v“ ——__.__. . - _. V——_— , .‘ .
wuwn‘gu-a..tu. ..,..,.., . «”ng . on. .- nu» ~. .-.‘ ... . . . ....o «.5... o . - . . M.....-.| LN...,...‘.... ,v “.02“. ... . n~|nvo- ‘v~-~v#a;vv'|‘:.m "k -.-:- .
x K I? ‘ wfimm

"r 33."

$
\

l

.
.

\

 

   

m M3123

llllllWI

 

 

 

 

 

 

 

 

 

 

 

 

111111111

LIBRARY

Michigan State
University

 

This is to certify that the

thesis entitled

Fruit Induced Dormancy in Apple
(Malus Domestica Borkh.) Seed

 

presented by

Chee-Keong Wan

has been accepted towards fulfillment
of the requirements for

Ph.D.

degree in Horticulture

 

 

February 25, 1980
Date

 

0-7839

Jude 2 AMI);

Major professor

 

a ”an

9* .

     
 
  

OVERDUE FINES:

 

  
   

 

‘1 ‘ , ‘2» 313’" 25¢ per day per itch
1 ,ij [éfl'mfi‘ f' , RETURNING LIBRARY MATERIALS:
1 4 r~ i , '
Mw-—'”~
1 2w.,:.'“\"/4<‘;'- ”A Place in book return to move

charge from circulation records

   

ffifiioikno
’ ;y 0165
dfikbrnurs ggmgn

“mm, *

‘,

“a“: 19%.. ’9- '”
i X’x MT I’m-2%? ‘

271

 

 

 

 

 

 

FRUIT INDUCED DORMANCY IN APPLE (MALUS
.DOMESTICA BORKH.) SEED

 

By
Chee-Keong Wan

A DISSERTATION

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

DOCTOR OF PHILOSOPHY
Department of Horticulture

1980

ABSTRACT

FRUIT INDUCED DORMANCY IN APPLE (MALUS
DOMESTICA BORKH.) SEED

 

By
Chee-Keong Wan

Apple (Malus domestica Borkh.) seeds after-ripened
(stratified) in the fruit germinated poorly in comparison
with those stratified on moist filter paper. EXperiments
were designed to test if the inhibitory effects of the
fruit were caused by restricted water uptake, the presence
of germination inhibitors either in the seed coat or the
locules (endocarp), or by production of volatiles.

After—ripening seeds in intact fruits strongly
inhibited germination on either moist paper or on a wire
mesh screen. Embryos excised from seeds after-ripened in
the fruits germinated almost as well as those from seeds
stratified outside the fruits on moist paper, but their
germination was markedly reduced on screen. The moisture
content of seeds stratified on screen was only slightly
higher than that of seeds after—ripened in the fruit. yet
the former germinated much more readily on both screen
and moist paper. Also, soaking seeds prior to after—
ripening and germination in locules did not permit
germination. Furthermore, the germination of stratified
seeds was reduced when placed in direct contact with the

endocarp. Thus an inhibitor in the endocarp interferes

Chee-Keong Wan

with the germination of seeds after-ripened in the fruit.

The inhibitor is unlikely to be abscisic acid (ABA)
because the final ABA content of the testa from seeds after-
ripened in the fruit, on screen, or on moist paper was not
correlated with germination capacity. Furthermore, the
level of ABA in the endocarp was too low to inhibit the
germination of fully stratified seeds.

After-ripening or stratifying seeds in the presence
of fruit-produced volatiles did not inhibit germination
to any appreciable extent. By contrast, germination was
inhibited when seeds were after-ripened in intact fruits.
Therefore volatiles produced by the fruit are not a major
factor responsible for fruit induced dormancy of apple seeds.

Germination capacity of seeds or embryos is indepen-
dent of the rate of ethylene evolution. Stratifying seeds
or treating embryos with ethephon promoted ethylene
production but had no consistent effect on germination.
Inhibition of ethylene biosynthesis by non-toxic levels of
8-hydroxyquinoline sulfate, aminoethoxyvinyl glycine, or
silver nitrate was not accompanied by a reduction in germin—
ation. The results do not support a role for ethylene in
apple seed germination or in the breaking of rest in

embryos.

Dedicated to
my wife, Mary, and children

ii

ACKNOWLEDGMENTS

I wish to express my sincere appreciation to Dr.
F. G. Dennis, Jr., my major professor, for his friendship,
counsel, guidance, and willingness to assist during my
program of research and preparation of this thesis. I am
also grateful for the advice and encouragement offered by
members of my Guidance Committee: Drs. Robert Carlson,
Harold Davidson, Lawrence Copeland, and William Tai.

The author gratefully acknowledges the support
of the Agriculture University, Malaysia for granting him
study leave and financial assistance which made this study
possible.

Finally, I thank my wife, Mary, and children for
their patience, understanding, and encouragement during

my graduate study at Michigan State.

iii

TABLE OF CONTENTS

Page
LIST OF TABLES . . . . . . . . . . . . . . . . . . vi
LIST OF FIGURES I I I I I I I I I I I I I I I I I viii

 

INTRODUCTION . . . . . . . . . . . . . . . . . . 1
LITERATURE REVIEW . . . . . . . . . . 2
Terms used in dormancy studies . . . . . . . . . 2
Role of external factors in apple seed dormancy. 3
Fruit I I I I I I I I I I I I I I I I I I I I 3
Seed coat I I I I I I I I I I I I I I I I I I 4
Gases . . . . . . . . . . . . . . . . . . . . 5
MOiSture I I I I I I I I I I I I I I I I I I 6
Temperature . . . . . . . . . . . . . . . . . 7
Light I o o I o o o o o .o I I I o c o o o o o 7
Growth regulators . . . . . . . . . . . . . . 8
Role of internal factors in apple seed dormancy. 9
Endogenous growth inhibitors . . . . . . . . 9
Endogenous growth promoters . . . . . . . . . ll
Enzymes I I I I I I I I I I I I I I I I I I I 13
Summary . . . . . . . . . . . . . . . . . . . . 13
SECTION I
FRUIT INDUCED DORMANCY IN APPLE (MALUS
DOMESTICA BORKH.) SEED.
I. ROLE OF WATER AND INHIBITORS

AbStraCt I I I I I I I I I I I I I I I I I I I I 15
Materials and method . . . . . . . . . . . . . 18
Results I I I I I I I I I I I I I I I I I I I I 26
DiscuSSion I I I I I I I I I I I I I I I I I I I “'0
Literature cited . . . . . . . . . . . . . . . . 47

iv

Page

SECTION II
FRUIT INDUCED DORMANCY IN APPLE (MALUS
DOMESTICA BORKH.) SEED.
II. ROLE OF VOLATILES

AbStraCt I I I I I I I I I I I I I I I I I I I I [49
Materials and methods . . . . . . . . . . . . . 51
Results I I I I I I I I I I I I I I I I I I I I 5“
DiSCUSSiOn I I I I I I I I I I I I I I I I I I I 66
Literature cited . . . . . . . . . . . . . . . . 68
SUMMARY AND CONCLUSIONS . . . . . . . . . . . . . . 7O
BIBLIOGRAPHY . . . . . . . . . . . . . . . . . . . 72

Table

LIST OF TABLES

SECTION I

Effects of after-ripening and germination
medium and of soaking in water prior to
after-ripening on germination of apple,
cv. 'Paulared', seeds . . . . . . . . .

Moisture content of seeds, cv. 'Paulared'

during after-ripening in intact fruit, on

screen, or on moist paper at 5°C . . .

Effects of after-ripening apple, cv."Paula-

red', seeds in fruit, on screen, and on

moist paper upon subsequent germination on

moist paper or screen . . . . . . . . .

Effects of after-ripening apple, cv. 'Paula-

red', seeds in fruit, on screen, and on

moist paper upon subsequent embryo germi-

nation on moist paper or screen . . . .

Effects of after-ripening and germination
medium and of soaking in water after 12

weeks of after-ripening on germination (%)

of apple seeds, cv. 'Paulared'. . . . .

Effect of various treatments on germination
of 'Jonathan' seed on surface of endocarp

of stored fruit . . . . . . . . . . . .

Effects of after-ripening method on the
amount of ABA in water diffusates from

apple, cv. 'Paulared', seed coats and on

germination of seeds . . . . . . . . .

Effects of soaking time and seed coat removal
on germination of seeds, cv. 'Jonathan',
after-ripened in the fruit for 12 weeks at
5°C, then germinated on screen . . . . . . .

vi

Page

27

29

30

32

35

37

39

#1

Table Page
SECTION II

1. Effects of volatiles from apple, cv.
'Paulared', fruit tissues on germina-
tion of seed and excised embryos . . . . . . . 55

2. Internal ethylene concn (ppm) in 'McIntosh’
fruits stored at OOC under different
storage atmospheres . . . . . . . . . . . . . 58

3. Effect of different low temperature (00C)
storage treatments and method of stratifi-
cation on the germination of seeds and
excised embryos after—ripened in
'McIntosh' fruits . . . . . . . . . . . . . . 59

4. Effects of treatment with ethephon and
inhibitors of ethylene biosynthesis
during moist stratification on ethylene
production of 'Paulared' apple seeds and
excised embryos during 24 hour period of
incubation at 2000. 1978 . . . . . . . . . . 62

5. Effects of treatment with ethephon and
inhibitors of ethylene biosynthesis
during moist stratification on germin-
ation of seeds and excised embryos. 1978 . . 64

6. Effects of treatment with ethephon and
inhibitors of ethylene biosynthesis during
moist stratification on ethylene production
and germination of 'Paulared' apple seeds.

1979 65

vii

LIST OF FIGURES

Figure Page
SECTION I

l. Fractionation procedure for free and bound
ABA in water diffusates from seed coats
and locules (endocarp) . . . . . . . . . . 24

2. Effects of method and time of after-ripening
and of germination medium on germination
of apple, cv. 'Paulared', seeds and
excised embryos . . . . . . ... . . . . . 33

SECTION II

1. Effects of stratification time at 0°C with
or without fruits on subsequent germination
of intact apple seeds and excised embryos,
cv. 'Paulared' . . . . . . . . . . . . . . 56

2. Effects of method of low temperature storage
(R=regular; CA=controlled atm; LP=low
pressure) on ethylene content of fruits
and on germination of seeds on moist paper
following after-ripening on sand or in
fruits . . . . . . . . . . . . . . . . . . 60

viii

Guidance Committee:

The thesis is organized in journal-style in
accordance with departmental and university requirements.
Two sections were prepared following the journal article
format of the Journal of the American Society for Horti-

cultural Science.

ix

INTRODUCTION

The phenomenon of seed dormancy has long concerned
nurserymen and intrigued plant physiologists. Extensive
studies have been devoted to gaining a better understanding
of the mechanisms involved in the after-ripening process.
Growth inhibiting and promoting substances appear to be
involved, but no single hormone seems to control the
germination process. Rather, the interaction or intricate
balance between growth substances probably regulates the
process (2, 62).

Most of the studies of rest and germination of apple
seeds have involved seeds removed from the fruits. The
inability of seeds to germinate within the fruits despite
prolonged storage at temperatures optimal for after-ripening
(5, 47, 65) has not been adequately eXplained. Various
factors have been suggested as the cause of inhibition of
germination in studies made by several workers (5, 11, 12,
45, 51).

The main goal of this study was to determine the role
of (a) moisture, (b) seed coat and/or locule inhibitors, and
(c) volatiles (chiefly ethylene),in the inhibition of
germination of apple seeds after-ripened in the fruit. An
understanding of the controlling factor(s) might provide
nurserymen with a practical tool for storing stratified seeds
for extended periods of time without danger of their

germinating prematurely.

LITERATURE REVIEW

This literature review deals primarily with the
phenomenon of rest in apple (Malus domestica Borkh.) seed,
although examples will be drawn from other species where
appropriate.

The seeds of many species of temperate-zone fruits
exhibit some form of dormancy at maturity, and will not
germinate when planted immediately after harvest even though
conditions are favorable for growth. Germination is prompt
and uniform once the seeds have undergone a period of moist
after-ripening (stratification) at low temperature. However,
apple seeds retained within fruits stored at temperatures
optimal for after-ripening do not germinate within the fruit
despite extended periods of storage, and germinate very

poorly on subsequent removal from the fruit (5, 47).

Terms used in dormancy studies

Dormancy is broadly defined as a temporary suspension
or arrest of active growth in plant organs under conditions
otherwise suitable for growth (39, 62). The types of
dormancy include: (a) quiescence (external, exogenous,
imposed, temporary, false, or summer dormancy), which results
from unfavorable external conditions: and (b) rest (internal,
endogenous, innate, true, primary, or constitutive dormancy)

due to internal conditions. Secondary dormancy refers to the

3

reimposition of dormancy in partially or fully after—ripened
seeds under conditions unfavorable for germination (1h).

The terms after-ripening and stratification are not
synonymous, although they have been used interchangeably in

the literature. After-ripening refers to changes that occur

 

within the seed during storage as a result of which germin-
ation can take place or is improved (37, 40). Stratification,
on the other hand, is the holding of seeds under moist
conditions at any temperature (40, 44). In thisthesis, it
will be used to mean holding moist seeds at cold tempera-
tures, except when otherwise noted.

Germination is defined as those processes which begin

 

with water uptake, and which terminate in the emergence of
the radicle or hypocotyl through the testa (7). In many
seeds the radicle is the part of the embryo which first
penetrates the seed coat. Therefore, germination is often

equated with radicle protrusion.

Role of external factors in apple seed dormancy

Fruit. Apple seeds extracted from the fruits at the
time of harvest and then stratified for 8 to 12 weeks under
moist conditions at temperatures of O0 to 10°C will
germinate on raising the temperature to 200C. On the
contrary, seeds removed from fruits which have been stored
for similar periods of time at temperatures close to the
optimum for after-ripening germinate very poorly (5, 47, 65).

Various reasons have been suggested for the poor germinability

of seeds after-ripened in the fruit. Bartlett (5) found
that germination percentage of apple seeds after-ripened

in the fruit for 24 weeks at 40C was less than that of seeds
which had been after-ripened outside the fruit. This
difference was ascribed to the fact that in the former case,
the seeds were not fully imbibed. Visser (65) also identi-
fied moisture as a factor responsible for the poor germin-
ability of seeds after-ripened in the fruits, but he did not
exclude other factors. Abscisic acid (ABA) which has been
identified in apple juice (45), seeds (4) and other

tissues (45) is considered by many to be involved in after-
ripening. Volatiles produced by the fruits reportedly
inhibit germination of embryos (17, 43) and stratified seeds
(51), and may prevent germination of seeds in apples stored
at low temperatures.

Seed coat. Seed coverings also restrict embryo
germination in apple. Removal of the testa permits the
germination of non- or partially after-ripened seeds
(18, 60). Visser (65) attributed the retarding effect of
the seed coat to mechanical resistance, as its removal did
not accelerate the after-ripening process. Other researchers
(42, 64, 65) have suggested that the impermeability of
the testa to water and gases, in particular, may delay or
inhibit germination of insufficiently after-ripened seeds.
However, the testa is permeable to water. The moisture

content of intact seeds soaked in water immediately after

removal from the fruits rose from 14 to 24% of the fresh
weightuflififlJ124 hours (47), and air—dried seeds regained 80

to 90% of their moisture content within 2 days of soaking (64).
The testa restricts the passage of oxygen to the embryo

(42, 66, 67) by as much as 90% (42), and upon its removal the
respiratory activity of the embryo increased three-fold

(66, 67). However, lack of oxygen is probably not a factor

in delaying or impairing germination, as rest of the embryo
can be broken by eXposure to pure nitrogen (64).

Come (12, 13) found that seeds taken out of fruits
which had been stored for 3 months at O0 to 40C germinated
very poorly unless the seed coats were removed. He concluded
that chlorogenic acid present in the seed coat, rather than
a deficiency of moisture, limited germination. Others (36,
49) suggest that germination inhibitors are metabolized
during moist after-ripening.

Stratification may alter the physical properties of
the testa, which restrains both embryo eXpansion and radicle
protrusion. Moist chilling may reduce the resistance of
the testa to radicle emergence, although no data are known
which support this concept. Once the seeds are completely
after-ripened, the seed coat is no longer an effective
barrier to germination. This could reflect either a change
in seed coat resistance, or a change in embryo vigor.

figggg. The testa restricts the diffusion of oxygen,
thereby limiting respiration and reducing or preventing seed

germination (14). Visser (65, 66, 67) ascribed the

inhibition of germination of partially after-ripened apple
seeds at relatively high temperatures to obstruction of
respiratory activity by the seed coats. However, Tissaoui
and Come (64) succeeded in breaking the rest of embryos
without cold treatment by exposure to pure nitrogen.

Volatile compounds produced by apple fruits may be
involved in the control of seed germination, for germination
of seeds and/or embryos is inhibited by the presence of
fruit tissues (43, 51, 53) or by the volatiles they produce
(17). Furthermore, enriching the atmosphere with ethylene
and other gases released from the fruits reportedly delays
‘the after-ripening process (30). These investigators (30,
51, 53) concluded that fruit-produced volatiles inhibit the
after-ripening process.

The germination of stratified seeds was reduced under
sub—atmospheric pressure (0.1 atm.), leading Kepczynski and
Rudnicki (30) to conclude that the internal ethylene
concentration of apple plays a regulatory role in the
breaking of their rest. Although ethylene is reportedly
required for the termination of rest and germination of
embryos (31), stratifying seeds in an atmosphere enriched
with ethylene (0.1 to 5%) had no effect on their germination
or on that of excised embryos (29). The conflicting results
obtained with ethylene raise doubts as to its participation
in the rest of apple seeds.

Moisture. Hydrated seeds subjected to low temperatures

for a duration of 8 to 12 weeks usually germinate promptly

7

and uniformly. Drying during stratification delays, stops,
or may cancel the effects of after-ripening (23). As noted
above, Bartlett (6) observed that the germination of apple
seeds after-ripened in the fruit was less than 50% of those
stratified on moist cotton wool outside the fruit, and
considered this to be the result of incomplete imbibition.
Visser (65) reported similar results but did not exclude
factors other than moisture content as causal factors.

Temperature. The most effective temperatures for after-

 

ripening apple seeds range from 00 to 1000 with an optimum
at 30 to 50 (1). Abbott (1) found that secondary dormancy
in apple seeds is dependent upon temperature. Germination
of partially stratified seeds was promoted at 50 to 13°C but
was decreased at 180 to 280C. A temperature of 170 was
defined as the "compensation temperature" at which the
germinability of partially stratified seeds remained unchanged.

Harrington and Hite (22) reported that apple seeds do
not after-ripen in dry storage or when kept moist at tempera-
tures of 200C or above. Visser (67) observed that non-
after—ripened seeds soaked in water and then stored under
moist conditions at 2500 were unable to germinate however
long the storage period.

Light. Light promotes or is required for germination
in a number of Species (8). In apple light partially over—
came dwarfism in seedlings obtained from non-after-ripened
seeds (68), and promoted germination of isolated embryos of

dormant seeds (60). In spite of these reports, the role of

light as a factor actively involved in the termination of
rest has not been positively established. Furthermore, light
has not been shown to be required for after-ripening, and
fully after-ripened apple seeds germinate equally well in
light or darkness.

Growth regulators. Evidence for the role of hormones

 

in the regulation of seed rest comes in part from studies
with exogenous growth substances. The dwarfed condition of
seedlings developing from non—after-ripened seeds (18) is
relieved by gibberellic acid (GAB) (6) which suggests that
gibberellins may be involved in rest. Gibberellins also
stimulate the germination of apple embryos excised from
non-stratified seeds UU9,27). Furthermore, Westwood and
Bjornstad (69), noted that seeds from GAB-treated apple
trees germinated 28% after partial chilling compared to only
8% for control seeds. However, Kopecky et al. (32) found
that soaking apple seeds in CA3 solutions prior to stratifi-
cation did not influence rest. This lack of response could
be due to the failure of the applied GA3 to penetrate the
seed coat, since excised embryos respond (19).

Cytokinins also can stimulate germination in dormant
seeds. Badizadegan and Carlson (3) demonstrated that
presoaking embryos excised from mature apple seeds in
N6benzyladenine (BA) solutions significantly increased their
germination, but the resulting seedlings were dwarfed, with
abnormal leaves and short internodes. A similar stimulatory

effect of BA and/or kinetin has been noted by other

investigators (19, 27, 34), as well as synergism with GA
in germination of excised embryos (27).

Ethephon (2-chloroethyl phosphonic acid), an ethylene
releasing compound, reportedly stimulates the germination of
apple seeds (29) and embryos (31). However, Halinska et al.
(20) found that adding ethephon (1.5 x 10—6M) to the
stratification medium inhibited apple seed germination
approximately 17 to 25% until the 70th day of stratification.
Similarly, Sinha and co-workers (56) reported that 250 ppm
ethephon was not effective in stimulating germination until
the seeds had been stratified for 60 days. This observation
was supported by Kepczynski and Rudnicki (29) who found that
stratifying intact seeds in the presence of 0.1 to 5%
ethylene promoted their germination only after 70 days of
stratification. These findings imply that ethylene only
stimulates germination of fully stratified seeds, and is

ineffective in breaking rest in apple seeds.

Role of internal factors in apple seed dormancy

Endogenous growth inhibitors. The endogenous inhibitor
most often presumed to control apple seed germination is
abscisic acid (ABA), which has been identified in extracts
prepared from resting apple seeds (4, 48). The compound
strongly inhibits wheat coleoptile section growth as well as
germination of stratified apple seeds and embryos (26, 27,

46, 47, 50, 52). The ABA level declines rapidly during

lO

stratification (4, 48) and this is accompanied by an increase
in the ability of the seeds to germinate. However, Balboa-
Zavala and Dennis (4) observed that the decline in ABA was
independent of temperature, and concluded that factors

other than ABA content control rest. The inability of seeds
to germinate in stored fruit has been ascribed to the ABA
content of the fruit tissue (45).

Little is known as to what happens to the ABA during
stratification. The rapid decline in ABA during stratifi-
cation could be due to leaching (36, 48) and/or inactivation
(46). Decarboxylation of 1L‘LC-ABA in apple seeds undergoing
low temperature stratification partially accounted for its
disappearance (49). However, decarboxylation was later
attributed to microbial contamination (38). Sondheimer et al.
(61) reported that dormant ash seeds metabolized ABA to
phaseic acid, dihydrophaseic acid, and an unidentified polar
metabolite, and that resting and non-resting seeds metabolized
ABA at the same rate.

Phenolic compounds such as phloridzin, phloretin, and
chlorogenic acid occur in apple tissues, including seeds
(13, 24, 46, 70), and pholoridzin retards the growth of
apple seedlings at a concentration of 10—4M (10). Come
(11, 12, 13) postulated that the phenolic compounds found in
the integuments of apple seeds regulate rest. He suggested
that the phenols absorb some of the oxygen passing through
the integuments and impede its penetration to the embryo.

Hence, the embryo is deprived of the oxygen required for

11

germination. But Dziewanowska et al. (16) and Pieniazek

and Grochowska (56) found that the amount of both phloridzin
and chlorogenic acid in apple seeds decreased or disappeared
completely at maturity (16, 70). Phloridzin by itself had no
effect on germination of embryos but strongly inhibited the
promotive effects of GA and/or BA (27).

Endogenous growth promoters. Extracts of apple seeds

 

contain auxin-like growth promoters (28, 36, 63), and
activity occurs in the seed coat, endosperm, and embryo
during after-ripening (28). Activity was very low during
the first 5 weeks of low temperature stratification, then
rose to a maximum in the 7th week (32). However, no
correlation was obtained between the level of auxin and the
termination of rest.

The rest breaking effect of chilling on apple seeds
has been attributed to the synthesis of gibberellins (GAs)
(57). GAs (A4' A7, and A9) were identified in extracts of
dormant and non-dormant apple seeds (59), although the GAs
identified may have been artifacts (15). The concentration
of GA“ rose very markedly in the 4th week of stratification
at 200, then decreased to the initial level between the 50th
and 60th day, although the GA7 content remained at a more or
less constant level during the entire period of stratification
(57, 58, 59). Kopecky et al. (32) observed similar changes
in GA content. The above observations do not establish a
close association between the level of GA“ and the termina-

tion of rest in seeds, for germination capacity did not

12

increase until several weeks after the GA” content had
declined (57). Halinska and Lewak (21) observed an increase
in the free GA level at the end of the after-ripening

9

period. They suggested that GA unlike GA“ and GA7, may

9.
be involved in the final phase of after—ripening including
germination.

Letham and Williams (33) identified 3 cytokinins
(zeatin, zeatin ribotide, and zeatin riboside) in extracts
of young apple fruits, while Zwar and co-workers (71) found
4 cytokinin—like components. Both bound and free cytokinin-
like compounds also occur in mature apple seeds (9).
Cytokinin-like activity in apple seeds rose during strati—
fication, reaching a maximum in the 5th week, but the
increased level of cytokinins was not directly correlated
with the ability of the seeds to germinate (9, 32).

It has recently been suggested that ethylene is one of
the hormones regulating rest in apple seeds (30, 31).
Kepczynski and Rudnicki (30) found that germination of
stratified seeds was reduced under sub-atmospheric pressure
(0.1 atm.), and this was attributed to a decrease in the
internal ethylene concentration of apple fruits. Endogenous
ethylene production by excised embryos increased during
after-ripening, and this was accompanied by an increase in
their ability to germinate (31). Kepczynski et al. (31)
concluded that endogenous ethylene is required for the

termination of rest and germination of embryos.

13

Enzymes. Nikolaeva and Yankelevich (40) suggested
that the breaking of dormancy and the ability of embryos to
overcome the inhibiting influence of seed coats was caused
by an'increase in enzyme activity. The activity of enzymes
(peroxidase, succinic dehydrogenase, lipases, and proteases)
in seeds and embryos increased seven-fold during chilling,
and any interruption of the cold stratification caused a
sharp decline in enzyme activity (41). Lewak et al. (35)
identified 3 phases in after—ripening of apple seed:
(1) removal of the primary cause of rest; (ii) high metabolic
activity; and (iii) initiation of germination. They
postulated that enzymes are involved in controlling the
second phase, and proposed that a gradual build-up of
hydrolytic enzymes could be responsible for breaking of
rest. The biosynthesis or release of hydrolytic enzymes is
presumably under hormonal control (2), and treatment of
dormant seeds with GAu or benzyladenine increases the
activity of lipase, phosphatase, and peroxidases (54, 55).
However, changes in enzyme activity during stratification
seem to be the result, rather than the cause, of breaking

rest in apple seed.

Summary

Mature apple seeds are in a state of physiological rest
which is terminated by a period of moist chilling (strati-
fication). Early studies emphasized the importance of

external factors, particularly the seed coat, in inhibiting

l4
the germination of the embryo. More recent work has
assumed rest to be under hormonal control, and to be
broken by changes in levels of inhibitors (such as ABA)
and promoters (GAS and cytokinin). The effects of exo-
genous growth substances support this hypothesis.

Seeds extracted from fruits held in storage at an
optimal temperature for after-ripening exhibit a very low
ability to germinate. Various reasons have been suggested
for this effect of fruits, including: (i) limited seed
water content, (ii) inhibitory effect of volatiles, and
(iii) inhibitory levels of ABA in fruit tissue. The
purpose of this research was to further examine these and
other factors in an attempt to explain fruit induced

dormancy in apple seed.

SECTION I

FRUIT INDUCED DORMANCY IN APPLE
(MALUS DOMESTICA BORKH.) SEED.
ROLE OF WATER AND INHIBITORS

FRUIT INDUCED DORMANCY IN APPLE (MALUS DOMESTICA BORKH.) SEED

I. ROLE OF WATER AND INHIBITORS

Abstract. The roles of inhibitors, especially abscisic
acid (ABA), in the seed coat and locules, and of seed water

content in fruit-induced dormancy of apple (Malus domestica

 

Borkh.) seeds were investigated. After-ripening seeds in

the fruit inhibited their subsequent germination on either
moist paper or on a wire mesh screen, whereas fruit volatiles
has little if any effect on seeds stratified on screen.
Soaking seeds either prior to or after stratification
promoted germination of seeds held in the fruit or on screen,
but did not overcome the effect of the fruit. Germination
capacity of seeds after-ripened on screen increased with
water content, although large increases in the former were
sometimes associated with small increases in the latter.
Seeds stratified on screen, then germinated on moist paper,
germinated better than seeds stratified on moist paper, then
germinated on screen, indicating that water content was more
crucial during germination than during after-ripening.
Germination capacity of embryos excised from seeds stratified
in the fruit was considerably lower than that of embryos from
seeds stratified on paper or screen, particularly when
germinated on screen; thus fruit-induced dormancy is not
simply a seed coat effect. The effect of the locule (endo-
carp) in inhibiting germination was blocked by aluminum foil,

indicating the involvement of a diffusible, water—soluble

15

16

inhibitor. However, the ABA contents of the endocarp and
of the testa were too low to account for the inhibition
observed. ABA content of the testa declined regardless of
whether seeds were held in the fruit, on screen, or on moist
paper. Soaking seeds after—ripened in the fruit in ABA
concentrations higher than that measured in the testa did
not reduce the promotive effect of soaking, indicating that
leaching of ABA was not responsible for this effect.
Finally, partial or complete removal of the testa indicated
that the effect of soaking was primarily mechanical, rather
than chemical. It is concluded that an inhibitor(s) in the
locule is the primary factor responsible for fruit induced

dormancy.

 

Apple seeds will not germinate until they have been
subjected to a period of after-ripening under moist condi-
tions at 00 to 10°C. However, seeds held in fruits at
temperatures close to the optimum for after-ripening germin-
ate very poorly on removal from the fruit even after extended
periods of storage (1, 14, 19).

The low germination of seeds after-ripened in apple
fruits has been attributed to: (a) insufficient moisture
content for after-ripening (1), (b) absence of free water
to leach out seed inhibitors (9), (c) fruit-produced
volatiles, including ethylene (17). A diffusible inhibitor

in locules (endocarp) could also prevent germination.

17

The content of abscisic acid (ABA) or an ABA-like
inhibitor declines rapidly during stratification of apple
seeds (2, 13, 15) and other (3, 7, 8) seeds which require
moist chilling to terminate rest. Application of exogenous
ABA prevents germination of seeds and embryos in which rest
has been broken (6, 13, 16, 17). Thus, the breaking of rest
has been ascribed to the decline in ABA content (9, 15, 20).
However, Balboa-Zavala and Dennis (2) found the decline in
ABA in apple seeds to be independent of temperature and
suggested that factors other than ABA control rest. Rudnicki
and Czapski (18) found that decarboxylation<xf(1-1uC)ABA in
apple seeds undergoing low temperature stratification
partially accounted for its disappearance. However,
Milborrow and Vaughan (11) reported that the amount of 14002
evolved from sterile apple seeds during stratification
amounted to less than 0.01% of the radioactivity supplied as
(2-14C)ABA. They concluded that the degradation observed by
-Rudnicki and Czapski (18) was probably brought about by
microflora in the medium.

Inhibitors, including ABA, occur in the juice of a
number of fleshy fruits (4, 10). ABA has also been identi-
fied in apple juice (6, 12), and Pieniazek and Rudnicki (14)
suggested that it prevents the germination of seeds after-
ripened in the fruit.

The testa also restricts germination, for its removal

permits germination of partially after-ripened embryos (5).

18

Visser (19) attributed the retarding effect of the seed coat
to mechanical resistance, as its removal did not accelerate
the after-ripening process. The objective of this study was
to determine the roles of water versus inhibitors in the
after-ripening and germination of apple seeds held in the

fruits during low temperature storage.

Materials and Methods

Plant material. Mature apple fruits of several
cultivars were collected from eXperimental or commerical
orchards. In some cases the fruits had fallen from the
trees and were collected from the ground. Seeds were either
removed from the fruits immediately or were removed after
storage of the fruits for varying periods of time at
50 1 10C. None of the seeds was dried before use.

Methods of after-ripening and germination. Several
methods of after-ripening and germination were used with
these seeds. Seeds held in the fruit were exposed to fruit
volatiles, but not to free water. Therefore leaching of
chemical inhibitors from the seeds was prevented, but
inhibitors in the lining of the locule (endocarp) might
prevent germination. Seeds were held on wire mesh over moist
paper or glass wool in petri dishes to simulate moisture
conditions in the locule, yet eliminate the possible effects
of volatiles and endocarp inhibitors. Finally, holding

seeds on moist filter paper in petri dishes also eliminated

19

the effects of volatiles and endocarp inhibitors, but
permitted both water uptake and leaching of inhibitors. In
some eXperiments, seeds were soaked before or after strati-
fication to test the effects of leaching and/or increased
water content, or embryos were excised to determine the role
of the testa in restricting germination.

Germination tests. Unless otherwise stated, germina-

 

tion tests employed 3 replicates of 20 seeds or embryos in
9—cm petri dishes lined with Whatman no. 1 filter paper,
which was moistened with distilled water. The petri dishes
were randomly placed in a growth chamber at 200 I 100 in
darkness. Seeds with visible radicle protrusion, and
embryos whose radicles showed geotropic curvature were
considered to be germinated. Germination was recorded for
a period of 10 days, and the results are exPressed as mean
percentages.

Measurement of seed moisture content. Seeds were

 

blotted with paper towels, weighed, held at 75°C for 72
hours, then reweighed. Water content is eXpressed as a per-
centage of initial fresh weight of the seeds.

Statistical analysis. The data were subjected to

 

analysis of variance, and the mean separation procedure of

Duncan's Multiple Range Test was used where appropriate.

20

Exp. 1. Effects of the locule, of water content, and
of fruit volatiles on after-ripening and germination of

apple seeds. If volatiles limit after-ripening and/or

 

germination of seeds held in fruits, seeds stratified on
screen in the same containers with fruits should germinate
poorly in comparison with similar seeds stratified in the
absence of fruits. On the other hand, if chemicals in the
locules inhibit stratification, seeds held on screen should
germinate better in the presence or absence of fruits than
those in the fruits. If water content during or after
stratification in the fruits limits germination, soaking
seeds prior to after-ripening should negate the effects.
Apple seeds, cv. 'Paulared', were collected from the
University Research Farm, East Lansing, in 1978 and from a
commercial orchard near Grand Rapids in 1979. To distin—
guish between the effects of (a) inhibitors in the locules,
(b) water content, and (0) fruit volatiles, seeds were
extracted from the fruits immediately after collection, and
after-ripened at 50 : 1°C: (a) in the locules of half-fruits
in loosely covered plastic containers, (b) on a wire mesh
screen over moist paper in the same containers, and (c) on
wire screen over moist paper in a similar container without
fruits. The relative humidity of the containers was main-
tained near 100% by lining the bottoms with a layer of
moistened glass wool. Half of the seeds in each treatment

were pre-soaked in distilled water for 24 or 48 hours, the

21

remainder were not soaked. Samples were transferred to a
growth chamber at 200 1 10C at 3 week intervals to evaluate
germination in darkness on the same medium used for after-
ripening (locules or screen). To assure that moisture was
not limiting germination in 1979, droplets of water were
placed on seeds held on screen for germination.

ExE. 2. Effects of after-ripening seeds in the fruit,
on screen, and on moistgpaper upon seed water content and
upon subsequentAgermination of seeds and embryos on moist
paper or screen. If water content during after-ripening
limits subsequent germination, after-ripening on screen
should be equivalent to after-ripening in the fruit, and
germination capacity should parallel water content. Previous
workers had reported that holding seeds in the fruit does
not affect the ability of the embryo to germinate, the
inhibitory effect being confined to the seed (4). This
concept was also tested in this eXperiment. Apple seeds used
in the 1978 eXperiment were extracted from 'Paulared' fruits
obtained from the University Research Farm, East Lansing.
The study was repeated in 1979 using fruits of the same
cultivar collected from a commercial source at Grand Rapids.
The seeds were after-ripened at 50 I 1°C: (a) in intact
fruit, (b) on moist filter paper, and (c) on screen over
moist paper. Samples were removed from storage at 3 week
intervals for germination tests, on both moist paper and

on screen, of both intact seeds and excised embryos.

22

Exp. 3. Separation of effects during after—ripening
from effects during germination. Treatments were arranged
in a factorial to attempt to separate the effects of moisture
supply and/or inhibitors during stratification versus
effects during germination. Seeds (cv. 'Paulared') were
held for 12 weeks at 5° : 1°C: (a) in intact fruits,

(b) on moist paper, or (c) on screen over moist paper, then
half the seeds in each treatment were soaked in distilled
water for 24 hours at 20° : 1°C. The seeds were test
germinated: (a) in the locules of half-fruits, (b) on moist
paper in a petri dish, and (c) on screen over moist paper in
a petri dish.

Exp. 4. Effect of blocking diffusion of locule
inhibitors on germination of seeds stratified on moist paper.
If a water-soluble inhibitor(s) prevents germination in the
fruit, blocking its diffusion to non-dormant seeds should
prevent its effect. Seeds (cv. 'Jonathan') stratified on
moist paper for 12 weeks were germinated on the endocarp of
fruit sections placed horizontally on moist paper. Seeds
were placed: (a) directly on the endocarp, (b) on moist
paper in contact with the endocarp, (c) on moist paper backed
by aluminum foil in contact with the endocarp, (d) on moist

paper, and (e) on screen over moist paper in petri dish.

23

Ex . . Effect of method of after-ripening seeds on
ABA content of the testa. If stratification in the fruit
presents leaching and/or metabolism of ABA, or allows a
continuoussnuuflgrfrom the endocarp, ABA content of the seed
coat should decline less rapidly in the fruit than outside
the fruit. Holding on a screen should prevent leaching, but
not metabolism, while holding on moist paper should prevent
neither. Seeds were removed from 'Paulared' fruits immedi-
ately after collection from a commercial orchard at Grand
Rapids in 1979. The seeds were then after-ripened: (a) in
intact fruits, (b) on a screen over moist paper in petri
dish, or (c) on moist paper in petri dish for 12 weeks.
Three samples of 20 seeds from each treatment were removed
after 12 weeks and germinated on moist filter paper. Two
samples of 50 seeds each were removed for ABA determination
at intervals of 3 weeks. The seed coat was separated from
the embryo and shaken in 25 ml of distilled water at 5°C for
7 days. The water was changed daily and pooled, and this
diffusate was processed (Fig. 1) to give an acidic fraction
(free ABA) and a base hydrolyzable fraction (bound ABA).

The ABA content of the locules at the beginning of
the eXperiment was also determined. Two replicates of 2 gram
each of the locule (endocarp) tissue were obtained from a
total of 10 fruits per replicate. All the flesh tissue was
scraped off and each sample was shaken in 50 ml of distilled
water for 24 hours at 5°C. The water diffusate was processed
into free and bound ABA fractions as described for seed coat

diffusates.

24

Water diffusate from
seed coat (175 ml)
or locule (50 ml)

Adjust to pH 8 with NH 0H,

partition 3X with 20 m CHZCl2

 

 

Aqueous phase CH2012

(discard)

Adjust to H 3 with HCOOH,

partition X‘With 20 ml CH20l2

 

 

l
CH2012 Aqueous phase

(free ABA) Adjust to pH ll,

hydrolyze for 1 hr at 60°C.
Adjust to H 3,

partition X with 20 ml CHZCl2

 

 

Aqueous phase CH2C12

(discard) (bound ABA)

Figure l. Fractionation procedure for free and
bound ABA in water diffusates from
seed coats and locules (endocarp)

25

The extracted free and bound fractions from both seed
coats and locules were evaporated to dryness and resuspended
in pure methanol. The fractions were methylated with
diazomethane, the methanol-diazomethane solution was evapor-
ated to dryness, and the residue was redissolved in ethyl
acetate. Aliquots of the ethyl acetate solution were injected
into a Packard 7300 gas liquid chromatograph equipped with
an electron capture detector (°3Ni foil) operated at 5 volts.
A glass column (152.4 x 0.35 cm i.d.) was packed with 1%
XE—60 on 100/120 mesh Gas Chrom Q. The column temperature
was 200°C, and inlet and detector temperature was 220°C.

The carrier gas was nitrogen at a flow rate of 40 ml/min

at 40 psig. Nitrogen scavenger gas was supplied to the
detector at 90 ml/min. The retention time for authentic
c,t-ABA was 1.5 min. A standard curve based upon peak

height was used for quantitation of c,t-ABA in the diffusates.

Exp. 6. Effects of moisture level,gseed coat resistanceL
and ABA upon germination of seeds. If leaching of ABA were
responsible for the promotive effect of soaking seeds.
inclusion of comparatively high concentrations of ABA in
water would prevent the effect. Furthermore, if chemical
inhibitors in the seed coat were responsible for limited
germination, partial seed coat removal should be less effec-
tive than total removal. If mechanical properties of the
seed coat limit germination, on the other hand, (a) removal

of the testa in contact with radicle should be just as

26

effective as total removal, and (b) soaking should have
little, if any, effect on germination of seeds from whichthe
testa has been partially or totally removed. To determine
the effects of soaking and partial or total testa removal in
the presence and absence of ABA, seeds were extracted from
‘Jonathan" apple fruits which had been harvested in
September 1979, and held at 5° 1 1°C for 12 weeks. Seeds
were then soaked in distilled water for 0, 24, 48, or 72
hours with or without the addition of 10 ppm ABA. Three
samples of 20 seeds per treatment were then test germinated
with the seed coat: (a) intact, (b) removed over the radicle
end, or (c) entirely removed (excised embryos). Seeds were
germinated on a wire mesh screen over moist filter paper in

petri dishes.

Results

Exp. 1. Effects of the locule, of water contentL and
of fruit volatiles on after-ripening and germination of
apple seeds. All seeds left in the locules failed to
germinate in both 1978 and 1979, whether they had been soaked
in water or not (Table 1). Germination of seeds held on
screen increased as after-ripening time increased from 6
(1979) or 9 (1978) to 12 weeks, and was promoted by soaking
in water prior to after—ripening in most instances. There—
fore, contact with the locules inhibited either after-

ripening or germination or both. Seeds held in containers

27

.Ho>oH mm one we pcaoamacm«m :odpthopCHc
.pmow :ofiumCASLow memhsc mcoom mo commune ow cocoa othmaosu
.am¢H 2“ mu: m: can .mmaa Cw an: am now nova; poaafipmflu :« cmxaom mooomh

.acoSHaohv mo

mmoHpumwmu Ammaav mxz n no Ammaav ox: o ho 3 poems coupaooo sewamcwahom
oz .Ho>oH Rn um smzo >2 menu new measaoo Canvas :oapmhanom :dwzx

 

 

 

 

.m.: c .m.: a .n.: .Amv x Am<v :oaaoahowcH
e s: c w s a n ma 5 0 oz
E Om E mm 5 a E mm 5 0 now
.Amv hope: a“ eoxmom
c me p mm c s c we c o mpazcu-mHmz ascend: comuom
a on m ma m m m an a o muflsuuumam: new: :oonom
m o p o m o u o p o magnum:mawz Ho amazoon
.Am<v wadsonuuauoam<
a on 0 NH 9 n 0 mm a o oz mwaapm:mamn
m cm a m: m o a mm a o no» wsozpwz coouom
a on e m n o c om a o oz mafiaum
n on n mm c n n mm m N no» umam: new: soonom
o o o o n o o o a o oz measuu
o o m o n o m o a o no» :mam: mo moasooq
ma m 0 NH a
szv mewcodmuuhopu¢ szv mcfisonaunpoum< noun: .:a scapmcaahom
:« so
Needs mesa . e

zwcfixwom MCMCondhnuoum<

 

:oapmcwsnmm pamopom

I|"l'lllll"lllIII'"l'IlIIII"|II'II'""II""Il|ll-.lllll'll""'l'|l""""-'lll'
Ill-‘5' I'I‘-l"-'Ill!III'I'Ill-I'III'l'|lll"""'I"'lll"|"'l"!l'|l'l'll'"ll'l

.mpoom ..ooumazmm.

.>o .madam zo COMQMCMEpmw so wsflconfluuumpmm o» hoMpQ nova; :H
mzflxnoh mo use Ezmooe :ozsmcmsaom paw wcflcoawunnmumm mo mvommmm .H manna

28

without half-fruits germinated significantly better than
those in containers with half-fruits in 6 of 7 comparisons
in which germination was appreciable, indicating a small but
measurable inhibition by Volatiles. Interaction between
soaking and method of after-ripening on germination was
significant in 2 cases, indicating beneficial effects of
soaking only when seeds were not held in locules. Moistening
the seeds during germination appeared to favor germination
(compare 1978 vs. 1979), but this was not critically tested.
Exp. 2. Effects of after-ripening seeds in the fruit,
on screen, and on moist paper upon seed water content and
upon subsequent germination of seeds and embryos on moist

paper or screen. Seeds held on moist filter paper contained

 

approximately 25% more water than did those held in intact
fruits, whereas seeds held on screen contained at most 11%
more (Table 2). After-ripening seeds in the fruit strongly
inhibited subsequent germination in comparison with after-
ripening on screen or on paper, and no germination occurred
when seeds were held on screen after removal from the fruits
(Table 3, Fig. 2A, C). Thus the fruit has an inhibitory
effect over and above any effect on water content. Seeds
held continuously on paper germinated only slightly better
in most instances than those held on screen during after-
ripening, then germinated on paper, and the latter germinated
significantly better than did seeds after-ripened on paper,

then germinated on screen. These results indicate that

not] 31"." c. 1 . I b.0uI-zfion‘..vrnx\vvbcln‘l->VI

29

Table 2. Moisture content of seeds, cv. 'Paulared',
during after—ripening in intact fruit, on
screen, or on moist paper at 5°C (1979)

———_————_—-—_______—__-—__———_—--———-————-——-—_._-———_—-———-——
—-—_——————_--—__.--__-----—-—-_-—--------—---------——-—-‘--—

Moisture content (%)y

 

After-ripening

 

 

method After—ripening (wk)
0 3 6 9 12
In intact fruit 47 a 46 c 44 c 44 c 47 b
On moist paper 47 a 57 a 57 a 57 a 58 a
On screen 47 a 49 b 49 b 49 b 48 b

 

yMean separation within columns by DMRT at 5% level.

30

new massaoo :azpflz cowvmhMAmm Chm:

.Ho>oH Rm one am panofimwcmMm cempowuopcmc

.Hmsea en

he exso an even

.quEHMoup mo mmmacpmth mCMCmnwh

 

 

 

 

 

 

lacuna mo Ammmav mg; n so Ammmav ex; 0 no 2 peaks popusooo COApMCAEhow ozN
t t t t t .Auv x Am¢v cowpowpoch
m mm m m m o m mm m H soouom so
a mm a am a MN 9 me u ma gonna pmfios co
.on cowuwcaspou
a am a a: e ea c we a Hm ceded hence no
m mm m on m 3H m m: m 0 season co
2 :H z o m N 2 ea w : pwzuh CH
afim<v wCMCmQflpnpopm<
2 mu 0 ma n o 3 mm 56 m :oopom
m ooa m mm m mm a mm m 0: Lemma unwoz Momma pmaos co
0 mm e m e o e NH e o :emcom ceded peace
a mm 9 mm m mm m we on ma nomad pmwoz uo>o Coopom :o
u o o o n o u o c o :oohom
e mm m o e m 0 am e m ceded peace peace eunuch :H
Na d 0 NH a
szv wcficodwpnpopm< szv MCMConu:Amwm< “so pecans
cowumcwsnou wcwconwhnhovm<
mmma mama

 

.vwzhu :w momma

..eeceasmd.

scapmcflshvw accused

.cmohom Lo nomad unwos
co cowpmcmsuow psosvonnzm com: comma pmfioe no can .Coohow co

.>o .oadam mCAConhnhmuzm mo apoowmu .n manna

31

moisture content was much more critical during germination
than during after-ripening. Interaction between method of
after-ripening and method of germination was primarily due
to quantitative, rather than qualitative, differences in
response (Fig. 2A, C). Similar results were obtained in
1978 and in 1979.

The germination on moist paper of embryos excised from
seeds held in the fruits was significantly lower than that
of those from seeds stratified on screen or moist paper for
the first 6 (1978) or 9 (1979) weeks of after-ripening, but
no differences were evident after 12 weeks (Table 4, Fig. 2B,
D). The difference was more pronounced when the embryos
were germinated on screen (data for 1979 only). Differences
between embryos after—ripened on screen versus moist paper
were small but significant for the first 4 to 9 weeks, with
the former being slightly inhibited in germination. Inter-
action in 1979 between after-ripening and germination methods
again indicated quantitative, rather than qualitative,
differences in response. Germination of embryos from seeds
after-ripened in the fruit was considerably lower at 6 and
9 weeks in 1979 than in 1978.

About 5 additional weeks of chilling were necessary to
induce 50% germination when embryos were germinated on screen
rather than on paper, germination paralleling that of intact
seeds held on moist paper (Table 4, Fig. 20, D). Thus the
restriction on germination imposed by lack of water and

the true state of embryo dormancy are better revealed when

:32

.Ho>oH fin oza pm u:hommH:wwm codpomuoHCH
t.
.Ho>oH am pm smza an mpom 6:8 messaoo Case“: Cowpmumdmm :mozq

 

s a t w I: I: :I :I .Auv x Am<v :oflpomumpch
mam mum mm no I: II I: I: season so
Lam pom emu no: I: I: I: I: gonna Pmmos co
”Auv :oflmeHEuou
and saw 5mm 5mm :: I: :I I: comma pmwos :0
5mm Emu Em: CEHN I: I: I: I: :mohom so
are CNN czfl CNH I: I: I: I: Hanan :H

.Am<v wCHCoQMQIEoum<

l-l'|'|l|'l"l-l‘|'II||II'II||IIII'I'lllllllllll‘II"llll'lllIIl"--|'l.l||-IIII'|"'-|I|'-|I

 

 

 

 

 

nmmm amp and no II I: I: I: coupon

mooH mooH mam mmm woos mood mom «mm ceded peso: honed peace so
comm om: am no II II :I :I :mmLom gonna umwos
mood and mom mm: coca wooa com nnm Lemma ammo: po>o :mouom :0
com to mo no I: II I: II Coopom .

sea on: new 0mm mood and gas eon ceded page: haste peeps“ :H

NH m e n ma m e a

szv mewcodfihIhoum< szv wadcodwpIuopm< “so eczema

mmmfi mmma :oflamcflseoo wcHQoQMLIEopk<

 

coapmcflsuow scooped
.Comhow no Momma
games :o :oflamczsaom ozznsm pcozzomnzn sod: Lemma unwoe £0 pcm .Coohom
:0 .pwstu Cw women ..cmhMasmm. .>o .oaaaw wCMCodeILoaMm no npommmm .2 canoe

Figure 2. Effects of method and time of after-
ripening and of germination medium on

germination of apple, cv. 'Paulared',
seeds and excised embryos.

 

100

80

IO

40

a
O N
O O O

cenumanou %

O
0

IO

33

 

- (A) I978: SEED

“tor-ripening
o in fruit
I on quII'I
"' I: on moist paper
Germination
— "my

  

'- (I) I97I8 EMIIYO

 

(C) I979: SEED

IO!-

20

 

....
.I
l

/

000m

 

 

4—

 

 

"unfit!
I
I

1
3 I

12

 

AFTER-RIPENING AT 5°C (and

 

34
water is limiting than when it is not. Taken as a whole,
the data from this experiment indicate that holding seeds
in the fruit does indeed affect embryo dormancy, contrary
to previous reports (4).

Exp. 3. Separation of effects duringpafter-ripening
from effects duringpggnmingpignJ Although all main effects
(after-ripening treatment, germination medium, and water
soak) were significant, interpretation was complicated by
the fact that all 4 interactions were also significant
(Table 5). Nevertheless, the data reveal a number of
facts about the control of seed germination by the fruit.

Seeds after-ripened and/or germinated in the locules
germinated poorly in comparison with those held outside the
fruits, and no germination occurred in seeds both after-
ripened and germinated in the locules (Table 5). Soaking
following after-ripening stimulated germination only in
seeds not stratified or germinated on moist paper. The
promotive effect of soaking seeds could be due either to
leaching of a water-soluble inhibitor, to increased water
content, or to weakening of the seed coat: the data do not
permit uneQuivocal resolution of this question. However,
the better germination of non-soaked seeds which were
stratified on screen, then germinated on paper (98%) in
comparison with similar seeds stratified on paper, then
germinated on screen (78%) suggests that water content is
the crucial factor. The few days in contact with moisture

in the former case should have had little effect on leaching

35

Table 5. Effects of after-ripening and germination medium
and of soaking in water after 12 weeks of after-
ripening on germination (%) of apple seeds, cv.

 

 

'Paulared'.
======SS=============================.========:==:1==;=======
After-ripening Soaked Germination medium

treatment (2° hrs) Locules Paper Screen
In intact fruit Yes 0 b 42 b 27 c
. No 0 b 27 c 0 d
On moist paper Yes 40 a 100 a 98 a
No 38 a 100 a 78 b
On screen over Yes 38 a 98 a 98 a

After-ripening (AR):

In intact fruits 18 g
0n moist paper 76 e
On screen 61 f

Germination medium (G):

Locules 20 t

Paper 77 r

Screen 56 s
Soaked (S):

Soaked 60 m

Not soaked 42 n

Interactions: (AR) x (S) *

(AR) x (G) *

(G) x (S) *

(AR) x (S) x (G) *

 

zMean separation within columns and sets by DMRT at 5%
level.

*Interaction significant at the 5% level.

36

of an inhibitor in comparison with the 12 weeks of exposure
in the-latter treatment. The "locule effect" was clearly

not merely a response to restricted water uptake, for
germination in the locule was inhibited in both soaked and
non-soaked seeds regardless of method of stratification.

This strongly suggests that the locule contains a germination
inhibitor.

Exp. 4. Effect of blocking diffusion of locule
inhibitors on germination of seeds stratified on moist
pgpgp. When fully stratified seeds were allowed to germin-
ate on the endocarp of fruit sections only 38% germinated
in comparison with 98% on screen (Table 6). When moist
paper was used between the endocarp and the seed, germin-
ation increased to 88%, possibly because of a dilution
effect. This was significantly less than that observed on
moist paper isolated from the endocarp by aluminum foil,
indicating a small but definite inhibitory effect of material
diffusing from the endocarp.

Exp. 5. Effect of method of after:pipening seeds
on ABA content of the testa. Methylated samples of both
the free and bound fractions contained a peak which had the
same retention time as authentic c,t-ABA. When 'blank'
samples (water only) were processed no peak was observed.
Exposure of methylated samples of c,t-ABA and of diffusates
to ultraviolet light (265 nm) for 16 hours greatly reduced

peak heights. The initial concentration of ABA in the

37

Table 6. Effect of various treatments on germination of
'Jonathan' seed on surface of endocarp of stored

 

fruit

Germination test Germination (%)Z
0n locules 38 c

On locules + moist paper 88 b

On locules + foil + moist paper 97 a

On moist filter paper in petri 100 a

dish

On screen in petri dish 98 a

 

ZMean separation within columns by DMRT at the 5% level

38

testa was approximately 9 ug/g (9 ppm) of free ABA and
0.8 ug/g (0.8 ppm) bound ABA.

The amount of ABA in the diffusates from the testes
of seeds after-ripened in the fruit, on screen, and on
moist filter paper declined considerably as after-ripening
progressed (Table 7). After 3 weeks of after-ripening free
ABA had declined to 20, 46, and 51% of the original level
in seeds stratified on moist filter paper, on screen, and
in intact fruits, respectively. The ABA level continued to
decline thereafter. After 12 weeks the percentage of free
ABA recovered was 0.3% for moist paper, 4% in fruit, and
16% on screen. The original content of bound ABA was one-
tenth of that of free ABA, but this also declined, except
in seeds after-ripened on screen. Only 27% of the seeds
held in the fruit germinated on moist filter paper after
12 weeks of after-ripening as opposed to 100% and 98%,
respectively, of those after-ripened on moist paper and on
screen (Table 7). Thus, germination capacity was not
correlated with ABA content of the seed coat diffusates.

The water diffusates obtained from locules, following

the removal of all fleshy tissue, contained 2.5,ng of free
ABA and 0.4 ug of bound ABA per gram fresh weight of tissue
(2.5 and 0.4 ppm, respectively) approximately one-fourth
of the concentration of free ABA and one-half of that of the
bound ABA found in the seed coat. The free acidic fraction

at a concentration equivalent to 0.25 ppm ABA did not inhibit

39

Table 7. Effects of after-ripening method on the amount of
ABA in water diffusates from apple, cv.'Paulared',
seed coats and on germination of seeds.

After-ripening method

 

 

 

 

 

 

 

 

 

After-r1 eni
(wklp ng In fruit On screen On paper Means
Free ABA (ng/seed coat)2
0 178 a 178 a 178 a 178 P
3 90 b 82 b 35 b 69 S
6 30 c 80 b 18 b 43 s
9 11 c 33 b 10 b 18 S
12 8 c 28 b 0.5 b 15 S
Means.... 63 m 81 m 48 m
Bound ABA4(pg/seed coat)
0 17 a 17 a 17 a 17 r
3 14 a 10 a 7 ab 10 r
6 20 a 21 a 4 ab 10 r
9 10 b 18 a 3 ab 8 r
12 5 b 17 a 0.2 b 7 r
Means.... ll m 17 m -7_;

Germination after 12 weeks (%)
Means.... 27 b 98 a 100 a

 

 

zMean separation within columns and sets by DMRT at 5% level

Means for 2 replicates of 50 seed coats each ABA content)
or 3 replicates of 20 seeds each (germination .

40

germination of fully stratified seeds, nor did 10 ppm of

ABA (data not shown). A concentration of 100 ppm c,t-ABA
resulted in 40% inhibition of germination of fully stratified
seeds. Therefore, ABA alone cannot account for the
inhibitory activity of the endocarp.

Exp. 6. Effects of moisture contentpiseed coat

 

resistanceigand ABA upon germination of seeds after-

ripened in the fruit. Response to imbibition varied with

 

seed coat treatment (Table 8). Germination of intact seeds
increased with soaking time, whereas that of seeds from
which the testa had been partially or wholly removed did
not. Significant interaction reflected this difference.
Although germination was higher following total removal of
the testa than following partial removal, the difference was
non-significant, suggesting that mechanical, rather than
chemical, characteristics of tmetesta restricted germin-
ation. Inclusion of ABA (10 ppm) in the water used for
soaking did not affect germination significantly (overall
means for ABA at O and 10 ppm were 83 and 78%, respectively)
regardless of seed coat removal. Thus leaching of ABA is

probably not responsible for the effect of soaking.

Discussion

The data presented in this study confirm the observa-
tions of others (1, 19, 19) that seeds after-ripened in the

fruit germinate poorly. Limited water content of seeds

#1

 

 

 

 

 

 

 

Table 8. Effects of soaking time and seed coat removal on
germination of seeds, cv. 'Jonathan', after-
ripened in the fruit for 12 weeks at 5°C, then
germinated on screen.

Germination (%)Z

Soaking Seed coat treatment Means
hr
Intact Removed over Entirely

radicle end removed
0 32 c 83 a 95 a 70 s
24 75 b 90 a 97 a 87 rs
#8 78 b 92 a 97 a 89 r
72 95 a 93 a 93 a 95 r
Means 70 n 90 m 97 m
Interaction (Soaking x Seed Coat Treatment) *

 

ZWithin sets means followed by same letter are not
significantly different at 5% level by DMRT.

*-
Interaction significant at 5% level.

42

during after-ripening does not fully explain the effect

of fruits on seed germination as has been suggested (1, 19).
The moisture content of seeds after—ripened on screen was
only slightly higher than that of seeds after-ripened in

the fruit (Table 2), yet the former germinated much more
readily than the latter on both screen and moist paper
(Table 3). Furthermore, seeds soaked prior to after—ripening
in the locules of half-fruits and germinated in situ failed
to germinate (Table 1). On the other hand, seeds after-
ripened on screen germinated moderately well without
exposure to free water and readily when soaked or held on
moist paper.

High water content is necessary for good germination
of both seeds and embryos (Tables 3, 4). Contact with
moist paper during germination appeared to reduce the
chilling requirement of excised embryos in comparison with
those held on screen. This difference probably indicates
that testa removal permits germination before the after-
ripening requirement is completely satisfied. When
environmental conditions are less than optimum (e.g. water
is limiting), germination is delayed. However, the
considerably longer periods of after-ripening required by
embryos held in the fruit are not easily exPlained on the
basis of differences in water content, and may reflect the
presence of higher levels of germination inhibitors in the
embryo itself. Embryos germinated on screen required 5

weeks more of stratification to attain 50% germination than

43

did those germinated on paper. This response approximated
that of intact seeds germinated on moist paper, and is
probably a better indication of true embryo dormancy than
is germination in the presence of free water.

Soaking seeds after-ripened in the fruits for 12
weeks promoted germination appreciably (Tables 5, 8).

This partially supports Bartlett's (1) proposal that poor
germination following after-ripening in the fruit resulted
from incomplete imbibition. However, inadequate moisture
cannot fully account for the inhibitory effect of the fruit.
This is further substantiated by the observation that both
soaking in water before or after stratification and eXposure
to moist filter paper stimulated germination except in seeds
stratified and germinated in the locule (Table 1).

An alternative explanation for these effects is that
germination inhibitors are leached from the seeds during
soaking or contact with moist paper. However, the data in
Tables 3 and 5 suggest that this is not the case. Seeds
after-ripened on screen, then germinated on paper germinated
significantly better than did those after-ripened on paper,
then germinated on screen. A few days of contact with moist
paper during germination in the former case were more
effective than 12 weeks of contact during after-ripening in
the latter. This suggests that water content, rather than
inhibiting substances in the testa, are the crucial factors

limiting germination.

44

The hypothesis that chemicals in the locules exert an
inhibiting effect on germination of seeds after-ripened in
the fruit is substantiated by the fact that germination of
seeds after-ripened outside the fruit is inhibited by contact
with the endocarp (Tables 5, 6). This inhibitory effect
was not alleviated completely even when seeds were soaked
in water for 24 hours. Furthermore, the germination of
fully stratified seeds was reduced somewhat when germinated
on a piece of moist filter paper in contact with the endocarp
(Table 6). Therefore, the locules must contain a diffusible
inhibitor which is responsible for the poor germinability
of seeds after-ripened in the fruit.

Abscisic acid (ABA) does not appear to be responsible
for inhibiting seed germination in the locules, as the
concentration (2.5 pg/g fresh wt. tissue) found was much
lower than the concentration of 20 to 40 ppm ABA needed in
the stratification medium to inhibit seed germination (16).
A concentration of 100 ppm c,t-ABA resulted in only 40%
inhibition of germination of fully stratified seeds in my
study. Therefore, other compounds are probably responsible
for inhibition of seed germination by the endocarp.

Diffusates from the testas of apple seeds contained
ABA as reported in previous studies (2, 15). Luckwill (9)
found that levels of inhibiting substances in the testas of
seeds stored dry at room temperature did not change with

time, whereas levels declined in seeds kept in a moist

45

medium at 400. He suggested that moisture in the strati-
fication medium was necessary to leach out the inhibitor.
Results of this study (Table 7), on the contrary, show that
ABA content of the testa diminished whether seeds were
eXposed to free water or not. Nevertheless, the most
rapid loss of ABA from the testa occurred in seeds after-
ripened on moist paper, indicating that leaching may have
contributed to some extent to the decline. The depletion
of ABA from the testas of seeds after-ripened in the
absence of free water suggests that it was metabolized.
However, bound ABA, a known metabolite, declined concomi-
tantly with free ABA.

Rudnicki (15) correlated the decline in ABA in seeds
during low temperature stratification with an increase in
their germinability. No direct relationship between ABA
content and germination was observed in this study (Table 7),
confirming the work of Balboa-Zavala and Dennis (2).
Although the level of ABA fell as rapidly in seed held in
the fruit as in those held on screen or moist paper, germin-
ation was not correlated with ABA content. These observations
suggest that germination of seeds after-ripened in the fruit
is not inhibited by the ABA present in the seed coat.

Soaking of seeds following after-ripening in the fruit
for 12 weeks improves germination only if the testa is
intact (Table 8). Removal of the testa or a portion thereof

eliminates the response. This suggest that the testa serves

46

as a mechanical barrier to germination, rather than as a

source of germination inhibitor(s).

10.

11.

12.

Literature Cited

Bartlett, C.E.C. 1961. The after-ripening of apple
seeds in the fruit during cold storage. Annu. Rept.
%gng Ashton Agr. Hort. Res. Sta., Bristol. 1961:
- 7.

Balboa-Zavala, 0., and F. G. Dennis, Jr. 1977. Abscisic
acid and apple seed dormancy. J. Amer. Soc. Hort.

801. 102: 633-637.

Bonamy, P. A., and F. G. Dennis, Jr. 1977. Abscisic
acid levels in seeds of peach. II. Effects of
stratification temperature. J. Amer. Soc. Hort.
Sci. 102: 26-28.

Evenari, M. 1949. Germination inhibitors. Bot. Rev.

15: 153-194.

Flemion, F. 1934. Dwarf seedlings from non-after-
ripened embryos of peach, apple, and hawthorn.
Contr. Boyce Thompson Inst. 6: 205-210.

Kaminski, W. 1968. Inhibitory effect of apple juice on
the germination of apple and cherry seeds and the
growth of apple seedlings. Acta. Soc. Bot. Polon.
37: 173-178.

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

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

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-

Milborrow, B. V. 1967. The identification of (+)-
Abscisin II, (+)-Dormin, in plants and measurement
of its concentrations. Planta 76: 93-113.

, and G. Vau han, 1979. Long term
metabolism of (+)-(2-1 C) abscisic acid by apple
seeds. J. Eth. Bot. 30: 983-995.

 

Pieniazek, J., and R. Rudnicki. 1967. The presence of
abscisin II in apple leaves and apple fruit juice.
Bull. Acad. Polon. Sci. 25: 251-25 .

47

13.

14.

15.

16.

17.

18.

19.

20.

48

, and M. J. Grochowska. 1967. The role

of the natural growth inhibitor (abscisin II) in
apple seed germination and the changes in the content
of phenolic substances during stratification. Acta.
Soc. Bot. Polon. 36:579-587.

 

, and R. Rudnicki. 1970. The inhibitory

 

effect of endogenous abscisic acid (ABA) on after-
ripening of apple seeds in the fruit. Bull. Acad.
Polon. Sci. 27: 707-711.

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

, and J. Pieniazek. 1973. The effect of
abscisic acid on stratification of apple seeds.
Bull. Acad. Polon. Sci. 21: 149—154.

 

, and . 1973. Apple fruit
volatiles and inhibitors of apple seed germination.
Bull. Acad. Polon. Sci. 21: 827-829.

 

, and J. gzapski. 1974. The uptake and
degradation of 1-1 C-abscisic acid by apple seeds
during stratification. Ann. Bot. 38: 189-192.

 

Visser, T. 1959. After-ripening and germination of
apple seeds in relation to the seed coats. Proc.
Kon. Ned. Wetensch. C57: 175-185.

Wareing, P. F., and P. F. Saunders. 1971. Hormones and
dormancy. Annu. Rev. Plant Physiol. 22: 261-288.

SECTION II

FRUIT INDUCED DORMANCY IN APPLE
(MALUS DOMESTICA BORKH.) SEED.
ROLE OF VOLATILES

FRUIT INDUCED DORMANCY OF APPLE (MALUS DOMESTICA BORKH.) SEED

II. ROLE OF VOLATILES

Abstract. The role of fruit volatiles, chiefly

ethylene, in preventing apple (Malus domestica Borkh.)

 

seed germination within the fruit was investigated. After-
ripening and germinating seeds in the presence of fruits

did not delay after-ripening appreciably, whereas germin-
ation was markedly reduced when seeds were stratified in

the fruits. Removal of the seed coat markedly promoted seed
germination in the presence or absence of fruits. Germin—
ability of seeds after-ripened in fruits was equally
inhibited in regular, controlled atmosphere, and low pressure
storage at 00C, although three-to five-fold differences in
internal ethylene concentration were observed. No
correlation could be established between the rate of ethylene
biosynthesis by seeds or excised embryos and their ability
to germinate. Addition of ethephon (2-chloroethylphosphonic
acid) to the stratification medium promoted ethylene
production by seeds, but had no consistent effect on
germination. The presumed inhibitors of ethylene action or
synthesis 8-hydroxyquinoline sulfate, aminoethoxyvinyl
glycine, and silver nitrate reduced ethylene production but
did not inhibit.germination at non-toxic concentrations.

It is concluded that fruit induced dormancy of apple seeds
is not controlled by an ethylene-induced response, nor is

ethylene essential for breaking rest in apple embryos.

 

49

50

After—ripening apple (Malus domestica Borkh.) seeds

 

in fruits inhibits their subsequent germination on removal
from the fruit (1, 9). Ethylene is produced by apple fruits
during low temperature storage and constitutes 70 to 80% of
the total volatile substances emanated (7). The volatiles
produced by the fruits (3, 10) or flesh (11) inhibit the
germination of fully after-ripened seeds (10, 11) and embryos
(8). Kepczynski and Rudnicki (5, 6) concluded that ethylene
was responsible for the inhibition of germination of

seeds after-ripened within apple fruits, although exogenous
ethylene did not affect after-ripening. Paillard (8) showed
that apple fruit emanations free of volatiles other than
ethylene did not inhibit embryo germination. Kepczynski

et al. (7) observed that the production of ethylene by
excised embryos paralleled their germination capacity during
after-ripening. Inhibition of ethylene production by
8-hydroxyquinoline sulfate and aminoethoxyvinyl glycine

was accompanied by an inhibition of embryo germination.
However, Halinska, et al. (4) observed that addition of
ethephon to the stratification medium inhibited apple seed
germination up to the 70th day, after which germination

was stimulated. Similarly, Sinha, et al. (12) reported that
ethephon (250 ppm) was not effective in stimulating germin-
ation until the seeds had been stratified for 60 days.

The present study was undertaken to determine the role of
fruit volatiles, chiefly ethylene, in preventing the germin-

ation of seeds after-ripened in the fruit.

5l
Materials and Methods

Exp. 1. Effect of fruit tissue volatiles on sgggiand
embryo_germination. This experiment was designed to test
whether after-ripening is delayed when apple seeds are
stratified in the presence of fruit-produced volatiles.
Apple fruits (cv. 'Paulared') were collected from a
research orchard at East Lansing and a commercial orchard
near Grand Rapids, in 1978 and 1979, respectively. Seeds
were extracted from the fruits soon after harvest and
placed in Open petri dishes lined with moist filter paper.
The seeds were then stratified at 50 i 1°C by placing them
in loosely covered plastic containers with and without fruit
tissue. Three samples of 20 seeds each were removed at
intervals of 3 weeks for germination tests. Germination of
both intact seeds and excised embryos on moist filter paper
was recorded after 10 days at 200 i 1°C in the presence or
absence of fruit tissues.

Exp. 2. Effect of ggs mixture and pressure during 19y
temperature storage on seed and embryo germination. If
ethylene is involved in after-ripening, germination should
be affected by low pressure and/or controlled atmosphere
storage, which reduce ethylene levels in the fruits. Apple
fruits (cv. 'McIntosh') were harvested on September 19, 1978,
from a research orchard at East Lansing. Seeds were after-

ripened either in the fruits or on moist sand in petri

52

dishes at 0°C under: (a) regular storage in ambient
atmosphere, (b) controlled atmosphere in 3% 02, 1% C02,
96% N2 and (c) low pressure at 50 mm atmosphere. Samples
were removed from storage after 2 and 5 months, and both
intact seeds and excised embryos were germinated on moist
filter paper at 200 i 1°C for 10 days at ambient atmosphere.

The internal ethylene concentration of the fruits was
determined prior to storage, and after 2 and 5 months of
storage, using the method described by Burg and Burg (4).
Gas samples were drawn from the fruits immediately after
removal from the storage chambers. The gas samples were
analyzed from 20 fruits per storage treatment, using a
Varian Aerograph series 1700 gas chromatograph equipped
with a flame ionization detector and a column (45 x 0.32
cm) of 60 to 80 mesh aluminum oxide operated at 600 or 80°C.

Exp. 3. Effect of treating seeds and embryos with
ethephon and ethylene biosynthesis inhibitors on ethylene
production and germination. If ethylene is required to
break the rest of seeds, the addition to the stratification
medium of chemical compounds which alter ethylene bio-
synthesis should affect germination. Seeds were removed
in the spring of 1979 from 'Paulared' fruits which had
been stored at 5°C i 1°C for 16 weeks. Secondary
dormancy was induced by holding the seeds at 320 i 2°C for
3 weeks under moist conditions. At the end of this period
the embryos were excised and test germinated to ensure they

were in a state of rest. Seeds were then soaked for 24 hours

53

in: (a) distilled water (control), (b) 100 ppm ethephon,
(c) 400 ppm aminoethoxyvinyl glycine, (d) 200 ppm
8-hydroxyquinoline sulfate, and (e) 50 ppm silver nitrate.
The concentrations of the chemicals used were determined to
be non-toxic in a preliminary study.

The eXperiment was repeated in the fall of 1979 with
fresh seeds of the same cultivar obtained from a commercial
orchard at Grand Rapids. Germination tests were conducted
on filter paper moistened with distilled water rather than
with the chemical solutions used previously.

Ethylene production was measured by placing 30 seeds
or embryos in a 25 ml flask lined with filter paper. The
paper was moistened with distilled water or the appropriate
test solutions. The flasks were closed with rubber serum
caps and incubated in the dark at 200 1 1°C for 24 hours.
Five gas samples were drawn from each flask with a 1 cc
syringe and ethylene content was determined by gas chromato-
graphy. One cc of ambient air was put back into the flask
after each time a sample of gas was drawn. Gas samples were
also taken from a flask lined with filter paper moistened
with distilled water without seeds and used to correct for
ambient levels of ethylene. All values were converted to
n1.g-1 of seeds hr‘l. Two replications of 30 seeds each
were used per treatment.

Germination tests. Unless otherwise stated, germination

tests were carried out with 3 replicates of 20 seeds or

54

excised embryos in 9-cm petri dishes lined with two layers
of Whatman no. 1 filter paper which was moistened with
distilled water. The petri dishes were randomly placed

in a growth chamber at 200 I 1°C in darkness. Seeds with
visible radicle protrusion, and embryos whose radicles
showed geotropic curvature were considered to be germin-
ated. Germination was recorded for a period of 10 days, and
the results expressed as mean percentages.

Statistical analysis. The data from the study were

 

subjected to analysis of variance, and the mean separation
procedure of Duncan's Multiple Range Test was used where

appropriate.

Results

Exp. 1. Effect of fruit tissue volatiles on seed
and embryo germination. Intact seeds began to acquire some
ability to germinate after 6 weeks of stratification
(Table 1, Fig. 1). At this time little or no inhibitory
effect of fruit tissue volatiles on seed germination was
evident, germination in the presence and absence of fruit
tissues being 28% and 37%. respectively. The presence of
fruit tissues significantly reduced germination of seeds
stratified for 9 weeks, but no difference was evident after
12 weeks.

Embryo excision markedly stimulated germination

after 3, 6, and 9 weeks of stratification (Table 1, Fig. 1).

.55

Table 1. Effects of volatiles from apple,

CV.

'Paulared',

fruit tissues on germination of seed and excised

embryos.

Germination (5‘)z

 

Stratificationy Seed

 

 

and coat Stratification (wk)
germination 3 6 9 12
Presence of Intact 0 c 28 b 52 b 97 a
fruit tissue Excised 85 b 97 a 98 a 100 a
Absence of Intact O c 37 b 93 a 100 a
fruit tissue Excised 93 a 97 a 100 a 100 a
Fruit tissue (F):
Presence 43 m 63 m 75 n 99 m
Absence 47 m 67 m 97 m 100 m
Seed coat (C):
Intact O s 33 s 73 s 99 r
Excised 89 r 97 r 99 r 100 r
Interaction (F) x (C): * n.s. * ‘ n.s.

 

ySeeds stratified in petri dishes at 5°C, seeds

germinated at 20°C for 10 days.

and embryos

zWithin columns and sets means followed by same letter are
not significantly different at the 5% level (DMRT).

”Interaction significant at the 5% level.

Figure 1.

Effects of stratification time at 000
with or without fruits on subsequent
germination of intact apple seeds and
excised embryos, cv. 'Paulared'.

GERMINA‘I’ION ' °lo

I00

0
O

a
O

N
O

56

euflguflflu

0 WITH FRUITS

O WITHOUT FRUITS
— SEED

mm EMBRYO

 

3 6 9 l2
STRA'I'IFICA'I'ION (WK)

57

Germination was reduced 8% by the presence of fruit tissues
after 3 weeks of stratification, but not thereafter.
Significant interactions were evident between fruit tissue
effects and seed coat effects at 3 and 9 weeks, for the
former were evident only in embryos after 3 weeks of
stratification, only in intact seeds after 9 weeks.

Exp. 2. Effect of gas mixture and pressure during
low temperature storage on seed and embryo germination.
Internal ethylene concentrations differed in fruits stored
under the different conditions (Table 2). Low pressure
storage was most effective in suppressing ethylene evolution,
and controlled atmosphere intermediate.

All excised embryos germinated, regardless of treat-
ment or sampling time. Therefore only data for intact seeds
are presented (Table 3, Fig. 2). Seeds after-ripened in
the fruits germinated poorly regardless of method of storage.
Higher germination was associated with a higher internal
ethylene concentration in the fruits (Table 2). However,
there was no correlation when fruits were held for 5 months
(Table 3). The percentage germination of seeds removed from
fruits held in low pressure storage (internal ethylene concn
23 ppm) was almost twice that for seeds from fruits held in
controlled atmosphere storage (ethylene concn 54 ppm).
Germination of seeds from fruits held in regular storage
was intermediate. Regardless of method of storage, the

percent germination rose in response to an additional

58

Table 2. Internal ethylene concn (PPm) in 'McIntosh'
fruits stored at 0°C under different storage
atmospheres.

_———————-————_—-—--—-————————--——---—-—————----—-.‘————-——-—
——————————~—————-——_———.——_-——-—--—-_——-——-—-—_———-——_—————————

Ethylene concn (ppm)Z

 

 

 

cifififi‘iin Storage (mo)

0 2 5
Regular 0.10 a 121 a 72 a
Controlled 0.10 a 81 b 54 a
atmosphere
Low pressure 0.10 a 28 c 23 b

 

zMean separation within columns by DMRT at 5% level.

.59

Table 3. Effect of different low temperature (0°C) storage
treatments and method of stratification on the
germination of seeds and excised embryos after-
ripened in 'McIntosh' fruitsz.

--—_— .. ------------—-—‘——---‘------—- ---------— —----——-

Germination (%)y

 

 

 

Storage Seeds
condition stratified Storage (mo)
in: 2 5
Regular Fruit 18 c 23 be
Moist sand 90 a 100 a
Controlled Fruit 3 c 17 0
atmosphere Moist sand 87 ab 100 a
low Fruit 5 c 33 b
Pressure Moist sand 63 b 100 a
Storage condition (A):
Regular 54 f 62 f
Controlled atm. 45 fg 59 f
Low pressure 34 g 67 f
Seeds stratified in (B):
In fruit 9 s 24 8
0n moist sand 80 r 100 r
Interaction (A) x (B): * n.s.

 

yWithin columns and sets means followed by the same letter
are not significantly different at the 5% level (DMRT).

*Interaction significant at the 5% level.

zAll seeds were germinated on moist paper in petri dishes
at 20°C.

Figure 2.

Effects of method of low temperature
storage (R=regular; CA=controlled atm;
LP=low pressure) on ethylene content
(0—0) of fruits and on germination
of seeds on moist paper following

after-ripenin on sand (On-"Q or in
fruits (AM-"ts

60

1.—

 

0

20.2928 3555 89933525» 39
5 a .3 (u a

   

 

.4 .-
...‘O ...-....

I
3:... ta! 2. .055

:2: z. ezuu o

.00.
9...
e

923 z. .330

 

O2: 2. ¢( :25 z QSNU

9.2.20! a 9:202 N

 

(mum‘s no (as) Nouvmwue

61

3 months of storage although the increase was small for
regular storage.

The germination of seeds stratified in moist sand in
the same storage chambers as the fruits was much greater
than that of seeds after-ripened in the fruits regardless
of method of storage or length of stratification period
(Table 3, Fig. 2). Low pressure storage reduced
germination after 2 months in sand, but all seeds germi-
nated after 5 months regardless of storage method. Poor
germination after 2 months was probably due to drying of
the stratification medium, however, rather than to storage
method pg; g2; Germination of neither seeds held in the
fruits nor seeds stratified in sand appeared to be

correlated with ethylene content of the fruits.

Exp. 3; Effect of treating seeds and embryos with \

ethephon and inhibitors of ethylene synthesis on ethylene
production and germination. Ethylene evolution from
control seeds declined with time in the first experiment,
but that from embryos remained relatively constant for the
first 9 weeks, then rose 8-fold at 12 weeks (Table 4).
Treatment with 100 ppm ethephon greatly stimulated ethylene
evolution in both seeds and embryos. In seeds, 1000 ppm
ethephon was less promotive than was 100 ppm, possibly
because of supra-optimal concentration. All other treat-
ments reduced ethylene evolution in both seeds and embryos,

with greater percentage reduction in the former. None of

‘62

8:83 ageing:
.383.“ 8535388208"
493 am 00 E5 13 9:54.00 5233 830.30% 502%

53 0030395»... 05 pagamws 80:20 3
Lori 23:05 a mom com um 083m 36.8 035 335 50.5 0982 mcoom 5 8265 mm: 822500 Ecooomu

 

 

 

 

930.0 200.0 n~0.0 900.0 0:0.0 0010 000.0 om0.0 00H
030.0 200.0 n»0.0 200.0 030.0 oma.0 o0H.0 om0.0 om mozw<
0H0.0 2H0.0 9H0.0 nm0.0 I I I I 00: 30><

2:0.0 nm0.0 0:0.0 nm0.0 0:00 0010 o0m.0 oom.0 com
I I I I 030.0 o:m.0 m0N.0 omm.0 00H xmczn0

I I I I 2H0.NH nmm.0m n05.0a 900.0 000A
89% 80.9. 39% «No.2 smmém £9; amt? «swam 2: 5850
200.0 90H.0 90H.0 000.0 000.0 000.0 00m.H 0:0.m I swam:

80.58 vmmaoxm mcoom 00.35

088

NH 0 0 a NH 0 0 = 5:00 0:85va

 

>23 cofiumocgmuum ampum A HIE. I045 8303093 .ENO

H

. 2.3 .008 as 83285 .8 828 8:2 in
05.30 8.058 69388 0:0 momma 3an .pmhmwgmm. .uo 83638.3 0:333 co 8303.03.83
838. 05.88 2802.183 8:228 .8 8833: can 8:858 :2: 295st so 8808 .2 Base

63

these reductions was statistically significant. However,
the reduction was found to be statistically significant
when the ethephon treatment was omitted (data not shown).

None of the chemicals stimulated germination
significantly. However, 8-HQS inhibited it in seeds, but
not in excised embryos (Table 5). The higher concentration
inhibited germination after 6 and 9 weeks of stratification
but not after 12 weeks.

Only seeds were used in the second experiment.
Ethylene production in control seeds was lower than in the
first eXperiment, and the decline was rapid, rather than
being gradual (Table 6). Ethephon again promoted ethylene
evolution, although production was considerably lower than
before (1978). All other chemicals again inhibited evolu-
tion of ethylene, but differences were non-significant at
5%. The reduction in ethylene production, however, was
significantly different from control when the ethephon was
omitted in the analysis (data not shown).

In this eXperiment, ethephon inhibited germination
at one or both concentrations after 6 and/or 9 weeks, the
higher concentration being less effective then the lower.
This differed from the response in the first eXperiment,
in which ethephon did not affect germination even at 1000
ppm. Response to 8-HQS and AgNO3 also differed, with no
significant inhibition being observed with the former, but

the latter being inhibitory at both concentrations after

6L;

.mcfiosdw Hacfl>sxocumocfis<>

0000030 mafiaocasasxoauszIwz

#:0500000 00 0030.303 600055000 0x003 NH 3 0 .000 0033030 00000 50.5 002.350 ng
.H0>0H mm 050 00 0.55 a: 05—300 :23: 003000000 200:»

6030083050 0:0 0:08.500”. 302.020 00
03.5 05:05 a .80 000 00 00.0000 380 0003 003.0.“ EQC 00550: 00000 5 000305 003 0050500 5:80.00

 

 

 

 

II a OCH 0 mm nu m: 0 mm OOH
m ooH m 00H 0 mm m 0: gm ma om mozw<
a on II II II II co: >0><
0 em 0 mm 0 mm 2 mm a m oom

II m 00H 0 mm mm Nm 0 NH 00H smazIm

II a 000 a 0m 0 mm a 00 0000
0 mm m ooH :0 cm 00 m: nu ma cod cocamcsm
0 mm m 000 am mm m N: 0 m0 I 00003

x8390 00300.0 00000 00005
a ma 0 0 a ”WWW 008500.00.

 

000300830000 00000 0x00: :0 83050500

 

.0053 .3055 @0388 0:0 00000 no 003055000 :0 0030083050
003: “05.50 3005:0003 023.930 0o 0.50310: 0.8 :020050 :33 000500000 00 00000.00 .0 0300.

65

.mcHoHHw chH>zxonuoocHe<z
.0000Hsm ocHHOCHsozxopczzIOx
.HgmH um 00 ES .3 0:530 550. 838800 50:»
.OOOH :H umm>00c 00000 HHugocm 00H=p0 2000 00>o=00 0:03 000000

 

 

 

 

 

 

a OOH 0 OO 0 m a O OOH.O ONO.O OmH.O ONO.O I OO:
O OOH 0 HO 000 mH 0 m OOH.O ONO.O OMH.O ONO.O I OON 30><

m OOH 00 OH 0 m m m oOOO omO.O OOO.O 00:O.O I OOH
a OOH :0 HO 0 m 0 O OOO.O OOO.O OOO.O OOOO.O I on mOzw<

0 OO O :0 on OH 0 m oOO.O ONO.O OO0.0 ONO.O I OON
0 OO 0 HO 00 OH 0 m OOO.O ONO.O OOO.O ONO.O I OOH xmamIO

m OOH :0 OO 00 O a O O:H.~ OOH.O~ mm~.m 000.: I Oom
m OOH o NH 0 O a O mOm.mH OO0.0H nmm.w ONO.m I omm cozaonum
O OO 00 HO 00 ON 0 m OON.H ONO.O 05H.O ONO.O mz.m I 00003

NHRO :oHumcupme 5H HI0:.HI0.H:O :oHuoOOopO 2:00 H5000
NH O 0 O NH O O O O 00:00 000500000

szv :oHanH0H00000
.NOHOH .mcoom 0HOOm .OmpaHamO. Oo :oHpmcfiguuw 0:0 :oHuoOOOLO acmHzcum co :oHumOH0Humpum

003.. 05.50 2800:00ng 05H200 00 28325 0:0 028200 5:. 0005080 00 300000 .0 308.

66

6 weeks of stratification, but not thereafter. AVG at
400 ppm also inhibited germination at 6 weeks, but not
at 9 or 12 weeks.

Correlation coefficients revealed no consistent
relationship between ethylene evolution and germination
in either seeds or excised embryos. Values ranged from

-O.50 (seeds, 4 wk) to +0.32 (seeds, 12 wk).

Discussion

Apple seeds removed from fruits held at low temperature
germinated poorly in comparison with those stratified in
moist sand under the same conditions (Table 3), yet embryos
excised from the former germinated readily on moist paper.
Several methods were tested to determine if ethylene or
other volatiles were responsible for inhibiting germination.

If volatiles inhibited germination, seeds stratified
in the presence of fruits should germinate poorly in
comparison with those removed from the fruits. The data in
Table 1 indicate little, if any, effect of the presence of
the fruits on germination.

If ethylene itself limits germination of seeds held
in the fruit, treatments which stimulate or inhibit
ethylene synthesis in fruits and/or seeds should inhibit
germination. Storage methods which inhibited ethylene
synthesis of fruits did not promote germination, and

sometimes inhibited it (Tables 2, 3). Although ethephon

67

greatly stimulated the rate of evolution of ethylene in
seeds and embryos (Tables 4, 6), it had no consistent effect
on their germination. Similarly, chemicals which inhibited
ethylene evolution, and therefore should have stimulated
germination, did not hasten germination, and in fact

reduced it in some cases in partially stratified seeds
(Tables 4, 5, 6).

These facts, taken together, do not support the
hypothesis that ethylene or other volatiles are controlling
factors in fruit-induced dormancy of apple seeds.

Paillard (8) inhibited the germination of apple
embryos by circulating emanations from 30 to 35 climacteric
apple fruits held in a closed container at 20°C through the
germination chamber. Passing the air through activated
charcoal, which presumably adsorbed volatiles other than
ethylene, prevented the inhibition. Paillard (8) probably
used excessive numbers of fruits in a closed system,
resulting in much higher concentrations of volatiles than
would be found with a single fruit under conditions of low

temperature storage.

10.

11.

Literature Cited

Bartlett, C. E. C. 1961. The after-ripening of apple
seeds in the fruit during cold storage. Annu. Rept.
Long Ashton Agr. Hort. Res. Sta., Bristol. 1961: 66—67.

Burg, S. P. and E. A. Burg. 1962. Role of ethylene in
fruit ripening. Plant Physiol. 37: 179-189.

Fidler, J. C. 1955. Volatile organic products of
metabolism of fruits, J. Sci. Food Agr. 6: 293—295.

Halinska, A., B. Zarska—Maciejewska, and S. Lewak. 1975.
Inhibitors as factors causing the uniform germina-
tion of apple seeds. Fruit Sci. Rept. Skierniewice,
Poland 2:23-49.

Kepczynski, J. and R. Rudnicki. 1975. Studies on
ethylene in dormancy of seeds. I. Effects of
exogenous ethylene on the after-ripening and
germination of apple seeds. Fruit Sci. Rept.
Skierniewice, Poland 2:25-41.

and . 1976. Studies on
ethylene in dormancy of seeds. II. The effect of
endogenous ethylene on after-ripening of apple seeds
in fruit stored at low temperature. Fruit Sci. Rept.
Skierniewice, Poland 3:27-33.

 

 

, , and A. A. Khan. 1977.
Ethylene requirement for germination of partly after-
ripened apple embryos. Physiol. Plantarum 90: 293-295.

Paillard, N. 197“. Influence des produits volatils emis
par les pommes sur la germination des embryons de
pommier. Physiol. Veg. 12: 739-799.

Pieniazek, J. and R. Rudnicki. 1967. The inhibitory
effect of endogenous abscisic acid (ABA) on after-
ripening of apple seeds in the fruit. Bull. Acad.
Polon. SCi. 27: 707-711.

Rudnicki, R. and J. Pieniazek. 1973. Apple fruit
volatiles as inhibitors of apfile seed germination.
Bull. Acad. Polon. Sci. 23: 5 8-553.

, and . 1975. The effect of

 

apple flesh on dormancy release and germination
of seeds. Bull. Acad. Polon. Sci. 23: 548-553.

68

69

12. Sinha, M. M., R. S. Pal, and D. N. Aswathi. 1977.
Effect of stratification and plant growth regulating
substances on seed germination and seedling growth
in apples. Progressive Hort. 9: 27-30.

SUMMARY AND CONCLUSIONS

SUMMARY AND CONCLUSIONS

Apple (Malus domestica Borkh.) seeds removed from

 

fruits held at low temperature (5°C) germinated poorly

on moist paper or on screen unless the embryos were excised.
Limited moisture content of seeds does not explain all of
the inhibitory effect of the fruit, for the moisture

content of seeds stratified on screen was only slightly
higher than that of seeds after-ripened in the fruits, yet
the former germinated much more readily than the latter on
both screen and moist paper. Furthermore, seeds after-
ripened and germinated in locules failed to germinate regard-
less of water content. Similarly, germination of seeds
which were after-ripened outside the fruits (moist paper)
was inhibited by placing them on the surface of locules
(endocarp). These results indicate that a chemical(s)
diffusing from the endocarp prevents germination.

Water content was more critical during germination
then during after—ripening for both seeds and embryos. This
is not due to leaching out of the inhibitors, as seeds
stratified on screen, then germinated on paper, germinated
as well as those stratified on moist paper, then germinated
on screen,in spite of the former having been subjected to
only a short period (several days) of leaching. Imbibing
seeds in water prior to stratification increased germin-
ation. However, imbibition improved germination only when

the testa was left intact. Removal of the testa or a

70

71

portion thereof eliminated the promotive effect of soaking
in water. The results suggest that seed moisture content

is only one of the several factors accounting for the effect
of soaking. The testa probably serves as a physical barrier
to germination, rather than as a source of germination
inhibitor(s).

Both free and bound ABA were found in diffusates from
the seed coat and locule. The ABA content of the testa
from seeds after-ripened in the fruits declined with time,
but seed germination was still inhibited. Also, the amount
of ABA contained in the diffusates from the endocarp was
insufficient to account for the inhibition of germination.
Therefore, the inhibitory effect of the fruit cannot be
attributed entirely to ABA.

Seed germination capacity was not appreciably reduced
by stratification in the presence of fruits or fruit tissues.
No correlation could be established between ethylene
evolution and germination in either seeds or excised embryos.
Stratifying seeds in the presence of chemical compounds
which either promoted or inhibited ethylene biosynthesis did
not affect germination provided the chemicals were used at
non—toxic levels. Thus fruit-produced volatiles do not
appear to play a major role in preventing germination in the

fruit.

BIBLIOGRAPHY

10.

11.

BIBLIOGRAPHY

Abbott, D. L. 1956. Temperature and the dormancy of
apple seeds. Rept. XVI Intern. Hort. Congr.,
Netherlands. Sec. 24: 746-753.

Amen, R. D. 1968. A model of seed dormancy. Bot.
Rev. 34: 1-34.

Badizadegan, M. and R. F. Carlson. 1967. Effects of
N6benzyladenine on seed germination and seedling
growth of apple (Malus sylvestris Mill.). Proc.
Amer. Soc. Hort. Sci. 91: 1-8.

Balboa-Zavala, C. and F. G. Dennis, Jr. 1977. Abscisic
acid and apple seed dormancy. J. Amer. Soc. Hort:

Sci. 102: 633-637.

Bartlett, C. E. C. 1961. The after-ripening of apple
seeds in the fruit during cold storage. Annu.
Rept. Long Ashton Agr. Hort. Res. Sta., Bristol.
1961: 66-67.

Barton, L. V. 1956. Growth response of physiological
dwarfs of Malus arnoldiana Sarg. Contr. Boyce
Thompson Inst. 18: 308-311.

Bewley, J. D. and M. Black. 1978. Biochemistry of seeds
in relation to germination. Springer—Verlag, Berlin,
New York. 306 pp.

Black, M. 1969. Light-controlled germination of seeds.
p. 193-217 In: Dormancy and survival. Academic
Press Inc., New York.

Borkowska, B. and R. Rudnicki. 197M. Changes in the
cytokinin level in stratified apple seeds. Proc.
XIX Intern. Hort. Congr., Warsaw. Vol. 1B: p. 496.

Borner, H. 1959. The apple replant problem. I. The
excretion of phloridzin from apple root residue.
Contr. Boyce Thompson Inst. 20: 39-56.

Come, D. 1965. influence de la temperature sur les taux
et la vitesse de germination des graines de Pommier
(Pirus malus L.). Comptes Rendus Acad. Sci. Paris
260: 1725-1728.

72

12.

13.

14.

15.

16.

17.

18.

19.

20.

21.

22.

23.

73

1965. Influence de la temperature sur la
germination des graines de pommier ayant subi leur
post-maturation au sein des fruits. Bull. Soc.
France Physiol. Vegetale 11: 73-80.

. 1967. L'inhibition de germination des graines
de pommier (Pirus malus L.) non dormentes. Role
possible des phenols tegumentaires. Comptes Rendus
Acad. Agr. France 53: 1359-1362.

Crocker, W. 1916. Mechanisms of dormancy in seeds.
Amer. J. Bot. 3: 99-120.

Dennis, F. G. Jr., G. C. Martin, P. Gaskin, and J.
MacMillan. 1980. Gibberellins in mature apple
seeds: Contaminants? Planta 147: 376-377.

Dziewanowska, K., M. J. Grochowska, and S. Lewak. 1974.
Changes in phloridzin and chlorogenic acid content
and in.indolylaceticacid oxidase activity during
development of apple seeds. Fruit. Sci. Rept.
Skierniewice, Poland 1: 3-9.

Fidler, J. C., and C. J. North. 1969. Production of
volatile organic compounds by apples. J. Sci. Food
Agr. 20: 521—526.

Flemion, F. 1934. Dwarf seedlings from non-after-
ripened embryos of peach, apple and hawthorn.
Contr. Boyce Thompson Inst. 6: 205-210.

Frankland, B. 1961. Effects of gibberellic acid,
kinetin, and other substances on seed dormancy.
Nature 192: 678-679.

Halinska, A., B. Zarska-Maciejewska, and S. Lewak.
1975. Inhibitors as factors causing the uniform
germination of apple seeds. Fruit Sci. Rept.

2: 23-29.

, and S. Lewak. 1978. The presence of bound
gibberellins in apple seeds. Bull. Acad. Polon.

Harrington, G. T., and B. C. Hite. 1923. After-
ripening and germination of apple seeds. J. Agr.
Res. 23: 153-161. '

Haut, I. C. 1932. The influence of drying on after-
ripening and germination of fruit tree seeds.
Proc. Amer. Soc. Hort. Sci. 29: 371-374.

24.

25.

26.

27.

28.

29.

30.

31.

32.

33.

74

Hutchinson, A., C. D. Taper, and G. H. P. Towers,
1959. Studies of phloridzin in Malus. Can. J.
Biochem. Physiol. 37: 901-910.

Isaia, A., and C. Bulard. 1978. Relative levels of
some bound and free gibberellins in dormant and
after- -ripened embryos of Pyrus malus cv. 'Golden
Delicious' Z. Pflanzenphysiol. 90: 409- 414.

Kaminski, W. 1968. Inhibitory effect of apple juice
on the germination of apple and cherry seeds and
the growth of apple seedlings. Acta Soc. Bot.
Polon. 37: 173-177.

, and J. Pieniazek. 1968. The effect of
different growth regulators on the germination of
apple cultivar 'Antonovka' dehusked seeds. Bull.
Acad. Polon. Sci. 16: 719-723.

Kawase, M. 1958. The after-ripening of dormant apple
seeds in relation to auxin content. J. Hort.
Assoc. Japan. 27: 256-264.

Kepczynski, J., and R. Rudnicki. 1975. Studies on
ethylene in dormancy of seeds. I. Effects of
exogenous ethylene on the after-ripening and
germination of apple seeds. Fruit Sci. Rpt.
Skierniewice Poland 2: 25- 41.

, and . 1976. Studies on ethylene in
dormancy of seeds. II. The effect of endogenous
ethylene on after-ripening of apple seeds in fruit
stored at low temperature. Fruit Sci. Rept.
Skierniewice, Poland 3: 27-33.

, and A. A. Khan. 1977 Ethylene
requirement for germination of partly after-
ripened apple embryo. Physiol. Plantarum 40:
293-295-

Kopecky, F., J. Sebanek, and J. Blazkova. 1975. Time
course of the changes in the level of endogenous
growth regulators during the stratification of the
seeds of "Paneske-ceske" apple. Biol. Plant.

17: 81-87.

 

Letham, D. S., and M. W. Williams. 1969. Regulators
of cell division in plant tissues. VIII. The
cytokinins of the apple fruit. Physiol. Plantarum

22: 925-936.

34.

35.

36.

37.

38.

39.

40.

41.

42.

43.

44.

45.

75

Lewak, S., and B. Bryzek. 1974. The influence of
cytokinins on the embryo photosensitivity and acid
phosphatase activity during stratification. Biol.
Plantarum 16: 334-3 0.

, A. Rychter, and B. Zarska-Maciejewska. 1975.
Metabolic aspects of embryonal dormancy in apple
seeds. Physiol. Veg. 13: 13-22.

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.

Mayer, A. M., and A. Poljakoff-Mayber, 1963. The
germination of seeds. MacMillan Co., New York.
236 pp.

Milborrow, B. V., and G. Vaughan. 1979. The long
term metabolism of (+)-(2-14C) abscisic acid by
apple seeds. J. EXpt. Bot. 30: 983-995.

Moore, T. C. 1979. Biochemistry and physiology of
plant hormones. Springer-Verlag, New York. 274 pp.

Nikolaeva, M.G. 1969. Physiology of deep dormancy
in seeds. Nat. Sci. Foundation, Washington, D.C.

219 pp.

, and B. B. Yankelevich. 1974. The changes in
enzyme activity in apple seeds during stratification
and induction of secondary dormancy. Proc. XIX
Intern. Hort. Congress, Warsaw, Vol. 13: 502.

, and D. A. Knape. 1974. Germination and gas
exchange of apple seeds depending on stratification
and degree of maturation. Proc. XIX Intern. Hort.
Congr., Warsaw. Vol. 1B: 497.

Paillard, N. 1974. Influence des produits volatils
emis par les pommes sur la germination des embryons
de pommier. Physiol. Veg. 12: 739-749.

Pellet, H. 1973. Seed stratification. Proc. Intern.
Plant Prop. Soc. 23: 266-275.

Pieniazek, J., and R. Rudnicki. 1967. The presence of
abscisin II in apple leaves and apfile fruit juice.
Bull. Acad. Polon. Sci. 25: 251-25 .

76

46. , and M. J. Grochowska. 1967. The role of the
natural growth inhibitor (abscisin II) in apple
seed germination and the changes in the content of
phenolic substances during stratification. Acta
Soc. Bot. Polon. 36: 579-587.

47. , and R. Rudnicki. 1970. The inhibitory effect
of endogenous abscisic acid (ABA) on after-ripening
of apple seeds in the fruit. Bull. Acad. Polon.
Sci. 27: 707-711.

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

49. , and J. Czaps i. 1974. The uptake and
degradation of 1-1 C-abscisic acid by apple seeds
during stratification. Ann. Bot. 38: 189-192.

50. , W. Kaminska, and J. Pieniazek. 1971. The
interaction of abscisic acid with growth stimula-
tors in germination of partially after-ripened
apple embryos. Biol. Plantarum. 13: 122-127.

51. , and J. Pieniazek. 1973. Apple fruit
volatiles as inhibitors of apple seed germination.
Bull. Acad. Polon. Sci. 21: 827-829.

52. , and . 1973. The effect of abscisic
acid on stratification of apple seeds. Bull.
Acad. Polon. Sci. 21: 149-15 .

53. , and . 1975. The effect of apple flesh
on dormancy release and germination of seeds. Bull.
Acad. Polon. Sci. 23: 547-553.

54. Rychter, A., and S. Lewak. 1971. Apple embryos
peroxidases. Phytochemistry 10: 2609-2613.

55. , R. Rudnicki, and S. Lewak. 1971. Regulation
of acid phosphatase activity by abscisic acid and
gibberellin in apple seeds during stratification.
Bull. Acad. Polon. Sci. 19: 211-214.

56. Sinha, M. M., R. S. Pal, and D. N. Awasthi. 1977.
Effects of stratification and plant growth
regulating substances on seed germination and
seedling growth in apples. Progressive Hort.
9: 27-30.

57. Sinska, I., and S. Lewak. 1970. Apple seeds
gibberellins. Physiol. Veg. 8: 661-667.

58.

59.

60.

61.

62.

63.

64.

65.

66.

67.

68.

69.

70.

71.

77

, and . 1974. Gibberellins biosynthesis
in apple seed dormancy. Proc. Intern. Hort. Congr.
XIX, Warsaw. Vol. 1B: p. 493.

_, P. G. Gaskin, and J. MacMillan. 1973.
Re- -investigation of apple seed gibberellins. Planta

11“ 359 34

Smolenska, G. S. and S. Lewak. 1971. Gibberellins
and photo- sensitivity of isolated embr os from non-
stratified apple seeds. Planta 99: 14-153.

Sondheimer, E., E. C. Galson, E. Tinelli, aEd D. C.
Walton. 1974. The metabolism of (8)-2-1
abscisic acid in ash seeds. Plant Physiol.
54: 803-808.

Taylorson, R. B., and S. B. Hendricks. 1977. Dormancy
in seeds. Annu. Rev. Plant Physiol. 28: 331-354.

Teubner, F. G. 1953. Identification of the auxin
present in apple endosperm. Science 118: 418.

Tissaoui, T., and D. Come. 1973. Levée de la dormance
de l'embryons de pommier (Pirus malus L.) en
l'absence d'oxygene et de froid. Planta 11: 315-322.

 

Visser, T. 1954. After-ripening and germination of
apple seeds in relation to the seed coats. Proc.
Kon. Ned. Wetensch C57: 175-185.

. 1956. The role of seed coat and temperature
in after-ripening, germination and respiration of
apple seed. Proc. Kon. Ned. Wetensch. 059:211-222.

. 1956. Some observations on respiration and
secondary dormancy in apple seeds. Proc. Kon. Ned.
Wetensch. C59: 314- 324.

Wareing, P. F., and P. F. Saunders. 1971. Hormones
and dormancy. Annu. Rev. Plant Physiol. 22:261-288.

Westwood, M. N., and H. 0. Bjornstad. 1969. Effect of
gibberellin A3 on fruit shape and subsequent seed
dormancy of apple. HortScience 3: 19-20.

Woodcock, D. 1947. Isolation of phloridzin from apple
seeds. Nature 159: 100.

Zwar, J. A., W. Bottomley, and N. P. Kefford. 1963.
Kinin activity from plant extracts. Aust. J. Biol.
Sci. 16: 407 15

nICHIan STATE UNIV. LIBRARIES
m(I("IllHHIIHWIWllWWIIWIWIIIUWI
31293107669479