THE ROLE OF ABSCISIC ACID ,_ ; :g -. 3 51,55:_-_;_.:;
. IN THE DORMANCY 0F
' .‘APPLEWUS “walls-5593‘ '

V :TDEssertatTon for the Degree of Ph D, .7 7 E
I ‘ ’ M'CHIGAN STATE UNWERSITY 5 A
ORLANDO BALBOA ZAVALA _
‘ 1975 _

 

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g This is to certify that the H,
“9.5 'T - I

thesis entitled

3 “T H
The Role of Abscisic Acid in the Dormancy of Ap
." (Pzrus_____ malus L.) Seeds
presented by
Orlando Balboa Zavala
has been accepted towards fulfillment

of the requirements for

Ph. D. degree in Horticulture

 

jor professor

Date June 23, 1975 :

0-7639

5:

 

 

 

 

Wm j

 

‘\ ABSTRACT

THE ROLE OF ABSCISIC ACID IN THE DORMANCY
0F APPLE (PYRUS MALUS L.) SEEDS

 

By

Orlando Balboa Zavala

The main goal of this research was to contribute to an understand-
ing of dormancy. Abscisic acid (ABA) was identified by gas-liquid
chromatography and mass spectrometry in extracts of methylene chloride
that has been partitioned against acidic methanolic extracts from dormant
apple seeds of 'Mclntosh'. Both 'free' and hydrolysable ('bound') ABA
were found in the water diffusate from the seeds, and in the methanolic
extracts of seed coats, cotyledons and embryonic axes. The concentra-
tion of 'free' ABA was highest in the embryonic axis, intermediate in
the seed coat, and least in the cotyledons.

Levels of both 'free' and 'bound' ABA of seeds declined during
stratification at both 5° and 20°C. Increasing the temperature to 25°C
for one week after 3 weeks at 5°C induced secondary dormancy, and
nullified the effect of prior chilling on germination. This treatment
also caused a marked reduction in 'free' and 'bound' ABA in the seeds
within 2 days.

During seed development within the fruit, two maxima occurred
in the level of 'free' ABA in the embryo, one in July-August coincident
with high germination capacity (62%) of excised embryos; the other at

maturity in late September, when excised embryo failed to germinate.

Orlando Balboa Zavala

However, germination capcity had declined to nil 2 weeks prior to
rise in the ABA content. Levels of 'bound' ABA remained relatively
low until the approach of maturity and then rose slightly. Hand
defoliation of branch units on July 30 - when embryos were nearly
full size - reduced the concentration of both 'free' and 'bound' ABA
in embryonic axes of seeds collected 5 weeks later, and favored the
germination of excised embryos. Lesser effects of defoliation were
noted at maturity 11 weeks after treatment, and germination of
excised embryos occurred regardless of treatment.

Drying the seeds on removal from the fruit increased their
content (nanograms per gram fresh weight) of both 'free' and 'bound'
ABA, the magnitude of the effect varying with time of harvest.

Application of succinic acid-2,2-dimethylhydrazide to growing
fruit lowered the ABA content of the embryo at harvest and reduced
the germination capacity of the seeds slightly. Inclusion of inhibitors
of gibberellin synthesis in the stratification and/or germination
medium had no effect on stratification requirement of intact seed,
provided the concentration used were not toxic.

The results of these studies do not support the view that
entrance to and release from dormancy in response to chilling are

controlled by endogenous levels of ABA.

THE ROLE OF ABSCISIC ACID IN THE DORMANCY
OF APPLE (PYRUS MALUS L.) SEEDS

 

By

Orlando Balboa Zavala

A DISSERTATION
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of
DOCTOR OF PHILOSOPHY

Department of Horticulture

1975

DEDICATED TO

MY WIFE, DAUGHTER, AND SON

ii

ACKNOWLEDGMENT

My grateful thanks are due to Dr. F. G. Dennis for his guidance
throughout this investigation and his helpful criticism during the
course of writing this thesis.

Special thanks are extended to Dr. M. J. Bukovac for his
guidance, helpful advice and for the use of the gas-liquid chromato—
graphy equipment, and to Dr. C. J. Pollard for his suggestions and
kindness, and Dr. J. A. Flore for serving on the Guidance Committee.

Special gratitude to Dr. E. A. Mielke for his assistance with
the GC-MS equipment.

Appreciation is extended to Dr. C. C. Sweeley for making the
mass spectrometer facilities available and to Mr. J. E. Harten for
his technical assistance in performing the analysis.

A very special thanks is extended to my wife Lilianafor her

love, encouragement, and understanding during this program of study.

LIST OF TABLES . . . . . . . . . . .
LIST OF FIGURES . . . . . . . . . . .
ABBREVIATIONS . . . . . . . . . . .
INTRODUCTION . . . . . . . . . . .
LITERATURE REVIEW . . . . . . . . . .
DEFINITIONS . . . . . . . . . .
EXOGENOUS CONTROL OF DORMANCY IN APPLE AND PEAR SEEDS

THE MECHANISM OF THE EFFECT OF CHILLING ON APPLE AND PEAR

SEEDS

TABLE OF CONTENTS

IMPERMEABLE SEED COAT . . . . .

MECHANICALLY RESISTANT SEED COAT. . .

RUDIMENTARY EMBRYO . . . . . .

IMMATURE EMBRYO OR MORPHOLOGICALLY MATURE

PHYSIOLOGICALLY DORMANT EMBRYO . . .

ENZYMES . . . . . . . . .
GROWTH PROMOTERS. . . . . . .
GROWTH INHIBITORS . . . . . .

INTERACTION OF PROMOTERS AND INHIBITORS .

METABOLISM OF ABA DURING CHILLING . .

SUMMARY . . . . . . . . . .

iv

Page

vi

10
12
13

13

Page

SECTION I: THE ROLE OF ABSCISIC ACID IN THE DORMANCY OF APPLE
(Pyrus malus L.) SEEDS. I. ROLE OF ABSCISIC ACID DURING THE

 

ONSET OF DORMANCY . . . . . . . . . . . 15
ABSTRACT . . . . . . . . . . . . lb
EFFECT OF SEED DEVELOPMENT ON ABA CONTENT AND GERMINA-

TION . . . . . . . . . . . . . l7
EFFECT OF DEFOLIATION . . . . . . . . . 22
EFFECT OF DRYING SEED . . . . . . . . . 22
LITERATURE CITED . . . . . . . . . . 25

SECTION II: THE ROLE OF ABSCISIC ACID IN THE DORMANCY OF APPLE
(Pyrus malus L.) SEEDS. II. ABSCISIC ACID LEVELS DURING

 

STRATIFICATION . . . . . . . . . . . . 26
ABSTRACT . . . . . . . . . . . . 26
INTRODUCTION . . . . . . . . . . . 26
MATERIALS AND METHODS . . . . . . . . . 27
RESULTS . . . . . . . . . . . . 31
DISCUSSION. . . . . . . . . . . . 39
REFERENCES. . . . . . . . . . . . 41

APPENDIX: THE ROLE OF ABSCISIC ACID IN THE DORMANCY OF APPLE
(gyrus malus L.) SEEDS. III. EFFECT OF GROWTH RETARDANTS ON

 

SEED DORMANCY AND ABA . . . . . . . . . . 43
ABSTRACT . . . . . . . . . . . . 43

EFFECT OF THE PRESENCE OF GROWTH RETARDANTS DURING
STRATIFICATION ON GERMINATION . . . . . . . 45
EFFECT OF SPRAYS ON SADH ON GERMINATION AND ABA CONTENT 45
LITERATURE CITED . . . . . . . . . . 48
SUMMARY AND CONCLUSIONS . . . . . . . . . . 49

BIBLIOGRAPHT............ 51

 

Table

LIST OF TABLES

Page
Section One

Effect of defoliation on July 30 on the level of 'free'
(F) and 'bound' (H) ABA (ng/g fresh wt.) in apple seed
cv. 'Golden Delicious'. Means for 2 samples of 25 seeds
each.

Effect Of drying, on the concentration of 'free' (F) and
'bound' (H) ABA (ng/g fresh wt.) in apple seed, cv.
'McIntosh'. Means for 2 samples of 25 seeds each.

Section Two

Effect of stratification time and temperature upon ABA
content of 'McIntosh' apple seeds and the water diffusate
from the seeds. Means for 2 replicates of 25 seeds each.

Appendix

Effect of tree sprays of SADH on germination (Z ) of intact
seeds (I) or excised embryos (R) of apple after chilling
either in the fruit or in moist sand. Values are means for
2 samples of 25 seeds each.

Effect of SADH treatment of trees on the ABA content of

apple seed cv. 'Jonathan'. Values are means for 2 samples
of 25 seeds each.

vi

LIST OF FIGURES

Figure Page

Section One

'Free' and 'bound' ABA during development of embryo of

apple seeds cv. 'McIntosh', in relation to their germination
capacity. Values are means for 2 replicate samples of 100
embryos each.

Section Two

Flow diagram of procedure used for preparation of fractions
containing 'free' and 'bound' ABA.

Concentration of 'free' and 'bound' ABA in the water
diffusate and in methanol extracts of seed coat, cotyledons
and embryonic axis of apple seed during stratification at

5 i2 and 20 i2°C. Each bar represents the mean of at

least 4 replicates of 25 seeds each. Per cent of germination
is indicated in parenthesis above each treatment.

Effect of inducing secondary dormancy with high temperature
upon concentration of ABA of apple seeds cv 'McIntosh'.

A. ABA content of seed during one week at 27 il°C after
treatment in A. In B, 0 represents the day the seeds were
returned to 5 i2°C as well as the day on which stratification
of the control, continuous chilling began. Each value is

the mean of at least 4 replicates of 25 seeds each. 'Bound'
ABA (o 0), 'free' ABA (x x), germination under
continuous chilling (x --- x); germination of seeds returned
to 5 i2°C after one week at 27 il°C (o —-- o).

 

vii

Guidance Committee:

The Paper-Format was adOpted for this dissertation in
accordance with Departmental and University regulations. The
dissertation body was separated into two sections and one
appendix. The first section and the appendix were prepared
for publication in HortScience. The second was styled for

publication in Physiologia Plantarum.

viii

ABBREVIATIONS

The following abbreviations will be used in this dissertation.

Names given in parentheses are additional common or trade names.

ABA abscisic acid
Alar succinic acid-2,2-dimethylhydrazide (SADH, B-9, B-995)
AMO-l6l8 4-hydroxy-5—iSOpropyl-Z-methylphenyltrimethylammonium

chloride l-piperidine carboxylate

BA N6-benzyladenine

CCC (2-chloroethyl) trimethyl ammonium chloride (Cycocel,
chlormequat)

GA denotes the series of gibberellins -— use of a subscript

denotes a specific gibberellin, as GAl

GLC gas—liquid chromatography

GC-MS combined gas—liquid chromatography and mass spectrometry
IAA indoleacetic acid

Zeatin 6-(4-hydroxy-3~methyl—2-butenylamino) purine

ix

INTRODUCTION

INTRODUCTION

The rest period of seeds of temperate woody perennials allows
these plants to endure low temperature stress. Knowledge of the
‘mechanism(s) underlying this phenomenon may lead to methods for
more rapid seed germination, and the production of fruit crops in
regions of insufficient chilling. The main goal of this research
was to contribute to an understanding of dormancy.

Apple seeds were chosen for this study because of their avail-
ability and the considerable body of existing information on this
species. Abscisic acid (ABA) and its glucose ester have been
identified in apple seeds and ABA is known to inhibit their germ-
ination. Furthermore, recent work has suggested that abscisic acid
(ABA), an endogenous growth inhibitor present in a wide range of
higher plants, is responsible for the rest period, and that chilling
reduces the level of ABA in seed tissue, allowing germination to
proceed. One of the advantages in studying ABA is that it can be
measured by gas-liquid chromatography without recourse to bioassay.

This study was designed to do the following: (a) to measure
the levels and distribution of ABA in apple seeds during their
entrance into dormancy and the breaking of dormancy by chilling;

(b) to determine the effect of temperature on the decline of ABA
during stratification; and (c) to determine the effect of raising

the temperature prematurely during chilling, and thereby inducing

dormancy, on the level of ABA. Preliminary experiments were conducted
with inhibitors of gibberellin synthesis to test the role of

gibberellin synthesis in the breaking of dormancy.

 

LITERATURE REVIEW

 

LITERATURE REVIEW

Introduction

This review will be concerned primarily with the literature
relating to dormancy in apple and pear seeds. The seeds of many
trees display some degree of dormancy (Kramer and Kozlowski, 1960),
which can be either advantageous or disadvantageous. For the
nurseryman it is disadvantageous, however, it may also be an
advantage because the nurseryman can keep or store the seed source.
However, seed dormancy is advantageous where survival is threatened
by adverse conditions.

Dormancy is usually separated into rest (also called constitutive
dormancy, primary dormancy, innate dormancy, internal dormancy or
endogenous dormancy) and quiescence (also called imposed dormancy,

external dormancy, exogenous dormancy or false dormancy).

Definitions:

The following definitions will apply in this dessertation:

Rest is defined as lack of germination capacity of the seed under
adequate environmental conditions such as temperature, moisture, light,
etc., and quiescence as dormancy imposed by the unfavorable levels of
any of these factors. Dormancy is defined as suspended growth of the

8Bed and will include either rest or quiescence. Stratification will

 

refer to the storage of the seeds in a moist environment, regardless
of temperature, and chilling to stratification at temperatures between
0 to 10°C. The chilling requirement delays germination until after
‘winter, when conditions again become favorable for growth (Kozlowski,
1971). In hot, dry climates, dormancy permits survival during

periods of moisture stress. Thus, dormancy serves a very useful

function in nature (Wareing, 1963).

Exogenous Control of Dormancy in Apple and Pear Seeds

The seeds of many Rosaceous species exhibit rest at harvest and
must undergo changes, usually called after—ripening, before germination
can occur. Seeds of most temperate zone Py£u§_species which enter a
state of dormancy can be made to germinate by continuous exposure to
low temperature (1 — 10°C) in a moist medium for at least 6 to 12 weeks.
These conditions resemble those which the seeds encounter in nature.
If chilling is insufficient, dwarfed and stunted plants are produced
(Flemion, 1934).

Light appears to play a minor role in breaking the dormancy of
apple seeds, although red light promotes the germination of isolated
embryos of dormant seed (Smolenska and Lewak, 1971). Oxygen, on the
other hand, may not be necessary, since embryos germinated more
rapidly when the rest was broken by N2 than by chilling (Tissaoui
and Come, 1973).

Secondary dormancy, which is as deep or deeper than primary
dOrmancy, may be induced in seeds partially after-ripened by exposure
to low temperature and then transferred to temperatures of 25 to 30°C.

Abbott (1955) showed that transferring excised embryos of apple to

 

temperatures of 25 to 28°C after 3 weeks at 3°C induced such a secondary
dormancy. The critical temperature lies between 18 and 21°C, higher
temperature reducing and lower temperature increasing the germination

of the seeds (Visser, 1956b).

Exogenous application of several growth promoters stimulates the
germination of excised embryos and partially after-ripened seeds of
apple and pear. Badizadegan and Carlson (1967) reported that BA alone
stimulated germination of excised embryos of dormant apple seeds.
Kaminski and Pieniazek (1968) also reported that BA, GAB’ and GA4+7,
singly or jointly, greatly enhanced the germination of dormant or
partially stratified embryos. Lewak g£_a1_ (1970) showed that BA,
IAA, and GA3 stimulated the germination of excised embryos, the
effect being light independent. The stimulating effect of GA included
in the stratification medium varies directly with the concentration
and inversely with the duration of cold stratification (Come and
Durand, 1970). Westwood and Bjornstad (1968) observed an increase in
germination of pear seeds when GA3 was included in the stratification
medium, but not when seeds were treated after stratification.

ABA counteracts the effects of GA3, CA or BA on both dormant

4+7
and partially stratified apple embryos (Rudnicki SE Ei" 1971). In
both dormant and nondormant pear embryos ABA inhibits the incorporation
Of 32? into several RNA fractions (Khan and Heit, 1969). GA is more
effective than BA in counteracting this inhibition, although the

Pattern of labeling of RNA varies depending on the promoter used.

Eflgogenous Control of Dormancy in Apple and Pear Seeds

A classification of types of dormancy was proposed by Crocker

 

(1916). He attributed dormancy to: (a) immaturity of the embryo;
(b) impermeability of the seed coat to water, (c) mechanical
resistance of the seed coat to embryo growth, (d) low permeability
of the seed coat to gases; (e) dormancy resulting from a metabolic
block within the embryo itself, (f) a combination of the above,
or (g) secondary dormancy. Kozlowski (1971) reclassified these
into five groups: (a) impermeable seed coat, (b) mechanically
resistant seed coat; (c) immature embryo, (d) rudimentary embryo;

(e) morphologically mature but physiologically dormant embryo.

Impermeable seed coat. When the seed coat is removed from dormant

 

apple seeds, some of the embryos germinate but the seedlings are
dwarfed (Flemion, 1934). The effects of low temperature stratification
appear to be cumulative, since partially stratified seeds give rise

to semidwarfed seedlings, whereas fully stratified seeds give rise

to normal plants (Flemion, 1934). Visser (1954, 1956b) found that

the seed coverings are barriers, not to water, but to oxygen uptake,
since in partially stratified apple seeds, the seed coverings, parti—
cularly the endosperm, reduced gas exchange to and from the embryo.
Removal of a small portion of the endosperm increased the respiratory
activity 3-fold. According to Visser, the barrier is more pronounced
at higher temperatures. Visser's work was substantiated by the
observation of Nikolaeva and Knape (1974) that the seed coat decreased
oxygen absorption by 90% and the endosperm alone by 70% in apple seeds,
regardless of the state of dormancy. However, Tissaoui and Come (1973)

were able to break rest by holding apple embryos in pure nitrogen and

 

concluded that oxygen was not required. From the above, one can
conclude that the seed coat in apple is permeable to water but

may not be readily permeable to oxygen. However, if oxygen is not
required to break rest and if permeability to oxygen does not
change during chilling, then one must look elsewhere for the

controlling factor in dormancy.

Mechanically resistant seed coat. Removal of the seed coat of

 

non-stratified apple seeds permits a low rate of germination (up to
20%) (Flemion, 1934, Smolenska and Lewak, 1971) suggesting that
mechanical resistance of the seed coat may be involved in dormancy.
However, radicle elongation is slow and the major site of dormancy
must lie within the embryo. Thus the seed coat apparently acts as a
physical barrier to the germination of the dormant embryo. Once
embryo dormancy has been overcome by chilling, the radicle is capable

of penetrating the seed coat and germination occurs.

Rudimentary embryo. Pyrus embryos are fully developed and

 

mature and stratification does not result in any gross morphogical

change.

Immature embryo or morphogically mature but physiologically

 

dormant embryo. The differences between anatomical and physiological

 

immaturity are subtle. However, several species have embryos which
are wall differentiated when the seed is diapersed, but which must
be imbibed in water to permit further growrh before germination can

occur (Villiers, 1972). This might be considered as either the final

 

step in seed development or the initial stage of germination. Pyrus
seeds typify physiological dormancy, for chilling affects only the

ability to germinate, rather than morphological changes.

The Mechanism of the Effect of Chilling on Apple and Pear Seeds.

Several hypotheses have been proposed to explain embryo dormancy,

some involving the activity of enzymes, others synthesis and/or

degradation of hormones.

Activity of enzymes such as peroxidase, succinic

Eanes .

dehydrogenase, lipase and proteases increased during cold stratification

 

0f excised embryos of apple, var. Antonowka, but remained unchanged
in non-chilled embryos (Nikolaeva and Yankelevich, 1974). 'Free' and
'bound' peroxidase increased during cold stratification in both intact
Seeds and isolated embryos of apple (Rychter and SZpakowics, 1974),
While phosphatase showed two peaks of activity (Rychter 35 31., 1971).
when isolated embryos were held in darkness at 25°C after different
Petiods of low-temperature stratification, GA3 and GA7 stimulated
ph03phatase activity between 10 and 50 days and 0A4 after 30 days of
ABA inhibited phosphatase activity but had little

af ter-ripening .

effect on its appearance. Changes in enzyme activity during cold

Stratification may be a result, however, rather than the cause of

the breaking of dormancy.

GrOWth promoters. Several gibberellins have been reported to occur

:1
1:1 immature Pyrus seeds, including CA4 and GA7 in apple (Dennis and

N
its ch, 1966) and GA45 in pear (Bearder 9.2 21., 1975). GA4, GA7 and

k

 

GA9 have been identified in mature apple seeds (Sinska g 31., 1973).
The participation of these compounds in seed dormancy is far from
clear however. 6A4 and GA7 are present in negligible amounts in
dormant seeds (Sinska and Lewak, 1970). However, GA4 increased

40,000-fold after 30 days of cold stratification only to fall to
its initial level before the seeds were capable of germination,

while GA7 remained-low during the entire process of stratification

Thus, the observed increase in

(Sinska and Lewak, 1970, 1973).
0A4 also

 

3A4 may or may not be a prerequisite for germination.
increases when isolated embryos are exposed to red light; in this

case a correlation was reported between the increase and germination

(Smolenska and Lewak, 1971). Excised embryos of stratified seeds

incorporated l4C—mevalonate into GA), and into GA7 if seeds were

allowed to dry (Sinska and Lewak, 1974).
Letham and Williams (1969) identified 3 cytokinins in the flesh

One resembled zeatin, a second zeatin

and seeds of apple fruitlets.
ri“beside and the third zeatin ribotide. Dorman apple seeds also

Q0l’ltain 'free' and 'bound' cytokinins with the bound forms reaching
the higher levels after 5 weeks of cold stratification. (Borkowska

and Rudnicki, 1974).
Luckwill (1948) found a promoter of tomato ovary enlargement in

e3‘Kt‘racts of immature apple seeds but only traces in dry seeds.

Following alkaline hydrolysis, or after stratification at 5°C, the

D resence of an auxin (called auxin 2) could be demonstrated (Luckwill,

l 957). Kawase (1958) also reported an increase in a promoter of

m

g

coleoptile elongation in the seed coat, endosperm and embryo of

 

10

apple seeds during after—ripening.

Growth inhibitors. A marked decrease in endogenous growth

inhibitors has been observed during cold stratification of both

apple (Luckwill, 1952; Kawase, 1958; Rudnicki, 1969) and pear

seeds (Strausz, 1970). Luckwill (1952) reported that dormant

seeds contained an inhibitor of Avena coleoptile section elongation

Which declined during after—ripening. The endosperm contained the

highest concentration of the inhibitor while the tests and embryo

contained progressively less, in the ratio 30:13:1. Later Luckwill

 

(1957) reported the presence of two inhibitors in immature apple

Seeds, Inhibitor l was shown to be toxic and therefore not biologi-

Cally important. 0n the other hand, inhibitor 2 was active over a

wide concentration range and reached the highest level at stage 3 of

Seed development (seed fully mature). In the seed coat of dormant

apple seed, Kawase (1958) found a strong inhibitor of Avena
coleoptile elongation which declined to its lowest level during

St:I‘atification at 5°C.

Phloridzin, a phenolic compound, is abundant in apple tissues

(Hutchinson et a1., 1959), and retards root growth of apple seedlings

in Water culture at a concentration of 10‘4M (Borner, 1959). Woodcock

( 1947) isolated phloridzin from apple seeds, and found that it decreased

from 8% of the fresh weight to 1% within 8 weeks after petal fall and

after 18 weeks it completely disappeared from the seed. However,

P.ZLeniazek and Grochowska (1967) demonstrated that phloridzin, phloretin

 

”—
u-l

 

our

...

v"

 

to.

-
o<w

_
u».-

“b

.~l

 

11

and chlorogenic acid were the main phenolic compounds present in

dormant apple seeds. Phloridzin was at its highest level in the
seed coat and dropped to negligible amounts during stratification,

while increasing in the embryo. Phloretin remained unchanged in
the seed coat during after-ripening, while embryos showed only
traces of phloretin after 11 weeks of cold stratification.

Chlorogenic acid, on the other hand, completely disappeared from

the seed coat and endosperm during stratification regardless of

temperature. Phloridzin alone had no effect on germination of apple
embryos but when applied with GA and/or BA greatly enhanced the

 

88mination capacity of dormant and nondormant embryos (Kaminski

and Pieniazek, 1968).
Come and Mittard (1970) reported a negative correlation between

percentage germination of seeds stratified within the fruit and the
phloridzin content of the seed coat following UV irradiation. Both

phlOridzin and chlorogenic acid were labile when exposed to UV light.

They concluded that these compounds absorb oxygen and thereby reduce

ltS availablity to the embryo.
Rudnicki (1969) identified ABA in dormant apple seeds, and reported

that all inhibitory activity disappeared from the water diffusate after

3 Weeks of chilling.
In pear seed, Strausz (1970) found a significantly higher
identified as ABA in the seed

Q<DIncentration of an inhibitor tentatively
During chilling, the

S
urface and seed coat than in the embryo.
remained unchanged in the

Q
Oncentration decreased in the embryo but

8
Qed coat. Although Okhawa (1974) was not able to identify ABA in

k

'7—
12

immature pear seeds, G. C. Martin and F. G. Dennis (personal communi-

cation) have recently done so.
As noted above, GA4 increased markedly during cold stratification

of apple seeds. Inclusion of ABA in the stratification medium completely
prevented this increase, and the GA!} which had accumulated during after-

ripening disappeared when ABA was added to the incubating medium at
20°C (Rudnicki _e__t__a_l_., 1972). According to these workers, ABA may
inhibit 6A4 synthesis and/or promote its breakdown.

Endogenous growth promoters

 

Interaction of promoters and inhibitors.

appear to be at their lowest levels in dormant apple seeds (Sinska
and Lewak, 1970; Smolenska and Lewak, 1971; Borkowska and Rudnicki,

1974) and progressively increase during chilling, while the reverse
is true for ABA (Rudnicki, 1969). These factors together with the
reSUltS of application of growth regulators to dormant and nondormant

Seeds suggest an interaction of promoters and inhibitors in controlling

Khan (1971) proposed that germination in some seeds

Seed dormancy.
is controlled by interaction between cytokinins, gibberellins and

inhibitors with cytokinins playing a permissive role. According to
However, inhibitors such

this hypothesis, germination requires GA.
as ABA can block the action of GA, and cytokinins are necessary to
This hypothesis does not hold for apple

Q0lit-Iteract this inhibition.
SQst, since both cytokinins and gibberellins stimulate germination

0 f excised apple embryos (Kaminski and Pieniazek, 1968; Badizadegan
Although

and Carlson, 1967), and ABA counteracts the effects of both.

ethiDgenous ABA decreases dramatically during 3 weeks of cold stratifi—

cation (Rudnicki, 1969) and at the same time, cytokinins and gibberellins

L,

are reaching their highest levels (Sinska and Lewak, 1970; Borkowska

and Rudnicki, 1974) apple seeds are incapable of germination at

this time.
Metabolism of ABA during chilling. Several workers have reported

a decline in the level of ABA or ABA—like inhibitors during cold
Stratification (Sondheimer 33331., 1968; Williams _e__t 31., 1973;
Rudnicki, 1969; Strausz, 1970), although the same decline occurred

during warm stratification in hazel (Cory_1us avellana L.) seeds

The question thus arises as to the fate of

 

(Williams £11., 1973).
Sondheimer 531531 (1974) reported the

ABA during 3 tratificat ion .
l4C—labelled phaseic acid, dihydrophaseic acid, and an

appearance of
Unidentified compound in dormant and nondormant ash seed (Fraxinus
They suggested that ABA is

americana L.) after feeding Z-lT‘C-ABA.
metabolized to phaseic acid and this in turn is converted to dihydro-
They supported this by isolating the labelled phaseic

phaseic acid.
acid and showing that it was converted to dihydrophaseic acid.

was the only identified product of l—lTC—ABA metabolism

However, CO2
in apple seed during stratification at 5°C (Rudnicki and Czapski, 1974).

These authors suggested that other metabolites could have been formed
One such product may be abscisyl-D—glucoside,

and remained undetected.
I:‘e130rted by Milborrow (1970) to be formed in several tissues, and

identified in apple seed by Bulard 3.2 31 (1974)

S
l"Jillnary

The failure of freshly harvested Pyrus seeds to germinate is caused

D
binarily by the dormancy of the embryo, which may be overcome by chilling.

 

k

l4

Raising the temperature to 25°C or more after dormancy is broken

completely negates the effect of previous chilling. Light has little

effect on dormancy. Both gibberellins and cytokinins are effective

in promoting the germination of excised embryos, particularly when

dormancy has been partially broken by chilling. ABA, on the other

hand, inhibits the germination of nondormant embryos and counteracts
the effect of promoters on dormant embryos.

The available evidence suggests that dormancy of apple and pear
seeds is associated with low levels of gibberellins and cytokinins and

Chilling apparently reduces the content of ABA

 

high levels of ABA.

and promotes the synthesis or release of promoters, as well as

certain hydrolytic enzymes.

Several aSpects of the role of ABA remain to be established, viz.:

(8.) its distribution within the seed; (b) its role in the induction

of both primary and secondary dormancy; and (c) the effect of tempera-

ture on its disappearance during stratification.

 

 

SECTION ONE

 

THE ROLE OF ABSCISIC ACID IN THE DORMANCY OF APPLE (Pyrus malus L.)
SEEDS. I. ROLE OF ABSCISIC ACID DURING THE ONSET OF DORMANCY

 

 

 

 

 

 

THE ROLE OF ABSCISIC ACID IN THE DORMANCY OF APPLE (Pyrus malus L.)
ROLE OF ABSCISIC ACID DURING THE ONSET OF DORMANCY

SEEDS O I 0

Abstract. Abscisic acid (ABA) levels were measured during the
growth and development of apple embryos of the cultivar 'McIntosh',
using gas-liquid chromatography. Two maxima in 'free' ABA were
The relatively high

observed; in mid—August and in late September.

rate of germination of excised embryos was noted when the first

 

maximum was observed; and there was negligible germination of the
embryos when the second maximum was reached. A similar lack of
correlation between germination capacity and ABA content was noted
in a second cultivar. Branch defoliation in late July, when

embryos had reached 75% of their final fresh weight, reduced the
ABA content of embryonic axes and favored the germination of

embryos sampled 5 weeks after treatment, but did not prevent the
induction of dormancy. Drying seeds on removal from the fruit markedly
increased the content of both 'free' and 'bound' ABA in the embryonic
axes of immature seeds, and in most portions of mature seeds. I

COD-elude that the level of ABA is not the primary factor controlling

eIllblr’yo dormancy during apple seed development.

 

\

Luckwill (5) reported the presence of two growth inhibitors in
i
rumattire apple seeds, but did not measure quantitative changes in their
co
ncentration during seed development. Rudnicki (9) identified ABA

1'
n mattire apple seeds and observed that the level of a diffusible

15

¥_

 

l6

inhibitor of wheat coleoptile elongation, assumed to be ABA, declined
during stratification at 2 - 4°C. I have subsequently shown that the
ABA content of the seed decline during stratification, but this
decline is temperature—independent, Suggesting that ABA concentration
does not play a primary role in the breaking of dormancy by chilling
(l). A question remains as to its role in the induction of dormancy.
Several workers have observed an increase of ABA content during the
late stage of seed development such as in peach (2) and in a dormant
ground-nut cultivar (10). The primary purpose of this investigation

was to determine if ABA levels in the developing seeds are correlated

 

with their germination capacity. Desiccation is known to reduce
germination capacity of hazel seeds (8) and other species (3) and

to increase the ABA content of several plant tissues (4, 7). Work

of Mielke and Dennis (6) on the level of ABA in sour cherry primordia
Suggested that defoliation might reduce the ABA content of seed
tissues and thereby affect their dormancy. I therefore tested the
effect of defoliation in late summer on the aCCumulation of ABA in
the seeds.

Samples of fruits, cv. 'McIntosh', were collected from mature
trees in a commercial orchard at Leslie, Michigan, those of 'Golden
Delicious' from an abandoned orchard at East Lansing, Michigan.
Branches approximately 10 cm in diameter at the base were used for
defoliation studies, leaves being removed July 30, 1974, when the
embryos had reached approximately 75% of their final fresh weight.
The seeds were removed within two hours of harvest, 150 to 300 seeds

being collected for each treatment. Two replicates of 25

 

 

l7

(defoliation and drying studies) or 100 seeds (maturation experiment)
were used for extraction of ABA experiment, and 4 samples of 25
seeds each were tested for germination in petri dishes containing
moist sand at 20°C and 200 foot—candles of fluorescent light. Tw0
samples were tested with seed coat intact, 2 with seed coat removed
(excised embryos). The protrusion of the radicle through the
seed coat (intact seed) or geotropic bending (excised embryos) was
used as criterion for germination.

Seed retained for ABA determination were dissected into seed

coat, cotyledons and embryonic axes immediately on removal from

 

the fruit (maturation and defoliation studies) or after 4 months of
storage over anhydrous CaC12 at 20°C (drying experiment). The seed
portions were immersed in absolute methanol, ground in a mortar and
pestle and left overnight at 4°C. The Supernatant was evaporated
under vacuum at 40°C, redissolved in 25 ml of phosphate buffer,
fractionated and the crude acidic ('free') and base hydrolyzable
('bound') ABA were methylated and injected in the GC using an
electron capture detector as described previously (1).

Effect of seed development on ABA content and germination. 'Free'

 

ABA content of McIntosh embryos (cotyledons plus embryonic axes) rose
gradually from July 13 to August 17, dropped in mid—August through
mid—September, then rose again at fruit maturity in late September

(Fig. 1). Level of 'bound' ABA rose reaching the highest level in

early August fell gradually then remained constant until early September.

The level then rose slightly at maturity, paralleling the sharp increase

 

 

18

in'free'ABA. Seeds with intact testa failed to germinate regardless

of the time of sampling. Germination of excised embryos was negligible
on the first sampling date, even though the embryos had reached

almost 90% of the final fresh weight. Germination reached a maximum
of 62% on August 3, declined to a plateau then fell sharply to nil

as maturity approached. There appear to be no relationship between
ABA content and the germination capacity of excised embryos until

the approach of fruit maturity in late September, when the former

rose as the latter fell. However, the decline in germination

 

capacity occurred 2 weeks prior to the rise in the ABA content.

The content of 'free' ABA in 'Golden Delicious' seeds dropped
to a level of l/3 to 1/8 the original content between July 30 and
September 5, depending on the tissue sampled (Table l). Decreases
were also apparent in 'bound' ABA levels, except in the embryonic
axes, which contained twice as much in September as in July. Between
September and October the content of 'free' ABA rose in the seed
coat and embryonic axes but fell in the cotyledons. The level of
'bound' ABA paralleled that of 'free' ABA in the seed coat, but
declined rather than increase in the embryonic axis. No change
occurred in the cotyledons during this period.

Again, no germination occurred in intact seeds. Percentage
germination of excised embryos declined steadily from 28% on July 30
to 6% and 0% on September 5 and October 20, respectively. Comparing
the ABA content of the embryos only (cotyledons and embryonic axis)

with the percentage of germination did not reveal any apparent

 

 

 

 

l9

 

Fig. 1. 'Free' and 'bound' ABA during development of apple seeds cv.
'McIntosh', in relation to their germination capacity. Values are
means for 2 replicate samples of 100 seeds each.

 

20

(96) NOIlVNllNHBQ

 

 

    

 

 

 

8 8 8 8 8 2 0
I T I I j I
X \ \ 4 m‘a
/ s
“m X 0 m\
8
5L /
/X /4 Ci 1 mt
x\ 4\ / I
Q’
X 4 0 '1 ”(V
/ / - \ b
X G ‘0 O 1 '-
1 s
x " \/0 - 0‘9
o - cox”
I \ t
c X G O [€91
.9
*-
2 \
.E x h‘
\-
a \ .,_,
l . <3 6 ’3“ cl) 0'“
g g _. a, .t N

(mm-5M»; 0/60) NONOO vsv

|974

Fig. l

 

 

 

Table l .

21

Effect of defoliation on July 30 on the level of 'free'

(F) and 'bound' (H) ABA (ng/g fresh wt.) in apple seed cv. 'Golden

Delicious ' . Mean

for 2 samples of 25 seeds each.

Collect-ion time (1974)

 

 

 

7/30 9/5 10/20
Tis3ues F H F H F H
Non—defoliated
Seed coat 321 109 112 219 459 786
COtyledons 373 64 44 17 23 23
Embryonic axis 1333 298 298 269 577 485
Defoliated
Seed coat - — 153 171 200 473
cotyledons - — 47 18 46 43
- 143 194 378 533

EmbrYCmic axis

 

 

 

 

22

correlation between the two.
Effect of defoliation. The most noticeable effect of defoliation
was the marked reduction in 'free' ABA in the embryonic axis in

September 5 (Table l). Smaller reduction in the 'bound' ABA were

noted in the axes in September but not in October. At maturity, the

Seed coat contained less and the cotyledons more ABA than did the

control. Defoliation increased the germination of excised embryos

Sampled on September 5 (28% vs. 6% for the control), but no
germination took place in embryos sampled on October 20 regardless
of treatment. Germination capacity was inversely correlated with

ABA content as shown above in the September sample only. Stratifica-

tion did not affect the germination capacity of the seed sampled on

October 20 (96% vs. 90% in the control) after 12 weeks at 5 i2°C.
Effect of drying seed. Drying increased both 'free' and 'bound'
ABA in the embryonic axes of immature seeds (seeds taken 8/24).

Although it increased 'bound' ABA in the seed coat and cotyledons

(Table 2), it reduced their 'free' ABA content slightly. Similar

treatment of mature seed (seed taken 9/21) did not affect 'free' ABA

in the embryonic axis, but 'free' and 'bound' ABA were increased

1. t0 30-fold in all other cases. No study of the effect of stratifi—

cation on seed germination was carried out.
In general ABA content does not parallel germination capacity in

th
ese comparisons. In 'McIntosh', a relatively early ripening cultivar,

no
Q0rrelation was observed until just prior to harvest. Even then,

 

 

 

23

Table 2. Effect of drying, on the concentration of 'free' (F) and
'bound' (H) ABA (ng/g fresh wt.) in apple seed, var. 'McIntosh'.
Means for two samples of 25 seeds each.

 

 

Sampling date, 1974

8/24 9/21

TiSSues F H F H

 

Fresh Seeds

 

Seed coat 218 131 936 593
Cotyledon 126 8 120 7
Embryonic axis 409 128 1283 750

Seed stored 4 months over CaC12_

 

Seed coat 189 401 1521 1414
Cotyledon 84 77 671 218
Embryonic axis 1260 1502 1221 2478

 

 

 

24

'bound' ABA levels paralleled those of 'free' ABA, suggesting that the
glucoside was not converted to the 'free' ABA during the seed develop-
ment. In 'Golden Delicious', a later—ripening cultivar, ABA content
was high in July, low in September except for 'bound' ABA in the
embryonic axis, and rose slightly in October, yet the germination
declined steadily during this period.

Assuming that ABA is synthesized in the leaves and translocated
to the seed, defoliation should reduce the content of ABA in the seed.
This was true for embryonic axis sampled 5 weeks after defoliation
and the germination of excised embryos surpassed that of embryos
from control branches. However, no germination occurred in mature
embryos regardless of treatment. Drying seed on removal from the
fruit generally increased their ABA content. The data obtained do
not, in general, support the hypothesis that the inability of apple
seed embryos to germinate as maturation proceeds is the result of the

accumulation of ABA in the tissues.

 

 

 

I-‘

10.

25

LITERATURE CITED

Balboa, 0., and F. G. Dennis, Jr. 1975. The role of abscisic

acid in the dormancy of apple (Pyrus malus L.) seeds. I.
Abscisic acid levels during stratification. Physiol. Plant

(in preparation).

Bausher, M. G., and R. H. Biggs. 1974. Relation to abscisic
acid and cytokinin content to developing peach seeds.
HortScience 9:296.

Crocker, W. 1938. Life—span of seeds. Bot. Rev. 4:235—274.

 

1973. Role of endogenous

Hiron, R. w. P., and s. T. c. Wright.
J. Exp. Bot.

aflbscisic acid in the response of plants to stress.
24-:769—781.

Liiclcwill, L. C. 1957. Studies of fruit development in relation
t0 [Jlant hormones. IV. Acidic auxins and growth-inhibitors in
-leaaxzes and fruit of the apple. J. Hort. Sci. 32:18-33.

MiEEJ.ke, E. A., and F. G. Dennis, Jr. 1975. Hormonal control
of lilower bud dormancy in sour cherry (Prunus ceraSus L.) III.
Ef15€2ct of leaves, defoliation and temperature on levels of
ab$<2isic acid. Physiol. Plant (in press).

Miillaorrow, B. V., and D. R. Robinson. 1973. Factors affecting
tklfii biosynthesis of abscisic acid. J. Exp. Bot. 24:537-548.

ROSS, J. D., and J. w. Bradbeer. 1971. Studies in seed dormancy.
V. The content of endogenous gibberellins in seed in Corylus
_aVellana L. Planta 100:288-302.

'Rthitiicki, R. 1969. Studies on abscisic acid in apple seeds.
Planta 86:62-68.

Slfeeramulu, N., and I. M. Rao. 1971. Physiological studies on

dormancy in seed ground—nut (Arachis hypogeae L.) III. Changes
in auxin and growth inhibitors content during development of the
SEeds of a dormant and non-dormant cultivars. Austr. J. Bot.

19:273-280.

 

 

SECTION TWO

THE ROLE OF ABSCISIC ACID IN THE DORMANCY OF APPLE (Pyrus malus L.)
SEEDS II. ABSCISIC ACID LEVELS DURING STRATIFICATION

 

 

 

 

26

THE ROLE OF ABSCISIC ACID IN THE DORMANCY OF APPLE (Pyrus malus L.)
II. ABSCISIC ACID LEVELS DURING STRATIFICATION

ABSTRACT

Gas—liquid chromatography (GLC) and mass spectrometry (GC—MS)
were used to identify abscisic acid (ABA) in extracts of dormant
apple (Eyrus malus L.) seeds, and levels of both'free'and base—

hydrolyzable ABA during stratification were quantified by GLC.

 

The concentration of ABA was greatest in the embryonic axis,
intermediate in the seed coat, and least in the cotyledons. ABA
content, expressed on a whole seed basis, declined to 20 to 25 per
cent of the initial level after 7 weeks of stratification, regardless
of whether the seeds were held at 5° or 20°C. Induction of

secondary dormancy by high temperature was associated with rapid

loss of both'free' and 'bound' ABA. The physiological significance

of these observations is discussed.
INTRODUCTION

Many seeds require cold stratification before being able to germinate,
and this effect has been attributed to a reduction in the levels of
endogenous growth inhibitors, such as, abscisic acid (ABA). ABA or
ABA-like compounds have been reported to decline during low temperature
stratification of seeds of apple (EX£E§.EEAE§ L.) (Rudnicki, 1969),

ash (Franxinus americana L.) (Sondheimer §£_31., 1968), plum (Prunus

 

 

27

domestica cv. Italian) (Lin and Boe, 1972), Scots pine (£1225
sylvestris L.) (Kopcewicz and Porazinski, 1973), peach (Prunus
persica), (Lipe and Crane, 1966) and hazel (Corylus ayellana L.)
(Williams et al., 1973). However, only in hazel seeds it has been
reported a decline of 60% in ABA level during stratification at
both 5 and 20°C, although germination occurred only after chilling.

If during the early phases of cold stratification, apple seeds
are transferred to temperatures of 25°C or higher, they usually

enter into secondary dormancy (Abbott, 1955; Visser, 1956). To

 

break this dormancy, the seeds require the same amount of cold
stratification as non—after-ripened seeds.

Rudnicki (1969) identified ABA in ethanol extract of apple
seeds, and used the wheat coleoptile elongation assay to meaSure the
amount of inhibitory activity in water diffusate of whole seeds
following various periods of low temperature stratification.
Rudnicki and Czapski (1974) followed the degradation of 1-14C-ABA
in apple seeds during stratification and determined its distribution
in the various tissues. Recently, Bulard et a1 (1974) have
identified the glucose ester of ABA in apple seeds, with retention
time of 7.28 min in 1% OVI at 200°C for ABA and 10 min for the

B-D-glucopyranose at 180°C.
MATERIALS AND METHODS

Plant material. Apple seeds, cv. 'McIntosh' and 'Red Delicious'

were removed from fruit collected in a commercial orchard near

 

 

28

East Lansing, Michigan in 1972, air—dried and stored over anhydrous
63012 at room temperature until used. Dry seeds were stratified

in darkness beginning 2 months after collection, in moist sand

at either 5 i2° or 20 t2°C. Four samples of 25 seeds each were
removed at every 3 week interval, 2 for extraction and ABA determina-
tion, and 2 for testing germination at 20° il°C, the protrusion

of the radicle through the seed coat was considered as an index of

germination.

Extraction and fractionation procedure. Two replicates of 25

dormant, dry seeds, or seed removed from the stratification medium

 

were shaken with 25 ml of distilled water at 5°C for seven days.

The water was changed daily, and pooled to give the fraction subsequently
called the diffusate. The seeds were then dissected into seed coat
(endosperm plus testa), cotyledons, and embryonic axis. The tissues
were ground in absolute methanol with a mortar and pestle and extracted
overnight at 2°C. The methanolic extract evaporated to dryness and

the residue redissolved in 25 ml of phosphate buffer, pH 7.3. This

was processed to give an acidic fraction ('free' ABA) and a base—
hydrolyzable fraction ('bound' ABA) (Fig. l). Fractionation of
extracts of both stratified and nonstratified seeds labeled with
2-14C-ABA reSulted in 80% of the radioactivity being recovered in

the acidic fraction, and 3% in the bound fraction. For identifica-
tion of ABA, 100 grams of whole dry seed were ground in absolute

methanol and processed as in Fig. l.

 

 

29

Residue from methanolic extract
dissolved in 25 ml phosphate buffer
pH 7.3.

Partition 3 to 5 times with 15 m1
of hexanes (b.p. 67.2 — 73.1°C).

 

 

Hexanes Water
(discarded)

Partition 3 x with
15 m1 CHZGIZ

 

 

Water CH2C12
(discarded)

Adjust to pH 3 with HCOOH
Partition 4 x with 15 m1

 

CH2C12
CHZClZ Water

Adjust pH to 11.0 with NH40H
('free' fraction) Hydrolyze for 1 hr at 60°C

Adjust to pH 3 with HCOOH
Partition with 4 x 15 m1
CH2C12.

 

Water CH2C12
(Discarded) ('bound' fraction)

Fig. 1. Flow diagram of procedure used for preparation of
‘ fractions containing 'free' and 'bound' ABA.

 

 

30

Gas chromatography. The residues from these two fractions were
resusPended in 1 m1 of diethyl ether/methanol (9:1 v/v) and
methylated with diazomethane following the method of Schlenk and
Gellerman (1960) as modified by Powell (1964). The ether—methanol
solution was evaporated, the residue was dissolved in ethyl acetate,
and aliquots were injected, without further purification into a
Packard 7300 gas—liquid chromatograph equipped with an electron
capture detector (65Ni foil) and operated either at 5 or 7.5 volts.
The column used (2 mm i.d. x 1.83 m) was packed with 3% SE30 (methyl
silicone) on 80/100 mesh Gaschrom Q (acid washed, dimethyl chloro-
silane). The column temperature was 180° and inlet and detector
temperature were 260 and 270°C, respectively. The carrier gas was
nitrogen at a flow rate of 40 ml/min at 40 psig. Nitrogen scavenger
gas was Supplied to the detector at 70 ml/min. The retention time for
ABA was 1.5 min. A standard curve based upon peak height was used

for quantitative determinations.

Gas chromatography — mass spectrometgy. The methylated 'free'

acid fraction from 100 grams of dormant seed without further purification
were subjected to combined gas—liquid chromatography-mass spectrometry
(GC-MS) using an LKB GC—MS, interphased with a POP 8/I computer.

The main spectra were determined at 70 eV. For GC a glass column,

i.d. 2 mm 6 ft long of 3% SP 2100 (methyl silicone) on supelcoport

(acid, washed silane treated diatomite 100/120 mesh) was used with a
helium flow rate of 20 ml/min. The column temperature was 200°C and

290°C for the detector. The retention time for ABA was 8 min.

 

 

 

31

Secondary dormancy. Apple seeds, cv. 'McIntosh', were stratified
at 5 12°C for 3 weeks. One group of seeds was subsequently transferred
to 27 11°C for one week, then returned to 5° i2°C for an additional
12 weeks. The remaining were held continuously at 5 i2°C. Four
samples of 25 seeds each were removed at interval for determination
of the germination capacity (both treatments) and ABA content (high
temperature only), as described above. Seeds held continuously at

5°C were not analyzed for ABA.

RESULTS

Identification of ABA. GLC traces of methylated 'free' and 'bound'

 

fractions both contained a peak that co-chromatographed with authentic
cis,trans—ABA. When nonmethylated samples of the 'free' and 'bound'
fractions were injected, no peak was observed. When either methylated
extracts or methylated cis-trans ABA were exposed to ultra-violet
light (365 nm) for 6 hr, the peak height was markedly reduced. 0n
GC-MS the retention time for both synthetic and for presumed ABA in
the extract was ca 8 min. Mass Spectrometry showed a molecular ion
(M+) at 278, and a base peak at 190. Major ions with respective
intensities as a percent, the base peaks were as follows: c, t-ABA—278
(12), 260 (37), 205 (6), 190 (100), 162 (53), 134 (53), 125 (50), 91
(31), unknown - 278 (11), 260 (31), 205 (5), 190 (100), 162 (33),

134 (34), 125 (31), 91 (17). c, t—ABA was only identified in the
'free' fraction.

Some samples, mostly the 'bound' fraction, showed peaks resembling

 

 

 

32

t,t—ABA in retention time on GC. However, no attempt was made to

identify these compounds.

Effect of temperature on germination and ABA content. Approximately
6 weeks of chilling at 5 i2°C was necessary for 50% seed germination
and 94% germination was obtained after 7 weeks at 5 iZC. No
germination occurred in seed held continuously at 20°C.

The concentration of ABA in the embryonic axis of nonstratified
'McIntosh' seeds was 100 times that found in the cotyledons (Table 1),

and 70-fold that in the seed coat (10,250, 120; and 150 ng/g fresh

 

weight). Similar relationships held in the 'Red Delicious' cv. (data
not shown). Eighty six to 90% of the 'free' acid was lost from the
embryonic axis during 7 weeks at 5°C and similar losses of 'bound'
ABA occurred (Table 1). A 30 to 40% loss of 'free' ABA occurred in
the seed coat and 36% loss in the cotyledons. Temperature did not
markedly affect these losses.

The ABA content of the water diffusate varied considerably among
treatments. The highest level of 'free' ABA in the diffusate was
observed following stratification at 5 i2°C for 7 Weeks, the concentra-
tion being 5 times that found in the diffusate from stratified seeds
for 3 weeks. 0n the other hand, the diffusate from seeds held at
20°C contained less 'free' ABA after stratification. In the case of
'bound' ABA in the diffusate, no change occurred at 20°C, but a 70%
decrease occurred at 5°C. Parallel data were obtained with 'Red

DeliciOus' seeds with the exception noted below (data not shown).

 

 

33 ,

Table 1. Effect of stratification time and temperature upon ABA content
of 'McIntosh' apple seeds and the water diffusate from the seeds. Means
for 2 replicates of 25 seeds each.

 

 

Treatment Diffusate Seed coat Cotyledon Embryonic axis

 

Free ABA (ng/g fresh wt.)

 

Dormant seeds 400 150 120 10,250
5° for 7 weeks 130 87 75 1,440
20 for 7 weeks 250 170 43 955

Bound ABA (ng/g fresh wt.)

 

 

Dormant seeds 200 150 35 8,500
5° for 7 weeks 60 130 60 27
20° for 7 weeks 200 82 39 480

Free ABA (ng/seed)

 

 

Dormant seeds 155 2 3 6

5° for 7 weeks 5 1.1 1.9 0.8

20° for 7 weeks 10.1 2.4 1.1 0.5
Bound ABA (mg/seeds)

Dormant seeds 8 1.8 0.9 4.9

5° for 7 weeks 2.2 1.7 1.5 0.1

20° for 7 weeks 5.1 1.1 1.0 0.2

 

 

 

 

 

34

Figure 2. Concentration of 'free' and 'bound' ABA in the water
diffusate and in methanol extracts of seed coat cotyledons, and
embryonic axis of apple seeds during stratification at 5 and
20°C. Each bar represents the mean of at least 4 replicates of
25 seeds each. Per cent of germination at 20°C is indicated in
parenthesis above each treatment.

ABA CONCN (ng/g fresh weight)

IOOO

800

600

400

200

IOOO

800

600

400

200

35

 

 

(O)

 

 

A. RED DELICIOUS

.‘Free' fraction

I 'Bound' fraction

Diffusote 'free' fraction
[:1 Diffusote 'bound' fraction

8. Mc INTOSH

 

STRATIFICATION TIME (wk)

Fig. 2

 

 

 

 

 

36

Figure 3. Effect of inducing secondary dormancy with high temperature
upon total ABA content of apple seeds. A. ABA content of seed
during one week at 27 il°C after 3 weeks at 5°C. B. ABA content
of seeds following their return to 5 i2°C following the treatment
in A. In B, 0 represents the day the seeds were returned to 5°
as well as the day on which stratification of the control,
continuous chilling began. Each value is the mean of at least

4 replicates of 25 seeds each. 'Bound' ABA (o 0); 'free'

ABA (x x); germination under continuous chilling (x — — - x);
germination of seeds returned to 5°C after 1 week at 27 il°C

(o — , — o).

 

ABA CONCN (ng/g fresh weight)

so 0 A
X
45 - x/x76
o___.___——-o
O l ' l I"
2 B 4 )7.
so - ,I ,x

40

20

37

 

 

 

 

 

/
- x x’ '/ -
\I/\ ./

/'0-.-/_.--6\ I

_ o—f— O I x

,/ 7\0
./ / \0 I
,x x,
/ ,v’
" -'—&”’ I l l
2| 42 63 82

STRATIFICATION TIME (days)

Fig. 3

IOO

80

60

4O

20

GERMINATION °/o

 

 

 

38

The concentration of ABA (ng/g fresh weight), including that
in the diffusate declined markedly within the first 3 weeks, with
smaller variations thereafter (Fig. 2). Again, no consistent effect
of temperature was evident. In contrast with the result obtained
with 'McIntosh', diffusate from 'Red Delicious' seed contained less

ABA after stratification, regardless of temperature.

Effect of induction of secondary dorman y. Contrary to
expectation both 'free' and 'bound' ABA declined markedly 2 days

after transferring seeds to 27 11°C after 3 weeks at 5°C (Fig. 3A).

 

The levels changed little during the remainder of the week at

27 11°C. On return to 5 i2°C, 'free' ABA rose to an intermediate
level and then declined while 'bound' ABA dropped to a lower level
and then remained relatively constant (Fig. 3B). Germination of
seeds in which secondary dormancy had been induced paralleled that
of the controls, indicating that raising the temperature after 3
weeks had indeed eliminated the effect of prior stratification at

5 i2°C.

 

39

DISCUSSION

ABA was identified in a methanol extract of dormant seeds, confirm—
ing previOus work (Rudnicki, 1969).
The concentration of 'free' ABA was highest in the embryonic
axis, and lowest in the seed coat. Levels declined from 150, 120 and
10,250 ng/g f.w. in seed coat, cotyledons and embryonic axis,
respectively, at 0 weeks, to 87, 75 and 1,440 ng/g after 7 weeks of r

chilling. Therefore, the decline in the embryonic axis was approxi—

 

mately 1/10 the original level and that in the seed coat and cotyledon
to a little over 1/2 the original levels. Levels of 'bound' ABA
declined in a parallel fashion Suggesting that the loss of 'free'
ABA was not the result of conjugation. Changes in the concentration
of 'free' ABA in the water diffusate were inconsistent. After
stratification, higher levels were found in the diffusate from
'McIntosh' seed held at 5°C lower levels in that from seeds held at
20°C. In the case of 'Delicious' seeds, however, less ABA was
present in the diffusate after 7 weeks of stratification, regardless
of the temperature. This inconsistency is surprising in view of
Rudnicki's (1969) observation that the inhibitor content of similar
diffusate declined markedly during low temperature stratification.

The decline in ABA is temperature—independent, yet chilling is
required to break dormancy. Hence, the changes in germination capacity

of the seeds cannot be ascribed to changes in ABA content alone.

 

 

40

5M ABA is required to completely inhibit

Furthermore, 1.5 x 10—
germination of stratified apple embryos (Rudnicki, 1969), while
our data show that endogenous ABA level in fully dormant embryos
is approximately 6 x 10'7M 335uming a 20% loss during fractionation.
Hence, Jacobs (1959) third rule (exact substitution) does not hold
for the inhibition of apple seed germination by ABA.
ABA levels fell during the induction of secondary dormancy
by high temperature after 3 weeks at 5 i2°C. Therefore, induction
of secondary dormancy cannot be attributed to changes in ABA level.
If ABA plays a role in the dormancy of apple seeds, it may
antagonize the germination-promoting effect of hormones synthesized
only during chilling (Sinska and Lewak, 1970; Borkowska and
Rudnicki, 1974). These may be essential for the synthesis of

promoters while ABA removal is temperature independent.

 

 

41

REFERENCES

Abbott, D. L. 1955. Temperature and dormancy of apple seeds. —-Proc.
XIV Int. Hort. Congress pp. 746-753.

Borkowska, B. & Rudnicki, R. 1974. Changes in the cytokinin level
in stratified apple seeds. -—Proc. XIX Int. Hort. Congress,
Warsaw p. 496.

Bulard, C., P. Barthe, G. Garello and M. T. LePage-Degivry. 1974.
Mise en evidence d'acide abscisique lie au B—D—glucopyranose dans
les embryons dormants de Pyrus malus L. -—C. R. Acad Sci. Ser. D
278:2145—2148. II———'——'—._

Jacobs, W. P. 1959. What substance normally controls a given biol—
ogical process? I. Formulation of some rules. ——Developmental
Biol. 1:527-533.

Kopcewicz, T. & Porazinski, Z. 1974. Influence of low temperature
on germination and endogenous regulation in Scots pine (Pinus
sylvestris L.) --Acta Soc. Bot. Pol. XLII 233—240.

Lin, C. F. & Boe, A. A. 1972. Effect of some endogenous and exogenous
growth regulators on plum seed dormancy. —-J. Amer. Soc. Hort.
Sci. 97:41—44.

Lipe, W. N. & Crane, J. C. 1966. Dormancy regulation in peach seeds.
--Science 153:541-542.

Powell, L. E. 1964. Preparation of indole extracts from plants for
gas chromatography and Spectrophotofluorometry. —-Plant Physiol.
39:836-842.

Rudnicki, R. 1969. Study of abscisic acid in apple seeds. --P1anta
86:63-68.

& Czapski, J. 1974. The uptake and degradation of 1-14C—abscisic
acid by apple seed during stratification. -—Ann. Bot. 38:189-192.

Schlenk, H. & Gellerman, J. D. 1960. Esterification of fatty acids
with diazomethane on a small scale. -—Anal. Chem. 32:1412-1414.

Seeley, S. D. 1971. Electron capture gas chromatography of plant
hormones with special reference to abscisic acid in apple bud
dormancy. ——Ph.D. Thesis, Cornell University, 128 pp.

 

42

Sinska, I. & Lewak, St. 1970. Apple seed gibberellins. ——Physiol.
Veg. 8:661-667.

Sondheimer, E., Tzou, D. S. & Galson, E. C. 1968. Abscisic acid
levels and seed dormancy. ——P1ant Physiol. 43:1443-1447.

Visser, T. 1956. Some observations on respiration and secondary
dormancy in apple seeds. —~Proc. Kon. Ned. Akad. Wetensch C
59:314—324.

Williams, R. W., Ross, J. D. & Bradbeer, J. W. 1973. Studies in
seed dormancy. VII. The abscisic acid content of the seed
and the fruit of Corylus avellana L. —-P1anta 110:303-310.

 

 

 

APPENDIX

 

43

THE ROLE OF ABSCISIC ACID IN THE DORMANCY OF APPLE (EX£E§.EELE§ L.) SEEDS
III. EFFECT OF GROWTH RETARDANTS ON SEED DORMANCY AND ABA CONTENT

Abstract. Inclusion of AND—1618 (4 hydroxy—S—isopropyl-Z—

methylphenyl-trimethylammonium chloride piperidine carboxylate)

in the stratification medium did not inhibit the germination of

intact apple (Py£p§_mélp§_L.) seeds or of excised embryos from

partially stratified seeds. Spraying trees with SADH (Succinic

acid—2,2-dimethylhydrazide) at 1000 ppm reduced germination

following stratification in some but not in all cases, and

reduced the ABA content of embryonic axes but increased that of

the cotyledons. These data suggest that the reduction in germina-

tion caused by SADH treatment are probably not an effect of

increased concentration of endogenous ABA.

 

During low—temperature after—ripening of apple embryos GA4 content
increases, but falls to the original level before dormancy is broken
(2). Red light promotes the germination of excised embryos of dormant
seeds, as well as the production of endogenous gibberellin (4) while
AND-1618 inhibits both responses following red light treatment. Sinska
and Lewak (3) also reported that AMO—l618 inhibits the synthesis of
0A4 in partially after—ripened apple embryos.

If the rise in GA4 content observed by Sinska and Lewak (2) plays

a role in the breaking of dormancy, prevention of the synthesis during

 

44

low temperature stratification Should reduce the germination capacity
of the seed. I therefore tested the effect of AND-1618 during
stratification on the germination of intact apple seeds. AND—1618
and CCC (2-chloroethyl)trimethyl ammonium chloride) were also tested
on germination of embryo of partially stratified seed.

SADH in contrast to AND-1618 and CCC does not inhibit GA

synthesis in Fusarium monoliforme Sheld (2). However, SADH appears

 

to inhibit some step(s) in the synthesis of GA precursors in peas
(6) and reduce the germination of hazel seeds (3). I therefore
compared seeds from sprayed vs. non-sprayed fruits as to their
germination capacity and ABA content.

Samples of mature fruits were collected from a commercial orchard
at Hartford and at Belding, Michigan. SADH was supplied at 500 or
1000 ppm in late July or early August, and the fruits were harvested
in mid or late September. Seeds were removed within 24 hours of
collection, or were left in the fruit during storage at 4 i2°C for
5 months, depending on the experiment. For stratification seeds were
held on moist sand for 12 weeks at 5 i2°C. Germination was induced
at 20 i2°C for 10 days, and values were converted to £10 (5). ABA
content was determined by dissecting seeds, previously soaked in water
for 24 hours, and extracting seed coats, cotyledons, and embryonic axes
with absolute methanol. The crude methylene chloride fraction ('free'
ABA) and the base hydrolyzable fraction ('bound' ABA) were analyzed
for ABA, using an electron capture gas chromatography as described

previously (1). The leachate was also processed for ABA, and was

 

45

called the diffusate.

Effect of the presence of growth retardants during stratification

on germination. Stratification of seeds in water containing the

 

growth retardants did not affect germination (94% germination in both

seeds treated or not treated with the growth retardants).

Effect of tree sprays of SADH on germination and ABA. SADH

 

treatment had no effect on the germination of excised embryos from
fruit stored for 5 months at 4 i2°C (Table 1). Germination of intact
seeds was much reduced in comparison with that of embryos, and SADH
possibly inhibited their germination in 'Red Delicious', but not in
the other cultivars. The germination in 'Jonathan' seeds stratified
in moist sand was also reduced by SADH treatment at the 6th and 9th
week but not at the 12th week. SADH thus appears to have a small
inhibitory effect on seed germination following stratification.

The ABA content of freshly harvested 'Jonathan' seeds was affected
by SADH treatment, but the response varied with the seed tissue sampled
(Table 2). Both 'free' and 'bound' ABA were higher in the cotyledon
but lower in the embryonic axis of seed, from sprayed trees. The
amount of 'free' ABA in the diffusate was increased by SADH treatment,
but the 'bound' ABA was not appreciably affected.

Assuming that the embryonic axis is the locus of dormancy in
these seeds, SADH appears to reduce ABA content concomitantly with

germination capacity, suggesting that the two are not casually related.

Table 1.

46

chilling either in the fruit or in moist sand.
means for 2 samples of 25 seeds each.

Effect of tree sprays of SADH on germination (210)
of intact seeds (I) or excised embryos (R) of apple after
Values are

 

 

500
1000

1000

Time at
SADH 4-5°C
(ppm) (wkS)

20
20
20

NOON

NOO“

 

 

 

 

(£10)
Cultivar
McIntosh Jonathan Delicious
R I R I R I
In the fruit at 4°C
982 110 532 46 742 4
724 50 588 48 538 28
744 12 572 22 526 0

In moist sand at 5°C

 

30
558
592

16
224
658

 

47

Table 2. Effect of SADH treatment of trees on the ABA content
of apple seed cv. 'Jonathan'. Values are means for 2 samples
of 25 seeds each.

 

 

ABA (ng/g Fresh wt.)

 

 

Embryonic
Treatment Cotyledons Seed Coat axis Diffusate
Alar, 1000 ppm
Free ABA 84 113 214 451
Bound ABA 157 207 336 512
Control
Free ABA 43 120 1298 121

Bound ABA 30 174 514 587

 

LITERATURE CITED

Balboa, 0., and F. G. Dennis. 1975. The role of abscisic acid
in the dormancy of apple (Pyrus malus L.) seeds. I. Abscisic
acid levels during stratification. Physiol. Plant. (In
preparation).

Ninnemann, H., J. A. D. Zeevart, H. Kende, and A. Lang. 1964.
The plant growth retardant CCC as inhibitor of gibberellin
biosynthesis in Fusarium monoliforme. Planta 61:229-235.

Ross, J. D., and J. W. Bradbeer. 1971. Studies in seed dormancy.
VI. The effect of growth retardant on the gibberellin content
and germination of chilled seeds of Corylus avellana L. Planta
100:303-308.

Sinska, I., and S. Lewak. 1970. Apple seed gibberellins.
Physiol. Veg. 8:661—667.

. 1974. Gibberellin biosynthesis in

, and
apple seed dormancy. Proc. XIX Int. Hort. Congress, Warsaw.

p. 493.

Smolenska, G. S., and S. Lewak. 1971. Gibberellins and the
photosensitivity of isolated embryos from non-stratified apple
seeds. Planta 99:144-153.

Timson, J. 1965. New method of recording germination data.
Nature 207:216—217.

Wylie, A. W., R. Kyugo, and R. M. Sachs. 1970. Effects of
growth retardants on biosynthesis of gibberellin precursors

in root tips of peas, PiSum sativum L. J. Amer. Soc. Hort. Sci.
95: 627-630.

I;

48

SUMMARY AND CONCLUS IONS

SUMMARY AND CONCLUSIONS

Abscisic acid was identified in a methanolic extract of dormant
seeds by gas chromatographydmass spectrometry. Stratification of the
seeds at 5 i2°C produced the same decline of the ABA level in the
2 cultivars studied. Yet, only chilled seeds germinated. Induction
of secondary dormancy by high temperature (27°C) was associated with
rapid loss of both 'free' and 'bound' ABA. Thus, the induction
of secondary dormancy cannot be ascribed to the rise in the abscisic
acid in apple seeds.

The 'free' ABA content of seeds sampled during their development
in the fruit showed two peaks, one in mid-August, the other at fruit
maturity. 'Bound' ABA remained relatively constant, paralleling the
increase of 'free' ABA at fruit maturity. Seeds with intact seed
coat failed to germinate regardless of the time of sampling. Excised
embryo germination was negligible in the first sampling date, reached
a maximum of 62% early August, and then fell to nil as maturity
approached. The decline in germination occurred 2 weeks prior to the
rise of the ABA level. Thus, there appears to be no relationship
between ABA content and germination of isolated embryos until the
approach of fruit maturity in late September.

Branch defoliation of 'Golden Delicious' trees in late July
affected markedly the 'free' ABA in the embryonic axis 5 weeks after
defoliation. Smaller reduction of the 'bound' ABA were noted in the

axis in both September and October samplings. Germination of excised

49

50

embryos was inversely correlated with ABA level in the September
sampling only. Chilling requirements of the seeds from defoliated
branches was not affected in seeds sampled on October 20. Drying
seed on removal from the fruit generally increased their ABA
content. The data obtained do not support the hypothesis that the
inability of embryos of apple seed to germinate as development
proceeds is the result of accumulation of ABA in the seed tiSSues.
Seeds from fruit of trees sprayed with succinic acid—2,2-dimethyl—
hydrazide showed less ABA content than those of the control. It also
reduced the germination capacity of the seeds from 88% in the control
to 54% in the seeds from fruit of trees sprayed, after 9 weeks of
cold stratification suggesting that the two are not casually related.
In a preliminary experiment, AND-1618 neither impaired the
germination of intact seeds when it was included in the stratification
medium nor did it affect the germination of isolated embryos of

partially stratified seeds in the fruit.

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95:627-630.

1 '-

 

 

 

IES

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