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Mitigation of Cold Stress |
By Dr.’s D. B. Fowler and A. E. Limin Crop Development Centre |
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Crop Plant Resistance
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1. Crop
Plant Resistance to Freezing Stress
Genetic control of low-temperature tolerance
Low-temperature
tolerance is a complex quantitative character that is expressed following exposure
of plants to temperatures that approach freezing. While a large number of
studies have been conducted, there is not a general consensus on the mode of
gene action controlling the expression of this character. Recessive, additive,
partial dominant and overdominant control have all been reported for genes
conditioning low-temperature tolerance. The limited resolution of differences
in low-temperature tolerance with field and controlled-freeze tests that employ
a single minimum stress level have been at least partially responsible for some
of the contradictory conclusions reported for genetic studies. For example,
consider a hypothetical genetic study where the differences in low-temperature
tolerance between two parents are controlled by additive gene action (Figure
6). In this example, if a single minimum temperature of a controlled-freeze
test or field trial was -13oC, most of the F1 and all of
the hardy parent population would survive suggesting that there was dominance for
low-temperature tolerance. Similarly, a single minimum temperature of -17oC
would suggest that low-temperature tolerance was a recessive character. In this
example, use of both -13oC and -17oC in a
controlled-freeze test could lead to the mistaken conclusion that
low-temperature tolerance was determined by dominant gene action under low
levels of stress and recessive gene action under high levels of stress. A
series of test temperatures that identifies the LT50 (temperature at
which 50 percent of the population is killed in a controlled-freeze test) of
each population reduces the likelihood of this type of error.
In
spite of the limitations imposed by screening methods, the results of numerous
research studies (Grafius 1981, Stushnoff et al. 1984, Blum 1988, Limin and
Fowler 1991) with field crops can be summarized to provide a general picture of
the genetics of low-temperature tolerance, the range of genetic variability for
gene pools within species, the potential sources of new exploitable genetic variability,
and the expression of superior low-temperature tolerance genes introduced into
alien genetic backgrounds. The results
of these studies demonstrate the complex and multigenic nature of the mechanism
controlling low-temperature tolerance.
a) Cytoplasmic factors have been implicated in the
control of low-temperature tolerance.
However, most studies have concluded that cytoplasmic differences are of
minor importance and, if involved at all, play a secondary role in the
low-temperature tolerance control mechanism.
b) Genes conferring different levels of
low-temperature tolerance are found within and among species. Plant breeders
have been able to successfully manipulate this variability to maintain cold
hardiness levels of cultivars within established production areas. Considerable
variability in the range of low-temperature tolerance also exists among
species; however, attempts at interspecific and intergeneric transfers have
produced discouraging results.
c) Although there are examples of nonadditive gene
action, low-temperature tolerance within species is controlled mainly by genes
with additive effects. In wheat, the
identification of a dominant gene(s) affecting low-temperature tolerance that
is tightly linked to vernalization (Vrn1) (Brule-Babel and Fowler 1988, Sutka
and Snape 1989) and prostrate growth type (Roberts 1990) genes on chromosome 5A
has proven to be an important exception to the additive gene action rule.
Within the Triticeae, the genes for vernalization are found on the 4th, 5th, and
1st group chromosomes (McIntosh et at. 1998) while the 4th and 5th group
chromosomes are most commonly associated with low-temperature tolerance (Sutka
1981, Law and Jenkins 1970). Subsequent
work with barley has identified a linkage between low-temperature tolerance and
vernalization genes on chromosome 7 (Hayes et al. 1993), which is homeologous
with the 5th chromosome group in wheat. These linkages have provided valuable
phenotypic markers for the investigation of low-temperature tolerance in Gramineae.
d) Superimposed upon both quality and quantity of
low-temperature tolerance genes is the effect of cell size. Smaller cell size amplifies the expression of
low-temperature tolerance genes during the acclimation process (Limin and
Fowler 1989). In winter wheat, although
cold-acclimated cells are smaller, cell size rankings of cultivars follow a
similar order for acclimated and nonacclimated plants indicating that
differences in this character are intrinsic to the cultivars and not just
low-temperature induced. Control of cell size has been localized to the Vrn region of the group 5 chromosomes in
wheat (Limin and Fowler 2001).
e) A high degree of genetic balance or harmony is
required for full expression of genes central to the low-temperature tolerance
mechanism. The unpredictable low-temperature tolerance of artificially
synthesized ABD genome hexaploid wheat (Limin and Fowler 1982) demonstrates the
nonadditivity and asynchronous behaviour of related but unintegrated genetic
systems. In synthetic amphiploids of the Triticeae tribe there appears to be a
chromosome (gene) dosage effect that favors the expression levels of
low-temperature tolerance genes from the parent species contributing the larger
chromosome number (Limin and Fowler 1991). Observations made at this level have
lead to the suggestion that some degree of genomic integration, which would
have been accomplished by recombination and (or) mutation followed by
selection, was necessary before maximum low-temperature tolerance was achieved
in naturally occurring polyploids of the Triticum
- Aegiolops group (Limin and Fowler
1989). These complex interactions should not be unexpected, as there is
evidence that certain regulatory mechanisms, such as species specific tRNA and
mRNA promoters and interacting transcription factors, may have coevolved in
eucaryotic genomes (Watson et al. 1987). However, the need for a highly
integrated genetic system to maximize gene expression within species does not
bode well for efforts to expand gene pools for low-temperature tolerance
through interspecific and intergeneric cytogenetic introgressions or the
production of transgenics using biotechnological techniques.
f) Specific gene interactions, such as homoeoallelic
dominance or threshold effects (Limin and Fowler 1991), may play a role in the
final expression of low-temperature tolerance genes introduced into alien
genetic backgrounds. As an example, the superior low-temperature tolerance of
rye is suppressed when combined in tetraploid (Limin et al. 1985) and hexaploid
(Dvorak and Fowler 1978) wheat backgrounds. These observations once again
emphasize the difficulties that can be expected to be associated with efforts
to provide breeding programs with superior low-temperature tolerance genes by
interspecific and intergeneric transfers using cytogenetic or transformation
procedures.
Developmental
Regulation
The
genetic system that determines low-temperature tolerance can be divided into three separate components for
discussion purposes. The master switches that are integrated into the
mechanism that regulates plant development e.g., vernalization and
photoperiod. Low-temperature
tolerance genes that have been identified in conventional genetic and
cytogenetic studies. Low-temperature induced genes that have been
identified through differential screening of cold-acclimated wheat cDNA
libraries (Figure 7).
The ability to
survive cold winters and continue growth at near freezing temperatures provides
a species with a competitive advantage by lengthening the effective growing
season or positioning the plant to capitalize on favorable weather periods
during the growing season. In order to cope with this stress, plants have
evolved reversible acclimation systems that are light and temperature
regulated. In areas with long, mild winters, a day length (photoperiod),
dormancy, or low-temperature (vernalization) requirement that prevents plants
from entering the extremely cold-sensitive reproductive growth stage until the
risk of low-temperature damage has passed are the most important adaptive
mechanisms. As a result, the evolution and selection of genetic options that
permit extensive modification of temperature sensitive metabolic processes and
critical structural components is of great concern in the successful adaptation
of plants that must survive a wide range of seasonal challenges.
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Low-temperature
tolerance in cereals is dependent upon a highly integrated system of
structural, regulatory, and developmental genes. In regions with cold winters, vernalization
requirement is an important adaptive feature that delays heading by postponing
the transition from the vegetative to the reproductive phase. Similarly,
photoperiod requirement is an adaptation that allows the plant to flower at the
optimum time. Time sequence studies have shown that low-temperature gene
expression is also developmentally regulated (Fowler et al. 1996b). In this
system, transition from the vegetative to the reproductive growth stage is a
critical switch that initiates the down regulation of low-temperature induced
genes (Fowler et al. 1996a, b, Mahfoozi et al. 2001). As a result, full
expression of cold hardiness genes only occurs in the vegetative stage and
plants in the
reproductive phase have a limited ability to cold acclimate. In addition,
plants that are still in the vegetative stage have the ability to re-acclimate
following periods of exposure to warm temperatures while plants in the
reproductive phase have only a limited ability to re-acclimate.
According to the developmental theory (Fowler et al.
1999), level and duration of gene expression determine the degree of
low-temperature tolerance. The developmental genes (vernalization, photoperiod)
act as the master switch controlling the duration of expression of
low-temperature induced structural genes (Fowler et al. 1996a, b, Mahfoozi et
al. 1998) while the level of
low-temperature tolerance is determined by the length of time and degree that
the structural genes are up-regulated. Vernalization requirements allow
low-temperature genes to be expressed for a longer period of time at
temperatures in the acclimation range (Fowler et al. 1996a, b). Similarly,
photoperiod sensitivity allows plants to maintain low-temperature genes in an
up-regulated state for a longer period of time under short day compared to long
day environments (Mahfoozi et al. 2000). In both instances, the delay in the
transition from the vegetative to the reproductive stage produces increased
low-temperature tolerance that is sustained for a longer period of time. This
observation also explains why a high level of cold tolerance has not been
observed in spring habit cultivars. Because low-temperature gene expression is
only up-regulated when the plant is in the vegetative stage, the genetic
potentials of spring habit cultivars are not given an opportunity to be fully
expressed leaving the impression that the spring habit Vrn1 allele has a dominant pleiotropic effect for frost
susceptibility.
Low-temperature induced
genes
Winter cereals
produce several proteins in response to low-temperature stress. Among these
low-temperature induced proteins, the dehydrin families have received the most attention
in recent low-temperature research (e.g. Wcor 410, Wcs120, dh5, and others). As
a group, dehydrins have a wide size range, have no similarity with any enzymes
or proteins of known function, are largely hydrophilic, and accumulate to high
levels during the late stages of embryogenesis or in response to
The
low-temperature induced dehydrin gene families have been studied extensively in
wheat where they have been mapped to the group 6 chromosomes (Limin et al.
1997, Danyluk et al. 1998). Expression studies with the low-temperature induced
Wcs120 and Wcor410 dehydrin gene families indicate that, even though there are
large differences in low-temperature tolerance, similar proteins are expressed
by spring and winter-habit cultivars within species (Houde et al. 1992, Fowler
et al. 1996a, Danyluk et al. 1998). Cold hardy genotypes just produce more of
the same dehydrins than tender genotypes. This indicates a common regulatory
control in the level of expression of these structural genes in both hardy and
non-hardy genotypes suggesting that the important regulatory factors affecting
all low-temperature tolerance associated genes may not be as multigenic in
nature as once thought. Further support for this conclusion can be drawn from
the report that a single transcriptional factor has been found to regulate the
expression of several low-temperature regulated genes in Arabidopsis (Thomashow 1999).
Low-temperature tolerance genes/Master switches
Because of the
large number of chromosomes that have been shown to influence low-temperature
tolerance in conventional, non-molecular genetic studies, it has been generally
assumed that a large number of genes with small effects and complex
interactions determine the phenotypic expression of low-temperature tolerance
in cereals. However, even with the availability of molecular mapping tools it
does not appear that cause and effect relationships will be easily established.
To date, molecular mapping studies have only succeeded in locating one
low-temperature tolerance gene (designated Fr1
by Sutka and Snape 1989) that appears to be tightly linked to the vernalization
gene, vrn1, of chromosome 5A in wheat
(Galiba et al. 1995). Earlier studies had identified a gene in this region with
a dominant effect for low-temperature tolerance that was normally expressed in
association with the recessive vrn1
allele for winter growth habit in wheat (Brule-Babel and Fowler 1988). Vrn1 is homoeoallelic to locus Sh2 in barley (Hayes et al. 1993) and Sp1 in rye (Brule-Babel and Fowler
1989), both of which have been linked to genetic differences in low-temperature
tolerance.
The group-5 chromosomes carry the vernalization alleles vrn1, vrn4, and vrn3 on
chromosomes 5A, 5B, and 5D, respectively.
Substitution of each of these chromosomes from a hardy winter cultivar
into a nonhardy spring cultivar reduced cell size without affecting growth
habit (Limin and Fowler 2001). Genes on the 5th group chromosomes are also
known to affect low-temperature tolerance. The Vrn1 region of chromosome 5A and homoeologous loci in wheat and
other cereals appear to play an especially important role in determining plant
responses to stress. Low-temperature tolerance and other stress tolerance
related characters that have been associated with Vrn1 or homoeologous chromosomal regions include: antifreeze
protein accumulation (Griffith et al. 1997), sucrose accumulation (Galiba et
al. 1997), ABA accumulation (Galiba et al. 1993), unsaturated phospholipid
synthesis (DeSilva 1978), prostrate growth habit (Roberts 1990), cell size
(Limin and Fowler 2001), low-temperature tolerance associated with the Wcs120 and Wcor410 gene families (Limin et al. 1997; Danyluk et al. 1998), LT50
(Brule-Babel and Fowler 1988, Sutka and Snape 1989), field survival, flowering
time, and fructan content (Hayes et al. 1993), regulation of drought induced
ABA accumulation (Quarrie et al. 1994), and stress tolerance to several
minerals (Manyowa and Miller 1991). These seemingly complex groupings of
tightly linked genes of diverse function can also be explained by the
pleiotropic action of regulators that synchronize multi-factorial physical,
biochemical and morphological responses of integrated environmentally-induced
genetic systems, i. e., master switches.
The
function of cells requires that single genes be expressed at different levels
and that different genes be expressed at different stages of development and in
different tissues. We know this to be intuitively correct because each cell in
our body has the genetic information to produce all our body parts yet growth
and development proceeds in an orderly, regulated fashion. Similarly,
developmental regulation of low-temperature tolerance gene expression provides
for a highly integrated system that
allows for maximum biochemical and physiological efficiency. An effective
system for regulation of low-temperature tolerance responses in plants would also permit multiple use of adaptive mechanisms
thereby allowing the successful plant to maintain its competitive advantage by
optimizing its metabolic activities. The vrn1 complex and related developmental regulators on chromosome 5A
of wheat appear to be an example of this type of highly integrated system
(Fowler and Limin 1997).
Role of transcriptional factors and promoters
Genes
are expressed through the transcription of DNA sequences to produce mRNAs,
which in turn provide the messages that are translated into proteins. Gene
expression is driven by a promoter, which is a DNA sequence generally found at
the front of the protein coding region of the gene. The promoter determines
where, when, and to what extent the gene is expressed. Regulatory elements of
various kinds that act as binding sites for unique transcription factors are
found throughout these promoters. Regulatory elements can activate or repress
transcription, depending on the specific situation. The transcriptional
machinery involves several protein factors that interact within the promoter to
determine the characteristics of gene expression including where and when the
gene is expressed. Promoter sequences
found further upstream will also attract specific proteins known as
trans-acting or transcription factors, which will activate or repress the
transcription machinery in the appropriate cells.
Extrapolation
of the present limited knowledge of the genetics of cold hardening in plants
suggests that low-temperature tolerance gene expression may be controlled by
regulatory elements that act as promoter binding sites for transcription
factors that activate the gene. The known low-temperature induced genes carry
the same regulatory sequences and they are likely regulated by the same
transcriptional factors (Jaglo-Ottosen et al. 1998). The transcription factors
vary with environmental conditions and developmental stage and modify gene
expression through a combination of transcriptional activation, amplification,
repression, and integration that is difficult to predict (Wray 1998). For
example, activation of the low-temperature induced promoter of cold regulated Arabidopsis genes (cor15a) can be overridden by developmental cues (Baker et al. 1994)
suggesting that developmental genes have an important low-temperature tolerance
regulatory role in determining the level of cold-induced gene expression.
Involvement of enhancer elements as well as negative and positive regulatory
regions for transcriptional regulation in the promoter of low-temperature
induced genes have been reported (Ouellet et al. 1998). Multiple DNA binding
(repressor) proteins have been associated with promoter regions of plants grown
in warm non-acclimating conditions, but these proteins were absent from the
promoter in low-temperature acclimated plants (Vasquez-Tello et al. 1998)
indicating a temperature regulated system. These observations point to a system
of developmental and pleiotropic regulation that is responsive to temperature
cues. They also suggest developmental
genes may act as master switches that are capable of regulating the level of
expression of cold hardiness genes.
Given
the close associations between the vernalization genes and the low-temperature
genes, it is possible that vernalization and low-temperature responses are
interrelated (Fowler et al. 1996b) and the vernalization (vrn) genes located on chromosome 5A may be pleiotropic, regulating
both phenological development and the expression of low-temperature tolerance
in wheat (Brule-Babel and Fowler 1988). Molecular studies designed to
investigate these interactions have demonstrated that the regulatory influence
exerted by the vrn complex over
low-temperature induced structural gene (Wcs120 and Wcor410) expression occurs
at the transcriptional level in winter cereals (Fowler et al. 1996a). The wheat group 5 chromosomes, which carry the Vrn genes, have also been found to
regulate the expression of a least four low-temperature regulated gene families
correlated with low-temperature tolerance (Sarhan and Danyluk 1998). These same
chromosomes (particularly 5A) induce higher levels of expression in many
low-temperature induced genes that are dispersed
across all 3 wheat genomes (Limin et al. 1997, Danyluk et al. 1998) indicating a single transcriptional activator on
chromosome 5A is able to target the low-temperature induced genes. Studies have
provided further evidence for regulation of dehydrin genes by group 5
chromosomes. In fact, it has been suggested (Campbell and Close 1997) that
barley Sh2 (=Vrn1 homoeoallelic wheat series) may be a dehydrin gene with major
regulatory and developmental influence as a result of autoregulatory activity
on other dehydrin genes.
The
above observations indicate that genes located on chromosome 5A act as a master
switch that plays the role of both developmental regulator and transcriptional
activator of low-temperature induced genes located on group 6 chromosomes
(Figure 7). The master switch theory accommodates the fact that group 5
chromosomes in wheat, and their homoeologous chromosomes in barley and rye,
carry both the strongest vernalization genes and the most important
low-temperature tolerance genes (Fowler et al. 1999). It also explains why so
many of the stress and low-temperature induced characters appear to be
regulated by genes associated with vernalization genes on chromosome 5 and
accounts for the often overlooked fact that vernalization and low-temperature
acclimation have similar temperature ranges for induction.
Interspecific gene transfer and transformation
While
much is known about plant low-temperature response, the maximum cold hardiness potential
of most crops has reached a stubborn plateau that has not been breached for
decades. In fact, all the efforts of modern science have been unable to produce
the super hardy cultivars needed to expand winter crop production into regions
requiring a level of cultivar low-temperature tolerance superior to that found
in the land races selected by early farmers. The search
for superior low-temperature tolerance genes has been expanded to include
attempts at interspecific and intergeneric transfers. There are considerable
differences in the maximum low-temperature tolerances found in different winter
cereals (Fowler et al. 1997, Fowler and Carles 1979, Limin and Fowler 1981) and
the possibility that genes can be transferred between species to increase the
genetic variability available to winter cereal breeding programs has been
explored. However, these attempts have
done little more than demonstrate the difficulties that must be overcome before
the full potential of superior species-specific cold-tolerance gene expression
can be captured through interspecific gene transfers in plant breeding
programs.
These observations indicate that, before we can
successfully exploit alien genetic variability for low-temperature tolerance,
we must first acquire a greater understanding of the complex genetic mechanisms
that plants have evolved for the efficient integration of low-temperature
responses into the daily processes of survival, growth, and reproduction. As emphasized
earlier, just about every morphological, physiological, and biochemical
characteristic that can be measured in the plant changes during low-temperature
acclimation. This observation in itself
suggests that low-temperature acclimation involves a large number of genes. Threshold induction temperatures, time-temperature
relationships for acclimation and deacclimation, effectiveness of regulators,
morphological adjustments to changes in light and temperature, and factors that
influence the plants transition from the vegetative to the reproductive phase
all appear to have an important influence on low-temperature gene regulation in
this system. Clearly, low-temperature tolerance gene expression is influenced
not only by environment, but also by the pleiotropic effects of other genes. Until biotechnology tools became
available it was difficult to separate the genes responsible for
low-temperature acclimation and cold tolerance from those associated with metabolic
adjustments to low temperature. Molecular genetics has provided researchers
with the ability to separate out cause-and-effect relationships and an
opportunity to explain some of the apparent contradictions that exist in the
low-temperature tolerance literature. This area
has been progressing rapidly and we have moved beyond molecular mapping and
protein accumulation studies into functional genomics and a more in depth
consideration of cause-and-effect relationships with an overall objective of
achieving a clearer understanding of the genetic cascade controlling gene
expression during acclimation. These investigations have
started with a search for the principle mechanisms that winter cereals use to
regulate low-temperature gene expression.
2. Breeding for Resistance to Freezing
Stress.
A large research effort has been concentrated
on solving the mysteries of low-temperature tolerance in plants. Some progress
has been made, but the impact of this research on plant breeding has been
minimal. While plant breeders have successfully maintained low-temperature
tolerance levels of crop plants within established production areas, attempts
to produce super-hardy cultivars using the existing genetic variability within
species have met with limited success in this century. The primary reasons for
this lack of success includes. a) Exploitable genetic variability for
low-temperature tolerance has been largely exhausted within the gene pools of
most species. b) A large number of genes with small effects and complex
interactions are assumed to determine the phenotypic expression of this
character making selection difficult. Although most studies indicate that
low-temperature tolerance within species is controlled mainly by genes with
additive effects; recessive, partial dominant, and overdominant control have
also been reported. c) Current
methodology for measuring low temperature tolerance gives poor resolution of
small phenotypic differences. d) Measures of
low-temperature tolerance lack the precision for single plant analysis and many
are destructive making selection procedures complicated. e) Poor expression of
low-temperature tolerance genes in alien genetic backgrounds has prevented the
expansion of gene pools through interspecific and intergeneric transfers e.g.,
the superior low-temperature tolerance of rye is suppressed in a wheat
background.
The
ability to acclimate or avoid low-temperature stress varies among species and
stages of crop growth. As a consequence, each plant-breeding situation is
unique and it is impossible to design a single approach that will satisfy the
requirements of all low-temperature tolerance breeding programs. For example,
the criteria used to select for low-temperature tolerance are different for
actively growing plants that must survive short periods of late spring frost
compared to plants that must withstand exposure to long periods of below
freezing winter temperatures. When pure, water can supercool or remain unfrozen
to its homogeneous nucleation point of approximately -40oC and, when
environmental sources of ice nucleation are absent, supercooling is an
important mechanism that determines the level of damage in actively growing
plants. Stage of plant development and physical barriers such as the stem
nodes, rachis and rachilla that slow ice nucleation and the spread of ice
crystals are some of the factors that determine the impact of supercooling on
the low-temperature tolerance of tender tissue. Supercooling is also an
important survival mechanism for certain cells in woody plants that must
survive very low winter temperatures and thermal analysis to detect exotherms
resulting from nucleation of supercooled cells have been used to identify
differences in the low-temperature tolerance in these species. However, while
supercooling is an important low-temperature tolerance mechanism in these
instances, winter survival in most plant tissues depends on an ability to
accommodate freezing temperatures and prevent damage to the cytoplasm by slowly
forming ice crystals in intercellular spaces. Consequently, slow freezing rates
and ice nucleation to avoid supercooling are important considerations when
conducting freezing tests on most plants that have the ability to
low-temperature acclimate. These few examples have been given to emphasize the
need for plant breeders to have a thorough understanding of the low-temperature
tolerance problems they are addressing. This short introduction also raises a
few basic questions that need to be answered to ensure the effectiveness of a
low-temperature tolerance-breeding program.
1) What are the climatic conditions of the
region that the breeding program targets?
2) What are the critical stress periods for
the species under selection?
3) What low-temperature tolerance or avoidance
mechanisms are employed and what are the sources of genetic variability in the
species under selection?
4) What are the most appropriate methods for
identifying superior individuals in breeding populations? Can artificial
environments that accurately reproduce the critical stresses experienced in
nature be created to facilitate selection programs? Are there low-temperature
tolerance prediction tests available that have been field validated for the
target region? Do the prediction tests provide measures of field reaction that
are accurate enough to be effective as selection tools?
5) Are the necessary resources available to
properly implement the selected breeding strategy?
A detailed consideration of breeding
strategies for different species in different environments is beyond the scope
of this paper. Consequently, the remainder of this discussion will focus
primarily on cereal crops as an example of the complex problems encountered
when breeding for low-temperature tolerance.
Prediction and controlled-freeze tests
The limitations inherent in field survival
trials have provided a continuing incentive for researchers to search for
rapid, more efficient methods of predicting low-temperature tolerance. Ideally, a low-temperature tolerance screen
should be simple, rapid, repeatable, and non-destructive. It should also provide an accurate, precise
measure of cold hardiness potential on single plants. The many changes that occur in morphological,
biochemical, and physiological characters during low-temperature acclimation
have provided fertile ground in the search for prediction tests and differences
in several characters, such as plant erectness in winter cereals, tissue water
content, cell size, etc., have been shown to be highly correlated with freezing
tolerance (Fowler et al. 1981, Limin and Fowler 2000). Use of these characters as screens satisfies
many of the criteria listed above.
However, while prediction tests have been used effectively for preliminary
low-temperature tolerance selection in breeding programs, high experimental
errors limit their usefulness in identifying small differences that are of
practical concern to plant breeders, especially where breeding programs target
high stress regions.
Controlled-freeze tests on plants that have
been cold acclimated in artificial environments are routinely used to measure
low-temperature tolerance in physiological and genetic studies. As indicated
earlier, rates of freezing and thawing and
recovery conditions must reflect the conditions that the plant will experience
in nature for controlled-freeze tests to have predictive value. Well designed
controlled-freeze tests that employ a single minimum temperature provide a
resolution of low-temperature tolerance differences similar to field trials and
the same level of caution must be exercised in interpreting results from these
two methods. Poor reproduction of cold
acclimation rates in repeat experiments makes selection of critical minimum
temperatures in controlled-freeze tests almost as difficult as the
identification of field sites that yield a high frequency of injury in the
target stress range. A series of test temperatures that establish the LT50
(minimum temperature that 50 percent of plants survive) of populations reduces
the difficulties associated with identifying critical selection temperatures.
Measures of LT50 provide the highest precision and heritability of
all low-temperature tolerance prediction tests (Fowler et al. 1981) but, they
require a sample of plants from a homogeneous population that can be tested
using a series of test temperatures or times. This restricts LT50
measurements to pure lines and limits their usefulness in plant breeding
programs that are normally dealing with segregating populations. Increased
availability of practical methods for doubled haploid production and asexual
propagation, which provide means for quickly
producing homogeneous populations, expand the
opportunities to use LT50 estimates for selection in plant breeding
programs.
The cold
tolerance of winter cereal crowns is reduced by prolonged exposure to
sub-lethal temperatures and, as a consequence, both temperature and exposure
time are important variables in controlled-freeze test procedures. The
predicable relationship between time and temperature (Equation 1) has meant
that survival time at a constant below freezing temperature (Figure 5; in Impact of Cold Stress) can also be used to
identify differences in plant low-temperature tolerance (Thomas et al. 1988).
When combined with the use of controlled
environments to simulate conditions for low-temperature acclimation,
controlled-freeze-tests provide for greater flexibility in the timing of
experiments than field trials. However, while controlled environments should
theoretically allow for more rigid control of experimental conditions,
comparative studies have shown that field trials usually provide more
repeatable results and have lower experimental errors (Fowler et al. 1981).
Consequently, regardless of available resources, field screens are routinely
used to provide the final measure of winter survival potential in most breeding
programs.
Marker Assisted
Selection
The selection for complex genetic traits, such
as low-temperature tolerance, can be simplified in plant breeding programs when
linked qualitative markers are identified. In wheat, the identification of a
dominant gene(s) affecting low-temperature tolerance that is closely associated
with the vernalization (vrn1)
(Brule-Babel and Fowler 1988, Sutka and Snape 1989) and prostrate growth habit
(Roberts 1990) genes on chromosome 5A has proven to be an important exception
to the additive gene action rule. Within the Triticeae, the genes for
vernalization are found on the 4th, 5th, and 1st group chromosomes (McIntosh et
at. 1998) while the 4th and 5th group chromosomes most commonly associated with
low-temperature tolerance (Sutka 1981, Law and Jenkins 1970). Subsequent work
with barley has identified a linkage between low-temperature tolerance and
vernalization genes on chromosome 7 (Hayes et al. 1993), which is homeologous
with the 5th chromosome group in wheat. These linkages have provided valuable
phenotypic markers for the investigation of low-temperature tolerance in
Gramineae.
The developing science of biotechnology has
provided an ever-increasing number of molecular markers that can be used to
assist selection in plant breeding programs. However, marker assisted selection
requires a detailed linkage map and there is still a dearth of markers for
low-temperature tolerance genes. For example,
molecular markers in the cereals have been limited to the chromosome regions
associated with the homoeoallelic genes Vrn1
in wheat (Galiba et al. 1995), Sp1 in
rye (Plaschke et al. 1993) and Sh2 in
barley (Laurie et al. 1995), regions that were previously known to have a
significant influence on plant cold hardiness. However, with as many as 15 out
of 21 chromosomes in wheat having an influence on low-temperature tolerance
(Stushnoff et al. 1984), there is still considerable work to be done before the
full potential of mapping assisted selection can be realized for this character
in most cereal breeding programs.
Advances in biotechnology have provided
additional opportunities for plant breeders to expand their attack on the
winter-hardiness barrier that has frustrated them for so long. The large number of
cold-induced genes associated with low-temperature tolerance and their products
that have been isolated in recent years have provided us with a snapshot of
these opportunities. For example, antibodies raised against WCS120 proteins produced by
low-temperature induced Triticeae genes (Houde et al. 1992) have provided an
immediate practical opportunity to simplify cold hardiness selection procedures
in breeding programs. Low-temperature induced expression of this gene family in
wheat, as measured by densitometry scanning of Western blots, closely follows
changes in LT50 thereby providing a direct means of quantifying
phenotypic differences in low-temperature tolerance of cereals (Fowler et al.
1996a).
The rapidly expanding area of functional
genomics offers even larger opportunities for the understanding and
manipulation of complex genetic systems. As expected in the Triticeae, these investigations have focused on the
major low-temperature tolerance genes associated with the vernalization loci.
Identification of the main genetic pathways responsible for low-temperature
gene expression in one species should provide a guide to charting the routes
and establishing the regulation of low-temperature gene expression in other
species, especially those that are closely related. Molecular procedures that
facilitate the analyses of genome structure and organization for purposes of
comparative molecular mapping should allow us to further expand our ability to
simultaneously exploit discoveries in a wide range of species. Transformation studies have demonstrated that some of
the low-temperature induced genes and their transcriptional factors may be
exploited to improve the low-temperature tolerance of tender genotypes
(McKersie and Bowley 1998, Kasuga et al. 1999, Thomashow 1999). Certainly, we now
have the tools to greatly improve our ability to breed for low-temperature
tolerance in plants. However, biotechnology has also brought the pharmaceutical
model to plant breeding, which includes patents and expanded intellectual
property rights that have greatly restricted plant breeders' freedom to
operate. These changes have not been unopposed and the full potential for biotechnology
to revolutionize the plant breeding world will have to wait for the new order
to evolve a more widely accepted and workable plant breeding environment.
Field Selection
In spite of the opportunities offered by
cold-hardiness indicators, controlled-freeze testing, and molecular markers,
most of today's winter cereal breeding programs still rely heavily on field
screening as the final measure of plant winter survival potential. Field-testing is simple, inexpensive, and
does not require access to specialized facilities or co-operating programs with
conflicting priorities. Unfortunately, the opportunity for selection in field
trials only occurs once a year, winters that provide critical selection
temperatures usually occur infrequently, and non-uniform stress levels due to
variable snow cover and other environmental factors often result in high
experimental errors that reduce within-trial selection efficiency.
Where adequate resources are available, a
number of measures can be implemented to increase the opportunity for effective
field selection for low-temperature tolerance. The frequency of test winters and degree of stress can often be
increased by growing trials at or outside the margin of the target production
region, thereby increasing the opportunity for selection and the level of
selection pressure (Fowler et al. 1993). Where low-temperature responses have been
characterized in detail, computer simulation models and historical weather
records may be used to assist in the identification of sites with a high
probability of low-temperature stress in the desired range (Savdie et al.
1991). Methods that control variation in snow cover may be employed to reduce
experimental errors and increase the probability of differential injury among
breeding lines of plant species with critical meristems near the soil surface.
The inclusion of cultivars with known low-temperature tolerance as reference
plots to monitor stress levels in trials and the use of a moving average in
data analyses provide further opportunities to a) adjust for non-uniform levels
of stress, b) maximize the information gleaned from trials with low levels of
differential injury, and c) pool results from different trials. The Field
Survival Index (FSI) is an example of how this approach has been used to obtain
objective measures of low-temperature tolerance in wheat and rye (Fowler et al.
1981).
Developmental Regulation
of Low-Temperature Tolerance
Because perennials survive for several years,
the staging and regulation of their vegetative/reproductive changes are much
more complex than are those of summer and winter annuals. In many cases both
vegetative and fully functional reproductive structures must survive the
winter, which greatly increases the range of injuries that affect different
parts of the plant and the complexity of breeding for low-temperature tolerance
in perennials. The biochemical, physiological, and morphological changes
associated with low-temperature tolerance clearly interfere with active growth
and to be successful a plant must be programmed to recognize and respond to the
environmental cues that signal seasonal changes. For these reasons,
low-temperature tolerance in perennials normally follows a yearly pattern of
environmental cues that include photoperiod and temperature changes. These cues
permit the plant to anticipate periods of stress while optimizing its ability
to take advantage of favourable periods for growth and reproduction.
Consequently, a large part of the plant breeder's task involves selection for the complex
genetic mechanisms that plants have evolved for the efficient integration of
low-temperature responses into the daily processes of survival, growth, and
reproduction.
In
general, the longer the plant life cycle the more complex and poorly understood
is the low-temperature response mechanism. However, while tree breeders have
some of the longest living plants to deal with, they have been much more
conscious of the linkage between phenological development and low-temperature
response in their selection programs than breeders working on species with
shorter life cycles. For example, cereal breeders normally restrict the
connection between phenological development and low-temperature tolerance to a
consideration of spring and winter growth habit. However, there is strong and
growing evidence of close links between the up-regulation of low-temperature
tolerance genes and vernalization requirement and photoperiod sensitivity in
cereals. For example, cereals with a vernalization requirement or short day photoperiod
sensitivity acclimate upon exposure to low-temperature until the point of
transition to the reproductive stage after which there is a loss in
low-temperature tolerance (Fowler et al. 1996a, Mahfoozi et al. 2000).
Consequently, the point of transition to the reproductive stage is pivotal in
the expression of low-temperature tolerance genes. This interaction also makes
all low-temperature tolerance associated characters or genes appear to be
associated with developmental genes (Fowler et al. 1999) and explains the
pleiotropic effects (growth habit and low-temperature tolerance) attributed to
developmental genes like vrn1 in
wheat (Brule-Babel and Fowler 1988, Sutka and Snape 1989, Roberts 1990).
The
linkage of low-temperature tolerance expression to phenological development
adapts the plant to the environment for which it was selected or in which it
evolved. For example, a high level of low-temperature tolerance is no longer
required after the onset of warm conditions in the spring when rapid growth and
reproduction begin. Consequently, satisfaction of dormancy, vernalization, and
photoperiod requirement results in a decline in low-temperature tolerance of
over-wintering plants. In fact, for
species adapted to regions with long, mild winters, a high level of freezing
tolerance is often less important than a rigorous photoperiod, dormancy, or
vernalization requirement that prevents plants from entering the extremely
cold-sensitive reproductive growth stage until the risk of low-temperature
damage has passed. Consequently, the
evolution of, and selection for, genetic options that permit extensive
modification of thermosensitive metabolic processes and critical structural
components should not come as a surprise, especially in winter annual and
perennial plants that must adapt to a wide range of seasonal challenges. It is this genetic system and its regulation
and complex interaction with the environment that makes breeding for
low-temperature tolerance a continuing challenge.
3. Crop
Management (See also Cold Protection)
The
availability of highly adapted cultivars with superior low-temperature
tolerance is considered a prerequisite for successful crop production in many areas
of the world. However, even the hardiest cultivars can be damaged by
low-temperature if proper attention is not paid to management practices,
especially in regions of marginal adaptation. Conversely, even the best
management systems will not be successful unless highly adapted cultivars are
available for production. In other words, genotype establishes crop potential
and proper management allows the farmer/grower to optimize this potential.
The role of crop management
Plant
survival and economic production on fringes of regions of adaptation for many
species has required the use of low-temperature avoidance systems and refined
management techniques. For example, the root system is the most susceptible
part of the plant to low temperature damage. Use of hardy rootstocks and
protective mulches are examples of how orchard managers and gardeners have
exploited this knowledge to over winter tender genetic stocks of economic and
ornamental value. Field location is also a particularly important consideration
in successful orchard and garden management. Protective techniques that include
the use of windbreaks, snow trapping, mulches, and transparent covers have a
moderating effect that help plants avoid low temperature extremes. In addition
to providing insulation, protective covers help the plant avoid large
temperature fluctuations and stresses due to alternate freezing and thawing.
Similarly, the shading effects of snow cover and other barriers protect
evergreens from winter burn and tree trunks from sun scald and desiccation
injury. Heaters, sprinklers, and wind machines are regularly used to protect
tender plant tissues from damage due to temperatures in the -2 to -5oC
range in high value commercial orchards. Similar methods have been investigated
for mid winter protection in regions where temperatures are much colder, but
success under these conditions requires sophisticated weather prediction and
crop monitoring systems.
Light
is important as an energy source and an environmental cue. Consequently, the timing
and method of pruning and training are important management tools that affect
winter hardiness through their influence on shading, the initiation and
differentiation of vegetative and floral meristem, and the general health of
the plant. Water supply affects growth, tissue water content, and the cooling
rate of the immediate environment. Excessive water on frozen soils can also
result in damage due to ice encasement.
Diseases
can affect the health of the plant and its general ability to tolerate low-temperature
stress. Similarly, proper nutrient balance is essential for the production of
healthy vigorous plants and deficiencies can be expected to have an adverse
effect on the low-temperature tolerance. Deficiencies and excesses of a number
of elements have been studied with mixed results that are probably related to
the size of the deficiency or excess of the element under study. High levels of
salts associated with soil salinity have been shown to reduce the winter
hardiness of some plants. Excessive nitrogen fertilization that stimulates
luxury growth prior to plant low-temperature acclimation has been reported to
prevent full expression of cold-hardiness potential while corrections of
deficiencies in phosphorous and potassium are generally associated with
increased cold hardiness.
As
emphasized earlier, plants have evolved mechanisms that allow them to
anticipate seasonal weather changes. Therefore, successful management of winter
crops requires an understanding of plant development, growth cycles and the
mechanisms used to survive periods of low-temperature stress. An understanding
of how plants respond to low-temperature stress at different growth stages can
also assist in the assessment of crop condition and production potential
throughout the growing season.
Management can succeed where genetics fails
The recent expansion of the western Canadian winter
wheat production area is an example of how management practices that modify the
plant microenvironment can be used to reduce the limitations imposed by low
temperature (see http://www.usask.ca/agriculture/plantsci/winter_cereals
). The lack of super-hardy cultivars limited winter wheat production in this region
when conventional production systems were employed, i.e., seeding into a tilled
seedbed. However, field studies demonstrated that only 8 to 10 cm of unpacked
snow was sufficient to maintain soil temperatures above the minimum survival
temperatures of critical crown tissue throughout the winter. This knowledge
stimulated efforts to develop snow-trapping techniques and it was quickly shown
that direct seeding into standing stubble (no-till) from a previous crop
provided an ideal means for maintaining uniform snow cover of this depth.
Success with this management system has allowed farmers to extend the winter
wheat production on the North American Great Plains north and east from a small
area along the Canada/USA border in southern
The
production of no-till winter wheat is straightforward and simple, but it
requires the use of management practices different from those commonly employed
by most prairie farmers. In western Canada, no-till winter wheat is seeded into
standing stubble from a previous crop between the end of August and the middle
of September, and harvested early the following August. Because of the short
growing season in this region and the requirement for standing stubble, winter
wheat is better suited to rotations that include early maturing spring crops.
Consequently, winter wheat growers must start planning for next year's crop
well before this year's crop is harvested. This necessitates a production
schedule that considers management decisions over a period of two or more crop
years. This usually means a reassessment of the entire crop rotation and
harvesting operation. Rotations have to be planned to include early maturing
crops, thereby ensuring that standing stubble is available at an early date.
Direct seeding equipment is a necessary component of this package and most
farmers have been reluctant to invest in seeding equipment solely for the
production of winter wheat. As a consequence, the successful adoption of winter
wheat has been closely linked to the level of acceptance of no-till spring
crop-production systems. This linkage also means that seeding equipment must be
selected carefully to ensure it provides the flexibility required to direct
seed a wide variety of crops under a wide range of conditions.
Plant establishment is the critical step in
the no-till winter wheat production system. Successful plant establishment has
required the acquisition of special management skills and the placement of a
high priority on stubble management and the seeding operation. Seeding date and
depth both have a large influence on the degree of success that can be achieved
in the production of winter wheat in western
Many of the soils in western
The results of field trials have demonstrated
that plant-available soil-nitrogen level does not normally affect the
winter-hardiness potential of wheat unless the nitrogen has been applied in the
seed-row at the time of planting. Urea (46-0-0) and ammonium nitrate (34-0-0)
are the two most common N forms that are seed placed and both can reduce
seedling number and size, especially when the soil is dry at seeding. The
effect of seed-placed urea is more insidious and damage is usually less of a
problem with ammonium nitrate. Also, the concentration of fertilizer
immediately adjacent to the seed is dependent upon fertilizer rate, row spacing
of the drill, and seed row opener design. High rates of phosphate fertilizer
will not offset the effect that seed-row banded nitrogen fertilizer has in
reducing winter hardiness. Placement of nitrogen fertilizers a minimum distance
of one 2.5 cm from the seed will minimize seedling damage and this has lead to
considerable recent research on "side-banding" seeding equipment and
other fertilizer placement options.
The management practices considered above all have a
direct influence on plant establishment and the ability of a cultivar to
realize its full winter hardiness potential.
These variables are all under the direct control of the producer
emphasising the important role that management skills play in the successful
production of winter wheat in western
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