiation
sites in section, since initiation sites might be displaced
by treatment effects, was accomplished by visually project¬
ing the phyllotactic spiral through an arc of approximately
136° beyond the last-formed primordium to the presumptive
site of I , thence continuing through another arc of 136°
to I2, and similarly on to I3.
Initiation sites were numbered in accordance with the
procedure established in the literature (Snow and Snow 1931).
The numerical order corresponds to the order in which the
sites were created; i.e., 1^ arose first, I2 second, and I3
third. Existing primordia were numbered in reverse order of
formation; i.e., the youngest as P^, the next as P2, the
next as P3, etc.
Sample points in each pine section consisted of five
points distributed as evenly as possible throughout the
peripheral zone and five points clustered in the central
mother cell zone.
Quantitative data, such as changes in phyllotactic
angles and the cytophotometric data, were analyzed by
conventional statistical methods for comparisons among two
or more means (Snedecor and Cochran 1967) (ref. Appendix.)
All stated differences were significant at the 95% level of
confidence.

Fig. 2 Location of cytophotometric sample points in
transverse section of apical meristems of
Lupinus albus, stained with Azure B. X 200.
1= sample points in initiation sites; Fz =
sample points in intervening peripheral zone
C= sample points in central mother cell zone
P'l' P2' P3' leaf primordia, numbered
in order from youngest to oldest.

36

RESULTS
Lupinus
Morphological Analyses of Treatments
Morphogenetic effects of growth regulators applied
to the apical meristem of lupine shoots were evaluated
by determining whether changes occurred in (1) the distance
from the shoot tip to the last-formed axillary bud,
and (2) the displacement of primordia. Analysis of
treatment effect on plastochron length was attempted.
Axillary buds appeared closer to the shoot tip only
in IAA-treated meristems (Table 2). The distance, expressed
in numbers of the intervening nodes, averaged 5.2 for IAA-
treated meristems, as compared to 8.4 for untreated
meristems and 9.0 for lanolin-treated ones. Kinetin and
GA treatments did not cause axillary bud formation to
occur significantly closer to the shoot tip than untreated
controls.
Length of the plastochron was variable. The plastochron
lengthened in successive crops as the seed from which
the crops were grown aged. The plastochron lengthened
within crops as the plants aged. Average plastochron
length by experiments ranged from 1.9 days to 5.9 days.

38
TABLE 2
Summary of treatment effects on morphology
of the shoot tip of Lupinus
Number of nodes from
apex to last-formed Angle (degrees)
axillary bud of divergence*
Treatments
Average**
Sx
Average**
Sx
Untreated
8.4
±0.9
134.5
±2.0
Lanolin only
9.0
±0.3
134.3
±3.0
IAA
5.2
±0.4
139.0
±7.2
Kinetin
7.6
±0.3
138.6
±2.3
GA
7.4
±0.4
137.6
±3.0
* Computed
only for
those
angles expected
to be affected
by treatment (ref. Table 1).
** Averaged over harvest dates and replications.

39
Treatment effects, when superimposed on the variation
caused by progressive ageing of seeds and plants, could
not be realistically evaluated. Average plastochron
length for each experiment was taken into account in the
analysis of treatment effects on displacement of primordia.
Primordia were displaced only by treatment with IAA.
The average angle of divergence for affected angles in the
IAA treatment did not differ significantly from that for
untreated meristems or lanolin controls, but variance,
indicated by the sample mean standard error, was greater
in the IAA treatment than in the other treatments and
controls (Table 2). The average and the variance for
angles not expected to be affected in IAA-treated meristems
did not differ from control values.
In the case of one meristem harvested after the first
elapsed plastochron following treatment with IAA, the angle
of divergence which had formed between and P2 was 90°
(Fig. 3), as compared to an average of 134.5° for untreated
meristems (Table 2) and 139° for unaffected angles in
treated meristems. The angle P^-P2 a^ter one elapsed
plastochron was expected to be smaller than average (Table 1).
However, in this meristem, contrary to Snow and Snow's
(1937) hypothesis that pirmordia adjacent to the treatment
site are displaced, the initiation site 1^ evidently was
displaced instead. P^ must not have been displaced
significantly, because when harvested the angle P2~P2

Fig. 3 Displacement of towards P2 following treatment
with 1.5 yg IAA in transection of apical meristem
of Lupinus albus, stained with alkaline fast
green. X 300.
A= apical meristem; P^, P2, P^, .... = leaf
primordia, numbered in sequence from youngest
to oldest.


42
was only 151° which was less than two standard deviations
from the average. If had been displaced, the angle
P2~l?3 would have been significantly greater than the average
(Table 1).
Another meristem, harvested eight days after treatment
with IAA, showed displaced 282° from P2 (Fig. 4). In this
case, enough time elapsed since treatment for ^ to develop
into P^, and 1^ into P2 (plastochron 2, Table 1). Hence,
I2 was displaced around the meristem away from the point
of treatment and away from 1^. 1^ was not displaced,
because the resulting angle of divergence between P2 and
P^ of 121.5° was less than a standard deviation from the
average. In this meristem, treatment with IAA caused reversal
of the phyllotactic spiral.
In view of the fact that morphological responses
in lupine were elicited only by IAA, cytochemical analyses
were restricted to the effects of this treatment.
Cytochemical Analyses of IAA Effects
Cytochemical analyses involved microphotometric
determinations of changes with time in concentration of
RNA, total protein, and unsaturated lipids in the cytoplasm
and histones in the nuclues.—^ Comparisons were made between
—^Results of experiments justifying the use of certain
cytochemical procedures and confirming the specificity of
the stains are listed in MATERIALS AND METHODS under the
sections headed Staining

Fig. 4 Displacement of P away from P2 following
treatment with 1.5 yg IAA in transection of apical
meristem of Lupinus albus, stained with alkaline
fast green. X300.
A = apical meristem; P^, P2, P^/ .... = leaf
primordia numbered in sequence from youngest to
oldest.

44

45
the initiation sites, the intervening peripheral zone,
and the central mother cell zone in lupine meristems.
RNA concentration in the initiation sites or in the
central mother cell zone of lupine apices was not affected
by treatment. However, IAA treatment caused RNA concen¬
tration of the intervening peripheral zone to increase by
29.5% on day two (Fig. 5). RNA concentration in the
intervening peripheral zone following the increase on day
two was comparable to that in the initiation sites.
Elevated RNA concentration in the intervening peripheral
zone tended to persist for the remainder of the study
period.
Evaluation of the possible effects of different
plastochron stages on RNA concentration in untreated
lupine meristems revealed that RNA in the three cytological
sites did not vary among plastochron stages (Table 3).
Protein concentrations in initiation sites and in the
intervening peripheral zone of IAA-treated lupine meristems
were increased relative to lanolin controls (Fig. 6). The
data indicate a tendency for protein concentration to
increase from day two through day eight, but differences
from control values were statistically significant only
for day eight. IAA did not significantly affect protein
concentration in the central mother cell zone. Uniformity
of protein concentration throughout the untreated apical
meristem, as previously reported for other species (Cecich

Fig. 5 Changes with time in RNA concentrations in the
intervening peripheral zone (•), expressed as a
percentage of RNA in the initiation sites ( ),
in IAA-treated meristems of Lupinus albus. Each
point is the average of three replications.

RNA CONCENTRATION (%)

TABLE 3
Average relative concentrations of RNA
during the plastochron in shoot apical meristems
in Lupinus*
-
Plastochron
stage
Initiation
sites
Intervening
peripheral
zone
Central
mother cell
zone
Means
Early
1.52
1.35
1.12
1.33
Middle
1.78
1.64
1.17
1.53
Late
1.65
1.48
1.11
1.41
Means
1.64
1.48
1.13
*
Averaged over replications and expressed in arbitrary units.

Fig. 6 Changes with time in protein concentration in
the initiation sites (A), and the intervening
peripheral zone (B) of IAA-treated meristems
(•) expressed as percentage of lanolin controls
( ) , of Lupinus albus. Each point is the
average of three replications.

PROTEIN CONCENTRATION (%)
O ro -fk O OOO OOOOOOOOO
U1
o

51
st al. 1927; Gifford and Tepper 1962a, 1962b;Riding and
Gifford 1973), was observed in lupine apices treated with
lanolin only. Apices treated with IAA, however, had
higher concentrations of protein in the initiation sites
and intervening peripheral zone than in the central mother
cell zone.
Concentrations of unsaturated lipids in the intervening
peripheral zone and central mother cell zone were affected
by IAA treatment (Fig. 7). Based on the assumption that
the optical density of the lipid-bound osmium oxidation
product is a linear function of the concentration of
unsaturated lipids, concentration of unsaturated lipid in
IAA-treated apices increased nearly twofold in the interven¬
ing peripheral zone by the sixth day and approximately
threefold in both zones by the eighth day.
Nucleohistone concentration throughout IAA-treated
lupine meristems was affected by treatment (Fig. 8).
Significant differences in nucleohistone concentration
between IAA- and lanolin-treated meristems were observed
on day two and day six. The differences on day two,
however, were due largely to unusually low control values
and probably do not represent real treatment effects. The
peak on day six apparently was a genuine treatment effect.
After day six, nucleohistone concentrations in treated
meristems declined sharply. Relative to lanolin controls,
fluctuations in nucleohistone concentrations following

Fig. 7
Changes with time in unsaturated lipid concentration
in the intervening peripheral zone (A), and
the central mother cell zone (B) of IAA-treated
meristems (•), expressed as percentage of
lanolin controls ( ) , of Lupinus albus.
Each point is the average of three replications.

LIPID CONCENTRATION (%)
Ln
CO

Fig. 8 Changes with time in nucleohistone concentration
in the initiation sites (A), the intervening
peripheral zone (B), and the central mother cell
zone (C) of IAA-treated meristems (!), expressed
as percentage of lanolin controls ( ), of
Lupinus albus. Each point is the average of three
replications. Because of an unusually low average
for lanolin controls, the percentage for day two
was based on the overall average for control.

55

56
treatment with IAA were similar for initiation sites,
the intervening peripheral zone, and the central mother
cell zone. The dramatic rise and fall of nucleohistone
concentration on the sixth day following treatment with
IAA coincided with the time of displacement of primordia
during the second plastochron.
Although IAA appeared to increase concentrations of RNA,
proteins, unsaturated lipids, and nucleohistones in lupine
meristems, the effect tended to diminish by the end of the
observation period. Diminution of effects should be
expected as the IAA that penetrated into the meristems
was diluted by growth and metabolic activities, including
translocation, and as the lanolin-IAA droplet was displaced
continually further away from the meristem by growth of
the shoot tip. Concentrations were rapidly approaching
control values after the eighth day for proteins (Fig. 6)
and for unsaturated lipids (Fig. 7) in affected cytological
zones. Diminishing effects of IAA on RNA concentration
in the intervening peripheral zone towards the end of the
experiment were perhaps equivocal (Fig 5). Nucleo¬
histone concentration decreased sharply after day six
(Fig. 8) and may have been approaching control values
towards the end of the experiment.

57
Pinus
Morphological Analyses of Treatments
Morphogenic effects of growth regulators applied to the
shoot apical meristerns of pine, as determined in this study,
were confined to the differentiation of primordia. Primordia
developed normally into foliar needles in all treatments,
except kinetin. Kinetin-treated meristems showed no response
to treatment until the eighth or tenth day at which time
differentiation of primordia into bud scales became evident
on some meristems (Fig. 9a). Out of 18 meristems harvested
on the eighth or tenth day following treatment with kinetin,
five produced bud scales. Six other kinetin-treated seed¬
lings which were not harvested all produced bud scales by day
14 and appeared at that time to be dormant. Immature bud
scales developing on kinetin-treated meristems appeared normal
in size, shape, coloration, and position on the meristem
(Figs. 9a and c). Mature scales on kinetin-treated meristems
were more brightly russet in color than scales subsequently
produced by unused plants (Fig. 9b) some weeks after the
study had been completed.
An attempt was made to determine whether the growth
regulators tested affected axillary bud formation.
Appearance of nascent axillary buds in the sectioned
portion of pine shoot tips, however, was too infrequent
to permit determination of treatment effects, although

Fig. 9 A. Dormant terminal bud following treatment
with 1.5 yg kinetin applied to the exposed
apical meristem of seedling of Pinus
elliottii. X14.
B. Dormant terminal bud of untreated seedling
of Pinus elliottii some weeks after the
study was completed. X14.
C. Excised bud scale from kinetin-treated shoot
tip of Pinus elliottii seedling. X23.
S = bud scale; L = young primary needle; N =
mature primary needle; B = axillary short-
shoot bud; SN = axillary short shoot with
secondary needles.

59

60
sectioning of the shoot tips extended 150 to 200 y below
the base of the apical meristem. Examination of sections
from approximately 50 meristems revealed only seven
meristems with axillary buds, and the bud-containing
meristems were distributed among almost all treatments.
No attempt was made to determine angles of divergence
or plastochron length in pine meristems because of the
difficulty in identifying the last-formed primordium.
Because kinetin was the only growth regulator tested
which elicited a morphogenetic response in pine meristems,
this treatment was the only one analyzed cytochemically.
Cytochemical Analyses of Kinetin Effects
Cytochemical analyses of pine meristems, as in the case
of lupine meristems, involved microspectrophotometric
determinations of changes with time in concentration of RNA,
total protein, unsaturated lipids, and nucleohistones.
Comparisons within pine meristems, however, were confined to
the peripheral and central mother cell zones.
Treating shoot apices of pine seedlings with kinetin
increased the concentration of cytoplasmic RNA only in the
central mother cell zone (Fig. 10). The increase was
significant only on day six, at which time RNA in treated
meristems exceeded controls by almost 300%. RNA concentra¬
tion in treated meristems had returned to control values
by day eight. The sharp rise and fall of RNA in treated

Fig. 10 Change with time in RNA concentration in the central
mother cell zone of kinetin-treated meristerns (•),
expressed as percentage of lanolin controls (—■*“—“) ,
of Pinus elliottii. Each point is the average of
three replications.

62

63
meristems preceded appearance of bud scales by two to four
days.
Kinetin affected protein concentration throughout the
pine meristem (Fig. 11). Protein was 20% to 30% more
concentrated in the peripheral zone of treated plants
than in controls on days two and six, respectively
(Fig. 11A). Concentrations then fell to approximately
20% less than control values on day eight. Protein in
treated meristems was still significantly less concentrated
than in controls on day ten.
In the central mother cell zone, protein concentration
in treated meristems differed from control only on day
eight (Fig. 11B). On this day, treated meristems had about
30% less protein than controls. Decline in protein
concentration in the central mother cell zone on day
eight corresponded to the decline in protein in the
peripheral zone on the same day.
Reduced concentrations of proteins in treated
meristems tended to correspond with the appearance of
bud scales and the onset of dormancy.
Unsaturated lipids were 25% less concentrated in the
central mother cell zone of kinetin-treated pine meristems
than in the lanolin controls (Fig. 12). Kinetin had no
apparent effect on unsaturated lipids in the peripheral
zone. Decreased lipid concentration in the central mother
cell zone was evident on the fourth day following treatment

Fig. 11 Change with time in protein concentration in the
peripheral zone (A) and the central mother cell zone
(B) of kinetin-treated meristems (t), expressed
as percentage of lanolin controls ( ) , of Pinus
elliottii. Each point is the average of three
replications.

PROTEIN
CONCENTRATION (%)
o o
00
o
ro
o
o
a>
o
CD
o
o
o
ro
o
o
Ln

Fig. 12
Change with time in unsaturated lipid concentration
in the central mother cell zone of kinetin-
treated meristems (i), expressed as percentage
of lanolin controls ( ■) , of Pinus elliottii ♦
Each point is the average of three replications.

LIPID CONCENTRATION (%)
O) oo 5 N o
ooo ooooo
o>

68
(Fig. 12), but was gradually returning towards control
levels afterwards.
Concentrations of nucleohistones increased in both
the peripheral zone and the central mother cell zone
following treatment of pine meristems with kinetin
(Fig. 13). Kinetin-increased nucleohistone concentrations
were significantly higher in both zones on day six and
rapidly decreased toward control values thereafter. Peak
nucleohistone concentration in kinetin-treated meristems
corresponded with peak RNA concentration in the central
mother cell zone (Fig. 10) and preceded appearance of bud
scales by two days.

Fig. 13
Change with time in nucleohistone concentrations
in the peripheral zone (A) and the central
mother cell zone (B) of kinetin-treated meristems
(•), expressed as percentage of lanolin controls
( ) , of Pinus elliottii.
Each point is the average of three replications.

HISTONE CONCENTRATION (%)
70
TIME (DAYS)

Coleus
Growth regulators, as tested in this study, did
not affect morphogenesis at the shoot tip of coleus.
Lateral buds were formed in the second or third leaf
axils in all treatments and controls. Primordia were
not displaced by any treatment, and primordia an all
treatments appeared to develop normally, except that
development was considerably retarded during the course
of the study as the plastochron increased substantially
during this time. The plastochron at the beginning of
the study appeared to be two to three days in length.
When the plants had been grown under the conditions
reported above for about 50 days, the plastochron was
estimated to be 40 days in length in untreated meristems.
Treatments did not appear to shorten the plastochron.
Cytochemical analyses originally planned for the coleus
meristems were abandoned when the treatments failed to
elicit a morphogenetic response.

DISCUSSION
Lupinus
Of the three growth regulators tested, only IAA
affected shoot meristems in lupine. Treatment with IAA
resulted in: (1) axillary buds forming closer to the apex
than normal, (2) displacement of primordia, and (3)
increases in concentrations of RNA, total protein, and
unsaturated lipids in the cytoplasm, and histones in the
nucleus, of cells in the subdistal and organogenic regions
of the apical meristem.
Initiation of axillary buds closer to the apex than
normal and displacement of primordia in IAA-treated lupines
in this study agree with the observations of other workers
(Ball 1944; Snow and Snow 1937). Ball (1944), working with
Tropaeolum, observed lateral buds on the flanks of auxin-
treated apices, and primordia were displaced as much as 40°
to 50° of arc from their normal positions on the meristem.
Snow and Snow (1937) observed primordial displacement on
lupine and Epilobium meristems treated with heteroauxin.
The three studies taken together, therefore, indicate that
IAA applied to the apical meristem of dicots disrupts the
normal physiological patterns within the apex and affects
72

73
physiological interactions between the apex and the rest of
the plant.
The only point of disagreement among these three studies
concerns the direction of primordial displacement. Snow and
Snow (1937) claimed that the direction of displacement tended
to be towards the treatment site. This claim was based pri¬
marily on observations in Epilobium, a decussate species, in
which the first pair of primordia to arise after treatment
were, without exception, displaced toward the site of treat¬
ment. Snow and Snow (1937) conceded that their data for
lupine, a species exhibiting spiral phyllotaxis, did not
provide much support for their claim.
Ball's (1944) observations of Tropaeolum, which also
features spiral phyllotaxis, as well as the present study of
lupine, does not support Snow and Snow's (1937) interpreta¬
tion. Greater variation in angles of divergence, indicated
by larger sample mean standard errors, was observed in auxin-
treated meristems than in controls for Tropaeolum by Ball
(1944), and for lupine in the present study. Average angles
of divergence for treated meristems in each of the three
studies did not differ significantly from that for untreated
meristems in the same species, except for Epilobium (Snow and
Snow 1937). Affected angles of divergence for treated
Epilobium meristems were always less than the normal 180°.
Greater variation around an average angle of divergence which
approximately equals that for untreated meristems means that

74
affected angles tended to be greater or smaller than the
normal, and there are about as many large angles as small
ones. Developmentally, these results support Ball's (1944)
conclusion that primordial displacement in auxin-treated
apices is random relative to the site of treatment. This
conclusion may apply to all dicotyledonous species with
spiral phyllotaxis.
Application of IAA to the apical meristem of lupine
appeared to confer initiation site capabilities to the
intervening peripheral zone for a short time. This was
demonstrated not only by displacement of initiation sites
to what must have formerly been the intervening peripheral
zone, but also by metabolic activity in the intervening
peripheral zone being increased to the same level as
observed in the initiation sites. Increased metabolic
activity in the intervening peripheral zone of IAA-treated
meristems was shown by increased concentrations of all of the
cytochemical substances observed relative to control
values (Figs. 5, 6, 7, and 8). Concentration of RNA and
unsaturated lipids increased in the intervening peripheral
zone, as compared to the initiation sites, in IAA-treated
meristems. Enhanced metabolism in the intervening
peripheral zone was additionally indicated by increased
concentration of protein, relative to the central mother
cell zone, in IAA-treated meristems. Increased histone
concentration in the intervening peripheral zone indicated

75
that not only the cytoplasm but also the nuclei in this
zone were affected by IAA treatment. Responses in RNA and
unsaturated lipids within two to four days after treatment
correspond weU with the duration of the first plastochron,
during which time some primordia were displaced. Responses
of all observed substances, except nucleohistone, through
day eight would extend IAA effects into the second and
third plastochron when other primordia were displaced. An
enlarged zone of initiation potential would help explain
not only the displacement of primordia observed in this
and other studies but also the enlarged leaf bases and
connation of primordia observed in Tropaeolum by Ball
(1944) and in lupine and Epilobium by Snow and Snow (1937).
Diminishing concentrations of nucleohistones after
the sixth day (Fig. 8) and of proteins and unsaturated
lipids after the eighth day (Figs. 6 and 7) indicate that
a single application of a minute droplet of IAA produces
a dramatic but transient effect. If treatment effects
had largely disappeared by the tenth day, only three or
four plastochrons could be affected. Primordia produced
thereafter in subsequent plastochrons would be expected
to arise in normal positions around the apex (Table 1),
and phyllotaxis would once again be characteristic for
the species. Although the present study did not monitor
treated plants beyond the tenth or twelveth day, Ball
(1944) reported that treated plants, exhibiting altered

76
phyllotaxis, soon reverted back to normal phyllotaxis.
One possible mechanism for the cytological effects
of IAA observed in this study involves interactions between
the growth regulator and nucleic acid metabolism, and,
conceivably, gene expression. Involvement of auxin in
nucleic acid metabolism was suggested 20 years ago (Skoog
1954), and support for the idea has been forthcoming
(Key and Shannon 1964; Key 1964). RNA concentrations in
the present study were affected relatively soon (two days)
after treatment (Fig. 5), while unsaturated lipid concentra¬
tions were not affected until day four or later (Fig. 7).
The change in RNA concentration in IAA-treated apices
was well within the normal response time for auxin-
mediated changes in nucleic acid metabolism (Kamisaka 1972;
Key and Ingle 1968). Changes in protein and unsaturated
lipid concentration at about the same time or after the
change in RNA would be expected if existing cytoplasmic RNA
had to be augmented by more of the same kind in order to
meet requirements for enhanced synthesis of existing
proteins and unsaturated lipids, and if different genetic
information had to be translated by new messenger RNA
species for the synthesis of novel compounds.
According to Swift (1966), cytoplasmic RNA determined
by the Azure B technique, which was used in this study,
consists largely of ribosomal RNA. Total cytoplasmic
protein, determined by the NYS technique, consists of

77
soluble and structural protein (Deitch 1966b). Much of the
structural protein in the cytoplasm is contained in the
ribosomes. Thus, close correspondence in fluctuations with
time between cytoplasmic RNA and proteins in the intervening
peripheral zone of IAA-treated lupines (Figs. 5 and 6B) would
be expected through their common association in ribosomes.
Changes in protein concentration unaccompanied by
obvious changes in RNA in the initiation sites of IAA-
treated meristems (Fig. 6A) might be explained by changes
in soluble proteins and structural, non-ribosomal
proteins. If ribosomal protein was involved, corresponding
changes in ribosomal RNA in these zones must have been
obscured by compensatory changes in other kinds of
cytoplasmic RNA.
Because unsaturated lipids in the cytoplasm are
largely contained in membranes, changes in unsaturated
lipid concentration would probably reflect changes in
membrane-related phenomena. Membrane changes regulated
by differential gene expression could have widespread
implications on the physiology of cells, tissues, and
organs, as well as on morphogenetic events.
Changes in nucleohistone concentrations (Fig. 8),
accompanied by changes in RNA, further suggest growth
regulator involvement within the nucleus. That auxin
binds nucleohistone, causing conformational changes in the
protein, has been suggested (Venis 1968). Thus, the

78
possibility exists that auxins, interacting with histones
may modulate the latter's gene-repressor functions.
Results of the present study indicate involvement of
the central mother cell zone in the functions of the apex.
The central mother cell zone responded to IAA treatment
with increased concentrations of unsaturated lipids and
nucleohistones (Figs. 7 and 8). Fluctuations in concentra¬
tions of these substances in the central mother cell zone
were identical to those in the intervening peripheral
zone for treated meristems. RNA and protein in the central
mother cell zone apparently were unaffected by IAA.
Pinus
Application of IAA, kinetin, or GA to shoot apical
meristems of pine seedlings in this study resulted in a
morphogenetic response, the appearance of bud scales and the
onset of dormancy only to the kinetin treatment. Bud
formation, as observed here, is a common response to
kinetin treatment (Paulet and Nitsch 1959; Schraudolf and
Reinert 1959; Sinnott and Miller 1957) in those plants which
have a natural tendency to form buds (Miller 1961). Bud

79
formation is regarded as one of the two primary activities
of kinetin (Cutter 1965). The mechanism by which kinetin
stimulates bud formation is not known, but results of the
cytochemical analyses from the present study shed some
light on the problem.
Cytochemical effects of kinetin were first apparent
the second day after treatment. At that time, protein
concentrations exceeded controls by about 18% in the
peripheral zone (Fig. 11A). This was followed by an
abrupt decrease in concentrations of unsaturated lipids
(Fig. 12) in the central mother cell zone on day four.
RNA in the central mother cell zone and nucleohistones
in both zones exceeded control levels on day six (Figs. 10
and 13), two days before bud scales were first observed.
The effect of kinetin on RNA and nucleohistones suggests
involvement of the growth regulator in nucleic acid
metabolism and/or gene expression, which might explain
the sharp decrease in protein concentration in treated
meristems on the eighth day following treatment (Fig. 11).
Kinetin has been shown to interact with events surrounding
transcription (Datta and Sen 1965; Matthysee 1969)+ transla¬
tion (Datta and Sen 1965), and enzyme activity (Mann et al.
1963; Steinhart et: al. 1964; Boothby and Wright 1962).
Decreased concentrations of unsaturated lipids in the
cytoplasm probably reflected reduced amounts of membrane
material. Reduction in unsaturated lipids and membranes

80
could be achieved if the enzymes catalyzing their synthesis
were adversely affected by kinetin. Kinetin-related changes
in enzymes can occur by any one of at least three
mechanisms. One, kinetin can act directly on specific
enzymes by directing their synthesis (Boothby and Wright
1962; Steinhart et ad. 1964) or by altering their activity
(Bergman and Kwienty 1958; Henderson et al. 1962). Two,
kinetin can act indirectly on specific enzymes by affecting
nucleic acid metabolism (Fox and Chen 1968) or gene
expression (Boothby and Wright 1962; Clum 1967). Three,
kinetin can affect general enzyme activity by altering
physical properties of the cell, conceivably via (1) or (2)
above, and thereby changing the availability of substrate
and the utilization of product (Mothes and Engelbrecht
1961). Thus, there is ample evidence, albeit circumstantial
that kinetin could influence lipid metabolism and membrane
structure in treated pine meristems. Direct evidence of
kinetin influencing lipid metabolism was not discovered
in the literature.
Examination of cytoplasmic membranes from intact,
kinetin-treated plants apparently has not been done. Nitsch
(1968) observed no effect of kinetin in the medium on ultra¬
structure of i_n vitro tissues, but kinetin can affect mem¬
brane morphology in excised roots (Vasil 1973). Whether
altered membrane morphology involved changes in lipid metab¬
olism was not determined (Vasil (1973). However, this infor

81
mation may not apply to intact plants, because kinetin effects
often differ between intact and excised tissues from the
same plant (Pilet 1968; Fletcher and Adedipe 1972).
Kinetin-activated fluctuations in RNA in the central
mother cell zone on day six (Fig. 10) probably reflected
ribosomal-related events, despite the apparent absence of
treatment effects on proteins in this zone. Effects on
ribosomal protein might have been obscured by compensatory
changes in other structural proteins or in soluble proteins
in the cytoplasm. That compensatory changes in concentrations
of different proteins could result in significant biological
effects without altering the total concentration of
cytoplasmic protein is an inherent problem in the study of
total protein and was discussed above (ref. p. 13) regarding
Nougaréde's (1967) hypothesis. A less likely explanation
for the fluctuations of RNA in the central mother cell zone
would be that the fluctuations resulted from massive
changes in concentration of messenger or transfer RNA.
In kinetin-treated apices, changes in RNA and nucleo-
histones on day six probably reflected activity within the
nuclei. Perhaps it was about this time that the physiological
state of treated meristems shifted from one favoring
growth to one favoring dormancy, and, as growth processes
slowed down and dormancy began to set in, novel enzymes
were needed in newly activated metabolic pathways. Genetic
information from previously unused segments of the

82
chromosomes would then be transcribed, and other segments
of the chromosomes would be repressed, perhaps by some of
the additional nucleohistones. Transcription of newly
derepressed genetic information could, conceivably, lead
to differentiation of primordia into bud scales and to
other events which, similarly, had never before occurred in
the brief lives of the pine seedlings used in this study.
A similar suggestion has been advanced to rationalize
the episodic annual growth habit of shoots in trees
(Romberger 1966).
Manifestations of the onset of dormancy in kinetin-
treated meristems included the rapid decline of RNA,
proteins, and nucleohistones (Figs. 10, 11, and 13) after
day six, the appearance of bud scales on day eight and ten,
and buds completely formed on all treated meristems on day
14. The rapid decrease in nucleohistones after day six
(Fig. 13) could correspond with declining metabolic activities
in the nuclei and elsewhere as dormancy was imposed. An
abrupt decrease in nucleohistone content in shoot meristems
has been correlated with cessation of elongation growth and
winter bud formation in white spruce (Cecich et al. 1972).
Indirect evidence indicates that as dormancy sets in
decreases would be expected in total RNA (Chen and Osborne
1970; Pilet 1970; Poulsen and Beevers 1970; Bex 1972) and
proteins (Varner and Johri 1968; Chen and Osborne 1970;
Pollard 1970; Poulsen and Beevers 1970; Paranjothy and
Wareing 1971; Rijven and Parkash 1971). Reduced levels of

83
RNA and protein could be at least partially accounted for by
reduced ribosomal activity (Chen and Osborne 1970), by
fewer polysomes (Poulsen and Beevers 1970; Paranjothy and
Wareing 1971; Bex 1972; Evins and Varner 1972), and by a
lower rate of polysome formation (Evins and Varner 1972) as
dormancy is imposed.
The central mother cell zone was involved in metabolic
functions of the pine seedling shoot meristem. Changes in
all the observed compounds occurred in the central mother
cell zone in response to kinetin treatment (Figs. 10, 11B,
12, and 13B). Fluctuations with time in RNA (Fig. 10) and
nucleohistones (Fig. 13) were the same in the central
mother cell zone. Responses of RNA (Fig. 10) and unsaturated
lipids (Fig. 12) to treatment appeared to be restricted
to the central mother cell zone. Involvement of the
central mother cell zone in metabolic functions of the
vegetative apex were also observed in lupine in this
study and in shoots of mature spruce trees (Cecich et al.
1972), despite Plantefol's (1946, 1947a, 1947b, 1948)
hypothesis to the contrary.
This study demonstrated that topical treatment of the
apical meristem can be successfully combined with
cytochemical analyses. The results add to our knowledge
of how developmental and cytological heterogeneity is
maintained within the apical meristem. Perhaps this study
will stimulate additional analytical research on development
within the vegetative shoot apical meristem.

APPENDIX
The average for angles of divergence expected to be
affected was compared with that for angles expected not
to be affected as paired samples from each plant in a
given treatment. A two-tailed "Student's" t-test was
used to evaluate mean differences.
The analysis of variance used to evaluate growth
regulator effects versus controls over five harvest dates
with three replications in each of the cytochemical
experiments was as follows:
Source of variation Degrees of freedom
Total 29
Treatment 1
Dates 4
Interaction 4
Error 20
The unit of variation was the mean per plant. If the F-
ratio for interaction was significant at the 5% level,
separate analyses were made comparing dates within each
treatment and comparing treatments within each harvest
date. The analysis for harvest dates within a treatment
was as follows:
84

85
Source of variation Degrees of freedom
Total
14
Between dates
4
Within dates
10
The analysis of treatments within a harvest date was a
"Student's" t-test, using the error mean square from the
two-way analysis above as an estimate of the standard
error.
If interaction was negligible, the error mean
square was used for testing date and treatment main effects.

LITERATURE CITED
Alfert, M. and I.I. Geschwind. 1953. A selective staining
method for the basic proteins of cell nuclei. Proc.
Nat. Acad. Sci. (U.S.) 39:991-999.
Baker, J.R. 1946. The histochemical recognition of lipine.
Quart. J. Microscopical Sci. 87:441-470.
Ball, E. 1944. The effects of synthetic growth substances
on the shoot apex of Tropaeolum majus L. Araer. J. Bot.
31:316-327.
Bergman, F. and H. Kwietny. 1958. Oxidation of kinetin by
mammalian xanthine oxidase. Biochem. Biophys. Acta
28:100-103.
Bersillon, G. 1951. Sur le point végétatif de Papaver
somniferum L.; structure et fonctionnement. Acad, des
Sci. Compt. Rend. 232:2470-2472.
Bex, J.H.M. 1972. Effects of abscisic acid on nucleic acid
metabolism in maize coleoptiles. Planta 103:1-10.
Boothby, D. and S.T.C. Wright. 1962. Effects of kinetin and
other plant growth regulators on starch degradation.
Nature 196:389-390.
Biinning, E. 1952. Morphogenesis in plants. Survey of Biol.
Prog. 2:105-140.
Cain, A.J. 1950. The histochemistry of lipoids in animals.
Biol. Rev. Trans. Camb. Phil. Soc. 25:73-112.
Cecich, R.A., N.R. Lersten, and J.P. Miksche. 1972. A
cytophotometric study of nucleic acids and proteins in
the shoot apex of white spruce. Amer. J. Bot. 59:442-
449.
Chen, D. and D.J. Osborne. 1970. Hormones in the trans¬
lational control of early germination in wheat embryos.
Nature 226:1157-1160.
Clowes, F.A.L. 1961. Apical meristems. Bot. Monog. 2.
Oxford, Blackwell.
Clum, H.H. 1967. Formation of amylase in disks of bean
hypocotyl. Plant Physiol. 42:568-572.
86

87-
Cutter, E.G. 1954. Experimental induction of buds from fern
leaf primordia. Nature 173:440-441.
1956. Experimental and analytical studies of
pteridophytes. XXXIII. The experimental induction of
buds from leaf primordia in Dryopteris aristata Druce.
Ann. Bot. N.S. 20:143-165.
1965. Recent experimental studies of the shoot
apex and shoot morphogenesis. Bot. Rev. 31:7-113.
Datta, A. and S.P. Sen. 1965. The mechanism of action of
plant growth substances: Growth substance stimulation
of amino acid incorporation into nuclear protein.
Biochem. Biophys. Acta 107:352-357.
Deitch, A.D. 1966a. Cytophotometry of nucleic acids. In:
Intro, to Quant. Cytochem.. G.L. Weid, ed. Academic
Press, New York. pp:327-354.
1966b. Cytophotometry of proteins. In: Intro.
to Quant. Cytochem. G.L. Weid, ed. Academic Press,
New York. pp:451-468.
Evans, L.S. and A.R. Berg. 1972. Qualitative histochemr.
istry of the shoot apex of Triticum. Can. J. Bot.
50:241-244.
Evins, W.H. and J.E. Varner. 1972. Hormonal control of
polyribosome formation in barley aleurone layers.
Plant Physiol. 49:348-352.
Fagerlind, F. 1954. The apical embryo- and shoot-meristem
in Gnetum, Ephedra and other gymnosperms. Svensk. Bot.
Tidskr. 48:449-470.
Flax, M.H. and M.H. Himes. 1950. A differential stain for
ribonucleic and desoxyribonucleic acids. Anat.
Record. 108:529.
1952. Microspectrophotometric analysis of meta-
chromatic staining of nucleic acids. Physiol. Zoo.
25:297-311.
Fletcher, R.A. and N.O. Adedipe. 1972. Hormonal regulation
of leaf senescence in intact plants. In: Plant Growth
Substances 1970. D.J. Carr, ed. Springer-Verlag,
Berlin. pp:571-580.
Fosket, D.E. and J.P. Miksche. 1966. A histochemical study
of the seedling shoot apical meristem of Pinus
lambertiana. Amer. J. Bot. 53:694-702.

88
Foster, A.S. 1938. Structure and growth of the shoot apex
in Ginkgo biloba. Torrey Bot. Club Bull. 65:531-556.
Fox, J.E. and C.M. Chen. 1968. Cytokinin incorporation in¬
to RNA and its possible role in plant growth. In:
Biochem. and Physiol, of Plant Growth Substances.
F. Wightman and G. Setterfield, ed. Runge Press,
Ottawa. pp:777-789.
Gifford, E.M., Jr., and H.B. Tepper. 1962a. Histochemical
and autoradiographic studies of floral induction in
Chenopodium album. Amer. J. Bot. 49:706-714.
1962b. Ontogenetic and histochemical changes in
the vegetative shoot of Chenopodium album. Amer. J.
Bot. 49:902-911.
Griffith, M.M. 1952. The structure and growth of the shoot
apex in Araucaria. Amer. J. Bot. 39:253-263.
Henderson, T.R., C.G. Skinner, and R.E. Eakin. 1962.
Kinetin and kinetin analogues as substrates and
inhibitors of xanthine oxidase. Plant Physiol. 37:
552-555.
Jensen, W.A. 1962. Botanical Histochemistry. Freeman &
Co., San Francisco.
Johnson, M.A. 1951. The shoot apex in gymnosperms.
Phytomorph. 1:188-204.
Kamisaka, S. 1972. Auxin-induced growth of tuber tissue of
Jerusalem artichoke. VII. Effect of cyclic 3',
5'-adenosine monophosphate on the auxin-induced cell
expansion growth. In: Plant Growth Substances 1970.
D.J. Carr, ed. Springer-Verlag, Berlin. pp:654-660.
Key, J.L. 1964. Ribonucleic acid and protein synthesis as
essential processes for cell elongation. Plant
Physiol. 39:365-370.
Key, J.L. and J. Ingle. 1968. RNA metabolism in response
to auxin. In: Biochem. and Physiol, of Plant Growth
Substances. F. Wightman and G. Setterfield, ed.
Runge Press, Ottawa. pp:711-722.
Key, J.L. and J.C. Shannon. 1964. Enhancement by auxin of
ribonucleic acid synthesis in excised soybean hypocotyl
tissue. Plant Physiol. 39:360-364.
Koch, L. 1891. Ueber Bau und Wachsthum der Sprosspitze der
Phanerogamen. I. Die Gymnospermen. Jahrb. f. Wiss.
Bot. 22:491-680.

Lance, A. 1954. Repartition de 1'acide ribonucléique dans
les méristemes apicaux de deux composées. Acad. des
Sci. Compt. Rend. 239:1238-1239.
1957. Recherches cytologeques sur 1'evolution de
quelques méristemes apicaux et sur ses variations
provoqueés par des traitements photopériodiques. Ann.
Sci. nat. Bot. Ser. XI. 18:91-422.
Mann, J.D., C.E. Steinhart, and S.H. Mudd. 1963. Alkaloids
and plant metabolism. V. The distribution and form¬
ation of tyramine methylpherase during germination of
barley. J. Biol. Chem. 238:676-681.
Matthysee, A.G. 1969. Interaction of effector substances
with chromosomal receptors. Abst. Int. Bot. Cong.
11th., Seattle. pp:143.
Miller, C.O. 1961. Kinetin and related compounds in plant
growth. Ann. Rev. Plant Physiol. 12:395-408.
Mothes, K. and L. Engelbrecht. 1961. Kinetin-induced
directed transport of substances in excised leaves in
the dark. Phytochem. 1:58-62.
Nitsch, J.P. 1968. Studies on the mode of action of
auxins, cytokinins and gibberellin at the subcellular
level. In: Biochem. and Physiol, of Plant Growth
Substances. F. Wightman and G. Setterfield, ed.
Runge Press, Ottawa. pp:563-580.
Nougaréde, A. 1967. Experimental cytology of the shoot
apical cells during vegetative growth and flowering.
Int. Rev. Cyto. 21:203-351.
Nougaréde, A., E.M. Gifford, Jr., and P. Rondet. 1965.
Cytohistological studies of the apical meristem of
Amaranthus retroflexus under various photoperiodic
regimes. Bot. Gaz. 126:281-298.
Paranjothy, K. and P.F. Wareing. 1971. The effects of
abscisic acid, kinetin and 5-fluorouracil on ribonucleic
acid and protein synthesis in senescing radish leaf
discs. Planta 99:112-119.
Paulet, P. and J.P. Nitsch. 1959. Stimulation chimique du
bourgeonnement chez Cardamine pratensis L. Bull. Soc.
Bot. France 106:425-441.
Pearse, A.G.E. 1968. Histochemistry: Theoretical and
Applied. Vol. 1., 3rd ed. Little, Brown & Co.,
Boston.

90
Pilet, P.E. 1968. Iri vitro and in vivo auxin and cytokinin
translocation. In: Biochem. and Physiol, of Plant
Growth Substances. F. Wightman and G. Setterfield, ed.
Runge Press, Ottawa. pp:993-1004.
1970. The effect of auxin and abscisic acid on
the catabolism of RNA. J. Exp. Bot. 21:446-451.
Plantefol, L. 1946. Fondements d'une theorie phyllotaxique
nouvelle. I. Historique et criteque. II. La.
phyllotaxie des monocotyledons. Ann. Sci. nat. Bot.
Ser. XI. 7:153-229.
1947a. La phyllotaxie des dikotylédons. IV.
Generalizations et conclusions. Ann. Sci. nat. Bot.
Ser. XI. 8:1-66.
1947b. Helices foliare, point végétatif et
stele chez les dicotylédones. La notion d'anneau
initial. Rev. gen. Bot. 54:49-80.
1948. La theorie des helices multiples foliaires.
Masson, Pairs.
1950. La phyllotaxie. Année Biol. 54 (Ser. III.
26) : 447-460.
Pollard, C.J. 1970. Initiation of responses in aleurone
layers by gibberellic acid. Biochem. Biophys. Acta
222:501-507.
Popham, R.A. 1951. Principal types of vegetative shoot
apex organization in vascular plants. Ohio J. Sci.
51:249-270.
Popham, R.A. and A.P. Chan. 1950. Zonation in the vegeta¬
tive stem tip of Chrysanthemum morifolium Bailey.
Amer. J. Bot. 37:476-484.
Poulsen, R. and L. Beevers. 1970. Effects of growth regu¬
lators on ribonucleic acid metabolism of barley leaf
segments. Plant Physiol. 46:782-785.
Richards, F.J. 1951. Phyllotaxis: Its quantitative
expression and relation to growth in the apex. Phil.
Trans. Roy. Soc. B. 235:509-563.
1956. Spatial and temporal correlations involved
in leaf pattern production in the apex. In: Growth
of Leaves. F.L. Milthorpe, ed. Butterworths Sci.
Publ., London. pp:66-75.

Riding, R.T. and E.M. Gifford, Jr. 1973. Histochemical
changes occurring at the seedling shoot apex of Pinus
radiata. Can. J. Bot. 51:501-512.
Rijven, A.H.G.C. and V. Parkash. 1971. Action of kinetin
on cotyledons of fenugreek. Plant Physiol. 47:59-64.
Romberger, J.A. 1963. Meristems, growth, and development
in woody plants. U.S.D.A. tech Bull. No. 1293.
1966. Developmental biology and the spruce tree.
J. Washington Acad. Sci. 56:69-81.
Schmidt, A. 1924. Histologische Studien an phanerogamen
Vegetationspunkten. Bot. Arch. 8:345-404.
Schoute, J.C. 1913. Beitrage zur Blattstellungslehre.
Rec. Trav. Bot. Neerl. 10:153-339.
1936. Fasciation and dichotomy. Rec. Trav. Bot.
Neerl. 33:649-669.
Schraudolf, H. and J. Reinert. 1959. Interaction of plant
growth regulators in regeneration processes. Nature
184:465-466.
Sinnott, E.W. and C.O. Miller. 1957. Chemical regulation
of growth and organ formation in plant tissues. Symp.
Soc. Exp. Biol. 11:118-131.
Skoog, F. 1954. Substances involved in normal growth and
differentiation of plants. Brookhaven Symp. Biol.
(BNL 258):1-21.
Snedecor, G.W. and W.G. Cochran. 1967. Statistical Methods.
6th. ed. Iowa State Univer. Press, Ames.
Snow, M. and R. Snow. 1931. Experiments on phyllotaxis. I.
The effects of isolating a primordium. Phil. Trans.
Roy. Soc. B. 221:1-43.
1933. Experiments on phyllotaxis. II. The
effect of displacing a primordium. Phil Trans. Roy.
Soc. B. 221:354-400.
1935. Experiments on phyllotaxis. III. Diagonal
splits through decussate apices. Phil. Trans. Roy.
Soc. B. 225:63-94.
1937. Auxin and leaf formation. New Phytol.
36:1-18.

92
Steeves, T.A., M.A. Hicks, J.M. Naylor, and P. Rennie.
1969. Analytical studies on the shoot apex of
Helianthus annuus. Can. J. Bot. 47:1367-1375.
Steinhart, C.E., J.D. Mann, and S.H. Mudd. 1964.
Alkaloids and plant metabolism. VII. The kinetin-
produced elevation in tyramine methylpherase levels.
Plant Physiol. 39:1030-1038.
Sunderland, M., J.K. Heyes, and R. Brown. 1956. Growth
and metabolism in the shoot apex of Lupinus albus. In:
The Growth of Leaves. F.L. Milthorpe, ed. Butter-
worths Sci. Publ., London. pp:77-90.
Swift, H. 1966. The quantitative cytochemistry of RNA.
In: Intro, to Quant. Cytochem. G.L. Weid, ed. Aca¬
demic Press, New York. pp:355-386.
Thielke, C. 1965. Strukturwechsel und Enzymmuster ans
Sprosschitel einiger Grasses. Planta 66:310-319.
Turing, M.A. 1952. The chemical basis of morphogenesis.
Phil. Trans. Roy. Soc. B. 237:37-72.
Vanden Born, W.H. 1963. Histochemical study of enzyme
distribution in shoot tips of white spruce (Picea
glauca (Moench) Voss). Can. J. Bot. 41:1509-1527.
Van Fleet, D.S. 1959. Analysis of the histochemical
localization of peroxidase related to the differentia¬
tion of plant tissues. Can. J. Bot. 37:449-458.
Varnell, R.J. and I.K. Vasil. 1972. Seasonal development
at the shoot tip of Podocarpus macrophyllus D. Don.
Amer. J. Bot. 59:657.
Varner, J.E. and M.M. Johri. 1968. Hormonal control of
enzyme synthesis. In: Biochem. and Physiol, of Plant
Growth Substances. F. Wightman and G. Setterfield, ed.
Runge Press, Ottawa. pp:793-874.
Vasil, I.K. 1973. Morphogenetic, histochemical and ultra-
structural effects of plant growth substances in vitro.
Biochem. Physiol. Pflanzen 164:58-71.
Venis, M.A. 1968. Auxin-histone interaction. In: Biochem.
and Physiol, of Plant Growth Substances. F. Wightman
and G. Setterfield, ed. Runge Press, Ottawa. pp:761-775.

93
Wardlaw, C.W. 1949. Experiments on organogenesis in ferns.
Growth 13, Suppl:93-131.
1950. Experimental and analytical studies of pter-
idophytes. XVI. The induction of leaves and buds in
Dryopteris aristata Druce. Ann. Bot. N.S. 14:435-455.
1955. Experimental investigations of leaf forma¬
tion, symmetry and orientation in ferns. Nature 175:
115-117.
1956. Experimental and analytical studies of
pteridophytes. XXXII. Further investigations on the
effect of undercutting fern leaf primordia. Ann. Bot.
N.S. 20:121-132.
1957. On the organization and reactivity of the
shoot apex in vascular plants. Amer. J. Bot. 44:176-187.
1960. The inception of shoot organization.
Phytomorph. 10:107-110.
1965a. The morphogenetic role of apical meristems:
Fundamental aspects (illustrated by means of the shoot
apical meristem). In: Handbuch der Pflanzenphysiologie.
W. Ruhland, ed. Springer-Verlag, Berlin. Vol. XV./I:
443-451.
1965b. The organization of the shoot apex. In:
Handbuch der Pflanzenphysiologie. W. Ruhland, ed.
Springer-Verlag, Berlin. Vol. XV./I:966-1076.
Wardlaw, C.W. and E.G. Cutter. 1956. Experimental and
analytical studies of pteridophytes. XXXI. The effect
of shallow incisions on organogenesis in Dryopteris
aristata Druce. Ann. Bot. N.S. 20:39-56.
West, W.C. and J.E. Gunckel. 1968. Histochemical studies
of the shoot of Brachychiton. I. Cellular growth and
insoluble carbohydrate. Phytomorph. 18:269-282.
Wetmore, R.H. 1943. Leaf and stem relationships in the
vascular plants. Torreya 43:16-28.
Wetmore, R.H. and C.W. Wardlaw. 1951. Experimental morpho¬
genesis in vascular plants. Ann. Rev. Plant. Physiol.
2:269-292.

BIOGRAPHICAL SKETCH
Ray Judson Varnell was born October 11, 1937, in
Shawnee, Oklahoma. He graduated from Classen High School
in Oklahoma City in May, 1955, and received a Bachelor
of Science degree in general forestry and a reserve
commission in the U.S. Air Force from Oklahoma State
University in January, 1960. He spent six months'active
duty as second lieutenant at Travis Air Force Base,
California in 1960. He entered the School of Forestry,
North Carolina State University, Raleigh, in October, 1960,
earning a Master of Science degree from there in January,
1963, on the in vitro culture of the female gametophyte of
Pinus taeda. He joined the U.S Forest Service as a
Research Forester at the Naval Stores and Timber Production
Laboratory, Olustee, Florida, in February, 1963, where
he was employed as a Forest Physiologist specializing in
flower production in southern pines. He spent 1966 and 1967
on a training assignment at the Forest Physiology Laboratory,
Plant Industry Station, Beltsville, Maryland, working on the
in vitro culture of apical meristems of Picae abies. He
resigned from the Forest Service in July, 1970, to enter the
University of Florida where he was supported by an
94

95
N.D.E.A., Title IV, Fellowship from September, 1970,
until September, 1973. He resigned a reserve captaincy
in the U.S. Air Force in November, 1971. From July, 1970,
until the present time, he has worked towards the Ph. D.
degree.
Ray Judson Varnell is married to Emily (nee) DiMilla
of Medford, Massachusetts. They have one child, Brian
Thomas, born August, 1973.
Ray Judson Varnell is a past member of Alpha Gamma Rho,
a national, social-professional fraternity and Alpha Zeta,
a national honorary agricultural fraternity. He is a current
member of the Botanical Society of America.

I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy.
(deceased)
Robert G. Stanley, Chairman
Professor of Forestry
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy.
Indra K. Vasil
Professor of Botany
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy.
Sherlie H. West, Cochairman
Professor of Agronomy
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy.
Rex L. Smith
Associate Professor of Agronomy

I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy.
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy.
Ray E. Goddard
Associate Professor of Forestry
This dissertation was submitted to the Graduate Faculty of
the College of Agriculture and to the Graduate Council,
and was accepted as partial fulfillment of the requirements
for the degree of Doctor of Philosophy.
August, 1974
Dean, college ot Agriculture
/
Dean, Graduate School

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