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fiv___._.—..—————

A MOLECULAR GENETIC STUDY OF WINTER HARDINESS
IN CANOLA (Brassica napus L.)

By

Moontae Song

A DISSERTATION

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

DOCTOR OF PHILOSOPHY

Department of Crop and Soil Sciences

1994

ABSTRACT

A MOLECULAR GENETIC STUDY OF WINTER HARDINESS
IN CANOLA (Brassica napus L.)

By

Moontae Song

Cold acclimation increases freezing tolerance of plants
and is involved in changes of gene expression. ‘This study was
conducted to determine how canola (Brassica napus L.) responds ‘
to cold acclimation to increase freezing tolerance both in
controlled environments and under field conditions. The
responses of two cold-regulated genes, BN28 and BN115, from B.
napus to cold acclimation were also examined to see whether
there was a correlation between the degree of gene expression,
freezing tolerance, and eventually to winter survival both in
controlled environment chambers and in the field.

Cold acclimation increased freezing tolerance of both
winter and spring cultivars, and the difference in freezing
tolerance among cultivars was due to their ability to
acclimate. Freezing tolerance of cultivars was highly
correlated with winter survival. Southern blot analysis
showed no difference in copy number of the genes among

cultivars tested. Though time-course increase in levels of

transcript in one of the genes was highly correlated with the
time-course increase in freezing tolerance, no correlation
existed at the cultivar level. However, this does not rule
out the possible roles of these cold-regulated genes in
freezing tolerance of canola.

Planting date is the most important factor which allows
canola to withstand freezing stress. Other agronomic
practices, such as application of plant growth regulators or
nitrogen and.phosphorus fertilization did not affect freezing

tolerance and winter survival of canola.

ACKNOWLEDGMENTS

I remember a conversation between me and Dr. Lawrence 0.
Copeland once I had done something that he did not want. I
asked him if he was angry. His answer was "Sometimes parents
get angry with their children but that does not mean they do
not love them." In an academic sense, Dr. Copeland was my
father and I was his child. Thus, I would like to express my
sincere gratitude and appreciation for his guidance, advice,
encouragement, and financial support during my graduate
program. Another nice person to whom my sincere thanks are
extended is Dr. Mike Thomashow who offered me the opportunity
to study molecular genetics of winter hardiness by providing
me with precious laboratory space, equipment and expensive
chemicals, as well as helpful guidance and insight to my
research.

Sincere thanks and appreciations are also extended to my
committee members, Dr. Donald Penner and Dr. Frank Dennis for
their time, valuable suggestions and review of this thesis.

I would also like to express my gratitude to my
colleagues and friends, Dr. Sabry Elias and Dr. Mohamed Ali
since we have worked together for so long since my arrival

here. Sincere thanks are expressed to those people in Dr.

iv

Thomashow's lab, Dr. Sarah Gilmour, Dr. Nancy Artus, Dr.
Stokes Baker, Dr. Lenny Bloksberg, Dr. Kevin O'Connele, Kathy
Wilhelm, Dean Lehmann, Ann Gustafson, and finally my friend
Brett McLarney and his wife Monica Miller for their kindness
and warm cooperation. Special thanks are given to Dr. Sarah
Gilmour, Dr. Stokes Baker, and Brett McLarney who willingly
taught me experimental techniques and offered me very precious
and helpful advice and encouragement. Also I would like to
extend my thanks and apologies to all the people in the
Department of Crop and Soil Sciences who have shown their
patience and generosity for my unintended rudeness and broken
English.

Finally, I would like to extend my gratitude and deepest
appreciation to my wife, You Soon Lee, and my son, Hyun Jun,

to whom this dissertation is dedicated.

THXBHJElDF‘CKJFHUENHflS

LIST or TABLES -------------------------------------- x
LIST or rrsunns ------------------------------------- xii
cnnrrnn I. erznnmunz nzvrzn ------------------------ 1
INTRODUCTION ------------------------------------ 1
FREEZING TOLERANCE, WINTER SURVIVAL, AND THE
EXPRESSION OF COLD-REGULATED GENES -------------- 3
Winter hardiness and screening method -------- 3
Freezing injury and cold acclimation --------- 5
Molecular genetics of cold acclimation ------- 9

EFFECT OF PLANTING DATE ON FREEZING TOLERANCE,
WINTER SURVIVAL, AND EXPRESSION OF COLD-REGULATED

GENES UNDER FIELD CONDITIONS -------------------- 14
Effect of planting date on crop growth and
agronomic performance ---------------------- 14
Development and maintenance of freezing tolerance
in the field ------------------------------- 16
Environmental factors affecting cold acclimation
---------------------------------------------- 17

Stress-induced gene expression under field
conditions --------------------------------- 19

PLANT GROWTH REGULATORS AND WINTER HARDINESS ---- 20

Synthetic plant growth regulators in winter
hardiness ---------------------------------- 20

Chemistry and functions of ethephon and mepiquad
chloride, and their possible role in winter

hardiness ---------------------------------- 22
THE GOALS OF TRIS RESEARCH --------------------- 25
nnrznznczs -------------------------------------- 26

vi

CHAPTER II. Correlation between winter survival, freezing
tolerance, and the expression of cold-induced
genes, BN28 and BN115, in canola (Brassica

napus L.).
ABSTRACT ---------------------------------------- 39
INTRODUCTION ------------------------------------ 41
MATERIALS AND METHODS --------------------------- 44
Plant materials and cold treatment ----------- 44
Tests for freezing tolerance ----------------- 45
Genomic DNA and total RNA isolation ---------- 46
Preparation of DNA probes ------------------- 47
Southern blot analysis ----------------------- 48
Northern blot analysis ----------------------- 48
Statistical analysis ------------------------- 49
RESULTS ----------------------------------------- 50
Winter survival ----------------------------- 50
Tests for freezing tolerance ---------------- 50
Nonacclimated freezing tolerance and cold
acclimation ability ----------------------- 60
Correlation between freezing tolerance and
winter survival --------------------------- 63
Effect of deacclimation on freezing
tolerance --------------------------------- 63

Southern blot analysis of BN28 and BN115 ---— 65
Northern blot analysis of BN28 and BN115 ---- 65
Effect of deacclimation on expression of

BN28 and BN115 ---------------------------- 7O
Correlation between freezing tolerance and
expression of BN28 and BN115 -------------- 74
Tissue-specific expression of BN28 and BN115- 77
DISCUSSION -------------------------------------- 79
REFERENCES -------------------------------------- 83

CHAPTER III. Effect of planting date on freezing tolerance,
winter survival, and responses of two cold-
regulated genes, BN28 and BN115, in canola
(Brassica napus L.).

anamor- ........................................ 87
Imonucr I ON ------------------------------------ 8 9
MATERIALS AND nzrnons ........................... 91

vii

Planting, management,and experimental design

--------------------------------------------- 91
Evaluation of freezing tolerance ------------ 91
Evaluation of winter survival --------------- 92

RNA extraction and Northern blot analysis---- 92
Nitrogen fertilizer rate and seeding rate

experiments ------------------------------- 92
Statistical analysis ------------------------ 94
RESULTS ----------------------------------------- 94
Weather conditions -------------------------- 94

Increase of freezing tolerance by acclimation
under field conditions -------------------- 96

Response of two cold-regulated genes, BN28 and
BN115 under field conditions --------------- 100

Effect of planting date on the expression of

the cold-regulated genes, BN28 and BN115 -- 107
Effect of planting date on winter survival--- 112
Correlation between freezing tolerance,

winter survival, and expression of BN28

and BN115 -------------------------------- 112
Effect of planting date and seeding rate on
winter survival --------------------------- 117
Effect of planting date and fall-applied
nitrogen on winter survival --------------- 117
nrscussrou -------------------------------------- 121
nsrznzuczs ------------------------------------- 124

Chapter IV. Effect of plant growth regulators, Terpal-c
and Cerone, on freezing tolerance and winter
survival of canola (Brassica napus L.).

ansrnacr ---------------------------------------- 127
turnonucrrou ------------------------------------ 128
narznrans nun uzrnons --------------------------- 130
Planting, management, and experimental
design ------------------------------------ 130
Application of plant growth regulators ------ 130
Evaluation of freezing tolerance ------------ 131
Evaluation of winter survival --------------- 131
Phosphorus experiment ----------------------- 131
nasanrs AND DISCUSSION -------------------------- 132

viii

Effect of plant growth regulators, Terpal—C
and Cerone on freezing tolerance and
winter survival --------------------------
Effect of fall—applied phosphorus on the
winter survival ---------------------------

ix

137

IJST‘CH?1DABHJES

TABLE

CHAPTER II

1. Winter survival of six winter canola cultivars in

1992-1993 and 1993-1994 -------------------------

2. Means of the nonacclimated freezing tolerance and
acclimation capacity of six winter and one spring
canola cultivar at different acclimation stages

3. Simple correlation coefficients between freezing
tolerance and winter survival of six winter

canola cultivars in 1992-1993 and 1993-1994 ------

CHAPTER III

1. Changes in transcript amount of BN28 by field

acclimation in six winter canola cultivars planted

Aug, 25, 1993 -----------------------------------

Page

105

2. Changes in transcript amount of BN115 in six winter

canola cultivars planted Aug. 25, 1993 ----------

3. Winter survival of six winter canola cultivars
planted Aug. 25, Sept. 10, and Sept. 25, 1993

4. Correlation between freezing tolerance and winter
survival of three winter canola cultivars planted
Aug. 25, Sept. 10 and Sept. 25, 1993 -----------

5. Correlation between transcript amount of BN28 and
BN115 and freezing tolerance of three winter

cultivars planted Aug. 25, Sept. 10, and Sept. 25,

1993 --------------------------------------------

6. Correlation between transcript amount of BN28 and
BN115 and winter survival of three winter

cultivars planted Aug. 25, Sept. 10, and Sept. 25,

1993 --------------------------------------------

106

115

116

Effect of planting date and seeding rate on winter
survival of canola in 1992-1993 ----------------- 118

Effect of planting date and seeding rate on winter
survival of canola in 1993-1994 ----------------- 119

Effect of fall-applied nitrogen and planting date on
winter survival of canola in 1992-1993 and 1993-
1994 -------------------------------------------- 120

CHAPTER IV

Effect of plant growth regulators on freezing
tolerance of three winter canola cultivars ------ 136

Effect of plant growth regulators on winter
survival of canola in 1992-1993 ----------------- 138

Effect of plant growth regulators on winter
survival of three winter canola cultivars in
1993-1994 -------------------------------------- 139

Effect of fall-applied phosphorus on freezing
tolerance of three winter canola cultivars in
1993-1994 --------------------------------------- 140

Effect of fall-applied phosphorus on winter

survival of three winter canola cultivars in
1992-1993 and 1993-1994 ------------------------- 141

xi

FIGURE

1.

10.

11.

12.

13.

14.

15.

LIST OF FIGURES

Page
CHAPTER II

Freezing curves of winter canola cv. Accord ------ 52
Freezing curves of winter canola cv. KWC4113 ----- 53
Freezing curves of winter canola cv. Ceres ------- S4
Freezing curves of winter canola cv. Duobul ------ 55
Freezing curves of winter canola cv. WRGB6 ------- 56
Freezing curves of winter canola cv. CDH3 -------- 57
Freezing curves of spring canola cv. Pactol ------ 58
Overall freezing curves of seven canola
cultivars ---------------------------------------- 59
LT” changes in six winter and one spring canola
cultivars by cold acclimation -------------------- 61
Effect of deacclimation on LT” changes in six
winter and one spring canola cultivars ----------- 66

Southern blot analysis of three restriction enzyme-
digested genomic DNA from seven canola cultivars
hybridized to BN28 ------------------------------- 67

Southern blot analysis of two restriction enzyme-
digested genomic DNA from seven canola cultivars
hybridized to BN115 ------------------------------ 68
Northern blot analysis of total RNA from leaves of

seven canola cultivars isolated at 0, 1, 5, 10-day
acclimation hybridized with BN28 and BN115

Standardization of RNA quantity in northern blot by
hybridization with rDNA probe, pRE12 ------------- 71

Overall means of seven cultivars in transcript

xii

16.

17.

18.

19.

10.

changes of BN28 and BN115 by cold acclimation ---— 72

Northern blot analysis for effects of deacclimation
on expression of BN28 and BN115 in seven canola
cultivars ---------------------------------------- 73

Comparison of time-course changes of LTS0 and
transcript amount of BN28 and BN115 by cold
acclimation ------------------------------------- 75

Correlation between freezing tolerance and

transcript amount in cultivar level -------------- 76
Northern blot analysis for tissue-specific

expression of BN28 and BN115 by cold acclimation

in two canola cultivars -------------------------- 76

CHAPTER III

Ten-day mean daily minimum temperatures at East
Lansing, Michigan during the winter of 1992, and
1993 -------------------------------------------- 9s

LT” change of six winter cultivars planted in the
field on Aug. 25, 1993 -------------------------- 97

LT” changes of three winter cultivars planted in
the field on Sept. 10, 1993 --------------------- 98

LT” changes of three winter cultivars planted in
the field on Sept. 25, 1993 --------------------- 99

Effect of planting date on overall LT50 changes of
six winter cultivars --------------------------- 101

Northern blot analysis of six winter cultivars
hybridized with BN28 and BN115 planted Aug. 25,
1993 -------------------------------------------- 103

Time-course changes in transcript amount of BN28
and BN115 in canola planted Aug. 25, 1993 ------- 104

Northern blot analysis of three winter canola
cultivars hybridized with BN28 planted Aug. 25,
Sept. 10, and Sept. 25, 1993 -------------------- 108

Northern blot analysis of three winter cultivars
hybridized with BN115 planted Aug. 25, Sept. 10,
and Sept. 25, 1993 ------------------------------ 109

Changes of transcript amount of BN28 in canola

xiii

11.

affected by different planting dates ------------- 110

Changes of transcript amount of BN115 in canola
affected by different planting dates ------------- 111

CHAPTER IV

Freezing curves of winter canola cv. WRG86 treated
with plant growth regulators --------------------- 133

Freezing curves of winter canola cv. Duobul treated
with plant growth regulators --------------------- 134

Freezing curves of winter canola cv. KWC4113 treated
with plant growth regulators --------------------- 135

xiv

CHAPTERI

LITERATURE REVIEW

INTRODUCTION

All oilseed rape belongs to the mustard family,
Brassicaceae, and includes three species of the genus,
Brassica, B. napus, B. campestris, and B. juncea, known as
rape, turnip rape and leaf mustard, respectively (Downey et
al., 1975; Downey, 1983; Shahidi, 1990). The name canola was
coined by Canadians in 1979 to apply to rapeseed cultivars
with low erucic acid and low glucosinolate. Such cultivars
may be further described as 'double low' or 'double zero'
rapeseed. Consequently, the term canola can be used to
describe any rapeseed cultivars with low erucic acid content
(2 % or less) in the oil and no more than 30 micromoles per
gram of glucosinolate in the defatted meal (Sahhidi, 1968).

Canola production is affected by cold temperature in
‘various ways. The cultivars of winter rapeseed and canola
evaluated in the United States have not consistently survived
the winter in the northern United States (Hang and Gilland

1982; Kephart et al. 1988; Auld et a1. 1989; Smith et

2
al.,1989; Copeland et al.,1990). Only in areas in which the
climate is moderated by proximity to large bodies of water has
consistent winter survival of canola been attained (Mahler and
Auld 1987, 1988, 1989). Canola production in most of the
northern United States has relied on spring cultivars that
have lower productivity than winter cultivars because of high
summer temperatures and pest problems. Because of lack of
cultivars with sufficient freezing tolerance, the safe area
for winter B. napus production is limited to latitudes below
33°N, though cultivars adapted to latitudes above 33°N have
been reported (Mahler and Auld 1987, 1988, 1989; Fribroug et
a1. 1989). However, in southern areas, winter cultivars are
often not exposed to sufficient cool temperature to satisfy
stringent vernalization requirements“ This eventually delays
flowering and results in heat stress during the seed filling

stage.

FREEZING TOLERANCE, WINTER SURVIVAL, AND THE
EXPRESSION OF COLD-REGULATED GENES

Winter Hardiness and Screening Methods

Winter hardiness of cereal grains and perennial forage
crops is of great concern to agronomists in cold northern
temperate regions of the world because winter injury severely
limits their production area. Consequently, winter hardiness
has been the subject of considerable investigation and the
development of cultivars with improved winter hardiness is of
primary concern. However, the introduction of new cultivars
that possess increased winter hardiness has not always been
successful (Grafius, 1974).

Winter hardiness implies avoidance of or tolerance to
the cumulative effects of winter that a plant encounters,
including freezing, heaving, smothering, desiccation, and
disease infestation. Climate, soil, plant, and cultural
practices interact to determine the degree of injury to a crop
following the rigors of winter (Steponkus, 1978). Tolerance
and avoidance mechanisms may reside at either the whole plant,
tzissue, or cellular level. Thus, the undertaking to increase
winter hardiness is indeed an ambitious objective, and one

'that may be insurmountable if approached in its entirety.

3

4

A common approach to screen winter hardiness is to
expose plants to the rigors of winter in a field situation,
although such tests suffer from a lack of control over the
severity and consistency of stress imposed on plants (Hill and
Salmon, 1927) . Consequently, Levitt (1980) indicated that
winter conditions that.result in differential survival occur
only once every ten years. Thus, it is often necessary to
grow materials for many years and in several locations to
achieve the comparisons desired (Andrews, 1960).

Because of the limitations inherent in screening for
winter hardiness under field conditions, there has been a
continuous search for rapid and efficient methods for
predicting freezing tolerance, and a number of tests have been
developed. These usually involve controlled freezing followed
by observation of plant recovery. However, this is often a
long process that is destructive and requires a large number
of plants (Bruce-Babel and Fowler, 1989). Plant tissue water
content also has been used to screen for freezing tolerance in
winter cereals. Water content of fully acclimated plants was
correlated with field evaluation, but was not able to detect
small difference between cultivars without excessive
replication (Brule-Babel and Fowler, 1989). Recently, the
method of choice for evaluating freezing tolerance is to
measure the leakage of ions from plant cells after freezing
stress by observing the loss in the integrity of cellular

membranes (Palta et al., 1977; Steffen et al., 1989). This

5

method has been used to study freezing injury and recovery in
onion bulbs (Arora and Palta, 1991), potato leaves (Palta and
Li, 1980), alfalfa cultivars (Sulc et al., 1991), Arabidopsis
(Gilmour et. al, 1988) and rapeseed (Teutonico et al., 1993) .

Though some of the results from artificial freezing
tests have been promising, ongoing breeding programs for
winter hardiness still rely heavily on field evaluations,
largely because breeders are not convinced about alternative

methods.

Freezing Injury and Cold Acclimation

Of all the factors related to winter hardiness, direct
freezing injury has been cited as the principal cause of
winter killing (Quisenberry, 1938) . At normal freezing rates
under natural conditions, ice nucleation occurs initially
outside the cell because the cell has a lower freezing point
than the extracellular water. The resultant lowering of
extracellular vapor pressure causes more water to leave the
cell to form extracellular ice, resulting in protoplasmic
dehydration and mechanical collapse of membranes (Levitt,
1980). During very rapid cooling, equilibrium freezing can
not occur and ice nucleation occurs inside the cell. This
intracellular freezing is usually lethal to the cold-
acclimated cell, since the presence of intracellular ice
disrupts the cellular membrane. However, freezing rates

sufficiently rapid to cause intracellular freezing are not

6
commonly encountered in the natural environment (Singh and de
la Roche, 1988). .

Though the plasma membrane is considered to be the
primary site of freezing injury (Siminovitch and Scarth 1938;
Levitt and Siminovitch, 1940; Palta et al., 1977; Steponkus
and Weiser, 1978; Singh 1979a), evidence that photosynthetic
capacity can be significantly impaired after a freeze-thaw
cycle has indicated that intercellular membranes may be at
least as sensitive to extracellular freezing as the plasma
membrane (Steffen and Palta, 1987).

In recent years, ultrastructural and biophysical
alteration of the plasma membrane during lethal extracellular
freezing has been elucidated. Irreversible conversion of the
planar membrane bilayer to tightly appressed multilamellar
vesicles has been observed in association with lethal
extracellular freezing (Gordon and Steponkus, 1984; Pearce and
Willison, 1985, Singh et. al, 1987). There is general
agreement that under conditions of extracellular ice
formation, decrease in temperature or the presence of ice
crystals per se is not responsible for freezing damage (Heber
and Santarius, 1973), and, that the jprocess of cellular
dehydration is the most disruptive and injurious component of
freezing injury.

The most dramatic effect of cold acclimation, although
involved in a variety of physical and biochemical changes, is

increased freezing tolerance after a period of exposure to low

7

nonfreezing temperature (Levitt, 1980; Sakai and Larcher,
1987) . Biochemical changes include alteration in lipid
composition, increased sugar and soluble protein content, and
the appearance of new isozyme forms (Levitt, 1980; Sakai and
Larcher, 1987; Steponkus and Lynch, 1989) . However, it is
uncertain whether these biochemical changes are associated
with increased freezing tolerance, or whether they are
manifestations of metabolic adjustments in response to cold
temperature. Some may contribute to the overall fitness of the
plant for low temperature survival, while others have specific
roles in bringing about increased freezing tolerance. Indeed,
it has been demonstrated that changes in membrane lipid
composition are directly related to the freezing tolerance of
plant cells (Steponkus et al., 1988; Steponkus and Lynch,
1989). In addition, there is evidence that proline and many
simple sugars (Santarius, 1973; Strauss and Hauser, 1986;
Carpenter and Crowe, 1988), as well as certain soluble
polypeptides from spinach (Volger and Heber, 1975) have
cryoprotective effects in vitro, though whether these
molecules contribute significantly to freezing tolerance in
vivo remains to be determined.

There are several reports of the effect of cold
acclimation on the physiology of Brassica plants, but these
studies do not indicate whether any of these changes are
required for freezing tolerance. For example, exposure of B.

napus to low temperature induced accumulation of reducing

8
sugars in leaves (Krause et al., 1982), altered the
phospholipase-D (Sikorska and Kacperska, 1982), induced seed
pigment synthesis (Johnson-Flanagan et al., 1991), inhibited
seed photosynthesis (Hodgins et al., 1989), and altered seed
chlorophylase and peroxidase activities (Johnson-Flanagan,
1939).

The cold acclimation of a given species is dependent on
two factors; genetic capacity of the species to acclimate in
order to withstand freezing temperature, and the conditioning
or expression of this heritable capacity. Cold acclimation
may be influenced by radiation, temperature, photoperiod,
precipitation, and developmental stage of plants,t with
different optimum conditions for different species and
cultivars (Steponkus, 1978) . Of all the environmental factors
influencing cold acclimation, temperature is most important
(Olien, 1967; Paulsen, 1968). Low, nonfreezing temperatures
are conducive to an increase in hardiness in the fall, and
warm temperatures are responsible for its decrease in the
spring. Generally, it is thought that. most plants will

acclimate as temperatures are gradually lowered below 10°C

(Alden and Hermann, 1971). However, during acclimation the
jprogressive decline in temperature from relatively high
temperatures in early fall, followed by low, nonfreezing
‘temperature in late fall and early' winter, followed, by
freezing temperature in winter is extremely important in the

acmlimation process. The importance of the progression of

9
temperature can be supported by the studies of Pomeroy and
Andrews (1976) . They found that high levels of hardiness can
be induced within only 4-6 days in wheat if the hardening
temperatures are preceded by warm temperatures, whereas 6-8
weeks at 2-4°C were required for maximum hardiness without

preceding warm temperatures (Andrews et al., 1974).

Molecular Genetics of Cold Acclimation

.Although the physiological and biochemical changes that
occur during cold acclimation could be brought about by
preexisting structural and enzymatic changes, it is also
possible, as first proposed by Weiser (1970), that cold ‘
acclimation involves changes in gene expression. Direct
evidence was obtained recently to show that low temperature
induced accumulation of specific mRNAs. The appearance of
novel transcripts during cold acclimation has been observed in
alfalfa (Mohapatra et al., 1989), wheat (Lin et al., 1990;
Houde et al., 1991), barley (Cativelli and Bartels, 1990; Dunn
et al., 1990), Arabidopsis (Hajela et al., 1990; Kurkela and
Frank, 1990; Nordin et al., 1991; Gilmour et al., 1992), rice
(Binh and Oono, 1992), and Brassica (Orr et al., 1992; I
Weretilnyk et al., 1993).

DNA sequences corresponding to these cold-regulated
(cor) transcripts have also been isolated and characterized by
differential screening of cDNA libraries constructed from

these species. These mRNAs appear rapidly upon exposure of

10

the plants to low temperatures, and deduced amino acid
sequences of the products of the some of these cold-regulated
genes have been identified. A number of cold-induced
transcripts hybridize to the transcripts of rab (responsive to
ABA) genes (Hahn and Walbot, 1989) or encode polypeptides
containing amino acid sequence motifs found in rab motifs
(Skriver and Mundy, 1990; Gilmour et al., 1992; Houde et a1. ,
1992), suggesting that they may play a role in the tolerance
to the cellular desiccation stress that accompanies
extracellular freezing. Other cold-induced transcripts encode
polypeptides containing stretches rich in alanine and glycine
(Kurkela and Franck, 1990; Orr et al. , 1992) , suggesting that
they may play an antinucleating role, although their functions
have not been elucidated. The product of one cold-regulated
gene has been reported to show cryoprotective properties in
vitro (Lin and Thomashow, 1992) .

In B. napus, two cold-regulated cDNA clones were
isolated and characterized (Orr et al. 1992; Weretilnyk,
1993) . BN28 is homologous to cor6.6 of Arabidopsis (Gilmour
et al., 1992) and encodes abundant mRNA of 0.45 kilobases,
induced by the exposure of the whole plant to 2°C, with the
transcript appearing within 6 hours of exposure (Orr et al.,
1992). The transcript disappears within 20 hours of the
return of plant to room temperature. ABA also induced the
expression of this gene but heat shock did not. The product

of cor6.6 from Arabidopsis is a 6.6 kDa polypeptide (Gilmour

11

et al., 1992) and has amino acid homology with KINI of
Arabidopsis, which again shares a sequence similarity with
antifreeze protein B from winter flounder. The functions of
the products of BN28, cor6.6 and kinl are not yet known.
However, based on regions of deduced amino acid homology to
flounder antifreeze protein, these genes may possess
antinucleation properties (Kurkela and Franck, 1990).

BN115 is another cold-regulated gene isolated from B.
napus (Weretinyk et al., 1993) which has sequence homology
with cor15 of Arabidopsis (Lin and Thomashow, 1992). This
gene encodes abundant mRNA of 0.8 kilobases induced by cold
temperature, with the transcript appearing within 1 day of
exposure. The transcript disappears within 20 hours of the
return of the plant to room temperature. In contrast to BN28,
ABA did not induce the expression of this gene, but
dehydration leads to a slight increase in transcript at room
temperature. Northern blot analysis showed that levels of
transcript increased within 24 hours of exposure to low
temperature, peaked after 3 days, and remain at elevated level
for the duration of the, cold treatment. BN115 reveals a single
open reading frame that potentially encodes a protein of 14.8
kDa which is matched to that of COR15 (14.7 kDa) of
Arabidopsis (Lin and Thomashow, 1992) . Immunological studies
have indicated that this COR15 is processed in plants to a
polypeptide of about 9 kDa, and that this polypeptide is

located in the soluble fraction of the chloroplast (Thomashow

 

12
et al., 1993). The amino terminal sequence of COR15 closely
resembles transit peptide target protein to the stromal
compartment of the chloroplast. A search of GenBank and EMBL
database did not reveal any proteins with significant
similarity to COR15.

Mohapatra et al. (1989) have addressed the question of
whether cold-regulated gene expression correlates with the
degree of freezing tolerance attained during cold acclimation
in alfalfa. Three cold-regulated cDNA clones were examined in
4 alfalfa species with different freezing tolerances. The
correlation coefficient between the levels of expression of
one of the three genes and the degree of freezing tolerance
attained by the cultivars was 0.99(***). The other two genes
also had a very high positive correlation, while no
correlation existed between the levels of freezing tolerance
of the cultivars and either the expression of the control
genes in the acclimated or nonacclimated plants. Likewise,
Houde et a1. (1992) found a positive correlation between the
level of expression of the wheat cold-regulated gene Wcs120
and freezing tolerance. Thus, linkage between expression of
at least some of the cold-regulated genes and the levels of
freezing tolerance implies that the cloned cold-regulated
lgenes might be used to aid in breeding for increased freezing
'tolerance of crop plants (Thomashow et al., 1990). However,
two cases exist in barley in which the levels of cor gene

expression were found not to correlate with freezing

13
tolerance; Levels of neither BLT4 (Dunn et al. , 1991) nor HVAl

(Sutton et a1. , 1992) were correlated with freezing tolerance.

EFFECT OF PLANTING DATE ON FREEZING TOLERANCE,
WINTER SURVIVAL, AND EXPRESSION OF COLD-
REGULATED GENES UNDER FIELD CONDITIONS

Effect of Planting Date on Crop Growth and Agronomic
Performance, Including Winter Survival.

Planting date is of critical importance to the
production of canola and directly impacts subsequent seed
yield, winter survival, insect damage, seed oil quality and
disease severity (Hang and Gilland, 1982; Auld et al., 1983;
Murray et al., 1984; Kephalt et al., 1988; Fribourg et al.,
1989; Raymond et al., 1990; Thomas et al., 1990). The optimum
planting date differs across production zones. Good cold
tolerance in winter cultivars requires that the seedling has
six to eight leaves and good crown development prior to onset
of severe winter condition (Karow and Pumphrey, 1986; Auld et
al., 1989). Winter canola is usually seeded approximately six
weeks before the first anticipated hard frost. Canola emerges
best when soil temperature is above 10°C (Kephart et al.,
1988). In northern parts of the United States, most winter
canola is seeded in late summer, following the harvest of an
early-maturing cereal crop.

In general, the earlier canola is planted, the better it

jperforms. Seeding in early rather than late August generally

14

15

improves winter survival and seed yield wherever winter canola
is grown (Noderstagaard, 1972; Huber, 1973; Kondratowicz and
Paprocki, 1974; Daniel et al., 1980). There are, however,
imany exceptions to this rule. Late planting in winter canola
is often unavoidable because of double cropping, soil water
availability, and other environmental factors (Thomas et al.,
1990). Planting too early creates pest and weed problems in
winter canola while planting too late may result in very small
plants which are more vulnerable to freezing damage (Thomas,
1984).

A significant cultivar x planting date interaction for
both seed yield and winter survival has been reported in
canola in which seed yield from late plantings was closely
associated with winter survival during years in which the
plants were exposed to a minimum temperature of -30 °C (Auld
et al., 1983).

Several studies have shown that delayed planting affects
not only the yield of rapeseed, but also the quality of oil
and protein produced (Kondra, 1977; Degenhardt and Kondra,
1981; Christensen, 1984). Delay in planting date reduces the
oil content (Scott et al., 1974; Kondra, 1977) and increases
protein content of the meal (DePauw et al. , 1978) . These
changes are probably due to delayed late spring growth of
plants planted in late fall and subsequent exposure of the
plant to increased temperatures during oil formation (Canvin

et al., 1965). However, another report indicated that oil

16
content and plant growth were not sensitive to planting date

(Auld et al., 1983).

Development and Maintenance of Freezing Tolerance in the Field

The development of freezing tolerance in plants during
the fall (acclimation) and its loss during spring
(deacclimation) roughly follows a cyclical pattern (Smith and
Nelson, 1985). In studies by Ruelke and Smith (1956) plants
began to develop freezing tolerance in early to mid-September
as measured by electrical conductance. The development of
tolerance continued through autumn until late November, and
reached a maximum shortly after permanent freezing of the soil '
surface after which weekly air temperature remained below
afreezing. A high level of freezing tolerance was maintained
from early December to mid-February, when snow cover provided
protection from freezing. Tolerance began to decrease in mid-
February with the onset of warmer temperatures and reduced
snow cover. Thereafter, it began to drop rapidly after the
snow'had disappeared and the soil surface thawed in late March
(Suneson and Peltier, 1938; Ruelke and Smith, 1956). Winter
kill usually occurs during late winter and early spring, when
the snow cover has disappeared and plants are exposed to
extreme temperature fluctuations above and below freezing. By
this time the plants have lost some freezing tolerance in
response to warmer temperature and may be unable to reharden

satisfactorily, or temperature drop is so rapid that they do

17
not have time to reharden (Smith and Nelson, 1985).
Environmental Factors Affecting Cold Acclimation of Plants

Cold hardiness of plants is known to be favored by (1)
short day length, (2) low temperature, (3) alternation of
temperature between warm (20°C) during the day and cold (0-
5°C) during' night, and (4) adequate radiation for' good
photosynthetic activity (Tysdal, 1933; Shin et al., 1967).
Temperature appeared to be of primary importance, but
metabolic processes associated with cold hardiness were also
influenced by photoperiod.

Low above-freezing temperatures are conducive to
acclimation in the fall, and warm temperatures for'
deacclimation in spring. Generally, most plants appear to
acclimate as temperatures are gradually lowered below 10°C
(Alden and Hermann, 1971) . Although there appears to be a
certain optimal temperature for cold acclimation for a given
species (Hodgson, 1964; Olin, 1967), the progressive decline
from relatively high temperature in early fall, to low above-
freezing temperatures in late fall and early winter, followed
by freezing temperature in winter is extremely important in
cold acclimation (Steponkus, 1978) . Each stage has a distinct
role in the overall process of acclimation in respect to
succeeding stages and the influence of other environmental
factors (Weiser, 1978). The importance of the progression of
‘temperatures is supported by reports by Pomeroy and Andrews

(1976). They found that high levels of hardiness could be

18
induced in only 4-6 days in wheat when hardening temperatures
were preceded by warm temperatures, whereas 6-8 weeks at
hardening temperature were required for maximum hardiness
(Andrews et al., 1974).

Two controversial points have been discussed by many
researchers on the role of light in cold acclimation. Dexter
(1933a, b) concluded that light only served to provide
photosynthate, and.in plants such as alfalfa, which have large
reserves, cold hardiness could be increased in the dark by
cold treatment alone. The suggestion that light per se is not
a requirement for the acclimation process is supported by
reports that the light requirement can be replaced by ‘
incubation in sugar solution (Tumanov and Trunova,1963;
Steponkus and Lanphear, 1967) . A second area of apparent
conflict in the literature in regard to the role of light in
acclimation relates to its function in a photoperiodic role in
addition to its photosynthetic role. Short photoperiod
generally stimulates acclimation, partly because of the annual
decline in photoperiod that accompanies natural acclimation,
but also because of reports that some species fail to
acclimate under long photoperiods (Hodgson and Bulba, 1956).
However, other reports (Turnova, 1965; Paulsen, 1968) indicate
that short photoperiods are not required for acclimation of
cereal grains, but are required for acclimation of perennial

such as alfalfa.

19
Stress-Induced Gene Expression under Field Condition

Although many studies have demonstrated the expression
of stress-induced genes under laboratory conditions (Mundy and
Chua, 1988; Close et. a1, 1989; Bartel et al., 1990, Hajela et
al., 1990; Kukela and Frank, 1990; Skriver and Mundy, 1990;
Bray, 1991; Fla et al. , 1991; Lang and Palba, 1992) , few
investigators have examined the expression of such genes under
field conditions. Kimpel and Key (1985) showed that mRNA
encoding heat shock proteins (HSP) accumulated in leaf tissues
of soybean experiencing high temperature in the field. Similar
studies were conducted with other crop species by Burke et al.
(1985). Both field experiments and growth chamber studies
using conditions designed to simulate a day of high-
temperature stress showed evidence that HSPs are expressed
under natural conditions (Chen et al., 1990; DeRocher et al.,
1991). Lorraine et al.(1993) examined the expression of HSPs
at the whole plant level in alfalfa under field conditions.
The fact that some of the HSPs are expressed in the seed and
flowering parts in the absence of heat shock suggests a more
widespread occurrence of HSPs in optimal growth environments
and their potential role in plant development.

Expression of cold-regulated genes like cor genes from
Arabidopsis (Hajela et al., 1990; Kurkela and Frank, 1990;
Nordin et al., 1991, Gilmour et al., 1992) and BN genes from
.Brassica napus (Orr et al.,1992; Weretilnyk, 1993) have not

Ibeen examined under field conditions.

PLANT GROWTH REGULATORS AND WINTER HARDINESS

Synthetic Plant Growth Regulators and Winter Hardiness

The :main. plant. growth. regulators (PGRs) that. have
increased winter hardiness are CCC (chloromequat chloride, an
antigibberrellin) and ethephon (2-chloroethyl phosphonic acid,
an ethylene-producer). Foliar sprays of CCC have been
reported to increase the freezing tolerance of winter wheat
(Gusta et al., 1982), potato (Chen and Li, 1976), winter rape
(Marth, 1965), and alfalfa (Paquin et al., 1976). Ethephon .
increased freezing tolerance of sweet cherry (Proebsting and
Mills, 1976), apple (Raese, 1977), apple and.pear (Ketchie and
Murren, 1976), and peach (Durner and Gianfagna, 1988).

However, new PGRs, which are mainly antigibberellins,
appear promising. Mefluidide and triazoles (triadimefon,
tridimenol, paclobutrazol, uniconazol, and tetcyclacis) have
been shown to increase tolerance to chilling (Whitaker and
Wang, 1987), heat (Pennypacker et al., 1982), drought
(Fletcher and Nath, 1984), and freezing (Paquin et al., 1976;
Fletcher and Hofstra, 1985), as well as to cause a transient
increase in endogenous ABA concentration in treated plants
(Wilkinson, 1982; Asare-Boaham et al., 1986). Triazole

Icompounds are thought to inhibit the biosynthetic pathway of

20

21
sterols and GAs (Grossmann, 1990).

Exogenous application of ABA to whole plants, parts of
plants or plant protoplast have increased frost tolerance in
the absence of cold treatment (Irving, 1969; Rikin et al.,
1975; Waldman et al., 1975; Chen et al., 1979; Orr et al.,
1986; Reaney and Gusta, 1987; Lang et al., 1989; Mohapatra et
al., 1989). A number of researchers have also demonstrated
that exposure to low temperature increased ABA concentration
in plants. There are two principal explanations for the
increase of endogenous ABA during cold acclimation. First, low
temperatures per se could cause an increase in ABA level (Daie
and Campbell, 1981). Second, the accumulation of ABA could be ‘
the result of induced water stress in plant (Eze et al.,
1983). The increase in endogenous ABA level following low
temperature exposure suggests a certain role for ABA in cold
hardiness. However, this conflicts with results obtained on
the effect of exogenous ABA on cold acclimation. Gusta et al.
(1988) found that ABA inhibited growth but had little or no
effect on enhanced freezing resistance of winter wheat crowns
if applied as a foliar spray or added to the nutrient
solution.

Some of the early interest in the use of PGRs on oilseed
:rape occurred.as.a:result.of experiments in Poland (Kacperska-
Palacz et al., 1969; Kacperska-Palacz and Wcislinska, 1972;
ZKacperska-Palacz and Egierszdorff, 1972) which suggested that

CCC improved cold hardiness of rape in controlled environment

22
chambers. The role of ABA in cold acclimation was studied in
cell suspension cultures.of.Brassica.napus (Orr et al., 1986).
ABA could promote freezing tolerance in the absence of low
temperatures. Recently, the effects of several plant growth
regulators on winter hardiness of canola under field
conditions were studied in Canada (Morrison and Andrews,
1993). Some of the triazole PGRs reduced plant size by
limiting cell expansion and increasing cell numbers. While
cold. hardiness and freezing 'tolerance ‘were increased. by
certain. growth regulators, these effects were not consistent
over time and did not result in increased winter survival.
This suggests that natural cold hardening may have eclipsed

the PGR-induced hardening.

Chemistry and Functions of Ethephon and Mepiquat Chloride, and
Their Possible Roles in Winter Hardiness

Terpal-C is a mixture of mepiquat chloride and ethephon,
while Cerone contains ethephon, iMepiquat chloride belongs to
a group of plant growth retardants known to inhibit GA
biosynthesis (Dennis et al., 1965; Lang, 1970). This group
of compounds includes AMO-1618, CCC (chlormequat chloride),
Phosphon D (chloroponium), and ancymydol, as well as mepiquat
chloride. Of all the compounds in this group, only CCC and
mepiquat chloride have found large-scale application in
agriculture.

Ethephon belongs to the group of compounds which, when

applied to plant materials, release ethylene as they

23

decompose. This group includes ethephon (2-chloroethyl
phosphonic acid), silaid (2-chloroethyl methylbis
phenylmethoxy silane), alsol (2-chloroethyl tris 2-
methoxyethoxy silane) and ACC (1-aminocyclopropane-1-
carboxylic acid). Though ethephon has many effects on plant
growth and development, the stunting of ethylene-treated
plants is now the basis of one of its most important uses,
that is, the prevention of lodging in small grains and other
crops (Reid, 1985). Ethephon not only reduces stem length,
but also stiffens the straw. Ethylene is also known to be
released when the plant is exposed to stress, e.g., the
stresses resulting from wounding and pathogen attack. Low ‘
temperature stress can be reduced by ethephon treatment in
some fruit trees by delaying the bloom in the spring and
increasing intrinsic winter hardiness of the buds (Durner and
Gianfagna, 1988).

The possible mechanisms of these PGRs in winter
hardiness have been illustrated by several studies in winter
cereals: 1) Some PGRs prevent the dehardening of plants in
mid-winter; The ‘ultimate level of :freezing ‘tolerance in
November was not increased by application of the chemicals.
However, they did reduce the loss of freezing tolerance in
mid-winter, which is considered to be the most critical period
(Gusta et al., 1990). 2) Certain PGRs promote regrowth of
reots in the spring, even if most of the root system is

'winter-killed (Robertson et al., 1992). 3) Some PGRs reduce

24

the elongation of the subcrown internodes, thereby avoiding
exposure to freezing air temperatures (Anderson et.al., 1985).
4) Some PGRs stimulate photosynthesis, resulting in increased
starch accumulation and enhanced winter hardiness (Astakhova
et al., 1988). 5) Certain PGRs inhibit degradation of
endogenous ABA, and thus, indirectly affect cold hardiness
(Gusta et al., 1990).

All of these effects are inconsistent from year to year
(Robertson et al., 1992). The reason for this inconsistency
is not known, but probable reasons are lack of uptake,
improper growth stage when applied, temperature at the time of
application, and severity of winter. A period of relatively
warm temperatures is generally required following application

to allow the PGRs to result in the desired chemical change.

THEGOALSOFTHETHESIS

The available data suggest that the changes in gene
expression that occur during cold acclimation may contribute
to the increased freezing tolerance of cold acclimated plants.
The correlation between the levels of expression of the cold-
regulated genes and the degree of freezing tolerance among
cultivars or species has been examined in several plant
species with several different cold-regulated genes. However,
the results are controversial, depending on the plant species
and the genes examined.

The primary goal of this research was to examine
expression of cor genes in a variety of cultivars that differ
in freezing tolerance, under both laboratory and field
conditions. A relationship between cor gene expression and
freezing tolerance and, eventually, winter hardiness would
provide us not only clues as to the functions of the genes,
but also molecular markers for selecting winter hardiness.

In addition to these genetic studies, effects of several
agronomic practices such as planting date, seeding rate,
nitrogen and phosphorus, and application of chemicals on

winter hardiness of canola were also studied.

25

REFERENCES

Alden, J., and R.K. Hermann. 1971. Aspects of the cold-
hardiness mechanism in plants. Bot. Rev. 37:37-142.

Anderson, 6., and G. Olsson. 1961. Cruciferous oilseeds. pp.
1-62. In: H. Kapper and W. Rudorf (eds.) Manual of Plant
Breeding. Vol. 5. Parey Press, Berlin.

Anderson, H.M., N.D.S. Huband, P.J. Murphy, and R.D. Child.
1985. Improving winter hardiness in winter oats by seed
treatment with tetcyclasis. Proc. British Crop Protection
Conf. Weeds 4C-25:569-573.

Andrews, J .E. 1960. Cold hardening and cold hardiness of
winter rye seedlings as affected by the stage of development
and temperature. Can. J. Plant Sci. 38:1-7.

Andrews, C.J., M.K. Pomeroy, and LA. de la Roche. 1974.
Changes in cold hardiness of overwintering winter wheat.
Can. J. Plant Sci. 54:9-15.

Arora, R., and J.P. Palta. 1991. A loss in the plasma membrane
ATPase activity and its recovery coincides with incipient
freeze-thaw injury and postthaw recovery in onion bulb scale
tissue. Plant Physiol. 95:846-852.

Asare-Boaham, 1N.K., G. ZHofstra, R.A. Fletcher, and E.B.
Dumbroff. 1986. Triadimefon protects bean plants from water
stress through its effects on abscisic acid. Plant Cell
Physiol. 27:383-390.

Astakhova, N.V., M.A. Bocharova, K.K. Boruakh, S.V. Klimov,
and T.I. Trunova. 1988. Effect of cartolin on chloroplast
structure and photosynthesis during hardening of winter wheat
to frost. Soc. Plant Physiol. 35:275-279.

.Auld, D.L., B.L. Bettis, and M.J. Dial. 1983. Planting date
and cultivar effect on winter rape production. Agron. J.
76:197-200.

.Anld, D.L., D.R. Brady, and K.A. Mahler. 1989. Rapeseed and
lcanola breeding in North America: U.S. potential. pp. 7-10.

26

27

5th Crucifier Genetic Workshop, Symposium I. Davis,
California.

Bartels, D., K. Schneider, G. Terstappen, D. Piatkowski, and
F. Salamini. 1990. Molecular cloning of absisic acid-modulated
genes which are induced during desiccation of the resurrection
plant Craterostigma plantagineum. Planta 181:27-34.

Bihn, L.T., and K. Oono. 1992. Molecular cloning and
characterization of genes related to chilling tolerance in
rice. Plant Physiol. 99:1146-1150.

Bray, E.A. 1991. Regulation of gene expression by endogenous
ABA during drought stress. pp. 81-98. In: W.J. Davis and H.G.
Jones (eds.) Abscisc Acid: Physiology and Biochemistry. Bios
Scientific Publishers, Oxford.

Bruce-Babel, A.L., and 0.3. Fowler. 1989. Use of controlled
environments for winter cereal cold survival evaluation:
Controlled freeze tests and tissue water content as prediction
test. Can. J. Plant Sci. 69:355-366.

Burke, J.J., J.L. Hatfield, R. Klein, and J.E. Mullet. 1985.
Accumulation of heat shock proteins in field-grown cotton.
Plant Physiol. 78:394-398.

Burke, M.J., L.V. Gusta, H.A. Quamme, C.J. Weiser. and P.H.
Li. 1976. Freezing and injury in plants. Annual Rev. Plant
Physiol. 25:507-508.

Canvin, D.T. 1965. The effect of temperature on the oil
content and fatty acid composition of oils from several oil
seed crops. Can J. Botany 43:63-69.

Carpenter, J.E., and J.H. Crowe. 1988. The mechanism of
cryoprotection of proteins by solutes. Cryobiology 25:244-255.

Cativelli, L., and D. Bartels. 1990. Molecular cloning and
characterization of cold-regulated genes in barley. Plant
Physiol. 93:1504-1510.

Chen, H.H. , P. Gavinlertvatana, and P.H. Li. 1979. Cold
acclimation of stem-cultured plants and leaf callus of Solanum
species. Bot. Gaz. 140:142-147.

Chen, H.H., and P.H. Li. 1976. Effect of photoperiod,
temperature and certain growth regulators on frost hardiness
of Solanum species. Bot. Gaz. 137:105-109.

Chen, Q., L. Lauzon, A. DeRocer, and E. Vierling. 1990.
Accumulation, stability and localization of a maze chloroplast
heat shock protein. J. Cell Biol. 110:1873-1883.

28

Christensen, J.0., W.G. Ledge, R.M. DePaew, A.W. Henning, J.S.
McKenzie, and J .B. Thomas. 1984. Effect of seeding date,
nitrogen and phosphate fertilizer on growth, yield and quality
of rapeseed in northwest Alberta. Can. J. Plant Sci. 65:275-
284.

Close, T.J., A.An Kortt, and.P.M; Chandler. 1989. A.cDNA-based
comparison of dehydration induced protein (dehydrin) in barley
and corn. Plant Mol. Biol. 12:475-486.

Copeland, L.O., and R.H. Leep. 1990. Canola production in
Michigan. Mich. State Univ. Ext. Ser. Bull. 1234.

Daie, L., and W.F. Campbell. 1981. Responses of tomato plants
to stress temperature. Plant Physiol. 67:26-29.

Daniels, R.W., D.H. Scarisbrick, and J.F. Chapman. 1980.
Toward a ten-ton crop of rapeseed. Arable Farming 7:30-33.

Degenhardt, D.F. and z.P. Kondra. 1981. The influence of
seeding‘ date and seeding' rate on seed yield and yield
components of five genotypes of Brassica napus. Can. J. plant
Sci. 61:175-183.

Dennis, D.T., C.D. Upper, and C.A. West. 1965. Studies in the
specifity and site of action of ancymidol, a plant growth
regulator. Plant Physiol. 62:571.

Durner, E.F., T.J. Gianfagna. 1988. Fall ethephon application
increases peach flower bud resistance to low temperature
stress. J. Am. Soc. Hort. Sci. 113:404-406.

DeRocher, A.E., K.Wx Helm, L.M. Lauzon, and.E.‘Vierling. 1991.
Expression of a conserved family of cytoplasmic low molecular
weight heat shock proteins during heat stress and recovery.
Plant Physiol. 96:1038-1047.

Dexter, S.T. 1933a. Effect of several environmental factors on
the hardening of plants. Plant Physiol. 8:123-139.

Dexter, S.T. 1933b. Decreasing hardness of winter wheat in
relation to photosynthesis, defoliation, and winter injury.
Plant Physiol. 8:297-304.

Downey, R.K. 1983. The origin and description of the Brassica
oilseed crops. pp. 1-18. In: J.K.G. Kramer, F.D. Sauer, and
WQJ. Pigden (eds.) High and Low Erucic Acid Rapeseed Oils.
.Academic Press, Canada.

Downey, R.K., G.R. Stringam, D.I. McGregor, and B.R.
Stefansson. 1975. Breeding rapeseed and mustard crops. pp.
157-183. In: J.T. Harapiak (ed.) Oilseed and Pulse Crops in

29
Western Canada. Western Cooperative Fertilizer Ltd., Canada.

Dunn, M.A., M.A. Hughes, R.S. Pearce, and P.L. Jack. 1990.
Molecular characterization of a barley gene induced by cold
treatment. J. Exp. Bot. 41:1405-1413.

Fletcher, R.A. and G. Hofstra. 1985. Triadimefon a plant
multi-protector. Plant Cell Physiol. 26:775-780.

Fletcher,R.A. and V. Nath. 1984 Triadimefon reduces
transpiration and increases yield in water stressed plants.
Physiol. Plant 62:422-426

Fowler, D.B., and L.V. Gusta. Selection for winter hardiness
in wheat. I. Identification of genotypic variability. Crop
Sci. 19:769-772.

Fribourg, H.A., C.R. Graves, G.N. Rhodes, Jr., J.F. Bradley,
E.C. Beranrd, M.A. Muller, R.B. Graves, M.L. Thorton, E.A.
Latka, and A.M. Plouy. 1989. Rapeseed-A.potential new crop for
Tennnessee. Univ. of Tenn. Agr. Exp. Stn. Bull. 669.

Gilmour, S.J., and M.F. Thomashow. 1991. Cold acclimation and
cold-regulated gene expression in ABA mutants of Arabidopsis
thaliana. Plant Mol. Biol. 17:1223-1240.

Gilmour, S.J., N.N. Artus, and M.F. Thomashow. 1992. cDNA
sequence analysis and expression of two cold-regulated genes
of Arabidopsis thaliana. Plant Mol. Biol. 18:13-21.

Gilmour., S.J., R.K. Hajela, and M.F. Thomashow. 1988. Cold
acclimation in Arabidopsis thaliana. Plant Physiol. 87:745-
750.

Gordon-Kamn, W.J., and P.L. Steponkus. 1984. The influence of
cold acclimation on the behavior of the plasma membrane
following osmotic contraction of isolated protoplast.
Protoplasma 123:161-173.

Grafius, J.E. 1974. Winter hardiness in barley: breeding for
resistance. Mich. Agric. Exp. Stn. Res. Rep. 247:16-20.

Grossmann, K. 1990. Plant growth retardants are as a tools in
physiological research. Physiol. Plant 78:640-648.

Gusta, L.V., B.J. O'Connor, and M.J.T. Reaney. 1990. The
effect of growth regulators on the winter survival of winter
wheat. pp. 531-536. In: R.P. Pharis and 8.8. Rood (eds.) Plant
Growth Substances 1988. Spring-Verlag, Berlin.

Gusta, L.V., D.B. Fowler, and N.J. Tyler. 1982. Factors
influencing hardening and survival in winter wheat. pp. 23-40.

30

In: P.H.Li and A. Sakai (eds.) Plant Cold Hardiness and
Freezing Stress: Mechanisms and Crop Implications, vol. 2.
Academic Press, New York.

Gusta, L.V., and M.G. MacHutchon. 1988. Freezing resistance in
spring and winter crops. pp. 899-907. In: S.K. Shiha, P.V
Sane, S.C Bhargava, and P.K. Agrawal (eds.) Procedings of the
International Congress of Plant Physiology, Vol. 2. Spring-
Verlag, Berlin.

Hahn, M., and V. Walbot. 1989. Effects of cold-treatment on
protein synthesis and mRNA levels in rice leaves. Plant
Physiol. 91:930-938.

Hajela, R.K., D.P. Horvath, S.J. Gilmour, and M.F. Thomashow.
1992. Molecular cloning and expression of cor (cold-regulated)
genes in Arabidopsis thaliana. Plant Physiol. 93:1246-1252.

Hang, A.N., and G.C. Gilland. 1982. Performance of spring and
winter Brassica (rape) in central Washington. Wash. State
Univ. Agric. Res. Bull. KB 0921.

a

Hill, D.D., and S.C. Salmon. 1927. The resistance of certain
varieties of winter wheat to artificially produced low
temperature. J. Agric. Res. 35:933-937.

Hodgins,R., K. Pomeroy, A.Horak, and A.M. Johnson-Flanagan.
1989. Inhibition of photosynthetic activity in canola
(Brassica napus cv. Westar) seeds following freezing. Plant
Physiol. 89(Suppl.):27.

Hodgson, H.J. 1964. Effect of photoperiod on development of
cold resistance in alfalfa. Crop Sci. 4:302-305.

Houde, M., J. Danyluk, J-F. Laliberte, R.S. Dhindsa, and F.
Sarhan. 1991. Cloning and characterization of four cDNAs
specifically’ induced. by low 'temperature in *wheat. Plant
Physiol. 96(Suppl.):161.

Houde, M., J. Danyluk, J-F. Laliberte, R.S. Dhindsa, and F.
Sarhan. 1991. Cloning, characterization and expression of a
cDNA encoding a 50 kDA protein specifically induced by cold
acclimation in wheat. Plant Physiol. 99:1381-1387.

Howell, G.S. , and F.G. Dennis Jr. 1981. Cultural management of
;perennial plants to maximize resistance to cold stress. pp.
175-204. In: C.R. Olien and M.N. Smith (eds.), Analysis and
Improvement of Plant Cold Hardiness. CRC Press, Boca Raton,

F1. '

Huber, W. 1973. Testing of winter rape varieties and yield
comparisons at different sowing date. Mitt. Schweizerische

31

Landwirsche 21:97-105.

Irving, R.M. 1969. Characterization and role of an endogenous
inhibitor in the induction of cold hardiness of Acer.negundo.
Plant Physiol. 44:801-805.

Johnson-Flanagan, A.M. 1989. The role of chlorophyllase in
canola (Brassica napus cv. Westar) seed degreening under
optimal conditions and following frost. Plant Physiol.
89(Suppl.):30.

Johnson-Flanagan, A.M. and J. Singh. 1987. Alteration of gene
expression during the induction of freezing tolerance in
Brassica napus suspension cultures. Plant Physiol. 85:699-705.

Johnson-Flanagan, A.M., M.R. Thiagarajah, and M.K. Pomeroy.
1991. The impact of freezing during maturation on storage
products in canola seeds. Plant Physiol. 81:301-308.

Kacperska-Palacz, A., and B. Wcislinska. 1972. The effect of
' CCC on the nitrogen compounds content in rapejplants and.their
frost hardiness: Relation to the condition of day-length and

temperature. Biol. Plantarum 40:39-47. ‘

Kacperska-Palacz, A., M. Blaziak, and B. Wcislinska. 1969. The
effect of growth retardant CCC and B-9 on certain factors
related to cold acclimation of plants. Bot. Gaz. 130:213-221.

Kacperska-Palacz, A., S. Egierrszdorff. 1972. Effect of cold
hardening and. CCC ‘treatment. on Ihydration and frost and
desiccation hardiness of plant tissue. Bot. Gaz. 133:355-360.

Ketchie, D.O., and C. Murren. 1976. Use of cryoprotectants on
apple and pear trees. J. Am. Soc. Hort. Sci. 101:57-59.

Kephart, K.D., M.F. Rice, J.F. McCaffery, C.A. Murray. 1988.
Spring rapeseed culture in Idaho. Idaho Agri. Exp. Stn. Bull.
681.

Kimpel, J.A., and J.L. Key. 1985. Presence of heat shock mRNA
in field grown soybeans. Plant physiol. 79:672-678.

Kondra, z.P. 1977. Effect of planted seed size and seed rate
on rapeseed. Can. J. Plant Sci. 57:277-280.

IKondratowicz, J., and S. Patrocki. 1974. The effect of sowing
date and of cultural methods in autumn on yield of rape in
(Dlszystn province. Akad. Roln-Tech. Olsztynie 9:119-133.

Krause, G.H., R.J. Klosson, and U. Troster. 1982. On the
mechanism of freezing injury and cold acclimation of spinach
.1eaves. pp. 55-75. In: P.H.Li and A. Sakai (eds.) Plant Cold

32
Survival and Freezing Stress. Academic Press, New York.

Kurkela, S., and M. Frank. 1990. Cloning and characterization
of a cold- and ABA-induced Arabidopsis gene. Plant Mol. Biol.
15:137-144. '

Lang, A. 1979. Gibberellin: Structure and metabolism. Annual
Rev. Plant Physiol. 21:537-594.

Lang, V. and E.T. Palva. 1992. The expression of a.rab-related
gene, rab18, induced by abscisic acid during the cold
acclimation process of Arabidopsis thaliana (L.) Heynh. Plant
Mol. Biol. 20:951-962.

Lang, V., P. Heino, and E.T. Palva. 1988. Low temperature
acclimation and treatment with exogenous abscisic acid induce
common polypeptides in Arabidopsis thaliana (L.) Heynh. Theor.
Appl. Genet. 77:729-734.

Levitt, J. 1980. Responses of Plant to Environment stresses
Vol.1. Chilling, Freezing and High Temperature Stresses, 2nd
ed. Academic Press, New York. 497 pp.

Levitt, J., and D. Siminovitch. 1940. The relation between
frost resistance and the physical state of the protoplasm.
Can. J. Res. Sect. C. 18:550-561.

Lin, C., and M. F. Thomashow. 1992. DNA sequence Analysis of
a complementary DNA for cold-regulated.Arabidopsis gene cor15
and characterization of the COR15 polypeptide. Plant Physiol.
99:519-525.

Lin, C., W.W. Guo, E. Everson, and.M.F. Thomashow. 1990. Cold
acclimation in.Arabidopsis and wheat. Plant Physiol. 94:1078-
1083.

Lorraine, D.H., and E. Vierling. 1993. Expression of low
molecular weight heat-shock proteins under field conditions.
Plant physiol. 101:1209-1216.

Mahler, K.A. and D.L. Auld. 1987. National Winter Rapeseed
Variety’Trial 1986-1987. Idaho.Agric. Exp. Stn. Misc. Ser. 113

Mahler, K.A. and D.L. Auld. 1988. National Winter Rapeseed
variety Trial 1987-1988. Idaho Agric. Exp. Stn. Misc. Ser.
120.

Mahler, K.A. and D.L. Auld. 1989. National Winter Rapeseed
variety Trial 1988-1989. Idaho Agric. Exp. Stn. Misc. Ser.
130.

IMarth, P.C. 1965 Increased frost resistance by application of

33

plant growth retardant chemicals. J. Agri. Food Chem. 13:331-
333.

Mohapatra, 8.8., L. Wolfraim, R.J. Poole, and R.S. Dhindsa.
1989. Molecular cloning and relation to freezing tolerance of
cold acclimation specific genes of alfalfa. Plant Physiol.
89:375-380.

Mohapatra, S.S., R.J. Poole, and R.S. Dhindsa. 1988. Absisic
acid regulated gene expression in relation to freezing
tolerance in alfalfa. Plant Physiol. 87:468-473.

Morrison, M.J., and C.J. Andrews. 1992. Variable increases in
cold hardiness induced in winter rape by plant growth
regulators. J. Plant Growth Regul. 11:113-117.

Mundy, J., and.N-H. Chua. 1988. Abscisic acid and water-stress
induce the expression of a novel rice gene. EMBO J. 7:2279-
2286.

Murray, G.A., D.L. Auld, L.E. O'Keeffe, and D.C. Thill. 1984.
Winter rape production practice in northern Idaho. Idaho Agr.
Exp. Stn. Bull. 634.

Noderstagaard,A. 1972. Combined sowing time and variety
experiments with winter rape. Tidsskr. Planteavl 76:597-603.

Nordin, K., P. Heino, and E.T. Palva. 1991. Separate signal
pathways regulate the expression of a low temperature-induced
gene in.Arabidopsis thaliana. Plant Mol. Biol. 16: 1061-1071.

Olien, C.R. 1967. Freezing stress and survival. Annual Rev.
Plant Physiol. 18:387-408.

Orr, W., B. In, T.C. White, L.S. Robert, and J. Singh. 1992.
Complementary DNA sequence of a low temperature-induced
Brassica napus gene with homology to the Arabidopsis thaliana
kinl gene. Plant Physiol. 98:1532-1534.

Orr, W., W.A. Keller, and J. Singh. 1986. Induction of
freezing tolerance in an embryogenic cell suspension culture
of Brassica napus by abcsisic acid at room temperature. J.
Plant Physiol. 126:23-32.

Palta, J.P. 1992. Mechanisms for obtaining freezing stress
resistance in herbaceous plants. pp. 219-250. In: H. T.
Stalker and.J.P. Murphy (eds.) Plant Breeding in the '90s. CAB
International, Walingford, England.

ZPalta, J.P., J. Levitt, and E.L. Stadelmann. 1977. Freezing
injury in onion bulb cells. I. Evaluation of the conductivity
method and analysis of ion and sugar efflux from injured

34

cells. Plant Physiol. 60:393-397.

Palta, J.P., and P.H.Li. 1980. Alteration in membrane
transport properties by freezing injury in herbaceous plants:
Evidence against rupture theory. Plant Physiol. 50:169-175.

Paquin, R., L. Belzile, C. Willemot, J.-C. St-Pierre. 1976.
Effects of some growth retardants and gibberellic acid on the
frost resistance of alfalfa (Medicago sativa). Can. J. Plant
Sci. 56 :79-86.

Paulsen, G.M. 1968. Effect of photoperiod and temperature on
cold hardiness in winter wheat. Crop Sci. 8:29-32.

Pearse, R.S., and J.M. Willison. 1985. Wheat tissues freeze-
etched during exposure to extracellular freezing: distribution
of ice. Planta 163:295-303.

Pennypacker, B.W., P.L. Sanders, L. Vaun Gregory, E.P.
Gilridge, and H. Cole. 1982. Influence of triadimefon on the
foliar growth and flowering of annual bluegrass. Can. J.
Pathol. 4:259-262.

Pla, M., J. Gomez, A. Goday, and M; Pages. 1991. Regulation of
the absisic acid responsive gene rab28 in maize viviparous
mutants. Mol. Gen. Genet. 230:394-400.

Pomeroy, M.K. , and C.J. Andrews. 1976. Ultrastructural changes
in shoot apex cells of winter wheat seedlings during ice
encasement. Can. J. Plant Sci. 55:529-535.

Proebsting, E.L.Jr., and H.H. Mills. 1976. Ethephon increases
cold hardiness of sweet cherry; J. Am. Soc. Hort. Sci. 101:31-
33.

Quisenberry, K.S. 1938. Survival of wheat varieties in the
great plains winter hardiness nursery. J. Am. Soc. Agron.
30:399-405.

Raese, J.T. 1977. Induction of cold hardiness in apple tree
shoot with ethephon, NAA and growth retardant. J. Am. Soc.
Hort. Sci. 102:789-792.

Raymond, P.L., D.C. Bullock, D.L. Thomas. 1990. Potential of
winter and spring rapeseed cultivars for oilseed production in
the southeastern United States. pp. 223-225. In: J.E. Simon
and J. Janick (eds.) Advances in New Crops. Timber Dioscorides
Press, New York.

lReaney, M.J.T., and L.V. Gusta. 1987. Factors influencing the
induction of freezing tolerance by abscisic acid in cell
suspension culture of Bromus inermis Leyss and Medicago sativa

L. P

Reid
rele
Rood
Berl

Riki
Harm
Rodi
(Red

Robe
role
vint
267.
Cold

Robe
hard
Plan

Ruel
resi
clov

Sake
Free

Sant
Chic
its
Plan

Seat

35
L. Plant Physiol. 83:423-427.

Reid, M.S. 1990. New aspects of the practical use of ethylene-
releasing compounds. pp. 595-603. In: R.P. Pharis and 8.8.
Rood (eds.) Plant Growth Substances 1988. Springer-Verlag,
Berlin.

Rikin, A., M. Waldman, A.E. Richmond, and A. Dovart. 1975.
Hormonal regulation of morphogenesis and cold-resistance. I.
Modification by abscisic acid and gibberellic acid in alfalfa
(Medicago sativa L.) seedlings. J. Exp. Bot. 26:175-183.

Robertson, A.J., G.C. Churchill, and L.V. Gusta. 1992. The
role of plant growth regulators on the freezing tolerance of
winter annual cereals and cell suspension culture. pp. 253-
267. In: P.H. Li and L. Christerson (eds.) Advances in Plant
Cold Hardiness. CRC Press, Boca Raton.

Robert, D.W.A., and M.N. Grant. 1968. Changes in cold
hardiness aCcompanying development in winter wheat. Can. J.
Plant Sci. 48:369-376.

Ruelke, 0.C., and.D. Smith. 1956. Overwintering trends of cold ‘
resistance and carbohydrate in red, ladinos and common white
clover. Plant Physiol. 31:364-68.

Sakai, A., and W. Larcher. 1987. Responses and Adaptation to
Freezing Stress. Springer-Verlag, Berlin. 321 pp.

Santarius, K.A. 1973. The protective effect of sugars on
chloroplast membrane during temperature and water stress and
its relationship to frost, desiccation and heat resistance.
Planta 113:105-114.

Scott, P.K., J.D. Ogunremi, and N.J; Mendham. 1973. The effect
of fertilizers and.harvest date onlgrowth and yield of oilseed
rape sown in autumn and spring. J. Agric. Sci. 81:287-293.

Shahidi, F. 1990. Rapeseed and canola: Global production and
distribution. pp. 3-15. In: F. Shahidi (ed.) Canola and
Rapeseed. Van Nostrand Reinhold Inc, New York.

Sicorska, E., and A. Kacperska. 1982. Freezing induced
membrane alteration: injury or adaptation. pp. 261-284. In:
P.H. Li and A. Sakai (eds.) Plant Cold Survival and Freezing
Stress. Academic Press, New York.

Simonovitch, D., and G.W. Scarth. 1938. A study of the
mechanism of frost injury in plants. Can. J. Res. Sect. C. 16:
467-481.

Singh, J. 1979a. Ultrastructural alterations in cells of

36

hardened and nonhardened winter rye during hyperosmotic
and extracellular freezing stress. Protoplasma 98:329-341.

Singh, J. 1979b. Freezing of protoplast isolated from cold
hardened and nonhardened winter rye. Plant Sci. Lett. 16:195-
201.

Singh, J. 1982. Alterations in rye cells during lethal
freezing' and. plasmolysis stress. Plant Physiol.
69(Suppl.):106.

Singh, J., and A. Laroche. 1988. Freezing tolerance in plants:
a biochemical overview. Biochem. Cell Biol. 66:650-657.

Singh, J., B. In, and A.M. Johnson-Flanagan. 1987. Membrane
alterations in winter rye and Brassica napus cells during
lethal freezing and plasmolysis. Plant Cell Environ. 10:163-
168.

Skriver, K., and J. Mundy. 1990. Gene response to ABA and
osmotic stress. Plant Cell. 2:503-512.

Smith, H.A., C.H. Rust, D.B. Baldridge, and R.M. Laughlin.
1989. Rapeseed (including canola): A.Montana speciality crop.
Mont. State Univ. Ext. Ser. Bull. 8909.

Smith, D., and C.J. Nelson. 1985. Physiological consideration
in forage management. pp. 322-330. In: M.F. Heath, R.F. Barns,
and.D.S. Metcalfe (eds.) Forage. Iowa State Press, Ames, Iowa.

Steffen, K.L., J.P. Palta. 1987. Photosynthesis as a key plant
responses to low temperature. pp. 67-100. In: P.H. Li (ed.)
Plant Cold Hardiness. Liss, New York.

Steponkus, P.K. 1978. Cold hardiness and freezing injury of
agronomic crops. Advances in Agronomy, 30:51-98.

Steponkus, P.L. , and D.V. Lynch. 1989. Freeze/thaw-induced
destabilization of the plasma membrane and the effects of cold
acclimation. J. Bioenergetics and Biomembranes 21:21-41.

Steponkus, P.L., and F.O. Lanphear. 1967. Light.stimulation of
cold acclimation: production of translocatable promoters.
Plant Physiol. 42:1673-1677.

Steponkus, P.L., M.‘Uemura, R.A. Balsamoq T. Arvinte, and D.V.
Lynch. 1988. Transformation of the cryobehavior of rye
protoplast by modification of the plasma membrane lipid
composition. Proc. Natl. Acad. Sci. USA. 85:9026-9030.

Strauss, G. and H. Hauser. 1986. Stabilization of lipid
bilayer vesicles by sucrose:during freezing. Proc. Natl. Acad.

37

Sci. USA. 83:2422-2426.

Sulc, R.M., K.A. Albrecht, J.P. Palta, and S.H. Duke. 1991.
Leakage of intracellular substance from alfalfa roots at
various subfreezing temperature. Crop Sci. 31:1575-1578.

Suneson, C.A., and G.L. Peltier. 1938. Effect of weather
variance on field hardiness of winter wheat. J. Am. Soc.
Agron. 30:769-778.

Teutonico, R.A., J.P. Palta, and T.C. Osborn. 1993. In vitro
freezing tolerance in relation to winter survival of rapeseed
cultivars. Crop Sci. 33:103-107.

Thomas, P. 1984. Canola Grower's Manual. Canola Council of
Canada. Winnipeg, Canada. 180 pp.

Thomas, D.L., M.A. Breve, P.L.Raymer, N.A. Minton, and
D.R.Sunner. 1989. Rapeseed. (Including' canola): a. Montana
specialty crop. Mont. State Univ. Ext. Ser. Bull. 8908.

Thomashow, M.F. 1990. Molecular genetics of cold acclimation
in higher plant. Adv. Genet. 28:99-131.

Thomashow, M.F., S.J. Gilmour, and C. Lin. 1993. Cold-
regulated genes of Arabidopsis thaliana. pp. 31-44. In: P.H.
Li and L. Christerson (eds.) Advances in Plant Cold.Hardiness.
CRC PreSs, Boca Raton.

Thompson, R.F., and W.G. Hughes. 1986. Breeding and varieties.
pp. 32-82. In: D.H. Scarisbrick and R.W. Daniels (eds.)
Oilseed Rape. Collins, London.

Tysdal, H.M. 1933. Influence of light, temperature, and soil
moisture on the hardening process in alfalfa. J. Agr. Res.
46:483-515.

‘Volger, H.G., and U. Heber. 1975. Cryoprotective leaf protein.
Biochem. Biophys. Acta. 412:335-349.

Waldman, M., A. Rikin, A. Dorvat, and A.E. Richmond. 1975.
Hormonal regulation of morphogenesis and cold acclimation. J.
Exp. Bot. 26:853-860.

‘Weiser, C.J. 1970. Cold.resistance and injury in woody plants.
Science 169:1269-1278.

Weretilnyk, E., W: Orr, T.C. White, B. In, and J. Singh. 1993.
Characterization of three related low-temperature regulated
cDNAs from winter Brassica napus. Plant Physiol. 101:171-177.

Whitaker, B.D., and C.Y. Wang. 1987. Effect of paclobutrazol

38

and chilling of leaf membranes in cucumber seedlings. Physiol.
Plant 70:404-411.

Wilkinson, R.E. 1982. Mefluidide inhibition of sorghum growth
and gibberellin precursor biosynthesis. J. Plant Growth Regul.
1:85.

Yamaguchi-Shinozaki, K., and K. Shinozaki. 1994. A novel cis-
acting element in an Arabidopsis gene is involved in
responsiveness to drought, low-temperature, or high-salt
stress. Plant Cell 6:251-264.

CHAPTER]

CORRELATIONS BETWEEN WINTER SURVIVAL, FREEZING
TOLERANCE, AND THE EXPRESSION OF COLD-REGULATED
GENES, BN28 AND BN115, IN CANOLA (Brassica napus L.)

ABSTRACT

Of the many factors influencing winter survival of
plants, freezing tolerance is one of the most important. Cold ‘
acclimation increases freezing tolerance and is involved in
changes of gene expression. This study was conducted to
determine the relationship between in vitro freezing tolerance
and winter survival of canola (Brassica napus L.) and to
determine the responses of two cold-regulated genes from B.
napus, BN28 and BN115, to cold acclimation. Six winter
cultivars and one spring cultivar that had previously
exhibited a broad range of winter survival were examined both
in a controlled environment chamber and under field
conditions.

Freezing tests showed that cold acclimation increased
the freezing tolerance of both winter and spring cultivars,

and that the differences in freezing tolerance among cultivars

39

40
were due to their ability to acclimate. Freezing tolerance
measured by freezing tests was highly correlated with winter
survival, suggesting that this test can be used to predict the
[comparative winter survival of canola cultivars.

Southern blot analysis revealed that the seven cultivars
examined had the same copy number of BN28 and BN115. Northern
blot analysis indicated that these genes were induced within
1 day by cold temperature and turned off immediately by
deacclimation. Though time-course increase in transcript in
one of the genes was highly correlated with the time-course
increase of freezing tolerance, no correlation was found at
the cultivar level. However, this does not rule out the ‘
possible role of cold-regulated genes in freezing tolerance of

canola.

INTRODUCTION

In most winter crop producing areas, freezing
temperature is the single most important factor limiting
winter survival in the field (Steponkus, 1978) . Freezing
tolerance is the ability of a plant to survive subfreezing
temperatures, and constitutes a major aspect of winter
survival. Screening for differences in freezing tolerance
among cultivars or breeding lines has been of great concern to
plant breeders for many years.

Field survival is the usual method of evaluating winter
hardiness. However, such results are often inconclusive
because of variability in weather conditions from year to year
(Levitt, 1980). Because of these yearly variations, trials
must be conducted for a number of years, and are therefore
extremely time-consuming (Andrew, 1960) . The development of
rapid techniques for screening plants for high acclimation
potential has led to the development of indirect screening
methods (Steponkus, 1978) . These methods have arisen largely
as a result of empirical correlations between some
physiological or anatomical characteristics of plants. One of
the most common methods for determining freezing tolerance is

the measurement of leakage of ions from plant cells after

41

42
freezing stress (Palta et al., 1977; Palta and Li, 1980;
Stephen et al., 1989; Sulc et al., 1991; Arora and Palta,
1991). The advantage of this method is that it is simple,
repeatable, rapid, and imposes a realistic freeze-thaw stress
on intact tissue (Teutonico et al., 1993).

Some genetic and phenotypic markers which segregate for
winter hardiness also have been used for screening. Prior
exposure of plants to low nonfreezing temperatures (cold
acclimation) increases freezing tolerance. Many physiological
changes are known to occur in plants during cold acclimation
(Levitt, 1980; Sakai and Larch, 1987; Steponkus and Lynch,
1989), leading'Weiser (1970) to suggest that they are mediated
by altered gene expression. Using recent molecular biology
techniques, several genes induced by cold temperature were
isolated and studied for their possible role in cold
acclimation (Mohapatra et al., 1989; Lin et al., 1990;
Cativelli and Bartels, 1990; Hajela et al., 1990; Kurkela and
Franck, 1990; Dunn et al., 1990; Houde et al., 1991; Nordin et
al., 1991; Gilmour et al., 1992; Orr et al., 1992; Bihn et
al., 1992; Weretilnyk et al., 1993). In addition to
functional studies of these genes, the link between the
expression of cold-regulated genes and freezing tolerance can
be used to aid in the breeding for winter hardiness, provided
that the expression of those molecular markers is highly
correlated with winter hardiness and/or freezing tolerance

(Mohapatra et al., 1989).

1.

43

The objectives of this study were to
Study the correlation between freezing tolerance and winter
survival and evaluate the reliability of in vitro freezing
tests for predicting winter survival of canola (B. napus).
Study the expression of two cold-regulated genes from
B. napus, BN28 and BN115, during cold acclimation and
deacclimation.
Determine whether the expression of cold-regulated
genes at the transcript level is correlated with

freezing tolerance, and eventually with winter survival

in the field.

MATERIALS AND NIETHODS

Six winter cultivars, CDH3, WRG86, Duobul, Ceres,
KWC4113, Accord, and one spring cultivar, Pactol, were used
for freezing tests while only winter cultivar was used for

field experiments.

Evaluation of Winter Survival

Seeds of the six winter cultivars were planted at the
rate of 5.6 kg/ha in a completely randomized block design with
3 replications on the Michigan State University Agronomy Farm
at East Lansing. Each replication consisted of a five-row
plot 6 m long and 0.92 m wide. Seeds were planted Sept. 7,
1992, and Sept. 6, 1993. The number of live plants in each
subplot (3 row x 1 m long each) was counted in the fall of
each year (Oct. 10, 1992, and Oct. 15, 1993) and again in the
spring of the next year (Apr. 15, 1993, and Apr. 15, 1994).
Percent survival was recorded as (no. of plants per plot in

spring / no. of plants per plot in fall) X 100.

Plant Materials, Growth and Cold Treatment for Freezing Tests
Plants were grown in plastic pots in a 1:1:1 mixture of

perlite:peat moss:fine vermiculite moistened with a nutrient

44

sch
were
24%
liq]
day:
r001
{Gil
tole
wit!

dear

i002

stag

rest.

“inc

45

solution as described by Sommerville and Ogren (1982) . Plants
were maintained in a controlled environment chamber at 22 to
24°C under constant illumination from cool white f luororescent
lights. Plants were irrigated with distilled water every 2
days. Wenty-day-old seedlings were moved to the 2°C cold
room with constant fluorescent light for cold acclimation
(Gilmour et al. , 1988) . Time-sequence experiments of freezing
tolerance were done with the nonacclimated control plants and
with plants acclimated for 1, 5, and 10 days. For the
deacclimation experiment, 10-days. For deacclimation
experiment, plants acclimated for 10 days were transferred to
room temperature and deacclimated for 1 and 3 days before the
freezing tests.

For RNA extraction from plants at each acclimation
stage, leaves and stems were excised at the soil level, frozen
in liquid N2, pulverized using a mortar and pestle, and stored
at -80°C prior to extraction. Genomic DNA was isolated from
control plants for southern blot analysis. Freezing tests and
RNA extraction were performed at three different times as

three replications.

Tests for Freezing Tolerance

Freezing tolerance of leaves was determined by the
method of Sukumaran and Weiser (1972) . Samples of three
excised leaves were placed in a stoppered culture tube

maintained in a low temperature bath (Masterline Model 2095,

Rom
addi
equi
aanu
with
-18°

thaw

acid
iqlf
2:10;

46
Forma Scientific) at 2°C. Freezing was initiated by the
addition of ice chips to each tube. After a 2 hour
equilibration period, the bath temperature was lowered
manually in 1°C increments every 30 min. Samples were
withdrawn at 1 hour intervals until the temperature reached
-18°C, and placed on ice for several hours, then removed and
thawed overnight in a cold room at 2°C. Freezing damage was
estimated by the electrolyte leakage test. Five ml of
distilled water were added to each tube and the samples were
shaken gently for 3 hours. Conductivity of the resulting
solution was measured using a conductance meter (YSI model
35). A value for 100 % leakage was obtained by freezing each ‘
sample at -80°C overnight, then reextracted in the original
solution. A plot of temperature versus percent electrolyte
leakage was used to determine the value for 50 % electrolyte

leakage, which was defined as LTm.

Genomic DNA and Total RNA Isolation

Total RNA was isolated by the method of Gilmour et al.
(1988). Frozen pulverized plant materials were extracted in
a buffer containing 100 mM Tris-HCl (pH 7.6), 100 mM NaCl, 50
mM EGTA, 1%(w/v) SDS, 10 mM DTT, 6 % (w/v) p-aminosalicylic
acid (sodium salt), and 1 % (w/v) tri-isopropyl naphthalene
sulfonic acid with an equal volume of buffer-saturated phenol:
chloroform: isoamyl. alcohol (25:24:1, PCI). .After

centrifugation to separate the phases, the aqueous phase was

47
extracted once more with PCI, and the nucleic acids were
ethanol-precipitated twice on ice with 2M LiCl, followed by a
final ethanol precipitation. The pellet was redissolved in
distilled water and stored at -80°C. Total DNA was prepared
from the supernatant left after LiCl precipitation of RNA from
total nucleic acids. The supernatant was diluted to 0.5 M
LiCl with distilled water and the DNA was precipitated with
ethanol. The DNA was then resuspended in a buffer containing

10 mM Tris-HCl (pH 8.0), 1 mM EDTA.

Preparation of DNA Probes

Two cold-regulated cDNA clones, BN28 and BN115, were ‘
obtained from Dr. Jas Singh, Plant Research Center,
Agriculture Canada, Ottawa, Ontario. Plasmid DNA was isolated
by alkaline lysis, followed by polyethyleneglycol (PEG)
precipitation. Whole plasmid was cut with the restriction
enzyme, EcoRI, and subjected to agarose gel electrophoresis.
Insert DNA from the gel slice was isolated using the Gene
Clean Kit. 32P-labelled probes were prepared using 64 ng of
template insert DNA by the random oligonucleotide primers

method.

Southern Blot Analysis
Total genomic DNA (5 ug) was incubated overnight with a
five-fold excess of a restriction enzyme. The digested DNA

was size-fractionated by electrophoresis in 1.0 % agarose gel

for 61

for 2<
NaPO,
(Schl:
buffer
the fj
to 24
washec
each.

and cu

48
for 640‘V-h. After electrophoresis, the gels were treated with
0.25 N HCl for 15 min, denatured with 0.5 N NaOH, 1.5 M NaCl
for 20 min, and finally neutralized with three washes in 25 mM
NaPO, buffer (pH 6.5) . DNA was transferred to Nytran filter
(Schleicher and Schuell) by capillary action with 25 mM NaPO4
buffer, pH 6.5. After transfer, the DNA was cross-linked to
the filters by UV irradiation. IFilters were hybridized for 16
to 24 hours according to Church and Gilbert (1984) , then
washed three times at 45°C in 0.1 X SSC, 0.1 % SDS for 20 min
each. Autoradiography was done using AR5 x-ray film (Kodak)

and Cronex intensifying screens (Dupont) at -80°C.

Northern Blot Analysis

Northern blot analysis was done as previously described
with some modification (Hajela et al., 1990). Fifty ug of
total RNA dissolved in 100 uL RNA loading buffer. From the
100 ul of sample solution, only 15 uL (7.5 ug of total RNA)
were loaded on the gel. Total RNA was fractionated on 1.2 %
denaturing formaldehyde agarose gel and northern blot was
prepared on a Nytran membrane (Schleicher and Schuell) using
20x SSPE as the transfer buffer. As a check for RNA
integrity, efficiency of transfer, and equivalency of loading,
the EtBr stained gel and membrane were carefully examined
visually under UV light. The RNA in the membrane was
immobilized by cross-linking with UV light. Prehybridization

and hybridization was done according to manufacturer's

inst
as
Corp
by s

and

Stat

49
instructions (Schleicher and Schuell). The transcript amount
was quantified using a Betagen 603 Blot Analyzer (Betagen
Corp.). Filters were stripped for subsequent rehybridization
by submerging in boiling 5 mM Tris-HCl (pH 7.8), 1 mM EDTA,
and 0.05 % SDS for 15 min. Stripped filters were rehybridized
with the radish rDNA, pRE12, and quantified as mentioned
before for the standardization of total RNA present in the
membrane. The northern blot experiments were conducted three

times as replications using three different RNA samples.

Statistical Analysis

Analysis of variance (AOV), least significant
difference (LSD), Duncan's multiple range test (DMRT), and
single correlation analysis were used to analyze the data
using the statistical package MSTAT (Michigan State

University, East Lansing, Michigan)

RESULTS

Winter Survival of Canola

In 1992-1993, winter survival of the most hardy
cultivar, Accord was significantly greater than that of the
other 5 cultivars. All except CDH3 had winter survival higher
than 80 % (Table 1). In 1993-1994, winter survival was lower,
and differences among cultivars were more pronounced than in

1992-1993.

Tests for Freezing Tolerance

Freezing assay was based on ion leakage as a measure of
freezing damage. The relationship between temperature and
percent ion leakage (freezing curves) for each of seven
cultivars is shown in Figs. 1 to 7. Freezing curves for the
overall mean values of seven cultivars are shown in Fig. 8.
The shapes of these curves were similar for all winter
cultivars as well as the spring cultivar, and in each case
acclimation shifted the entire freezing curve to lower
temperatures, although the specific temperature range varied
among cultivars. Freezing tolerance of all cultivars was
increased within 1-day of exposure to cold temperature, and

appeared to be at a maximum within 5 days.

50

51

Table 1. Winter survival of six winter canola cultivars
in 1992-1993 and 1993-1994.

 

 

 

Cultivars Winter survival (%)
1992-1993 1993-1994

ACCORD 100 a? 72 a
KWC4113 86 ab 70 a
CERES 82 b 59 b
DUOBUL 80 b 65 b
WRGSG 81 b 57 be
CDH3 75 b 41 c

 

T Means followed by the same letter within a column
are not significantly different at the 0.05
probability level

52

 

 

  

 

 

 

 

 

 

 

100
80-
“£1
E 60-
<3
III
A 40-
0
B\ CONTROL
IDAY
20- SDAY
10 DAY
0 I I I I I I I I
o 2 4 6 8 1o 12 14 16 18

TEMPERATURE (- °C)

Figure 1. Freezing curves of winter canola cv. Accord
acclimated for 0, 1, 5, and 10 days, respectively.
Each data point is a mean of three replications with

nine sample leaves in a replication. Vertical bar
indicates mean t SE.

53

 

 

 

 

 

 

 

 

 

 

 

100
80 I -
f
m 60- /_
<¢ 40-
m
A —o— CONTROL
‘ —-—— 1 DAY
56 20- / —o— 5 DAY
'1‘” —e— 10 DAY
0 I I I I I I I I
o 2 4 6 8 1o 12 14 16 18

TEMPERATURE (- ° C)

Figure 2. Freezing curves of winter canola cv. KWC4113
acclimated for 0, 1, 5, and 10 days, respectively.
Each data point is a mean of three replications
with nine sample leaves in a replication. Vertical
bar indicates mean t SE.

54

 

 

 

 

 

 

 

 

 

 

 

 

 

 

100
80- Cg I.
5
60- ..
é TEE
E
40-
1.]
s“ CONTROL
1DAY
20- 5 DAY
10 DAY
0 I 7 I I I I I I
o 2 4 6 8 1o 12 14 16 18

TEMPERATURE (- ° C)

Figure 3. Freezing curves of winter canola cv. Ceres
acclimated for 0, 1, 5, and 10 days, respectively.
Each data point is a mean of three replications with
nine sample leaves in a replication. Vertical bar
indicates mean t SE.

55

 

100

 

 

 

 

  

 

 

 

 

 

 

6‘
g 60- -
<
m
'4 40-
8°
,. —o— CONTROL
20- ——-— 1DAY
J ——o— 5 DAY
—._ 10 DAY
0 I I I I I I I I
o 2 4 6 8 1o 12 14 16 18

TEMPERATURE (- °C)

Figure 4. Freezing curves of winter canola cv. Duobul
acclimated for 0, 1, 5, and 10 days, respectively.
Each data point is a mean of three replications
with nine sample leaves in a replication. Vertical
bar indicates mean 1 SE.

 

56

 

 

   

 

 

 

 

 

 

 

 

100
80-
S
g 60-
4:
LO
,4 40-
8°
. CONTROL
20.. —|— 10AY
—o—— 5 DAY
—.— 10 DAY
0 I W I I T I I I
o 2 4 6 8 1o 12 14 16 18

TEMPERATURE (- °C)

Figure 5. Freezing curves of winter canola cv. WRG86
acclimated for 0, 1, 5, and 10 days, respectively.
Each data point is a mean of three replications with
nine sample leaves in a replication. Vertical bar
indicates mean t SE.

57

 

 

100
J
. 1'—
80“ "
0‘
60"

% LEAKAGE

4o— ' ./

/ —o— CONTROL
__-/ —-—. 1 DAY

20- " —o— 5 DAY
1/

 

—e-— 10 DAY

 

 

 

 

 

 

I I I I I I I

T
0 Z 4 6 8 10 12 14 16 18

TEMPERATURE (- ° C)

Figure 6. Freezing curves of winter canola cv. CDH3
acclimated for 0, 1, 5, and 10 days, respectively.
Each data point is a mean of three replications with
nine sample leaves in a replication. Vertical bar

indicates mean t SE.

58

 

 

 

 

 

 

 

 

 

 

 

 

 

100
49
80-
1‘3
60-
<
M
E
40--
p]
53 CONTROL
1DAY
20- 5DAY
10 DAY
0 I I I I I I I I
o 2 4 6 8 1o 12 14 16 18

TEMPERATURE (- °C)

Figure 7. Freezing curves of spring canola cv. Pactol
acclimated for 0, 1, 5, and 10 days, respectively.
Each data point is a mean of three replications with

nine sample leaves in a replication. Vertical bar
indicates mean 1 SE.

59

 

 

 

 

 

 

 

 

 

 

 

100
I: ’§
80- 9 I “I;
an 60 .
<1
fig 40-
m
A —a— CONTROL
G q -—+—— 1 DAY
5 2° —-— 5 DAY
-—4»—- 10 DAY
0 I I I I I I I I
0 2 4 6 8 10 12 I4 16 18

TEMPERATURE (— ° C)

Figure 8. Freezing curves of canola acclimated for 0, 1, 5,
and 10 days, respectively. Each data point
represents the overall mean of seven cultivars
tested in Figs. 7. Vertical bar indicates mean t
SE.

 

60

LT,0 values were determined from the freezing curve of
each cultivar (Fig. 9). Nonacclimated control plants had an
LT,0 value of approximately -2°C, while LT,0 values in plants
exposed to cold temperature were increased 1-2°C per day. LT,0
values of control plants did not differ among cultivars.
However, as soon as acclimation began, three groups of
cultivars having different LT,0 values had appeared. Although
significant differences in LT,o value occurred among groups, no
differences occurred within groups. KWC4113 and Accord
belonged to the most hardy group, having the minimum LT,0
values of -10°C and -11°C, respectively, when fully
acclimated, and the spring variety, Pactol, belonged the least
hardy group having a minimum LT,0 value of -5.5°C. Another
spring canola cultivar, Legend, had the same L'I',0 value as
Pactol (data not shown). Other cultivars, Ceres, Duobul,
WRGB6, and CDH3, belonged to the medium group having LT50

values of -7°C to -8°C.

Nonacclimated Freezing Tolerance and Cold Acclimation Capacity
Freezing tolerance can be divided into two separate

factors: Nonacclimated freezing tolerance and acclimation

capacity (Table 2). Nonacclimated freezing tolerance is

LTso values of plants which were not acclimated. Acclimation

capacity is the ability to increase freezing tolerance by cold

acclimation (LT50 value of the acclimated stage - LT50

 

61

 

 

 

 

 

 

 

 

 

 

 

 

1 2
1 o -
8 - -1
A 4‘
P —
I 5‘ I
V m
8
5..
,4 4 - ACCORD
KWC41 13
CERES
DUOBUL
2 ‘ WR686
CDH3
PACTOL
0 I I l I I I
- 1 1 3 5 7 9 1 1

ACCLIMATION DURATION (DAYS)

Figure 9. LT” value changes in six winter canola cultivars and
one spring canola cultivar (Pactol) during cold-
acclimation, ThejplantS‘were acclimated for 0, 1, 5,
10 days. Vertical bar indicates mean i SE.

62

Table 2. Means of nonacclimated freezing tolerance and
acclimation capacity of six winter cultivars and
one spring cultivar at different acclimation

 

 

 

stages.

Nonacclimated Acclimation capacity(°C)
Cultivars freezing

tolerance (°C) 1 day 5 day 10 day
ACCORD -2.0 ab 2.8 a 8.0 a 8.8 aT
KWC4113 -2.2 a 1.8 ab 7.6 a 7.8 a
CERES -2.0 ab 1.3 b 5.8 b 5.9 b
DUOBUL -1.8 ab 1.6 ab 5.7 b 5.7 b
WRGB6 -1.8 ab 1.6 ab 4.7 be 5.0 bc
CDH3 -2.2 a 1.5 ab 5.4 b 4.8 bc
PACTOL -l.7 b 0.7 b 3.4 c 4.0 c

 

T Means followed by the same letter within a column are
not significantly different at the 0.05 probability

level.

 

63
value of the nonacclimated stage). Nonacclimated freezing
tolerance showed no significant differences among cultivars
except for the spring cultivar, Pactol. However, acclimation
capacity at 5 and 10 days showed clear differences among
cultivars. Accord had the highest increase in LT,0 value,
8.8°C in 10 day acclimation, while that of spring variety,

Pactol, increased by only 4°C.

Correlation between Freezing Tolerance and Winter Survival
High correlations were found between in vitro freezing
tolerance and winter survival of cultivars in all three years
(Table 3) . Nonacclimated (control) freezing tolerance was not
correlated with winter survival in any year, however, freezing
tolerance at 1, 5, and 10 days of acclimation was highly
correlated with winter survival every year. Futhermore, the
value of the correlation coefficient increased with
acclimation time. The correlation coefficient between
freezing tolerance and winter survival in 1993-1994 increased
from 0.89(**) after 1«day oflacclimation.to 0.95(***) after 10

days of acclimation.

Effect of Deacclimation on Freezing Tolerance
Comparison of changes in LT” value of cultivars from

nonacclimated control plants, 10-day acclimated plants, and 3-

 

64

Table 3. Simple correlation coefficients between freezing
tolerance and winter survival of six winter canola
cultivars in 1992-1993 and 1993-1994.

 

 

 

 

 

Freezing Winter survival
tolerance
1992-1993 1993-1994
in -
Control 0.33ns 0.54ns
1-day acclimation 0.93** 0.89** i
5-day acclimation 0.86** 0.90**
10-day acclimation 0.92** 0.95**
T (*) and (**) simple correlation coefficient significant

at 0.05 and 0.01 probability levels, respectively. (ns)
not significant at the 0.05 probability level. -

65
day deacclimated plants are shown in Fig. 10. Generally,
deacclimation decreased freezing tolerance as rapidly as
acclimation increased it. More hardy cultivars lost freezing
tolerance more rapidly, but still retained higher LT,O values
than less hardy cultivars after 3 days of deacclimation, with
a 3.0°C difference between the hardiest cultivar and the least

hardy cultivars.

Southern Blot Analysis of SN 28 and EN 115

Autoradiograms for genomic DNA of seven cultivars cut
with several restriction enzymes and probed with BN28 (Fig.
11) and BN115 (Fig. 12) showed five to ten bands, depending on
the restriction enzyme and probes used. Two of the seven
cultivars that were digested with EcoRI and proved with BN28
showed a restriction fragment length polymorphism in one of
the minor band loci. However, based upon various restriction
enzyme-digestion patterns, there was no differences in copy
number or position of bands among the cultivars tested, even

between the spring and winter cultivars.

Northern Blot Analysis of BN28 and BN115

Total RNA isolated from control, 1-day, 5-day, and 10-
day acclimated plants of each cultivar was fractionated in
denaturing formaldehyde agarose gel. RNA was transferred to a

Nytran membrane and probed with BN28 and BN115 (Fig. 13). The

66

12

 

10-

 

 

     

     

/
' 6- ‘x
V , ;. .
y.’ .
g - . _, .
x 2 .
//
.1 v' '
E“ 4 ,- ;.
. : "3 I
, I" ’ {z I K .- ‘, -’ _ - /
I X J 1 V n , . 4‘ . I
, /. , , , 2 .. , , / .
. - ' r ' 7 7 . l
.2: / // ~ , , .- ,7 p
1;, .1 / x, , 1' , r, / z , , . I‘ I.»
-’ .'I , 4 - .’ . , " . .
. f. «I I ' i - . 1' 1' "
, .- / /_ .’ -' ix _/ . ,1 _ ,. . _- .
"1 'l’,-" /"A " ‘ "I 4” ’.//:'. 1’, .'.//' ' I." / ' ;
”*4M’é -~.n.m n 1» R” .,
r, . .- V. , l . . , . , I I
. / I C .'// x .r Ij/II/ - .l* ,.-',l // z . / l,/ _ // .
. ' ’ ' _/ /I' / - ‘ ’ . I ~ , - "I ,.
. / J/ . 'I‘llé / :4 / {/ 4./

           

o 1

 

123123 123123123 23
PACTOL CDH3 WRG86 DUOBUL CERES KWC4113 ACCORD

CULTIVARS

Figure 10. Effect of deacclimation on LT,o values of seven
canola cultivars. Plants were cold-acclimated for
10 days, then deacclimated for 3 days. Vertical
bar indicates mean t SE. Lane 1: control
(nonacclimated); 2: 10-day acclimation,
3: 3-day deacclimation.

 

67

EcoRI ECORV

a b c d e f g a b c d e f g

e-zugf-I

   

mic-6
..1,51é

I“.aml|e.-¢a

Figure 11. Southern blot analysis of the three restriction

 

I

I

.,~ l

. r ‘ v- I"
P O I-
.1. " .

OCQOe-e

enzymes, EcoRI, EcorV, and Hind III,-digested
genomic DNA from seven canola cultivars
hybridized to BN28. Lane a:WRG86; b:CDH3;

c:Duobul; d:Ceres; ezKWC4113;

szccord; g:Pactol

 

68

EcoRI HInd HI

 

a b c d e f g a b c d e f g

   

Figure 12. Southern blot analysis of the two restriction
enzymes, EcoRI, and Hind III,-digested genomic
DNA from seven canola cultivars hybridized to
BN115. Lane a:WRG86; b:CDH3; c:Duobul; d:Ceres;
e:KWC4113; f:Accord; g:Pactol.

69

BN28 BN115

 

 

a b c d a b c

D

' 1
.1

CDH3 ’ ' 0
WRG86 ’ 0 .
DUOBUL . . .

CERES '9.

4

.00'01
O) ’41..“fl'l" 1".1‘1'IDRIE'DVIIII'W‘

e ..O -. «O.

 

 

- . '1’,‘
‘fi'

 

!
1
1

r
. ‘~. -
‘9‘

 

KWC4113 .. O O

ACCORD {5e 0 O

PACTOL Q O O‘

:Frlfiiure 13. Northern blot analysis of total RNA from leaves
of seven canola cultivars isolated at O-day (lane
a), 1-day (lane b), 5-day (lane c), and 10-day
(lane d)-acclimation stages and probed with BN28
and BN115.

70

radioactivity bound to the membrane was measured by a Betagen
Blot Analyzer. For the standardization of RNA quantity in
Northern blot, the blot was stripped and rehybridized with
rDNA, pRE12, which is expressed constitutively in plants (Fig.
14) . Changes of transcript amount (representing the overall
means of seven cultivars) by cold acclimation are shown in

F 19. 15. RNA transcripts for both genes were undetected in

control plants; however, in plants exposed to cold
temperature, the amount of transcript increased rapidly and
remained at elevated levels as long as the plants remained at
(3016 temperatures. The two genes showed differences in their
exPression pattern.) BN28 showed a rapid increase in the amount
0f transcript at 1-day (60 % of that of maximum expression)
and a steady increase until 10 days of acclimation. BN115
reached its maximum expression level within 1 day of exposure
to Cold temperature and maintained that elevated level as long

as Plants remained at cold temperature.

attfict of Deacclimation on Expression of BN28 and BN115

Even 1 day of deacclimation decreased the expression of
BN28 and BN115 in most of seven cultivars examined, although
s°me cultivars with 1 day of deacclimation showed some
actrivity remaining. Three days of deacclimation totally
elj~lllinated the transcript of both genes in all cultivars (Fig.
16) , showing that both were completely turned off by

a‘Qacclimation and that degradation of the transcripts occurred

 

KWC4113
WRG86 ACCORD
DUOBUL PACTOL
' A.
CERES

 

Figure 14. Standardization of Northern blot analysis by
hybridizing with rDNA probe, pRE12. The blot was
the membrane presented in Fig. 14, which was
stripped and rehybridized with the rDNA probe.

72

 

 

 

 

 

 

 

 

 

83
f...
E 100-
o
2
E 80-
E
CLr2) 60 -
2
<1
6::
E—
m 40-
: —'O'- BN115
55 —o— sun
—3
m 20-
a:
GI I I I I I
o 2 4 6 8 1o

ACCLIMATION DURATION (DAYS)

Flg- 15. Changes in transcript amount of BN28 and BN115
during cold-acclimation. The data is a graphical
presentation of Fig. 13 and represents overall
mean values of seven cultivars. Vertical bar

indicates mean 1 SE.

 

73

BN28 BN115

 

 

a b c a b c

WRG86

DUOBUL

CERES

KWC4113

ACCORD

PACTOL

 

Figure 16. Northern blot analysis for effect of deacclimation

on the expressions of BN28 and BN115 in seven
canola cultivars. Lane a: 10-day acclimation;
l-day deacclimation after lo-day acclimation;
3-day deacclimation after lo-day acclimation.

b:
c:

 

74

very rapidly at room temperature.

Correlation between Freezing Tolerance and Expression of 3828
end 38115

Correlation between time-course increase in LT” values
for seven cultivars and time-course increase in relative
transcript amount of BN28 and BN115 was examined (Fig. 17).
Each point is the overall mean of seven cultivars. Time-course
increase in transcript amount of BN28 was highly correlated
with time-course increase in LTso value (r=0.92**) . As the
transcript of BN28 increased due to longer acclimation period,
the 1'_.'I'_.,0 value also increased. However, the increase in
transcript amount of BN115 was not significantly correlated
(0-79ns) with the time-course increase in L'I'so value.

Correlations were also examined for each cultivar (Fig.
13) . Expression of BN28 and BN115 at a specific acclimation
stage was not significantly correlated with freezing tolerance
at: that time or with winter survival at the cultivar level.
The relative transcript amount of BN28 at 10 days of
acclimation was Duobul > Pactol > WR686 > Accord > Ceres >
c1383 > KWC4113, while the freezing tolerance at this time was

Accord > KWC4113 > Ceres > Duobul > WRGBG > CDH3 > Pactol.

Figure 17 .

‘ ”K 120
as
8 —O— BN1 1 5

S —D— BN28

E 1 00 - 7253/
7/ ,7.

< 2/ %
2:; /4://2/$/?é

8 0 "' Zfiy/fl/f/// 74.1;

////////§//

5 6 o - ”jig/7%
3331/2/13 ///”

3 4° ' :// /// xx

Iyw I¢ ;

E 20 - E?/ L

2 /

:5 o_ z/

5

75

 

       
     

  
        
  

      
   

    
    
 

 

 

ACCLIMATION DURATION (DAYS)

Comparison of time-course changes of L'I',0 value and
transcript amount of BN28 and BN115 during cold
acclimation. The bar indicates LTso value and the
line indicates the transcript amount. The data
are overall mean value for seven cultivars.
Vertical bar indicates mean t SE.

 

76

 

 

E
[.3
E 100-
<
g 80-
o
2
E 50-
E
m 40-
z
<
E
u 20—
Z
[—
< _
.J
k}
z

I LT50
-o—BN28
-D—BN‘I 1 5

  

5 (I: , / :1; - /:I .. v / 713 , ,' ’." ,
1234567 12345670

CONTROL 1 DAY 5 DAY 10 DAY

Figure 18. Correlation between freezing tolerance and

transcript amount of BN28 and BN115 by cold
acclimation in cultivar level at specific time of
measurement. The bar indicates LT5o value and the
line indicates the transcript amount. Vertical bar
indicates mean t SE. Lane 1: Pactol; 2: CDH3;

3: WRG86; 4: Duobul; 5: Ceres; 6: KWC4113; 7:
Accord.

 

77
Tissue specific-Expression of 8828 and 88115

The tissue-specific expression of BN28 and BN115 was
examined in two representative winter cultivars, the hardy
cultivar, KWC4113, and the tender cultivar, WRGSG (Fig. 19).
Both genes were expressed more strongly in the leaf than in
the stem, and there was no detectable expression in root

tissue.

 

78

 

 

BN28 BN115
3 1 2 3
WRG86 . O
KWC4113

 

Figure 19. Northern blot analysis for tissue-specific
expression of BN28 and BN115 by cold acclimation
in two winter cultivars, WRGB6 and KWC4113.

Lane 1: leaf; 2: stem; 3: root.

DISCUSSION

The primary observations from freezing tolerance studies
are as follows: (1) cold acclimation increases the freezing
tolerance of both spring and winter cultivars with different
acclimation capacities. (2) There is no difference in
nonacclimated freezing tolerance among cultivars, however,
differences in acclimation capacity among cultivars determine
their freezing tolerance after cold acclimation. (3) Freezing
tolerance highly correlates with winter survival, though small
differences in freezing tolerance of cultivars with similar
winter survival were not separated by freezing tests. (4)
Deacclimation decreases the freezing tolerance very rapidly;
however, different cultivars still have different levels of
freezing tolerance after deacclimation.

In this study, three were three groups of cultivars with
different levels of freezing tolerances (Fig. 9). This
included twol groups of ‘winter cultivars and one spring
cultivar. Although cultivars within a group had different
winter survivals, their freezing tolerance to freezing tests
was not significantly different. This suggests a lack of
precision in freezing tests for predicting differences in

winter survival of cultivars with a narrow differences in

79

E "' "fir-Y ‘
.l‘ .

 

80

freezing tolerance. It may also indicate that factors other
than freezing tolerance itself are involved in winter survival
under field conditions (Thompson and Hughes, 1986). However,
differences in freezing tolerance between groups, and their
correlation with winter survival, suggest that freezing tests
can be used to detect differences in winter survival of
cultivars with broad differences in freezing tolerance. This
agrees with observations by Teutonico et al. (1993).

The observation that freezing tolerance in the
nonacclimated state and acclimation capacity are not
correlated, and that only acclimation capacity has a high
correlation with winter survival, suggests that they are '
regulated independently. It also suggests that acclimation
ability is more important to winter survival. This agrees with
observations reported for these two traits in potato (Palta,
1992) and Brassica species (Teutonico et al., 1993).

All cultivars showed a rapid decrease in freezing
tolerance during a short period of deacclimation. However,
differences in freezing tolerance among cultivars occurred
after deacclimation. Winter survival may be determined by how
much a certain cultivar retains freezing tolerance after the
oCcurrence of warm temperatures in the middle of winter and
the speed of reacclimation prior to subsequent lethal freezing
temperatures. As shown in this experiment, the hardy cultivars
increased more rapidly in freezing tolerance during cold

acclimation and retained a higher freezing tolerance after

81

deacclimation than less hardy cultivars. This suggests that
the retention of freezing tolerance after deacclimation and
Ithe speed of reacclimation may play a role in winter survival.

BN28 and BN115 are two genes from Brassica napus induced
by cold temperature (Orr et al., 1992; Weretilynk et
al.,l993). The reason for cultivar differences in the level of
expression are not known. If the regulation of thisexpression
occurs at the levels of transcription, then the possible
reason may include differences in (1) copy number of genes
involved; (2) promoter sequences resulting in different

‘transcriptional efficiency; (3) the availability' and

efficiency of promoter-binding protein and other ‘

transcriptional factors; and (4) changes in mRNA processing
and stability (Mohapatra et al., 1989).

Southern blot data (Fig. 11, 12) showed that these genes
are a multigene family. This multigene family may consist of
either the same genes having multiple copies in the genome or
slightly different copies of genes having very similar
molecular weight of transcript as discussed by Weretilynk et
al. (1993). The lack of difference in copy number and even in
restriction fragment length polymorphism (RFLP) of these two
genes among cultivars is not surprising considering the narrow
genetic bases of breeding stocks and.the recent development of
canola cultivars (Sahhidi, 1990).

Expression of these two genes was induced within 12

hours of exposure to cold temperature and maintained their

" n'.

82

expression as long as plants were kept at cold temperatures.
The expression pattern of the two genes was slightly
different: BN28 was induced to 60 % of full expression with a
1-day exposure and increased steadily to full expression with
10 days of acclimation, while BN115 was expressed fully with
a 1-day exposure and maintained full expression with cold
temperature. These results are consistent with those reported
for Brassica napus cv. Jet neuf (Weretilnyk et al., 1993) and
Arabidopsis thaliana L. Heyn (Hajela et al.,l990) for BN115,
but inconsistent with that of Arabidopsis for BN28.

Transcript level of the genes was not positively
correlated with freezing tolerance in cultivars at the
specific time of acclimation (Fig. 18). However, the
observations that these genes are turned on in response to
cold temperature and maintain the expressions under cold
temperature (Fig. 13) and the time-course increase of BN28
expression is coincident with the time-course increase in
freezing tolerance (Fig. 17) do not allow us to rule out the
possible role of the genes in freezing tolerance. One possible
explanation is that these genes represent a minimum
requirement for the plants to gain freezing tolerance, though
the difference in freezing tolerance among cultivars can be
explained by other genes associated with these two genes.
Considering that the gene expression was determined in the
transcription level in this experiment, examination of the

translation expression level may provide further information.

IUEFTHIEDNCEEB

Andrews, J .E. 1960. Cold hardening and cold hardiness of
winter rye seedlings as affected by the stage of development
and temperature. Can. J. Plant Sci. 38:1-7.

Arora, R., and J.P. Palta. 1991. A loss in the plasma membrane
ATPase activity and its recovery coincides with incipient
freeze-thaw injury and postthaw recovery in onion bulb scale
tissue. Plant physiol. 95:846-852.

Bihn, L.T., and K. Oono. 1992. Molecular cloning and
characterization of genes related to chilling tolerance in
rice. Plant Physiol. 99:1146-1150.

Cativelli, L., and D. Bartels. 1990. Molecular cloning and
characterization of cold-regulated genes in barley. Plant
Physiol. 93:1504-1510.

Church, G.M., and W. Gilbert. 1984. Genomic sequence. Proc.
Natl. Acad. Sci. USA. 81:1991-1995.

Dunn, M.A., M.A. Hughes, R.S. Pearce, and P.L. Jack. 1990.
Molecular characterization of a barley gene induced by cold
treatment. J. Exp. Bot. 41:1405-1413.

Gilmour, S.J., N.N. Artus, and M.F. Thomashow. 1992. cDNA
sequence analysis and expression of two cold-regulated genes
of Arabidopsis thaliana. Plant Mol. Biol. 18:13-21.

Gilmour., S.J., R.K. Hajela, and M.F. Thomashow. 1988. Cold
acclimation in Arabidopsis thaliana. Plant Physiol. 87:745-
750.

Hajela, R.K., D.P. Horvath, S.J. Gilmour, and M.F. Thomashow.
1992. Molecular cloning and expression of cor (cold-regulated)
genes in Arabidopsis thaliana. Plant Physiol. 93:1246-1252.

Houde, M., J. Danyluk, J-F. Laliberte, R.S. Dhindsa, and F.
Sarhan. 1991. Cloning and characterization of four cDNAs
specifically induced. by low 'temperature in *wheat. Plant
Physiol. 96(Suppl.):161.

83

84

Kurkela, S., and M. Frank. 1990. Cloning and characterization
of a cold- and ABA-induced Arabidopsis gene. Plant Mol. Biol.
15:137-144.

Levitt, J. 1980. Chilling, Freezing, and High Temperature
Stresses, Ed.2. Academic Press, New York. 497 pp.

Levitt, J., and D. Siminovitch. 1940. The relation between
frost resistance and the physical state of the protoplasm.
Can. J. Res. Sect. C. 18:550-561.

Lin, C., and M. F. Thomashow. 1992. DNA sequence analysis of
a complementary DNA for cold-regulated Arabidopsis gene corlS
and characterization of the COR15 polypeptide. Plant Physiol.
99:519-525.

Lin, C., W.W. Guo, E. Everson, and.M.F. Thomashow. 1990. Cold
acclimation in Arabidopsis and wheat. Plant Physiol. 94:1078-
1083.

Mohapatra, 8.8., L. Wolfraim, R.J. Poole, and R.S. Dhindsa.
1989. Molecular cloning and relation to freezing tolerance of
cold acclimation specific genes of alfalfa. Plant Physiol.
89:375-380.

Nordin, K., P. Heino, and E.T. Palva. 1991. Separate signal
pathways regulate the expression of a low temperature-induced
gene in Arabidopsis thaliana. Plant Mol. Biol. 16:1061-1071.

Orr, W., B. In, T.C. White, L.S. Robert, and J. Singh. 1992.
Complementary DNA sequence of a low temperature-induced
Brassica napus gene with homology to the Arabidopsis thaliana
kinl gene. Plant Physiol. 98:1532-1534.

Palta, J.P. 1992. Mechanisms for obtaining freezing stress
resistance in herbaceous plants. pp. 219-250. In: H. T.
Stalker and J.P. Murphy (eds.) Plant Breeding in the '905. CAB
International, Walingford, England.

Palta, J.P., J. Levitt, and E.L. Stadelmann. 1977. Freezing
injury in onion bulb cells. I. Evaluation of the conductivity
method and analysis of ion and sugar efflux from injured
cells. Plant Physiol. 60:393-97.

Palta, J.P., and P.H.Li. 1980. Alteration in membrane
transport properties by freezing injury in herbaceous plants:
Evidence against rupture theory. Plant Physiol. 50:169-175.

Shahidi, F. 1990. Rapeseed and canola: Global production and
distribution. pp. 3-15. In: F. Shahidi (ed.) Canola and
Rapeseed. Van Nostrand Reinhold Inc, New York.

85

Sakai, A., and W. Larcher. 1987. Responses and Adaptation to
Freezing Stress. Springer-Verlag, Berlin. 321 pp.

Sommerville, C.R., and W.L. Ogren. 1982. Isolation of
photorespiration mutants in Arabidopsis thaliana. pp. 129-138.
In: E. Edelman, R.B. Hallick, and N.-H. Chua (eds.) Methods in

Chloroplast Molecular Biology. Elsevier, Amsterdam.

Steffen, K.L., J.P. Palta. 1987. Photosynthesis as a key plant
responses to low temperature. pp. 67-100. In: P.H. Li (ed.)
Plant Cold Hardiness. Liss, New York.

Steponkus, P.K. 1978. Cold hardiness and freezing injury of
agronomic crops. Advances in Agronomy 30:51-98.

Steponkus, P.L. , and D.V. Lynch. 1989. Freeze/thaw-induced
destabilization of the plasma membrane and the effects of cold
acclimation. J. Bioenergetics and Biomembranes 21:21-41.

Steponkus, P.L., M. Uemura, R.A. Balsamo, T. Arvinte, and D.V.
Lynch. 1988. Transformation of the cryobehavior of rye
ProtOplast by modification of the plasma membrane lipid
composition. Proc. Natl. Acad. Sci. USA 85:9026-9030.

Sukumaran, N.P., and C.J. Weiser. 1972. An excised leaflet
test for evaluating frost tolerance. HortScience 7:467-468.

Sulc, R.M., K.A. Albrecht, J.P. Palta, and S.H. Duke. 1991.
LeEllfage of intracellular substance from alfalfa roots at
Vaili‘.‘.l.c>us subfreezing temperature. Crop Sci. 31:1575-1578.

gentcnico, R.A., J.P. Palta, and T.C. Osborn. 1993. In vitro
reezing tolerance in relation to winter survival of rapeseed

cultivars. Crop Sci. 33:103-107.

ihomashow, M.F. 1990. Molecular genetics of cold acclimation
n higher plants. Adv. Genet. 28:99-131.

Thomashow, M.F., S.J. Gilmour, and C. Lin. 1993. Cold-

E?gu1ated genes of Arabidopsis thaliana. pp. 231-44. In: P.H.
1 and L. Christerson (eds.) Advances in Plant Cold Hardiness.

CRC Press, Boca Raton.

ghompson, R.F. , and W.G. Hughes. 1986. Breeding and varieties.
p- 32-82. In: D.H. Scarisbrick and R.W. Daniels (eds.)

o O
llseed Rape. Collins, London.

V .
Bilger, H.G., and U. Heber. 1975. Cryoprotective leaf protein.
ochem. Biophys. Acta. 412:335-349.

W ~ .
sglser, C.J. 1970. Cold resistance and injury in woody plants.
ience 169:1269-1278.

86

Weretilnyk, E., W. Orr, T.C. White, B. In, and J. Singh. 1993.
Characterization of three related low-temperature regulated
cDNAs from winter Brassica napus. Plant Physiol. 101:171-177.

CHAPTERII

EFFECTS OF PLANTING DATE ON FREEZING TOLERANCE,
WINTER SURVIVAL, AND RESPONSES OF TWO COLD-
REGULATED GENES, BN28 AND BN115, IN CANOLA

(Bmssica napus L.)

ABSTRACT

Planting date is the most important factor in determing
winter survival of crop plants. The objective of this study
was to explore the effect of planting date on the development
Of freezing tolerance and winter survival of canola. In
ad(ii‘tion, the expression of two cold-regulated genes, BN28 and
BN115, under field condition was also explored.

Six winter cultivars were planted at three different
dates during the fall on the Michigan State University
Research Farm at East Lansing, MI. Freezing tolerance was
determined by ion leakage tests, while expression of the genes
was determined by northern blot analysis of total RNA taken at
the same times.

Planting date as well as cultivar treatment had a
Siglilificant effect on freezing tolerance and winter survival.
A different pattern in development of freezing tolerance was
observed for different planting dates. There was a high

87

88
correlation between freezing tolerance and winter survival
suggesting that freezing tolerance is the single most
important factor in determining winter survival. The
expression of cold-regulated genes was detected within 15 days
after planting. Although time-course increase in expression of
the cold-regulated genes was matched with increase in freezing
tolerance, the difference of expression in cultivar level at
specific times of measurement was not correlated with freezing
tolerance.
The effect of fall-applied nitrogen and seeding rate

combined with planting date on winter survival was also

examined .

INTRODUCTION

Each crop should be planted at the time of the year when
its stages of development will be appropriate for the expected
environments. Temperature and moisture are of particular
importance, and in many species, light is also very important.
Numerous studies have been conducted to determine the best
planting dates for various overwintering crops. In general,
the earlier a crop can be planted, the better will be its
Productivity (Auld et al., 1983, 1989) .

Although there is considerable information available on
the effect of planting date on the performance of winter crops
(Canvin, 1965; Noderstaggard, 1972; Huber, 1973; Kondratowicz,
1974 ; Kondra, 1977; Degenhardt and Kondra, 1981; Christensen
et, al . , 1984) , most are concerned with agronomic factors such
as yield, crop quality, and winter survival. Limited
infC-T'Ili‘lnation is available on the mechanism of cold hardiness
and increase in freezing tolerance as affected by different
planting dates. Differences in planting date and exposure of
plants to different environmental conditions at different
Stages of growth influence their capacity for winter

hardiness, An understanding of how plants respond to different

e
nvil‘onmental factors (e.g. , photoperiod and temperature, and

89

 

fl‘
di

ha

re
sh
Ch
al
Br

Sh

fu
fi

ex

De
is
en
ha

in

9f

to

33:.

a1.

9O
fluctuation of temperature during the day and night) at
different growth stages provides us clues for improving cold
hardiness by both cultural practice and breeding strategies.
Recently, a number of genes have been described that
respond to severe environmental stresses such as drought, heat
shock, low temperature, or high salt concentration (Mundy and
Chua , 1988; Close et al. , 1989; Bartel et al. , 1990; Hajela et
al- , 1990; Kukela and Frank, 1990; Skriver and Mundy, 1990;
Bray, 1991; Pla et al., 1991; Lang and Palba, 1992; Yamaguchi-
Shinozaki, 1994) . Though laboratory manipulation of these
genes provides some clues for their regulation and possible
functions, little is known as to how they are expressed under
field condition. A few investigators have explored the
expression of heat shock protein genes under field conditions
(Burk et al., 1985; Kimpel and Key, 1985; Chen et al., 1990;
DeRocher et al., 1991; Lorrain et al., 1993) , however, there
is no information on how cold-regulated genes respond to field
environments. Data from field experiments combined with what
has been learned from laboratory experiments provide some
11.181th in the functions and regulations of these genes.

The objectives of this research were 1) to determine the
effect of planting date on the development of freezing
tolerance and eventually, on winter survival of canola and 2)
to study the response of two cold-regulated genes, BN28 and
BN115 , isolated from B. napus (Orr et al. , 1992; Weretilynk et

a1 .
‘ ' 1993) under field conditions at different planting dates.

(KI

Pla

Uni
plc
dat
to
of

rat

Eva

{31,11
fre.
30

Plat

Plar

I

MATERIALS AND METHODS

The same six winter cultivars used in the previous study

(WRG86, CDH3, Duobul, Ceres, Accord and KWC4113) were used in

thi s study .

Planting, Management and Experimental Design

This study was conducted at the Michigan State
University Agronomy Farm in East Lansing, Michigan. A split-
Plot design was used with three replications. Three planting
dates, Aug. 25, Sept. 10, and Sept. 25in 1993, were assigned
to the main plot. Six cultivars were assigned to the subplots
of each main plots. Seed of each cultivar was planted at the

rate of 5.6 kg/ha in a five-row plot 6 m long and 92 cm wide.

B"'l-II-iaation of Freezing Tolerance

Six cultivars planted on Aug. 25 and three of the six
cultivars planted on Sept. 10 and Sept. 25 were subjected to
fI‘eezing tests. Freezing tests were performed 15 (Sept. 10),
30 (Sept. 25), 45 (Oct. 10), and 75 (Nov. 13) days after
planting (DAP) for the Aug. 25 planting. For the Sept. 10
plat"thing, freezing tests were performed at 15 (Sept. 25): 30

(0
Ct. 10), and 60 DAP (Nov. 13), and for the Sept. 25

91

 

pla

te
fr

St

92
planting, at 15 ~(Oct. 10) and 45 DAP (Nov. 14).

Three different plants were collected from each plot in
the field and transferred to the laboratory immediately. One
leaf disc was taken from each of the 3 plants and the 3 discs
were placed in a stoppered culture tube as a replication.

Freezing tests were performed as previously described (Chapter

I)-

Ev. luation of Winter Survival

Winter survival was evaluated as previously described

(Chapter I) .

m Extraction and Northern Blot Analysis

Sampling was done at the same time as for freezing
tests. Leaf and stem parts of plants were cut in the field,
1EITJZen in liquid N2, then kept in a box with dry ice before
stoIli’age at -80 °C in the laboratory. RNA extraction and
noI‘tihern blot analysis with BN28 and BN115 were performed as
previously described (Chapter I). Three different northern
blot analyses were conducted with different RNA samples

e)‘tili‘acted from each replication.

N
1till-‘ogen Rate and Seeding Rate Experiment
To explore the effect of fall-applied nitrogen and
s e o o e
ee(iing rate on winter surVival of canola, the susceptible

c
ultivar, Duobul, was planted at the same dates as in the

93
previous experiment. For each planting time, 0, 20, and 40
Kg/ha of actual nitrogen (as urea) was applied. Winter
survival was evaluated as previously described. Another

experiment was established with seeding rates of 4, 8, and 12

kg / ha at each of three planting dates. The winter survival of

all treatments was evaluated.

Statistical analysis

Analysis of variance, least significant difference,
Duncan's multiple range test, and simple correlation analysis
were used to analyze the data using the statistical package

MSTAT (Michigan State University, East Lansing, Michigan).

I9!

19E
19E
195
tax

COI

199
(at
maj
ten

mic

RESULTS

Weather Conditions
The winter of 1993-1994 was colder than that of 1992-

1993, with a minimum air temperature of -29.0°C (Jan. 19,
199 4) , compared with a low of only -22.0°C for the winter of
199 2-1993 (Fig. 1). The coldest 10-day mean daily minimum
temperature in 1992-1993 was -16.5°C (end of Feb. 1993)
compared to -22°C in 1993-1994 (middle Of Jan. 1994).

The overall pattern of air temperature changes in the
1993- 1994 growing season was as follows: warm temperatures
(above 10°C of lo-day mean daily minimum temperature) were
maintained until the middle of the September. Then, the
temperature decreased slowly from 10°C to freezing through the
Inid<ile of September to the beginning of November. Freezing
temperatures occurred at the beginning of November, developed
to around -2°C until there was a sudden temperature drop to
~14 - 2°C in the end of December. The coldest time of the year
occLlrred in mid-January, and these severe conditions lasted
until the end of February. In March, the temperature was
still maintained at freezing levels through the end of the
month, then slowly increased to 0°C until the beginning of

April.

94

95

 

 
  

 

 

 

 

 

 

20
A —0— 992-1993
E) 10- —o-— I993-1994
E ..
f...
<
a.
5
E_ -20-

~30

 

 

51525 51525 51525 51525 S 1525 51525 51525 51525 51525
AUG SEPT OCT NOV DEC JAN FEB MAR APR

Figure 1. Ten-day mean daily minimum temperatures at East
Lansing, MI during the winter in 1992-1993 and 1993-
1994.

96
Increase of Freezing Tolerance under Field Conditions

For the canola planted Aug. 25 (Fig. 2), no increase in
freezing tolerance was detected until 30 DAP (Aug. 25 - Sept.
25) . Thereafter, a rapid increase in freezing tolerance
occurred during 30 to 45 DAP (Sept. 25 - Oct. 10) followed by
gradual increase until 75 DAP (Oct. 10 - Nov. 10). There were
3 distinct groups of cultivars with different LT,o values. CDH3
and WRG86 belonged to the least hardy group, Duobul and Ceres
belonged to medium hardy group, and Accord and KWC4113
belonged to the most hardy group. The very hardy cultivars,
KWC4113 and Accord, attained their maximum LT,o values of
-20 °C, while that of the susceptible cultivar, CDH3 was -15°C
on Nov. 13. However, whether the increase in freezing
tolerance would be continued to the next growth period (after
NOV- 13) was not determined in this experiment.

For the Sept. 10 planting (Fig. 3), a rapid increase in
f:"-"eezing tolerance occurred during 15 to 30 DAP (Sept. 25 -
Oct; . 10) followed by very steady increase during 30 to 60 DAP
(Oct. 10 - Nov. 10). However, the maximum freezing tolerances
of Cultivars in these plots were much less than that of the
Same cultivars planted Aug. 25. For example, KWC4113 planted
Sept. 10 attained an LT,0 value of -15°C whereas the same
cultivar planted Aug. 25 attained -20°C.

For the Sept. 25 planting (Fig. 4), most of the freezing

t
01erance retained at the end of hardening process was gained

97

 

21-

 
 
 
 
 
 
   

 

—-s—— ACCORD
13- —A— KWC4113
—e— CERES

—o— DUOBUL

 

 

 

 

 

 

 

 

15- —I—- WRGBG
A CDH3
o
12'-
\L
c
E—‘u" 9'
I-J
5..
3..
O I f I I I I
10 20 30 4O 50 60 7O 80

DAYS AFTER PLANTING

Figure 2. Changes in freezing tolerance of six winter canola
cultivars planted Aug. 25, 1993 . Freezing tests were
conducted at 15, 30, 45, and 75 days after planting.
Bar indicates mean t SE.

98

 

 

 

 

 

 

 

 

1 5 -
12 -
A
C.)
O 9 _
\t,
c
In
E_ 6 ..
h-;:
—A— KWC4113
—o— DUOBUL
3 - ——o—— CDH3
0 I F l l I
10 20 3o 40 so 60

DAYS AFTER PLANTING

Fight-e 3. Changes in freezing tolerance of three winter canola
cultivars planted Sept. 10, 1993. Freezing tests
were conducted at 15, 30, and 60 days after
planting. Bar indicates mean t SE.

,VII‘ 0

99

 

 

 

 

 

 

 

 

15"
12-
/—-\
C)
o
I 9'
V
O
In
9
r4 5‘
-fi&—- KMKMJI3
-—<>—- DUOBUL
3- —a— CDH3
0 u u 1
‘IO 20 30 40 50

DAYS AFTER PLANTING

Flgure 4. Changes in freezing tolerance of three winter canola
cultivars planted Sept.25, 1993 . Freezing tests were

conducted at 15 and 45 days after planting. Bar
indicates t SE.

mm
stea

cul

10
6::
inc
pia:
resx
betw
Plax

free

100

during the first 15 DAP (Sept. 25 to Oct. 10), followed by
steady increase to a maximum of -13°C for the most hardy
cultivar, KWC 4113, and only -8.5°C for less hardy cultivars.

The effect of planting date on freezing tolerance is
shown in Fig. 5. Each data point represents the overall means
of six or three cultivars shown in Fig. 2, Fig. 3, and Fig. 4.
A dramatic increase in freezing tolerance in all three
planting dates occurred during the period of Sept. 25 to Oct.
10 during which the 10-day mean daily minimum temperature
dropped from 8.9°C to 2.7°C. Before that time, no significant
increase in freezing tolerance occurred in either of the plots
Planted Aug. 25 or Sept. 10. Though all three planting dates
resulted in a very steady increase in freezing tolerance
between Oct. 10 and Nov. 15, the increase in the Aug. 25
Planting was more rapid. Planting date significantly affected
freezing tolerance with maximum LTso values of -18°C, -14°C,

and ~12°C ,for plantings made Aug. 25, Sept- 10. and Sept. 25,

respectively .

:E‘Ponaos of Two Cold-Regulated Genes, BN28 and 88115, under
-11! conditions

To determine the response of two-cold regulated genes
11 . . . . .
rider field conditions, total RNA from Six cultivars planted
A
“9 - 25 were extracted from 15, 30, 45, and 75-day old plants.

N
orthern blot analysis was performed with BN28 and BN115

101

 

 

     

 

—9— AUG. 25
—0— SEPT.10
—D— SEPT.25

 

 

 

 

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15‘
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U I
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to
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I I I I I
AUG. 25 SEPT. 10 SEPT. 25 OCT. 10 NOV. 15

Changes in freezing tolerance of winter canola
affected by different planting dates. Data
represent overall means of six cultivars planted
Aug. 25, and overall means of three cultivars
planted Sept. 10 and Sept. 25. Freezing tests
were performed at every 15-day or 30-day interval,
depending on planting date. Vertical bar indicates
mean t SE.

 

102

probes (Fig. 6). The general pattern of expression of the two
genes is shown in Fig. 7. Each data point is the overall mean
for six cultivars. Both genes were turned on within 15 days of
planting; however, the expression patterns of the two genes
were different. The transcript amount of BN28 increased
gradually throughout the experimental period, while that of
BN115 increased rapidly within 15 DAP followed by a steady
increase until the end of the experiment (Nov. 13) . At this
Point, both genes retained their full transcript level. One
interesting observation is that both the genes were turned on
well before the onset of increase in freezing tolerance.

The expression of these genes in each cultivar was
exv‘fiunined to determine the correlation between the freezing
tolerance of a specific cultivar and transcript level (Table
1 and Table 2) . There were no specific patterns of the gene
e)(‘pression to explain the difference in freezing tolerance
aHuang cultivars. Though most cultivars showed increases in
B5'28 expression over time, the differences among cultivars on
a specific sampling date were inconsistant. In the case of
Bl«115, there was no difference in gene expression at either
all'iong sampling dates or cultivars on a specific sampling

date .

 

103

BN115

 

 

a b c d

9000
9090
mo

CDH3

 

WRG86 '

DUOBUL .

 

CJHREEI CI 1.Ni".I‘I
KWC4113 o g . Q .
ACCORD

06.8

 

Figure 6. Northern blot analysis of six winter canola
cultivars planted Aug. 25, 1993 in the field. Total
RNA were isolated Sept. 10 (lane a), Sept. 25 (lane
b), Oct. 10 (lane c), and Nov. 13 (lane d). The
membrane was hybridized with BN28 and BN115.

RELATIVE TRANSCRIPT AMOUNT (%)

:FjJJure 7.

104

 

 

 

 

 

0..
8 —O— BN115
—D— BN28

60-

40-

20-

0

 

 

 

I I r I I
AUG.ZS SEPT.10 SEPT.ZS OCTJO NOV.13

DATE OF MEASUREMENT

Time-course changes in transcript amount of BN28
and BN115 in canola planted Aug. 25, 1993. The
data represent the overall mean of six cultivars
and is a graphical representation of Fig. 6.
Vertical bar indicates 1 SE.

 

105

Table 1. Changee in transcript anount of BN28 in 6 winter
canola cultivars planted Aug. 25, 1993. Data are
numerical presentation of Northern blot in Fig. 6.

 

Relative transcript amount (%)

 

 

 

 

Cultivars

Sept. 10 Sept. 25 Oct. 10 Nov. 13
ACCORD 37a‘I’ 28ab 683 100a
KWC4113 19a 16b 62ab 100a
CERES 20a 13b 37b 66c
DUOBUL 10a 18b 33b 82b
WRGSG 10a 13b 35b 780
CDH3 13a 31a 20c 80bc
T Means followed by the same letter within column are not

significantly different at the 0.05 probability level.

Ithl!

Cult

ACCO
KWCI
C K2
DUCB
HESS

CDH3

106

Table 2. Changes in transcript amount of BN115 in 6 winter
canola cultivars planted Aug. 25, 1993. Data are
numerical presentation of Northern blot in Pig. 6

 

Relative transcript amount (%)

 

 

 

 

Cultivars

Sept. 10 Sept. 25 Oct. 10 Nov. 13
.ACCORD 43bT 58b 58bc 50bc
IKWC4113 46b 35c 47c 55b
CERES 35b 44c 46c 46c
DUOBUL 57a 100a 100a 70ab
WRG86 49a 95a 95a 100a
CDH3 60a 56ab 72ab 72ab
T Means followed by the same letter within a column are

not significantly different at the 0.05 probability
level.

m:
Gene

am
f r

p
2h
U

f 0
DA

45

9) .
aff
(BI-I:
Phas
Pla!
DAP
Sep1
C011
Pla:

the

thr
'I’it

ah

107

Effect of Planting Date on the Expression of Cold-Regulated
Genes, BN28 and BN115.

The effect of planting date on the expression of BN28
and BN115 was examined by northern blot analysis of total RNA
from 3 winter cultivars at 3 different planting dates (Aug.
25, Sept. 10, and Sept. 25). Total RNA was isolated at 15
(Sept. 10), 30 (Sept. 25), 45 (Oct. 10), and 75 DAP (Nov. 13)
for the Aug. 25 planting; 15 (Sept. 25), 30 (Oct. 10), and 60
DAP (Nov. 13) for the Sept. 10 planting; and 15 (Oct. 10) and
45 DAP (Nov. 13) for the Sept. 25 planting.

Total RNA was probed with BN28 (Fig. 8) and BN115 (Fig.
9) . The graphical representation of the gene expression .
affected by different planting dates is shown in Fig. 10
(BN28) and Fig. 11 (BN115). There was both a slow and rapid

Phase of increase in expression of BN28. In the Aug. 25
Planting, the increase in expression of BN28 was slow until 30
DAP: then increased rapidly from 30 to 45 DAP. Both in the
Sept. 10 and Sept. 25 plantings, the rapid increase phase was
coincident with that of the Aug. 25 planting regardless of
Plant ages. The maximum expression was attained at the end of
the experiment (Nov. 13) and there was no difference in
transcript amount of BN28 at the end of experiment among the
three planting dates. BN115 reached its full expression
within 15 DAP in two planting dates and later planting induced
a higher expression than earlier planting at the end of the

experiment (Nov. 13) .

 

108

 

 

WRG86

DUOBUL

KWC4113

 

Figure 8. Northern blot analysis of 3 winter canola cultivars
planted Aug. 25, Sept.

10, and Sept. 25, 1993,
respectively. Total RNA from leaves of plants was

isolated Sept. 10 (lane a), Sept. 25 (lane b), Oct.

10 (lane c), and Nov. 13 (lane.d), respectively. The
membrane was hybridized with BN28.

 

109

BN115

AUGJS

SEPTJO sgprgs
c d
WRG86

     

DUOBUL .'..I: ‘ ; .
KWC4113 61...... O

Flgure 9. Northern blot analysis of 3 winter cultivars
planted Aug. 25, Sept. 10, and Sept. 25, 1993,
respectively. Total RNA from leaves of plants was
isolated Sept. 10 (lane a), Sept. 25 (lane b), Oct

10 (lane c), and Nov. 13 (lane d), respectively The
membrane was hybridized with BN115.

 

110

 

 

 

 

 

 

 

 

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I I f I 1
AUG.ZS SEPTJO SEPT.ZS OCT.‘IO NOV.13

DATE OF MEASUREMENT

Flgure 10. Changes in transcript amount of BN28 affected by
different planting dates. Data represent overall
means of three cultivars and a graphical
presentation of the northern blot in Fig. 8.
Vertical bar indicates mean t SE.

 

111

 

 

 

 

 

 

 

 

 

 

g —o-— AUG.ZS L

100- —o— SEPTJO j},
g —-a— SEPT.ZS T
o
2 80-
<
.5; a}
a: -
o 60
3

I
Z
S 20-
E
o

 

~AUG.25 SEPTJO $07.25 oc1".1o "ml/.13

DATE OF MEASUREMENT

Figure 11. Changes in transcript amount of BN115 affected by
different planting dates. Data represent overall
means of three cultivars and a graphical
presentation of the northern blot in Fig. 9.
Vertical bar indicates mean t SE.

112

Effect of Planting Date on Winter Survival of canola

Although both cultivar and planting date significantly
affected winter survival, the effect of planting date was
greater than that of cultivar (Table 3). Earlier planting
(Aug. 25) resulted in above 80 % survival except for the least
hardy cultivar (66 %), while later planting (Sept. 25)
resulted in no survival for four of the six cultivars.

The hardy cultivars, Accord and KWC4113, showed 40 and
20 % survival, respectively, even when planted Sept. 25, while
other cultivars showed no survival. However, plant growth
prior to freezing was much poorer than that of earlier

plantings.

Correlation between Freezing Tolerance, Winter Survival, and
Expression of BN28 and BN115

A significant correlation was observed between freezing
tolerance and winter survival of cultivars in these studies
(Table 4) except for the Sept. 10 sampling date from the Aug.
25 planting. Correlation coefficients ranged from 0.83(*) to
O.96(**).

Though the time-course increase in transcript amount of
BN28 was correlated with time-course increase in freezing
tOIerance, 'no significant correlation occurred between the
gene expression and freezing tolerance (Table 5), and

eventually, with winter survival (Table 6) in cultivar level

 

113

Table 3. Iinter survival of six winter canola cultivars,
planted Aug. 25, Sept. 10, and Sept. 25, 1993.

 

Planting date

 

 

 

Cultivars
Aug. 25 Sept. 10 Sept. 25
--winter survival (%)--
ACCORD 100.0aT 72.0a 40.0a
KWC4113 100.0a 70.0a 20.03
CERES 84.0b 59.0b 6.0b
DUOBUL 93.0b 65.0b 0.0C
WRGBG 79.0bc 57.0b 0.00
CDH3 ' 66.06 41.0C 0.00

 

I Means followed by the same letter within a column are
not significantly different at the 0.05 probability level.

114

Table 4. Simple correlation coefficients between freezing
tolerance and winter survival of canola cultivars
planted Aug. 25, Sept. 10, and Sept. 25, 1993.

 

 

Planting Date Sampling Date r

Aug. 25 Sept. 10 0.54ns

Sept. 25 0.89*

Oct. 10 0.85*

Nov. 15 0.94**
Sept.10 Sept. 25 0.88*

Oct. 10 O.96**

Nov. 15 0.83*
Sept.25 Oct. 10 0.87*

Nov. 15 0.93**

 

 

T (*) and (**) simple correlation coefficient significant
at 0.05 and 0.01 probability levels, respectively.
(ns) not significant at the 0.05 probability level.

115

Table 5. Simple correlation coefficientsf between

transcript amount of SNZS and SN115 and freezing
tolerance of canola cultivars planted Aug. 25,

Sept. 10, and Sept, 25, 1993.

 

 

 

r

Planting Date SN 28 EN 115
Aug. 25 0.59nsT -0.37ns
Sept.10 0.47ns -0.33ns
Sept.25 0.60ns -O.40ns

 

T Correlation coefficient was estimated between transcript
amount at Nov. 13 and freezing tolerance at Nov. 13.

i (*) and (**) simple correlation coefficient,
significant at 0.05 and 0.01 probability levels,
respectively. (ns) not significant at the 0.05
probability level.

116

Table 6. Simple correlation coefficients? between
transcript amount of SNza and SNiis and winter
survival of canola cultivars planted on Aug. 25,
Sept. 10, and Sept. 25.

 

 

 

 

r

Planting date SN 28 EN 115
Aug. 25 0.49nsi -0.27ns
Sept.10 0.15ns -0.33ns
Sept.25 0.30ns -0.34ns

 

T Correlation coefficient between winter survival and
transcript amount at Nov. 13.

i (*) and (**) simple correlation coefficient,
significant at 0.05 and 0.01 probability levels,
respectively. (ns) not significant at the 0.05
probability level.

117
at specific time of measurement. Transcript amount of BN115
was negatively correlated with freezing tolerance and winter

survival, but no values were significant.

Effect of Planting Date and Seeding Rate on winter Survival
of Canola

Planting date and seeding rate had significant effects
on winter survival of Duobul in both 1992-1993 and 1993-1994
(Table 7, 8) . As the seeding rate increased, the winter
survival decreased. However, there was no survival from the
Sept. 25 planting from any of the three seeding rates during

1993-1994.

Effects of Planting Date and Pall-Applied Nitrogen on Ninter
Survival

The effects of planting date and fall-applied nitrogen
on winter survival were examined in the less hardy cultivar,
Duobul, in both 1992-1993 and 1993-1994 (Table 2.9). Twenty
and forty kg/ha of nitrogen (as urea) were applied after
seedling emergence. Only planting date had a significant
effect on winter survival in both years; nitrogen had no

significant effect.

 

118

Table 7. Effect of planting date and seeding rate on
winter survival of canola cultivar, Duobul,

in 1992-1993.

 

 

 

 

Planting date Seeding rate (kg/ha)
4 8 12 Mean
---winter survival (%)--
Aug. 25 81 80 46 69aT
Sept. 10 83 80 39 67a
Sept. 25 69 60 46 58b
Mean 77a 73a 43b
F- test: Planting date (A) 8.7 **1
Seeding date (8) 10.6 **
Interaction (A X B) 0.5 ns

 

I Means followed by the same letter within a column and

rows are not significantly different at the 0.05
probability level.

i (*) and (**), significant at 0.05 and 0.01 probability
levels, respectively. (ns) not significant at the 0.05

probability level.

 

119

Table 8. Effect of planting date and seeding rate on
winter survival of canola cultivar, Duobul,
in 1993-1994.

 

Planting date Seeding rate (kg/ha)

 

4 8 12 Mean

 

---winter survival (%)--

 

Aug. 25 87 80 56 74aT
Sept. 10 55 47 39 47b
Sept. 25 o o 0 0c
Mean 47a 42a 31b
F- test: Planting date (A) 21.7 **¢
Seeding rate (B) 12.6 **
Interaction (A X B) 0.5 ns

 

Means followed by the same letter within a column and

rows are not significantly different at the 0.05
probability level.

i (*) and (**), significant at 0.05 and 0.01 probability

levels, respectively.

probability level.

(ns) not

significant at the 0.05

_'—.' r j
r'

 

-11.!

120

Table 9. Effect of fall-applied nitrogen and planting date
on winter survival of canola cultivar, Duobul, in
1992-1993 and 1993-1994.

 

Winter survival(%)

 

 

 

 

Nitrogen ----1992-l993--- ----- 1993-1994 -----
(kc/ha)
Aug. Sep. Sep. Mean Aug. Sep. Sep. Mean
25 10 25 25 10 25

Control 88 91 45 74 90 73 0 54
20 90 81 42 71 87 68 0 51
40 86 90 53 76 93 55 0 49
Mean 88a 87a 46b 90a 65b 0c
F-test:

Planting date (A) 31.5(**) 114.8(**)
Nitrogen (B) 0.4(ns) 1.2(ns)

A x B 0.8(ns) 0.8(ns)

 

 

i (*) and (**), significant at 0.05 and 0.01 probability
levels, respectively. (ns) not significant at the 0.05
probability level.

DISCUSSION

At all three planting dates, a phase occurred when a

rapid increase in freezing tolerance occurred regardless of'

plant age. Between Sept. 25 and Oct. 10, the minimum air
temperature decreased from 10°C to 4°C and freezing tolerance
of plants increased rapidly. This is consistent with the
findings of Kacperska-Palacz (1978) showing that alfalfa and
rapeseed initiated cold-hardening at a temperature of 5°C,
apparently without the transition to a state of readiness to
harden seen in woody plants (Sakai and Larcher, 1978).
However, earlier planting resulted in greater freezing
tolerance compared to later planting. The most hardy cultivar
planted Aug. 25 reached an LT” value of -20°C in mid-November,
while that of the Sept. 25 planting reached only -13°C. In
addition, planting date greatly affected how plants attained
freezing tolerance. For all three planting dates the first
phase of rapid increase in freezing tolerance was followed by
a steady increase from mid-October to mid-November (Fig. 5).
However, the pattern during the second phase differed among
planting dates. The earlier planting showed a gradual increase
in freezing tolerance until mid-November, which presumably

continued until December. However, the later planting showed

121

 

 

122
less increase in freezing tolerance than the earlier planting
in this period. This may be explained by differences in
growth; earlier plantings grow more and accumulate more
photosynthate reserves which increase cold acclimation. This
suggestion is supported by published reports that reduction of
carbohydrate reserves reduced cold hardiness in over-wintering
plants (Levitt, 1956). Another possibility is that the gradual
exposure to cold temperature is more effective than the sudden
exposure. This is supported by a report by Pomeroy et al.
(1974) that high levels of hardiness in wheat could be rapidly

induced in 4-6 days if the hardening temperature was preceded

by warm temperature, while previous reports (Pomeroy and ‘

Fowler, 1973; Andrew et al., 1974) showed that 6-8 weeks of
hardening temperature were required for maximum hardiness
without previous exposure to warm temperature.

Growth itself may affect the winter survival of canola.
Earlier planting promotes greater fall foliar growth, which
covers the growing point, thus protecting it. Later planting
does not provide adequate foliar coverage; thus, the growing
point remains vulnerable to freezing damage. This is
consistent with data that snow cover increases winter survival
of canola under severe winter conditions in Michigan's Upper
Peninsula (Copeland, unpublished data).

The process of cold acclimation and development of
freezing tolerance is a prime example of the interaction

between genetic factors and the environment. Aside from the

 

 

 

123
effect of planting date, different cultivars showed different
freezing tolerance and winter survival. The most hardy
cultivars showed acceptable levels of winter survival when
planted at dates too late for less hardy cultivars.

The study on the expression of the two cold-regulated
Genes, BN28 and BN115, revealed no clue about their roles in
cold acclimation. Though there is no correlation between
freezing tolerance and expression of these genes at the
cultivar level at specific sampling times, observations that
these genes are turned on in response to cold temperature in
the field and that the time-course increase in BN28 expression
is coincident with increase in freezing tolerance (Fig. 7 and
Fig. 10) suggests that they may have a role in winter
hardiness. Expression of the genes was examined only on the
transcript level in this study. Future studies on
translational level expression may explain cultivar
differences in freezing tolerance.

In conclusion, planting date is the most important
factor determining the freezing tolerance of canola, and
freezing tolerance is highly correlated with winter survival.
However, in case of unavoidable late planting, a choice of a
hardy' cultivar' may' compensate for the loss of freezing
tolerance due to later planting. The possibility of the two
cold-regulated genes for use as molecular markers for

selection for winter hardiness is not promising.

 

 

 

REFERENCES

Andrews, C.J., M.K. Pomeroy, and LA. de la Roche. 1974.
Changes in cold hardiness of overwintering winter wheat.
Can. J. Plant Sci. 54:9-15.

Auld, D.L., B.L. Bettis, and N.J. Dial. 1983. Planting date
and cultivar effect on winter rape production. Agron. J.
76:197-200.

Auld, D.L., D.R. Brady, and K.A. Mahler. 1989. Rapeseed and
canola breeding in North America. pp. 7-10. 5th Crucifier
Genetic Workshop, Symposium I. Davis, California.

Bartels, D., K. Schneider, G. Terstappen, D. Piatkowski, and
F. Salamini. 1990. Molecular cloning of abscisic acid-
modulated genes which are induced during desiccation of the
resurrection plant.Craterostigma plantagineum. Planta 181:27-
34.

Bray, E.A. 1991. Regulation of gene expression by endogenous
ABA during drought stress. pp. 81-98. In: N.J. Davis and H.G.
Jones (eds.) Abscisic.Acid: Physiology and Biochemistry. Bios
Scientific Publishers, Oxford.

Burke, J.J., J.L. Hatfield, R. Klein, and J.E. Mullet. 1985.
Accumulation of heat shock proteins in field-grown cotton.
Plant Physiol. 78:394-398.

Canvin, D.T. 1965. The effect of temperature on the oil
content and fatty acid composition of oils from several oil
seed crops. Can J. Botany 43:63-69.

Chen, Q., L. Lauzon, A. DeRocer, and E. Vierling. 1990.
Accumulation, stability and localization of a chloroplast heat
shock protein. J. Cell Biol. 110:1873-1883.

Christensen, J.0., W.G. Ledge, R.M. DePaew, A.W. Henning, J.S.
McKenzie, and J.B. Thomas. 1984. Effect of seeding date,
nitrogen and phosphate fertilizer on growth, yield and quality
of rapeseed in northwest Alberta. Can. J. Plant Sci. 65:275-
284.

124

 

125

Close, T.J., A.A, Kortt, and.P.M; Chandler. 1989. A.cDNA-based
comparison of dehydration induced protein (dehydrin) in barley
and corn. Plant Mol. Biol. 12:475-486.

Degenhardt, D.F. and z.P. Kondra. 1981. The influence of
seeding date and seeding rate on seed, yield and yield
components of five genotypes of Brassica napus. Can. J. plant
Sci. 61:175-183.

DeRocher, A.E., R.Wu Helm, L.M. Lauzon, and E. Vierling. 1991.
Expression of a conserved family of cytoplasmic low'molecular
weight heat shock proteins during heat stress and recovery.
Plant Physiol. 96:1038-1047.

Gilmour, S.J., N.N. Artus, and M.F. Thomashow. 1992. cDNA
sequence analysis and expression of two cold-regulated genes
of Arabidopsis thaliana. Plant Mol. Biol. 18: 13-21.

Gusta, L.V., B.J. O'Connor, and M.J.T. Reaney. 1990. The
effect of growth regulators on the winter survival of winter
wheat. pp. 531-536. In: R.P. Pharis and S.B. Rood(eds.) Plant
Growth Substances 1988. Spring-Verlag, Berlin.

Hajela, R.K., D.P. Horvath, S.J. Gilmour, and M.F. Thomashow.
Molecular cloning and expression of cor (cold-regulated) genes
in Arabidopsis thaliana. Plant Physiol. 93:1246-1252.

Huber, W. 1973. Testing of winter rape varieties and yield
comparisons at different sowing date. Mitt. Schweizerische
Landwirsche 21:97-105.

Kondra, z.P. 1977. Effect of planted seed size and seed rate
on rapeseed. Can. J. Plant Sci. 57:277-280.

Kondratowicz, J., and S. Patrocki. 1974. The effect of sowing
date and of cultural methods in autumn on yield of rape in
Olszystn province. Akad. Roln-Tech. Olsztynie 9:119-133.

Kurkela, S., and M. Frank. 1990. Cloning and characterization
of a cold- and ABA-induced Arabidopsis gene. Plant Mol. Biol.
15:137-144.

Lang, V. and E.T. Palva. 1992. The expression of a.rab-related
gene, rab18, induced by abscisic acid during the cold
acclimation process of Arabidopsis thaliana (L.) Heynh. Plant
Mol. Biol. 20:951-962.

Lorraine, D.H., and E. Vierling. 1993. Expression of low
molecular weight heat-shock proteins under field conditions.
Plant physiol. 101:1209-1216.

Mundy, J., and N-H. Chua. 1988. Absisic acid and water-stress

 

 

126

induce the expression of a novel rice gene. EMBO J. 7:2279-
2286.

Noderstagaard,A. 1972. Combined sowing time and variety
experiments with winter rape. Tidsskr. Planteavl 76:597-603.

Nordin, K., P. Heino, and E.T. Palva. 1991. Separate signal
pathways regulate the expression of a low temperature-induced
gene in Arabidopsis thaliana. Plant Mol. Biol. 16: 1061-1071.

Orr, W., B. Iu, T.C. White, L.S. Robert, and J. Singh. 1992.
Complementary DNA sequence of a low temperature-induced
Brassica.napus gene with homology to the Arabidopsis thaliana
kinl gene. Plant Physiol. 98:1532-1534.

Pla, M., J. Gomez, A. Goday, and.M. pages. 1991. Regulation of
the abscisic acid responsive gene rab28 in maize viviparous
mutants. Mol. Gen. Genet. 230:394-400.

Robertson, A.J., G.C. Churchill, and L.V. Gusta. 1992. The
role of plant growth regulators on the freezing tolerance of
winter annual cereals and cell suspension culture. pp. 253-
267. In: P.H. Li and L. Christerson (eds.) Advances in Plant
Cold Hardiness. CRC Press, Boca Raton, Fl.

Sakai, A., and W. Larcher. 1987. Responses and Adaptation to
Freezing Tolerance. Spring-Verlag, Berlin. 321 pp.

Skriver, K., and J. Mundy. 1990. Gene response to ABA and
osmotic stress. Plant Cell. 2:503-512.

Yamaguchi-Shinozaki, K., and K. Shinozaki. 1994. A novel cis-
acting element in an Arabidopsis gene is involved in
responsiveness to drought, low-temperature, or high-salt
stress. Plant Cell 6:251-264.

Weretilnyk, E., W; Orr, T.C. White, B. Iu, and J. Singh. 1993.
Characterization of three related low-temperature regulated
cDNAs from winter Brassica napus. Plant Physiol. 101:171-177.

 

 

 

CHAPTERIII

EFFECTS OF TWO PLANT GROWTH REGULATORS, CERONE
AND TERPAL-C, ON FREEZING TOLERANCE AND WINTER
SURVIVAL OF CAN OLA (Brassica napus L.)

ABSTRACT

One of the most attractive short-term solutions
postulated for increasing freezing tolerance of winter crops
may be application of chemicals. This research was conducted
to determine the effects of two plant growth regulators,
Terpal-C and Cerone, as well as fall-applied phosphorus, on
freezing’ tolerance and. winter survival of canola. Three
cultivars were planted on the Michigan State University
Agronomy FarmIat East Lansing, MI. on Sept. 10, 1992 and 1993.
Chemicals were applied to one-month-old plants when they
reached the 5 leaf stage. Ion leakage tests were conducted 25
days after chemical treatment. Winter survival was evaluated
by counting the plant standing in the fall and spring.

Neither the chemicals nor the phosphorus have any effect
in increasing freezing tolerance and winter survival. Instead,
one of the chemicals, Terpal-C, inhibited natural cold

hardening.

127

 

 

 

INTRODUCTION

Numerous efforts have been made to overcome the freezing
problems of crop plants in agriculture, including breeding
efforts to develop more hardy cultivars and in modification of
cultural practices to ensure winter survival. Most of the
hardy cultivars of wheat grown today contain major genes for
cold hardiness (Gusta et al., 1990). Consequently, little or
no progress has been made in enhancing the genetic potential
of cold hardiness in winter wheat. Searches for cost-
effective strategies in plant freezing protection have focused
on short-term solutions because of the limited gene pool and
difficulties in breeding procedures for cold hardiness (Fowler
and Gusta, 1979).

One of the most attractive short-term methods for
increasing freezing tolerance may be the application of
chemicals (Marth, 1965; Chen and Li, 1976; Paquin et al.,
1976; Proebsting and Mills, 1976; Raese, 1977; Ketchie and
Miller, 1978; Gusta et al., 1982; Dunner and Gianfagna, 1988;
Morrison and Andrews, 1993). One group of chemicals, called
cryoprotectants, increases hardiness, per se, regardless of
stage of development, while a second group, primarily growth

regulators, modifies the stage of development, thus indirectly

128

 

 

 

129
modifying freezing tolerance. The basic idea is that because
of the general negative correlation between growth rate and
cold hardiness, one might expect growth-inhibiting chemicals
to favor the development of hardiness and growth promoters to
reduce hardiness (Howell and Dennis, 1980).

The development of freezing tolerance is controlled by
plant hormones. ABA is known to be involved in cold
acclimation of plants (Irving, 1969; Rikin, 1975; Waldman,
1975; Chen et al., 1979; Orr et al., 1986; Reany and Gusta,
1987; Lang et al., 1988; Mohapatra et al., 1989) and,
coincidently, with expression of certain cold-regulated genes
(Thomashow, 1990). Another plant hormone involved in cold
hardiness is gibberellic acid (GA) which has an antagonistic
effect on freezing tolerance. Waldman et al. (1975) suggested
that ABA induces cold acclimation by inhibiting the synthesis
of GA, and demonstrated that the exogenous application of ABA
dramatically reduced GA level in cold hardy alfalfa cultivars.
The primary synthetic plant growth regulators that increase
winter hardiness have either antigibberellin or ethylene-
producing characteristics. Though the role of these plant
growth regulators in winter hardiness at the molecular and
cellular level is uncertain, it is assumed that they prevent
the loss of freezing tolerance (Gusta et al., 1990).

The objective of this study was to determine the effects
of two plant growth regulators, Terpal-C and Cerone, on

freezing tolerance and winter survival of canola.

 

 

 

MATERIALS AND METHODS

Three winter canola (Brassica napus L.) cultivars, WRGS6,

Duobul, and KWC4113, were used in this study.

Planting, Management and Experimental Design

This study was conducted at the Michigan State
University Agronomy Farm in East Lansing, Michigan. A split-
plot design with cultivar as the 'main plot and growth
regulator as subplots was used with three replications. Seed
was planted at the rate of 5.6 kg/ha in a 5-row plot 6 m long
and 92 cm wide. The planting date for the first year was

Sept. 5, 1992 and for the second year, Sept. 6, 1993.

Application of Plant Growth Regulators

Two plant growth regulators, Terpal-C (BASF) and Cerone
(Union Carbide) , were used for this experiment. Chemicals were
applied with a compressed CO, sprayer one month after planting
(Oct. 13, 1993) when most of the plants reached the 5 leaf
stage. The chemicals were diluted with distilled water at the
rate of 300:1, then applied at the rate of 1840 g/ha for
Terpal-C and 620 g/ha for Cerone. Control plots were sprayed

with distilled water.

130

 

131

Evaluation of Freezing Tolerance
In 1993-1994, freezing tests were conducted on plant
samples collected just before chemical application (Oct. 10)
and 25 days after chemical application (Nov. 5). Five
different plants were collected from each plot in the field
and immediately transferred to the laboratory. One leaf disc
was taken from each plant and 3 leaf discs from the same plot
were placed in a stoppered culture tube representing one
replication. Freezing tests were conducted as described in

Chapter I.

Evaluation of Winter Survival
Winter survival for both 1992-1993 and 1993-1994 studies

were evaluated as described in Chapter I.

Phosphorus Experiment

The same cultivars were planted on the same date at a
different experimental site. At planting time, 0, 100, and 200
kg/ha of actual phosphorus (as foam of 46 % ingredient of
fertilizer) were applied to provide 3 different P53 levels.

Winter survival was evaluated as previously described.

 

 

 

RESULTS AND DISCUSSIONS

Effect of Plant Growth Regulators, Terpal-C and Cerone on
Preesing Tolerance and Winter Survival of Canola

Typical freezing curves were developed from the two
sensitive cultivars, WRGS6 (Fig. 1) and Duobul (Fig. 2), and
the hardy cultivar, KWC4113 (Fig. 3). All three cultivars
showed similar responses to the growth regulator treatment.

Cerone had little or no effect in increasing freezing
tolerance. However, Terpal-C decreased the freezing tolerances
of all cultivars compared to that of control on Nov. 10 (Table
1) . Interestingly, this treatment seemed to inhibit the normal
development of freezing tolerance.

Both Terpal-C and Cerone treatment had no significant
effect on the winter survival of canola in either year (Table
2 and Table 3). The only effect on winter survival was among
cultivars, and only during the second year. No interaction
occurred between growth regulator treatment and cultivar in
either year.

The results from freezing tests are not consistent with
those of.Morrison and Andrews (1992) who reported that Terpal-
C increased freezing tolerance. Terpal-C application to canola

in the field increased freezing tolerance of canola by 1-2 °C

132

 

 

133

 

 

 

 

 

 

 

 

100
—a— comocmm
—-— CON(NOV.S)
- —o— TERPAL-C(NOV.5) ,. .
80 —o— CERONE(NOV.S) .—
If}
CD so- /
i /'
33 4o /
In}
s - - /
. 9?;;> .._ .
0 I I T y I I I I I

 

0 Z 4 6 8 10 12 14 16 18

TEMPERATURE (- ° C)

Figure 1. Effect of plant growth regulators, Terpal-C and
Cerone, on freezing tolerance of winter canola cv.

WRG86. Freezing tests were conducted before
chemical treatment (Oct. 10) and 25 days after
chemical treatment (Nov. 5). Vertical bar
indicates mean t SE.

 

134

 

100

 

—o— comocm 0)
—I— CON(NOV.S)

—o— TERPAL-C(NOV.5) , .-
CERONE(NOV.S) 1"

+.

 

 

 

O!
O
I

.b
O
I

% LEAKAGE

 

 

 

20 7 . ° . . '
4.252%
0 . , , . . . r . .

0 2 4 6 8 10 12 14 15 13

TEMPERATURE (- ° C)

Figure 2. Effect of plant growth regulators, Terpal-C and
Cerone, on freezing tolerance of winter canola cv.
Duobul. Freezing tests were conducted before
chemical treatment (Oct. 10) and 25 days after
chemical treatment (Nov. 5). Vertical bar
indicates mean t SE.

 

135

 

 

 
 
 

 

 

 

 

 

 

 

 

100
—o— CON(OCT.10)
—I— CON(NOV.S)
—o— TERPAL-C(NOV.5)
3° " CERONE(NOV.5)
8
a: «60-
a
h; «40-
S“
20-
o I I l I l I I I I
0 2 4 6 8 10 12 14 16 18
TEMPERATURE (- °C)
Figure 3. Effect of plant growth regulators, Terpal-C and

Cerone, on freezing tolerance of winter canola cv.
KWC4113. Freezing tests were conducted before
chemical treatment (Oct. 10) and 25 days after
chemical treatment (Nov. 5). Vertical bar
indicates mean t SE.

 

 

136

Table 1. Effect of plant growth regulators, Terpal-C and
Cerone, on freezing tolerance of three canola
cultivars, WRG86, Duobul, and EWC4113.

 

 

 

 

 

 

 

 

LTso (-°C)
Chemical
WRGS6 Duobul KWC4113 Mean
ControlT(Oct. 10) 10.3 9.5 12.1 10.6b
Control (Nov. 5) 11.9 12.0 13.8 12.6a
Terpal-C (Nov. 5) 9.2 10.4 11.4 10.3b
Cerone (Nov. 5) 11.7 12.6 13.6 12.3a
Mean 10.8c 11.2b 12.7a
F-test: Variety (A) 11.74 (**)1
Chemical (B) 12.75 (**)
A x B 0.75 (ns)
1 Two controls as before chemical treatment (Oct. 10) and

after chemical treatment (Nov. 5).

i (*) and (**); significant at 0.05 and 0.01 probability
levels, respectively. (ns) not significant at 0.05
probability level.

 

137
in December compared to the control. Whether decrease in
freezing tolerance by Terpal-C treatment in this experiment is
due to its effect on the retention of physiological immaturity
or the inhibition of natural cold-hardening by the chemical
application should be elucidated in future.

In spite of the decrease of freezing tolerance by
Terpal-C, it did not affect winter survival. This may be
additional evidence that its effect is not the increase in
freezing tolerance but the prevention in loss of freezing
tolerance during the middle of severe winters (Gusta et. a1,

1992) .

Effect of Pall-Applied Phosphorus on the Winter Survival of
Canola

In the 1993-1994 experiments, the effect of phosphorus
on freezing tolerance of three canola cultivars was examined
by ion leakage tests on Nov. 15. Phosphorus treatment did not
affect freezing tolerance (Table 4) and winter survival of
canola in either year (Table 5).

The experimental evidence of an association between
phosphorus and winter hardiness is limited and somewhat
controversial. Levitt ( 1980) indicated that there were as
many investigators who found no association between phosphorus
and winter hardiness as there were who found a positive

association. The role of phosphorus in winter hardiness is

 

 

138

Table 2. Effect of plant growth regulators, Terpal-C and
Cerone, on winter survival of three canola
cultivars in 1992-1993.

 

 

 

 

 

 

 

Chemical Winter survival(%)

WRGS6 Duobul KWC4113 Mean
Control 82.0 80.3 86.0 83.0
Terpal-C 92.0 81.0 90.3 88.0
Cerone 83.0 91.0 95.6 89.0
Mean 80.5 84.3 90.8

F-test: Variety (A) 1.160 (ns)?
Chemical (B) 0.270 (ns)
A x B 1.470 (ns)

1 (*) and (**); significant at 0.05 and 0.01 probability

levels, respectively. (ns) not significant at 0.05
probability level.

139

Table 3. Effect of plant growth regulators, Terpal-C and
Cerone on winter survival of three canola
cultivars in 1993-1994.

 

 

 

 

 

 

 

Chemical Winter survival(%)
WRGS6 Duobul KWC4113 Mean
Control 60.0 65.0 76.5 67.0
Terpal-C 63.5 58.0 73.5 65.0
Cerone 58.0 59.0 78.5 65.1
Mean 60.3b 60.6b 76.1a
F-test: Variety (A) 3.200 (*)1
Chemical (B) 0.270 (ns)
A x B 1.470 (ns)
1 (*) and (**); significant at 0.05 and 0.01 probability

levels, respectively. (ns) not significant at 0.05
probability level.

 

140

Table 4. Effect of fall-applied phosphorus on freezing
tolerance of three winter cultivars in 1993-1994

 

Treatment Cultivars

 

190, (kg /ha) WRGBG DUOBUL KWC4113

 

---LT” at Nov. 15---

 

Control 11.5 11.2 13.0 11.9
100 10.9 11.8 12.9 11.8
200 9.4 11.1 11.9 10.8
Mean 10.6c 11.4b 12.6a
F-test : Variety (A) 4.34 (*)T

Phosphorus (B) 0.78 (ns)

A x B 0.94 (ns)

 

T (*) and (**); significant at 0.05 and 0.01 probability
levels, respectively. (ns) not significant at the 0.05

probability level.

 

141

Table 5. Effect of fall-applied phosphorus on winter
survival of three winter cultivars in 1992-1993
and 1993-1994.

 

Winter survival (%)

 

 

 

 

 

 

1(2PO4 ----1992-1993--- ----- 1993-1994 -----
(kg/ha)
WRG Duo- KWC4 Mean WRG Duo- KWC Mean
86 bul 4113 86 bul 4113
Control 81 80 86 83 56 52 73 60
100 97 97 87 93 42 65 82 63
200 91 83 79 84 55 56 75 63
Mean 89 86 84 51b 57b 76a
F-test:
Variety (A) 1.35(ns)T 4.8(*)
Phosphorus (B) 0.88(ns) 1.2(ns)
A x B 0.73(ns) 0.8(ns)
T (*) and (**), significant at 0.05 and 0.01 probability

levels, respectively. (ns) not significant at the 0.05
probability level.

 

142
also controversial. Phosphorus is known to favor accumulation
of photosynthate, which can be used in the hardening process.
Phosphate accumulated in the plant also enhances the regrowth
of roots in the spring to enable recovery of the damage by
winter freezing (Robertson et al., 1992).

Either phosphorus does not have any relation to freezing
tolerance and winter survival of canola, or the winters were
not adequately severe to reveal the difference induced by
phosphorus treatment. Finally, the effect of phosphorus may

have been masked by natural cold-hardening.

 

 

 

REFERENCES

Chen, H.H. , P. Gavinlertvatana, and P.H. Li. 1979. Cold
acclimation of stem-cultured plants and leaf callus of Solanum
species. Bot. Gaz. 140:142-147.

Chen, H.H., and P.H. Li. 1976. Effect of photoperiod,
temperature and certain growth regulators on frost hardiness
of Solanum species. Bot. Gaz. 137:105-109.

Durner, E.F., T.J. Gianfagna. 1988. Fall ethephon application
increases peach flower bud resistance to low temperature
stress. J. Am. Soc. Hort. Sci. 113:404-406.

Fowler, D.B., and L.V. Gusta. Selection for winter hardiness
in wheat. I. Identification of genotypic variability. Crop .
Sci. 19:769-772.

Gusta, L.V., B.J. O'Connor, and M.J.T. Reaney. 1990. The
effect of growth regulators on the winter survival of winter
wheat. pp. 531-536. In: R.P. Pharis and S.B. Rood (eds.) Plant
Growth Substances 1988. Spring-Verlag, Berlin.

Gusta, L.V., D.B. Fowler, and N.J. Tyler. 1982. Factors
influencing hardening and survival in winter wheat. pp. 23-40.
In: P.H.Li and A. Sakai (eds.) Plant Cold Hardiness and
Freezing Stress: Mechanisms and Crop Implications, vol. 2.
Academic Press, New York.

Gusta, L.V., and M.G. MacHutchon. 1988. Freezing resistance in
spring and winter crops. pp. 899-907. In: S.K. Shiha, P.V
Sane, S.CiBhargava, and P.K. Agrawal (eds.) Proceedings of the
International Congress of Plant Physiology, Vol. 2. Spring-
Verlag, Berlin.

Irving, R.M. 1969. Characterization and role of an endogenous
inhibitor in the induction of cold hardiness of Acer negundo.
Plant Physiol. 44:801-805.

Ketchie, D.O., and C. Murren. 1976. Use of cryoprotectans on
apple and pear trees. J. Am. Soc. Hort. Sci. 101:57-59.

Lang, A. 1979. Gibberellin: Structure and metabolism. Annual

143

 

 

144
Rev. Plant Physiol. 21:537-594.

Lang, V., P. Heino, and E.T. Palva. 1988. Low temperature
acclimation and treatment with exogenous abscisic acid induce
common polypeptides in Arabidopsis thaliana (L.) Heynh. Theor.
Appl. Genet. 77:729-734.

Levitt, J. 1980. Responses of Plant to Environmental stresses
Vol.1. Chilling, Freezing and High Temperature Stresses, 2nd
ed. Academic Press, New York. 497 pp.

Marth, P.C. 1965 Increased frost resistance by application of
plant growth retardant chemicals. J. Agri. Food Chem. 13:331-
333.

Mohapatra, 8.8., L. Wolfraim, R.J. Poole, and R.S. Dhindsa.
1989. Molecular cloning and relation to freezing tolerance of
cold-acclimation-specific genes of alfalfa. Plant physiol.
89:375-380.

Morrison, M.J., and C.J. Andrews. 1992. Variable increases in
cold hardiness induced in winter rape by plant growth
regulators. J. Plant Growth Regul. 11:113-117.

Orr, W., B. Iu, T.C. White, L.S. Robert, and J. Singh. 1992.
Complementary DNA sequence of a low temperature-induced
Brassica napus gene with homology to the Arabidopsis thaliana
kinl gene. Plant Physiol. 98:1532-1534.

Orr, W., W.A. Keller, and J. Singh. 1986. Induction of
freezing tolerance in an embryogenic cell suspension culture
of Brassica napus by abscisic acid at room temperature. J.
Plant Physiol. 126:23-32.

Paquin, R., L. Belzile, C. Willemot, J.-C. St-Pierre. 1976.
Effects of some growth retardant and gibberellic acid on the
frost resistance of alfalfa (Medicago sativa). Can. J. Plant
Sci. 56: 79-86.

Probsting, E.L.Jr., and H.H. Mills. 1976. Ethephon increase
cold hardiness of sweet cherry; J. Am. Soc. Hort. Sci. 101:31-
33.

Raese, J.T. 1977. Induction of cold hardiness in apple tree
shoot with ethephon, NAA and growth retardant. J. Am. Soc.
Hort. Sci. 102:789-792.

Reaney, M.J.T., and L.V. Gusta. 1987. Factors influencing the
induction of freezing tolerance by abscisic acid in cell
suspension culture of Bromus inermi's Leyss and Medicago sativa
L. Plant Physiol. 83:423-427.

 

145

Rikin, A., M. Waldman, A.E. Richmond, and A. Dovart. 1975.
Hormonal regulation of morphogenesis and cold-resistance. I.
Modification by abscisic acid and gibberellic acid in alfalfa
(Medicago sativa L.) seedlings. J. Exp. Bot. 26:175-183.

Robertson, A.J., G.C. Churchill, and Luv. Gusta. 1992. The
role of plant growth regulators on the freezing tolerance of
winter annual cereals and cell suspension culture. pp. 253-
267. In: P. H. Li and L. Christerson (eds.) Advances in Plant
Cold Hardiness. CRC Press, Boca Raton.

Thomashow, M.F. 1990. Molecular genetics of cold acclimation
in higher plant. Adv. Genet. 28:99-131.’

Waldman, M., A. Rikin, A. Dorvat, and A.E. Richmond. 1975.
Hormonal regulation of morphogenesis and cold acclimation. J.
Exp. Bot. 26:853-860.

 

 

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