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IIIWIWIWiiiilkfii‘lifTIVTflTiTII 9 5 / 4 6’ at 67 :1

HERARY
Michian State
L University

 

 

 

This is to certify that the

dissertation entitled

Characterization of Secondary Dormancy

in Apple Seeds
presented by

Jocelyn Ann Ozga

has been accepted towards fulfillment
of the requirements for

Ph.D. degree in HortiCU1tUY‘e

Major pr fessor

 

Date 11/ 17/88

MS U is an Affirmative Action/Equal Opportunity Institution 0-12771

 

 

PLACE IN RETURN BOX to remove this checkout from your record.
TO AVOID FINES return on or before date due.

 

F—_————————'——1
DATE DUE DATE DUE DATE DUE

 

 

 

 

 

 

 

 

 

 

 

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WV

*5

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usu is An Affirmative Action/Equal Opportunity Institution

 

CHARACTERIZATION OF SECONDARY DORMANCY IN APPLE SEEDS

BY

Jocelyn Ann Ozga

A DISSERTATION

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Horticulture

1988

L“

ABSTRACT
CHARACTERIZATION OF SECONDARY DORMANCY IN APPLE SEEDS
BY

JOCELYN ANN OZGA

When stratified apple seeds are stressed, secondary
dormancy is induced. High temperature, low oxygen tension,
high levels of ethylene and exogenous ABA were characterized
as stress factors in inducing secondary dormancy.

Exposure to 25-35°C inhibited subsequent germination of
stratified apple seeds at 20°; however, only 35° inhibited
embryo germination. As time of stratification at 5°
increased, the seeds and embryos became less sensitive to
high temperature induction of dormancy. High temperature
did not completely negate the effect of a prior exposure to
low temperature unless full germination potential of the
seed had been achieved prior to the heat treatment.

Anaerobiosis reportedly breaks the dormancy of non-
stratified apple embryos. Low 02 treatments (0.3-0.5% and
<0.16% 02) were tested for effectiveness on both non-
stratified and stratified apple seeds and embryos, and on

stratified seeds in which secondary dormancy had been

induced by exposure to 30°C for 6 days. A small promotive
effect was observed in non-stratified, but not in stratified
embryos, however, the treatments inhibited germination of
stratified seeds. Neither treatment broke secondary
dormancy in intact seeds. And germination of embryos excised
from them was often inhibited.

Exposure to 10'6 to 10'3 H ABA or to 3 or 6 days at
30°C inhibited stratified seed and embryo germination, and
the effects of the two treatments were additive. However,
no correlation was found between endogenous ABA content of
seed tissues (testa, cotyledons, or embryonic axis) and
either the duration of incubation at 5°, or the induction of
secondary dormancy by heat stress (30°C for 0, 3 or 6 days).

The observations made by others that ethylene (C2H4) is
required for germination of stratified embryos of apple was
not confirmed. Neither removal of ethylene from the
atmosphere nor inhibition of ethylene action by
norbornadiene or silver thiosulfate appreciably affected
germination of fully stratified seeds or embryos at 20° or
30°C. The addition of ethylene either had no effect or
reduced germination of fully stratified seeds or embryos;
therefore, C2H4 does not appear to be essential for

germination of apple seeds or embryos.

ACKNOWLEDGEMENTS

I would like to express my deep appreciation to my
major professor, Frank Dennis, for his encouragement,
direction, and participation in the completion of this
thesis and for being a great human being.

I would also like to thank my family for their support
during the making of this thesis.

My deepest gratitude belongs to my husband Dennis, for

all his assistance, encouragement, sacrifice, and love.

iv

TABLE OF CONTENTS

Page

LIST OF TABLES viii
LIST OF FIGURES x
INTRODUCTION 1
LITERATURE REVIEW 1
Introduction 1
Definition of dormancy 1

Apple seed morphology 3
Conditions which induce secondary dormancy 3
Mechanisms of secondary dormancy induction 5

Limited availability of oxygen to embryos 5

Levels of growth inhibitors 8

Abscisic acid 8

Ethylene 12

Changes in membranes 13

Maintenance of secondary dormancy 13
Release of secondary dormancy 14

Summary 15
Literature Cited 16

SECTION I 21

The Effect of Temperature on the Induction and Release

of Secondary Dormancy in Apple Seeds 21

Introduction 22
Materials and Methods 22
Experiment 1. Induction of secondary dormancy 23
Experiment 2. Release from secondary dormancy 23
Results 24
Experiment 1. Induction of secondary dormancy 24

Experiment 2. Release from secondary dormancy 25

Discussion 26
Literature Cited 27
SECTION II 32

Germination Responses of Apple Seeds vs. Embryos to High

Temperature and Low Oxygen Tension 32
Abstract 33
Materials and Methods 34

Seed source and methods of stratification and

germination 34

Experimental conditions 35

Evaluation of germination 36

Statistical analysis 36

Results 36
Discussion 39 ,

Literature Cited 40

SECTION III 50

The Role of Abscisic Acid in Heat Stress Induced

Secondary Dormancy in Apple Seeds 50
Abstract 51
Materials and Methods 52

vi

Seed source and methods of stratification
Experimental conditions
Evaluation of germination
Extraction, purification, and quantification
procedures
Identification of ABA by GC-MS
Statistical analysis
Results
Discussion
Literature Cited
SECTION IV
Is Ethylene required for Apple Seed or Embryo
Germination
Abstract
Materials and Methods
Results
Discussion
Literature Cited
APPENDIX
The Effect of ABA on Apple Seed Germination
Introduction
Materials and Methods
Results
Discussion
Literature Cited

BIBLIOGRAPHY

vii

52

53

53

53

54

55

55

57

59

63

63

64

65

68

70

71

77

77

77

77

78

80

80

87

LIST OF TABLES

TABLE Page
SECTION I

Table 1. The effect of time of stratification of seeds at
5° C and subsequent exposure to higher temperatures
for 1 week on germination of seeds and embryos
during 10 days at 20° C. 28

Table 2. The effect of stratification at 5°C before and
after exposure to 30° for 6 days on germination of
apple seeds and embryos after 12 days at 20° . 29

SECTION II

Table 1. The effects of stratification time and subsequent
exposure to high temperature and/or low oxygen
tension (< 0.16%) on germination of apple seeds and
embryos (Experiment 1). 42

Table 2. The effects of stratification time and subsequent
exposure to high temperature and/or low oxygen
tension (0.3-0.5%) on germination of apple seeds and
embryos (Experiment 2). 43

SECTION III

Table 1 Germination (Sum 10) of seeds ostratified at 5° or
20°C and after exposure to 30° . 60

Table 2. Effects of stratification temperature and
subsequent exposure to 30°C on ABA concentration in
embryonic axes, cotyledons, and seed coats of apple
seeds. 61

SECTION IV
Table 1. Effects of temperature and removal of C2H4 from the

atmosphere on germination of partially and fully
stratified apple seeds and embryos. 72

viii

Table 2. Effects of temperature and addition of C H to the
atmosphere on germination of partially and gully
stratified apple seeds and embryos. 73

Table 3. Effect of temperature on C H4 levels in air within
jars containing fully stratifiied apple seeds or
embryos. Gas samples removed after 3 days at 20° or
30° C. 74

Table 4. Effects of temperature and inhibition of C2H4
action by silver thiosulfate (STS) on germination of

partially and fully stratified apple seeds and
embryos. 75

Table 5. Effects of temperature and norbornadiene (NB) on

germination of fully stratified apple seeds and
embryos. 76

APPENDIX

Table 1. The effect of stratification at 5°C and subsequent

exposure to 30° and/or exogenous ABA on germination
of apple seeds. 81

Table 2. The effect of ostratification at 5°C and subsequent

exposure to 30° and/or exogenous ABA on germination
(Sum 10) of apple embryos at 20° . 82

ix

LIST OF FIGURES

FIGURE Page
SECTION I

Figure 1. The effect of stratification at 5°C, before and
after exposure to 30°C for 6 days, on germination of
apple seeds and embryos. 30

SECTION II

Figure 1. Germination response of dried and non-dried apple
seeds to temperature and oxygen level. Seeds dried
prior to stratification at 5 C (Dried), or held in
fruits at 5°C for 23 weeks prior to treatment (Non-
dried). 44

Figure 2. The effect of stratification time and subsequent
exposure to 30°C and low oxygen tension (< 0.16%) on
germination (Sum 12) of apple seeds and embryos. 46

Figure 3. The effect of stratification time and subsequent
exposure to 30°C and low oxygen tension (0. 3- 0. 5%)
on germination (Sum 12) of apple seeds and embryos.

48

SECTION III

Figure 1. Mass spectrum of Me-ABA in a methylated extract of
seed coats of apple (A), and of authentic Me-ABA
(B). 62

APPENDIX

Figure 1. The effect of ABA on germination (Sum 14) of
seeds at 20° and 30° oC following 9 weeks of
stratification at 5°. 83

Figure 2. The effecto of ABA on germination (Sum 14) of
embryos at 20° and 30°C following 9 weeks of
stratification at 5°. 85

INTRODUCTION

LITERATURE REVIEW
11111112122112]:

Environmental factors play an important role in seed
germination. Firstly, imbibition of the seed is necessary
to initiate an active metabolism. Such metabolic activity
requires 02 and will occur only within certain temperature
limits. However, many seeds are unable to germinate even
when placed in the presence of water and oxygen at
permissible temperatures. This inability to germinate,

known as dormancy, will be addressed in this review.

MW

Dormancy is the suspension of growth of any plant
structure containing a meristem. Classification into
primary and secondary dormancy is based on the phase in the
life-cycle of a seed in which the induction occurs. Primary
dormancy prevents germination during development and
maturation on the mother plant and may continue for some
time after shedding or harvesting of the seeds. Secondary
dormancy develops after dispersal or harvest in seeds that
are primarily non-dormant or have emerged partly or fully

from primary dormancy (Karssen, 1980/81). Under natural

1

2
conditions, secondary dormancy increases the probability

that once the seed germinates, the seedling will survive.

primary dormancy ___%> breaking of.___). germination

dormancy
inhibition of induction of
germination secondary dormancy

No evidence exists that proves that a seed entering
secondary dormancy returns to a physiological state
identical to its state during primary dormancy. Visser
(1956b), stored stratified apple seeds for various periods
of time at 25°C. As storage time increased, longer periods
at a dormancy breaking temperature (3°C) were required to
restore the initial germination capacity of the embryos.
This was also the case in Impatiens glandulifigza seeds
(Mumford, 1988). These data support the hypothesis that
primary and secondary dormancy are not identical states.
However, if dormancy is quantitative, longer periods merely
increase the depth of dormancy; therefore, secondary
dormancy could be argued to be identical to primary
dormancy. Other researchers did conclude that primary and
secondary dormancy were identical (Thornton, 1945: Abbott,
1955); however, they presented no firm evidence to verify
their claim.

This thesis will emphasize the factors and mechanisms

involved in the induction and release of secondary dormancy

in apple seeds.

W

The mature apple seed consists of the following parts

(Luckwill, 1952: Harrington, 1923):

1. Embryg: embryonic axis with two large fleshy
cotyledons containing the bulk of the stored food
reserves.

2. Engggpezm_1aygz: a whitish membranous layer of

endosperm tissue to which a very thin nucellus layer

adheres closely.

3. Inne;_integumgnt: a thin, translucent brownish

layer of tissue without any opening.

4. Quter_integument: a thick, brown and fibrous layer

with the open micropyle.

The outer integument constitutes approximately 33%, the
inner integument and endosperm layer 11%, and the embryo 56%
of the seed fresh weight (Harrington, 1923).

The inner and outer integuments collectively are called

the seed coat in this thesis.

WNW

Induction of secondary dormancy under natural
conditions in the field has been observed in seeds of many
wild species of plants. The increase in dormancy is often
part of cyclical changes that follow a seasonal pattern

(Karssen, 1980/81).

4
In controlled conditions, induction of secondary

dormancy occurs in seeds when exposed to one or more of a
number of environmental stresses. High temperature induces
secondary dormancy in seeds of many species (Stokes, 1964).
Abbott's (1955) experiments with apple seeds demonstrated
that exposure to temperatures above 17°C induced secondary
dormancy in partially chilled seeds: temperatures below 17°C
were effective in breaking dormancy. At 17°C, defined as
the "compensation temperature", germination capacity of the
partially chilled seeds remained the same. This implied
that both dormancy breaking and dormancy inducing processes
are in equilibrium at that temperature. Imbibition in an
osmoticum imposes secondary dormancy in seeds of lettuce
(Kahn, 1960) and Chengpgdium bong§;henzigu§ (Khan and
Karssen, 1980). Low oxygen tension induces secondary
dormancy in seeds of Xanthinm Qanaggngg (Davis, 1930) and

embryos of apple (Malgg 99mg§tiga) (Come and Tissaoui,
1968). In addition, exogenous abscisic acid inhibits

germination in non-dormant apple embryos (Rudnicki and
Pieniazek, 1973), and induces secondary dormancy at 10'4 M
after a 15 day exposure (Durand et a1., 1973).

Induction of secondary dormancy can be prevented in

certain cases. Sigymbrigm foiginalg seeds escaped light-

induced, and Pglyggnum persigaria dark-induced, dormancy
when treated with nitrate (Karssen,1980/81a and b). In some

cases simultaneous treatment with light or growth regulators

(e.g. gibberellin) counteracts the dormancy-inducing factors

5
such as high temperature and osmotica (Khan and Karssen,

1980; Khan, 1980/81). High levels of oxygen and ethylene
prevent secondary dormancy in Xanthium (Esashi et al.,
1978); in contrast, apple embryos did not enter a dormant
phase when subjected to high temperature under anaerobic
conditions (Bulard, 1986). Lettuce and Bumgx gzigpug seeds
escaped high temperature and dark-induced dormancy when held
under anaerobic conditions (Karssen, 1980/81a: LeDeunff,

1973).

uechanisms_2f_eec9ndarx_d2rmanex_indncfien

Various hypotheses have been proposed to explain
secondary dormancy in seeds. One of the earliest hypotheses
suggested that a limited supply of oxygen to the embryo was
responsible for germination inhibition and secondary
dormancy induction (Crocker, 1906). In 1930, Davis
suggested that a decrease in growth promotors or increase in
inhibitors may have a role in secondary dormancy induction,
and, in 1982, Bewley and Black proposed changes in membranes
as a possible mechanism in the regulation of dormancy.
Limited_axai1abili;¥_2f_9xxgen_t2_emhrxes

Crocker (1906) found that upper seeds of Xanthigm
gangfigngg germinate readily on exposure to an atmosphere of
100% 02: seeds held in air did not. He concluded that the
inhibition of germination was due to the seed coat
restricting the supply of oxygen to the embryo; seeds

displayed no dormancy if the seed coat was removed. Davis

6
(1930) was able to induce dormancy in the cocklebur embryo

by embedding the moist seeds in clay or immersing them in
agar for two months at 27°-30°C under conditions where
presumably a low supply of oxygen was available to the
seeds. The dormancy was subsequently overcome by removal of
the seed coats or moist storage at 5° for three months.

Visser (1956b) studied the effect of high temperature
on respiratory activity (02 consumption and C02 production)
in apple seeds. Partially chilled seeds were successively
subjected to temperatures from 3° to 30°C for 2 hours at
each temperature. With increasing temperature the
respiratory intensity of both seeds and embryos increased.
However, at 25-30° the respiratory quotient of the intact
seeds was higher than that of the excised embryos. Visser
Suggested that restriction of 02 uptake by the seed coat
increased with temperature. If intact, partially stratified
seeds were germinated at 25°, about 60% of seeds entered
secondary dormancy. However, if the seed coat was removed
and the endosperm ruptured, particularly at the radicle end,
high germination levels were obtained (Visser, 1956a).

From these data, Visser proposed that the comparatively
high respiration, combined with the lower 02 availability to
the embryos in intact seeds at high temperatures, are the
primary factors responsible for the development of secondary
dormancy.

Secondary dormancy was induced in 5 days at 25°C in

partially stratified Tatarian maple seeds (Nikolaeva, 1969).

7
A period greater than 8 days ( < 92 days) at low

temperatures was required for seeds to respond to the
transfer to higher temperatures in the above manner. After
transfer of partially stratified seeds to high temperature
(25°) for 2 hours, a sharp intensification of respiration
(02 consumpted) followed and the RQ value rose above unity.
The initial high respiration and R0 fell after 5 days, and
after 16 days, they had dropped to the level of dormant
seeds. Nikolaeva proposed that secondary dormancy was
induced by high respiration rates of the embryo under
conditions of greatly impeded gas exchange, which reduced
the supply of 02 to the embryo.

Come and his colleagues (1972), following this line of
inquiry, suggested that oxygen is consumed by the testa
enclosing the embryo. In apple, oxygen consumption by the
testa was attributed to the oxidation of various phenolics
present such as phloridzin, chlorogenic acid, and para-
coumarquuinic acid. They suggested that when apple seeds
are exposed to elevated temperatures, oxygen becomes less
soluble in water and oxidation of the phenolic compounds in
the seed coat increases. These factors reduce availability
of 02 to the embryo, resulting in induction of secondary
dormancy.

Come and Tissaoui (1968,1972) directly measured the
effect of low 02 tension on apple embryos. A 42 day
exposure to 02 tensions of <0.16% had no effect on

germination of partially stratified embryos, but exposure to

8
higher 02 levels (0.16 to 0.84%) induced secondary dormancy.

However, a 42 day period of 02 deprivation for apple embryos
is not physiologically meaningful when only 8 days at 30°C
were required for secondary dormancy induction in embryos
(Perino and Come, 1977). Moreover, embryos exposed to 0.5%
02 for 7 days do not enter into secondary dormancy (Come and

Ralambosoa, 1979).

WWW
Ab§91§12_agid& Since its chemical identification in 1965

(Ohkuma et a1.), abscisic acid has become the most studied
growth inhibitor in plants. Attempts to correlate the level
of ABA with the induction of dormancy in seeds have resulted
in conflicting evidence. Freshly harvested seeds of species
that exhibit primary dormancy, such as ash (Ergxings
angrigana) and hazelnut ($911135 ayellana) (Sondheimer et
al., 1968; Williams, et al., 1973) contain relatively high
levels of ABA.

Balboa-2avala and Dennis (1977) studied the seed
maturation of two apple cultivars with respect to their ABA
levels and germinability. In developing 'Golden Delicious'
seeds, ABA levels in the embryonic axis, as measured by
electron capture gas chromatography (EC-GC), were highest
when germinability was highest, with both declining as the
seed matured. In 'McIntosh', ABA levels remained relatively
constant (400-600 ng/g fresh wt.) during the period when

germinability declined from 60% to 0%. ABA levels

9
subsequently rose to very high levels (1300 ng/g fresh wt.),

after the seeds had become dormant. However, the
concentrations of ABA in embryonic axes reported by Balboa-
Zavala and Dennis (1977) are approximately 500 times greater
than values obtained using gas chromatography combined with
mass spectrometry (GO-MS) (Subbaiah, 1987), making these
data questionable.

The most consistent evidence available for the
involvement of abscisic acid in the induction of seed
dormancy comes from the studies on ABA-deficient mutants of
Arabiggpgig thaligna (L.) Heynh. Mutant lines of
AIQEIQQESIS characterized by high transpiration rates and a
lack of seed dormancy were found to contain low levels of
ABA in leaves and mature seeds in comparison with the wild-
type plants (Karssen, et al., 1983). In the wild-type
seeds, the ABA concentrations reached 200-500 ng per g fresh
weight, whereas in the mutants the concentration never
exceeded 10 ng per gram. The wild-type seeds were dormant
at maturity but the mutant seeds were not. Reciprocal
crosses of wild type (Aba/Aba) and ABA-deficient mutants
(aba/aba) were made in order to produce seeds with the
Aba/aba embryo genotype in both a wild-type and an Aha-type
mother plant. Dormancy did not develop when both parents
were ABA-deficient, but it developed fully in the
heterozygous F1 seeds, irrespective of the genotype of the
mother plant. Therefore, Karssen et a1. (1983) concluded

(a) that the development of dormancy is regulated by the

10
genotype of the embryo and is not a maternal effect, and (b)

that the probability that ABA and dormancy induction are not
causally related is very low because genetic analysis has
shown that only a single gene is involved in this mutation
(Koornneff et a1. 1982). The data of Karssen et a1. (1983)
are very strong evidence that embryonic ABA is associated
with primary dormancy in Arabidgpgis; however, can we
conclude that induction of dormancy is the same in
herbaceous species as in a woody species, or in primary
dormancy as in secondary dormancy?

The possibility that ABA is responsible for the
initiation of secondary dormancy is equivocal. Balboa-
Zavala and Dennis (1977) also measured ABA levels in
partially stratified apple seeds in which secondary dormancy
was induced by exposure to 1 week at 27°C. ABA levels
decreased during the period of high temperature in whole
seeds; however, ABA levels in the embryonic axis,
cotyledons, and seed coats were not measured separately.

ABA prevents only the very last phase of germination,
that involving cell expansion, in Qhengpgdigm alpgm
(Karssen, 1976). Radicle growth in this seed can be divided
temporally into 3 stages. The radicle splits the outer
integument in stage 1, but the inner testa remains intact;
in stage 2, further growth occurs, still inside the intact
inner integument, then, in stage 3, the radicle bursts
through this coat. Application of ABA to germinating seeds

does not stop them from reaching stages 1 and 2, but

11
prevents the onset of stage 3. Studies with 14C-labelled

ABA demonstrated that a substantial amount of applied
inhibitor entered the seeds within 16 hours from the start
of inhibition, well before seeds had reached stage 1.

Studies on Sinapis alba (Schopfer et al., 1979) and
fiaplgpgppgg gzggilig (Galli et al., 1980) also suggest a
late action of abscisic acid. ABA remains an effective
inhibitor as long as it is applied just before the
initiation of radicle elongation.

The late action of ABA could explain the inhibition of
germination of fully stratified apple seeds, since they are
poised for radicle elongation before exposure to dormancy
inducing conditions. However, secondary dormancy can also
be induced in partially stratified seeds that would not be
poised for radicle elongation in the same conditions.

At which level, and via what mechanism, ABA exerts its
inhibitory influence in the developing or mature seed is an
interesting line of inquiry. Galli, et al. (1979) observed
that ABA at 10’5M completely inhibited the emergence of
radicles from Haplgpappug gragilig seeds, and this effect
was prevented effectively by fusicoccin (FC), a fungal toxin
which promotes H+/K+ exchange in plant membranes. However,
FC had limited effect in preventing ABA inhibition of post-
germinative growth. ABA at this later stage of germination
inhibited DNA synthesis as demonstrated by the incorporation
of [3H] thymidine into nuclei. Because FC did not reverse
this inhibition, the authors suggested that two different

12
mechanisms are involved in the ABA inhibition of H&_g;agili§

germination. One mechanism, which is reversed by PC,
interferes with KI uptake at the level of cell membranes;
the other, which is not, interferes with the synthesis of
nucleic acids and/or proteins.

Studies by Jacobsen and Beach (1985) on the inhibition
of Ga3-induced synthesis of “-amylase by ABA in barley
(figrgegm yulgaze) aleurone cells has led to the conclusion
that ABA antagonizes GA action in two major ways, one
preventative and one promotive. It prevents GA action at
the level of transcription by suppressing GA-induced mRNA
synthesis. The promotive action involves the ABA-induced
increase in mRNAs and protein, which in one case is proposed
to be related to the production of an -amylase inhibitor
(Ho, 1988).

Ethylene. Another growth regulator with a possible role in
secondary dormancy induction is ethylene. Sinska and Gladon
(1984) suggested that ethylene was required for germination
of apple embryos. If this is the case, inducers of
secondary dormancy may act in apple seeds by reducing
ethylene levels, resulting in inhibition of germination.
Secondary dormancy of sunflower seeds (figlignthng annugg L.)
induced by 5 days at 45°C, was partially prevented when
seeds were presoaked at 5° for 22 hours with 2-
chloroethylphosphonic acid (ethephon). However, C2H4 (55
ul/l) and ACC (2.5mM) were not effective (Corbineau, et al.,

1988).

13
W

A body of information supports the possibility that
processes controlled by the state of membranes are involved
in the regulation of dormancy (Bewley and Black, 1982).
Membranes undergo sharp transitions in their physical state
at particular temperatures. Enzymes associated with them
also undergo abrupt changes in activity. Factors which
induce dormancy may do so through changes in membranes and
membrane-bound enzymes. However, the nature of these

processes remains speculative.

W

Investigations on the mechanisms involved in the
maintenance of secondary dormancy have led to an
understanding of the processes that occur during this
period, but they do not define which process or processes
are responsible for this state. In the Visser study (1956b)
when partially chilled apple seeds were stored for up to 8
weeks at 25°C under moist conditions, both respiratory
activity and germination capacity in excised embryos
decreased. Therefore, high respiratory activity in the
embryos was not necessary for the maintenance of secondary
dormancy.

Autoradiographic studies have shown that protein
synthesis occurs in the axis, scutellum, coleorhiza, and
aleurone layer of dormant wild oat grains. The amount of
synthesis in the dormant embryos, however, is not

substantially different from that in the embryos of after-

l4
ripened grains in the first 12-24 hours of inhibition (Chen

and Varner, 1970; Cuming and Osborne, 1978). In dormant
grains, an analysis of membrane proteins indicated that they
continuously turn over at a relatively high rate, although
no new classes of membrane proteins were produced (Cuming
and Osborne, 1978a and b). The authors suggested that this
continuous generation and recycling of cellular membranes is
necessary to maintain the metabolic integrity of the
hydrated cells of the dormant embryos.

Evidence that chemical inhibitors are responsible for
the maintenance of dormancy is equivocal. In some cases,
dormant seeds have higher levels of abscisic acid (ABA) than
non-dormant seed, as in Eraxinug (Sondheimer et al., 1968).
However, no such differences occur between non-dormant Ayena
satiya and the highly dormant A;_fatua (Berrie et al., 1979)
or among species and strains of Eyrug with different degrees

of dormancy (Dennis et al., 1978).

W):

Secondary dormancy of apple seeds is broken by low
temperature stratification. Although no reports on
alternative methods for breaking secondary dormancy have
been published for apple seeds, factors such as light and
combinations of growth regulators reportedly overcome
secondary dormancy in other species. High temperature
dormancy in lettuce can be relieved by a combination of 20%
oxygen and 40-80% carbon dioxide (Thornton, 1936) or

exposure to respiratory inhibitors (Khan and Zeng, 1985).

15
Osmotically induced dormancy of sheneeedim Mentions

is overocme by light and by gibberellin A4 and A7 (Khan and
Karssen, 1980). Secondary dormancy induced by darkness and
high temperature in lettuce seeds is broken by various

combinations of light, gibberellin, cytokinin, and ethylene

(Bewley, 1980; Khan, 1980/81).

Summer!

Secondary dormancy in seeds reflects a general
insensitivity to external and, perhaps, internal inducers of
germination. The non-specificity of both the inducing
agents and the inhibitory condition suggests a very general
change in cell metabolism or subcellular structure, but what
these changes may be is not known. Since this dormancy can
be fairly easily manipulated under laboratory conditions,
further investigation in this field could prove very
fruitful in understanding the biochemical basis of dormancy.

The goals of this thesis were to a) confirm and extend
the observations of the effects of temperature on the
induction and release of secondary dormancy in apple seeds,
b) define the roles of apple seeds and embryos with respect
to the induction of secondary dormancy by low 02 tension, c)
define the role of abscisic acid in heat stress induced
secondary dormancy in apple seeds, and d) define the role of
C2H4 in apple seed germination and secondary dormancy

induction.

16
Liggrgggzg Qitgg

Abbott, D.L. 1955. Temperature and the dormancy of apple
seeds. Rept. XIV Intern. Hort. Congr., Netherlands.
2A:746-753.

Balboa-Zavala, O. and F.G. Dennis, Jr. 1977. Abscisic acid
and apple seed dormancy. J. Amer. Soc. Hort. Sci.
102:633-637.

Berrie, A.M.M., Buller, D., Don, R. and W. Parker. 1979.
Possible role of volatile fatty acids and abscisic acid
in the dormancy of oats. Plant Physiol. 63:758-764.

Bewley, J.D. 1980. Secondary dormancy (skotodormancy) in
seeds of lettuce (Lagtgga gatiya cv. Grand Rapids) and
its release by light, gibberellic acid and
benzyladenine. Physiol. Plant. 49:277-280.

Bewley, J.D. and M. Black. 1982. Physiology and Biochemistry
of Seeds, Vol.2. Springer-Verlag, New York. pp.207-212.

Bulard, C. 1986. Conditions of induction of secondary
dormancy in apple after breaking of primary dormancy by
anaerobiosis and low temperature. Physiol. Plant.
67:279-284.

Chen, S.S.C., and J.E. Varner. 1970. Respiration and protein
synthesis in dormant and after-ripened seeds of Aygna
fiatga. Plant Physiol. 46:108-112.

Come, D. and T. Tissaoui. 1968. Induction d'une dormance
embryonnaire secondaire chez le pommier (Riggs malgs
L.) par des atmospheres tres appauvries en oxygene.
C.R. Acad. Sci. Paris 266:477-479.

Come, D. and T. Tissaoui. 1972. Interrelated effects of
inhibition temperature and oxygen on seed germination.
p.157-168. In: W. Heydecker (ed) Seed Ecology.
Pennsylvania State University Press, Univ. Park, PA.

Come, D. and J. Ralambosoa. 1979. Influence simultanee de la
lumiere et de l'oxygene sur la germination de l'embryon
de pommier non-dormant. C.R. Acad. Sc. Paris 289:869-
871.

Corbineau, F., Rudnicki, R.M., and D. Come. 1988. Induction
of secondary dormancy in sunflower seeds by high
temperature. Possible involvement of ethylene
biosynthesis. Physiol. Plant. 73:368-373.

Crocker, W. 1906. Role of seed coats in delayed germination.

17

Cuming, A.C. and D.J. Osborne. 1978a. Membrane turnover in
imbibed and dormant embryos of the wild oat (Aygna
issue L.) I. Protein turnover and membrane replacement.
Planta 139:209-217.

Cuming, A.C. and D.J. Osborne. 1978b. Membrane turnover in
imbibed and dormant embryos of the wild oat (Avega
fiatug L.) II. Phospholipid turnover and membrane
replacement. Planta 139:219-226.

Davis, W.E. 1930. Development of dormancy in seeds of
cocklebur (Xanthigm). Amer. J. Bot. 17:77-87.

Dennis, F.G. Jr., Martin, G.C., Gaskin, P. and J. MacMillan.
1978. Hormones in pear seeds. II. Levels of abscisic
acid, dihydrophaseic acid, and their metabolites in
relation to seed dormancy in several Eyrus species. J.
Amer. Soc. Hort. Sci. 103:314-317.

Durand, M., Thevenot, C. and D. Come. 1973. Influences de
l'acide abscissique sur la germination et la levee de
dormance des embryons de Pommier (Riggs Helge L.). C.
R. Acad. Sci. Paris D 277:53-55.

Esashi, Y., Okazaki, M., Yanai, N., and K. Hishinuma. 1978.
Control of the germination of secondary dormant
cocklebur seeds by various germination stimulants.
Plant and Cell Physiol. 19:1497-1506.

Galli, M.G., Miracca, P., and E. Sparvoli. 1979. Interaction
between abscisic acid and fusicoccin during germination
and post germination growth in naplgpgppug gragilig.
Plant Sci. Lett. 14:105-111.

Galli, M.G., Miracca, P., and E. Sparvoli. 1980. Lack of
inhibiting effects of abscisic acid on seeds of
W 911219.111: (Nutt-) Gray pregeminated in
water for short times. J. Exp. Bot. 31:763-770.

Harrington, G.T. 1923. Respiration of apple seeds. J. Agr.
Res. 23:117-131.

Ho, T.D. 1988. Regulation of gegfi expression by ABA in
barley aleurone layers. 13 International Conf. Plant
Growth Substances. p.23 (Abstract)

Jacobsen, J.V. and L.R. Beach. 1985. Evidence for control of
transcription of CQ-amylase and ribosomal RNA genes in
barley aleurone protoplasts by gibberellic acid and
abscisic acid. Nature 316:275-277.

18

Kahn,A. 1960. An analysis of "dark-osmotic inhibition" of
germination of lettuce seeds. Plant Physiol. 35:1-7.

Karssen, C.M. 1976. Uptake and effect of abscisic acid
during induction and progress of radicle growth in

seeds of Chengpggium album. Physiol. Plant. 36:259-263.

Karssen, C.M. 1980/81a. Environmental conditions and
endogenous mechanisms involved in secondary dormancy of
seeds. Israel J. Bot. 29:45-64.

Karssen, C.M. 1980/1981b. Patterns of change in dormancy
during burial of seeds in soil. Israel J. Bot. 29:65-
73.

Karssen, C.M., Brinkhorst-van der Swan, D.L.C., Breekland,
A.E., and M. Koornneef. 1983. Induction of dormancy
during seed development by endogenous abscisic acid:
studies on abscisic acid deficient genotypes of

Arabiggpgis thaliana (L.) Heynh. Planta 157:158-165.

Khan, A.A. 1980/1981. Hormonal regulation of primary and
secondary seed dormancy. Israel J. Bot. 29:207-224.

Khan, A.A. and C.M. Karssen. 1980. Induction of secondary
dormancy in Qhengpgdium bonus-henricus L. seeds by
osmotic and high temperature treatments and its
prevention by light and growth regulators. Plant
Physiol. 66:175-181.

Khan, A.A. and G. Zeng. 1985. Dual action of respiratory
inhibitors: inhibition of germination and prevention of
dormancy induction in lettuce seeds. Plant Physiol.
77:817-823.

Koornneeff, M., Jorna, M.L., Brinkhorst-van der Swan,
D.L.C., and C.M. Karssen. 1982. The isolation of
abscisic acid (ABA) deficient mutants by selection of
induced revertants in non-germinating gibberellin

sensitive lines of Arabiggpsis thaliana (L.) Heynh.
Theor. Appl. Genet. 61:385-393.

Le Deunf, Y. 1973. Interactions entre l'oxygene et la
lumiere dans la germination et l'induction d'une
dormance secondaire chez les semences de 33mg; gzigpgg
L. Note C.R.Acad. Sci. Serie D 276:2381-2384.

Luckwill, L.C. 1952. Growth-inhibiting and growth-promoting
substances in relation to the dormancy and after-
ripening of apple seeds. J. Hort. Sci. 27:53-65.

19

Mumford, P.M. 1988. Alleviation and induction of dormancy by

temperature in Impatiens glandulifera Royle. New
Phytol. 109:107-110.

Nikolaeva, M.G. 1969. Physiology of deep dormancy in seeds.
p. 146-162 Israel Program for Scientific Translations
Press, Jerusalem.

Ohkuma, K., Addicott, F.T., Smith, O.E., and W.E. Thiessen.
1965. The structure of abscisin II. Tetrahedron Lett.
29:2529-2535.

Perino, C. and D. Come. 1977. Influence de la temperature
sur les phases de la germination de l'embryon de
Pommier (21:35 malug L.). Physiol. Veg. 15:469-474.

Rudnicki, R. and J. Pieniazek. 1973. The effect of abscisic
acid on stratification of apple seeds. Bull. Acad. Pol.
Sci. 21:149-154.

Schopfer, P., Bajracharya, D., and D. Plachy. 1979. Control
of seed germination by abscisic acid. I. Time course of
action in Sinapis alga L. Plant Physiol. 64:822-827.

Sinska, I. and R.J. Gladon. 1984. Ethylene and the removal
of embryonal apple seed dormancy. HortScience. 19:73-
75.

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

Stokes, P. 1964. Temperature and seed dormancy, pp746-803,
In: Handbuch der Pflanzenphysiologie, Volume 15.
Ruhland, W. (Ed.), Springer, Berlin.

Subbaiah, T. 1987. Abscisic acid relationships in apple seed
dormancy. Ph.D. Thesis, Cornell University.

Thornton, N. 1936. Carbon dioxide storage. IX. Germination
of lettuce seeds at high temperatures in both light and
darkness. Contrib. Boyce Thompson Inst. Plant Res.
8:25-40.

Thornton, N. 1945. Importance of oxygen supply in secondary
dormancy and its relation to the inhibiting mechanism
regulating dormancy. Contr. Boyce Thompson Inst. Pl.
Res. 13:487-500.

Visser, T. 1956a. The role of seed coats and temperature in
after-ripening, germination and respiration of apple
seeds. Proc. K. Ned. Akad. Wet. 59:211-222.

20

Visser, T. 1956b. Some observations on respiration and
secondary dormancy in apple seeds. Proc. K. Ned. Akad.
Wet. 59:314-324.

Williams, P.M., Ross, J.D., and J.W. Bradbeer. 1973. Studies
in seed dormancy. VII. The abscisic acid content of the
seeds and fruits of gorylug ayellana L. Planta 110:303-
310

SECTION I

The Effect of Temperature on the Induction and Release of

Secondary Dormancy in Apple Seeds

21

The Effect of Temperature on the Induction and Release of

Secondary Dormancy in Apple Seeds

mm

Partially stratified apple seeds can be induced into
secondary dormancy by high temperature and release from this
dormancy by restratification at low temperature (Abbott,
1955: Visser, 1956). Visser (1956) held apple seeds at 25°C
to induce secondary dormancy following cold stratification.
As the time of storage at 25°C increased, longer periods at
3°C were required to restore the initial germination
capacity of the excised embryos. However, he did not
establish whether high temperature completely negated the
promotive effects of chilling. In order to further
characterize the conditions inducing and breaking secondary
dormancy, seeds stratified at 5°C for 6,8, and 12 weeks were
exposed to temperatures ranging from 15 to 40°, then
germinated at 20°. In a second experiment, seeds were
stratified for varying periods of time prior to exposure to

30°, the restratifed at 5°.

He£§£iel§_end_nethgd§
Apple seeds (Melee gemeegiee cv. Golden Delicious) were
removed from fruit at harvest, dried, and stored at 5°C.
Subsequently, seeds were soaked overnight in water, rinsed,
placed in Petri plates containing filter paper wetted with

0.5% Captan solution, then held in darkness at 5°C for 0 to

22

 

23
12 weeks before exposure to higher temperatures. Embryos

were considered germinated when the radicle reached a length
of 3 mm or longer. Seeds induced into secondary dormancy by
high temperature were restratified at 5°C to check
viability.

Both experiments were arranged in a completely random
design. An analysis of variance (ANOVA) was performed and

Duncan's Multiple Range Test (DMRT) used to determine mean

separation.
Experiment 1. Imduetien e: seeemdegy dermaney, Seeds were

stratified for 6, 8, or 12 weeks, then placed at 15, 20, 25,
30, 35, or 40°C for 1 week prior to transfer to 20° for
germination (5 replications of 10 seeds each). Embryos were
dissected from half the seeds on transfer to 20°.
Germination was evaluated during a 10-day period at 20° in
darkness.

WWW Seeds were
stratified at 5°C for 3, 6, or 9 weeks. Control seeds were
germinated at 20° immediately on removal from 5°. All other
seeds were transferred from 5° to 30° for 6 days, then
returned to 5° for 0, 3, 6, or 9 weeks. Following the last
exposure to 5°, all seeds, or embryos dissected from
similarly treated seeds, were placed at 20° for germination.
Six replications of 10 seeds or embryos were used per
treatment.

The results of both experiments are presented as the

sum of the mean daily percentage germination after 12 days

 

24
(Sum 12: 1200 indicates 100% germination on the first day),

according to the method of Timson (1965).

W- Germination

of seeds increased with stratification time, reaching nearly
100% after 12 weeks at 5°C, provided subsequent temperature
did not exceed 30° (Table 1). In contrast, almost all
embryos germinated, even after only 6 weeks of
stratification, at all temperatures except 35°.

Exposure to temperatures greater than 20°C inhibited
germination of seeds stratified for 6 or 8 weeks, whereas a
temperature of 35° was required to inhibit those stratified
for 12 weeks (Table 1). Germination at 35° was negligible
in seeds stratified for 6 or 8 weeks. Embryo germination
was inhibited at 35°, regardless of stratification time.
Both seeds and embryos were killed by exposure to 40° for 1
week, although radical protrusion occurred in a few cases
(data not shown). More than 90% of the seeds induced into
secondary dormancy by 25, 20, and 35°C germinated on
restratification at 5° (data not shown).

Interaction between duration of stratification at 5°C
and temperature of exposure after stratification was
significant at P < 0.01 for seed germination, indicating
that inhibition occurred at 25° and 30° only when
stratification time was less than 12 weeks (Table 1). This

interaction was not significant for embryos excised

25
following exposure to 15, 20, 30, and 35°.

s an . Germination
capacity of both seeds and embryos increased as the duration
at 5°C increased from 0 to 9 weeks. Although the seed
versus embryo comparison was not tested critically, as
separate ANOVAs were performed, removal of the seed coat
consistently increased germination capacity except in seeds
stratified continuously for 9 weeks.

High temperature treatment prior to stratification did
not appreciably affect response to stratification in either
seeds or embryos (compare first column vs. first line in
each set of data), although Sum 12 at 6 weeks was
significantly higher for both following the 30°C treatment
(Table 2). In contrast, exposure to 6 days at 30° following
stratification reduced germination capacity in both seeds
and embryos. Exposure to high temperature eliminated the
effects of prior chilling only in seeds stratified for 9
weeks: those chilled for 3 or 6 weeks following high
temperature treatment germinated consistently better than
those chilled for the same periods of time, but not exposed
to high temperature (Table 2: Figure 1). Interaction was
not tested because of missing data, but is clearly evident
in Figure 1, which suggests that high temperature inhibition

of germination was more effective in seeds in which the

chilling requirement had been saturated.

 

26
n. .

The data for experiment 1 confirm those of Abbott
(1955) and Visser (1956) in demonstrating that high
temperatures induce secondary dormancy in apple seeds.
Temperatures of 25° and 30°C were equally effective in
inhibiting germination of seeds, but neither was effective
in embryos following stratification for 6 weeks or longer.
Holding seeds at 35°, on the other hand, reduced subsequent
germination at 20° of both seeds and of embryos dissected
from them. As stratification proceeded, the seeds and
embryos became less sensitive to high temperature induction
of dormancy. The data in Table 1 suggest that seeds are
more sensitive to the induction of dermancy by high
temperature than are embryos. This is probably more
apparent than real, however, for the seed coat acts as a
physical barrier to germination of the embryos. Seed coat
removal therefore allows germination to occur despite
relatively unfavorable conditions. Had an osmoticum been
used to restrict germination, inhibitory effects on embryo
germination might have been apparent at 25° and 30°.

In experiment 2, exposure to 30°C, which induced
secondary dormancy in seeds and embryos stratified for 3 or
6 weeks, did not completely negate the promotive effect of
the previous stratification period. In contrast, exposure
to high temperature eliminated the effects of prior chilling
in seeds stratified for 9 weeks.

These data indicate that high temperature may not

27
completely negate the promotive effect low temperature

exposure has on seed or embryo germination unless full
germination potential of the seed has been achieved prior to

the heat treatment.

Literature—Cited

Abbott, D.L. 1955. Temperature and the dormancy of apple
seeds. Rept. XIV Intern. Hort. Congr., Netherlands.
2A:746-753.

Timson, I. 1965. New method of recording germination data.
Nature 207:216-217.

Visser, T. 1956. Some observations on respiration and

secondary dormancy in apple seeds. Proc. K. ned. Akad.
Wet. 59:314-324.

28

Table 1. The effect of time of stratification of seeds at
5°C and subsequent exposure to higher temperatures for
1 week on germination of seeds and embryos during 10
days at 20 .

 

 

Weeks at Subsequent Germination (%)
5°C temp. (°C)
Seeds Embryos
"2"""""’I§""'"m""SESSSYZSEIE'"ISB;""
20 49bc (48) 100a
25 26d (8) 100a
30 22de (2) 96a
35 2f (2) 34c
""S'mm'mig """"""" SEQ'Z?§§"""'IEB§ """
20 89a (87) 100a
25 40bcd (16) 100a
30 54b (16) 100a
35 6ef (4) 34¢
""I'émmm’IE """"""" ;§;’Z;§§"""'133; """
20 100a (100) 100a
25 96a (82) --
30 94a (62) 100a
35 32cd (32) 54b
Time at 5°C X Temperature ** n.s.

-- Missing treatment

2 % germination after 7 days prior to transfer to 20°

Y Within seeds or embryos, means followed by the same
letter are not significantly different from one
another by DMRT, P < 0.05.

** Significant at the 5% level : n.s. = not significant

29

Table 2. The effect of stratification at 5°C before and
after exposure to 30° for 6 days on germination of
apple seeds and embryos after 12 days at 20° .

 

Sum 12

 

 

 

 

Stratification Stratification at 5°C (wks) after
at 5°C (wks) exposure to 30 °C
before
exposure to
control2 0 3 6 9
§§§Q§ (Sum 12)
o 4eY 0e 8e 637C 951b
3 319 208 - 7330 11323
6 459d 108 456d - 1107a
9 923b 0e 97$ 483d 896b
Em (Sum 12)
0 483g 277h 668f 1052bC 1139ab
3 663f 527g - 1125ab 1194a
6 936d 8088 1157a - 11753
9 1113ab 216h 979Cd 1104ab 1183a

 

2

not exposed to 30°C

Y Within seeds or embryos, means followed by the same
letter are not significantly different from one another by

DMRT, P < 0.05.

30

Figure l. The effect of stratification at 5°C, before and
after exposure to 30°C for 6 days, on germination of
apple seeds and embryos.

31

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eesmoaxe 823 .925 n outsobm xlx

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SECTION II

Germination Responses of Apple Seeds vs. Embryos to High

Temperature and Low Oxygen Tension

32

Germination Responses of Apple Seeds vs. Embryos to High

Temperature and Low Oxygen Tension

beegzeeg. Anaerobiosis is reported to break the dormancy
of non-stratified apple embryos. Both anaerobiosis (<0.16%
02) and low 02 treatments (0.3-0.5% 02) were tested for
effectiveness on both non-stratified and stratified apple
seeds and embryos, and on stratified seeds in which
secondary dormancy had been induced by high temperature. A
small promotive effect was observed in non-stratified, but
not in stratified embryos, and the treatments inhibited
germination of stratified seeds. Neither treatment broke
secondary dormancy in intact seeds, and often inhibited the
germination of embryos excised from them. The possible
reasons for the difference in response between seeds and

embryos to low 02 tension are discussed.

33

34
Tissaoui and Come (6) using apple seeds immediately

removed from the fruit, reported that 7 days of anaerobiosis
at 20°C broke dormancy in non-stratified embryos, and that
42 days of anaerobiosis did not induce secondary dormancy in
stratified embryos (1,2). Come and Ralambosoa (3) also
reported that 7 days of exposure to 0.5% 02 did not induce
secondary dormancy in non-dormant embryos. However, effects
of low 02 tension on the dormancy status of intact apple
seeds has not been reported. We repeated the work of
Tissaoui and Come on embryos, and observed the germination
response of dormant and non-dormant intact seeds following

exposure to high temperature and low oxygen tension.

W

-e“ :0- file H‘ 100.807 :7 2 O 1 o! ,1. o-guolt 0!
Seeds were removed from 'Golden Delicious' fruit at harvest,
dried, and stored at 5°C. Subsequently, the seeds were
soaked overnight in water, rinsed, placed in 150 X 15 mm
Petri plates (400 seeds/plate) on filter paper wetted with
0.5% Captan solution, and stratified at 5° in the dark for
0,3,6,and 9 weeks. Half the seeds were then exposed to 6
days at 30°C to induce secondary dormancy. In one
experiment, non-dried seeds were removed from fruit stored
at 5° for approximately 23 weeks and used without drying.
To test germination following various treatments, seeds or
embryos were held on moist filter paper in Petri plates at

20°C. Embryos were considered germinated whenever radicle

35
length was 3mm or more.

Experimemee1_eengieiene. In experiment 1 (<0.16% 02), non-
stratified seeds, partially stratified seeds, and seeds
exhibiting secondary dormancy were placed in a 125 ml flask
(30 seeds per replication: 3 replications per treatment)
containing 10 ml Captan solution (0.5%). The flasks were
placed on an apparatus made with copper tubing and
humidified nitrogen gas was passed through the flasks at
0.4-0.5 ml/min for 6 days at 20°. Oxygen content of the
effluent gas stream was measured daily using a flow-through
02 electrochemical cell and was always below 0.16%. After 6
days the seeds were removed from the low 02 environment,
embryos were dissected from half the seeds, and both seeds
and embryos were placed at 20° in air for germination.

In experiment 2 (0.3-0.5% oxygen), non-stratified
seeds, partially stratified seeds, and seeds exhibiting
secondary dormancy were placed in 60 X 15 mm Petri plates (6
replications of 15 seeds per treatment) on filter paper
moistened with 4 ml Captan solution (0.5%). The plates were
placed in a desiccator (2.5 L volume), the air was
evacuated, and the desiccator was flushed with nitrogen gas
3 or more times. The final oxygen concentration varied from
0.3 to 0.5%. The desiccators were placed at 20° or 30°C for
6 days. Seeds were then removed from the desiccators,
embryos dissected from half the seeds, and the seeds and

embryos were placed at 20° in air for germination.

36
Emelee;193_eg_ge:mineeien. The results are presented as the

sum of the mean daily percentage germination after 12 days
(Sum 12: 1200 indicates 100% germination on the first day),
according to the method of Timson (5).

Seeds induced into secondary dormancy by high
temperature or low 02 were restratified at 5° for 12 weeks
to check viability.

Stetietieel_ene1yeie. Experiments were arranged in a
completely random design. A one-way analysis of variance
was performed and Duncan's Multiple Range Test (DMRT) was
used to determine mean separation. A 3 X 2 X 2 factorial
analysis (stratification time X temperature X atmosphere)

was performed and interactions tested.

W

Stratification at 5° in air increased Sum 12 in both
seeds and embryos germinated at 20° (Tables 1 and 2).
Subsequent exposure to 30° almost completely inhibited
germination of seeds and reduced that of excised embryos in
9 of 12 comparisons. The effect upon embryos was more
pronounced in the first experiment than in the second.

Exposure to oxygen levels less than 0.16% (Table 1) at
20° for 6 days reduced germination of seeds and embryos
stratified for 9 weeks, but promoted germination of non-
stratified embryos. Oxygen tension between 0.3-0.5% was
also inhibitory to seed germination following 6 and 9 weeks

stratification, but did not affect embryos excised from

37
similarly-treated seeds (Table 2).

Low 02 levels were ineffective in overcoming secondary
dormancy induced by high temperature: in fact, germination
of embryos was inhibited in 4 of 6 cases (Tables 1 and 2).

The response of non-stratified embryos to low 02
tension was much less than expected based upon the data of
Tissaoui and Come (6). However, they did not dry the seeds
before use. We therefore compared the response of non-dried
seeds that had been immediately removed from fruit stored at
5°C for approximately 23 weeks with those of seeds which
were dried on removal from the fruit and stratified for 9
weeks in Petri plates. In general, germination was lower in
dried than in non-dried seeds, although germination of
control embryos was similar (Fig. 1). Holding seeds of both
lots, or embryos of dried seeds, in air at 30°C markedly
reduced subsequent germination at 20°. Exposure to low
oxygen did not stimulate germination significantly in either
seeds or embryos regardless of seed source, and was
ineffective in breaking secondary dormancy. In fact,
anaerobiosis significantly reduced germination is seeds held
at 20°.

Low oxygen tension (0.3-0.5% 02) did not affect the
germination capacity of embryos from seeds held in Petri
plates at 20°C (non-stratifying temperature) for 3, 6, and 9
weeks (data not shown).

Seeds induced into secondary dormancy by high

temperature or low 02 remained viable, as they germinated on

38
restratification at 5°C (data not shown).

Significant interaction (P < 0.01) between
stratification time (at 5°) and temperature following
stratification in experiment 1 indicated that seed and
embryo germination at 20°C increased with increasing
stratification time, whereas exposure to 30° for 6 days
inhibited germination of embryos greatly and seeds totally,
regardless of the length of stratification. In experiment
2, seeds responded as in experiment 1 (significant
interaction at P < 0.01), however, there was no significant
interaction between stratification time and temperature for
embryos.

Interaction between stratification time and atmosphere
for seeds in experiment 1 and 2 was also significant (P
<0.01). Germination capacity of seeds held in air increased
with increasing time of stratification, whereas germination
of seeds exposed to 6 days of anaerobiosis was totally
inhibited regardless of stratification time. This
interaction was also significant (P < 0.05) for embryos in
experiment 2.

Temperature X atmosphere interaction was significant (P
< 0.01) for seeds in both experiments. Germination capacity
at 20° increased with increasing stratification time at 5°,
but little or no increase occurred when stratified seeds
were exposed to 30°. This interaction was also significant
(P <0.01) for embryos in experiment 1. Exposure to high

temperature reduced germination capacity of embryos except

39
after 3 weeks of stratification. In experiment 2,

significant interaction (P < 0.05) in embryos was due to a
greater inhibition of germination of embryos exposed to 30°
followed by low 02 than of embryos exposed to 30° alone.
Finally, the triple interaction (stratification time X
temperature X atmosphere) was significant for seeds (P <
0.01) and embryos (P < 0.05) in experiment 1 and seeds (P <
0.01) in experiment 2, indicating that the effect of low 02
in inhibiting germination increased with time of
stratification in seeds held at 20°, but the effect was
small or negligible in seeds held at 30° (Figure 2 and 3).
Low 02 did not affect embryo germination at 20°C, however,
at 30°, germination was inhibited and embryos stratified for
3 and 6 weeks were more sensitive to low 02 inhibition than

those stratified for at 9 weeks (Figure 2).

Diseneeien

Stratified seeds responded differently from excised
embryos following exposure to low oxygen tension. The
endosperm acts as a mechanical barrier to germination and
the seed coat contains compounds that are inhibitory to
germination (4). Thus excising embryos from non-stratified
or partially stratified seeds markedly improves germination.
Low 02 greatly reduced germination of seeds but only
slightly reduced or had no effect on germination of embryos
after 6 and 9 weeks stratification. This response may be

mediated by the outer seed coverings. Oxygen-requiring

4O
enzymes may break down endosperm tissue near the radical and

the low 02 tension could severely reduce their activity. On
the other hand, embryo vigor may be reduced by high
temperature: thus isolated embryos are capable of
germination but those enclosed in the seed coat are not.

Our results differ from those of Tissaoui and Come (6)
in that the dormancy-breaking effect of anaerobiosis on non-
stratified embryos was small. This could be due to seed
source, differences in depth of dormancy, or negation of
response by drying.

Anaerobiosis (< 0.16%) did not break high temperature-
induced secondary dormancy in apple seeds. Since (a)
exposure of stratified seeds to 30°C for 6 days does not
completely negate the promotive effects of previous
chilling, and (b) anaerobiosis does not stimulate
germination in stratified embryos, anaerobiosis may only be
effective in stimulating germination of embryos that have
neither been dried nor exposed to dormancy-breaking
temperatures.

mm

1. Come, D. and T. Tissaoui. 1968. Induction d'une dormance
embryonnaire secondaire chez le pommier (gimme melee
L.) par des atmospheres tres appauvries en oxygene.
C.R. Acad. Sci. Paris 266:477-479.

2. Come, D. and T. Tissaoui. 1972. Interrelated effects of
imbibition temperature and oxygen on seed germination.
p.157-168. In: W. Heydecker (ed) Seed Ecology.
Pennsylvania State University Press, Univ. Park, PA.

3. Come, D. and J. Ralambosoa. 1979. Influence simultanee de
la lumiere et de l'oxygene sur la germination de
l'embryon de pommier non-dormant. C.R. Acad. Sci. Paris
289:869-871.

41

4. Luckwill, L.C. 1952. Growth-inhibiting and growth-
promoting substances in relation to the dormancy and
after-ripening of apple seeds. J. Hort. Sci. 27:53-65.

5. Timson, I. 1965. New method of recording germination
data. Nature 207:216-217.

6. Tissaoui, T. and D. Come. 1973. Levee de dormance de
l'embryon de pommier (Eieee melee L.) en l'absence
d'oxygene et de froid. Planta 111:315-322.

42

Table 1. The effects of stratification time and subsequent
exposure to high temperature and/or low oxygen tension
(< 0.16%) on germination of apple seeds and embryos
(Experiment 1): see text for sequence of treatments.

 

 

 

 

 

 

 

Sum 12
Strat. Seeds Embryos
(wks at Exposure
5°C) to 30°C *ir Low 02 mean Air Low 02 mean
02 - 0d 0d 1509 309de
3 - 21ch 27cd 568C 514c
+ .2Q .2Q 5212 1§§£Q
mean 11j 13j 12t 5481 350j 449s
6 - 157b 92b0 73Gb 721b
+ __19 .2Q 211s: _2§Q
mean 821 46ij 64s 5021 410j 4568
9 - 450a 58cd 937a 806D
+ __29 .2Q 1959 lflfifie
mean 226h 29ij 128r 67lh 557i 6l4r
Means for atmosphere:
106m 30n 574m 439n
Means for - 134p 713p
exposure
to 30° + lq 300g

 

2 Data excluded from main effect means

Y Within seeds or embryos, means followed by the same letter
are not significantly different from one another within
sets (a,b,c,d,e,f for all treatments: m,n for atmosphere:
p,q for 30° treatment; r,s,t for stratification time: and
h,i,j for atmosphere within stratification time) by DMRT,
P < 0.05.

43

Table 2. The effects of stratification time and subsequent
exposure to high temperature and/or low oxygen tension
(0.3-0.5%) on germination of apple seeds and embryos
(Experiment 2); see text for sequence of treatments.

 

 

 

 

 

 

 

Sum 12
Seeds Embryos
Strat. Exposure
(wks at to 30°C Air Low 02 mean Air Low 02 mean
5°C)
0 - 4e 0e 483e 450e
3 - 31eY 19e 663cd 737c
+ .91. .22 §1§de 51311
mean 15k 9k 12t 605k 635k 6205
6 - 459b 252c 936b 975b
+ .123 ,__Qe 2112 1119
mean 264i 126j 1958 940ij 875j 907r
9 - 923a 165d 1113a 1028ab
+ §]§ 16g 9532 77 c
mean 487h 105j 296r 1028i 900j 964r
Means for atmosphere: 256m 80n 858m 803n
Means for - 308p 909p
exposure
to 30° + 28g 752g

 

2 Data excluded from main effect means.

Y Within seeds or embryos, means followed by the same letter
are not significantly different from one another within
sets (a,b,c,d,e,f for all treatments; m,n for atmosphere;
p,q for 30 treatment; r,s,t for stratification time: and
h,i for atmosphere within stratification time) by DMRT, P<
0.05.

44

Figure 1. Germination response of dried and non-dried apple
seeds to temperature and oxygen level. Seeds dried
prior to stratification at 5 C (Dried), or held in
fruits at 5°C for 23 weeks prior to treatment (Non-
dried).

omEo om_molzoz

 

 

  

 

45

 

 

 

 

      

   

                                      

 

    

 

                          

 

 

          

 

 

 

 

mega 88m means“. 8H8
I! x -n o
v". I
x I
"A. m :08
m u
v". I
x ” .100?
H "a“ a
v”. I
. vol I 41000
x I
m m H :08
a No 30..
L w +oofiuoon mm.
H No 3383 I . 89
can a
08 U :89

 

 

e 0.59.".

Zl W08

46

Figure 2. The effect of stratification time and subsequent
exposure to 30°C and low oxygen tension (< 0.16%) on
germination (Sum 12) of apple seeds and embryos.

SUM 12

FIGURE 2

 

 

 

 

 

 

1000
o—ozocut was
0—0 acre-0,
”W's—am“
A—A 300L000:
000--
400-- . /
A
200“. \
A
0 i 3 i
SEEDS
800«-
000--
O
400«-
200"
—O
O 3 6 9

WEEKS AT SC

48

Figure 3. The effect of stratification time and subsequent
exposure to 30°C and low oxygen tension (0.3-0.5%) on
germination (Sum 12) of apple seeds and embryos.

SUM 12

49

FIGURE 3

 

  
 
 

 

 

 

 

 

 

 

 

 

120000—0200“! ms
O—O 206 Low 02
‘Mde ‘-‘ mm
5—5 300 Lee 02
800 / A
400«-
200v
0 : :
1000" SEEDS
O
GOO-r
600--
O
400-»
200--
AA
0 K)?
0 3 6 9

WEEKS AT SC

SECTION I I I

The Role of Abscisic Acid in Heat Stress Induced Secondary

Dormancy in Apple Seeds

50

The Role of Abscisic Acid in Heat Stress Induced Secondary

Dormancy in Apple Seeds

eesgeect. Exposure of stratified apple seeds to a
temperature of 30°C induces secondary dormancy. To
determine if a rise in abscisic acid (ABA) content was
associated with the loss in germination capacity, stratified
seeds (3, 6, or 9 weeks at 5°) were held at 30° for 0, 3, or
6 days. Seeds were dissected into seed coat, cotyledons,
and embryonic axis and analyzed for ABA content using HPLC
and capillary column electron capture gas chromatography
(EC-GC). Stratification at 5° either had no effect or
increased ABA content in embryonic axes, cotyledons and seed
coats. Exposure to 30° after stratification either did not
affect or decreased ABA content in embryonic axes and seed
coats: in contrast, cotyledonary ABA was increased. Seed
coats, cotyledons, and embryonic axes stratified for 3, 6,
or 9 weeks at 20° contained the same or higher levels of ABA
in comparison with non-stratified seeds or seeds stratified
at 5°. ABA changes were not correlated with germination
capacity during stratification or after exposure to 30°.
These data suggest that changes in ABA are unrelated to

changes in dormancy status.

51

52
Since its chemical identification in 1965 (Ohkuma, et

al.), abscisic acid has become the primary growth inhibitor
studied in plants. In dormant seeds of apple, Pieniazek
and Rudnicki (1967) first detected the presence of an ABA-
like inhibitor, which Rudnicki (1969) later confirmed as
ABA. Attempts to correlate the levels of ABA with the
induction of dormancy in seeds have resulted in conflicting
evidence. The data of Karssen et al. (1983) provide very
strong evidence that embryonic ABA is associated with
primary dormancy in Aeeeigeeeie Lheiieme. In contrast,
Balboa-Zavala and Dennis (1977) measured ABA levels in
stratified apple seeds during the induction of secondary
dormancy. They found that ABA levels in whole seeds
decreased during the period of high temperature; however,
levels in the embryonic axes, cotyledons, and seed coats
were not separately measured. In order to clarify the role
of ABA in the process of secondary dormancy induction, we
have measured the levels of ABA in seed coat, cotyledons,
and embryonic axes of seeds statified at 5°C, then induced

into secondary dormancy by high temperature.

te ' s d
SEMWWLW Seeds were
removed from Frazier Spur 'Golden Delicious' fruit at
harvest, dried, and stored at 5°C. Subsequently, seeds were
soaked overnight in water, rinsed, placed in 150 X 15 mm
Petri plates (400 seeds/plate) on filter paper wetted with

0.5% Captan solution, and held at 5° or 20° in the dark for

53
0, 3, 6, or 9 weeks.

Ernerimental.cenditien§- After stratification, seeds (15
per replication, 3 replications per treatment) were rinsed
and placed in 100 X 15 mm Petri plates containing the same
solution used for stratification, and held at 30°C for 0,3,
and 6 days. Seeds were then dissected into embryonic axes
(125: 54 mg), cotyledons (100: 2.1 g) and testa plus
endosperm (seed coat) (100: 1.4 g) and analyzed for ABA
content (3 replications per treatment). Seeds held at 20°
were not exposed to 30°.
Eyelee;iee_ej_ge;mime;ien. The results are presented as the
sum of the mean daily percentage germination after 10 days
(Sum 10: 1000 indicates 100% germination on the first day),
according to the method of Timson (1965).

'1 .0- 2.91 1!! 0.1-11. ° - 01 .- - 1-2-.
All seed tissues were lyophilized, ground in liquid N2, then
extracted with 80 and 100% acetone [acetone containing 1%
acetic acid and 10 mg/l butylated hydroxy-toluene (BHT)].
i3H-ABA (approximately 5000 cpm) was added to each sample
for determination of recovery. The tissues were extracted
by shaking gently in darkness at 4°C for 12-16 hours. The
supernatant solution was drawn off with a Pasteur pipette
and more solvent added: this was repeated twice. The
extracts were dried and redissolved in aqueous 1% acetic
acid. All samples were filtered, then purified by reverse
phase HPLC on a uBondapack C18 (10um particle size), 10 X 8

cm cartridge column (Waters Associates). The samples were

54
eluted by means of a convex gradient (curve 5 on the Waters

Associates Model 600 Solvent Programmer) from 0 to 50%
ethanol in aqueous 1% acetic acid. The retention time of
ABA was 18.5 min. at a flow rate of 2 ml/min, determined by
UV absorption of authentic ABA at 254 nm. The fraction
containing ABA was dried and methylated with ethereal
diazomethane. Quantification of the methyl ester of ABA
(Me-ABA) was performed with a Varian 3700 gas chromatograph
equipped with a 63Ni-electron capture detector. Samples
were dissolved in ethyl acetate containing an internal
standard (dieldrin), and analyzed on a Durabond DB-l (J&W
Scientific, Inc) gas capillary column (30m X 0.32mm X
0.25um). Injections (1 ul) were splitless. Temperatures of
the injector and detector were 250° and 290°C, respectively.
The column temperature was increased from 60 to 165° in
approx. 2.2 min., then increased to a final temperature of
240° at a rate of 5°/min. Carrier gas (He) linear velocity
was 32 cm/sec., and N2 was used as the detector make-up gas
with a flow rate at the detector of 30 ml/min.
ieemLifiiee;ien_efi_5§A_§y_§Q;n§. GC-MS verification of ABA
in each seed tissue sample was carried out using a Hewlett-
Packard 5890 Gas Chromatograph coupled to a Hewlett-Packard
5970 Mass Selective Detector. Samples were purified by HPLC
as described above, methylated, then dissolved in ethyl
acetate and injected onto a ChromPAK (12.5m X 0.25mm X 0.19
um) capillary column, for GC-MS analysis. Injections (1 ul)

were on-column, with the injector port maintained at 250°C,

55
and the column carrier gas (He) flowing at a rate of 1

ml/min. Initial column temperature was 80°, immediately
increasing 20°/min to a final temperature of 230°. The Ms
was operated at an ionization potential of 70eV with a
source pressure of 6-7 X 10'5 Torr.

egeeieeieei eneiyeie. The experiment was arranged in a 2-
factor (treatment X time) factorial design. An analysis of
variance was performed and Duncan's Multiple Range Test
(DMRT) was used to determine mean separation. Simple
correlations were run to measure the strength of the
relationship between endogenous ABA content and germination
capacity during stratification at 5°C or after exposure to 3

and 6 days at 30°.

Besuits

Control seeds and those stratified at 20°C germinated
very poorly or not at all: stratification at 5° greatly
increased Sum 10 values, although no difference was evident
between 3 and 6 weeks (Table 1). As expected, holding
chilled seeds at 30° for 3 or 6 days consistently reduced
germination, the effect increasing with time at 30°.

GC-MS of the extracts of seed coats, cotyledons, and
embryonic axes indicated the presence of ABA. Major
fragments and intensities were in close agreement with those
of synthetic cis,trans-ABA (Fig. 1).

The concentration of ABA in the embryonic axis was

similar to that in non-stratified seeds, regardless of

56
treatment, with three exceptions (Table 2). The

concentration was considerably higher after 6 weeks at
either 5° or 20°, and subsequent exposure to 30° decreased
ABA levels. This resulted in a significant temperature X
time interaction. Concentrations in other samples, although
consistently lower than those in control (non-stratified)
axes, did not differ significantly.

In the cotyledons, ABA concentration was significantly
higher in seeds held at 20°C than in all other treatments
(Table 2). The concentration at 5°, was not significantly
different from control values. Holding seeds at 30° affected
ABA content significantly only in seeds stratified for 6
weeks. Significant temperature X time interaction reflected
the change in ABA content at 200 vs. lack of change in other
treatments.

In seed coats, ABA concentration was consistently
higher at 20°C than in non-stratified seeds or in seeds held
at 5° for 3 or 6 weeks. The concentration also appeared to
rise in seeds held at 5°, but the rise became significant
only after 9 weeks (Table 2). Transfer of stratified seeds
to 30° reduced ABA content after 3 and 9 weeks of
stratification at 5°. Interaction was again significant.

ABA changes were not correlated with germination
capacity during stratification or after exposure to 30°C

(Table 1 and 2).

57
1111911111211

Stratification at 5°C or subsequent exposure to 30° did
not greatly affect ABA content of embryonic axes (70-180
ng/g dw) or cotyledons (13-26 ng/g dw). Subbaiah (1987),
using GC-MS, also reported that ABA levels remained fairly
constant during stratification at 5° in embryonic axes (50-
100 ng/g dw) and cotyledons (50-70 ng/g dw) of 'Northern
Spy' apple seeds. Balboa-Zavala and Dennis (1977) measured
ABA levels (fresh weight basis) during stratification at 5°
in seed coats, cotyledons, and embryonic axes of 'Delicious'
and 'McIntosh' apple cultivars. Since the dry weight of the
embryos is approximately 33% of its wet weight (Harrington,
1923), levels in the cotyledons (44-108 ng/g fw) reported by
Balboa—Zavala and Dennis were approximately 5-6 fold higher
than those observed by Subbaiah: however, ABA levels for
embryonic axes (9032-1582 ng/g fw) were approximately 100-
200 times greater than levels observed in this study or by
Subbaiah (1987). The very high values for embryonic axes
values reported by Balboa-Zavala and Dennis are probably
inaccurate due to the very small sample assayed
(approximately 37 mg fresh weight), minimal sample
purification, and reduced sensitivity in quantification due
to the use of a packed GC column with an extremely short
retention time for ABA (1.5 minutes). Artifacts could
easily have affected the values obtained. Much larger
samples of cotyledons (320 mg) and seed coats (245 mg) were

extracted and the ABA values reported for these tissues are

58
in the approximate range reported in this study and by

Subbaiah (1987).

ABA levels were not affected by 3 or 6 weeks
stratification at 5°C: however, a large increase occurred in
seed coats from seeds stratified for 9 weeks. This was
unexpected and its significance is not known. Subbaiah
(1987), using 'Northern Spy' and 'Lande' apple cultivars,
found high levels of ABA in the testa and endosperm
(equivalent to seeds coat in this paper) of non-stratified
seeds. Stratification at 5oC decreased ABA levels
substantially: this trend was also reported by Balboa-Zavala
and Dennis (1977) in 'Delicious' and 'McIntosh' cultivars.

The same or higher levels of ABA were found in seed
coats, cotyledons, and embryonic axes after 3, 6, and 9
weeks at 20° than in non-stratififed seeds or seeds
stratified at 5°. In contrast, Subbaiah (1987) and Balboa-
Zavala and Dennis (1977) reported that ABA levels dropped
significantly in seeds held at both 20° and 5°. The reason
for this contradiction is not known.

There was no consistent change in ABA levels in any of
the seed tissues during exposure to 30°C after
stratification at 5°. However, because of the higher
metabolic rate at 30°, a study of turnover rate of ABA and
its metabolites would be required to fully evaluate the role
of ABA in this heat-induced stress process.

In conclusion, our data do not support the hypotheses

that changes in ABA content are responsible for the breaking

59
of dormancy by chilling or for the induction of secondary

dormancy by high temperature.

1.111211111111211

Balboa-Zavala, O. and F.G. Dennis, Jr. 1977. Abscisic acid
and apple seed dormancy. J. Amer. Soc. Hort. Sci.
102:633-637.

Harrington, G.T. 1923. Respiration of apple seeds. J. Agr.
Res. 23:117-131.

Karssen, C.M., Brinkhorst-van der Swan, D.L.C., Breekland,
A.E., and M. Koornneef. 1983. Induction of dormancy
during seed development by endogenous abscisic acid:
studies on abscisic acid deficient genotypes of

Aeeeieepeie eneiiene (L.) Heynh. Planta 157:158-165.

Ohkuma, K., Addicott, F.T., Smith, O.E., and W.E. Thiessen.
1965. The structure of abscisin II. Tetrahedron Lett.
29:2529-2535.

Pieniazek, J. and R. Rudnicki. 1967. The presence of
abscisin-II in apple leaves and apple fruit juice.
Bull. Acad. Pol. Sci. 15:251-254.

Rudnicki, R. 1969. Studies on abscisic acid in apple seeds.
Planta 86:63-68.

Subbaiah, T. 1987. Abscisic acid relationships in apple seed
dormancy. Ph.D. Thesis, Cornell University.

Timson, I. 1965. New method of recording germination data.
Nature 207:216-217.

60

Table 1 Germination (Sum 10) of seeds stratified at 5° or
20°C and after exposure to 30°.

 

Seed germination (Sum 10)

 

Time (weeks)

 

 

Temperature 0 3 6 9 Mean
20°2 47 0 0
) 150eY
5° 574b 591b 742a 636r
5°,3 days 30° -- 308d 373cd 46lbc 381s
5°,6 days 30° -- .112 .gée, 7§e 68t
Mean 307m 351mm 427n
m at e n.s. n.s. n.s.

 

z Data from the 20° treatment were not included in the
factorial analysis

Y Means followed by the same letter are not significantly
different from one another within sets (a,b,c,d,e for all
treatments: m,n for time: and r,s,t for temperature) by
DMRT, P < 0.05.

n.s. = not significant

61

Table 2. Effects of stratification temperature and
subsequent exposure to 30°C on ABA concentration in
embryonic axes, cotyledons, and seed coats of apple

 

 

 

 

 

 

seeds.
ABA content (ng/g dw)
Time (weeks)
Stratification = ,
temperature (°C) 0 3 6 9 Mean
Embrxéfilc.§iis
20° 62c 401a 88c 184h
) 106C
5° 82c 179b 70c 1101
5°, then 3 days 30° 89c 102c 83c 911j
5°, then 6 days 30° fig; .882 gee 703
Meen 723 192r 77s
Temperature_x_time ** ** **
‘ mm
20° 58b 106a 54b 73h
) 26cdef
5° 21def 13f 15ef 16j
5°, then 3 days 30° 25cdef 40c 29cde 311
5°. than 6 days 30° 152d 112:1 25.201: 311
Mean 353 47r 313
Temperature_x_rime ** ** **
5221.22131
20° 3445 528a 449ab 440a
) 115cde
5° 188cd 204c 436ab 2761
5°, then 3 days 30° 55e 210c 113cde 125j
5°. than 6 days 30° 1545 15955; 15914:. 1281
neen 1658 273r 290r
** ** **

Temperature_x_11me

 

2 Within seed tissues, means followed by the same letter are
not significantly different from one another within sets
(a,b,c,d,e,f for all treatments: r,s for time: and h,i,j
for stratification temperature) by DMRT, P < 0.05.

** Significant at the 1% level.

62

 

 

Retention time: (10.299 min)

 

 

 

 

 

 

 

 

1 A
1.5554 90
4
3 j 1,3 162
S 1.055‘1 55 91 /
'0 I
:5, 5.0011 / ’ 222 2180279
a? 0 -‘k‘kLk—‘IZL‘! :11“. . . .1“. .l - :-“:-f‘-::/-°-.*
108 158 200 250
HaSS/Charge
Retention time: (10.233 min)
3.055% Age 3
g 2 0:55 134 162
: ° 1 / /
«I J 9]
'° J 67 / 205 260
g ӣ51 / t I l L / \ 273
G: 01 ‘LJLlflul .. .1..Ai . .. . . .
100 150 200 250
Mass/Charge

 

 

 

Figure 1. Mass spectrum of Me-ABA in a methylated extract of
seed coats of apple (A), and of authentic Me-ABA (B).

SECTION IV

Is Ethylene required for Apple Seed or Embryo Germination ?

63

Is Ethylene Required for Apple Seed or Embryo Germination ?

APSLIQQE

The observations made by others that ethylene (C2H4) is
required for germination of stratified embryos of apple
(Melee eemeegiee Borkh.) was not confirmed. In addition,
silver thiosulfate and norbornadiene (1000 ul'liter'l),
which inhibit C2H4 action, either slightly promoted or did
not affect seed or embryo germination. The addition of 166
or 332 ul'liter"1 CZH4 significantly inhibited rate and
final percent germination of fully stratified seeds.
Removal of atmospheric C2H4 or addition of C2H4 (up to 332
ul'liter'l) did not reduce the inhibitory effect of high
temperature (30°C) on seed or embryo germination. We
conclude that the presence of atmospheric C2H4 at
concentrations greater than 20 nl‘liter'"1 is not essential

for germination of apple seeds or embryos.

64

65
Ethylene has been implicated in germination of certain

seeds. As little as 3.5 ul'liter'1 can stimulate
germination in dormant Virginia-type peanut seeds (3). C2H4
at 100 and 1000 ul'liter'l hastened apple embryo germination
(2), but did not affect final germination when used at 100
to 50,000 ul'liter"1 (1). Sinska and Gladon (4) reported
that the presence of mercuric perchlorate (Hg(ClO4)2)
completely inhibited germination of partially and fully
stratified apple embryos, and that addition of ethephon
during stratification stimulated germination of partially
stratified (30 days at 4°C) embryos.

We tested the effects of removal of atmospheric C2H4,
addition of C2H4, and inhibition of C2H4 action on apple

seed and embryo germination at both 20° and 30°C.

Material§_and.neth2d§

Partially stratified seeds and embryos were obtained by
removing seeds from 'Golden Delicious' fruits that had been
stored at 5°C for 1 year. The seeds were leached under
running tap water (12°) for 1 day, then stratified in the
dark for 1 month at 5° in petri dishes on filter paper
moistened with about 0.5% Captan fungicide.

Fully stratified seeds and embryos were obtained by
removing seeds from 'Golden Delicious' fruits at harvest,
drying them, and storing at 5°C. Subsequently, the seeds
were soaked overnight in water, rinsed, placed in 100 X 15

mm Petri plates (120 seeds per plate) provided with two

66
filter papers wetted with 0.5% Captan solution, and

stratified at 5° in the dark for 52 days.

Seeds or embryos, 15 per replicate dish, six
replications per treatment (4 replications in norbornadiene
treatment), were placed in 60 X 15 mm Petri dishes
containing two filter papers moistened with Captan solution.
All seeds and embryos except those used for one treatment
(Table 1), were germinated in 483 ml glass jars with air-
tight lids.

Partially stratified seeds were germinated in the jars
for ten days at 20°C under a 16 hr photoperiod of 150
umols's'1°m-2. Fully stratified seeds were held in the jars
in darkness for 3 days (at 20° or 30°), then removed and
germinated at 20° in the dark.

Purafil (permanganate-alumina) or solutions of
Hg(ClO4)2 in 2M HClO4 were contained in 20 and 10 ml
beakers, respectively, within the jars. Norbornadiene (NB),
as a liquid, was placed onto a filter paper wick attached to
the jar inner wall and the jars were quickly sealed. The
quantity of NB used was calculated to yield upon
volatilization concentrations of 1000 and 3000 ul'liter'l.
C2H4 was injected into specified jars and the concentrations
within the jars verified by gas chromatography.

Partially stratified seeds were soaked in silver
thiosulfate (STS) solutions for 1 hr to inhibit ethylene

action, then rinsed with water, transferred to Petri dishes

containing Captan solution and placed inside jars.

67
Alternatively, STS solution was added directly to the filter

papers in dishes containing fully stratified seeds or
embryos, then replaced with Captan solution after 3 days
(Table 3).

In separate experiments, seeds were held in jars at 20°
or 30°C, and levels of C02 and 02 were monitored (six
replications of 15 seeds each per temperature). C02 levels
did not exceed 0.5 i 0.1% at 20° or 30° and 1.3 i 0.4% at
20° after 3 and 10 days, respectively. 02 levels did not
fall below 17.2 i 5.2% at 20 or 30° and 19.7: 1% at 20°
after 3 and 10 days, respectively. Therefore, C02 and 02
levels should not have affected C2H4 action or synthesis by
these seeds.

Gases were sampled through a rubber septum in the jar
lid using a 1 ml syringe. Quantification of C2H4 was
performed with a Varian series 1400 gas chromatograph
equipped with a flame ionization detector and a 60/80 mesh
activated alumina column (100 cm), using N2 (30 ml'min'l) as
the carrier gas. Temperatures of the injector, column, and
detector were 130, 100, and 150°C, respectively. Identity
of C2H4 was confirmed by comparison of retention time of
sample with that of an ethylene standard.

The results are presented both as final percentage
germination and as the sum of the mean daily percentage
germination after 10 days (Sum 10: 1000 indicates 100%
germination on the first day), according to the method of

Timson (5).

68
Most experiments were arranged in a completely random

design. A randomized complete block design was used with
fully stratified seeds in Table 1 and embryos in Table 2,
using seed size as blocks. An analysis of variance was
performed and Duncan's Multiple Range Test (DMRT) was used
to determine mean separation. Only main effect means are
presented for experiments in which interaction was not
significant at P < 0.05. Regression analysis was also
performed where appropriate,and standard deviations from the
mean were calculated for partially stratified embryo

treatments in Table 1.

gesules
Removal of ambient C2H4 with Hg(ClO4)2 or Purafil did

not significantly (P < 0.05) affect percent germination or
Sum 10 of partially or fully stratified apple seeds or
embryos at 20°C (Table 1). Germination was significantly
lower at 30° than at 20°, and the removal of atmospheric
C2H4 with Purafil did not affect the response (Table 1).
Therefore only main effects are presented.

The addition of up to 72 ul'liter'1 C2H4 to the
atmosphere did not significantly (P < 0.05) affect the rate
or final percentage germination of partially stratified
seeds (Table 2) using ANOVA analysis: however, regression
analysis of Sum 10 values showed a significant positive
effect of C2H4 on germination. C2H4 (166 and 332 ul'liter'

1) significantly inhibited final germination and Sum 10 of

69
fully stratified seeds using either method of analysis

(Table 2). The inhibitory effect was not as prominent in
fully stratified embryos, in which only the Sum 10 value was
affected (Table 2). Germination of seeds was consistently
lower at 30°C than at 20°, regardless of treatment (Tables
1,2).

Ethylene concentration in jars containing fully
stratified control seeds was not significantly affected by
temperature (Table 3). However, control embryos held at
30°C for three days evolved significantly more C2H4 than did
those held at 20°. Embryos evolved more ethylene than did
intact seeds.

Inhibition of C2H4 action by exposure to silver
thiosulfate (STS) at 1, 0.1, or 0.01 mM for one hr did not
significantly (P < 0.05) affect percentage germination or
Sum 10 at 20°C of partially stratified seeds relative to
controls using ANOVA (Table 4). Although a significant
increase in germination capacity with increasing STS levels
was detected using regression analysis (P < 0.01), only
seeds exposed to the highest concentration of STS (1 mM)
germinated better than the controls. Exposure of fully
stratified seeds for 3 days to STS solutions (0.1 mM) did
not affect final germination or Sum 10 of seeds or final
germination of embryos: however, a small but significant
reduction of Sum 10 was evident in embryos (Table 4).

Inhibition of C2H4 action by exposure to norbornadiene

at 1000 and 3000 ul'liter"1 did not significantly (P < 0.05)

7O
affect percentage germination or Sum 10 of fully stratified

embryos or percent germination of seeds using ANOVA or
regression analysis (Table 5). Although both ANOVA and
regression analysis indicated a significant reduction in Sum
10 of seeds at 20°C, only seeds exposed to 3000 ul’liter'1
were inhibited compared to the control. The reduction in
germination capacity at 3000 ul'liter'1 was probably due to
NB's toxic effect (5). The lack of effect at 30°, which
resulted in significant temperature x norbornadiene

interaction, may be a reflection of the lower germination

capacity at this temperature.

Diseneeien

In summary, neither removal of ethylene from the
atmosphere nor inhibition of ethylene action by NB or STS
appreciably affected germination of fully stratified seeds
or embryos at 20 or 30°C. The addition of ethylene either
had no effect or reduced germination of fully stratified
seeds and embryos. Using regression analysis, a significant
positive effect of C2H4 on germination on partially
stratified seeds was detected. However, STS, an inhibitor
of ethylene action, also significantly increased both
percent germination and Sum 10 in these seeds.

If C2H4 does indeed promote the germination of apple
embryos, treatments which stimulate C2H4 evolution in
embryos should stimulate germination: however, we observed

the opposite. Control embryos evolved significantly more

71
C2H4 at 30° than at 20°C, yet Sum 10 values were either not

affected or were significantly lower at 30° (Table 3). In
no case did any of the chemical treatments reduce the
inhibitory effect of high temperature (30°) on germination
of apple seeds or embryos. Therefore we conclude that the
presence of C2H4 at concentrations greater than 20
nl‘liter'1 (detection limit with our equipment) is not
essential for germination of partially or fully stratified
apple seeds or embryos. In addition, C2H4 concentrations

1

equal to or greater than 166 ul'liter' may reduce

germination of seeds and embryos.
Wired

1. Kepczynski, J. and R.M. Rudnicki. 1975. Studies on
ethylene in dormancy of seeds. I. Effect of exogenous
ethylene on the after-ripening and germination of apple
seeds. Fruit Sci. Rpts. 2:25-41.

2. Kepczynski, J., R.M. Rudnicki and A. A. Khan. 1977.
Ethylene requirement for germination of partly after-
ripened apple embryo. Physiol. Plant. 40:292-295.

3. Ketring, D.L. and P.W. Morgan. 1969. Ethylene as a
component of the emanations from germinating peanut
seeds and its effect on dormant Virginia-type peanut
seeds. Plant Physiol. 44:326-330.

4. Sinska, I. and R.J. Gladon. 1984. Ethylene and the
removal of embryonal apple seed dormancy. HortScience
19:73-75.

5. Sisler, E.C. and S.F. Yang. 1984. Anti-ethylene effects
of cis-2-butene and cyclic olefins. Phytochemistry
23:2765-2768.

6. Timson, I. 1965. New method of recording germination
data. Nature 207:216-217.

72

Table 1. Effects of temperature and removal of C H from the
atmosphere on germination of partially and iuily
stratified apple seeds and embryos.

 

 

Seeds ___Embrxes____
% Sum % Sum
Treatment Germ.z 10 Germ.z 10
Eartiall¥.§tratified
Control
in jar 42aY 251a 97ax 788ax
no jar 41a 217a -- --
Hg (c104)2 49a 266a 100ax 8835x
Purafil 52a 250a -- --
Eull¥_§tratified
Temperature_:_uain.effects
20°C 97m 729m 99m 708m
30°C 75n 36ln 85n 486n
Purafil_:_nain.szeet§
Control 86r 531r 94r 606r
Purafil 86r 559r 90r 589r

 

2After 10 days at 20°C.

YMean separation within columns among treatments (ab),
temperatures (mn), and Purafil vs. none (rs) by DMRT, P <
0.05.

xOnly 10 embryos/replication, 6 replications: otherwise 15
embryos/replication.

73

Table 2. Effects of temperature and addition of CZH to the
atmosphere on germination of partially and fuliy
stratified apple seeds and embryos.

 

._§eeds___ __Embrxes__

 

CZHi.
app 1ed Temp. % Sum % Sum
(ul'liter'l) (°C) Germ.z 10 Germ.z 10
E l'aJJ ! !'E° 3
o 20 42aY 251a -- --
7.4 45a 246a -- --
72 51a 351a -- --
Enllx_§tratified
Temperatnre_:.nain_effects
20 89m 636m 100m 815m
30 59h 273n 99m 786m
Qzfii.:_flein.gffifig£§
0 86r 532r 99r 848r
166 708 4308 --
332 66s 4028 99: 7525

 

2 After 10 days at 20°C.

Y Mean separation within columns among treatments (ab),
temperatures (mm), and ethylene concentrations (rs) by
DMRT, P < 0.05.

74

Table 3. Effect of temperature on C H4 levels in air within
jars containing fully stratified apple seeds or
embryos. Gas samples removed after 3 days at 20° or

 

 

 

30°C.
C2H4 (nl'liter'l)
Temperature Seeds‘ Embryosz
(°C)
20° 30 i 20Y 95 i 25
30° 20 i 10 450 i 110

 

z Fifteen seeds or embryos per 483 ml jar: l2 jars
(replications) per treatment. Approximate weight = 60
mg/seed, 30 mg/embryo.

Y Standard error.

75

Table 4. Effects of temperature and inhibition of C H4
action by silver thiosulfate (STS) on germinat1on of
partially and fully stratified apple seeds and embryos.

 

 

___§esds___ ___Embrxee_._
STS Temperature % Sum % Sum
(mM) (°C) Germ.z 10 Germ.z 10
Partiallx_stratified
0 20 42abY 251ab -- --
0.01 34h 184b -- ~-
0.1 49ab 238ab -- --
1 60a 349a -- --

ue-
20 96m 688m 99m 796m
30 71n 342n 92n 654n
§T§_:_Main_effects
0 86: 530: 96: 764:
0.1 81r 500: 96: 6868

 

2 After 10 days at 20°C.

Y Mean separation within columns among treatments (ab),
temperatures (mn), and STS concentrations (rs) by DMRT, P
< 0.05. '

76

Table 5. Effects of temperature and norbornadiene (NB) on
germination of fully stratified apple seeds and

 

 

embryos.

‘_H.Seed§.___ ___merxcs__

Temp. NB _ % Sum % Sum

(°C) (ul'liter 1) Germ.z 10 Germ.z 10

20 0 80aY 494a 98a 924a
1000 87a 517a 100a 906a

3000 Zia 115b 21a 151a

Mean 80m 462m 98m 895m

30 0 295 77c 96a 884a
1000 385 118c 95a 766a

3000 11b 1922 25a 5255

Mean 33n 101n 95m 848m

Means.f2r.un

0 54: 286:s 97: 904:

1000 62: 317: 98: 836:

3000 53: 2423 96: 875:
Temperature.x.NB nos. * n-S- n.s.

 

2 After 10 days at 20°.

Y Mean separation within columns among all treatments (abc),
temperatures (mn), or norbornadiene concentrations (rs) by
DMRT, p < 0.05.

* Significant at 5%.

APPENDIX

Appendix

The Effect of ABA on Apple Seed Germination

Intreductien

Exogenous abscisic acid inhibits germination of non-
dormant apple seeds (Rudnicki and Pieniazek, 1973) and
embryos (Rudnicki, 1969). High temperature also inhibits
germination of these seeds (Abbott, 1955: Visser, 1956). In
order to understand the relationship between the inhibitory
effect of ABA and that of high temperature on germination,
apple seeds were exposed to ABA either during or after

induction of secondary dormancy by high temperature.

Materials.and_nethed§

Apple (Melee eemeeeiee cv. Golden Delicious) seeds were
removed from the fruit at harvest, dried, and stored at 5°C.
Subsequently, the seeds were soaked overnight in water,
rinsed, placed in 100 X 15 mm petri plates containing filter
paper wetted with 10 ml 0.5% Captan solution, and stratified
at 5°.

In experiment 1, seeds stratified for 9 weeks were
transferred to plates containing 0.15 or 0.3 X 10 mM ABA in
Captan solution and placed at either 20° or 30°C for 3 days
(30 seeds per plate: 3 replications). Seeds were then
rinsed with water and the embryos dissected from half of the

seeds: both were placed in 0.5% Captan solution and

77

78
germinated at 20°C in the dark.

In experiment 2, seeds stratified for 0, 3, or 6 weeks
were exposed to 30°C for 0, 3, or 6 days (15 seeds per
plate: 4 replications). Seeds, and embryos dissected from
similarly treated seeds, were subsequently transferred to
plates containing 0, 10'6, 10'5, 10'4, or 10'3M ABA and
placed at 20°C for 10 days for germination. Stratification,
exposure to high temperature and germination were carried
out in darkness in all cases.

The results are presented as the sum of the mean daily
percentage germination after 10 or 14 days (Sum 10 or Sum
14: 1000 and 1400, respectively, indicates 100% germination
on the first day), according to the method of Timson (1965).

Standard deviations (SD) from the mean were calculated
for experiment 1. Experiment 2 was arranged factorially for
embryos, and seeds were in a completely random design. An
analysis of variance was performed and Duncan's Multiple

Range Test (DMRT) used to determine mean separation.

Beeulta
In experiment 1, exposure to 30°C for 3 days
significantly inhibited subsequent germination at 20° of
stratified seeds and embryos (Fig. 1 and 2). ABA
consistently inhibited germination of both seeds and embryos
regardless of prior temperature treatment. Seed germination
was completely inhibited at 0.15 mM, but some germination

occurred in embryos even at the high ABA concentration. The

79
concentration of ABA required to equal the effect of high

temperature was 0.15 mM in embryos but less than this in
seeds.

In experiment 2, germination of seeds increased with
stratification time, reaching a Sum 10 value of 275 after 6
weeks at 5°C (Table 1). ABA at concentrations of 10'5M or
higher inhibited germination of seeds regardless of
stratification time. When stratified seeds were exposed to
30°C for 3 or 6 days, germination was almost completely
inhibited, exogenous ABA having little effect.

The germination capacity of embryos was much greater
than that of seeds after the same stratification period (Sum
10=827) (Table 2). Exposure to 30°C for 6 days promoted
germination in non-stratified embryos, but inhibited it in
stratified embryos (Table 2). Exposure to ABA subsequent to
heat treatment further reduced embryo germination. ABA
consistently inhibited germination regardless of
stratification time, but the inhibitory effect increased
with length of stratification. ABA at 10-5, 10’4 and 10'3 M
totally inhibited germination, except for slight germination
(Sum 10 varied for 0 to 44) of embryos from seeds stratified
for 6 weeks at 5° (data not shown).

Interaction between stratification time and exogenous
ABA concentration was significant at P < 0.01, for ABA at
10'6M was inhibitory only in stratified embryos (3 and 6
weeks at 5°C). Time of exposure to 30° X duration of

stratification was significant at P < 0.01: in this case

80
chilling consistently increased Sum 10 in seeds exposed to

30° for 0 or 3 days, but had little or no effect on those
exposed for 6 days.
Dimesien

Exposure to 10'6 to 10"3 M ABA or to 30°C inhibited
germination of stratified seeds and embryos, and ABA
application during or after exposure to 30° further
inhibited germination. Because the induction of secondary
dormancy by high temperatures was not correlated with an
increase in endogenous ABA levels in the seed coat,
cotyledons, or embryonic axes of apple seeds (chapter 3 of
this thesis), the mechanism of action of high temperature

appears to differ from that of ABA.
I'le ! :0! :

Abbott, D.L. 1955. Temperature and the dormancy of apple
seeds. Rept. XIV Intern. Hort. Congr., Netherlands.
2A:746-753.

Timson, I. 1965. New method of recording germination data.
Nature 207:216-217.

Rudnicki, R. 1969. Studies on abscisic acid in apple seeds.
Planta 86:63-68.

Rudnicki, R. and J. Pieniazek. 1973. The effect of abscisic
acid on stratification of apple seeds. Bull. Acad. Pol.
Sci. 21:149-154.

Visser, T. 1956. Some observations on respiration and
secondary dormancy in apple seeds. Proc. K. Ned. Akad.
Wet. 59:314-324.

81

Table 1. The effect of stratification at 5°C and subsequent
exposure to 30° and/or exogenous ABA on germination
of apple seeds.

 

 

Sum 10
""""""" i3??§§'""""""
"6253 at ”3‘63?“ ""3""’"1333""'1333""1333
0 0 7c2 -- -- --
3 0 174b 127b 142b --
3 220 34c 10c --
6 15C 13c 0c --
6 0 275a 318a 1935 14c
3 39C 31C 49C 9c
6 0c DC DC 0c

 

2 Means followed by the same letter are not significantly
different from one another by DMRT, P < 0.05.

82

Table 2. The effect of stratification at 5°C and subsequent
exposure to 30° and/or exogenous ABA on germination
(Sum 10) of apple embryos at 20°.

 

 

 

 

Sum 10
""""" ;;;'?§§""""'
Weeks at Days at --------------------------
5°C 30°C 0 10"6 mean
0 0 1952 154 1751
3 1207 174 1901
6 112 221 .2llk
mean 239p 212pq 225c
3 O 530 161 345jk
3 603 253 428j
6 215 .11 .2111
mean 503n 153q 328b
6 0 827 579 703h
3 725 379 5521
6 121 112 .212k1
mean 660m 357o 508a
Means for ABA 467f 241g
Means for 0 517r 298t 408d
days at 30°C
3 512r 268t 390d
6 3725 156u 264e

 

Means followed by the same letter are not significantly

different from one another within sets (a-c for weeks at
5°: d,e for days at 30°: f-g for ABA: h-l for weeks at 5°
X days at 30°: m-q for weeks at 5° X ABA: and r-u for days

at 30° x ABA) by DMRT, p < 0.05.

83

Figure 1. The effect of ABA on germination (Sum 14) of
seeds at 200 and 30°C following 9 weeks of
stratification at 5°.

84

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85

Figure 2. The effect of ABA on germination (Sum 14) of
embryos at 20° and 30°C following 9 weeks of stratification
at 5 .

86

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BI BLI OGRAPHY

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seeds. Rept. XIV Intern. Hort. Congr., Netherlands.
2A:746-753. -

Balboa-Zavala, O. and F.G. Dennis, Jr. 1977. Abscisic acid
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Berrie, A.M.M., Buller, D., Don, R. and W. Parker. 1979.
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87

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89

Ho, T. D. 1988. Regulation of gees expression by ABA in
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Kepczynski, J., R.M. Rudnicki and A. A. Khan. 1977.
Ethylene requirement for germination of partly after-
ripened apple embryo. Physiol. Plant. 40:292-295.

Ketring, D.L. and P.W. Morgan. 1969. Ethylene as a
component of the emanations from germinating peanut
seeds and its effect on dormant Virginia-type peanut
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9O

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