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ONION DORMANCY IN RELATION TO TEMPERATURE, APPLIED
GROWTH SUBSTANCES AND AN ENDOGENOUS
GROWTH INHIBITOR

presented by
Sauveur Maho tiere

has been accepted towards fulfillment
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ABSTRACT
ONION DORMANCY IN RELATION TO TEMPERATURE,

APPLIED GROWTH SUBSTANCES AND AN
ENDOGENOUS GROWTH INHIBITOR

BY

Sauveur Mahotiere

The relationship between growth and dormancy character-
istics for two varieties of onions, the level of an endogenous
inhibitor present during the dormancy of these varieties, the
effect of wounding and‘oxygen treatment on dormancy release and
the effect of temperature and chemicals in promoting growth of
shoots excised from dormant bulbs were investigated.

For field studies,"MSU 4535'; a genotype with a long dor-
mant period matured later than ‘DowningYs Yellow Globe' with
an intermediate dormant period. The initial inhibitor content
of [MSU 4535' was higher than that of ‘Downing's Yellow Globe',
and it declined in both cultivars during storage. The inhibitor
resembled abscisic acid (ABA) in several respects but was not
ABA itself. Dormancy release in onion is temperature-dependent
with 7.5-12.5‘C being generally more effective than the range
of 0-5° and 20-30°C. Dormancy was more intense in large bulbs
than in medium‘or small ones. Transverse wounding was effec-

tive in breaking dormancy in onion but increasing the oxygen

Sauveur Mahotiere

tension was not. Gibberellic acid (GA3 and GA4/7),

and naphtaleneacetic acid (NAA) had no effect but kinetin did

ethephon,

break dormancy. Shoot apices excised from dormant bulbs
responded to temperature in a manner similar to the entire
bulb but a much shorter exposure was required. Kinetin and
sucrose were very effective in promoting growth of the shoot
apices when applied alone but had no effect in combination
with temperature. ABA applied before or after temperature
treatment nullified the temperature effect. 4-Hydroxy-5-
isopropyl-2-methylphenyltrimethylammonium chloride l-piperi-
dine carboxylate (Amo 1618) was effective in reducing growth
of the excised onion shoot apices only if applied prior to
temperature treatment. 'Sucrose fully overcame the effect of
Amo 1618 but not that of ABA. Kinetin slightly counteracted
the effect of ABA and Amo 1618. Low temperatures (0-5°C)
were more effective in increasing reducing sugars in onion
shoots than high temperatures (lo-30°C). ABA and Amo 1618

nullified this effect.

ONION DORMANCY IN RELATION TO TEMPERATURE,
APPLIED GROWTH SUBSTANCES AND AN
ENDOGENOUS GROWTH INHIBITOR

BY

Sauveur Mahotiere

A THESIS

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

DOCTOR OF PHILOSOPHY

Department of Horticulture

1972

In memory of my

daughter,

Cassandre.

ii

ACKNOWLEDGMENTS

The author wishes to acknowledge the patient guidance
and assistance of Dr. R. C. Herner.

His gratitude is extended to Dr. F. C. Dennis for his
constructive suggestions and criticisms throughout the execu-
tion of this work and the use of his laboratory and equip-
'ment.

Special thanks are also extended to Drs. D. H. Dewey,

C. J. Pollard and H. G. Vest for their suggestions and kind-
ness. The assistance and suggestions of fellow graduate
students, especially E. A. Mielke,-R. Amezquita, and E. M.
Sfakiotakis, as well as those of J. E. Harten, mass spectrom-
eter technician, are greatly appreciated.

To all the professors who directly or indirectly con-
tributed to his scientific training, he expresses his sincere
gratitude.

Thanks also are extended to his wife, Marlene, for her

patience, help and moral support.

iii

TABLE OF CONTENTS

Page
INTRODUCTION. . . . . o . . . . . . . . . . . . . . . . 1
LITERATURE REVIEW . . . . . . . . . . . . . . . . . . . 2
The Concept of Rest and Dormancy . . . . . . . . . 2
Onion Bulb Formation and Induction of Dormancy . . 3
Exogenous Control of Dormancy in Onion . . . . . . 6
Anatomical and Biochemical Changes During the
Breaking of Onion Bulb Dormancy . . . . . . . 9
MATERIAL AND METHODS. . . . . . . . . . . . . . . . . . 14
Field Study of Growth and Maturation . . . . . . . 14
Effects of Temperature During Storage of Whole
Bulbs 0 O O O I I O O O I O I O O O O O I O O 15
Effect of Wounding and Oxygen Treatment of Un-
chilled Bulbs on Sprouting and Rooting. . . . 20
Growth of Excised Apices . . . . . . . . . . . . . 21
Level of Acidic Inhibitor in Onion Shoots During
Storage . . . . . . . . . . . . . . . . . . 25
Characterization of the Inhibitor. . . . . . . . . 28
RESULTS AND DISCUSSION. . . . . . . . . . . . . . . . . 32
Field Studies of Growth and Maturation . . . . . . 32
Effect of Temperature During Storage of Whole
Bulbs 0 O O O O O O O O O O O O O O O O O I 3 6
Effect of Wounding and Oxygen Treatment of Un-
chilled Bulbs on Sprouting and Rooting. . . . 50
Growth of Excised Apices . . . . . . . . . . . . 52
Levels of Acidic Inhibitor in Shoots During .
Storage 0 O O O O O O O O O O O O O O O O O 8 5
Characterization of the Inhibitor. . . . . . . . . 9O
CONCLUS IONS O O O O O O O O O I O O O I O O O O O O O O 9 8

LITERATURE CITED 0 O O O I O O I O O O O O O O O I O O O 100

iv

LIST OF TABLES

TABLE

1.

Conditions of gas chromatography and mass spec-
trometry used in the characterization of the onion
inhibitor. 0 O O O O O C O I O O I O I O O O O O O

Bulbing indices of Downing's Yellow Globe and MSU
4535 during development, 1970. . . . . . . . . . .

Effect of intermittent warm-cold temperature on
sprouting and rooting, and the effect of root re-
moval on sprouting of Abundance onion bulbs
planted at 20°C in moist peat moss . . . . . . . .

Effect of root removal and kinetin treatment on
sprouting of Spartan Banner onion bulbs planted at
20°C in moist peat moss. . . . . . . . . . . . .

Effect of wounding and 100% oxygen on sprout and
root emergence of MSU 4535 and Abundance onion
bulbs O O O O O O O I O O O O O O O O O O O O O O 0

Effect of exposure of onion shoots, half-cut bulbs
and whole bulbs to temperature (10°C) for differ-
ent period of time on the subsequent growth of
excised shoots planted in moist sand in the dark
at 20°C for 96 hours . . . . . . . . . . . . . . .

Effect of exposure of onion shoots, half-cut bulbs
and whole bulbs to 10°C for 96 hours on the subse-
quent growth of excised shoots planted in moist

sand in the dark at 20°C for 96 hours. . . . . . .

Effectiveness of different temperatures in stimu-
lating growth of excised onion shoot after 96
hours of exposure. . . . . . . . . . . . . . . . .

Effect of high temperature (30°C) in inducing
secondary dormancy in onion shoots(cv Spartan
Banner) and its reversal by subsequent low temper—
ature treatment (10°C) . . . . . . . . . . . . . .

Page

31

35

48

49

51

61

61

65

67

LIST OF TABLES--Cont1nued

TABLE

10.

ll.

12.

13.

14.

15.

16.

Cumulative effect of low temperature treatment
(10°C) in stimulating growth of excised onion
shoots (cv Abundance) after planting in moist
sand in dark at 20°C. . . . . . . . . . . . . . .

Effect of growth regulators on growth of excised
onion shoots (cv Spartan Banner). Shoots were
not Ch’illed O I O O O O O O O I I O O I O O 0 O 0

Effects of Amo 1618 and chilling on the growth of
excised onion shoots (cv Abundance). Amo 1618
was applied before and after chilling . . . . . .

Effect of sucrose, Amo 1618 and chilling on
growth of excised onion shoots (cv Downing's
Yellow Globe) . . . . . . . . . . . . . . . . . .

Effect of ABA, kinetin + ABA, sucrose + ABA with
and without chilling on growth of excised shoots
(cv Spartan Banner) . . . . . . . . . . . . . . .

Reducing sugar levels (percent fresh weight) in
Spartan Banner onion shoots after 96 hours of
exposure to 5 different temperatures. . . . . . .

Reducing sugar levels (percent fresh weight) in
Spartan Banner onion shoots as affected by
temperature, Amo 1618 (100 ppm) + temperature and
ABA (1 ppm) + temperature. ABA and Amo 1618 were
applied before exposure of onions shoots to 10°C
for 96 hours. . . . . . . . . . . . . . . . . . .

vi

Page

69

71

74

76

79

84

84

LIST OF FIGURES

FIGURE

1.

2.

10.

Photograph of excised shoot apices of dormant
Spartan Banner onion . . . . . . . . . . . . . . .

Bulb development of Downing's Yellow Globe and MSU
4535 as measured by the diameter of the leaf base.

Effect of storage temperatures on subsequent
sprouting of MSU 4535 and Downing's Yellow Globe
onion bulbs exposed to 20°C in dry air . . . . . .

Effect of storage temperatures on subsequent
sprouting of Spartan Banner onion bulbs exposed at
20°C with their base in moist peat moss. . . . . .

Effect of storage temperatures on rooting of
Spartan Banner onion bulbs planted in moist peat
moss at 20°C after storage for 8 and 16 weeks. . .

Root growth and development of Spartan Banner
onion bulbs planted in moist peat moss at 20°C
after 8 weeks of temperature treatment . . . . . .

Effect of size on sprouting of Spartan Banner
onion bulbs planted in moist peat moss at 20°C
after storage for 8 weeks at 10°C. . . . . . . . .

Effect of size on rooting of Spartan Banner
onion bulbs planted in moist peat moss at 20°C
after storage for 8 weeks at 10°C. . . . . . . . .

Effect of storage temperature on growth of intact
inner sprout leaves of MSU 4535 and Spartan Banner
onion. 0 O I I O I O I O I O C O O O O O O O O O O

Elongation of excised sprout leaves as influenced
by previous storage temperature. Excised sprout
leaves were planted in moist sand in the dark at
20°C and growth measurement was recorded at dif—
ferent periods . . . . . . . . . . . . . . . . . .

vii

Page

19

34

38

41

43

43

45

45

54

57

LIST OF FIGURES--C0ntinued

FIGURE

11.

12.

13.

14.

15.

16.

17.

18.

19.

Elongation of Sprout leaves excised from Spartan
Banner onion bulbs after exposure to various
storage temperatures for 8 and 16 weeks. The
sprout leaves were planted in moist sand for 96
hours in the dark at 20°C. . . . . . . . . . . .

Effect of temperatures and time of exposure on
subsequent growth of onion shoot planted in
moist sand at 20°C in the dark for 96 hours. . .

Effect of IAA and chilling on growth of shoots
(Spartan Banner). Chilled shoots were held at
10°C for 96 hours. . . . . . . . . . . . . . . .

Effect of kinetin and Amo 1618 with and without
chilling on growth of onion shoots (cv Spartan
Banner). Chilled shoots were held at 10°C for
96 hours. Kinetin and Amo 1618 were applied
before chilling. . . . . . . . . . . . . . . . .

Elongation response of Genesee wheat coleoptile
sections to eluates from thin layer chromato-

grams of the acid fraction of onion shoot apices
excised from whole bulbs before and after stor-

age 0 O O O O I O I O O O O I O O O O O O O O O O

Elongation response of Genesee wheat coleoptile
sections to eluates from thin layer chromato-
grams of the acid fractions of shoot apices. . .

Response of wheat coleoptile sections to eluates
of thin layer chromatograms of onion shoot ex-
tract (CV MSU 4535) O O O O O O O O O O O O O O 0

Response of wheat coleoptile sections to eluates
from TLC of: AzABA; B:methylated acid fraction
of onion extract; C:methylated ABA . . . . . . .

Response of wheat coleoptile sections to eluates
from charcoal-celite (1:2) columns of A) 8 ng of
ABA and B) 20 gram-equivalents of the acidic

fraction of an onion extract . . . . . . . . . .

viii

Page

60

64

73

78

87

89

92

92

92

LIST OF FIGURES—-Continued

FIGURE Page
20. Gas chromatographic lines of methylabscisate
and an acidic inhibitor from onion bulbs after
thin layer chromatography and methylation. . . . 95
21. Mass spectrograph line diagrams of authentic
methylabscisate, of peak 1, and peak 2 in
methylated sample of inhibitory zone from thin 9
7

layer chromatogram . . . . . . . . . . . . . . .

ix

INTRODUCTION

The onset of dormancy in onion bulbs is associated with
senescence of the leaves.’ Dormancy is important from many
aspects.‘ The shelfelife of the bulbs depends upon the degree
of dormancy of the bulb which, in turn; is affected by culti-
var, and by cultural and storage practices; In addition,
dormancy permits the survival of the species under adverse
conditions. Dormancy may govern the production of both seeds
and~bulbs. The time of flower and bulb formation is controlled
by the degree of dormancy of the bulbs and the temperature to
which they are exposed‘during'storage.‘ Onion bulb dormancy is
thus Of interest to farmers, retailers, consumers, physiolo-
gistsvand breeders.

For these reasons, a good understanding of the dormancy
phenomenon in this horticultural commodity is both interesting
and useful.

-In this thesis, I have investigated the following:

1. Effects of temperature and growth substances in

breaking dormancy of whole bulbs or isolated shoot
apices.

2. The levels of an endogenous inhibitor during the
dormancy of onion bulbs.

LITERATURE REVIEW

The Concept of Rest and Dormancy

Wareing and Saunders (136) define dormancy as a state in
which growth is temporarily suspended. This phenomenon is
caused either by external or internal conditions. To avoid
confusion between the two states of growth suspension, some
authors (74,120,132) term the former "dormancy? and the latter
"rest". Vegis (132) presented an extensive review of rest and
dormancy with temperature models for phases of rest. He dis-
tinguished three phases:

1. Early rest or predormancy.

2. Middle rest or true dormancy.

3, The state of potential maximum growth activity or

post dormancy.

Buch and Smith (21), however, used the terms rest and
dormancy interchangeably and questioned the real meaning of
the above distinction. Emilson (45), in his study on the
potato, also makes the distinction between rest and dormancy,
but notes that dormancy includes and may coincide with the
rest period.

Both states of growth suspension have been demonstrated

in onion bulbs (59,61,125,135). Wareing's and Saunders'

definition (136) fits this study which will be concerned
with internal conditions controlling true dormancy or rest
in onion bulbs. The general term of dormancy, however, will

be used.

Onion Bulb Formation and
Induction of Dormancy

 

 

Anatomical Changes

 

Before the onset of bulbing, the formation of leaf blades
is inhibited and bladeless sheaths are produced. These, along
with the sheaths of previously formed bladed leaves, increase
in thickness, resulting in swelling of the bulb (1). This
swelling occurs by increase in cell size and development of
intercellular spaces without cell division (50,123). During
bulbing, the apparently mature (vacuolated) cells of the inner-
most leaves swell laterally and become isodiametric (30,108).
During the differentiation into sheath and blade, a rapid
increase in the diameter of the growing region occurs (54).
Thus, the-base of the newly formed leaf is pushed farther and
farther away-from the center. At the same time, the leaf
blade and the sheath continue to increase in thickness so
that by the-time-the younger leaf is differentiated, no more
periclinal walls are formed.: Further increase in the thickness
of the sheath to form the fleshy bulb-scale is due to the
formation and enlargement of intercellular spaces and to the

growth of the cells.

Hormonal Changes Associated With
Photoperiodism

 

 

The increase in cell size during bulbing suggests that
auxins are involved in the-bulbing process (30). The investi-
gations of-Leopold (78) indicated that‘the auxin content of
plants varied with the photoperiod under which the plants were
grown. Cook (34) studied the effect of daylength on the auxin
content of 8 different plant species including Zinnia and
Xanthium, and found that the plants grown under long days con-
tained more auxins and were considerably larger than those
grown under short day conditions. Clark and Heath (31) ob-
served a rapid increase of IAA following the transfer of young
seedling onions from short days to long days. The maximum
auxin content occurred after 3*5 long-days, thereafter a
sharp decrease was observed. Tsukamoto (130), noted a remark-
able decrease in the auxin activity from the end of the bulb-
forming period to the lifting period.

Considering the bulbing ratio (i.e.,

maximum diameter of the base
minimum diameter of the neck

 

Clark and Heath (31) observed that a measurable increase in

the bulbing ratio occurred 14 days after the long-day treat-
ment, at which time the level of auxin in the long-day treated
plants had fallen to a value below that of the control. Thus,
IAA apparently initiated the bulbing process but continuation
of bulbing was independent of the IAA level (30). Gibberellins

may also be involved in photoperiodism. Cleland and Zeevaart

(33) found that the total amount of extractable GA in Silene
plants was-25-75% higher under long days than under short
days (46). ‘Thes 'findings support Digby and Wareing's (39)
observation-that Betula leaves exposed to long days contained
more GA than those exposed to short days.' However, despite
the fact that onion is a long day plant, Tsukamoto (130)
demonstrated that the GA level-was lower in onion plants
grown under long days than under short days.

The above discussion suggests that bulb formation in
onion is induced under long day conditions. Magruder and
Allard (84), using 18 of the most important varieties of
onions grown in the United States, found that increasing the
length of the photoperiod hastened the maturity of all the
varieties studied.

One of the factors controlling the induction of dormancy
in woody plants is daylength.' In the majority of the species
so far studied,]rnm;days promote vegetative growth and short
days bring about the cessation of growth and the formation
of resting buds (135). According to Wareing (133), dormancy

in Acer pseudoplatanus is controlled by the daylength to which

 

the mature leaves are exposed. There exists, however, some
evidence that in certain plant species dormancy is induced by

long phot0periods. "Lunularia cruciata and onion fall into

 

this category (50,131).
Hemberg (51,52) appears to have first suggested that in-

hibitors are involved‘in bud dormancy. In several woody

species, higher amounts of growth inhibitors have been ex-
tracted from leaves and buds*under short days than under long

day conditions (90,99,100).‘ However, in the case of Lunularia

 

cruciata, more inhibitors were accumulated under long days
than under short days (131). Tsukamoto's study (130) also
demonstrated that storage leaves and inner leaves of onion
contained more inhibitors under long days than under short

days.

Exogenous Control of Dormancy in Onion

 

Temperature and Relative Humidity

 

When dormancy is broken, the primordial bladed leaves
at the center of the onion bulb may emerge as-sprouts (1,58).
Under conditions of high relative humidity, the non-dormant
bulb may root (143) and in that case, root emergence precedes
sprout emergence (1,61). Removal of the roots can delay
sprouting (1).

From a commercial standpoint, a sprouted and/or rooted
onion is undesirable. Thus, keeping the onion in a dormant
or nearly dormant condition has been the goal of many re-
searchers. Their investigations have been aimed at observing
the sprouting and rooting tendency of the onion bulbs at dif-
ferent temperatures after a certain period of time in storage.
Jones (61) planted bulbs which were periodically removed from

storage and demonstrated that they sprouted and rooted more

quickly, the longer they were held in storage. The most
critical factors during storage were temperature and relative
humidity. If bulbs were not removed from storage, Sprouting
was usually greater at 10°C (50°F), than at 5°C (41°F) and or
at 0°C (32°F). Jones (62) stored bulbs for 4 months at 5
temperatures ranging from 3.7 to 30°C. Sprouting during
storage was progressively greater with increasing temperature
up to 16 to 20°C and then decreased at 30°C. Thompson (126),
however, observed less sprouting in storage at 15.5°-21.1°C
(GO-70°F) than at either 5 or 10°C (41 or 50°F). Wright et_al.
(143) stored bule‘at'O, 5 and 10°C under controlled humidity,
and found that sprouting increased with increasing temperature
but was little affected by humidity. The effectiveness of
'intermittent temperature in breaking dormancy of onion bulbs
was reported by Boswell (15) who observed that storage at 0°C
followed by 10°C caused more rapid growth than storage at 10°C
for the entire period. 'Rooting was greatly favored by high
humidities but little affected by temperature. More recently,
Abdalla and Mann (1) demonstrated the presence of a rest
period in onion which after harvest disappears at all storage
temperatures, the intermediate temperatures being in general
more effective than the extremes.‘ However, some investigators
have reported that extreme temperatures (BO-40°C) were more
effective than intermediate or lower temperatures in breaking

dormancy in onion (1,82). Exposure of onion bulbs to freezing

temperature resulted in decay upon subsequent exposure to
high temperature (14). Quick vacuum cooling was also unsuccess—

ful in breaking dormancy in onion (60).

Controlled Atmosphere Storage

 

Controlled atmosphere storage was shown to be a good way
of controlling onion dormancy.” Chawan and Pflug (35) working
with Downing Yellow Globe and Abundance onions concluded that
controlled atmosphere storage in general improved the appear-
ance and keeping quality of onions.v The best storage condi-

and 3% 0 or 5% CO and 5% 0

tions were found to be 10% CO 2 2 2

2
at 5°C.

Choice of Variety

 

Magruder §E_al. (85) on the basis of two years of tests
in seven areas of the United States, grouped onion varieties
according to their~suitability'for-storage.‘ Despite great
variability in the production, maturity, time between harvest
and storage, and conditions of storage, there was a remarkable
agreement in storage rating quality within varieties. Among
varieties, however, there was great variation in storage life
and generally those varieties which rooted and sprouted quickly
were the poorest keepers; -Bulbs stored at high temperatures

'lost marketability faster than those stored at low temperatures.

Chemical and Physical Treatments

 

Besides environmental control and the choice of varieties,

chemicals have been tested as a way of controlling dormancy.

Maleic hydrazide applied before harvest retards sprouting and
improves the marketability of the product (57,58,59,l4l).

So far, use of chemicals for prolonging dormancy has been
more successful than-for shortening it.‘ Boswell (l4) exposed
onion bulbs to an atmosphere saturated with ether at room
temperature but failed to break rest.

‘Contrary to its effects on sprouting of gladiolus corms,

 

ethylene chlorhydrin has not given any positive result in
onions. Loomis (82) suggested that the effect of ethylene
chlorhydrin in breaking the rest period may be associated with
hydrolysis of starch reserves, and that this and similar com-
pounds will not be*effective in plant organs which contain
little or no starch such as most bulbs.

The application of exogenous gibberellic acid induces
bud-break inea number of woody species (135) and its effective-
ness in shortening the rest period of potato tubers is well
documented (81,105,106). Thomas (125), however, could not
GA

break dormancy in onion bulbs with GA or naphthylene-

3' 4/7
acetic acid (NAA). However, transverse and longitudinal wound-
ing was very effective in promoting both sprout and root

elongation (14,82).

Anatomical and Biochemical Changes During
the Breaking of Onion Bulb Dormancy

 

 

Visible sprout and/or root formation are sometimes con-

sidered as unique indicators of the termination of the rest

10

period.' Such an approach may be misleading, for, the invisible
anatomical and physiological changes leading tO‘the sprouting
and rooting phenomena may be of importance for the understanding
of dormancy and for its control. According to Chouard (28),
all organs or their primordia are present during dormancy but
either do not grow or grow slowly. 'He also noted that the fre-
quency of mitosis decreased with the inhibition of growth.
Abdalla and Mann (1) found that in onion the sprouts resulted
from the elongation of leaves which were formed before harvest,
suggesting that sprouting was independent of leaf initiation
during storage. The same investigators (1) observed mitotic
figures in the shoot apex from twenty days before harvest until
fifty days after storage at 0, 15, and 30°C. Mitotic activity
decreased during storage while the cells had the capability to
enlarge. They concluded that sprouting was independent of cell
division but was associated with cell enlargement in the inner
leaves.

Color changes have been*observed during and after the rest
period in the‘shoot apices of onion bulbs. :During rest, the
apices are white but they turn yellow or green when rest is
broken (58). The response of the onion bulb to storage sug-
‘gests some biochemical changes (125). .Bennett (9), studying
the level of total, reducing and non-reducing sugars under 0,

5 anui'lSOC storage temperature, could not show any significant
change in the soluble sugar level. With increasing temperature,

however, the level of reducing sugars was lower and that of

11

non-reducing sugars higher. With eleven temperatures ranging
from ()1xa 40°C, the same conclusion was reached, i.e., low
temperature favored accumulation of reducing sugars. Also,
the aminonitrogen content of onion bul s stored at higher
temperatures was generally higher (144).

*Recently, Poovaiah gt 31. (102), studying peroxidase
activity in onion bulbs with long vs short dormant periods,
found that peroxidase activity was higher in the short dormant
than in the long dormant bulbs.

Virtually all processes connected with growth, develop-
ment and metabolism in plants are governed one way or another
by hormones (71). There is evidence that bulbs do not escape
this rule. In fact, a possible relationship between tempera-
ture treatment and the level of gibberellinhlike activity
has been reported in tulips. Exposure to 5°C for 9 weeks
resulted in an increase in the activity of both free and bound
extractable gibberellins (4). The occurrence of gibberellin-
like substances in onion in free and bound form has been
reported, but their presence was not associated with any physio-
logical process (5). Thomas (125), however, studying the GA
level of Rijnsburger onions stored at 5°C, observed a decrease
‘in gibberellin activity before sprouting and an increase as
sprouting commenced; 'Tsukamoto (130) and Kato (66) obtained
similar results by storing onions at room temperature, i.e.,
the gibberellin-like.substance decreased after harvest and
increased gradually until the period prior to sprout leaf

elongation.

12

Abscisic acid accumulation has been correlated with
induction of rest period in buds of woody plants (44,134,80).
Its level has been reported to fluctuate during the rest
period, suggesting that ABA alone does not account for the
rest phenomenon (36). Sondheimer et al. (117), however,
found that the most striking effect of chilling on Fraxinus
seeds was a decrease in ABA level. Regarding onion bulb
dormancy, few studies exist concerning the role of inhibitors
in controlling the rest period. An accumulation of inhibitor
tentatively identified as abscisic acid in onion bulbs was
observed shortly before harvest (130). Interestingly, this
inhibitor increased immediately after digging and then de-
creased during storage~at room temperature. Thomas (125),
working with'long and'short'dormantrtypes of onions stored at
5°C also observed a decrease in the inhibitor level at the
end of dormancy. 'Furthermore,'the'long-dormant type contained
more of this inhibitor than the short dormant type. He did not
decisively identify the inhibitory substance. ‘Fresh juice
extracted from dormant onion applied to young seedling onions
inhibited their growth and this inhibition declined progres-
sively with time. Here again, this inhibitory substance has
‘not been identified (67), A substance inhibiting seed germin-
ation and identified as allyl sulphide was present in large
quantities in dormant onions and in lesser amounts in sprouting
bulbs (68). 'In a previous study, however, the same investi-
gators (66) found that the concentration of some inhibitory

‘substances in'dormant*onion showed an opposite trend.

13

In summary, the existing literature provides the follow-
ing information: Onion bulbs exhibit both rest and dormancy.
The bulbing process results from cell enlargement and is under
photoperiodic and hormonal control. The end of the bulbing
process, which coincides with the induction of rest, is associ-
ated with low levels of auxins and gibberellins and a high
level of inhibitors.

Dormancy of onions is affected by genotype, temperature,
relative humidity and CO2 and 02 levels. Maleic hydrazide
prolongs dormancy, while GA, IAA, ethylene chlorhydrin and
ether vapor have been ineffective in breaking it. The breaking
of dormancy is associated with cell enlargement, and with
yellowing and greening of the shoot apices, increases in reduc-

ing sugar, peroxidase activity and gibberellin-like substances,

and a decrease in inhibitors.

MATERIAL AND METHODS

Field Study of Growth and Maturation

 

The growth and bulbing of cv Downing's Yellow Globe
onions were compared with that of cv MSU 4535 to determine if
a correlation existed between these characteristics and
storageability. MSU 4535 is an inbred line of long dormancy,
while Downing's Yellow Globe has an intermediate dormant period.
Seeds were sown at the MSU Muck Farm, May 29, 1970. Forty
plants of each variety were randomly selected and staked 5
weeks after emergence. Diameters of the neck and the base were
measured then and at weekly intervals thereafter until the
onset of senescence. The Downing's Yellow Globe variety
started maturing about one week before MSU 4535, but for con-
venience, both were harvested on October 16, when the tOps
had dried and fallen. The bulbs were cured in ventilated
crates held in a workroom at ambient temperature for two weeks
at which time they were cleaned and sorted for storage experi-
ments.

In 1971, the varieties Abundance, Downing's Yellow Globe,
and Spartan Banner, short, intermediate and long dormant type

respectively, were grown. The seed was planted May 5 and the

14

15

bulbs were harvested on October 11, according to the same
criteria as the previous year. The order of maturing from
earliest to latest was Abundance, Downing's Yellow Globe and
Spartan Banner. The curing procedure was the same-as previous-
ly described except that forced air was blown through the
crates to hasten drying and reduce losses from decay.

Constant temperature rooms were used for storage.

Effects of Temperature During Storage
of Whole Bulbs

 

Continuous Temperature

 

The first year, the sprouting response-of Downing's
Yellow Globe and MSU 4535 onions to storage at 0, 10 and 20°C
was investigated. The bulbs were put in crates and placed in
controlled temperature rooms. ‘The relative humidity was not
controlled. Random samples of 40 uniform bulbs were removed
after 2, 4, 8 and 16 weeks and 20 were planted With,their
bases in water at.20°C- The remainder was put in paper bags
and kept in dry air at the same temperature. The controls
were either held with their bases in water or they were put
in paper bags at 20°C from the beginning. Percentages of
bulbs rooting and sprouting were recorded weekly. In this
study sprouting and rooting are defined as the appearance of
leaves and roots at the top-and the base of the bulbs,

respectively.

16

When a long time was required for rooting and sprouting,
immersion of the bases in water often resulted in rotting.
However, as dormancy ended, root initiation occurred in a
matter of days. Also, because Downing's Yellow Globe and MSU
4535 have relatively short dormant periods, sprouting sometimes
occurred in samples stored for a long period of time at higher
temperatures. Furthermore, a 20-bu1b sample did not permit
detection of small differences*between treatments.

For these reasons, some modifications were adopted during
the second year. Spartan Banner onions, a relatively long
dormant type, were used in 1971. Bulb samples were removed
from the storage rooms after 8 and 16 weeks at 0, 5, 10, 20
and 30°C. Three or four replicates of 20 bulbs each were used.
Moist peat moss was substituted for water and the dry treatment
was omitted. The rooting and sprouting data were recorded
daily until 50 percent of the bulbs had rooted and sprouted.

To determine variability due to size, the bulbs were
classified as follows:

Small (4-5 cm in diameter)

Medium (7-8 cm in diameter)

Large (10~ll cm in diameter)
These bulbs were stored at 10°C for 8 weeks and then planted
as usual. The controls were planted immediately after curing.

Effects of 0, 5, 10, 20 and 30°C on root development of
Spartan Banner were also studied. Bulbs were stored for 8
weeks and planted at 20°C in moist peat moss. Four replicates

of 10 bulbs each were used. ‘Ten days after planting, the roots

17

were carefully removed, washed, and dried with filter paper.
Their length and weight were measured.
Effect of Storage Temperature on
Growth of Apices in situ

During the first year, six sound and uniform MSU 4535
and Downing's Yellow Globe onion bulbs were removed from
storage after 5 and 8 weeks at 0, 10 and 20°C. At 10 and 20°C,
sprouting occurred in the Downing's Yellow Globe bulbs and not
enough bulbs were available for removal after-8 weeks. The
scales-were removed to expose the shoot apex (see-Figure l-A),
the shoot apices were then measured to assess the effect of
'storage temperature on their elongation inside the bulb.
During the second year, a similar experiment was performed with
Spartan Banner, using temperatures of 0, 10 and 30°C for 8
weeks. In both cases, apices excised from unchilled bulbs
were used as controls. The apices, after measurement, were
>grown according to the procedure described later (p. 21).
Effects of Intermittent Temperature,
Root Removal and Substitution by
Kinetin on Sprout1ng

These experiments were performed to answer the following
questions: 1) Is continuous chilling necessary to break dor-
mancy in onion? 2) Does rooting govern sprouting and if so,

how? Abundance and Spartan Banner bulbs were used.

18

Figure 1. Photograph of excised shoot apices of dormant
Spartan Onion.

A. Shoot apices held at 10°C for 96 hours.
B. Shoot apices held at 10°C for 96 hours,
then grown in dark at 20°C in moist sand

for 96 hours.

Distance between bars = 1 inch.

 

 

19

ONION snoorl
PREPARATIONS

3m“)

INITLAL Lrnarn

 

ONION SHOOT
PREPARATIONS

:N ”(a

FINAL LENGTH

    

B

20

Treatment for Abundance~

 

- Control, not stored

weeks at 10°C

weeks at 10°C, 2 weeks at 30°C

weeks at 10°C, 2 weeks at-30°C, 3 weeks at 10°C
weeks at 10°C, then derotted daily after planting.

U1bwuh$H
l I
mcucam

Treatment for Spartan Banner

 

l - Control, not stored

2 - 8 weeks at 0°C

3 — 8 weeks at 0°C, injected with half a-milliliter
of water and derooted daily

4 - 8 weeks at 0°C,injected with half a milliliter of
100 ppm kinetin solution and derooted daily.

After removal from storage, bulbs were planted in moist

peat moss at 20°C and examined daily until 50% had sprouted.

Effect of Wounding and Oxygen Treatment
of Unchilled Bulbs on Sprouting
and Rooting

 

 

 

According to some authors (109,113), dormancy in seeds is
related to oxygen deficiency. The effectiveness of wounding in
promoting sprouting has been-attributed to release of inhibi-
tory gases~(l4) and oxygen penetration‘to the growing point
(82). These experiments were designed to test this hypothesis
on onion bulbs. The sprouting and rooting procedure as well as
the number of bulbs.used was the same as described previously,
and MSU 4535 and Abundance were used. Two kinds of wounding
were tested: transverse and longitudinal. In the first case,

one fourth of the upper portion of the bulb was removed and

21

in the second, the same proportion was removed from one side.
Care was-taken to avoid damage of the base and the inner
shoots. The wounded bulbs were.placed in water at 20°C.
Intact bulbs served as controls. To test the oxygen tension,
intact bulbs were exposed to a flow Of 100 percent of oxygen
for 48 hours at the rate of 472 cubic centimeters.per minute.
The controls and the oxygen-treated bulbs were placed in

water at 20°C.

Growth of Excised Apices

 

Effects of Temperature Upon Subsequent
Growth of Excised Shoot Apices

 

After assessing the effect of storage temperature upon
the growth of the apices in EEEE (p. 17), these excised shoot
apices were washed 3 times with distilled water and then
planted in moist sand in the dark at 20°C. Shoot length was
measured after 24, 48, 72, 96 and 120 hours. The controls
were shoot apices excised from dormant bulbs kept at ambient
temperature.' In 1970, only six MSU 4535 and Downing's Yellow
Globe shoots'were used per treatment for lack of materials.
In the 1971 Spartan Banner experiments,.four replicates of
five apices each were used per treatment and the shoot apices
were grown in moist sand for 96 hours (see-Figure 1-B, page

19).

22

Effect of Cutting Bulb Prior to Low
Temperature Treatment Upon Subse-
quent Growth of Excised Apices

 

 

Greenhouse-grown Spartan Banner bulbs were used in the
first experiment. Intact bulbs and bulbs with the upper half
removed-were exposed-to 10°C for 24, 48 and 72 hours. Excised
apices were also included. The shoot apices were~then excised
from the treated-bulbs, washed with~distilled water and planted
as previously described. After 96’hours, the final length was
recorded.‘ A second experiment was performed according to the
above procedure. However, the Spartan Banner bulbs used were
grown in the field, and five replicates of 5 shoots each were
used. The data were analyzed by using analysis of variance

and Duncan's multiple range test (41).

Effect of Temperature and Time of
Ex osure Upon Subsequent Growth
of Isolated Apices

 

Excised shoot apices were washed and exposed to 0, 5, 10,

12.5 and 20°C for 96 hours, and then treated as above.

Effect of~Higthemperature'in Blocking
tfie Responsento'Loeremperature~

 

The excised‘shoots«were-submitted to the following treat-

ments:

23

1 - Controls, no treatment
2 - 10°C for 96 hours

3 - 10°C for 96 hours, 30°C for 24 hours or 20°C for
24 hours

4 — 10°C for 96 hours, 30°C or 20°C for 24 hours, 10°C
for 48 hours

5 - 10°C for 48 hours
6 - 20°C for 96 hours

The apices were then grown as previously described.

Effects of Chemical Treatment

 

The following chemicals were used either alone or in
combination:* sucrose (10 percent); ethephon (100 ppm);
kinetin (100 ppm); GA3 or GA4/7
1618 (100 ppm) and IAA (l or 10 ppm). Distilled water served

(100 ppm); ABA-( 1 ppm); Amo

as a control. The possible antagonistic effects of sucrose
and kinetin on ABA or Am0'1618-treatment-weremalso investi-
gated.

The apices were soaked in the test solution for 10 hours.
They were then either planted directly at room temperature or
exposed to 10°C for 96 hours and then planted at room tempera-

ture.

Effects of Temperature, ABA, and Amo
1618 on Levels of Reducing Sugars
in Onion Shoots

 

 

 

‘Onion-shoot apices (var. Spartan Banner) were exposed to

0, 5, 10, 20 or 30°C for 96 hours. Additional apices were

24

dipped into solutions of ABA (1 ppm), or Amo 1618 (100 ppm)
for 10 hours before exposure to 10°C for 96 hours. Exposure
to temperature was carried out in the dark to prevent develop-
ment of chlorophyll. For extraction of reducing sugars the
method of McCready gt 31° (87) was followed with some modifi-
cations. One gram of the treated material was cut into small
pieces and ground in a mortar with 4 m1 of hot 80 percent
ethanol. The mortar was rinsed twice with 2yml of hot 80 per-
cent ethanol. The extract was transferred to a centrifuge
tube, stirred, allowed to stand for 20 minutes, and then
centrifuged for 10 minutes at 10,000 rpm. The supernatant
solution was then*decanted into a flask.

Reducing sugars were determined by the Nelson Method
(32,89). Aliquots of 0.2 ml of the supernatant solution in a
test tube was mixed with 1 ml of Nelson’s reagent, prepared
just before use by mixing 25 ml of solution A consisting of:

12.5 grams of NaZCOé

12.5 grams of potassium sodium tartrate
10.0 grams of NaHCO3
100.0 grams of NaZSO4

500.0 ml of distilled water

with 1 m1 of solution B consisting of:

7.5 grams of CuSO4.5HZO
50.0 ml of distilled water

1 drop of concentrated H2804

The tubes were placed in a boiling water bath for exactly 20

minutes and cooled by plunging them immediately in a cold

25

water bath. One ml of arsenomolybdate reagent.was added to
each test tube-and color was developed by shaking vigorously.
The volume was adjusted to 10 ml with distilled water and
read at 540 nm in a colorimeter.
Preparation of arsenomolybdate reagent:
Solution A

25 grams of (NH4)6 MO7024-4H20

450 ml of distilled water
21 ml of concentrated H2S04
Solution B

3 grams of NazHASO4-7H20

25 m1 of distilled water.
Solutions A and B were mixed and stored together at 37°C for
24 hours during which time the reagent turned yellow.
A freshly prepared solution of glucose (100 mg/l) was used
as a standard. Four replicates were run for each treatment.
The-data were statistically analyzed and Duncan's new multiple

range test was used for comparing the treatment means (41).

Level of Acidic Inhibitor.in10nion Shoots
‘During Storage

 

 

Extraction

 

The-samples were extracted-and the acid.fraction prepared
according to the procedure used by Aung and De Hertogh(4) for
the extraction of gibberellins.“ Shoot apices excised from

MSU 4535 and Downing's Yellow Globe bulbs were removed from

26

storage after 2, 4, 8 and 16 weeks at 10°C or 16 weeks at
0°C. After 8 weeks at 10°C, all the Downing's Yellow Globe
bulbs had sprouted, so no data are presented for Downing's
Yellow Globe stored at 10°C for 16 weeks.

After removal from storage, the-onions were frozen and
kept at -18°C until extracted. The bulbs were allowed to
thaw for 4 hours. The shoots were then excised, frozen in
liquid nitrogen, homogenized with absolute-methanol in a
Waring blender, and extracted with constant shaking for 48
hours at room temperature. The extractions were run in
duplicate for each treatment. After filtration through
double Whatman paper No: 3 and Celite 535, the methanolic
extracts were-concentrated in 33339 at 40°C. The dry residues
were resuspended in 0.33 M KH2P04 buffer at pH 8 and petroleum
ether. ‘The aqueous phase was partioned 5 times with fresh
petroleum ether and the petroleum phase similarly partitioned
5 times with fresh buffer. The petroleum ether phase was dis-
carded. The aqueous phase was further-partitioned 3 times
with ethyl acetate, the latter being discarded.‘ The pH of the
aqueous layer was adjusted to 2.5 with HCl, and extracted 5
times with ethyl acetate. After the excess water had been
frozen out, the ethyl acetate fraction was evaporated to dryness
in_v§gug and the residue resuspended in absolute ethanol. This

ethanolic solution constituted the free acidic fraction and

was stored at 5°C until used.

27

Chromatography

 

Duplicate aliquots of 10 grams of the acidic fraction were
evaporated in_zagug, dissolved in 0.2 ml of absolute ethanol,
and streaked on 100 micron thick silica gel thin-layer chroma-
tograms (Eastman Kodak Co.). Each aliquot was applied to a
section of a plate 6'cm wide, leaving a third section for use
as a blank control. The plates were developed (ascending) to
a height of 15 cm in iSOpropan01:ammoninmxhydroxide:water
(10:1:1, v/V). The developing tank?was lined with filter paper
and equilibrated for 48 hours before use. .Four chromatograms
(i.e., 2 per extraction), were developed for each treatment.
After drying, the chromatograms were cut into ten 1.5 cm sec-
tions representing 1 Rf unit, and eluted with absolute ethanol
for 12 hours. The eluates were evaporated to dryness in 32329,
and aliquots were removed for assay with wheat coleoptile sec-
tions.

Once the zone of inhibition had been established by bio-
assay, eluates from the active region of the chromatogram were
combined, and aliquots representing 2 gram-equivalents fresh
weight of tissue were removed. Relative levels of inhibitors
were determined by assaying these aliquots with wheat coleoptile

sections.

Wheat Coleoptile Bioassay
The general procedure followed was that described by
Nitsch and Nitsch (91). Genesee-wheat seeds were used through-

out the experiment. The seeds were germinated in the dark at

28

25°C. When the coleoptiles were 2.5-3.0 cm long, 4-mm sec-
tions were cut 3 mm from the tip under green light. Five
sections were placed in a test tube containing 0.3 ml of 2

3M citrate buffer

percent sucrose, lO-ZM phosphate and 5 x 10-
(pH 5.0). A clinostat prevented geotropic curvature.
A microscope provided with a micrometer eyepiece was used to

measure the sections after 24 hours incubation in the dark.

Characterization of the Inhibitor

 

Thin Layer and Column Chromatography

 

An aliquot of the free acidic fraction (p. 27), equivalent
to 8 grams of onion shoot, was chromatographed on silica gel
thin-layer plates with fluorescent indicator. ‘Four different
solvent systems were used:

- Isopropanol:ammonia: water (10:1:1 v/v/v)
- Benzene:acetic acid:water (8:3:5 v/v/v)

- l-Butanol:n-propanol:ammonia:water (2:6:l:2 v/v/v/v)
- l-Butanol:acetic acid:water (40:11:29 v/v/v)

bWNI—d

For further identification, onion extract and synthetic
ABA were separately chromatographed in solvent 1. Standard
ABA was located by examining the plates under ultraviolet light,
and the corresponding zone on the extract plate was scraped and
methylated with diazald(N-methyl—N-nitroso-p-toluene-sulfonamide)
according to Schlenk and Gellerman (112) as modified by Powell
(103). Diethylether was substituted for the methylene chloride.

The methylation procedure involved:

29

1 - Dissolving the sample in a 1:9 methanol:ether
solution.

2 - Generating diazomethane from a test tube containing
1.5 ml of carbitol(2-(2-ethoxy-ethoxy)ethanol),
1.0 ml of 60% potassium hydroxide, and 1.5 ml of a
saturated solution of diazald(N-methyl-N-nitroso-p-
toluenesulfonamide) in ether into the sample tube in
a stream of nitrogen saturated with ether.

3 - Stopping the reaction upon appearance of a yellow
color in the sample tube.

4 - Evaporating the methanol:ether solution of the sample
tube under a stream of nitrogen.

Both the methylated extract and methylated synthetic ABA
were chromatographed on silica gel thin-layer plates in
solvent 1. The location of synthetic Me-ABA was detected under
UV light. Eluates of this region were bioassayed with wheat
coleoptile sections. Non-methylated ABA was chromatographed
as a control.

Synthetic ABA and the acidic fraction were also chromato-
graphhd on charcoal-celite (1:2) columns (charcoal:Darco G-60:
Celite-535:Johns-Manville, Lompac, California. One hundred
fifty m1 of 60 percent acetone in water was used for elution.
Ten-ml fractions were collected, and evaporated under reduced
pressure at 40°C, and the residues were assayed with wheat

coleoptile sections.

Gas-liquid Chromatography and Mass
Spectrometry

 

Eluate from the zone of the highest biological activity
(Rf 0.4-0.7) on TLC silica gel plates developed in solvent 1,
and the active tubes (4 to 10) from the charcoal celite column

were methylated as previously described.

30

Methylated samples were dissolved in chloroform before
injection into the Packard 7300 series gas-liquid chromato-
graph with flame ionization or electron capture detector.
One g ~equivalent of tissue and 1 ng authentic ABA were in-
jected.

The samples were then dissolved in hexane and analyzed
in LKB 9000 Gas Chromatograph-Mass Spectrometer whose condi-

tions are also described in Table l.

31

 

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mamas omafloo pm 4

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UoOHN

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luouuuumso msu ca coma muuosouuoomm mode can armoumouoeomzo mom mo.m:0flvfio:oo

.H.OHQMB

RESULTS AND DISCUSSION

Field Studies of Growth and Maturation

 

The time of occurrence of maximum linear growth coincides
with that for maximum bulbing index for each variety (Figure
2, Table 2). Senescence begins simultaneously for varieties
and for linear growth and bulbing index.~ These changes
occurred in Downing's Yellow Globe about one week earlier than
in MSU 4535. MSU 2935, a long dormant line, originally was
included in this study. Unfortunately, the season was too
short'and this variety was not harvested because of immaturity
at harvest time. The growth curve for this variety (data not
shown) was a straight line.

No comparison of the growth of Abundance, Downing's
Yellow Globe, and Spartan Banner was made in summer 1971.
However, Abundance was first to mature, followed by Downing's
Yellow Globe.' At the time of harvest, when frosts were im-
minent, 50 percent of the'Spartan Banner tops were still
green, necessitating careful selection of bulbs with dry tops.

Good keeping onions thus appear to have a longer growing
period than poor keeping varieties. Tronickova's (128,129)

data supports this conclusion. 'Abdalla and Mann (1) observed

32

33

Figure 2. Bulb development of Downing's Yellow Globe and
MSU 4535 as measured by the diameter of the
leaf base. Measurements were started 38 days
after sowing. Each point is the average of 40
plants.

Diomeler oI the leaf base

(In In)

30

no
0

8

O

34

 

W
7’6 7'13 7’20 7’26 8’4 8(lo 8’w 9’23 3'3: 9’8

Date

Figure 2

35

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one mo HouoEuHc EsEHCHE o» omen on» mo

ow mo some one we ouomfim comm .Aamv xooc
HouoEMHo EDEonE mo oaumm ”xoocfl mcwnasma

 

 

 

mm.a mm.H om.H oh.H mv.a om.a NH.H mmmv sz
vm.a mm.a oo.~. om.a mn.H mv.H mH.H macaw
Boaaow m.mcflc3oo
mxm Hm\m mm\m aa\m oa\m sxm maxs
mama Sawflum>

 

 

.ucoEmoHo>oo mcflusc mmmv sz cam cacao BoHHo» m.mcHQBOQ

.osma
mo mmoaocfi maflnasm .~ «Home

36

that under California conditions, the variety "Excel", a
short dormant type, matured one month earlier than Australian
Brown 5, a long dormant one.

Dormancy in onion is obviously genetically controlled.
.Correlations between growth rate and keeping quality are
possibly due to the synthesis-of some substances during the
growing period which accumulate in the bulbs and prolong the
dormant period. Katofs (65) data showing that the auxin
content increased earlier in an early variety than in a mid-

season variety are also a plausible explanation.

Effect of Temperature During Storage
of Whole Bulbs

 

Continuous Temperature

 

The sprouting response of Downing's Yellow Globe and MSU
4535 held at 2 temperatures are compared in Figure 3. Downing's
Yellow Globe sprouted at a faster rate than MSU 4535. Under
dry conditions, 10°C appeared to be more effective than 0°C
in breaking dormancy in MSU 4535, while for Downing's Yellow
Globe 0°C was more effective. Considerable rotting and de-
terioration occurred in the bulbs placed in water after storage,
thus the data are not reported.’ In general, 10°C was more
effective in breaking dormancy in MSU 4535 bulbs placed in
water than either 0 or 20°C. Conversely 0°C was more effective
for Downing's Yellow Globe while 10 and 20°C tended to delay

sprouting.

Figure 3.

37

Effect of storage temperatures (O-lO°C) on
subsequent sprouting of MSU 4535 and Downing's
Yellow Globe onion bulbs exposed to 20°C in dry
air. The controls were put to sprout in dry air
immediately after curing. Incomplete graphs
indicate that bulbs had sprouted in storage
before transfer to 20°C. Each point is based on
a 20-bulb sample.

IO

Weeks for 50% Spmuling
O

b

O

38

cv:Yel low Globe
control

 

42w|rs
:—- ”I. —————— 4 II
. Owls

II

.16

 

0 IO
Temperature'C

Figure 3

39

A similar experiment with Spartan Banner was undertaken
in 1971. In contrast to the results obtained with MSU 4535
and Downing's Yellow Globe, all temperatures were effective
in promoting Sprouting. After 16 weeks, the most rapid
sprouting occurred in bulbs stored at 20°C, the slowest in
those stored at 0°C (Figure 4).

Root formation as well as root development in response
to temperature showed the same trends as Sprouting (Figures
5 and 6) but the differences between temperatures were less
marked. Early Sprouting was associated with better root
develOpment at 20 and 30°C.

The smaller the bulbs, the more quickly they sprouted
and rooted when transferred to moist conditions at 20°C
(Figures 7 and 8).

Regardless of variety and temperature, increasing the
storage period reduced the time required for rooting and
sprouting.~ However, differences were-observed in Sprouting
of the varieties. Dormancy in onion-is therefore a hereditary
property, with the beginning, duration and the-end of rest
genetically controlled.

Magruder gt El' (85), reported differences in keeping
quality among onion varieties. Abdalla and Mann (1) found
that Australian Brown 5 and Excel responded differently to
identical temperature. Although the internal conditions of the

onion bulbs at the time of storage as well as-the duration of

Figure 4.

40

Effect of storage temperatures (0-30°C) on
subsequent sprouting of Spartan Banner onion
bulbs exposed at 20°C with their base in moist
peat moss. Each point is the average of 3
replicates of 20 bulbs each. The controls were
planted in the moist peat moss immediately after
curing. Vertical bars = Standard error.

on
O

50 percent sgrourkrg
3 o o 3

Do sfor
I: 3

8

41

 

 

 

 

S . Bo nn e r
control

f—* I

I .............. 8 wk,

"-4 I
I 1°... ..O’.1 low,
”“4...
o 5 no 20 so

Temperature '

Figure 4

42

Figure 5. Effect of storage temperatures (0-30°C) on
rooting of Spartan Banner onion bulbs planted
in moist peat moss at 20°C after storage for
8 and 16 weeks. The controls were planted
immediately after curing. Vertical bars =
Standard error.

Figure 6. Root growth and development of Spartan Banner
onion bulbs planted in moist peat moss at 20°C
after 8 weeks of temperature treatment (0-30°C).
Root measurement was made 10 days after planting.
Vertical bars = Standard error.

m9
0

Days for supercenr
roo

Weiglmgms)

43

control

 

 

._l

5 IO 20 30
Temperature'c

Figure 5

 

 

lengrhmn)

Temperotu re 1:

Figure 6

Figure 7.

Figure 8.

44

Effect of size on sprouting of Spartan Banner
onion bulbs planted in moist peat moss at 20°C
after storage for 8 weeks at 10°C. The controls
were planted immediately after curing.

A = Small size bulbs (4-5 cm diam.)
B = Medium Size (7-8 cm diam.)
C = Large size (10-11 cm diam.)

Vertical bars = Standard error.

Effect of size on rooting of Spartan Banner onion
bulbs planted in moist peat moss at 20°C after
storage for 8 weeks at 10°C. The controls (before
storage) were planted immediately after curing.

A = Small Size bulbs (4-5 cm diam.)
B = Medium size (7-8 cm diam.)
C = Large Size (10-11 cm diam.)

Vertical bars = Standard error.

    
  
 

3

8

retreat sprouting

8

Raptor 50

 

 

 

"l

 

 

 

 

 

 

 

 

 

 

A

Figure 7

 

 

 

45

El control

it: after 8 wks
ot IO’C

I] control
SLO. I°¥t

.8

«b
0
___q____

8

 

 

5

Days for so percent rooting

 

 

 

mun-00.0.01

 

iii
ABC

Figure 8

O

46

the storage are important, most of the varieties have Specific
temperature requirements to break their dormancy.

The size of onion bulbs affects flowering and seed produc-
tion. The plants developing from small bulbs tend to remain
vegetative while plants from medium and large Size bulbs are
more inclined to produce flowers and seeds (2,3,88,115).

Small Size bulbs also produce flowers earlier than large bulbs
(3). Size may also affect dormancy. Small excised plugs from
potato tubers sprout earlier than large ones (107). Possibly
the lower resistance of small bulbs to temperature change
accounts for this observation. Aura’s finding (6) that small
onion bulbs required a shorter heat treatment to prevent
flowering than did large onesrsupports this hypothesis.

Rooting precedes sprouting." Since it appears to have a
major effect on Sprout emergence, temperature may modify
onion shoot dormancy through its effect'on root dormancy. The
data in Figures 5 and 8 Show that there is root dormancy in
the onion which disappears with time. 'This dormancy, in gen-
eral, is affected similarly to shoot dormancy by temperature
except that it is broken earlier. The effect of storage
temperature on root growth'as expressed by length and weight
(Figures 6 vs 4) again'suggests~that'temperature influences
onion shoot dormancy through its effect on the root, in the
sense that whenever'root initiation and growth are promoted,
shoot growth is also enhanced:' Conversely, frequent observa-

tions revealed that in general unsound bulbs may Sprout without

47

forming roots and did not subsequently root. Apparently, the
growing Sprout depletesthe food reserves at the expense of
the root primordia. Lathrop and Mecklenburg's (75) data
indicating in Taxus Sp that with the increase in shoot growth
after dormancy there iS'a concomittant decrease in root re-
generation potential, supports this hypothesis.

Effects of Intermittent Temperature, Root

Removal and Treatment with Kinetin on
Sprouting

 

Table 3 shows the effetts of intermittent temperature and
root removal on the subsequent sprouting of Abundance onions.
The controls were placed in the rooting medium without any
prior temperature treatment.~ High temperature (30°C) delayed
Sprouting or induced secondary dormancy when applied after a
low temperature treatment. ‘However, the effects of high temper-
ature were completely offset by an additional 3 weeks of 10°C.
Rooting Showed the same trend as sprouting. Root removal
delayed sprouting. A Similar effect is shown in Table 4 for
root removal, but the effect of the root could be partially
restored by kinetin application.

Abdalla (1) also Showed that root removal could delay
sprouting of onion. Shoot elongation may be dependent upon
the supply of water and nutrient elements from the roots.

In our experiment, however, the derooted onion bases were in
direct contact with the moist medium, so water deficiency prob-

ably cannot account for the result. Nutrient supply probably

48

Table 3. Effect of intermittent warm~cold temperature on
Sprouting and rooting, and the effect of root
removal on sprouting of Abundance onion bulbs
planted at 20°C in moist peat moss (days to 50%
Sprouting or rooting).

 

 

 

Treatment Sprouting Rooting
Control, not 35.2 i 2.69 13.7 i 6.14
stored

8 wks at 10°C 17 3 i 2.07 3.00 i 0.82
8 wks at 10°C; 30 4 i 2.85 15.5 i 0.97
2 wks at 30°C

3 wks at 10°C; 8.66 + 1 69 2.00 i 0.00

2 wks at 30°C;
3 wks at 10°C

8 wks at 10°C,
then derooted
daily.

 

 

23.67 + 1.67

 

 

 

49

Table 4. Effect of root removal and kinetin treatment on
sprouting of Spartan Banner onion bulbs planted
at 20°C in moist peat moss (days to 50% sprouting).

 

 

 

Treatment Sprouting
Control, not stored 32.70 i 2.51
8 weeks at 0°C 12.50 i 1.03
8 weeks at 0°C; 21.50 i 1.07

injected with water
and derooted daily

8 weeks at 0°C; 16.50 i 1.00
injected with kinetin
and derooted daily

 

 

 

 

50

is not a critical factor because in the early stage of shoot
growth, the fleshy scales surrounding the shoot apices can
provide the necessary nutrients. Thus the root may be provid-
ing something that neither the medium nor the scales can pro-
vide.

The fact that kinetin could partially substitute for the
roots suggests that the latter provide growth substances.
Went (139) hypothesized that a Specific shoot factor,
caulocaline, was synthesized in the roots. There is now
direct and indirect evidence that the roots are the source
of cytokinin for the aerial parts of many plant Species
(22,63,83,70,ll4). A similar Situation may exist in onion,
but further study is needed to characterize this hormonal

substance.

Effect of Wounding and Oxygen Treatment
of Unchilled Bulbs on Sprouting
and Rooting

 

While transverse wounding was more effective in promoting
sprouting of both cultivars than was longitudinal wounding,
the latter promoted rooting while the former did not (Table 5).
Oxygen application did not alleviate onion dormancy; it even
prolonged it in Abundance. Thus, lack of oxygen cannot be
responsible for dormancy in onion. A Similar conclusion was
reached for Phacelia tanacetifolia seeds (25) and for potato
(lll). Consequently, some other explanations should be

offered for the wounding effect.

51

 

 

 

 

 

 

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m m m Ha Honucou

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oocoocsn< mmmv sz

 

 

 

 

 

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oocomuofio noon can vacuum :0 sommxo wooa coo mcflocso3 mo uoommm. .m canoe

 

Wounding of plant organs leads to numerous metabolic
changes, including an increase in respiration (11,119),
renewed capacity for protein synthesis (42,43), increase in
mitochondrial number (77), RNA synthesis (76) and phospho-
lipid synthesis (121). The onset of renewed growth may be
related to changes in hormonal balance (16,56,116). Slicing
of apple resulted in an increase of ethylene synthesis (79).

'Similar changes probably occur in onion following wounding.

Growth of Excised Apices

 

Effect of Storage Temperature on‘
Growth of Apices in sitn

 

 

Growth of shoot apices during storage of whole bulbs was
greatest at 10°, least at 0°, and intermediate at 20 or 30°C,
for all cultivars (Figure 9). 'Response~of Downing's Yellow
Globe is omitted as-it was identical with that of MSU 4535.

Abdalla and Mann‘(1), performing'a Similar experiment
with the cultivar Excel found that 0 and 30°C retarded growth
of the'intact'shoot."PeriodiC'observatio 'of sprout leaf
elongation inside the bulbs can be‘a fairly accurate way to
ascertain the degree of dormancy without waiting weeks or
months for visible sprouting. 'These results confirm that at
'intermediate temperatures of 10-20°C onion bulbs tend to
sprout in storage at a higher“rate’than“at'low (0°) or high

temperature (30°C).

Figure 9.

53

Effect of storage temperature on growth of
intact inner sprout leaves of MSU 4535 and
Spartan Banner onion. Each point is the mean
of 6 sprout leaves excised from 6 uniform
bulbs for MSU 4535 and 20 for Spartan Banner.

54

4535

 

  

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3 8
4:
‘-
2’
34 SJonner
lot
3
30':
:2 fl’.’—").<:

() i\-‘L--l--‘l--JLIII
. . l6
'I'Ime:wks In storage

Figure 9

55

Growth of Excised Apices

 

Effective chemical treatment of onion bulbs is difficult
because of the protected position of the apices, i.e., the
potential sprout leaves. In addition, one bulb can contain
up to 3-6 shoots, making accurate injection of solutions
difficult. Furthermore, variability exists in the degree of
dormancy in the same lot of bulbs, requiring the use of many
onions to reduce experimental error. Since color change of
the Shoots is a fairly accurate index of dormancy (58), the
use of excised'shoots allows selection of uniform material.
Although shoot preparation requires some Skill and is time
consuming, response to treatment can be obtained more quickly
than with entire bulbs. Finally, this system in many instances
constitutes a check for the response of whole bulb to some
treatments.

Effect of Storage Temperature Upon
Subsequent Growth of Excised

Apices

Five weeks of storage markedly promoted growth of Sprout
leaves fpllowing their excision from the bulbs (Figure 10).
Temperatures of 0 and 10°C were equally effective on Downing's
Yellow Globe while 20°C was much less effective. Results were
similar for MSU 4535 except that 10°C was only Slightly more

effective than 20°C. Eight weeks at 0°C produced maximal

growth in both cultivars.

Figure 10.

56

Elongation of excised Sprout leaves as influenced
by previous storage temperature. Excised Sprout
leaves were planted in moist sand in the dark at
20°C and growth measurement was recorded at dif-
ferent periods. Each point represents the average
of 6 excised sprout leaves.

MSU 4535
Downing's Yellow Globe

A
B

200

160

I20

A on
0 O

0

Percent initial length
3
0

Growth
5
0

I20

80

40

57

A

........ 8 win at 0°C
_. kas at 0°C
___ 5 wks at 10°C
-—- 5 wks at 20‘C ...-
.. ._ control,not stored ...;

   

’.‘e-e-e- 0 -0-0-0-

0

‘0’
0‘.

.—o‘

 

 

4-
......m Swim at 0°C
_ Sula at UC ...
--- SWIG at IO'C 0,:
--.- swim cnoc
_ . _ control .not stored ,’
.- ’1
I
I
I
I
I
I
I
I I’
r ’1
. I
I
I
I
I
’I
I
." ’l”
..' ’a
... I
. 0'2"” ............... .-
24 48 72 96 I20
Time:hrs

Figure 10

58

Data for Spartan Banner (Figure 11) Show similar trends.
After 8 weeks storage, apices from bulbs stored at 0°C grew
the most, while 30 and 10°C were much less effective. After
16 weeks storage, however, 0 and 30°C were equally effective.
Growthcnfshoots $2.32EE was least at 0° (Figure 9). Yet
this storage temperature was most effective in promoting subse-
quent growth of excised Shoots at 20°C. Thus, 0°C is probably
effective for storage of onions, not because it keeps them
dormant, but because it prevents or retards Shoot growth.
A similar result is reported by Boswell (15) with bulbs
planted in the field. Bulbs stored at 0°C produced more vigor-
ous plants than those stored at higher temperatures. The ef-
ficiency of the 0°C treatment in promoting subsequent growth
of the excised Shoots may be attributed to lower utilization
of food reserves, during the low temperature treatment as a
result of reduced respiration. These materials were, there-
fore, available when the excised Shoots were placed at 20°C.
Effect of Cutting Bulbs Prior to Tempera-

ture Treatment Upon Subsequent Growth of
Excised Apices

 

 

 

Shoot apices directly exposed to the low temperature
treatment grew more when planted in moist sand at 20°C than
those excised from similarly treated halfecut or intact bulbs
(Tables 6 and 7). Although the growth of the shoots excised
from the half-cut Xi intact bulbs in experiment 2 did not

differ signifiCantly, the former did grow more. Thus the

Figure 11.

59

Elongation of sprout leaves excised from
Spartan Banner onion bulbs after exposure to
various storage temperatures for 8 and 16
weeks. The sprout leaves were planted in
moist sand for 96 hours in the dark at 20°C.
Each point is the average of 20 Sprout leaves.
Vertical bars = Standard error.

60

 

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w 8
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Figure 11

61

Table 6. Effect of exposure of onion Shoots, half-cut bulbs
and whole bulbs to temperature (10°C) for different
period of time on the subsequent growth of excised
shoots planted in moist sand in the dark at 20°C
for 96 hours (cv Spartan Banner grown in greenhouse).
Growth: percent increase over initial length.

 

 

Experiment 1*

 

Time of Exposure (hrs)

 

Treatment 0 24 48 72

 

Excised shoots 40.2:5.6 76.03:3.01 9o.5:3.2o 101.0012.7o
Half-cut bulbs 55.5o:2.3o 61.25:1.75 70.80:3.00

Whole bulbs 4o.55:4.oo 45.23:2.8 46.70:3.60

 

*Means and standard error for 10 shoots.

Table 7. Effect of exposure of onion shoots, half-cut bulbs
and whole bulbs to 10°C for 96 hours on the subse-
quent growth of excised shoots planted in moist sand
in the dark at 20°C for 96 hours (cv Spartan Banner
grown in the field). Growth: percent increase over
initial length.

 

 

Experiment 2*

 

 

 

Treatment
Control, , Excised
not chilled Half-cut bulb Whole bulb shoot
20.50a 40.57b 34.22b 49.00d

 

*Means followed by the same letter are not significant at the
5% level.

62

onion scales may play a role in dormancy. However, the Shoot
apex appears to be the primary receptor of the temperature
stimulus.

Effect of Temperature and Length of

Exposure Upon Subsequent Growth off
Isolated Apices

 

 

The period of exposure, as well as temperature, affect
the subsequent growth of the Shoots (see Figure 12). The in-
creasing order of effectiveness, whatever the period of
exposure, is 0, 5 and 10°C. Response at each temperature in-
creased with increasing time of exposure. Two additional
experiments were designed to determine the optimum temperature
(Table 8). In experiment 1, growth was maximal at 5 and 10°C,
intermediate at 0°, and minimal at 20°C. The second experi-
ment indicated that the temperature range 7.5-12.5°C was much
more effective than the control and 20°C.

By comparison of these data with those for apices ip,§itu_
(Figure 9), the effectiveness of 10°C and the ineffectiveness
of 20°C are again confirmed. A contradiction, however, arises
from comparison with Figures 10 and 11. In Table 8, 0° is less
effective than 10°C, while in Figures 10 and 11 it is more
effective. However, the data in Figures 10 and 11 were ob-
tained with Shoot apices excised from bulbs subjected to
temperature treatment for 5 to 16 weeks while those in Table 8
are for shoot apices exposed for a short period of time. Thus

the environmental conditions of the apices were not the same.

63

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64

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musos om umumm Aumcamm amunmmm >ov yoonm coaco cmmfloxm Mo Anumcma Hmwuwcw
mo usmoummv £u3oum unflumasfiflum :H mmnsumnmmfimu ucmummmww mo mmmcm>fluommmm

.m GHQMB

66

In excised apices, the synthesis of materials necessary
for growth apparently occurs at a faster rate at 10°C than at
O or 5°C, but since the exposure is brief their utilization

in situ is limited. Thus they are available for growth when

 

the shoots are transferred to a suitable environment. During
long periods of storage, on the other hand, the turnover rate
of the synthesized material ifl.§iEE‘WOUId be higher. Shoot
apices excised from bulbs previously stored at 20°C were
partially depleted of reserves and consequently the growth is
less.

Comparison of the temperature responses of shoot apices
with those of entire bulbs (Table 8 XE Figures 3 and 4) shows
that, in general, intact bulbs and shoot apices respond simi-
larly to temperature except that the shoot apices are less
responsive to high temperature (20°C). In both cases, 10°C
appears to be most effective in promoting growth. However,
20°C is also effective for intact Spartan Banner bulbs
(Figure 4).

The possible physiological importance of the scales should
not be minimized. Besides the reserve material that they pro-
vide for the growth of the shoots, they probably participate
in the dormancy-breaking process, perhaps by serving as insula-
tion.

Effect of High Temperature in Blocking
the Response to Low Temperature

 

The effect of intermittent temperature treatments on the

subsequent growth of the shoot apices is illustrated in Table 9.

67

Effect of high temperature (30°C9 in inducing

 

 

 

 

Table 9.
secondary dormancy in onion shoots (cv Spartan
Banner) and its reversal by subsequent low temper-
ature treatment (10°C). Growth is expressed as
percent increase over initial length after 96 hours
in moist sand in dark at 20°C.

Growth*

Treatment Experiment 1 Experiment 2

Control, shoot a a

not stored 20.15 20.35

0 b b

96 hours at 10 C 39.78 44.92

96 hours at 10°c+ 16.25a ~--

24 hours at 30°C

96 hours at 10°c+ 55.20C ---

24 hours at 20°C

96 hours at 10°C b

24 hours at 30°C --- 38.47

48 hours at 10°C

 

*Means followed by the same letter are not significantly
different at the 5% level.

68

Exposure to a temperature of 30°C following the low temperature
treatment induced secondary dormancy of the shoot apieces
(Experiment 1), but this secondary dormancy was easfily reversed
by a relatively short exposure to low temperature (Experiment
2). Exposure to 20°C following the low temperature treatment
was promotive. Comparison with the response of intact bulbs
(Table 3) shows a striking similarity.

The cumulative effect of the low temperature treatment
is illustrated in Table 10. Again the data indicate that low
temperature need not be applied continuously to be effective,
provided that the high temperature is not high enough to cause
injury. In fact, interruption of the low temperature treat-
ment by a period at 20°C resulted in some promotion of growth.
Response increased with length of exposure to low temperature.
Thus low temperature treatment appears to favor synthesis of
growth promoters in the tissue. Increasing the exposure in-
creases the accumulation of material, stimulates subsequent
growth when placed at 20°C. The failure of rapid vacuum cool-
ing, as practiced by Jaffe and Isenberg (60), to elicit sig-
nificant growth supports this hypothesis.

In the intermittent temperature experiment, again the
shoots behave the same way as the whole bulbs. Boden (12)
found that alternating temperature promoted germination of

dormant Eucalyptus pauciflora seeds. Some evidence for an

 

antagonistic effect of high temperature on peach bud dormancy

has been reported (10,96,138). However, high temperature

69

Table 10. Cumulative effect of low temperature treatment
(10°C) in stimulating growth of excised onion
shoots (cv Abundance) after planting in moist
sand in dark at 20°C. Growth is expressed as
percent increase over initial length.*

 

 

 

Treatment ~ r~‘- ' Growth
a

Control 24.23

96 hours at 10°C 45.49bC

48 hours at 10°C- c
24 hours at 20°C- 51.27
48 hours at 10°C

48 hours at 10°C 39.15

96 hours at 20°C 18.36a

 

*Means followed by the same letter are not significantly
different at the 5% level.

70

(30°C) is effective in inducing secondary dormancy of onion
only when applied after a low temperature treatment (Table 9).
A temperature of 20°C, although ineffective by itself, pro-
motes growth when applied either after or between the low
temperature treatment. This finding does not agree with the
statement of Vegis (132) that "even a moderate rise in tempera-
ture at an early stage of post-dormancy can induce secondary

dormancy" (p. 191).

Effects of Chemical Treatment

 

A preliminary experiment with intact bulbs indicated

no effect of NAA, ethephon or GA on Spartan Banner or

3
Abundance, whereas kinetin hastened sprouting in both.

The effect of kinetin, IAA, GA GA4/7, and ethephon on

3'
excised onion shoots are presented in Table 11. Kinetin was
again the only chemical effective in promoting growth.
Ethephon and IAA were somewhat inhibitory, while GA3 and

GA had no effect.

4/7
The effects of IAA and low temperature in a factorial

experiment are shown in Figure 13. At a concentration of 1

ppm, IAA inhibited growth of nonchilled apices but promoted

growth of chilled apices. Interaction was highly significant.
To determine if Amo 1618 would interfere with the response

to chilling, this chemical was applied either before or after

a low temperature treatment (Table 12). The former treatment

markedly reduced shoot growth in comparison with the chilled

control while the after-storage treatment had no effect.

71

Table 11. Effect of growth regulators on growth of excised
onion shoots (cv Spartan Banner). Shoots were
not chilled. Growth: percent initial length.*

 

 

 

Treatment Growth
Water Control 18.64a
Kinetin, 100 ppm 37.13b
IAA: 10 ppm 17.19a
6A3, 1000 ppm 18.83a
GA4/7 1000 ppm 18.67a
Ethephon, 100 ppm 14.83a

 

*Means with the same letter are not significantly different
at the 5% level.

Figure 13.

72

Effect of IAA and chilling on growth of shoots
(Spartan Banner). Chilled shoots were held at
10°C for 96 hours. IAA (1 ppm) was applied
before chilling. The main effect and chilling
x IAA are significant at the 1% level. IAA is
not significant at either 1% or 5% level.

73

Figure l3

74

Table 12. Effects of Amo 1618 and chilling on the growth
of excised onion shoots (cv Abundance). Amo
1618 was applied before and after chilling.
Growth: percent initial length.*

 

 

 

Treatment Growth
Water control 14.40a
96 hours at 10°C 42.54°

Amo 1618, then
96 hours at 10°C 26.50

96 hours at 10°C c
Amo 1618 after- 44.34

 

*Means followed by the same letter are not significantly
different at the 5% level.

75

These results suggest that gibberellins were synthesized
during the chilling treatment.

In preliminary experiments, sucrose was more effective
than either glucose or fructose in promoting growth of non-
chilled excised apices. Sucrose was as effective as chilling
(Table 13) in promoting growth of non-chilled onion shoots
but had little effect on chilled shoots. Amo 1618 was not
tested on non-chilled shoots, but it inhibited growth of
chilled shoots. However, the synergistic effect of sucrose
plus Amo 1618 was greater than that of sucrose alone (Table
13).

The effects of kinetin (Figure 14) were similar to those
of sucrose (Table 13) in that both promoted growth of non-
chilled shoots, but had no effect on chilled shoots. Kinetin
greatly overcame the inhibitory effect of Amo 1618 of non-
chilled shootstnn:was only partially effective on chilled
shoots. Sucrose was not tested in combination with Amo 1618
on non-chilled shoots but it completely reversed the effect
of the latter on chilled shoots, the response being even
greater than that obtained with sudrose alone (Table 13).

Contrary to the results with Amo 1618, ABA was effective
in inhibiting growth even when applied after temperature treat-
ment. Sucrose was without effect on ABA action, and kinetin
was only slightly effective in overcoming the ABA effect

(Table 14).

76

Table 13. Effect of sucrose, Amo 1618 and chilling on growth
of excised onion shoots (cv Downing's Yellow Globe).
Growth: percent initial length.*

 

 

 

Treatment Growth
Water control 20.35a
Sucrose, 10% 38.99b
96 hours at 10°C 39.18b

Sucrose — 96 hours
at 10°C 44.55

Amo 1618 - 96 hours a
at 10°C 19.75

Amo 1618 - Sucrose c
96 hours at 10°C 54.77

 

*Means followed by the same letter are not significantly dif-
ferent at the 5% level.

77

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78

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79

Table 14. Effect of ABA, kinetin + ABA, sucrose + ABA
with and without chilling on growth of excised
shoots (cv Spartan Banner). Growth: percent
initial length*

 

 

 

Chemical treatment** - Not chilled ‘ Chilled
None 16.03ab 37.62C
ABA, 1 ppm before chilling 9.01a 7.69a
After chilling --- 11.15ab
Kinetin, 100 ppm 40.23C ---
ABA + kinetin 13.99ab 18.35b
ABA + sucrose, 10% 7.12a 7.28a

*Means followed by th- same letter are not significantly
different at the 5% level.

**Applied before chilling except where otherwise noted.

80

The interaction of auxin with chilling (Figure 13)
deserves some consideration, for similar results were obtained
by Guthrie with potato tubers (49). Evidence exists that
auxin action depends on the presence of gibberallins (92).
Thus, IAA may synergise with gibberellins, synthesized during
the low temperature treatment.

' Kinetin breaks dormancy of buds and seeds of some species
(48,53,72,l40), but its mechanism of action is at present
unknown. It reportedly promotes nucleic acid and protein syn-
thesis (93), but more critical studies suggest that its ef-
fects are due to suppression of protein degradation (73,122).
Cytokinins may also affect carbohydrate metabolism in plants
(13), thus making available reserve materials for growth.
Similar processes may occur in onion shoots treated with
kinetin. The finding that sucrose could substitute for the low
temperature treatment in promoting growth (Table 13) seems to
support this hypothesis. In addition, the finding that kinetin
can remove the block to growth imposed by inhibitors and
increase some promoters (27,53) introduces a new dimension in
the interpretation of the role of this hormone in the dormancy
release process.

Gibberellic acid breaks dormancy in buds and seeds (40,
48,86,99,104). However, it fails to do so in onions (55,125).
Many cases, in fact, are known where GA3 fails to break dor-
mancy (17,18,137). However, this failure does not exclude the

participation of gibberellins in the dormancy breaking process,

81

for they may act synergistically with other hormones.
Furthermore, some three dozen different gibberellins are now
known (37), and each one may have a specific physiological

role. Thus, gibberellins other than GA and GA4/7 may be

3
involved in the breaking of dormancy in onion.

Ethylene, a fruit-ripening hormone (19,20,104), is now
generally accepted as a plant hormone ranking in importance
with the auxins, gibberellins and cytokinins as a controlling
factor in many physiological processes, including sprouting
of corms (122) and germination of some species of seeds (8,
127). However, ethephon, an ethylene-releasing compound
(35), did not break dormancy in onion. Ethylene chlorhydrin
does not break dormancy in onion, and the failure has been
attributed to insufficient starch (82). Whether or not the

presence of critical levels of starch in the tissue is a

sine qua non for the effectiveness of ethylene remains to be

 

determined experimentally. However, its efficiency in the
case of potato and gladiolus corms (38,82) seems to support
such a hypothesis.

Perhaps onion dormancy may be partially controlled by
carbohydrate level, which is, in turn dependent upon hormonal-
ly regulated enzymatic activity. Promotive effect of sucrose
on growth of tomato roots and seed germination has been
reported (26,124).

The growth retardants CCC and Amo 1618 are both potent

inhibitors of GA biosynthesis and most of their physiological

82

effects are attributed to this property (7,18,23,69,94).

In some cases, chilling predisposes a tissue to synthesize
gibberellins but actual synthesis takes place after transfer
to a higher temperature (110). That Amo 1618 nullified the
chilling effect when applied before but not after temperature
treatment suggests that a growth promoter is synthesized dur-
ing the low temperature treatment and exerts its effect when
the tissue is transferred to conditions suitable for growth.
This hypothesis is supported by Thomas' finding that CCC had
no effect on sprouting of onion previously subjected to low
temperature (125).

The fact that sucrose is more effective than kinetin
(Table 13 Kg Figure 14) in overcoming the effect of Amo 1618
is another indication of possible involvement of gibberellins.
GA has been reported to promote synthesis and/or release of
hydrolytic enzymes leading to the degradation of starch in
certain seeds (29,97,101). Abscisic acid inhibits shoot
growth whether applied before or after chilling (Table 14).
Kinetin only partially overcomes the ABA effect while sucrose
is ineffective. Thus the modes of action of Amo 1618 and ABA
appear to differ. The failure of kinetin to completely over—
come the ABA effect may indicate that either the concentration
of ABA applied was too high or the penetration of the kinetin
was insufficient. Similar results were obtained when duckweed

cultures (Lemna minor) were grown in media containing ABA and

 

the cytokinin, benzyladenine (95).

83

Effects of Temperature, ABA, and Amo
1618 on the Levels of Reducing
Sugarsiin Onion Shoots

 

 

 

The levels of reducing sugars in the shoots were inversely
proportional to the temperature of incubation (Table 15). At
30°C, a net loss occurred in comparison with the control. Both
Amo 1618 and ABA prevented the rise in reducing sugar at 10°C
(Table 16). High temperature is known to enhance utilization

of reserve materials. In Phacelia tanacetifolia seeds the

 

respiratory quotient was very high at 28°C, a temperature at
which little or no germination occurred (25). Whether the
decrease in reducing sugars in onion is due to a faster rate of
utilization or a reduced rate of synthesis remains to be de-
termined. Other studies on onion show an accumulation of
reducing sugars at low temperature (9,144). Recently, Fontes
and Ozbun (47) and Kacperska-Palacz and Wcislinka (64), work-
ing with broccoli and rape plants, demonstrated low temperature
enhancement of reducing sugar level.

The fact that both Amo 1618 and ABA prevent the increase
in reducing sugars during chilling is indirect evidence that
these chemicals block the synthesis or activation of some
hydrolytic enzymes which in turn may be under hormonal control.
This hypothesis is supported by the ability of CCC to prevent
accumulation of reducing sugars in rape leaves during chill-

ing (64).

84

Table 15. Reducing sugar levels (percent fresh weight) in
Spartan Banner onion shoots after 96 hours of
exposure to 5 different temperatures.*

 

Temperature Treatment (°C)

 

None 30°< 20° 10° 5° 0°

 

b d

14.04 11.30a 15.48 19.71C 22.50 25.866

 

*Means followed by the same letter are not significantly dif-
ferent at the 5% level.

Table 16. Reducing sugar levels (percent fresh weight) in
Spartan Banner onion shoots as affected by
temperature, Amo 1618 (100 ppm) + temperature and
ABA (1 ppm) + temperature. ABA and Amo 1618 were
applied before exposure of onions shoots to 10°C
for 96 hours.

 

Chemical treatment

 

Chilling (hr) -- 8 Control Amo 1618 ABA
0 13.85a --- ---
96 20.77b 12.88a 13.64a

 

*Means followed by the same letter are not significantly dif-
ferent at the 5% level.

85

Levels of Acidic Inhibitor
in Shoots During Storage

 

The levels of inhibitor in the acidic fraction of shoot
extracts, as measured by wheat coleoptile bioassay are shown
in Figures 15 and 16. Three facts emerge from these data:

1. Inhibitor content was higher in MSU 4535 than in
Downing's Yellow Globe, which are long and inter-
mediate dormant types, respectively.

2. The inhibitor decreased during storage at 10°C and
at 0°C, but never completely disappeared. Comparison
of Figures 15 and 16 XE Figure 3 shows a fairly good
parallel between the degree of dormancy and the in-
hibitor level.

3. Growth promoters were observed in some samples, but
their levels could not be related to dormancy.

Thus, a true inverse correlation appears to exist between
the level of inhibitor and the degree of dormancy in onion.
However, the following questions remain:

a) Are the results valid? In the bioassay used, the
possibility exists that promoters having the same Rf as the
inhibitor could have been responsible for the results ob-
tained. This study has demonstrated the effectiveness of
kinetin as a substitute for low temperature in breaking dor—
mancy in entire onion bulbs and in shoot apices. The demon-
stration by Staden 25 El- (118) of a zeatin-like compound in

stratified Acer saccharum seeds lends support to a role of

 

 

Figure 15.

86

Elongation response of Genesee wheat coleoptile
sections to eluates from thin layer chromatograms
of the acid fraction of onion shoot apices ex-
cised from whole bulbs before and after storage.
Each chromatogram is the average of 4 replicates
of 10 gram-equivalents of shoot.

MSU 4535

Downing's Yellow Globe

A
B

87
200 A
'00 PT W
1

200

’8‘.

 

O

4wlaJO'C

 

§°

[q CE1

Omn'c

8

 

Gremlin.“ control

0

 

 

1001Il FLI MWksJO’C Nonmplu

 

 

 

 

O.
200(
0.
0 oi: filo if 01: fl?
Rf Rf

Figure 15

 

88

Figure 16. Elongation response of Genesee wheat coleoptile
sections to eluates from thin layer chromatograms
of the acid fractions of shoot apices. Each
point is the average of 4 replicates of 2 gram-
equivalents of shoot each.

Percenl inhibilion

0|
0

h
O

00
O

N
C

d
O

89

4535
Yellow Globe
2 4 8 lb

Time in sloroge(WI(s)

Figure 16

9O

endogenous cytokinins in the dormancy breaking process.
Interestingly, the zeatin-like compound found by Staden gt 31.
(118) had the same Rf value as abscisic acid (142) in the
solvent used for paper chromatography. The possibility of an
interplay between inhibitors and promoters should not be
excluded. The data of Thomas (125) and Tsukamoto (130) as well
as my own suggest this possibility.

b) Is the decrease observed in the inhibitor level the

cause or the effect of dormancy release? The fact that the

long:dormant type contains more inhibitor than the intermedi-

ate one suggests a causal relationship.

Characterization of the Inhibitor

 

Thin layer chromatograms developed in isopropanol:
ammonia:water (10:1:1 v/v/v) showed the Rf of the inhibitor
(0.4-0.7) was identical with that of ABA. Identical Rf's
were also observed in 3 additional solvent systems (Figure l7)°
When methylated samples of the inhibitor and ABA were chroma-
tographed and bioassayed, activity was found at Rf 0.6 to 0.9
in both cases (Figure 18). Inhibitory activity was detected
in fractions 5 to 11 following elution of both ABA and the
acidic fraction from charcoal-celite columns (Figure 19).

Extracts were next subjected to gas liquid chromatog-

raphy (GLC) and combined GLC-mass spectrometry (GLC-MS).

Figure 17.

Figure 18.

Figure 19.

91

Response of wheat coleOptile sections to eluates
of thin layer chromatograms of onion shoot ex-
tract (cv MSU 4535). The origins are on the left.
The position of the synthetic ABA as observed
under UV are indicated by vertical bar.

A = Isopropanol:Ammonia:Water 10:1:1 v/v/v

B = Benzene:Acetic acid:Water 8:3:5 v/v/v

C = 1—Butanol:n-propanol:Ammonia:Water 2:6:l:2
v/v/v/v

D = l-Butanol:Acetic acid:Water 40:11:29 v/v/v

Response of wheat coleoptile sections to eluates
from TLC of: A:ABA; B:methylated acid fraction
of onion extract; C:methylated ABA. Plates de-
veloped in isopropanol:ammonia:water 10:1:1.
Vertical bar indicates the position of authentic
ABA under UV light.

Response of wheat coleoptile sections to eluates
from charcoal-celite (1:2) columns of A) 8 ng of
ABA and B) 20 gram-equivalents of the acidic
fraction of an onion extract. Each column was
eluted with 150 ml 60% acetone and 10 ml fractions
were collected.

92

 

 

 

mmmm «mm»

.228 Barn;

in

oi:

 

 

Rl

Figure 17

l t]
B
I
Figure 18

.A

m u

.9559 .05 .2395

n_H_

J.

 

 

«new

3.55.3553!

o.

'0

A

 

Tube Number

Figure 19

93

Aliquots of the crude acidic fraction were partially purified
either by TLC in isopropanol:ammonia:water (10:1:1) (Sample A),
or by column chromatography on charcoal:celite (1:2) (Sample
B), as previously described, before methylation. Under the
conditions used (Table l) the retention time of authentic

cis,trans-methyl abscisate was 2.0 minutes (Figure 20). A GLC

 

trace of sample A following methylation (Figure 20) gave slight,
if any, evidence of ABA, although one component of the extract
had a retention time slightly greater than that of ABA. Sample
B gave similar results, and no ABA was detectable in either
sample when the electron capture detector was used in place of
flame ionization (data not shown).

GLC-MS line diagrams of methyl ABA and the two major com-
ponents of the methylated extract are shown in Figure 21.
The identity of the latter could not be established.

Thus, the inhibitor observed in the onion extract re-
sembled ABA chromatographically and biologically. However, no
evidence could be obtained for the presence of ABA using gas

chromatography or mass spectrometry.

94

Figure 20. Gas chromatographic lines of methylabscisate
(left) and an acidic inhibitor from onion bulbs
after thin layer chromatography and methylation
(right). Conditions are given in Table l.
Quantities used were 1 ug Me-ABA and 1 gram-
equivalent (fresh weight) of onion extract.

Response

 

 

Me-ABA

 

 

95

 

 

 

 

 

 

 

I 2
Reienlion Time

Rf :o.4.o.7
Peck l

3 Peak 2

e

o

o.

3

a
4 0 l 2 3 4
:min. Releniion Time:min.

Figure 20

96

Figure 21. Mass spectrograph line diagrams of authentic
methylabscisate (top), of peak 1 (middle),
and peak 2 (bottom) in methylated sample of
inhibitory zone from thin layer chromatogram.

(For retention time of peaks 1 and 2, see
Figure 20).

Hm musmflm

 

97

 

«I

—I

j
—a
l
5
Ti

 

z...

CONCLUS IONS

The following results were obtained in this thesis:

Onions having an intermediate or long dormant period
matured later in the summer than those with a short one.
Dormancy in onions declined with time. The change was gene-
tically controlled, temperature-dependent with an intermediate
temperature (5 to 15°C) being more effective than the extremes
(0 or 30°C). Small onion bulbs rooted and Sprouted faster
than large ones. High temperature induced secondary dormancy
in onion bulbs, but this dormancy was quickly overcome by
subsequent low temperature treatment. Root dormancy in onion
was weaker than shoot dormancy, and the latter appeared to
depend on the former. Wounding the scales significantly re-
duced root and shoot dormancy, but oxygen treatment did not.

During storage, the shoot apices grew inside the bulbs.
Intermediate temperatures (5-15°C) were more promotive than
the extremes (0-20,30°C) in the onion varieties tested. Shoot
apices excised from dormant bulbs responded to temperature
and exogenous growth substances similarly to entire bulbs.
Sucrose and kinetin were very effective in substituting for
intermediate temperature treatment in the release of dormancy

in onion shoots but no additive effect of these substances and

98

99

temperature could be obtained. Amo 1618 was very effective in
nullifying the effect of intermediate temperature when applied
prior to the temperature treatment. Applied after, it was
without effect. Sucrose was very effective in overcoming the
effect of Amo 1618 but kinetin was not. Abscisic acid re-
stored dormancy in temperature-treated onion shoots and neither
kinetin nor sucrose significantly alleviated this effect.
Temperature treatment increased the level of reducing
sugars of onion shoots and lower temperature were more effec-
tive than higher temperature. Amo 1618 and ABA applied before
the temperature treatment nullified the effect of low tempera-
ture on the accumulation of reducing sugars. The inhibitor
level was higher in MSU 4535, a long dormant type, than in
Downing's Yellow Globe, an intermediate dormant type, and it
declined as dormancy was broken. Chromatographically and
biologically, the inhibitor behaved like ABA, but gas liquid
chromatography and mass spectrometry indicated that insuffi-

cient ABA was present to account for the biological activity.

1)

2)

3)

4)

5)

6)

7)

8)

9)

10)

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