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THESIS

11111 111:1l 11111

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

                   

 

 

 

     

 

This is to certify that the

thesis entitled
PHYSIOLOGICAL STUDIES OF DORMANCY
AND GERMINATION OF
APPLE (MaZus Sylvestris MILL.) SEED

presented by

Morteza Badizadegan
has been accepted towards fulfillment

of the requirements for

Ph.D. (kgmeinHorticulture

4;;EZ£*H%§Z’(€Ix/<CWLM

Major professor

 

Date w

0-169

 

 

 

 

 

 

 

 

 

ABSTRACT

PHYSIOLOGICAL STUDIES OF DORMANCY
AND GERMINATION OF
APPLE (MaZus Sylvestris MILL.) SEED

by Morteza Badizadegan

The purpose of the research reported in this thesis
was to determine the factors and mechanisms which control
dormancy in apple seeds. In addition, attempts were made to
develop a technique for producing normal seedlings in a short
time from dormant seeds which normally require 70 to 80 days
of stratification prior to germination.

Gibberellic acid (GA), N6benzyladenine (BA) and
thiourea at different concentrations were tested on both in—
tact seeds and excised embryos. None of these chemicals was
effective in stimulating germination of the intact seeds.
However, it was found that EA at concentrations of 10 to 20
ppm significantly increased germination of the embryos.

To study the chemical and physical effects of the
exembryo (combination of all tissue layers covering the em-
bryo) in controlling dormancy, different parts of the exem—
bryo layers were ruptured or removed. The seeds were treated

with BA and GA, singly and in combination, and the response

of the seeds to these chemicals was measured. It was found

 

 

 

Morteza Badizadegan

that a combination of GA + BA was much more active in stimu-
lating germination of the seeds with partially ruptured
exembryos than either one alone. From these and other ex-
periments it was hypothesized that the dormancy of the apple
seeds was mainly due to (1) presence of one or more growth
inhibiting substances in the embryos and particularly in the
exembryos, (2) restriction of aerobic respiration of the em-
bryos caused by the endosperm layer and the inner integument.

These two factors, chemical and physical, seemed to be inter—

 

related. The combination of the GA + BA appeared to reverse
or inactivate the effect of the inhibitors contained in par—
tially ruptured seeds so that the embryos germinated.

Other experiments showed that apple seedlings could
be produced in a relatively short time by rupturing the exem-
bryo by hand, treating the seeds with BA or GA + BA, and then
planting them in the soil. It was also shown that when pro-
ducing apple seedlings on a large scale, the slow process of
rupturing the exembryos by hand could be substituted by
soaking the seeds for 2 1/2 to 3 1/2 hours in concentrated
sulfuric acid prior to the chemical treatment. All such
seedlings produced in the laboratory or in the greenhouse
were dwarfed with very short internodes and thick dark green
leaves.

The dwarf seedlings were stimulated to grow at a
nearly normal rate with applications of GA. Repeated appli—

cations of GA were necessary to prevent reversion of the

elongated seedlings to the dwarf form.

 

 

 

 

PHYSIOLOGICAL STUDIES OF DORMANCY
AND GERMINATION OF
APPLE (Malus Sylvestris MILL.) SEED

By

Morteza Badizadegan

A THESIS

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

DOCTOR OF PHILOSOPHY

Department of Horticulture

1967

 

 

 

\7’l/J‘
. // . J ‘T "

 

   

 

DEDICATED
to
my beloved country IRAN,
which supported my graduate

studies in

the United States

 

 

 

 

 

DEDICATED

to
my beloved country IRAN,
which supported my graduate

studies in

the United States

 

 

 

 

ACKNOWLEDGMENTS

The author wishes to express his sincere grati-
tude and appreciation to Dr. R. F. Carlson for his encourage-
ment given throughout the graduate program, for his guidance
in conducting this research, for his financial assistance,
and especially for his helpful suggestions during preparation
of the manuscript.

Sincere thanks are also extended to the other mem-
bers of the guidance committee, Dr. H. Davidson, Dr. H. D.
Foth, Dr. A. L. Kenworthy, and Dr. C. J. Pollard for their

valuable guidance and suggestions during the entire period

of the graduate program.

 

 

TABLE OF CONTENTS

ACKNOWLEDGMENTS
LIST OF TABLES

LIST OF FIGURES

INTRODUCTION
LITERATURE REVIEW

General

Changes Taking Place in the Seeds
During After— Ripening

Structure of Apple Seed .

Apple Seed Dormancy and Germination

Growth Promoting Substances and Seeds

Growth Inhibiting Substances and Seeds

Relation between Growth Promoting and
Growth Inhibiting Substances and Seeds

Production of Seedlings from Embryos
of Dormant Seeds .

Nature of the Seedlings Grown from
Embryos of Dormant Seeds .

MATERIALS AND METHODS

1. Growth Abnormalities
2. Effect of N6Benzy1adenine° on Germination
of Embryos of Dormant Seeds
A Experiments with Fresh Seeds
Results and Discussion
1. Laboratory Experiments
2. Greenhouse Experiments
B. Experiments with Dry Seeds
Results and Discussion .
3. Chemical Effects of the Exembryo in.
Seed Dormancy
A. Effect of BA and GA with Part
of the Exembryos Removed .
Results and Discussion

 

Page

Vi

viii

A

#OVO‘LD

 

 

 

 

B. Effect of BA and a Combination of BA
and GA on Seeds with Ruptured
Exembryos . .

4. Physical Effects of the Exembryo
in Seed Dormancy

A. Effect of a Combination of GA and
BA on Seeds with the Outer
Integument Totally Removed .

Results and Discussion . .

B Effect of Vacuum Treatment with a
Combination of GA + BA on Seeds
with Outer Integument Totally
Removed . . . . . .

Results
Results and Discussion .
5. Chemical Treatments for Ruptur1ng. the
Exembryos
Results and Discussion
6. Chemical Treatments for Removing the

Physiological Dwarfness of the Seedlings.

A. Growing of the Seedlings
B. Gibberellic Acid Treatment
Results and Discussion . . .

DISCUSSION

LITERATURE CITED

 

58
61

61
63

Table

 

LIST OF TABLES

The effect of different concentrations
of N benzyladenine on stimulation of
germination of embryos excised from
McIntosh and Wealthy apple seeds

Per cent germination of N6benzyladenine
treated and control excised embryos
of different maturity seeds in rela—
tion to the number of days following
treatment . . . . . . . . . .

The effect of N6benzyladenine treatments
on germination of excised embryos of
McIntosh and Wealthy seeds and on
fresh and dry weights of the seedlings
produced .

The effect of N6benzyladenine treatments
on germination of the excised embryos
10 days after planting in soil .

Per cent stand at 10, 20, and 30 days
after planting of seedlings grown from
excised embryos of McIntosh and
Wealthy seeds treated with Nébenzyla-
denine solutions and planted in soil

The effect of pre-soaking at 5 daily
intervals in water on the germination
of the embryos excised from dry McIn—
tosh seeds treated with N benzyladenine
and distilled water . . . . .

The effect of N6benzyladenine, gibberel-
lic acid, alone and a combination of
the two on germination of McIntosh
seeds in which part of the exembryo
was removed from the micropilar end.

vi

Page

34

36

39

40

43

50

53

 

 

 

8. The effect of different gombinations of

gibberellic acid and N benzyladenine
on germination of McIntosh seeds in
which part of the exembryo was re-
moved from the micropilar end

9. Total growth of the green cotyledons
out of the exembryo of 10 McIntosh
seeds ruptured at the chalazal end
and treated with distilled water,
N6benzgladenine and gibberellic° acid
plus N benzyladenine . .

10. Germination of McIntosh seeds with the
outer integuments removed, the re-
maining of the exembryo punctured at
chalazal end and then vacuumed in
gibberellic acid plus N6benzyladenine
or distilled water° These seeds were
placed under germination conditions
for 20 days, then the remaining of
the exembryos were ruptured at Cha—
lazal end and the seeds grown for 10
days . . . . .

ll. The effect of the acid treatment on
germination of McIntosh seed 10 days
after treating with a combination of
gibberellic acid on shoot growth of
the apple seedlings measured 40 ndays-
after first spraying . .

12. The effect of the number of applica—
tions and concentration of gibberel-
lic acid on shoot growth of the apple
seedlings measured 40 days after
first spraying . . . . . .

 

55

60

66

7O

74

 

 

 

Figure

1.

 

 

LIST OF FIGURES

Abnormalities in growth of apple seed-
lings . . . . . . . . . . . . . . .

Correlation of per cent germination and
N benzyladenine concentrations in em-
bryos excised from immature and mature
McIntosh and mature Wealthy seeds

Per cent germination of apple embryos ex—
cised from mature McIntosh seeds fol—
lowing treatments with N6benzyladenine

The effect of N6benzy1adenine on germi-
nation of embryos excised from mature
McIntosh apple seeds

The effect of different N6benzyladenine
concentrations on germination of apple
embryos excised from mature Wealthy and
mature and immature McIntosh seeds

McIntosh seedlings grown from embryos ex—
cised from mature seeds treated with
different concentrations of BA and
planted in soil

Per cent seedling stand of mature McIntosh
and Wealthy seeds which were excised
then treatgd with different concentra—
tions of N benzyladenine and then
planted in soil .

McIntosh seedlings grown from excised em—
bgyos treated with distilled water or
N benzyladenine . . . . . . . .

The effect of pre-soaking dry McIntosh
seeds in water for 1 and 4 days, then
treating the excised embryos with dis—
tilled water or Nébenzyladenine on ger-
mination of the embryos . . . . . .

viii

Page

35

37

41

42

46

49

 

 

Figure

10.

ll.

12.

14.

 

 

Germination of McIntosh seeds with part
of exembryos removed from micropilar
end following chemical treatments

The effect of distilled water, N6benzy1a-
denine, gibberellic acid and gibberelic
acid plus N6benzy1adenine on germina—
tion and growth of McIntosh Seeds with
part of exembryo removed from micro—
pilar end . . . .

The effect of water and gibberellic acid
plus N benzyladenine on germination of
McIntosh seed with different parts of
the exembryos ruptured or removed

The growth of McIntosh seedlings treated
with distilled water or gibberellic
acid . . . . . . . . . . .

The effect of application of different
concentrations of gibberellic acid on
shoot growth of physiologically dwarfed
McIntosh apple seedlings .

ix

Page

54

56

57

75

77

 

INTRODUCTION

Apple cultivars (MaZus sylvestris Mill.) are
grown commercially in about 35 states of the United States
and many other countries.

In 1964 F. W. Burrow (4), the executive vice-
president of the International Apple Association stated
that the United States apple industry contributes about
250 million dollars to the economy at the grower level
and nearly one billion dollars at the retail level.

A leader of the pome fruits, the apple plays a
very important part in the diet, health, and economy of
this and many other countries. It is grown and used more
than any other single fruit species.

This well known fruit of many cultivars is grown
on trees produced by grafting a selected scion onto suit—
able rootstocks. The reason for this is that apple seed—
lings are exceedingly variable and not true to variety.
This disadvantage, however, does not rule out the neces—
sity of the production of seedlings. Apple seedlings play
an important role in the fruit industry for the following
reasons:

1. Although the trend in the United States is toward

planting of dwarf or compact apple trees, a large

 

 

 

 

 

 

number of apple trees are still produced on apple

seedling rootstocks.

2. Apple seedlings are used when trees are dwarfed
by intermediate stem systems.

3. In breeding new apple varieties, seedlings are
produced from seed.

4. Apple seedlings are also used in biochemical or
physiological studies in plant science labora-

tories.

Accordingly, the production of apple seedlings is
and will continue to be an important phase of apple tree
propagation and research.

It is a known fact that when freshly harvested,
or dry stored, viable mature seeds of apple are placed
under favorable germination conditions they do not germi-
nate. The apple seed has a long dormant period often re—
ferred to as the rest period.

Dormancy in general is defined as ”a state of
reduced physiological activity.” This dormancy condition
may be due to different factors or a combination of two or

more factors, such as:

l. dormancy of the embryo.
2. impermeability of seed coats to water or gases.
3. mechanical resistance of the seed coats.

4. germination inhibitors.

 

 

 

Whatever the cause or causes, apple seeds are
dormant and must be held moist at a low temperature (about
41° F or 5° C) for 70 to 80 days before complete germina-
tion is possible. After this conditioning period, growth
of the seedling is normal.

The changes taking place in the seed during this
80 day period which enable it to germinate promptly under
normal growing conditions are collectively known as ”after-
ripening.” After—ripening can occur only with time, under
suitable conditions and can not be caused by any other
known means. Considerable time and equipment are necessary
for furnishing optimum conditions for after—ripening of the
seeds.

For the nurseryman or for the scientist this after—
ripening requirement is a problem, in that it is time con-
suming, costly and often bothersome. The nurseryman has to
plan ahead in order to produce his apple seedlings.

This research was undertaken to determine the fac-
tors and mechanisms which cause dormancy in apple seeds.

In addition, an attempt was made to develop a technique for
producing normal seedlings in a short time from dormant

seeds which normally require 70 to 80 days of stratification

prior to germination.

 

 

 

 

 

 

 

LITERATURE REVIEW
General

Seed propagation is one of the major means of re-
producing plants. Due to this fact, man for many years has
been interested in the subject of seed physiology. Eckerson
(25), studying the physiological and chemical changes occur-
ring in the seeds during after—ripening in 1913, published
one of the first major papers of this century on seed physi-
ology. Different areas of this field including dormancy,
after-ripening, germination, etc., have been extensively
studied. Results of various investigations have been re—
viewed (3, 55, 105, 118) and several books (7, 21, 28, 69,
111, 112, 113) have been written on the subject of seed phy—
siology.

Germination of the seed of the higher plants has
been defined as “consecutive number of steps which cause a
quiescent seed, with a low water content, to show a rise in
its general metabolic activity and to initiate the formation
of a seedling from the embryo.” The exact stage at which

germination ends and growth begins is extremely difficult

to define (69).

 

 

 

In many species, although the embryos are com-
pletely developed, the seeds are dormant and do not germi-
nate when placed under favorable germination conditions
(70). A period of stratification under moist and cold con—
ditions is necessary in order to make it possible for these
seeds to germinate.

In general, no visible anatomical or morphologi—
cal changes occur in the embryo during after-ripening. In
these dormant seeds it must be assumed that the process of
after-ripening is the result of chemical or physical changes
which occur within the seed or seed coat (69). The embryos
of such seeds will not grow when the seeds first ripen,
even if the seed coats are removed. Among the many species
whose seeds exhibit dormancy of this type are: (MaZus)

apple, (Prunus) peach, (Pinus) pine, etc. (70).

Changes Taking Place in the Seeds
DuringgAfter—Ripening

 

 

Studies measuring changes taking place in seeds
being stratified have shown increased water—absorbing power,
increased enzyme activity, increased acidity and the trans-
formation of insoluble, osmotically inactive substances to
soluble, osmotically active ones. That is, a change from
fats to subars and insoluble proteins to soluble proteins,

amino acids and other nitrogenous compounds (19, 25). How-

ever, it has been po1nted out by Haut (44) that such changes

 

 

 

also occur during germination and may not represent after-
ripening changes but the increased ability of the seeds to
germinate with stratification. In some species studied,

after-ripening involved morphological growth of the embryo.

One such example is the ginko seed (41).

Structure of Apple Seed

 

Luckwill (65) and Harrington (42) have shown that
the mature apple seed is made up of the following parts:
1. The embryo, with two large fleshy cotyledons con-
taining the bulk of the stored food reserves;

2. The endosperm layer, a delicate whitish membranous

 

layer of endosperm tissue to which a very thin
nucellus layer adheres closely;

3. The inner integument, a thin, translucent brownish

 

layer of a very dense structure without any opening;

4. The outer integument, a thick, brown and fibrous

 

layer with the open micropile.

The inner and outer integuments collectively are
called the testa. In this thesis the term ”exembryo” is
used to describe the combination of all the tissues outside
of the embryo of the seed. This includes the endosperm

layer (plus the nucellus layer attached to it), the inner,

and the outer integuments.

 

 

 

 

 

 

 

 

Apple Seed Dormancy and Germination

 

Since the apple seed is dormant, a period of after—
ripening under moist and cold conditions for 70 to 80 days
is essential to start the dormant embryo into growth (1, 5,
43, 65, 105). The physiology of the apple seed has been
under investigation for many years. Harrington (43) in 1923
studied the after—ripening and germination of the apple seeds
and indicated that the dormancy is located in the embryo.
He recorded that the naked embryos failed to germinate nor—
mally in his studies. However, removal of the testa caused
some of the dormant embryos to produce weak growth. He also
found that the respiration rate of the dormant apple seed
was low. The respiration rate of the seeds capable of ger-
mination was higher and became increasingly so with advancing
germination. Removal of the seed coats increased respiratory
intensity and accelerated germination (42). Crocker (18) in
1928 reported that apple seeds germinate almost equally well
after 1/2, 1 1/2 and 2 1/2 years of dry storage provided they
are properly after-ripened. Contrary to the common belief,
he showed that a perlod of dry storage does not lengthen the
period of low temperature requirement.

A comprehensive study of several rosaceous seeds,
including apple and peach, was made by Crocker and Barton in
1931 (20). They indicated that apple seeds after—ripen in

the apple during cold storage and such seeds retain their

after-ripened conditions even after one month of dry storage.

 

 

 

Apple seeds lose their vitality slowly and 2 l/Z-year old
seeds are capable of producing a perfect stand.

In weeping crabapple, Nickell (81) found that
the seed coats played a most important role in the delay of
germination of after-ripened seed. If the seed coats were
removed and conditions were favorable, germination took
place immediately. The most extensive study on apple seed
dormancy and after-ripening was made by Luckwill in 1952
(65). Looking for an inhibitor, he made extracts from dif-
ferent parts of the seed and tested their inhibitory effect
on the growth of isolated sections of oat coleoptiles. He
found a rough ratio of 30 13:1 concentration of the inhibi-
tor in the endosperm layer, the testa and the embryo, re—
spectively. Luckwill suggested that the total dormancy of
the embryo within the seed is partly due to the presence of
the endosperm and the testa. The inhibitory effect of the
endosperm and the seed coat is chemical. This was illus-
trated by the fact that the excised embryos grew without
difficulty, but when the testa and the endosperm layer were
ruptured or partially removed, still no germination occur—
red. He further found that after 85 days of seed storage,
the inhibitor disappeared from both seeds in dry conditions
and seeds stratified at 4° C. The after-ripened seeds ger—
minated 100 percent. However, the dry stored seeds with no

inhibitors were still dormant and the exc1sed embryos from

such seeds did not germinate (65).

 

 

Luckwill concluded that the main difference between
after-ripened and non after-ripened embryos was not the dis—
appearance of the inhibitor, but the appearance of growth—
promoting substances in the after-ripened seeds just prior
to germination. The experiments with excised embryos indi-
cated that the inhibitor could not be the only or even the
dominant factor involved. He concluded that the factors
controlling the dormancy of apple seeds, to a large extent,
reside in the embryo itself, and stressed that the moist
condition which is one of the requirements for the success—
ful after—ripening of apple seeds, is probably necessary to
remove growth—inhibitors from the testa.

Visser (120, 122) studied the role of seed coats
in after—ripening and germination of apple seeds. He noted
that both the endosperm layer and the testa hindered the
germination of insufficiently after-ripened seeds. He indi'
cated that the hinderance caused by the integuments was due
mainly to mechanical resistance, whereas, the part played
by the endOSperm in both after—ripening and germination,
may be a physical obstruction to respiration. He also sug-
gested that the inhibitors, if present in the seed coats,
play no part in germination and after—ripening, but if
present in the embryo, their role is probably secondary.

In a different paper, Visser (121) reported that

seed from the more mature apples at harvest had a shorter

after—ripening period. Somewhat conflicting with this

 

 

 

10

statement, Zagaja (126), in 1962, reported that the after-
ripening requirements of immature apple embryos of varieties
studied are similar to those of the more mature embryos.

Two acidic substances capable of inhibiting the
germination of rape seeds were isolated from dormant apple
seeds by Evans (26). Evans also stated that extracts of
non-dormant apple seeds were also able to inhibit germina—
tion of rape seeds. Two Russian investigators found that
water extracts from unstratified apple seeds inhibited the
germination of stratified seeds (84). They isolated a num-
ber of compounds from apple seeds including tryptophane, a
potent inhibitor of germination. They noted that when the
seed coats became more permeable, small quantities of tryp—
tophane were dischanged from the seeds.

Apple seeds were after-ripened in the apple during
cold storage (16, 20, 43). When cold storage conditions
were prolonged beyond the optimum period, the seeds decreased

in germination capability (16).

Growth Promoting Substances and Seeds

 

Breaking dormancy of seeds frequently is of eco-
nomic importance (69). In addition, this also provides a
tool for studying the physiology of seed dormancy. Many

attempts have been made to break dormancy of seeds by other

means than the slow and cumbersome method of cold treatment.

 

 

 

11

Prior to the use of plant growth regulators in
breaking seed dormancy two types of germination promotors,
namely, nitrogenous (nitrates and nitrites) and sulfur
(cysteine,glutathione and thiourea) compounds were known
(105).

Nitrates have long been known as powerful agents
in germination, and both nitrates and nitrites have been
found to promote germination of seeds of four varieties of
tobacco (55, 105).

Thiourea is known to be particularly effective in
promoting germination of some light-requiring or temperature
inhibited seeds, such as lettuce (69, 103, 105). Further—
more, other sulfur containing compounds also stimulate ger-
mination of lettuce seeds (104). Tukey and Carlson (109)
were able to break dormancy of peach seeds with thiourea.
Subsequent growth of the seedlings, however, was inhibited.
Thiourea has been found to be in the seeds of at least one
species, Lahurnum anagyroides (69). Although less effective
than thiourea, ethylenechlorohydrin has been shown to have
similar effects on germination (69).

Many other substances, including indolyl acetic
acid, water and ether extracts of yeast, peat extracts and
the cis- and trans-forms of 2:5 tetrahydrothiophene dichloric
acid were tested over a wide range of concentrations in an

attempt to induce germination of dormant apple seed. However,

results were negative in all cases (65).

 

 

 

 

12

Yabuta and Sumiki (125), in 1938 reported on the
isolation and characterization of gibberellin from the fun-
gus GibbereZZa fujikuroi (Sawada) Wollenw. The best known
of the gibberellins, and commercially produced by fermenta-
tion from fungal cultures, is gibberellic acid (GA3) (22).
Several other gibberellins from GA1 through GA13 and their
specificity are known (69). Gibberellin-like substances
have been isolated from higher plants and they can be con-
sidered as normal constituents of green plants (66, 71, 87,
91, 114, 118, 124). Lona (1956) (64), and Khan (1957) (50),
were among the first investigators to show that gibberellic
acid could replace red light in lettuce seed germination of
a number of light-requiring and light-indifferent seeds (51,
55, 69, 82). Donoho and Walker (24) in 1957 were able to
break dormacy of peach seeds which already had 35 days of
stratification and which normally require 60 to 100 days
stratification. Their data shows 80 per cent germination
in seeds treated With 10 ppm GIBREL (Potassium salt of GA3)
in agar medium broke the dormancy of peach seeds. Gibberel-
lic acid at 100 ppm had no effect in stimulating germination
of intact apple seed or seed in which the endosperm layer
was left intact and the testa removed. Some stimulation was
obtained when isolated embryos were treated with the GA (34).

Miller et a7. (76, 77, 78) reported on the isola—
tion of an adenine derivative, called kinetin, from deoxyri—

bonucleic acid. This substance was characterized as

6-furfury1amino purine (76). Other adenine derivations

 

 

 

13

similar in structure to kinetin were found. These compounds
were originally named kinins by Skoog (77, 78) but later
they were renamed cytokinins (99).

Cytokinin activity has been found in green plants
and in their seeds (54, 74,75, 114, 117). Miller (74)
isolated substances from Zea mays kernels which showed
kinetin-like properties. Letham and his associates (61) in
1964 identified the structure of a cytokinin, named zeatin,
which naturally occurs in young maize seed (75). Zeatin,
like kinetin is an adenine derivative.

Kinetin and its analogues have been shown to stimu-
late germination of lettuce (40, 60, 71, 73, 93, 94, 97) car—
pet grass and clove seeds (95). Among the 6-(substituted)
purines which were tested, 6-benzy1amino purine was especially
active in stimulating germination of seeds in a number of
species (60, 94, 95, 97). Van Overbeek (114) in 1966 indi-
cated that perhaps the best known synthetic cytokinin is
N6benzy1adenine.

The effect of auxins on germination has long been
in dispute. In general it must be concluded that IAA (in—
dole acetic acid) can, under very special conditions, stimu—
late germination, but normally it has little or no effect
(69). Another thing is that synergism has been noticed
between different factors affecting germination as, for

example, the combination of thiourea and light and/or tempera-

ture has been shown to have more effect on germination than

 

 

 

%

14

when thiourea is used alone (69). The following combina-

tions also act synergistically: GA and KNO or light (55);

3
light, KNOS, GA and kinetin or 6-substituted purines (ll,

72, 32, 95, 96, 97).

Growth Inhibiting Substances and Seeds

 

Occurrence of germination inhibitors in seeds and
their possible role in dormancy and germination has been
studied and reported by several workers (8, 10, 26, 27, 65,
79, 84, 85, 120). Evenari (27) listed about 100 species of
plants from which germination inhibitors have been isolated.
In more than 60 of these species the inhibitor was found in
seeds or fruits, and in 22 species the seeds were specifically
mentioned to contain inhibitors.

In recent years several growth regulating substan-
ces have been isolated from different plant tissues and their
role in plant growth control determined. An inhibitor named
naringenin, which apparently controls dormancy, was isolated
from dormant flower buds in 1959 (45, 46). Furthermore, Liu
and Corms (63) in 1961 isolated an abscission accelerating
substance from cotton bur and they named it ”abscisin.” A
few years later, Ohkuma at al. isolated from young cottom
fruit a different hormone, which they called "abscisin II.”
In 1964 Wareing (cited by 114) reported on isolating inhibi-

tors from leaves of woody plants which were kept under short—

day conditions. These inhibitors which were named ”dormins”

 

 

 

15

caused a vegetative bud to change into bud scales. One in-
hibitor in particular was of high potency and appeared to

be identical to abscisin II. Cornforth et a7. (17) isolated,
in crystalline form, a dormin from sycamore and established
its identity with abscisin II. The latest development came
from Lipe and Crane (62) in 1966, when they extracted an in-
hibitor from peach seeds which they indicated inhibited ger-
mination in this species, and that this substance was similar
to dormins.

Relation between Growth Promotingfiand

 

Growth Inhibiting Substances in Seeds

 

Thus far it has been shown that both growth pro-
moters and growth inhibitors occur naturally in seeds.
Attempts have been made to study interactions of these sub-
stances in terms of dormancy and germination, although to
date, no concrete data has been published on such a rela—
tionship.

In 1949, Hemberg (47), for the first time, hy-
pothesized that endogenous growth inhibitors may regulate
dormancy in potatoes. Many other investigators have tested
the validity of this hypothesis and have come up with some
promising results, some of which are discussed in the fol-
lowing paragraphs.

The balance between endogenous inhibitors and pro-

moters in lettuce seed were changed by application of cou—

marin or thiourea and these changes were correlated with

 

 

16

the germination response of the seed (l3). Khan and Tolbert
(54) were able to inhibit germination of lettuce seed by
coumarin and xanthatin. This inhibition was reversed by a
combination of red light plus kinetin. The onset of dor—
mancy in seeds of Avena fatua was prevented by soaking the
end of oat stems when in the seed milk stage in gibberellic
acid (11). Seeds of this species have been shown to contain
gibberellin—like substances. Exogenous applications of GA
increased GA content and prevented onset of dormancy. There
was a close correlation between the disappearance of some
germination inhibitory substance and the ability of peach
seeds to germinate during the stratification period (62).
The action of this germination inhibitor was antagonistic

to the GA effect in wheat coleoptile sections.

Dormancy in Avena fatua involves a reduction in
accumulation of sugar and sugar utilization by endogenous
inhibitors. The action of these inhibitors in oat can be
reversed by GA,and the loss of dormancy during after-ripening
is correlated with gradual disappearance of the inhibitor.
Evidence has been found for the presence of a natural gib—
berellin in seeds of the oat species (80). According to
this report, the gibberellin was absent in freshly matured
dormant embryos but increased in amount with the length of
the after-ripening period (92).

In recent years there has been an increasing num—

ber of observations which lead to the suppOSition that natu-

rally-occurring growth inhibitors may play an important role

 

  

 

17

in the origin and the control of dormancy in seeds (10, 19,
27, 65, 79, 119, etc.). Most of the seed inhibitors seem to
be located in the structures covering the embryo of these
seeds (55, 118). Furthermore, it is assumed that dormancy
is due to an inhibitor and that dormancy-breaking is the
result of the counteraction of the inhibitor by some stimu—
lator (69).

There is considerable evidence that the gibberel-
lin is the promoter part of the inhibitor—promoter complex.
These are: (1) the isolation of GA—like compounds from
runner bean (66), lettuce (71), and many other seeds;

(2) the change in the amount of GA-like compounds during
after—ripening and its correlation with the ability of the
seed to germinate (35, 69); and (3) the germination stimu-
latory effect of the GA on dormant seeds. The gibberellin
may play an importnat part in the stratification and germi-
nation of dormant seeds (11, 30, 48, 69, 102, 115, 118, 119).

Investigations have been carried out to determine
the mode of action of factors involved in seed dormancy.
Van Overbeek (114) indicated that in lettuce seed, light
causes formation or activation of hydrolytic enzymes. These
weaken seed coats, then the radicle breaks through and seed
germinates. It has been pointed out that the inhibitors

and the promoters in the seeds antagonize the action of

each other (11, 62, 80, 102).

 

       

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18

Simpson (92) indicated that gibberellin possibly
promotes the synthesis of, or activates preformed enzymes
necessary for the utilization of the endosperm as the sub-
strate for germination and growth. Harber and Luippoid
(38, 39, 40) attempted to find out whether GA and kinetin
treatments induce initiation of cell division, cell elonga—
tion, or both. They suggested that cellular expansion is
both necessary and sufficient, whereas cell division is
neither necessary nor sufficient for initiation of lettuce
seed germination. The stimulating effects of GA and kinetin
on germination produced a direct effect on cellular expan-
sion, whereas the apparent effects on cell division were
the results rather than the cause of such stimulation.

Recently it has been postulated that in seeds of
cereals, GA is secreted by the embryo. This secretion in-
duced the formation of stable messenger RNA which in turn
caused formation of hydrolytic enzymes. These enzymes ini—
tiated starch digestion in the endosperm from which trypto-
phane was formed. Tryptophane formed auxin and as a result
growth occurred (114, 115, 116). Furthermore, the gibberel—
lin also activates enzymes that promote cell wall degradation.
Weakening of the cell walls increases the suction of the

cells, thus, water uptake is increased and cells elongate

(12, 67, 114).

 

18

Simpson (92) indicated that gibberellin possibly
promotes the synthesis of, or activates preformed enzymes
necessary for the utilization of the endosperm as the sub-
strate for germination and growth. Harber and Luippoid
(38, 39, 40) attempted to find out whether GA and kinetin
treatments induce initiation of cell division, cell elonga-
tion, or both. They suggested that cellular expansion is
both necessary and sufficient, whereas cell division is
neither necessary nor sufficient for initiation of lettuce
seed germination. The stimulating effects of GA and kinetin
on germination produced a direct effect on cellular expan—
sion, whereas the apparent effects on cell division were
the results rather than the cause of such stimulation.

Recently it has been postulated that in seeds of
cereals, GA is secreted by the embryo. This secretion in—
duced the formation of stable messenger RNA which in turn
caused formation of hydrolytic enzymes. These enzymes ini—
tiated starch digestion in the endosperm from which trypto—
phane was formed. Tryptophane formed auxin and as a result
growth occurred (114, 115, 116). Furthermore, the gibberel—
lin also activates enzymes that promote cell wall degradation.
Weakening of the cell walls increases the suction of the

cellS, thus, water uptake is increased and cells elongate

(12,67, 114).

 

 

19

Production of Seedlings from
Embryos of Dormant Seeds

The tissues enveloping the embryos of dormant
seeds are often involved in various ways in regulation of
germination (55, 65, 105, 120, 121, 123). If these tissues
are ruptured or removed some germination often occurs (12,
43, 55, 118). Use has been made of this possibility in
studying seed physiology, and methods have been suggested
for producing seedlings by culturing the excised embryos
(98, 123). After-ripening requirements of immature embryos
of fruits were studied by culturing the excised embryos on
an agar medium and subjecting them to different temperatures
(126). Many investigators have grown seedlings from excised
embryos and studied the behavior of the seedlings in relation
to the after—ripening requirement of the seeds (12, 29, 30,
67, 90, 101). Plancher (88), a German worker, germinated
dormant apple embryos in closed Petri dishes at high humidity
in an attempt to raise seedlings for biological studies in a
shorter time. He got 32.6 per cent germination. Tukey (106),
in 1933, applied the nutrient culture technique to the ger—
mination of early sweet cherry embryos, which usually abort,
and later reported successful culture of peach, apple, pear,
and plum embryos (107, 108). Blake (9) modified Tukey's
methods and cultured over 1,000 hybrid peach seedlings.
Lammerts (57) suggested that embryo culture may be effectively

used in large scale commercial fruit breeding to greatly

 

 

20

shorten the breeding cycle of deciduous trees and markedly
increase the germination of hybrid seeds.

Tukey (107) pointed out that the state of develop-
ment of the embryo when removed from the fruit is a limiting
factor in the use of his method and suggested that the best
stage to remove embryos is when the fruit is ripe. Immature
embryos of apple and pear failed to develope as vigorously
in culture as did those of Prunus, (peach and appricofl (109).
No development in culture was observed from embryos excised
prior to 39 days from full bloom. Several other workers
(23, 36, 49, 52, 53) also have reported on embryo culture

methods for Prunus Species.

Nature of the Seedlings Grown from

 

Embryos of Dormant Seeds

 

Seedlings grown from dormant embryos are dwarfish,
the internodes are thicker, deeper green, shorter, and often
much deformed, whereas seedlings grown from after—ripened
embryos are normal (15, 19, 41, 69, etc.). These dwarf
characteristics may persist for a long time, or until the
conditions of the seedling are changed, such as in after-
ripening. A lateral bud of the epycotyl often is uninhibited
and will grow. The seat of dwarfness in seedlings obtained
from non after-ripened embryos is in the growing tip of the
epicotyl (19). This was shown by the grafting technique.

When the growing tip of a normal seedling was grafted onto

the stem of a dwarf seedling, the result was a normal seedling.

 

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21

But the reverse grafting gave a dwarfish seedling. This
showed that the root system of dwarf seedlings are capable
of normal growth (19). Physiological dwarfism of seedlings
from non after-ripened seeds is regarded as a consequence
of insufficient chilling (118). Pollock (89, 90), working
with embryos of several peach varieties, showed that seed-
lings produced from non after-ripened embryos may be dwarfed
or normal, depending on the temperature to which the seeds
are exposed during the first eight to nine days of germi-
nation. Within this period, germination at 19 or 22° C
resulted in severe dwarfing.

In a preliminary study of dwarfism in peach seed-
lings, Flemion et a7. (32), in 1965, detected a virus in
some of the physiological dwarfs tested. However, they did
not determine if there was any relation between presence of
the virus and physiological dwarfism in the seedlings.

A recent report on cause(s) of dwarfism in seed—
lings came from Lipe and Crane (62) in 1966. They pointed
out that physiological dwarfing in peach seedlings is caused
by a certain concentration of an inhibitor which they iso-
lated from peach seeds. The concentration of the inhibitor
is not high enough to prevent germination of the seed but
enough to induce dwarfing in the seedlings.

An effective condition for after-ripening the ter-
minal bud of dwarf seedlings is a period of 6 to 9 weeks at

a low (about 41° F) temperature (19, 29, 31, 41, 57, 109,

110). High temperatures and increased light also have been

 

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22

shown to be effective in eliminating dwarfing of these seed—
lings (19, 30, 41, 57, 58, 60, 87, 121). Apparently normal
seedlings were obtained from unchilled seeds by removing the
cotyledons and growing the rest of the embryo on nutrient
agar (33).

A typical action of gibberellin on higher plants
is an enhancement of stem elongation (100). This material
has been used for eliminating dwarfness in higher plants.
Brian and Hemming (14), in 1955, showed that gibberellin
significantly increased the rate of shoot elongation in
seedlings of dwarf pea. Stimulation of stem elongation
with gibberellin has been shown in single—gene mutants of
maize (86), henbane (Hyoscyamus niger) (59), crabapple (6),
peach (12, 56) and many other plants (12, 67, 68).

The response to gibberellin in removing dwarf
growth is not universal among plants, and the effect will
vary with the age of the plant. Young plants and shoots
respond more readily than mature plants and plant parts
(100). In many cases repeated applications of gibberellin
are necessary in order to prevent the seedlings from re-
verting to the dwarf condition (30). In one case it has
been reported that subsequent growth of dwarf seedlings of
peach, apricot and plum was normal when treated with six

weekly water sprays of potassium salt (Gibrel) of the gib-

berellic acid at 200 ppm (12).

 

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MATERIALS AND METHODS

Seeds from apples (MaZus sylvestris Mill.) of 1965-
1966 seasons were extensively used in these experiments. In
1965 seeds from Red Delicious and McIntosh, and in 1966 seeds
from McIntosh and Wealthy were used. The apples from which
these seeds were collected were grown at the Michigan State
University Horticulture Farm, except for a group of McIntosh
from the 1966 season, which were obtained from a commercial
orchard.

In 1965, apples were collected from trees on seed-
ling rootstocks 35 years of age and from trees on clonal
rootstocks 10 years of age. Apples were harvested at random
from all sides of all the trees of each group at lO-day inter-
vals starting 40 days before harvesting time and continued
to maturity.

After the fruit was collected, one half was stored
at 30° F and the other half was used shortly after harvest.
The seeds were removed from the fruit, air dried and then
kept in covered jars at room temperature. Seeds from the
stored apples were used at a later date.

In 1966 three different groups of apples were col—

lected and used as follows: Group I: The McIntosh apples

were harvested from the same trees as in 1965. One harvest

 

 

24

was made 23 days before maturity. Group II: Wealthy apples
from the same location as the first group were harvested at
maturity. Group III: McIntosh apples were obtained from a
commercial orchard at Hartford, Michigan. The trees were

on seedling rootstocks and 30 years of age. These apples
were picked at maturity, stored at 61° F and used as needed
over a three month period.

It has been shown by many seed physiologists that
there are three main groups of substances which are active
in breaking dormancy of seeds, and that these stimulate seed
germination. These groups are: (l) sulfur compounds;

(2) gibberellins; and (3) cytokinins.

To explore the possibility of the effect of these
compounds in breaking dormancy and in stimulating germination
of apple seeds, one representative chemical from each group
was selected for use in these experiments. They were thiourea,
gibberellic acid and N6benzy1adenine. The structural formulas

and discriptions of these chemicals are as follows:

1. Thioureal or thiocarbamide is an
urea (carbamide) molecule in which l/NHZ
the ketone group has been replaced C=S
by a sulfur atom. Its molecular ‘\NH
weight is 76.12. It has been used 2
in breaking dormancy of lettuce
(103), peach (109) and other seeds. Thiourea

 

1The thiourea and the benzyladenine were obtained
from Nutritional Biochemical Corp., 21010 Miles Ave., Cleve-
land, Ohio, U.S.A.

 

 

 

25

2. Gibberellic acid2 (GAS)
C19H2206. It is a fun—
gal product which also A
has been isolated from
plant tissues. Its mo-
lecular weight is 346. HO OH

kA=CH

Its germination stimula— 2
. 1 1 CH3 COOH

ting act1v1ty has been

shown in lettuce (50,

64), peach (24) and

other seeds (51).

Gibberellic acid

3. N6benzy1adenine3 (BA) or

6-benzy1amino-purine is

an anologue of kinetin

in which the furfuryl c————————g

group has been replaced HG/ §%—I‘—NH

by a benzyl group. Its \\ / E

molecular weight is 225.2. g_—g c H
It is a synthetic prod- Né/ \\C//N\\
uct. Its germination HG 1 /9H
stimulating effect has V§N// \\N
been shown in lettuce 6 .

seeds (94, 95, 97)“ N benzyladenine

Stock solutions of these chemicals were made by dissolving the
proper amounts of each crystalline compound in a few drops of

a solvent and bringing this to 100 mls. Appropriate amounts

 

2The gibberellic acid was obtained from AMDAL Co.,
Agricultural Div , Abbott Laboratories, North Chicago, 1111—
nois, U.S.A.

3Obtained from Nutritional Biochemical Corp. (See
footnote 1).

 

         
 

 
 

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26

of distilled water were added to obtain the different concen-
trations of the stock solutions. The stock solutions were
made every two to three weeks and were kept under refriger—
ation. Distilled water, 95 per cent ethanol and 3N sodium
hydroxide were used to dissolve the thiourea, the GA and the
BA, respectively.

At the beginning of these experiments the previous-
ly mentioned chemicals were used at three different concen-
trations, namely, 1 x 10‘s, 1 x 10 4, and 1 x 10_5 molar.
Most of the seeds in these experiments were germinated in
Petri dishes. Two layers of Whatman No. 11 filter papers
were placed in each heat sterilized Petri dish. The filter
papers were moistened either with distilled water (control)
or with the chemical solutions.

The fresh or dry intact seeds from the different
harvests were treated with the three concentrations of each
chemical. Three methods of application were used, namely:
(1) soaking the embryos in the chemicals for various periods
of time, then washing and placing them under germination con-
ditions in Petri dishes; (2) putting the seeds directly in
Petri dishes and moistening the filter papers with the chemi—
cal solutions as required for proper moisture; and (3) soaking
as in number 1 and then placing the seeds in Petri dishes and
applying the appropriate chemicals to the Petri dishes as in

number 2. Such treated seeds were also placed either on

filter papers or were planted in sterilized sand placed in

 

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27

Petri dishes. The Petri dishes containing the seeds were
kept at room temperature under diffuse light. These treat-
ments were repeated with seeds of different maturity from
several harvests. The treated seeds were observed every day
for any sign of germination. None of these seeds showed any
sign of germination for twenty days after the treatments.
Due to the failure of the intact seeds to respond
to the chemical treatments, work with them was discontinued.
Instead, the intact seeds were excised prior to treatment.
This was done by making an incision through the chalazal end
(end opposite to the micropilar end) of the seeds and remov—
ing the exembryo carefully by hand. Then the excised embryos
were treated similarly as described for the intact seeds. A
few days after the treatments, cotyledons of the embryos
treated with BA started turning green and enlarged. This

response was best shown in embryos treated with BA at 1 x 10—5

and 1 x 10‘4

molar concentrations. By the sixth day some
germination activity was observed in BA treated embryos and
very little in the untreated embryos.

Since BA showed promise in activating germination
of dormant seeds, most of the following experiments were con—
ducted using this chemical.

During the first year of this research many seed
experiments were performed in the laboratory and in the green-

house, and these experiments again were repeated on seeds

from apples of the 1966 season.

 

u

 

 

28

Since this work consisted of several separate ex—
periments, the methods used in each experiment are described
along with the results.

In taking the records, germinated embryos were
those which produced a radicle of 3 mm. or more in length.
Changes such as separation, greening and enlargement of the
cotyledons; elongation of epicotyl and hypocotyl; and a
radicle less than 3 mm. in length were considered as germi-

nation activities.

1. Growth Abnormalities

 

Crocker (l9) and others have reported that when
the excised embryos from dormant seed of some species, in—
cluding apple and pear, were placed on moist filter papers,
only the cotyledon in contact with the paper enlarged and
turned green, whereas the other cotyledon remained unchanged.
This phenomenon was also observed in these experiments both
with water and BA treated embryos (Figure 1).

In these studies a different abnormality was ob—
served in the hypocotyl section of the seedlings. In normal
seedlings hypocotyls and radicles were relatively long but
small in diameter, whereas, in other seedlings these parts
were short and the hypocotyl rather thick. In other seed—

lings the radicle grew very little, then stopped growing,

showing a short thick hypocotyl (Figure l).

 

 

 

29

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30

The single cotyledon enlargement apparently was
caused by the inability of the chemical solution to move
from one cotyledon to the other. Similar results have been
reported by Crocker (19). The thickening and shortening of
the hypocotyl apparently was due to the differential effect
of BA on the hypocotyl and the radicle.

It was found that the second abnormality (hypocotyl
thickening) was caused by high concentrations of the BA solu-
tions. The higher the concentrations (over 1 x 10‘5 molar
BA), the thicker and the shorter the hypocotyl. Microscopic
cross sections were made of hypocotyls of different thicknes-
ses and these were examined and compared microscopically.

The examinations showed that it was the size and not the num—
ber of the cells which contributed to the thicker hypocotyl.
There were eleven rows of cells in the cortex area of the
hypocotyl sections, but the cells were larger in sections
taken from chemically treated thicker hypocotyls.

Experiments were carried out to find methods for
avoiding these abnormalities. For preventing one—cotyledon
growth of the excised embryos it was found that the embryos
should be soaked in solutions rather than solutions being
applied to the Petri dishes. A twenty—four hour soaking
period prior to putting the embryos on filter papers was
found to be adequate for the embryos to absorb enough solu—

tion and to grow normally.

 

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'L:

_-" 1:- {11' “Cf-'1 .

 

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. 1 I I

 

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31

The formation of abnormally thick hypocotyls was

5 molar concentra-

prevented by using BA solutions at l x 10—
tion, or below. One application of the solution to the seed
in the Petri dish was adequate. The abnormality of thick hy-
pocotyls was also avoided by soaking the seed for 24 hours

in the optimum concentration of the BA solution.

2. Effect of N6Benzyladenine on Germination
of Embryos of Dormant Seeds

 

 

A. Experiments with Fresh Seeds

 

Preliminary experiments with apples in 1965 showed
that treatments with N6benzy1adenine (BA) solutions signifi—
cantly increased the per cent of germination of excised em—
bryos from dormant apple seeds. These experiments were
repeated in 1966 using the three previously mentioned groups
of apples. The first group was of the McIntosh variety from
the Horticulture Farm, harvested 23 days before the commer-
cial picking date. The second group was the Wealthy variety
also from the same source but harvested at maturity. Although
Hartman and Kester (41) have indicated that for various
reasons seeds of some varieties, including Wealthy, have oc—
casionally given unsatisfactory results, Wealthy seeds were
included to find their response to the treatments. The third
group comprised of mature McIntosh apples obtained from a

commercial fruit farm.

 

32

The seeds from apples of each group were tested
immediately or soon after harvest. Thus, three separate
experiments were carried out at three different times and
the results were recorded accordingly.

The excised embryos were grown under two different
conditions, in Petri dishes at room temperature and in flats
of soil in the greenhouse.

Six different concentrations of BA, namely 0, 5,
10, 15, 20 and 25 ppm were tested with these apple seeds.
Each concentration was replicated and randomized five times
using 10 excised embryos per replicate in these experiments
in the laboratory and in the greenhouse. The volume of each
concentration for embryo soaking was 5 ml. in small 4—ounce
covered jars. Sterilized Petri dishes were used for embryo
germination in the laboratory. Two layers of Whatman No. II
filter paper were used in each Petri dish. For the green—
house experiments clean wooden flats containing steam steri-
lized soil were used. This provided a uniform seed germi-
nation and growing media.

For preparing the excised embryos, apples were cut
vertically and the seeds were extracted. While the seeds
were still moist an incision was made with a razor blade in

4 Then the

the chalazal end of the seed through the exembryo.
exembryo layers were carefully removed by hand so as not to

injure the radicle. The excised embryos thus obtained were

 

4For a definition of the term, exembryo, see page 6.

 

 

1.533151st

Ems ear-.11 fits-31511111.) nun-H 1.; in}; .

:.-. -‘ Fin‘Iii-i't-ié’V-f' Errata.

. :‘m... I: ,

 

 

 

33

kept moist until used. Ten excised embryos were placed in
each jar containing the solution. The jars were kept at
room temperature and were shaken occasionally to aerate the
solutions.

In the case of the lab experiments, after 24 hours
soaking, the BA solution was drained off and the excised em-
bryos rinsed and placed on filter papers moistened with
3.5 ml. distilled water. The Petri dishes were placed on
the bench in the laboratory at room temperature and were kept
moist throughout the entire length of each experiment.

In the case of the greenhouse experiments, the ex—
cised embryos from each jar containing different concentra—
tions were planted in a row in the flat of soil. The em—
bryos were planted with the cotyledons near the soil surface
and the soil lightly watered as required during germination
and growth of the seedlings in the greenhouse.

Germination data for excised embryos placed in
Petri dishes were recorded for 10 days, and for those planted

in soil, for 30 days after planting.

Results and Discussion

 

1. Laboratory experiments: The germination of non—

 

treated embryos was gradual in all seed groups tested in
Petri dishes. The cotyledons showed some greening and en—
larged slightly after 10 days. Very few of the radicles and

epicotyls elongated. Treated embryos, however, showed rapid

I
r--.

'. ’ il’féfidhasian sis-fade. wit? '

 

 

—_____________________________________fIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII

34

germination and development of embryo parts. In fact,
treated embryos germinated a minimum of two days ahead of
untreated embryos. Statistical analysis of the data showed
a highly significant difference in the per cent of germina—
tion of treated over that of control embryos (Table 1).
There was also a linear correlation between BA concentrations
and per cent of germination. This correlation, however, was
highly significant in the case of embryos from mature
McIntosh seeds, but not significant in the case of embryos
from immature McIntosh and mature Wealthy seeds (Table 1,
Figure 2).

Table l. The effect of different concentrations of N6benzy—

ladenine on stimulation of germination of embryos
excised from McIntosh and Wealthy apple seeds.

 

 

Germination (Per cent)

 

 

 

 

 

N6benzy1adenine McIntosh Wealthy
(ppm) Immaturea Mature Mature
0 14 28 28
5 18 28 24
10 26 52 56
15 26 62 52
20 46 72 68
25 32 66 50
LSD 5% 14 19 2
8Seeds from apples harvested 23 days before ma—
turity.

 

35

100‘
Mature McIntosh embryos
_ -————u—— Mature Wealthy embryos
__"__n__ Immature McIntosh embryos
80-

Per cent Germination

 

l I l J I

O 5 10 15 20 25

 

N6benzy1adenine (ppm)

Figure 2. Correlation of per cent germination and N6benzy1a-
denine concentrations in embryos excised from
immature and mature McIntosh and mature Wealthy
seeds.

** Highly significant correlation.

_

 

_ :6 '61. 6.14.9 day 3' r! 2' ,I-h'. 11.1: Ins-fl.

I l I

-e I'I-T'li‘? .'_'.":‘Jt ~.m-' =1;'?ai'.1——........._. -

“" '. :‘i-.3iIfii--".:

 

 

 

 

 

36

Theper centgermination of the excised embryos in both treated
and controls increased with the increase in number of days

following treatments (Table 2).

Table 2. Percent germination of N6ben2y1adenine (20 ppm)
treated and control excised embryos of different
maturity seeds in relation to the number of days
following treatment.

 

 

Germination (Per cent)

 

 

 

Days
Following Immature McIntosh Wealthy Mature McIntosh
Treatment

BA BA BA
Control (20ppm) Control (20ppm) Control (20ppm)

1 O O 0 0 0 0

2 0 4 0 0 0 6

3 0 l6 0 0 0 l6

4 2 22 0 16 6 42

5 4 32 0 24 14 52

6 8 38 10 40 14 70

7 8 42 12 48 22 70

8 12 42 14 60 28 72

9 14 46 22 66 28 72

10 14 48 28 68 28 72

 

This growth increase started earlier in treated than in the
untreated embryos of all three seed groups (Table 2, Figure 3).
The seedlings obtained as a result of the BA treatments ap—
peared normal (Figure 4). Radicles grew to a relatively long
length. There were no lateral roots formed on these seed—
lings while they were in the Petri dishes, but when the seed—
lings were planted in the soil they formed lateral roots and
resumed growth. The epicotyl grew slightly in the Petri

dishes, and when the seedlings were planted in the soil the

 

 

37

N6benzy1adenine

Control

 

 

 

 

100*
80—
e
o _
-H
p
to
a
E 60 —
H
<1)
(3
P
a _
O)
U
2:
m 40”
20-
0
Figure 3.

Days Following Treatment

Per cent germination of apple embryos excised
from mature McIntosh seeds following treatment
with N6benzy1adenine at 20 ppm.

 
       
 

 
   

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39

epicotyls continued growing for a while and then stopped and
remained as dwarfed seedlings.

In measuring the response of the excised embryos
to the BA treatments, on the basis of fresh and dry weights
of the seedlings, it was found that the fresh weight signifi—
cantly followed the same pattern as the germination percen—
tage which was based on the number of embryos froming radi-
cles. The dry weight changes, however, did not correspond

to the per cent of germination of the embryos (Table 3).

Table 3. The effect of N6benzyladenine treatments on germi—
nation of excised embryos of McIntosh and Wealthy
seeds and on fresh and dry weights of the seed-
lings produced.

 

 

 

 

 

 

N6benzy1— Germi— Ave.Fresh Ave. Drya
- adenine nation Wt. of 10 Wt. of 10
Variety (ppm) (Per cent) Seedlings Seedlings
(mg) (mg)
McIntosh 0 28 572 212
5 28 637 218
10 52 720 220
15 62 805 212
20 72 904 218
25 66 889 221
LSD 5% 19 180 -——
Wealthy 0 28 598 213
5 24 687 213
10 56 853 214
15 52 892 211
20 68 932 210
25 50 922 208
LSD 5% 2 121 ——-

 

aSeedlings were dried in 1659 F oven for 5 days.

 

   

 
     

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40

The change in fresh and dry weights showed that
seedling growth in these embryos was mainly as a result of
water absorbtion. Under favorable conditions for photosyn—
thesis, organic materials could have been formed in the seed-

lings and thus a further change in dry weight.

2. Greenhouse experiments; Four to six days after

 

planting the excised embryos, the cotyledons of the treated
embryos started emerging from the soil and turned green.

By the tenth day many of the BA-treated embryos had germi-
nated, whereas very few of the untreated embryos germinated

(Table 4).

Table 4. The effect of N6benzy1adenine treatments on germi-
nation of the excised embryos 10 days after plant-
ing in soil.

 

 

 

Germination (Per cent)

 

 

N6benzy1adenine McIntosh Wealthy
(ppm) ——‘_———— __
Immature Mature Mature
0 0 15 5
5 5 55 25
10 10 70 60
15 20 70 65
20 30 80 80
25 10 8O 6O

 

Here again the immature McIntosh embryos reSponded less to
the treatments than did the mature ones (Figures 5, 6).
Although mature McIntosh and Wealthy embryos planted in soil

reSponded similarly to the treatments, subsequent counts

is. J!L!.’;"-'r s.‘ as minim: as. I: emulate“.
:r -- * 12.“! -: :-)J'71'Jr;-:
l ;.-‘ .'. ' -' ' ' 1W .

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41

 

 

 

100 -
---—- Mature McIntosh embryos
_ Mature Wealthy embryos
m__u_._. Immature McIntosh embryos
80 - o-—~————-—*
/
a //
O ._ o — 0/
.g /
E / /°
5' 60 — / . .
0)
u
p
a
0)
L)
In
CD
a
O
/'/ \
./’ \
/o/ \‘
/,/‘ \\
-/°/’ \°
1 I I I
0 5 10 15 20 25
N6benzy1adenine (ppm)
Figure 5. The effect of different Nébenzyladenine concen-

trations on germination of apple embryos excised
from mature Wealthy and mature and immature
McIntosh seeds 10 days after planting in soil.

       

  

r’. ..-. :1 I .-'.-'. arirmflj'j .

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42

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.Emm mH new .599 OH Amy .Emm m HNV .Hopms HHV nqu woumohu mwoom manpme
Eoum womHoxo moxhpEo Scum czoem mwcHHwoom smoucHoz wHo zap p:om->uhom

.o ousmHm

 

 

 

 

43

showed that a large number of Wealthy seedlings-failed to
survive. However, this was not true with mature McIntosh
seedlings, which showed a high per cent of survival in all
treatments. Those embryos treated with concentrations of
10 and 20 ppm BA were the most vigorous seedlings (Table 5,

Figures 6, 7).

Table 5. Per cent stand at 10, 20, and 30 days after plant-
ing of seedlings grown from excised embryos of
McIntosh and Wealthy seeds treated with N6benzy1—
adenine solutions and planted in soil.

 

 

Survival (Per cent)

 

 

N6benzy1adenine McIntosh Wealthy

(ppm) _—_ —_

10 20 30 10 20 3O

0 15 35 30 5 10 5

5 55 60 55 25 3O 25

10 7O 80 80 6O 50 20

15 70 90 90 65 65 25

20 80 9O 90 80 55 25

25 80 90 90 60 45 15

 

Failure of some Wealthy seedlings to survive the BA treat—
ments in these experiments further supports the information
given by Hartman and Kester (41) that Wealthy seeds are not
as suitable for seedling production as the McIntosh seeds,
when obtained from mature apples. This is shown by the

higher per cent of germination and good vigor and stand of

the McIntosh seedlings, as compared with the Wealthy variety.

 

 
   

 
 

"fluihgtdifii'h

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I", {:I;i‘[3‘.=I-,-I I_' .I‘ +-a.--_..-_.: -- 11 y'f'fiw 1'56 Iran: 0-;

. 1:" '1 29‘!

 

 

-- Z'IJIIT

 

Seedling Stand (Per cent)

 

44

100—

(

 

 

 

 

Figure 7.

5 10 15 20 25

N6benzy1adenine (ppm)
Mature McIntosh

Per cent seedlings stand of mature McIntosh and Wealthy

Mature Wealthy

N6benzy1adenine (ppm)

seeds which were excised then treated with different con—

centrations of N6benzy1adenine and then planted in soil.
Records were taken 10, 20 and 30 days after planting the

treated embryos in soil.

 

 

an age... .

 

 

45

The McIntosh seedlings had several large normal
green leaves on stems which grew slightly, then formed a
dormant terminal bud and stOpped elongation. Thus a dwarf
seedling was formed—~a phenomenon observed in seedlings pro-
duced from dormant seeds. This will be discussed later.
The root systems,however, did not stop growing. To compare
the extent and amount of root growth of seedlings produced
from chemical treatments with that of controls, several
seedlings were dug and roots washed (Figures 6, 8). The
McIntosh seedlings produced from BA—treated embryos had a
superior root system when compared to Wealthy seedlings
similarly treated. The untreated seedlings had a rather
thick taproot with very few lateral roots on it, indicating
a root growth retardation. The seedlings from the BA-treat-
ment of 10 ppm, however, had very long narrow taproots with
many large lateral roots. In general, the whole root system
of treated seedlings was very vigorous and seemed to be ef—
ficient in supporting a vigorous top, whereas the root system

in controls seemed poor and lacked in vigor.

It can be concluded from these experiments that
excised embryos from mature and dormant seeds of certain
varieties of apple could be germinated and seedlings pro-
duced as a result of treatments with N6benzy1adenine. These
seedlings, however, are dwarf and do not grow normally.

This indicates that there is a lack of some growth regulator

in these seedlings which is naturally produced in the embryo

__ 1

 

    
  

 
 

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-:r; ajgéfhsea nl'barraadu wunafiunaflq 3+

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'l

rI3::':__=-'_”_--.\ -' i '.' ,.- I:‘.".‘ -'-:I':.-FIP. THEE-.9105?

—-';' .. 1. . - . ;- =I'...:.-.1l. ;,':.. .1155.

 

46

 

Figure 8. Forty-four day old McIntosh seedlings grown
from excised embryos treated with, top:
distilled water (control) and bottom:
Nébenzyladenine at 10 ppm.

 

 

—<—

47

of the seeds placed under stratification. In these tests it
was apparent that N6benzy1adenine replaced this factor and
probably played a similar role. The role of N6benzy1adenine
may be that of an antagonizer of growth inhibitors present
in the embryos or, in other words, it is an anti-growth-
inhibitor chemical.

This finding has been used as a tool to study the
role of the exembryo layers in controlling the dormancy of
the embryo and their relationship to the growth control regu-

lations involved in the process of germination.

B. Experiments with Dry Seeds

 

All the seeds used in section I of this group of
experiments were fresh seeds obtained directly from the
apples. Since there have been reports of changes in the
inhibitor content of the seeds during dry storage, experi-
ments were conducted to see if the seeds stored dry for some
time would respond to the BA treatments as well as the fresh
seeds (65).

In one experiment 10 month old dry seeds were
soaked in water for 24 hours and then excised. The excised
embryos were then soaked for 24 hours in BA solutions and
then placed in Petri dishes as in previous experiments. Re-
sults of this test showed that there was no significant dif-
ferences in germination between the untreated and treated

embryos. In other experiments including one in which the

  

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__ . éfi-ffiafiaivxflfbda“ iéigflé‘ WHIP . 91:01 .IIIII

 
  
 
   
 

rr'; 9 .c-:T.-.=_-r!rri Iii-1.401;: “r: ran..- I'WIIIHIIIIIK 5F.“ PEI"
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E’_"|‘_.|_.I:Ij I!” I

 

48

excised embryos from dry seeds were soaked for periods from
1 to 5 days before treating with BA, embryos failed to ger-
minate. However, in the final experiment, it was noticed
that the excised embryos from dry seeds deteriorated rapidly
when placed in the Petri dishes. Apparently this was due to
contamination or poor aeration during the soaking period.
The deterioration of the embryos was minimized by changing
the technique of handling the dry seeds during germination.
McIntosh seeds stored dry at room temperature for

10 months were used. At daily intervals for five days,

 

thirty—five seeds were placed in a jar containing 3 ml dis—
tilled water. On the sixth day all the seeds soaked for l
to 5 days were excised. Five excised embryos from each
group were placed in each of 3 glass jars containing 5 ml
solution of BA at 10 ppm. Twenty—four hours later, the ex—
cised embryos were placed in Petri dishes and were grown, as

previously, at room temperature for 7 days.
Results and Discussion

The excised embryos soaked for l and 2 days and
then treated with the BA did not show much germination ac—
tivity. This was also true for the control. Seeds exposed
to the 3 and 4 day pre-soak period appeared to be more ac—
tive, as was shown by the darker green and enlarged cotyle—
dons. Radicles and epicotyls were formed in many of these
embryos, whereas the control embryos did not show similar

response (Figure 9, Table 6).

ia'vl Iil ,I..:I‘-inmI:.-'It:-:;Ixé 5.17:?th

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50

Table 6. The effect of pre-soaking at 5 daily intervals in
water on the germination of the embryos excised
from dry McIntosh seeds treated with N6benzy1ade-
nine and distilled water.

 

 

Germination (Per cent)

 

Pre—Soaking

 

(Days) N6benzyladenine Distilled Water
(10 ppm) (Control)
1 47 26
2 66 33
3 80 13
4 86 26
5 53 20

 

The hydrated excised embryos which had imbibed water for 3
and 4 days apparently were able to reSpond to the BA—treat-
ment more actively than seeds which were not hydrated as
much. In other words, embryo germination activity was not
complete until the seed was thoroughly pre—soaked with water
and the embryos soaked in the BA solution” This pre-soaking
requirement seems to correlate with or have the same basis
as that of stratification in which the seeds must be kept
moist during the after—ripening periodo

The pre—soaking of the intact seeds in distilled
water for a 3 to 4 day period proved better than soaking the
excised embryos for the same length of time, possibly for
the following reasons: (1) the thick seed coats prevented
decay of the embryos; and (2) reSpiration in the intact
seeds was much lower than in excised embryos“ The aeration

of the intact seeds while under the soaking conditions, was

 

'.‘I-J

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15‘1}J I'nZH' Hui; 1mi-

  
     

 

51

not critical. The three or four day pre-soaking period ap-
parently facilitated the separation of the endosperm layer
from the embryo. This resulted in less damage to the radi-

cle when the exembryo was removed.

3. Chemical Effects of the

Exembryo in Seed Dormancy

 

 

A. Effect of BA and GA with Part
of the Exembryos Removed

 

 

The fact that N6benzyladenine significantly in-

creasedthe per centof germination of excised embryos of
dormant seeds might have been due to a destruction of the
seed inhibitors. The presence of growth inhibitory sub-
stances in the embryos has been mentioned by Luckwill (65)
as a possible causal factor in seed dormancy. He also found
that the integuments and particularly the endosperm layer
contain a much larger amount of the inhibitor than the em-
bryo itself. The effect of BA and GA on partly excised em—
bryos was studied as follows:

Seeds from McIntosh apples obtained from a com-
mercial orchard and stored at 61°F forSl days were used.
With a sharp razor blade an incision was made through the
exembryo laterally around the seed to 2 to 3 millimeters
from the micropilar end of the seed° The exembryo layers
were removed from the micropilar end of the seed by hand

so the radicle and a small part of the cotyledons were

    
      

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13:9“ am :3. sgfiwfi- ' saga "141' ”WWW! E

:-_..==.:.-- _. -.; - --.‘-=::n Iii-[-

 

 

52

exposed. The rest of the cotyledons were still covered with
about three quarters of the exembryo.

Ten seeds were treated with each of distilled
water (control), BA at 20 ppm, GA at 100 ppm and a combina-
tion of GA at 50 ppm plus GA at 10 ppm solutions. Each
treatment was replicated 5 times. Two filter papers were
placed in each of 20 sterilized Petri dishes, then the seeds
were placed in the dishes and the papers were moistened with
3.5 ml of the respective solutions, then one drop of the
same solution was placed on tOp of each seed to make sure
the entire seed surface was in contact with the solution.
The Petri dishes were left on the bench in the laboratory
and were kept uniformly moist during the entire length of
the experiment. Seeds were observed for germination activi-

ty every day and growth was measured after 10 days.

Results and Discussion

 

Untreated partially excised seeds did not germi—
nate although they appeared sound and in good condition.
The cotyledons of these seeds did not show any greening nor
any elongation of the radicles during the first 10 days.
However, seeds treated with BA or GA started turning green
and showed some germination activity the second day in
Petri dishes, and elongation of the radicles was noticed
in only a few seeds. The combination treatment of both GA

and BA, however, showed a very active synergistic effect

 

 

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53

in stimulating seed germination. This was also manifested
in rapid seedling development of all such treated seeds.
Exposed parts of the cotyledons in many of the treated
seeds turned green on the second day and some showed radi-
cle elongation as early as the third day after treatment.
By the fifth day 30 per cent of the seeds had germinated
(Figure 10). Germination data collected from all the seeds
on the tenth day after treatment showed a highly signifi—
cant difference between the combination of GA and BA treat—

ment and the control (Table 7).

Table 7. The effect of N6benzyladenine, gibberellic acid,
alone and a combination of the two on germination
of McIntosh seeds in which part of the exembryo
was removed from the micrOpilar end. At 10 and
20 days after treatment.

 

 

Germination (Per cent)

 

 

 

Treatments
10 Days 20 Days

Dgstilled Water (Control) 0 0
N benzyladenine 20 ppm 10 26
Gibberellic Acid 100 ppm 10 30

Gibberellic Acid 50 ppm, +
N6benzy1adenine 10 ppm 46 S8
LSD 5% 17 30

 

At this time the radicles and hypocotyls of the germinated
seeds were about 10 to 30 mm in length. Epicotyls in some
seeds had emerged from between the cotyledons. The green

cotyledons remained within the embryo. Observations made

 

      
 
  
   
 

    

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54

 

 

 

 

 

 

80'
— —-uu—»u-Gibberellic acid (50ppm)+N6benzyladenine(10 ppm)
——-———-—-N6benzyladenine (20 ppm)
60' Gibberellic acid (100 ppm)
g —————~—~Distilled water (control)
‘3
m _
a
-.-4 ,/0- —o— --——o—---—o
(3 40 — ’l/
:2 ,/
8 _ o/
s. ,,/
CD /'
m /;
20 — /'
J/
/
'— ’/ /a///°———°———O———O———:7D
/
/ /°/ v—0 0 o
0 U ' ’2// fl/’€\/- . W W
l I l l l I l l I l
0 2 4 6 8 10
Days Following Treatment
Figure 10. Germination of McIntosh seeds with part of

 

exembryos removed from micropilar end fol—
lowing chemical treatments.

 

55

20 days after the treatments showed a slight increase in
the per cent of germination of the treated seeds (Table 7).
At this time the seedlings also had grown to a much larger
size and many of the cotyledons had emerged from the ex—
embryo. Epicotyls had elongated and the seedlings appeared
normal (Figure 11, Figure 12 left).

In order to determine the influence of GA and BA
in combination at various concentrations, apple seeds were

prepared as before and treated as recorded in Table 8.

Table 8. The effect of different combinations of gibberel-
lic acid and N benzyladenine on germination of
McIntosh seeds in which part of the exembryo was
removed from the micropilar end.

 

 

 

Gibberellic Acid + N6benzyladenine Germination
(ppm) (ppm) (Per cenfl

25 + 7 5 20

25 + 10 16

50 + 7 5 40

50 + 10 52

75 + 7 5 46

75 + 10 SO

 

Although not Significant, the germination data recorded 10
days after the treatments showed some response to the GA +
BA treatments (Table 8). This was also noticed in daily
checks made on the germination activity of the seed. This
indicated that the concentration of the chemicals in the
combination was not as critical as their presence in the

solution.

 

.eHo mama om ma none:
30H Eouuop :H macaw ustu umouxo pHo mxmp OH mmcHHpoom HH< .pco amHHm
-oauHE anw po>oth moszono mo pawn csz mpoom cmOchoz wo cuzoam paw
coHpmcHanw do HSOH Eonuonv Ema OH pw ochopmeNConoz + Ema om pm pHom

uHHHoaoanw paw HH:MHs mouO Eng OOH um pHom oHHHoHonnHm .Hpoucoo mouv

Egg ON on ocHzopmewmonoz .Huon mouv popes poHHHume mo puowwo use .HH ossmHm

.gvm.

‘\fi

.u.¢¢. Icvr «of:

 

 

 

 

.mzmp OH HEN .ON .HH ”ohm ppm: ow pon Eosw mmcHHpoom

23 Ho mowm .2: .Hoo>oEo.H mpCoEsmoucH guano 3:3: .98 Hugo HwNmHmco

pm @9359? mo>HLono Copcouv “Hose .HmHHQOHUHE Eogw Hoo>oEoLH moxhnEo

L8 we 2mm Him: ”mpoom amoucHoz :o HSOH Eoupofi Ema OH pm ochopmH
135362 + an: Om um HoHum UHHHoHoanm paw Tsos mod .3me we poowwo 9:. .NH Saw;

a :uew \éac flu «flag... a.

“:2 ..: . .2...

 

58

These experiments clearly showed that in the ab-
sence of the exembryo and the inhibitors contained therein,
BA alone, at certain concentrations, increased germination
of the embryos by 1.8 to 2.5 times. This chemical, at the
concentration used, was not capable of eliminating the
total inhibitory effect of the exembryo. The combination
treatment of the two hormones (GA and BA) was more effec-
tive than when each was used alone. This is in contrast to
the suggestion made by Luckwill (65) and Visser (122) that
the inhibitors present in the seed coats do not play a role

in seed dormancy. Contrary to this belief, these inhibitory

 

substances in the exembryo certainly did play an important
part in controlling dormancy of the seeds. The influence
of the inhibitors in the exembryo is antagonized under
normal after-ripening of the seed. In other words, during
after-ripening there are produced certain endogenous mater—
ials which antagonize the inhibitory substances in the seed.
N6benzyladenine or a combination of GA and BA applied exo—
genously appears to act in a similar manner, that is, to
antagonize the inhibitory substances in the exembryo and

perhaps in the embryo.

B. Effect of BA and a Combination of BA
and GA on Seeds With Ruptured Exembryos

 

 

The first sign of germination in stratified apple

seeds is the elongation of the radicle and its emergence

through the micropilar Opening of the outer integument.

 

  

...' ¢- . ..:‘---
F-Ls—F-“aiéii‘ . .. _.
coir-3.1 S'fiflf‘lg begins“ Tier-Ida". n55 _ .
.-.-. I.§v'—:1r-9n';- um“. , 1...: .1 .'-.- M a:

"mi - - ' -' ‘-' r-..'.--.' ‘----'::‘.:~"_':.’ :flrifl'

'. 7! drift-i

 
 
   
   
  

 

 

lIIIIIIII:____________________::JIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII

59

This indicates that the chemical and/or physical restrictions
exerted upon the radicle by the exembryo in the dormant seed
have been altered by the stratification treatment.

In an effort to further similate stratification
with the use of chemicals, the physical restrictions of the
exembryo were modified. This was done by removing a small
part of the exembryo from the micrOpilar end, in the last
two experiments. The combination treatment of the GA and
BA actually broke the dormancy and stimulated the germina-
tion of these seeds.

In other tests, the influence of the total endo-
genous inhibitors in the seed were explored by rupturing
(to remove possible physical restrictions), but not remov—
ing the exembryos. The procedure was as follows:

Mature apple seeds of the McIntosh variety stored
at 61° F for 25 days again were used. An incision was made
with a sharp razor blade through the exembryo in the chala-
zal end of the seeds. The razor blade was pushed down be-
tween the cotyledons for about 2/3 of the length of the
seed and moved towards the outside of each cotyledon in an
attempt to separate the cotyledons slightly in order to
facilitate passage of the solutions towards the radicle.
The chemical treatments included distilled water (control),
BA at 20 ppm, and a combination of GA at 50 ppm + BA at
10 ppm. Five replicates of each treatment were made. The
seeds were treated in the same manner as in experiment

number 2.

——_ 1

  

--..._.-~'; ._ _.,-_v_
_' 5" Fetal-W

:I sl‘m'i'rs'ifia'

 
  
     
 
 
 

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.. -- .2 i . Its-1;",:J'ii.l_.'..':'1'!-l'ié: radians“:- mil" 31%..”

"7" ' - H".E"T.: -.. .;;.'.1 . ' .-- [-'. :uhnzi'sii') In lured-X

60

Ten days after the treatments the cotyledons of
the treated seeds had turned to a dark green color and were
partly emerged from the exembryo (Figure 12, center). This
condition was much more advanced in the GA plus BA treat-
ment than in the BA treatment. Only a few of the untreated

seeds showed such germination activity (Table 9).

Table 9. Total growth of the green cotyledons out of the
exembryoa of ten McIntosh seeds ruptured at the
chalazal end and treated with distilled water,

N benzyladenine and gibberellic acid plus N6 benzy-

 

 

 

 

 

ladenine
Cotyledon Elongation
Treatments
lO Daysb 20 DaysC
D%stilled Water (Control) 0.4 mm 7.9 mm
Nbenzyladenine (20 ppm) 2.7 24.9
Gibberellic Acid (50 ppm), +
N6benzyladenine (10 ppm) 10.2 38.4
LSD 5% 3.4 13.5

 

aPart of this might have been due to radicle
growth inside the exembryo.

bTen days under diffuse light.

CFollowed by 10 days of continuous light (100 w.,
120 v., white bulb).

After the first measurement of the seedlings the
Petri dishes were placed under continuous light (100 w.,
120 v., white bulb) to stimulate growth of the seedlings.
Ten days later (20 days from the chemical treatment) the

germination activity of the seedlings again was measured.

   

 

mnmmn

 

  

air": 1' .‘=.'-:' 263-112: , ti. fine-1143313.; saunthulgmms;

"(5' *3”? - :E"f11!..-£ 9143:”?

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—

61

The radicles had elongated in several cases, forming normal
seedlings. The difference between GA plus BA treatment and
the control was highly significant (Table 9). The data
shows that the embryos of dormant apple seeds can be_germi-
nated even in the presence of inhibitors, providing they

are treated with the prOper chemicals. Although the coty—
ledonary growth of the seeds was apparent, the radicles did
not elongate even when treated with GA plus BA. This partly
could have been due to the failure of the chemicals, under
conditions of this experiment, to reach the radicles. On

the other hand, it might have been that, under these condi—

 

tions, the chemical concentrations were not at optimum.
Here again the combination treatment of GA and BA showed

greater stimulatory response than BA alone.

4. Physical Effects of the Exembryo
in Seed Dormancy

 

A. Effect of a Combination of GA
and BA on’Seeds With the Outer
Integument Totally Removed

 

 

 

Besides the chemical effect, the exembryo has been
mentioned to have some physical influence in the dormancy
and the germination of the apple seed (42, 120, 122). This
influence may be due to mechanical resistance of the seed

coats or as a result of impermeability of the exembryo to

the respiratory gases.

 

  
  
 

9'. _ 1__ ' "131‘s 111113.11- .

   
   
  
 
  

|F|'

' ' - - 3.1-; "In: “1

: :4 H23 sane; B131. nnrJhu; i _;
- - '- - " -1-.1ax='-v:::--'1.q'sum m:

.1 E -.'-."-‘.'_._'_,-. . ' .. _ I".
1' ”'1“ hate,

writ-fl .

_' [s Thu”

 

 

62

The outer integument of the apple seed is a thick
brown fibrous layer with a micrOpilar opening. This layer
comprises 33 per cent of the fresh weight of the seed,
whereas the intact, thin and translucent inner integument
and the intact endosperm layer together comprise only 11
per cent of the fresh weight (43). 1

Being a thick fibrous layer, the outer integument
can hinder the emergence of the seedling. Having a large
micropilar opening, the outer integument seems to play a
minor part in controlling respiration of the embryo. The
inner integument and the endosperm layer, both being dense
and thin but without any opening, may be more effective in
hindering reSpiration than resisting the expansion of the
embryo.

Using the combination of gibberellic acid and
N6benzyladenine, the physical role of the inner integument
and the endosperm layer in controlling the dormancy of the
seed was further studied.

McIntosh apples stored at 61° F for 29 days were
used. To prepare the seeds, 3 very shallow cut was made
with a razor blade through the outer coat at the chalazal
end of the seed. Caution was taken not to damage the in—
tegrity of the inner integument and particularly the endo-
Sperm layer. The outer integument was then removed by hand.

To facilitate chemical absorbtion by the embryo,

they were soaked in the solution for l, 2, and 3 days. Each

soaking period was replicated 5 times. The seeds were all

 

 
  

swam ."-' "

 
  

.basa an: in addiaw daafii.9drm '

 
  

 
 

:..I§ga1-ni 'rsmz- ?r:s.au.’-I;=:-'-::I fume. MM”-

      
 
 

' -.-.' -.'- :5 :-:..- .2 -- .'.'= _--'.-'.-- " " .-.:-',;.-24:Il:-.nr.-r- 1.15-
“I ' ." _':|' '-!II_'I ':::| j

 

I==::—____________________________________________________________________:::|Ill
63

uniformly prepared and 10 seeds were placed in each steri-
lized jar containing 5 ml of GA at 50 ppm plus BA at 10 ppm.
At the end of the soaking periods the solutions were drained
and the seeds were rinsed with distilled water. Then the

‘ seeds were placed on two moistened filter papers in steri-
lized Petri dishes. The Petri dishes were kept at room tem—
perature and the seeds were checked for germination activity

at regular intervals.

Results and Discussion

 

After 10 and 20 days the seeds showed no germina-

 

tion activity except in a few cases in which the endosperm
layer presumably had been slightly cut Open during the re-
moval of the outer integument. In these seeds, the green

cotyledons had elongated and emerged from the remaining of
the exembryo layers (Figure 12, right).

The failure of the seeds with intact inner integu-
ment and endosperm layers to respond to the treatment prob-
ably was due either to the failure of the chemical to pene—
trate these structures or to the restriction of the
respiration of the germinating embryo by the remaining
layers of the exembryo. This is supported by the observa-
tion that a small rupture in these barriers made the treat—
ment effective and the seeds germinated with the embryo

emerging from the remaining of the exembryo. This observa-

tion also indicated that the mechnaical hindrance of these

 

 
     

    
 
  

imll-fl'wtfi‘t‘: v.1“! 9"”)qu .

LUJfiw J .L”;.“:,.f;iw bfififlfififi

  

, _._ .J JqL. -: _,_ .-_,~. .— a; in banalq"

   

nunui.iiiai

' .'._": Enufl-I _ .

 

fi

64

structures was negligible in comparison to other effects,

such as the exchange of gases and the chemical influence.

Bo Effect of Vacuum Treatment with
a Combination of GA + BA on Seeds
with Outer Integument Totally
Removed

 

 

In previous experiments seeds with outer integu-
ments removed failed to germinate under different treat-
ments and it was determined that the intact inner integu—
ment and the endosperm layer prevented germination of the
embryos.

To further pursue this study, the outer integu-
ments were removed from 100 seeds, as described for the
previous experiment. In addition, a sharp needle was in-
serted through the chalazal end of these seeds between the
cotyledons. This was done carefully so as not to rupture
the remaining exembryo layers. Fifty seeds thus prepared
were placed in a sterilized 250 ml beaker containing 30 ml
distilled water. The remaining 50 seeds were placed in
another beaker containing 30 ml of a solution of GA at
50 ppm plus BA at 10 ppm. The two beakers were then placed

in a glass dessicator under vacuum for one hour to remove

 

most of the air from the seeds. Ten seeds from each beaker
were then placed in each of five sterilized Petri dishes.
The filter papers were moistened with 3.5 ml of the same

solution and then each seed was moistened with one drOp of

the solution. Ample amounts of the solutions were placed

 

 

65

in the Petri dishes to insure that the solution would be
absorbed. The Petri dishes were then kept at room tempera-

ture and the seed response was checked at daily intervals.

Results

The results obtained were similar to those of the
previous experiment. Only a few of the seeds in which the
inner exembryo layers were unintentionally ruptured germi-
nated, whereas the other seeds did not show any signs of
germination. Twenty days after the treatment one seed from
the GA plus BA treatment had germinated and several others
showed elongation of the cotyledons which were partially
emerged from the inner exembryo. Again these were mainly
from ruptured seeds.

The question arose at this point whether or not
the treated seeds had absorbed enough of the chemicals to
germinate, or if the physical effect of the inner integument
and the endosperm layers was still a barrier to germination.

To determine more of these underlaying effects of
the inner integument and the endosperm layer, all control
and treated seeds were ruptured at the chalazal end with a
razor blade. This was done to eliminate the physical inhi-
bition exerted by these structures. No more chemicals were

added to the seeds at this time and other conditions remained

the same.

 

"TEE-115w? ,E'If' 'W. -‘ .

t. .

.aIs-i'tvu‘u; 11.1.1.1; fi'hgniszif'F‘T-‘I'HEFF I

 

66

Results and Discussion

 

A few days after rupturing the chalazal end, the
vacuum-GA plus BA-treated seeds started showing some germi—
nation activity. The cotyledons turned green and grew out
of the remaining exembryo layers. Germination, in per cent,
on the tenth day after rupturing was significantly higher

in treated than in controls (Table 10)°

Table 10. Germination of McIntosh seeds with the outer in—
teguments removed, the remaining of the exembryos
punctured at chalazal end and then vacuumed in
gibberellic acid plus N6benzyladenine, or distil-
led water. These seeds were placed under germi—
nation conditions for 20 days, then the remaining
of the exembryos were ruptured at chalazal end
and the seeds grown for 10 days.

 

 

 

 

Treatments Germination (Per cent)
Distilled Water (Control) 2
Gibberellic Acid (50 ppm), +

N6ben2yladenine (10 ppm) 24
LSD 5% 18.5

 

Germinated seeds had well colored and enlarged cotyledons.
The radicles, the hypocotyls and the epicotyls were rela-
tively long and the seedlings appeared normal.

Results of this experiment strongly indicated
that the endosperm layer and the inner integument of the

dormant apple seed play an important physical part in the

control of the dormancy This was shown by the observation

 

 

  

  

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. ..' — - -

. 55711:“ -_';1..‘ 1:1?

.. ".

  

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-=w““4 fiMfi? ¢KEWéfla'bsr*BTa-aheaa 5%

 
 

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12111- “31.1%"
. 14' ' -'-'- . '- ' - .. - -: m1 ”ambit:

:E"."r-711‘ .-

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211'. '1'

 

67

that although the embryos had absorbed enough of the chemi-
cals and were potentially able to germinate, they did not
do so until the integrity of the inner integument and the
endOSperm layers were altered. Consequently, it can be
stated that the physical effect in germination of the inner
integument and the endosperm layer is due to restriction of
respiration. This was also clearly shown when a small open-
ing was unintentionally provided for seed respiration, the
cotyledons expanded and burst these layers and emerged.
This physical influence of the inner integument
and the endOSperm layer is in addition to their chemical
effect, because in this test enough of the chemicals pro-
moting germination were absorbed by the embryos to counter-
act the effect of the inhibitors present in these tissues.
Whether or not the effects are physical or chemi-
cal in nature, or both, the exembryo layers of the dormant
apple seed play a very important part in controlling dor—
mancy, and this control must be altered before the seeds

will germinate.

5. Chemical Treatments for

 

Rupturing the Exembryos

 

In the experiments already described, it has been
established that the exembryo layers play a very important
part in controlling the dormancy of the apple seed. Also

it has been shown that the inherent chemical and physical

control in the seed must in some way be altered before the

 

 

 

 

68

seed will germinate. The commonly used method for doing
this is cold stratification. However, this method takes
time and often is cumbersome. A faster and simpler method
for making the seeds germinate is frequently desirable for
producing apple seedlings from dormant seeds. For example,
in apple propagation, a rapid germination method would eli-
minate the stratification requirement.

Instead of pre-soaking the seed and removing the
exembryo by hand from the seed, concentrated sulfuric acid
was used to rupture or scarify the exembryo layers.

Ten ml concentrated sulfuric acid was placed in
each of six sterilized 250 ml glass beakers. Fifty-five
dry seeds of the McIntosh variety were then placed in each
beaker and stirred intermittently with a glass rod. The
acid soaking periods used were: 1, 1 1/2, 2, 2 1/2, 3 and
3 1/2 hours. At the end of each soaking period, the acid
was drained and the seeds were washed with running water
for 10 minutes.

Since a 3 to 4 day pre—soaking in water is essen-
tial for dry seeds before chemical treatments, the seeds
were handled as follows: The seeds were divided into lots
of 10 seeds and placed in 4-ounce sterilized glass jar
containing distilled water. The seeds were kept in the
water for three days, and the water was changed each day.

On the third day the water was substituted with 5.5 ml of

a solution of GA at 50 ppm plus BA at 10 ppm. Jars were

 

 

 

 

   

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.-.--i _. . - ':3-.. l :1..- .. 1:19-31 fiiifiqfi - p
I I

.. ...gr--'.;.u':'r;-

 

 

é

69

aerated by frequent stirring. Twenty—four hours later, the
seeds were placed in Petri dishes, as in previous experi-
ments° The seeds in Petri dishes were kept at room tempera-

ture for 10 days.

Results and Discussion

 

At the end of each acid treatment the exembryo Y}
layers still were on the seeds. By washing, however, de- :.j
pending on the length of the acid treatment, the exembryo
layers separated and the embryos appeared. Seeds acid
treated for l and l l/2 hour periods had very few ruptured k
exembryo layers. In the seeds acid treated for periods
longer than 1 l/2 hour, the exembryo layers were more or
less ruptured. The seeds acid treated for 2 l/Z, 3, and
3 l/2 hours completely lost their exembryo layers during
the washing process.

The seeds with ruptured or removed exembryo layers

started turning green and showed germination activity the
third day. Radicles and epicotyls elongated in many of
these seeds. Seeds without ruptured exembryos did not ger-
minate and this was apparently due to lack of respiration.
These seeds soon deteriorated. Seeds which were acid
treated for 2 l/2 to 3 l/2 hours gave the highest germina-
tion (Table 11).

The pre—germination treatment outlined is an ef—

fective method of making quick germination test, and of

 

growing seedlings from completely dormant apple seeds.

 

       
 

—'h‘-:-Far.§n*§" «we: 3.3 Jq.a,2I-'a:nw 111111.15

 

70

Table 11. The effect of the acid treatment on germination
of McIntosh seed 10 days after treating with a
combination of gibberellic acid (50 ppm) and
N6benzyladenine (10 ppm).

 

 

 

Acid Treatment Germination (Per cent)
1 hour(s) 6
1 1/2 " 30
2 " 62
2 l/2 " 82 ‘TN
" 88 j

3 1/2 " 86 51

 

For example, the method is useful in apple propagation and 133
in testing seeds prior to planting in the nursery. However,
acid treated seeds must be handled under very clean condi-
tions following acid treatment because when the partly rup—
tured exembryo layers are left on the embryos, there is a
tendency for the seeds to decay much faster than if they

are removed from the embryos. Aeration of the acid treated
seeds during the soaking process also is extremely important.
In other tests, the seeds were soaked in water prior to the
acid treatment, but this neither reduced contamination, nor
improved seed germination. Failure to provide Optimum sani-

tation and aeration for the acid treated seeds might result

 

in complete failure in germination.
The possibility of the sowing the sulfuric acid-
BA treated seeds directly in the soil was studied. It was

found that such seeds planted in sterilized soil germinated

 

as well as in Petri dishes. However, seedlings produced

 

 
     
   
 

.1._.—__ _ . -. .. “rm“..“wwm . ..

.- _._—_-_ .. _-_-...nmi-—'-.-h_a-£---—
1 ' .'_- _- 'r'_-'.-\-1_._I ml}. 1.;1' ' _". ..'."
. . .... . --_.. -.. .

-' 1:111:61"?
..,

71
were anomalous in the form of rosette terminal leaves and
dwarf growth. The apical bud became dormant.
To overcome the anomalous seedling condition, the

seedlings were treated with gibberellic acid as described

in the following section.

6. Chemical Treatments for Removing

 

the Physiological Dwarfness
of the Seedlings

 

When the intact mature dormant seeds of peach,
apple and other rosaceous species are placed under favor—
able germination conditions, they fail to germinate. How-
ever, when the exembryos are ruptured or removed, some of
the viable embryos germinate, but produce anomalous seed-
lings. After a period of initial growth the epicotyls be—
come dormant and due to the failure of the internodes to
elongate, a rosetted dwarfish seedling is formed (41, 19,
etc.). This physiological dwarfness of the seedlings has
been reported in peach seedlings produced by thiourea treat-
ment of intact seed (109), and in seedlings produced by
agar nutrient culture of the embryos of many other species
(107, 108, 110, etc.).

As previously mentioned, all the seedlings pro-
duced by the BA or the GA plus BA treatments of the dormant
seeds in these experiments also were anomalous.

Exposure of the seedlings to 41° F for six weeks

stimulated normal growth (19). Other factors including

 

 

     
 

  

- '.d I 3.11% 5:_,|.¢jg’¢m.f_

. . '. i""1=":j'l.'.'_f {:1.” 1:," h

 

 

72

high temperatures, illumination, and treatments with gibber
rellic acid also have been mentioned to be effective in
removing seedling dwarfness (6, 30, 56, 57, 58, 86, etc.).
Studies were carried out to explore the possibili—
ty of the use of the gibberellic acid in eliminating the
dwarfness or anomalous characteristics of apple seedlings
grown from the dormant seed chemically treated prior to

germination.

A. Growing of the Seedlings

 

Two hundred one—year old dormant McIntosh seeds
were washed and soaked in 4 m1 of water for 3 days. On the
third day, the exembryos were removed and 15 excised embryos
were placed in each of 10 jars containing 5 ml of BA solu-
tion at 10 ppm. The embryos were soaked for 24 hours at
room temperature. At the end of the soaking period, the
embryos were drained, washed and planted in sterilized soil
in 6—inch clay pots and kept in the greenhouse. The germi—
nation percentage recorded at 10 and 20 days after planting

was 72 and 85.5,re5pectively.

B. Gibberellic Acid Treatment

 

The concentration of the GA and the number of ap-
plications necessary to remove the dwarfness were simultane-

ously studied in a factorial experiment With split plot de-

sign. Four concentrations of the GA, namely, 0

, 100, 200

 

 

      

ru.l_
flu: Fe ma
' .m

.- r; a a

mm133'_

‘.

      

 

:-

and 300 ppm were applied for l, 2, 3 and 4 times at S-day
intervals. Treatments were randomized and replicated 5
times.

Five-inch clay pots were filled with steam steri-
lized soil, and a 20-day old McIntosh seedling was trans-
planted into each pot. The plants were uniformly watered

as required under greenhouse conditions.

 

The GA—solutions were made by dissolving the

prOper amount of the crystalline GA in a few drops of 95 (a;
per cent ethanol and adding distilled water to make the aft
different concentrations. E

One drOp of a liquid detergent was added as a
wetting agent to each concentration in the small hand
sprayer, prior to spraying the newly transplanted seedlings.
The plants were sprayed by wetting the entire leaf surface
of the seedlings.

In the preliminary experiments the young apple

 

seedlings were severely affected by powdery mildew
(Podosphaera Zeucotricha). To control this disease a Kata-
thane solution at 350 ppm was Sprayed on all the seedlings
three days after each GA application.

Shoot growth of the seedlings was measured 40

days after the first treatment.

 

Results and Discu551on

 

The seedlings showed measurable shoot elongation

four days after the first application of GA. The control

 

 

_'744A_______"""""""""""""""""""""""""""""""""IIII

74

seedlings, however, remained unchanged with dark green1
leaves and very short internodes. After the second and
third application significant differences in shoot growth
were observed° The seedlings which received repeated ap-
plications grew more than those with one‘application° The
internodes were very long and the leaves were lighter green
than the controls (Figure 13).

Shoot growth of the seedlings measured 40 days
after the first spray application clearly showed that the

internodes in inhibited dwarf seedlings elongated (Table 12).

Table 12, The effect of the number of applications and con—
centration of gibberellic acid on shoot growth
(cm) of the apple seedlings measured 40 days
after first spraying.

 

 

Number of Applications a
GA Conc. (ppm) Conc. Means

l 2 Z 4

 

 

Conc. x ApplicationsC

 

Control 1.9 2.3 1.6 2.1 2.0
100 3.6 6.6 9.5 13.8 8.4
200 7.6 10.5 12.1 16.6 11.7
300 5.0 8.5 13.5 17.7 11.2
Appln. Meansb 4.5 7.0 9.2 12.6

 

aLSD 5% = 1.3 cm

bLSD 5% = 1.5 cm

c . .
LSD 5%, for applications at the same GA conc. =
2 cm; for concentrations at the same application = 2 cm.

 

 

      

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em.- .--.-.': i .13 1‘. l '.1- 1;:' -‘ --'.' .'t. minim: wee-n

1 =1 5'“. a

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76

Statistical analysis of the data showed that the differen-
ces between the shoot growth of the treated and the control
seedlings were highly significant. Each additional appli-
cation, in all cases, significantly increased the shoot
elongation (Figure 14). All GA concentrations significantly
increased elongation of the stems over that of the control.
There, also, was a significant difference between the effect
of the GA at 100 and 200 ppm.

The data obtained indicates that the gibberellic
acid apparently stimulated the growth of the physiologically

dwarf apple seedlings. The stimulatory effect was directly

 

dependent on the concentration of the GA solution and also,
to a larger extent, on the number of times the chemical was
applied. On termination of the repeated applications, the
terminal bud diverted back to its dormant stage. The inter-
nodes stayed short and the plant was dwarfed again. It is
assumed that repeated applications may have kept the seed-
lings growing indefinitely. Blommaert and Hurter (12),

however, produced normal seedlings of dwarf seedlings of

 

peach,apricot and plum after six weekly water sprays of

 

Gibrel (potassium salt of the GA) at 200 ppm.

 

The effect of the GA was mainly to counteract

 

apical bud inhibition and in so doing, caused elongation
of the internodes. The number of leaves on each plant,

however, was not significantly altered by the GA treatment.

 

  

 
 

‘ i. a... .3. .'...s: _
I 44qu lsn-ai..‘ib£m

 

77

 

18—
———————— Gibberellic acid 200 ppm
— Gibberellic acid 100 ppm
_. ..... __ Control
/0
16 _ /
/
_ //// o
E 0/
V 12 — /
FE: /
g o//
a _ //
:3 // °
8 8 - ./
‘65:) D
4 — 0
__ P"-’-——_’°“\~\~\ ’—’__a—o
-\°,__
| L l 1
0 l 2 3 4
Number of Applications
Figure 14. The effect of four applications at S—day

 

intervals of three concentrations of gib-
berellic acid on shoot growth of physiologi—
cally dwarfed McIntosh apple seedlings forty
days after spray application.

 

 

'-

78

The stems in treated plants were narrow and deli—
cate. The leaves were thin and yellowish-green in color.
The control seedlings, however, had thick stems with rather
thick dark green leaves. This might be an indication that
the new growth of the treated seedlings was at the expense
of the food materials stored in the cotyledons and t0ps of
the seedlings before GA treatment.

The gibberellic acid probably antagonized the in-
hibitory effect of substances which in turn act on the en—
Zyme activity in the plant. New enzymes may have been
formed or present inactive ones activated. As a result of
this new enzyme activity structure of the cell walls became

modified, so that water was absorbed and the cells were able

to elongate.

 

 

 

DISCUSSION

From the results of these experiments it appears
that the dormancy of the apple seeds is caused mainly by
two factors.

The first factor is the presence of growth inhibi—
tory substances in different parts of the seed. This is
contradictory to the suggestions made by Luckwill (65) and
Visser (122) that the inhibitor does not play any important
part in the dormancy of the apple seed, but the hypothesis
is in accordance with the recent work of Lipe and Crane
(62) who stated that peach seed dormancy is caused by the
presence of an inhibitor in the seed. The controlling role
of the inhibitors in the apple seed was shown by the fact
that BA inactivated these inhibitors. The BA treatments
were very active in breaking the dormancy of the excised
embryos and inducing germination. When partly ruptured
exembryos were felt on the embryos the stimulatory effect
of the BA, at the same concentrations, was not as effective
in stimulating the germination process. The controlling
effect of the inhibitors on germination also was observed
in seedlings. Cotyledons in many of the untreated excised
embryos turned green and some of the radicles elongated.

The seeds with partly ruptured exembryos, however, showed

79

 

 

 

 

 

80

very little germination activity. Although BA was effec-
tive in antagonizing or reducing the activity of the inhi-
bitors in the embryos, it was not effective in antagonizing
the inhibitors present in the exembryos. This may have
been due to the presence of high concentration of inhibitors
in the exembryos and very little in the embryo itself. A
combination of GA plus BA was much more active in stimula-
ting the germination of the embryos than BA alone, even in
the presence of partly ruptured exembryos. This indicated
that the dormancy of the seed is caused by a more complex
phenomenon than the reactions between one inhibitor and one
promotor.

The second factor involved in dormancy of the
apple seed is the restriction of aerobic respiration of the
embryo by the inner integument and eSpecially by the endo-
sperm layer. This was shown by removing the outer integu—
ments and applying the chemical under vacuum. As long as
the remaining layers of the exembryos were intact no germi—
nation occurred, however, when all these layers were rup-
tured, many of the embryos germinated. The exembryo layers
seemed not to exert much mechanical resistance to the expan—
sion and emergence of the embryo. For example, when the
outer integument was removed, none of the seeds germinated,
but when a small part of the exembryo was removed from the

micropilar end, most of the seeds germinated.

 

 

 

7%

In order to cause germination of the intact dor-
mant apple seeds, the retarding effect of the inhibitors
in the embryo and exembryo must be inactivated. In addition,
the physical conditions of the exembryo layers which re-
strict aerobic respiration of the embryo also must be al-
tered. Both of these changes are brought about by cold
and moist treatment of the seeds during the normal after- if:
ripening period. The questions arise as to how these
phenomena occur and what their relationships are to the
after—ripening conditions. It seems possible that these
physical and chemical changes are interrelated with the
formation of the endogenous growth promoting substances
in the seeds.

Germination of the excised BA-treated embryos
occurred only when they were in the hydrated state. The
moist conditions in after-ripening also seem to be neces—
sary for this purpose. The hydrated condition of the seed
appears to be necessary for the develOpment and/or use of
the growth-promoting substances. In addition, the moist
conditions seem to make the exembryo layers more permeable
to exchange of gases. On the other hand the cold tempera-
tures used during after—ripening may be necessary for the

production of the growth promoting substances. Synergistic

 

effect of the GA plus BA showed that these growth promoting

substances might be of different kinds, including gibberel-

lin and cytokinin types. It seems possible that the growth

 

 

 

82

promoting substances, applied exogenously or-produced endo-
genously in the seed during after—ripening, antagonize or
neutralize the seed germination inhibitors. This, then,
may increase the enzyme activity of the seed. As a result
of this enzyme activity, reserved insoluble food materials
such as sugars, starches, etc., may be changed to soluble
materials. The increased enzyme activity also may have an
influence on cell walls of the exembryo layers, making them
permeable to respiratory gases.

The seedlings obtained by chemical treatment in

these experiments were dwarf, whereas those grown from

 

after-ripened seeds were normal. The dwarfing characteris—
tics of the seedlings were temporarily removed with GA ap-
plications, thus causing normal growth. The GA also
increased the germination stimulating effect of the BA—
treated embryos. This could mean that GA played a single
role in both cases and that it may be one of the hormones
produced in the seeds under after—ripening conditions.

From these experiments it could be hypothesized
that the dormancy of the apple seed is due to the presence

of some growth inhibitory substances. Further, the ex-

 

embryo layers exert a restriction on the aerobic respiration
of the embryo. Under favorable conditions for after—ripening,
a complex of reactions occur, such as the inactivation of
seed germination inhibitors and possibly the appearance of

growth stimulating hormones. These may include gibberellins

_

 

 

 

83
and cytokinins. These hormones may antagonize the effect
radicle and epicotyle, and as a result the enzyme activity

of the seed increases and the conditions become suitable

for germination of the seed and a normal seedling is pro—

duced.

of the inhibitors in all parts of the seed including the
\
1

 

 

 

 

 

 

 

10.

ll.

12.

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_ ,

 

 

 

 

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_

 

 

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._._.-—_

F—__-

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