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

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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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Agronomy thesis Ph. D
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Thesis (Ph. D.)--University of Florida, 1974.
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Includes bibliographical references (leaves 86-93).
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by Ray Judson Varnell.
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Typescript.
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Vita.

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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,

whor 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.


iii















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 ...... 60









Coleus ...... ...................................... 71

DISCUSSION .............. ................... ... ......... 72

Lupinus .... .... ....... ...... .... ................ 72

Pinus ....... ..... ......... .... .......... ... ...... 78

APPENDIX ... .... ......... ....... .... ........ ... ...... 84

LITERATURE CITED ............................... ........ 86

BIOGRAPHICAL SKETCH ................. .... ............. 94















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 pg

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


vii









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









which disrupt the normal course of development in the

apical meristem. 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








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. 1B).

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; O =
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.























B









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 pa 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









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 (Binning

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









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, Plantefols (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 "m4risteme

d'attente" (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 "meristeme

medullaire," which is the same as the rib meristem.

The concept of an inactive "meristeme d'attente'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









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 al. 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,









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.










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; Nougarede

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).









The data of Steeves et al. (1969) suggest a longer G1

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 1954;

Gifford and Tepper 1962a, 1962b). Hence, the RNA content

and the physiological activity of a cell appear to be

associated.

Nougarede (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

Nougarede'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










Nougarede'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








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.









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









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 260C day- and 180C 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.








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 40C. 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










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 pg of hormone.

Growth regulator was applied to lupine meristems at

the site where the next primordium was expected to arise,

the so-called Il site. This site was determined by visually

projecting the phyllotactic spiral around the apical meristem

through an arc of 1360 beyond the last-formed primordium.

The arc of 1360 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 I1 sites or on

the summit of the meristem. The I1 sites in coleus, a decus-

sate species, are located 900 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.










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 p 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.










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, destined 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








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 700C 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 350C, 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 destined 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 temperature, did not reduce affinity of

pine tissue for NYS.

Enzymic extraction of protein reduced NYS staining









of both pine and lupine tissues. Protease, at 1% concen-

tration, in 0.2 M cacodylate buffer, at pH 7.2, at 350C,

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 270C

for 30 minutes, followed by fresh pyridine at 600C 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 900C 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.









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.









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 I1 (Table 1).

In the case of one elapsed plastochron, Il would have

become P1, and P1 would have become P2 (Table 1). It follows

from (2) above that, if treatment at Il was effective, after

one elapsed plastochron, P2 would be displaced towards P1,

resulting in a smaller-than-average angle of divergence

P1-P2. Displacement of P2 towards P1 would further result

in P2 being further away from P3 and a larger-than-average

angle P2-P3.

A similar rationale applied to the situation of more

than one elapsed plastochron. The only added factor was

that 12 may have been displaced if treatment at I1 was

effective. Then, after two or more elapsed plastochrons

and after 12 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 Il would not affect I3, and 13 would be located

1360 of arc around the meristem from I2, regardless of

whether 12 was displaced.






















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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 P2-P3 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 Il, 12, and 13, the three areas of intervening

peripheral zone between the initiation sites, and three









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

1360 beyond the last-formed primordium to the presumptive

site of I thence continuing through another arc of 1360

to I2, and similarly on to 13.

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., Il arose first, 12 second, and 13

third. Existing primordia were numbered in reverse order of

formation; i.e., the youngest as P1, 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.

I= sample points in initiation sites; Fz =
sample points in intervening peripheral zone;
C= sample points in central mother cell zone;
B1' 2' P3, ....= leaf primordia, numbered
in order from youngest to oldest.






36
















P





2

~48~3 P~1114~,p


~TL~f~p

P64















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.















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** S~


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.









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 P1 and P2 was 90

(Fig. 3), as compared to an average of 134.50 for untreated

meristems (Table 2) and 1390 for unaffected angles in

treated meristems. The angle P -P2 after 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 I1 evidently was

displaced instead. P1 must not have been displaced

significantly, because when harvested the angle P2-P3































Fig. 3 Displacement of P1 towards P2 following treatment
with 1.5 pg IAA in transaction of apical meristem
of Lupinus albus, stained with alkaline fast
green. X 300.

A= apical meristem; P1, P2, P3, .... = leaf
primordia, numbered in sequence from youngest
to oldest.








41










































I l



I j





,...,../
46r









was only 1510 which was less than two standard deviations

from the average. If P1 had been displaced, the angle

P2-P3 would have been significantly greater than the average

(Table 1).

Another meristem, harvested eight days after treatment

with IAA, showed P1 displaced 2820 from P2 (Fig. 4). In this

case, enough time elapsed since treatment for 12 to develop

into P and Il into P2 (plastochron 2, Table 1). Hence,

12 was displaced around the meristem away from the point

of treatment and away from Il. I1 was not displaced,

because the resulting angle of divergence between P2 and

P3 of 121.50 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 pg IAA in transaction of apical
meristem of Lupinus albus, stained with alkaline
fast green. X300.

A = apical meristem; P, P2, P3' .... = leaf
primordia numbered in sequence from youngest to
oldest.












































.CL qLf'

3);r, L* v
I r ),F'c









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 (0), 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.






















SI10
i3


z
z 100


I-
z
W 90
z


--- -* -0-


*

**------.--
*


I


80o


I p I I I I, p


0 1 2 3 4


5 6 7 8 9 10 II 12

TIME (DAYS)


Ii






























m
E
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wo

04



0
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0


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Ln





























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





























200

180

160

140

120

100

200

180

160

140

120

100


2 4 6 8 10
TIME (DAYS)









et 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 (1), expressed as percentage of
lanolin controls (----), of Lupinus albus.
Each point is the average of three replications.




















300 A





OR
200 -


*
Z 100-
0



S300 B
Z
O
z
0
0 200 -


_i j


100

2 4 6 8 10
TIME (DAYS)



























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 (6), 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.











































2 4 6 8
TIME (DAYS)









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.









Pinus

Morphological Analyses of Treatments

Morphogenic effects of growth regulators applied to the

shoot apical meristems 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 pg 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











r N
^XH 01
N W zl









sectioning of the shoot tips extended 150 to 200 p 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 meristems (0),
expressed as percentage of lanolin controls (---),
of Pinus elliottii. Each point is the average of
three replications.


















































2 4 6

TIME (DAYS)


280

260

240

220

200

180

160

140

120

100


z
0
I-


w
01

to
z
0
0

z


,K









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 (*), expressed
as percentage of lanolin controls (----, of Pinus
elliottii. Each point is the average of three
replications.













































100-

80o -

60 -
f01 I-
2 4 6 8 10
TIME (DAYS)
































Fig. 12 Change with time in unsaturated lipid concentration
in the central mother cell zone of kinetin-
treated meristems (0), expressed as percentage
of lanolin controls (---), of Pinus elliottii.
Each point is the average of three replications.






67
















S160

" 140 *
140
z
0 10 -
O
100
sI-
2 80 \ -,
w
Z 60-
0
o 40 -
cl 0
-J 2 4 6 8 10
TIME (DAYS)








(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
(0), expressed as percentage of lanolin controls
(----), of Pinus elliottii.
Each point is the average of three replications.





















240
A.
220

200

180

160

140

120

100 .*--- --..

300 f B


200



100


I I I I
3 2 4 6 8
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 500 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








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









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









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 wel 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









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









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









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








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









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 al. 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 in 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-









nation 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

Nougarede'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








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









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:










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.















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