EVALUATION OF ΑLPHA-AMYLASE ACTIVITY AND FALLING NUMBER FOR
SOFT WHITE AND SOFT RED WHEAT VARIETIES ADAPTED TO MICHIGAN
By
Chong Yu

A THESIS
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of
MASTER OF SCIENCE
Plant Breeding, Genetics and Biotechnology-Crop and Soil
2012

ABSTRACT
EVALUATION OF ΑLPHA-AMYLASE ACTIVITY AND FALLING NUMBER FOR SOFT
WHITE AND SOFT RED WHEAT VARIETIES ADAPTED TO MICHIGAN
By
Chong Yu
Pre-Harvest Sprouting (PHS) is a major threat to Michigan soft white wheat, and recent
epidemics were experienced in 2008 and 2009. Alpha-amylase is an important component of
PHS and the falling number (FN) test is used by industry to identify sprouted wheat that is
unacceptable for various food products. The objectives of this study were to evaluate wheat
cultivars adapted to Michigan for the activity of α-amylase and FN values around physiological
maturity (PM) in natural conditions and multiple artificial PHS induction treatments. Twentyfour soft winter wheat genotypes with varying levels of susceptibility to PHS were planted in αlattice designs in two locations in three years. Spikes were collected three days before PM, at
PM, and three days post PM. Treated samples were freeze-dried, processed and evaluated for αamylase activity and FN values. Genetic differences existed for both responses at all maturity
categories in the absence of PHS induction. A clear trend was observed in the reduction of αamylase and the increase in FN during the maturation in non-PHS conditions. For the study of
PHS induction methods, genetic differences among 24 cultivars existed for both responses at PM
in all misting treatments. After-ripening period had a significant effect on breaking dormancy for
some lines, and an effective protocol for PHS screening at MSU was identified. ‘Jupiter’,
‘Lowell’ and ‘Caledonia’ were highly susceptible, while ‘MSU E5024’ and AgriPro ‘W1062’
were two white wheat cultivars that showed consistent tolerance to PHS in this study.
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ACKNOWLEDGMENTS
I would first like to thank my major advisor, Dr. Janet Lewis for her careful instructions
and generous support. I learned a lot from her work attitude, professional communication and
team management.
I would also like to thank my committee members, Dr. Russ Freed, Dr. Perry Ng and Dr.
James Kelly. Dr. Freed gave me tremendous help on my thesis writing and professional guidance.
Dr. Ng is a brilliant cereal chemist and his research philosophy and abundant experience
impacted me greatly on my graduate study. I also appreciate Dr. Kelly’s insightful suggestions,
which enable my thinking and writing more critically and logistically.
I appreciate Dr. Edward Souza for his encouragement and support in the academic
communication.
Thanks to Lee Siler, Randy Lorenz and Sue Hammer for your great help in the field and
greenhouse work. It was really nice to work with you.
Thanks to MSU sugar beet breeding program for generous help to provide the facility and
space in my research. I also want to bring special thanks to Dave Main for his help in the
freezing dry process.
Finally, I want to thank other graduate students, Swasti Mishra, Yuanjie Su, Esteban
Falconi and the undergraduate student who have worked in wheat breeding program. I have been
experienced wonderful and joyful time with you and I appreciate your help and advice.

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TABLE OF CONTENTS

LIST OF TABLES ......................................................................................................................... vi
LIST OF FIGURES ..................................................................................................................... viii
CHAPTER I .................................................................................................................................... 1
GENERAL INTRODUCTION ....................................................................................................... 1
INTRODUCTION ...................................................................................................................... 1
The Importance of Wheat: from World to Michigan .............................................................. 1
Losses from Pre-harvest Sprouting ......................................................................................... 2
Evaluation of Pre-Harvest Sprouting ...................................................................................... 3
Objectives of the Study ........................................................................................................... 3
REVIEW OF LITERATURE ..................................................................................................... 5
Wheat Domestication and Breeding ....................................................................................... 5
Wheat Classification ............................................................................................................... 6
Seed Morphology .................................................................................................................... 6
Plant Development and Physiological Maturity ..................................................................... 7
Seed Dormancy ....................................................................................................................... 8
Enzymes in Seed Development .............................................................................................. 9
Hormones in Seed Development .......................................................................................... 10
Process of PHS ...................................................................................................................... 11
Environment Effects on PHS ................................................................................................ 11
PHS on Wheat and Product Quality...................................................................................... 12
PHS Assessment ................................................................................................................... 13
Physical Control of PHS ....................................................................................................... 15
Genetic Control of PHS ........................................................................................................ 16
LITERATURE CITED ............................................................................................................. 18
CHAPTER II ................................................................................................................................. 26
EVALUATION OF ΑLPHA-AMYLASE ACTIVITY AND FALLING NUMBER FOR SOFT
WHITE AND SOFT RED WHEAT VARIETIES ADAPTED TO MICHIGAN IN NATURAL
CONDITONS ............................................................................................................................... 26
ABSTRACT .............................................................................................................................. 26
INTRODUCTION .................................................................................................................... 27
MATERIALS AND METHODS .............................................................................................. 30
Plant Materials ...................................................................................................................... 30
Field Design .......................................................................................................................... 31
Physiological Maturity (PM) Determination and Harvesting ............................................... 31
Post-Harvest Processing........................................................................................................ 31
Determination of α-Amylase Activity .................................................................................. 32
Determination of FN ............................................................................................................. 33
Statistical Analyses ............................................................................................................... 34
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RESULTS ................................................................................................................................. 34
Planting Time, Flowering Time and PM Time ..................................................................... 34
Alpha-amylase Activity Test and FN test ............................................................................. 34
Genotype Comparisons and Relative Grouping at PM-3 ..................................................... 35
Correlation between α-Amylase Activity and FN Values .................................................... 36
DISCUSSION ........................................................................................................................... 36
LITERATURE CITED ............................................................................................................. 49
CHAPTER III ............................................................................................................................... 49
EVALUATION OF ΑLPHA-AMYLASE ACTIVITY AND FALLING NUMBER FOR SOFT
WHITE AND SOFT RED WHEAT VARIETIES ADAPTED TO MICHIGAN FOLLOWING
MULTIPLE PHS INDUCTION TREATMENTS ........................................................................ 54
ABSTRACT .............................................................................................................................. 54
INTRODUCTION .................................................................................................................... 55
MATERIAL AND METHODS ................................................................................................ 58
Plant Materials, Filed Design and Plot Sampling ................................................................. 58
Post-harvest Treatment ......................................................................................................... 58
Sample processing, α-amylase and Falling Number measurements ..................................... 59
Statistical Analysis ................................................................................................................ 59
RESULTS ................................................................................................................................. 60
PHS Severity vs. Maturity Categories: PM-3, PM, PM+3 ................................................... 60
Moderate Misting Treatments at PM in 2010 and 2011 ....................................................... 61
PHS Severity, ARP and Misting Treatments: Within Genotype Comparisons .................... 61
PHS Severity, ARP and Misting Treatments: Within Treatment Comparisons ................... 63
Genotype Ranking ................................................................................................................ 64
Scatter Plot of the Experimental Data between α-Amylase Activity and FN Values .......... 65
DISCUSSION ........................................................................................................................... 65
SUMMARY .............................................................................................................................. 69
LITERATURE CITED ............................................................................................................. 97

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LIST OF TABLES

Table 1.2. Field location information by year, soil type, fertilizer & herbicide application and
plot size for the trials..................................................................................................................... 40
Table 2.1. Number of days between planting date to PM, flowering date and PM date for
different locations in 2010 and 2011 ............................................................................................ 41
Table 2.2. Absorbance of a series of diluted standard solutions (p-nitrophenol in 1% tri-sodium
phosphate) at 400 nm and corresponding EmM value .................................................................. 41
Table 2.3. The α-amylase activities for 24 wheat varieties at PM-3 in the absence of PHS were
ranked from smallest to the largest; the FN values were ranked from largest to the smallest...... 42
Table 3.1. Post-harvesting treatment design of trial 2011 ............................................................ 71
Table 3.2 Genotypes were shown in each treatment category when the pair wise comparisons
between severe misting (20 s per 2 min, last for 72 h) and non-misting were significant (p<0.05)
....................................................................................................................................................... 71
Table 3.3 Genotypes were shown in each treatment category when the pair wise comparisons
between moderate misting (45 min per 6 h, last for 48 h) and non-misting were significant
(p<0.05) ......................................................................................................................................... 72
Table 3.4 Genotypes were shown in each treatment category when the pair wise comparisons
between the group with ARP treatment and without ARP treatment were significant (p<0.05).. 72
Table 3.5. The α-amylase activities and FN values for 12 white wheat varieties at PM for misting
and ARP treatment, were ranked from smallest to the largest; the FN values were ranked from
largest to the smallest .................................................................................................................... 73
Table 3.6. The α-amylase activities and FN values for 12 red wheat varieties at PM for misting
and ARP treatment, were ranked from smallest to the largest; the FN values were ranked from
largest to the smallest .................................................................................................................... 74
Table 3.7. The summary of hit times and remarks for the genotypes listed in the top-three group
of the percentile rank for white wheat in table 3.5 ....................................................................... 75
Table 3.8. The summary of hit times and remarks for the genotypes listed in the top-three group
of the percentile rank for red wheat in table 3.6 ........................................................................... 75
Table 3.9. The summary of hit times and remarks for the genotypes listed in the bottom-three
group of the percentile rank for white wheat in table 3.5 ............................................................. 76

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Table 3.10. The summary of hit times and remarks for the genotypes listed in the bottom-three
group of the percentile rank for red wheat in table 3.6 ................................................................. 76
Table 3.11. Pedigree and awn information for white wheat ......................................................... 77
Table 3.12. Pedigree and awn information for red wheat ............................................................. 78

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LIST OF FIGURES

Figure 2.1. The α-amylase activity (CU/g) at three days before PM (PM-3), PM and three days
after PM (PM+3) for 24 Michigan varieties, using grain frozen immediately after harvesting.
LSD values are included at each time point (p<0.05). .................................................................. 43
Figure 2.2. Falling Number values at three days before PM (PM-3), PM and three days after PM
(PM+3) for 24 Michigan varieties, using grain frozen immediately after harvesting. LSD values
are included at each time point (p<0.05). ..................................................................................... 44
Figure 2.3. The α-amylase activity (CU/g) at three days before PM (PM-3), PM and three days
after PM (PM+3) for 24 Michigan varieties, using grain frozen immediately after harvesting.
LSD values are included at each time point (p<0.05). Red color represents red wheat lines, and
white color represents white wheat lines. ..................................................................................... 45
Figure 2.4. Falling Number values (s) at three days before PM (PM-3), PM and three days after
PM (PM+3) for 24 Michigan varieties, using grain frozen immediately after harvesting. LSD
values are included at each time point (p<0.05). Red color represents red wheat lines, and white
color represents white wheat lines. ............................................................................................... 46
Figure 2.5. Correlation of α-amylase activity to FN for 24 wheat varieties................................. 47
Figure 2.6. Correlation of α-amylase activity to FN for 12 red wheat varieties. ......................... 47
Figure 2.7. Correlation of α-amylase activity to FN for 12 white wheat varieties. ...................... 48
Figure 3.1. The α-amylase activity (CU/g) at three days before PM (PM-3), PM and three days
after PM (PM+3) for 24 MI cultivars with artificial misting treatment immediately after
harvesting in 2010. ........................................................................................................................ 79
Figure 3.2. The Falling Number values (s) at three days before PM (PM-3), PM and three days
after PM (PM+3) for 24 MI cultivars with artificial misting treatment immediately after
harvesting in 2010. ........................................................................................................................ 80
Figure 3.3. The α-amylase activity (CU/g) at three days before PM (PM-3), PM and three days
after PM (PM+3) for 24 MI cultivars with non-misting treatment (ambient control) immediately
after harvesting in 2010. ............................................................................................................... 81
Figure 3.4. The Falling Number values (s) at three days before PM (PM-3), PM and three days
after PM (PM+3) for 24 MI cultivars with non-misting treatment (ambient control) immediately
after harvesting in 2010. ............................................................................................................... 82
Figure 3.5. The α-amylase activity (CU/g) of genotype ‘Lowell’ at PM for misting and nonmisting test in 2010. The letter indicated the significantly different group, at p<0.05 criteria. ... 83
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Figure 3.6. The Falling Number values (s) of genotype ‘Lowell’ and ‘Jupiter’ at PM for misting
and non-misting test in 2010 and 2011. The letter indicated the significantly different group, at
p<0.05 criteria. .............................................................................................................................. 83
Figure 3.7. The α-amylase activity (CU/g) for 24 MI cultivars with severe misting treatment (20
s per 2 min for 72 h), after five days ARP storage in 2011. Left side showed red cultivars and
right side showed white cultivars. In each part, the cultivars are ordered and LSDs are provided
(p<0.05). ........................................................................................................................................ 84
Figure 3.8. The α-amylase activity (CU/g) for 24 MI cultivars with severe misting treatment (20
s per 2 min for 72 h), immediately after harvesting in 2011. Left side showed red cultivars and
right side showed white cultivars. In each part, the cultivars are ordered and LSDs are provided
(p<0.05). ........................................................................................................................................ 85
Figure 3.10. The α-amylase activity (CU/g) for 24 MI cultivars with moderate misting treatment
(45 min per 6 h for 48 h), immediately after harvesting in 2011. Left side showed red cultivars
and right side showed white cultivars. In each part, the cultivars are ordered and LSDs are
provided (p<0.05). ........................................................................................................................ 87
Figure 3.11. The α-amylase activity (CU/g) for 24 MI cultivars with non-misting treatment
(ambient control), after five days ARP storage in 2011. Left side showed red cultivars and right
side showed white cultivars. In each part, the cultivars are ordered and LSDs are provided
(p<0.05). ........................................................................................................................................ 88
Figure 3.12. The α-amylase activity (CU/g) for 24 MI cultivars with non-misting treatment
(ambient control), immediately after harvesting in 2011. Left side showed red cultivars and right
side showed white cultivars. In each part, the cultivars are ordered and LSDs are provided
(p<0.05). ........................................................................................................................................ 89
Figure 3.13. The Falling Number (FN) values (s) for 24 MI cultivars with severe misting
treatment (20 s per 2 min for 72 h), after five days ARP storage in 2011. Left side showed red
cultivars and right side showed white cultivars. In each part, the cultivars are ordered and LSDs
are provided (p<0.05). The horizontal line at 250 indicated the commercial acceptance FN for
soft wheat. ..................................................................................................................................... 90
Figure 3.14. The Falling Number (FN) values (s) for 24 MI cultivars with severe misting
treatment (20 s per 2 min for 72 h), immediately after harvesting in 2011. Left side showed red
cultivars and right side showed white cultivars. In each part, the cultivars are ordered and LSDs
are provided (p<0.05). The horizontal line at 250 indicated the commercial acceptance FN for
soft wheat. ..................................................................................................................................... 91
Figure 3.15. The Falling Number (FN) values (s) for 24 MI cultivars with moderate misting
treatment (45 min per 6 h for 48 h), after five days ARP storage in 2011. Left side showed red
cultivars and right side showed white cultivars. In each part, the cultivars are ordered and LSDs

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are provided (p<0.05). The horizontal line at 250 indicated the commercial acceptance FN for
soft wheat. ..................................................................................................................................... 92
Figure 3.16. The Falling Number (FN) values (s) for 24 MI cultivars with moderate misting
treatment (45 min per 6 h for 48 h), immediately after harvesting in 2011. Left side showed red
cultivars and right side showed white cultivars. In each part, the cultivars are ordered and LSDs
are provided (p<0.05). The horizontal line at 250 indicated the commercial acceptance FN for
soft wheat. ..................................................................................................................................... 93
Figure 3.17. The Falling Number (FN) values (s) for 24 MI cultivars with non-misting treatment
(ambient control), after five days ARP storage in 2011. Left side showed red cultivars and right
side showed white cultivars. In each part, the cultivars are ordered and LSDs are provided
(p<0.05). The horizontal line at 250 indicated the commercial acceptance FN for soft wheat. ... 94
Figure 3.18. The Falling Number (FN) values (s) for 24 MI cultivars with non-misting treatment
(ambient control), immediately after harvesting in 2011. Left side showed red cultivars and right
side showed white cultivars. In each part, the cultivars are ordered and LSDs are provided
(p<0.05). The horizontal line at 250 indicated the commercial acceptance FN for soft wheat. ... 95
Figure 3.19. Scatter plot of α-amylase activity (CU/g) and Falling Number values (s) for all the
data points collected in 2009-2011 studies. .................................................................................. 96

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CHAPTER I
GENERAL INTRODUCTION
INTRODUCTION
The Importance of Wheat: from World to Michigan
Wheat is the one of the world’s most important food crops and is produced widely around
the globe. In 2011, world production of wheat was 690 million tons, making it the second most
produced cereal after corn (861 million tons) (IGC, 2012). Wheat is grown more widely than any
other commercial crop and holds the greatest world trade (FAO, 2002). Globally, it is the second
major human food crop after rice. Wheat can be ground into flour, semolina or used as malts and
groats, all of which are used in a wide variety of human foods, such as bread, pasta, cakes,
cookies and so on. Wheat, which provides the majority of carbohydrates, is the main source of
daily food in a lot of countries. Wheat is also the best grain to deliver gluten protein to the human
diet. Starch and gluten from wheat also can be processed into animal feed, paper, adhesives and
biofuels (Graybosch et al., 2009).
Wheat production is a major contributor to Michigan’s economy and it is also a
significant component of farming and the Agri-food industry. According to the National
Agriculture Statistics Service, the production value of 226,720 ha Michigan wheat reached $164
million in 2009. Soft red and soft white winter wheat produced in Michigan are used in various
products such as ready-to-eat cereals, pastries and baked goods, and soup thickeners. An
economic review showed the total value of soft wheat food products manufacturing in Michigan
was greater than $3.9 billion in 2002 (Peterson et al., 2006). Soft white wheat is primarily grown
around the Great Lakes region of the U.S. and Canada (Michigan, New York, and Ontario) area
and the Pacific Northwest. However, production of white wheat has dramatically declined in
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New York and Ontario in the past 15 years, leaving Michigan as the only major producer of
white wheat in the Eastern U.S. Soft white wheat is especially critical for cereal producers due to
its less bitter taste and better color preference in the food processing compared to the red type. It
is a primary raw material source of many whole grain wheat products, which become more
popular to public with their growing fiber nutrition demand. Because of the quality difference
and considerable shipping cost of the Pacific Northwest soft white wheat, a local material source
is important for Eastern U.S. cereal industries. As a result, the continued presence of soft white
winter wheat in Michigan is not only a critical and profitable role for the state economy, but is
essential to the sustaining of the wheat industry. Increased protection of the white wheat crop and
promotion of improvements in production will result in major economic benefits for Michigan.
Losses from Pre-harvest Sprouting
Pre-harvest sprouting (PHS) in wheat (Triticum aestivum L.) is a phenomenon whereby
seeds germinate while still in the ear in the field. It usually happens when there is high relative
humidity and cool temperatures prior to harvest. Pre-harvest Sprouting results in functional
changes to the wheat flour and severely limits the end-use applications for wheat flour and
results in substantial economic loss to farmers and food processors. In 2008 and 2009, Michigan
wheat growing region experienced higher than average rainfall prior to harvest, resulting in high
levels of PHS. The occurrence of PHS dramatically reduced the seed quality by the degradation
of starch and protein, which leads to an economic loss for both farmers and end-users. Millers
discount prices on PHS damaged grains when they bought wheat from farmers. In Canada, wheat
with over 5% sprouted frequency can only be used as animal feed (Official Grain Grading Guide,
2006). The sprouted grains produce lower flour yield and decreased flour quality. Flour obtained
from PHS grains loses tolerance for intense mixing and the baked products have poor volume
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and sticky crumb structure. While PHS affects both red and white wheat, PHS is a particularly
serious threat to the soft white wheat.
Evaluation of Pre-Harvest Sprouting
Pre-harvest sprouting can be evaluated by several different methods. “Sprout Count” is a
visual assessment of sprouted seeds, with sprouting occurring while seeds are in the head.
“Germination Index” is an assessment of germination of seeds following harvest, threshing, and
treatment with water. “Falling Number” (FN) is an industry-standard method used to quantify
PHS by measuring the viscosity of the flour slurry. “Alpha-amylase activity test” directly
measure the activity of the enzyme, which plays a major role in degrading endosperm starches
and provides the source for seed germination. Unfortunately, although sprout count is the most
easily assessed, it often does not effectively reflect the extent of internal damage to the seed
(damage to the starches which affect grain/flour functionality), since many physiological
processes occur prior to the external evidence of visual symptoms.
Objectives of the Study
As PHS susceptibility is a major threat to the wheat industry in Michigan and little
information exists regarding PHS resistance for adapted cultivars, further evaluation research is
needed to determine the resistance of adapted cultivars. In addition, a reliable and effective
method for PHS evaluation is needed in Michigan to screen breeding germplasm for PHS
resistance. Considering the limitations of visible evaluation methods, α-amylase and FN test that
directly reflect changes in the seed irrespective of external symptoms are chosen to identify
germplasm adapted to Michigan that have good resistance to PHS. Cultivars adapted to Michigan
were assessed under natural condition (in the absence of PHS induction) to identify the base
enzyme level during maturation with genetic variation. Multiple artificial weathering treatments
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were designed to evaluate the efficiency of the misting treatments and the variety performance in
the different methods.

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REVIEW OF LITERATURE
Wheat Domestication and Breeding
Recent evidence reveals that wheat domestication began over 10,000 – 12,000 years ago
in the Fertile Crescent of the Near East (Gustafson et al., 2009). Modern wheat cultivars include
two polyploid species: allohexaploid wheat (2n = 6× =42 chromosomes) (Triticum aestivum) and
tetraploid wheat (2n = 4× = 28 chromosomes) (Triticum turgidum). The genome designations for
hexaploid and tetraploid wheat are AABBDD and AABB, respectively. Wheat is a self pollinated
crop and the chromosomes in A, B, D genomes are considered to be homoelogous, which are
from a common ancestor (Gustafson et al., 2009). The A genome originates from the diploid
species T. urartu (Huang et al., 2002). The B genome is hypothesized to originate from Aegilops
speltoides (Triticum speltoides) (Johnson, 1972; Friebe et al., 1996; Sasanuma et al., 1996).
Tetraploid durum wheat (AABB) (T. turgidum) evolved from the natural hybridization of the wild
donors of genome A and B. Hexaploid wheat (AABBDD) derived from the crossing between the
tetraploid wheat T. turgidum (AABB) and the wild diploid goat grass Aegilops tauschii (Aegilops
squarrosa) (DD) (Kihara, 1954; Lubbers et al., 1991). A single locus Ph1 on chromosome 5B,
enables homologous chromosomes to pair and prevent homoelogous chromosomes from pairing
during meiosis, to produce functional progeny (Poehlman and Sleper, 1995).
As early as the 19

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century in France and Germany, the practice of hybridization

followed by selection initiated the wheat breeding, which is currently improved to contain
multiple elements world widely (Baenziger and DePauw, 2009). The genetic resources for
breeding future cultivars could come from classical and biotechnological techniques, such as
hybridization, mutations and tissue culture. In addition, transgenic technologies are now being
used to develop the germplasm with traits including herbicide tolerance, pathogen resistance,
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abiotic stress tolerance and improved nutritional quality (Vasil, 2007). Synthetic wheat created
by integrating source plants of genome A, B and D, followed by chromosome doubling, has been
proven to be a successful method to improve genetic variantion (Villareal et al., 1994). Doubled
haploid technology is used to reduce the time needed to obtain homozygous lines for breeding
and genetic studies (Guzy-Wrobelska et al., 2007). Mapping the location of a desirable gene in
wheat allows breeders to use marker-assisted selection (MAS) in early breeding generations to
identify progeny with the gene of interest (Knox and Clarke, 2007). With the advent of next
generation sequencing, SNP marker discovery has the potential to be a significant impact the
wheat crop improvement in future (Berkman et al., 2012). A team of UK scientists has produced
the first draft of wheat genome sequence (covered 95% of the all wheat genes), which is
expected to have more annotated data and SNP identification in near future (BBSRC, 2010).
Wheat Classification
Hexaploid wheat is classified by kernel color, kernel texture and vernalization (cold
temperature) requirements. Color is divided primarily into red and white, where “red” refers to
the reddish brown color of the seed coat, while “white” wheat lacks this brown color and is a
yellowish and tan color (McFall and Fowler, 2009). Wheat texture is classified as soft or hard.
Winter wheat is planted in the fall; stays dormant through the vernalization period and is
harvested the next summer. Spring wheat is planted in the spring and harvested in the early fall,
without a vernalization period.
Seed Morphology
The major components of the wheat seed are the embryo, endosperm, and the seed coat.
The embryo consists of the embryo axis, scutellum and epiblast. The scutellum is connected to
the endosperm, which provides nutrition during germination. The fully developed endosperm
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contains starch and the aleurone layer. Starch dominates up to 75% of the endosperm and
proteins usually occupy 6% to 16%. In addition, bioactive vitamins and nucleic acids in the seeds
assist the enzyme synthesis during early maturity (Xiao et al, 1995). The testa (seed coat) is
combined with the pericarp (fruit coat) in cereal grains, all of which are derived from ovular
integuments. The testa is not only critical to provide protection for the embryo, but also assists in
transportation of water, nutrition sources and other bioactive compounds from the maternal
vascular system to storage places such as vacuoles and starch (Bewley and Black, 1994). The
color and texture of the seed coat can be considered distinguishable characteristics of the seed.
Plant Development and Physiological Maturity
There are four stages of wheat growth after germination: tillering, stem extension,
heading, and ripening. Tillering is the period when the plant starts to protrude shoots and tillers.
In the second stage, stem elongation results in visible nodes and the wheat head begins to
enlarge. Reproductive organs become mature and flowering is initiated after the heading phase.
The seeds are filling and become firmer and drier during the ripening stage. The crop is ready to
harvest after ripening. The time between germination to harvest varies by wheat species and
environmental conditions, but it generally takes around nine months for winter wheat (Atwell,
2001).
The wheat kernel undergoes three phases spanning approximately four weeks to reach
maturity. Endosperm cells accumulate in the milk stage and the dry weight also increases
dramatically thereafter. During the third stage, the kernel approaches the end of grain filling and
become solid. The kernel development can be affected by adverse environment conditions in
these periods (Robson et al., 1995).

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Physiological maturity (PM) is a time when crop seeds have reached maximum dry
weight, after which the kernel merely loses water (Hanft and Wych, 1982). Zadoks et al. (1974)
pointed out that the caryopsis development, included PM stage, is particular important for yield
potential evaluation, quality and other environment affect risks, which suggested that the visual
methods to determine PM in the field is valuable. Although this time point is defined by
maximum dry weight, indirect measurements have been used to identify the time of PM. Grain
moisture has been suggested as an indirect indicator, but measuring moisture to predict the time
of PM is not appropriate because it varies in the range of 20% to 40% due to varieties and
growing conditions (Wong and Baker, 1986).

Theoretically, the most precise method to

determine the time of PM is to track the dry weight, but field operation challenge drives people
to use visual inspections to predict PM. Physiological maturity occurs very close to the time at
which there is a complete loss of chlorophyll color from the glumes (Hanft and Wych, 1982;
Falcinelli and Giannoni, 1985). The color judgment method is widely used in PHS research on
wheat (Paterson and Sorrells, 1990; Humphreys and Noll, 2002; Hughes et al., 2010).
Seed Dormancy
A healthy seed is considered dormant if it does not germinate when it is in optimized
conditions (such as presence of water and oxygen, suitable temperature and light) and is absent
of inhibitory chemicals (Bewley and Black, 1994). Dormancy at harvest is a critical trait for most
cereal crops to reduce undesired germination under cool moist conditions.
There are two types of dormancy in plants. In “coat-imposed” dormancy, the seed coat
constrains the protrusion of the radicle, gas exchange and light filtration, and thereby prevents
germination. In “embryo” dormancy, the dormancy is directly related to embryo itself (Bewley
and Black, 1994). Environment factors, such as temperature, could affect the embryonic
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dormancy during wheat development (Walker-Simmons and Sesing, 1990). Phenolic
compounds, especially proanthocyanidins (PAs) were found in Arabidopsis that could increase
the seed coat thickness to improve coat-imposed seed dormancy (Bradford and Nonogaki, 2007).
Kruger (1989) indicated that seed coat of red-grained wheat restricts embryo expanding and
prevents degradation of the germination inhibitor. The effect of seed coat color on dormancy
varies by the environmental conditions during kernel development and dormant white wheat was
obtained by crossing red and white wheat (Torada and Amano, 2002).
There are multiple treatments that can release a seed from dormancy. After ripening
period (ARP) refers to a period of time that the seeds broke dormancy in dry conditions after
reaching PM and this effect depends on time length, moisture, temperature and oxygen level
(Bewley and Black, 1994). Tavakkol-Afshari and Hucl (2002) observed that the durum wheat
lost dormancy within 2 weeks of ARP in room temperature. Low temperature, usually below 10
°C, can break down wheat dormancy after 12 h (Bewley and Black, 1994). In sprouting tests,
most soft whet cultivars showed inherent dormancy and needed to have ARP to initiate
germination (Thomason et al., 2009).
Enzymes in Seed Development
Starch consists of linear amylose with mainly α-1, 4 bonds and branched amylopectin
linked with α-1, 4 and α-1, 6 bonds. Starch hydrolytic enzymes such as α-amylase become
increasingly active in germination and further catalyze endohydrolysis of α-1, 4 bonds in the
starch (Van der Maarel et al., 2002). Beta-amylase cleaves the second α-1, 4 bonds after αamylase action and de-branching enzyme is necessary to break down α-1, 6 bonds (Bewley and
Black, 1994). Because β-amylase is not active prior to germination and is active under limited
conditions, α-amylase is widely researched in cereal studies. Two α-amylase isozymes are
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present in developing wheat grains: α-AMY-1 (also termed “germination amylase”) and α-AMY-2
(also termed “pericarp” or “green” amylases) (Olered, 1976). Flintham and Gale (1988)
summarized three phases of α-amylase formation during seed development: (1) pre-mature state
with relative high moisture in the absence of germination; (2) excess deposition of α-amylase in
the endosperm cavity after maturity; and (3) germination after breaking down the dormancy.
Another source of α-amylase presence is that the residual pericarp α-amylase does not degrade
with ripening (Olered, 1976; Lunn et al., 2001). Upon germination, α-AMY-1 was the first
synthesized product and α-AMY-2 formed at later stage (Lunn et al., 2001). However, without
germination, the formation of α-AMY-1 isozymes, also called late maturity amylase (LMA), is
activated by a cool temperature shock (e.g. 18 °C day and 12 °C night) during the middle to later
stages of grain development and ripening (25-30 days after anthesis) (Mares and Mrva, 2008).
Hormones in Seed Development
The plant hormones abscisic acid (ABA) and gibberellic acid (GA) control seed
dormancy antagonistically. Abscisic acid plays an important role in seed dormancy development
and maintenance (Walker-Simmons, 1987; Hilhorst and Karssen, 1992). Abscisic acid
accumulates in the embryo during embryo development and decreases after maturity (WalkerSimmons, 1987). A series of publications reported the effect of ABA on embryos separated from
the remainder of the seed and found that genetic and environmental conditions affected embryo
sensitivity to ABA (Walker-Simmons, 1988; Walker-Simmons and Sesing, 1990). The effect of
GA is focused on the production of α-amylase in aleurone tissues in cereal grains and α-amylase
transcription activation is associated with the GA regulation (Gubler et al., 1995). The ABA and
GA content ratio is essential in seed dormancy and the germination process (Bewley and Black,
1994).
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Process of PHS
High relative humidity during the time between PM and harvest lead to precocious
germination of the grain prior to harvest, with the consequence of yield loss and decreased grain
quality. Absence of germination inhibitors, water and gas infiltration to the seeds, and activation
of germination enzyme are major features involved in the PHS process (Xiao et al., 2005).
Moisture differences between mature seeds and available water in the environment provide the
potential of the water movement (Xiao et al., 2005).
Environment Effects on PHS
Susceptibility to PHS is dependent on both genotype and environmental conditions such
as temperature and moisture (Nielsen et al., 1984; Barnard and Smith, 2009). In a study by
Nielsen et al. (1984) , high temperature before maturity reduced sprouting resistance.
Conversely, it is observed that cool temperature during the maturation period lead to higher
levels of dormancy (Reddy et al., 1985). Similarly, greater dormancy was observed for cultivars
maintained at 9 °C or lower during grain ripening (Mares, 1993). It was reported that 15 to 20 °C
is the desired temperature range to study the dormancy issue for spring wheat cultivars (Nyachiro
et al., 2002). However, Nakatsu et al. (2007) found that some genotypes are more likely to
activate α-amylase and to have PHS under lower (15-17 °C) temperature in ripening period.
In addition to the effect of temperatures during maturation, the moisture greatly impacts
the occurrence of PHS. In a study by Mares (1993), rainfall during the 20-day period before
harvesting caused major variation on seed dormancy level. Conversely, drought and high
temperature during grain filling has been shown to increase seed dormancy and non-dormant
plants can be induced to show a dormant phenotype (Biddulph et al., 2005). In general, dry
conditions combined with cooler temperatures just prior to maturity may also result in seed with
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higher dormancy, and cool and moist conditions after maturity are inclined to break dormancy
and lead to PHS (Thomason et al., 2009).
Artificial misting is widely used to simulate rainfall in the field in PHS research (Mares,
1993; Humphreys and Noll, 2002; Hughes et al., 2010). FN results declined rapidly when wheat
plants received 22mm of rainfall after harvest (Mares, 1993). From FN data, the use of artificial
rain gives comparable results to natural rain conditions in the field for screening genotypes for
resistance to PHS (Humphreys and Noll, 2002). Multiple wetting and drying cycles help water to
penetrate the seed coat quickly (Thomason et al., 2009). However, there is no standard protocol
to induce PHS post harvest in artificial environments.
PHS on Wheat and Product Quality
PHS can induce a series of physiological and biochemical changes, including the
activation of plant hormones, α-amylase and proteases. The activities of α-amylase and protease
degrade the seed starch and protein, respectively. If such seed is used for milling and baking, the
seed is deemed as having poor grain quality and large economic losses are incurred.
In addition to end users, farmers are dramatically impacted by PHS damage. Sprouting
not only results in loss of yield and lower test weights, but also farmers are paid less for sprouted
grain due to the damage to the grain functional milling and baking characteristics (Xiao et al.,
2005). If the damage is severe enough, the grain may be degraded to animal feed. Sprouted seed
may also be unsustainable for planting, due to lower germination probabilities (Kruger, 1989).
Many grain quality characteristics are damaged in sprouted wheat.

When milled,

sprouted wheat has lower flour yields and higher ash content (Kruger, 1989). Reduced
extensibility and maximum resistance showed in the properties of the dough from sprouted
grains proved its weakness and would be detrimental to the end baking products (Buchanan and
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Nicholas, 1980). The increased α-amylase activity deteriorates many baked products. Sticky
crumb, collapsed loaves and compact interiors were observed in bread products made from
sprouting damaged flour (Derera and Bhatt, 1980). Darken crusts were also observed, caused by
caramelization of sugars (Bewley and Black, 1994). In addition, degraded starch has a strong
ability to absorb water, but once released, the water would be too much to damage the intact
flour and dextrin (Xiao et al., 2005). The hydrolysis of the protein and starch in the damaged
flour resulted in noodle products with less elasticity, dark color and weak strength (Xiao et al.,
2005). Generally, pan bread is more sensitive to high α-amylase activity compared to flat bread
and bun, but sound flour definitely produces better food (Xiao et al., 2005).
PHS Assessment
Because of its high impact on wheat quality and the value of wheat grain, grain elevator
and mill owners commonly evaluate PHS of the grains, when they purchase wheat from farmers.
There are various approaches widely used around the world to assess for PHS and grain
dormancy. Three main categories of these methods are visual evaluation, flour quality test and
biochemical test.
A germination test of the seeds is widely used to detect seed dormancy directly (Reddy et
al., 1985; Kruger, 1989). Seeds are placed in a petri dish and incubated under moisture condition
for a controlled time. The numbers of germinated seeds are visually observed and can be directly
calculated as germination percentage or the length of time to obtain the specific level of
germination (Kruger, 1989).
The weighted germination index (WGI) gives higher weight to the seeds that germinate
easily under standard condition and is calculated from the following formula: WGI = (7×n1 +

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6×n2 +…1×n7) ⁄ total days of test × total grains, where n1, n2, … n7 are the number of grains that
had germinated on day1, day 2, …day7, respectively (Reddy et al., 1985).
An intact-head sprouting test is preformed by treating heads with an artificial misting
system or water saturation and, after a period of days, counting the sprouted grains. This method
has been historically used in Sweden and China (Xiao et al., 2005). The spikes are selected at a
fixed maturity time, usually at PM to reduce the effect of maturity on dormancy (Paterson and
Sorrells, 1990). A sprouting score is used to rate the spike PHS severity after misting treatment
(Paterson and Sorrells, 1990). However, a count of sprouted grains often does not effectively
reflect the extent of starch damage inside the seed, since enzymatic processes occur prior to the
external evidence of sprout extrusion. Sprouting tests may be affected with other mechanisms
associated with the spike itself, such as husk germination inhibitors and spike morphology (King
and Richards, 1984; Kato et al., 2002).
Falling Number test originated in Europe in 1960s and it has been approved as a standard
evaluation method for sprouting damaged cereals (Hagberg, 1960). This test measures the
weakness of the starch structure by recording the time required for the plunger to fall through a
heated flour-water slurry. Typically, undamaged starches will cross-link to form a thick structure
when mixed and heated with water, causing the plunger to fall slowly through the slurry. If the
starches from the flour are degraded by α-amylase generated from PHS, cross-linking will be
reduced according to the degree of degradation, and the plunger will fall more quickly through
the slurry. Generally, 250 seconds is a widely accepted standard of acceptable grain quality for
soft wheat flour using a FN test (Xiao et al., 2005). The FN value has been found to correlate
well with the α-amylase activity (Perten, 1964). However, the correlation between sprouting

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scores and FN is not good and FN can provide more reliable estimation of PHS damage
(Humphreys and Noll, 2002).
Rapid Visco-Analyzer (RVA) measures the starch viscosity by monitoring the stirring
counter-force in real-time. The RVA test is widely used in Europe and Australia in wheat
research as it needs only a small amount (3-4 g) of sample and short determination time (13-15
min) (Xiao et al., 2005).
McCleary and Shehan (1987) developed the α-amylase activity assay and it showed great
correlation with FN and other α-amylase assays. The level of α-amylase activity has been widely
used as an evaluation method of PHS in wheat, barley and rye (Singh et al., 2008; Masojc and
Milczarski, 2009).
Physical Control of PHS
Spike morphology influences water absorption and drying in the spike, which affects
PHS tolerance. The process of wetting is slower in awnless wheat compared to awned wheat,
resulting in higher PHS resistance in awnless wheat (King and Richards, 1984). The existence
and thickness of epicuticular waxes also impacts the water uptake rate (King and Von WettsteinKnowles, 2000). King and Von Wettstein-Knowles (2000) also reported that wheat heads that
remain upright were more likely to resist sprouting by shedding more water than lodged ones.
Wheat with long and weak heads is more tolerant to PHS compared with the individual with
short and strong heads (Zanetti et al., 2000). The position of the kernels also shows a relationship
with PHS resistance. The seeds in the top of the spike germinated firstly, then the bottom ones,
and finally germination proceeded to the central part of the spike (Hardesty and Elliott, 1956).

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Genetic Control of PHS
Seed dormancy is a heritable trait and is controlled by dominant or recessive nuclear or
cytoplasmic genes (Foley and Fennimore, 1998). The gene related to dormancy was found on
chromosome 3D and it is also expected to associated with ABA sensitivity (Mares et al., 2002).
Seed coat color was proposed to have a relationship with seed dormancy and usually red
kernel is associated more with dormancy (Xiao et al., 2005). Three R1 genes (R-A1, R-B1, R-D1)
control the level of seed coat red color, and only if all the R1 genes have recessive alleles, the
seed coat color is white (Metzger and Silbaugh, 1970; Flintham, 2000). Himi et al. (2002)
reported that the R1 genes increase the seed dormancy by increasing the sensitivity to ABA. The
association between genes controlling seed coat color and dormancy may result from a
pleiotropic effect or tight linkage with genes directly controlling seed dormancy (Groos et al.,
2002). However, Himi et al. (2002) reported that the R1 genes effect on dormancy was relatively
small since they found that white wheat with R1 gene derived by mutation showed a low level of
dormancy. Gene dosage effects can explain the variation of seed coat color in the wheat kernel
(Wang et al., 1999), but the differences in dormancy individuals that carry all three R1 dominant
alleles can not be explained (Noll et al., 1982). The R1 genes effect on PHS resistance varied by
the evaluation measurement, such as sprout count, FN test and germination index (Groos et al.,
2002). R1 genes have been mapped on the long arm of group 3 chromosomes (3A, 3B and 3D)
(McIntosh et al., 1998). Genetic analysis has identified a potential marker for the dormancy
genes on chromosome 3D in white wheat populations (Mares et al., 2002). PCR markers of
Tamyb10 genes, the homologous to TT2 gene that control the proanthocyanidin (related to seed
coat pigment synthesis) production in Arabidopsis, have also been mapped at the same locus as
R1 genes in wheat (Himi et al., 2011).
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There are three major genes controlling the α-amylase expression in wheat: α-AMY-1, αAMY-2 and α-AMY-3. Alpha-AMY-1 has high isoelectric point (pI) and is induced by GA during
germination. However, during the later stage of maturity, this kind of enzyme can also be
detected and usually it is called LMA (Mares and Mrva, 2008). The α-AMY-1 gene is located on
chromosome 6A, 6B and 6D. The recent association mapping study on LMA has confirmed that
the existence of LMA gene on long arm of chromosome 6 (Emebiri et al., 2010). Alpha-AMY-2
has low pI and the gene is located on chromosome 7A, 7B and 7D. Alpha-AMY-3 only exists on
the out layer of pericarp with high pI and the few gene copies were observed on chromosome 5
(Xiao et al., 2005). In addition, the barley α-amylase inhibitor gene was effectively expressed in
wheat and could be used in developing transgenic PHS resistant wheat (Xiao et al, 1995).
It is important to identify linked molecular markers to assist selection of PHS resistance
plants in breeding. PHS resistant QTLs have been mapped to wheat chromosome 1, 2, 3, 4, 5, 6,
7 and all the QTLs are on the short arm except the ones on chromosome 4A long arm (Flintham
et al., 2002). These results suggest that the PHS resistance comes from multiple loci and the
hypothetical gene could be found at similar QTL in wheat (Flintham et al., 2002).
In addition, the comparison genetics study found the homologous gene of Vp1, which
encodes a transcriptional factor affecting maize (Zea Mays) dormancy, in wheat and this TaVp1
gene has been mapped close to R loci (Bailey, McKibbin et al. 1999). The TaVp1 gene is also
validated to be associated with wheat seed dormancy and sprouting (Chang et al., 2010a; Chang
et al., 2010b; Chang et al., 2011).

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Sasanuma, T., N.T. Miyashita and K. Tsunewaki. 1996. Wheat phylogeny determined by RFLP
analysis of nuclear DNA interspecific variations of five Aegilops sitopsis species." Theor.
Appl. Genet. 92:928-934.
Singh, R., M. Matus-Cadiz, M. Baga, P. Hucl and R.N. Chibbar. 2008. Comparison of different
methods for phenotyping preharvest sprouting in white-grained wheat. Cereal Chemistry.
85:238-242.
Tavakkol-Afshari, R. and P. Hucl. 2002. Variation of seed dormancy and after-ripening in
tetraploid wheat (Triticum durum, T. turgidum, T. turanicum, T. carthlicum, T.
polonicum). Journal of Agricultural Science and Technology. 4:23-36.
Thomason, W.E., K.R. Hughes, C.A. Griffer, D.G. Parrish and W.E. Barbeau. 2009.
Understanding Pre-harvest Sprouting of Wheat. Virginia Cooperative Extension.
Publication:424-060.
Torada, A. and Y. Amano. 2002. Effect of seed coat color on seed dormancy in different
environments. Euphytica. 126:99-105.
Van der Maarel, M., B. Van der Veen, J.C.M. Uitdehaag, H. Leemhuis and L. Dijkhuizen. 2002.
Properties and applications of starch-converting enzymes of the alpha-amylase family.
Journal of Biotechnology. 94:137-155.
Vasil, I.K. 2007. Molecular genetic improvement of cereals: transgenic wheat (Triticum aestivum
L.). Plant Cell Reports. 26:1133-1154.
Villareal, R.L., A. Mujeebkazi, E. Deltoro, J. Crossa and S. Rajaram. 1994. Agronomic
variability in selected triticum-turgidum × t-tauschii synthetic hexaploid wheats. Journal
of Agronomy and Crop Science. 173:307-317.
Walker-Simmons, M. 1987. ABA Levels and Sensitivity in Developing Wheat Embryos of
Sprouting Resistant and Susceptible Cultivars. Plant Physiology 84:61-66.
Walker-Simmons, M. 1988. Enhancement of Aba Responsiveness in Wheat Embryos by HighTemperature. Plant Cell and Environment 11:769-775.
Walker-Simmons, M., and J. Sesing. 1990. Temperature Effects on Embryonic Abscisic-Acid
Levels during Development of Wheat-Grain Dormancy. Journal of Plant Growth
Regulation 9:51-56.
Wong, L.S.L. and R.J. Baker. 1986. Selection for time to maturity in spring wheat. Crop Science.
26:1171-1175.
Xiao, S.H., Yan, C.S. and S. Tang. 2005. Wheat Pre-harvesting Research. China Agriculture
Scientific and Technology Press. pp:2-180.

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Zadoks, J. C., T. T. Chang and C.F. Konzak. 1974. Decimal code for growth stages of cereals.
Weed Research. 14:415-421.
Zanetti, S., M. Winzeler, M. Keller, B. Keller and M. Messmer. 2000. Genetic analysis of preharvest sprouting resistance in a wheat × spelt cross. Crop Science 40:1406-1417.

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CHAPTER II
EVALUATION OF ΑLPHA-AMYLASE ACTIVITY AND FALLING NUMBER FOR
SOFT WHITE AND SOFT RED WHEAT VARIETIES ADAPTED TO MICHIGAN IN
NATURAL CONDITONS

ABSTRACT
Michigan growers have experienced severe pre-harvest sprouting (PHS) problems on
wheat in 2008 and 2009. Alpha-amylase is an important component of PHS and the falling
number (FN) test is used by industry to identify sprouted wheat that is unacceptable for use in
various food products. The objective of this study was to evaluate wheat cultivars adapted to
Michigan for the activity of α-amylase and the corresponding FN values around physiological
maturity (PM) time under natural conditions (in the absence of PHS induction). Twenty-four
soft winter wheat genotypes (12 red and 12 white) with varying levels of susceptibility to PHS
were planted in an α-lattice design in two locations from 2008 to 2010. Spikes were collected
three days before PM, at PM, and three days post PM. Samples were freeze-dried, threshed,
milled and evaluated for α-amylase activity and FN values. Genetic differences existed for both
responses at all time points and treatments. A clear trend was observed in the reduction of αamylase and the increase in FN during the maturation in non-PHS conditions. The genetic
variability identified in natural α-amylase accumulation could be a standard for PHS research. It
is conceivable that PM and 3 days post PM are reasonable maturity time-points to evaluate the
effects of inducing PHS through artificial misting of heads, due to the converging base level for
α-amylase activity and FN values.

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INTRODUCTION
Soft white and soft red winter wheat production is economically important to the farming
and food industries in Michigan. Due to its light color, less bitter taste and specific flour quality,
soft white wheat is the basis of a wide variety of food products, including pastries, crackers,
biscuits, cakes, cookies and breakfast cereals. An economic study by Peterson et al. (2006)
showed the total value of these soft wheat products manufacturing in Michigan was greater than
$3.9 billion in 2002. Soft white wheat is primarily grown in Great Lakes area and Pacific
Northwest. However, declining soft wheat production in Ontario and New York has increased
the importance of Michigan soft wheat in Great Lakes region.
Pre-harvest sprouting is the premature germination of wheat seeds while still in the wheat
head in the field. Germination causes a sequence of physiological processes, which include
changes in plant hormones and hydrolytic enzymes. Gibberellic acid (GA), in the soaked grain,
will induce and increase synthesis and secretion of α-amylase and proteases (Gale and Lenton,
1987; Cornford et al., 1987). Starches and protein are hydrolyzed by increased α-amylase
activity and subsequently flour quality is lower, which results in sticky crumb, compact interiors
and off colors in baking products, which have harmful consequences for millers and end-users
(Edwards et al., 1989). In addition, farmers are dramatically impacted by reduced yield and large
discount for damaged grains (Xiao et al., 2005).
Physiological maturity (PM) is defined as the time when the seed reaches its maximum
dry weight for wheat or grain crops (Hanft and Wych, 1982). Over cultivars and years, kernel
moisture ranges from 32.4 to 43.6% at PM, which suggests the involvement of genetic and
environment factors (Clarke, 1983). Zadoks et al. (1974) pointed out that the caryopsis
development, included PM stage, is particular important for yield potential evaluation, quality
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and other environment affect risks, which suggested that the visual methods to determine PM in
the field is valuable. PM occurs very close to the time at which there is a complete loss of
chlorophyll color from the glumes (Hanft and Wych, 1982; Falcinelli and Giannoni, 1985). The
color judgment method is widely used in PHS research (Paterson and Sorrells, 1990; Humphreys
and Noll, 2002; Hughes et al., 2010)
Two α-amylase isozymes are present in developing wheat grains: α-AMY-1 (also termed
“germination amylase”) and α-AMY-2 (also termed “pericarp” or “green” amylases) (Olered,
1976). Both isozymes act by cleaving the α-1, 4-bond in starch, and their activity varies during
different stages of formation and maturation, dormancy, and germination of the grain. Kruger
(1989) reported that in red hard spring wheat kernels sampled in various stages ranged from
early development stage to full maturity, the level of these α-amylases increases after pollination
and then decreases to a low level when reaching full maturation. In addition, when immature
seeds were harvested and bench-dried, the level of α-amylase would further decrease, while
seeds that were frozen following harvest maintained their original α-amylase level (Kruger,
1989). In the dormant stage after maturation, the levels of α-amylase level are very low and
varied in different cultivars (Kruger, 1989). Upon germination, α-AMY-1 was the first
synthesized product and α-AMY-2 formed at a later stage (Lunn et al., 2001). However, in the
absence of germination, the formation of α-AMY-1 isozymes, also called late maturity amylase
(LMA), is activated by a cool temperature shock (e.g. 18 °C day and 12 °C night) during the
middle to later stages of grain development and ripening (25-30 days after anthesis) (Mares and
Mrva, 2008).
A healthy seed is considered dormant if it does not germinate when it is in optimized
conditions (Bewley and Black, 1994). It is critical for plant species to determine the best
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germination time and space. Pre-harvest Sprouting in wheat is associated with inadequate seed
dormancy, which is affected by the seed coat and embryo (Kruger, 1989). Seed coat color and
grain dormancy are pleiotropic effects of three R genes (R-A1, R-B1, R-D1) located at
homoeologous loci on chromosomes 3A, 3B and 3D of hexaploid wheat (Flintham, 2000).
Dominance at any one of these loci will result in red seed coat color (Metzger and Silbaugh,
1970). Wheat grain dormancy is a multigenic trait controlled by R genes as well other genes, at
least one of which has a major effect (Flintham, 2000). Red wheat is generally more resistant to
PHS than white wheat (Groos et al., 2002), though variation for PHS resistance exists within
both red and white wheat (Flintham, 2000). Embryonic resistance is determined by the degree of
sensitivity of the embryo to factors in the grain that inhibit germination, such as abscisic acid
(ABA) (Gimbi and Kitabatake, 2002). Several researchers have reported the effect of ABA on
embryos separated from the remainder of the seed and found that genetic and environmental
conditions affected embryo sensitivity to ABA (Walker-Simmons 1987; Walker-Simmons 1988;
Walker-Simmons and Sesing, 1990). ABA has antagonistic effects to Gibberellic acid (GA) and
the ratio of the ABA and GA content is essential in seed dormancy and germination processes
(Bewley and Black, 1994). Endogenous α-amylase inhibitor is active against native α-amylase.
During germination, GA-induced de novo synthesis of α-amylase could be inhibited by the αamylase inhibitor, which is induced by ABA (Mundy, 1984).
Various methods are widely used to evaluate PHS and grain dormancy. Visual methods
include “sprout count”, a visual assessment of sprouted seeds in the head (this measure has been
used historically in the MSU Wheat Breeding Program), and “germination index”, which is a
calculation of the percent of seeds germinated after treatment with water. The Hagberg falling
number (FN) test (Hagberg, 1960), which is the AACCI standard evaluation method for
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sprouting-damaged cereals (AACC International, 2000), measures the functional integrity of the
starch structure. Since germination results in the starch being degraded by α-amylase, starch
properties are reduced in PHS damaged wheat. Furthermore, α-amylase can be quantified
directly using a chromogenic method (McCleary and Sheehan, 1987). The visual sprout count
method is an unreliable indicators of PHS damage as assessed by baking tests, while FN and αamylase activity tests are reliable (Moot and Every, 1990).
The objective of this work was to determine FN values and α-amylase activity at different
levels of maturity under natural conditions (in the absence of PHS). Michigan wheat breeding
germplasm has been evaluated, historically, for sprout count, but not for FN values or α-amylase
activity, which are considered to be of greater importance for end-users. This study for the wheat
varieties in the absence of PHS is also critical to understand the base value around PM and
facilitate the prediction and selection of elite red and white genotypes adapted to Michigan with
enchanced levels of dormancy performance.
MATERIALS AND METHODS
Plant Materials
In 2008, 10 soft white and 10 soft red winter wheat cultivars (Tables 1.1) adapted to
growing in Michigan were selected for a study of α-amylase activity and falling number (FN).
The selection was based on the goal of evaluating varying levels of PHS resistance in both white
and red genotypes, according to previous sprouting performance studies (unpublished data). In
2009 and 2010, two additional soft white and two soft red winter wheat cultivars were added to
the trials (Table 1.1).

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Field Design
Field trials were planted in a three-replication α-lattice design at each location. An αlattice design is a replicated design that uses incomplete blocks within each of the replicates to
further strengthen the analysis by reducing the effect of field variability (Yau, 1997). Trials were
planted in two locations each of the fall of year from 2008 to 2010, and the field information was
provided in Table 1.2. In the fall of 2008, cultivars were planted with 1.27 cm in seed spacing,
®

using a Hege

95 planter at Michigan State University Agronomy Farm. The rest of the
®

plantings were done by ALMANCO Heavy Duty Drill planter at a rate of 15 million seeds per
ha.
Physiological Maturity (PM) Determination and Harvesting
Flowering notes were taken when anthers were extruded for 50% of the plants for each
plot. Three maturity time-points were identified for each plot: three days prior to PM (PM-3),
PM and three days after PM (PM+3). The maturity time-points were determined by visualization
of the chlorophyll loss for the plot. The following definitions were used: at PM-3 60% of heads
in the plot had lost their chlorophyll; at PM 80% of the heads had lost their chlorophyll; PM+3
was three physical days passed the occurrence of PM. At each of the three time points, 100
spikes were sampled at from each plot. The samples were transported on ice to the greenhouse.
They were stored in the freezer to maintain the metabolism level until they were freeze-dried.
Post-Harvest Processing
®

All spike samples were freeze-dried in a Genesis 12EL (The Virtis Company, Gardiner,
NY) in 2009, and a Tri-Philizer TM MP, (FTS Systems, Warminster, PA) in 2010 and 2011.
Samples were then threshed using the gas-motored thresher (ALMACO LPR91001, Allen
Machine Company, Nevada, IA). Threshed samples were subsequently cleaned using an air
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column cleaner (CB-2A, AGRICULEX Inc., Guelph, Ontario, Canada) with the ventilation scale
set at 5.1. After cleaning, whole grain flour was obtained from approximately 40 g sample of
grain using a UDY

®

Mill (UDY Cyclone Sample Mill, UDY Corporation, Fort Collins, CO)

with a 0.5 mm sieve.
Determination of α-Amylase Activity
Alpha-amylase activity was measured according to AACCI Approved Method 22-02.01
(Ceralpha method) (AACC International, 2000). The enzyme extraction and assay was
performed using the Megazyme

®

kit (K-CERA 08/05, Megazyme International Ltd. Ireland)

with a modified protocol developed in USDA Soft White Wheat Quality Lab (Wooster, Ohio).
Specifically, 3.0 g of flour and 20 mL of extraction buffer (50mM malic acid, 87.5 mM sodium
hydroxide, 50mM sodium chloride, 2mM calcium chloride, 3mM sodium azide, pH 5.2) were
added into a 50 mL centrifuge tube followed by vigorous stirring and incubation at 42 °C (Water
Bath 20L, Fisher Scientific, Pittsburgh, PA) for 20 min with 5 min interval mixing. The mixture
®

was then centrifuged at 1,500 g (SORVALL RT7, Kendroll Laboratory Produces, Newton, CT)
for 15 min at 35°C. Twenty µL aliquots of Ceralpha substrate (non-reducing-end blocked pnitrophenyl maltoheptaoside, BPNPG7) solution were dispensed into each well of a 96 well plate
and pre-incubated at 42 °C. Twnty µL of α-amylase extract was directly added to the bottom of
the well. For each whole-grain sample, three replicates were conducted adjacent to each other on
the 96 well plate. Since the enzyme reaction needed to be performed at 42 °C for exactly 20 min,
30 seconds intervals were kept between each group of three aliquots pipetted. At the end of the
20 min period for each well, 300 µL of the stopping reagent was added. Non-enzymatic control
was obtained by following the order of adding the substrate after the stopping reagent. The
absorbance of each well was read using a spectrophotometer (Synergy
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®

HT Multi-Mode

Microplate Reader, BioTek, Winooski, VT) at 400nm, using a 340 uL distilled water as a
control. The enzyme extract was diluted if the absorbance values were greater than 1.2, due to
the possible saturation for the limited amount of substrate. The spectrophotometer was
standardized and the extinction coefficient (EmM) was determined with a dilution series of pnitrophenol standard solution (Cat. 104-1, Sigma) in 1% tri-sodium phosphate. The absorbance
of the solution was measured and then the concentration (mM/L) of the solution was divided by
the absorbance to obtain the EmM.
Determination of FN
In 2009, flour moisture content was determined following AACCI Approved Method 4415.02 (AACC International, 2000), by calculating the loss of water for each sample after heating
at 130 °C for 1 hour in the oven (ISOTEMP OVEN, Fisher Scientific, Pittsburgh, PA). In 2010
and 2011, flour moisture content was determined using Near-Infrared Spectroscopy (NIR) (Multi
Purpose Analyzer, Bruker Optics, Billerica, MA). The appropriate amount of flour weight
necessary for each FN test was calculated based on 7.0 g of flour at 14% moisture. According to
AACCI Approved Method 56-81.03 (AACC International, 2000), a weighted flour sample was
placed in the viscosity test tube with 25 ml of distilled water. Manual hand shaking (10 shakes)
®

and automated shaking (SHAKEMATIC , SM-1095, Perten Instruments, Kungens Kurva,
Sweden) were used in 2009 and 2010-2011 respectively, to form a flour-water slurry without
visualized dry flour in the bottom of the test tube. Immediately, the test tubes were placed into
the FN apparatus to measure the seconds needed for the plunger to drop from the top of the tube
to bottom by gravity force. The FN-1400 (Perten Instruments, Kungens Kurva, Sweden) was
used in 2009 and the FN-1700 (Perten Instruments, Kungens Kurva, Sweden) was used is 2010
and 2011. Two replicates for each sample were performed in this test.
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Statistical Analyses
Data were analyzed using SAS statistical software 9.2 (SAS Institute, Cary, NC).
Analysis of variance (ANOVA) was carried out to check the effect of fixed factors and least
square (LS) mean of α-amylase activity and FN values for each variety at each time points was
generated using the mixed models. Data were logarithm transformed to reduce the heterogeneity
of variance. Fisher’s least significant difference (LSD) was calculated for each maturity category.
Pairwise comparisons were performed using the Tukey’s Honestly Significant Difference (HSD)
test at a significance level of 0.05. All data points for α-amylase activity and FN values were
fitting into a linear regression model to check the correlation coefficient for the two methods.
RESULTS
Planting Time, Flowering Time and PM Time
Some of the varieties (‘MSU E2043’, ‘Jupiter’, ‘Coral’) consistently showed relatively
later maturity than others (data not showed). The summary of the time interval between planting
date to PM and flowering date to PM for each location at 2010 and 2011 (Table 2.1), indicated
that it took around 268 days (range of all genotypes) for these adapted soft red and soft white
winter cultivars to reach PM, and 32 days after anthesis (DAA) average is required for both red
and white wheat to reach PM.
Alpha-amylase Activity Test and FN test
Before the implementation of the chromogenic method to determine α-amylase activity,
the linear absorbance changes for diluted standard solution were checked to define the EmM of
the following test. The absorbance at 400 nm of a series of diluted standardize solution and
corresponding EmM is shown in Table 2.2. The average EmM 14.23 was used for further data
conversion and analysis.
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The effects of location, year, replication and blocks were not significant and the effects of
genotype, maturity and genotype × maturity (PM-3, PM, PM+3) were significant (p < 0.05) for
both α-amylase activity and FN test. Both the genotypes and PM maturity category contributed
the variance of the α-amylase activity and FN values. The significant effect of genotype ×
maturity interaction indicated that the experimental varieties performed differently at all three
PM times.
The means of each genotype for α-amylase activity and FN values at the three maturity
categories around PM are presented in Figures 2.1 & 2.2. It is clear that all 24 cultivars showed a
decreasing trend of α-amylase activity from PM-3 to PM+3. The date range of PM-3 to PM+3
was corresponding to approximately the period from 27 DAA to 37 DAA. The decrease between
PM-3 to the time of PM was far greater than from PM to PM+3. There were significant
differences among the 24 cultivars in terms of the α-amylase activity at all three time points,
even though α-amylase activity levels converged towards similar values at PM+3 for all 24
genotypes. When examining the red and white cultivars, categorically, similar trends were
observed and there were no specific differences in the pattern of red vs. white wheat (Fig 2.3).
For FN test, all 24 cultivars showed an increasing trend in FN from PM-3 to PM+3. In
contrast to the converging trend observed over time for α-amylase activity, no strong
convergence of values was observed for FN. The analysis of red vs. white wheat, categorically,
also showed similar trends to each other (Fig 2.4).
Genotype Comparisons and Relative Grouping at PM-3
Pair-wise comparisons were made at PM-3 to determine significant differences between
genotypes for both α-amylase activity and FN values. The least significant differences (LSDs) at
three time points around PM for α-amylase activity were 0.122, 0.056, and 0.016, respectively
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(p<0.05) (Figure 2.1 & 2.2). The LSDs at these time points for FN values were 22.46, 22.12, and
13.60, respectively (p<0.05) (Figure 2.1 & 2.2).
In addition, α-amylase activity and FN values for 24 genotypes at PM-3 were selected to
assign relative groups (low, middle and high) by percentile, due to only PM-3 had greatest
differentiate strength of significant differences between genotypes (Table 2.3).
Correlation between α-Amylase Activity and FN Values
The linear regression for α-amylase activity and FN values is shown in Figure 2.5. The
correlation coefficient (r2) was 0.5743. The r2 for red and white wheats were 0.5216 and 0.6881,
respectively (Figure 2.6 & 2.7).
DISCUSSION
In this study, there was no heavy rainfall or visual PHS during the field sampling period,
and consistent low α-amylase activity and high FN values at PM and PM+3 supported that all
the wheat lines were absent of PHS. These results are in agreement with a study by Nishikawa
and Watanabe (1988), which also showed progressively decreasing α-amylase activity in five out
of six varieties at 14 to 35 DAA, due to imminent germination.
From the results it was clear that, irrespective of genotype, α-amylase accumulated in the
seeds during the maturation process and decreased steadily as PM was neared and surpassed.
This changing pattern is in agreement with the previous study on α-amylase activity in wheat
kernels approaching maturity after pollination (Marchylo et al., 1980; Gale and Ainsworth, 1984;
Reddy et al., 1985; Nishikawa and Watanabe, 1988). The increasing trend across the maturity
categories (PM-3 to PM+3) for FN values, which indirectly reflected the α-amylase activity, was
also consistent with another study on two wheat lines around PM, which is determined by the
water content instead of visual judgment (Clarke, 1983).
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Kruger (1989) indicated that the high level of α-amylase was found mainly in the
pericarp during early stage of the kernel development. In our study, the highest α-amylase
activity was observed at three days before PM, which is consistent with the physiological change
that energy consumption is reduced when seed reaching PM. Physiological maturity was defined
as the time when the kernel had the maximum weight. The decreasing enzyme activity
contributed to the net starch deposition (Reddy et al., 1985). The converging trend of low αamylase activity at PM and thereafter suggested seeds reducing starch degradation to provide
energy, since the seeds had already reached maturity.
The decreasing enzyme activity during maturation was possibly due to multiple reasons:
enzymatic protein degradation, insolublization and inhibition of the enzyme. Two
immunochemical studies supported the theory that protein degradation caused the low activity of
α-amylase (Daussant and Renard, 1976, 1987). The barley α-amylase inhibitor (BASI) (Afshari
et al., 2011) were clearly observed 14 DAA and increased until 28 DAA without any further
changes (Hill et al., 1995). In addition, the reduction of BASI results in the release the α-amylase
protein in the germination study (Hill et al., 1995). The evidence of present endogenous αamylase inhibitor in wheat seeds may explain the reason for decreasing α-amylase activity when
seeds approach PM (30 to 34 DAA in our study) (Mundy, 1984; Weselake et al., 1985). The
effect of α-amylase inhibitors isolated from sprouting resistant wheat lines suggested the role of
α-amylase inhibitor in PHS (Abdulhussain and Paulsen, 1989). The variability of α-amylase
activity of 24 genotypes at PM-3 might be associated with the function level of α-amylase
inhibitor.
Significant differences in α-amylase activity were observed among the 24 cultivars,
indicating a variation in genotypes for this enzyme. The genotypic distinction, especially prior to
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PM, gives us the expectation that genetic differences would also be observed following the
induction of sprouting, although the relative rank might be very different. Further studies need to
be conducted to determine if the genetic variation observed in the absence of sprouting is
associated with the variation in conditions that induce PHS.
In the absence of sprouting conditions, the wheat genotypes have relatively low levels of
α-amylase at PM and thereafter in our study. The levels of α-amylase activity fall dramatically
when wheat reaches full maturity (ready to harvest) and some lines showed no detectable αamylase activity (Kruger, 1972). This result indirectly suggested that the low level of α-amylase
activity was maintained from PM to the farmer’s harvest time (7-10 days after PM). Because the
α-amylase activity is highly converged at PM and PM+3, they are expected to be good starting
time points to evaluate the effects of induced heads sprouting via misting in PHS research.
The correlation coefficient of the linear relationship between α-amylase activity and FN
in our study is low compared the value of 0.998 in one study (Mathewson and Pomeranz 1978).
The figure showed several outliers when FN value was low (<200) or α-amylase activity was
high (>0.4). The plot also indicated that the correlation between α-amylase activity and FN
values was poor when α-amylase activity was low (<0.2). In addition, recent research indicated
that the correlation between alpha-amylase activity and FN is best for samples with FN values
between 200 and 300 seconds (Souza et al., 2011). These evidence support the statement that
starch quality became the determine factor for FN with low α-amylase activity (Ringlund, 1983).
Hucl (1994) reported low repeatability of FN due to cultivar by year interaction. It was also
suggested that FN should not be used as the only method to detect the PHS damage because it
did not provide quantified and accurate protein composition and quality changes due to
weathering (Barbeau et al., 2006).
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Table 1.1. Soft white and soft red winter wheat cultivars and related references for PHS
evaluation trial
Cultivar
Ambassador
Aubrey
Caledonia
Coral
Crystal
Envoy
Jupiter
Lowell
MSU D8006
MSU E2043
MSU E5024
W1062
Arena
Hopewell
Hyland Emmit
MCIA Oasis
OH04-264-58
Pioneer Brand 25R47
Pioneer Brand 25R62
R045
R055
Red Ruby
Roane
Tribute

Cultivar References
Lewis et al., 2010b
Private company†
Sorrells et al., 2004
Lewis et al., 2010a
PVP‡ 200800367
NR§, MSU
NR, MSU
PVP 009500040
PVP 200500308
NR, MSU
NR, MSU
PVP 200900411
N/A
Campbell et al., 2001
NR, Hyland Seeds
NR, Ohio State University
NR, Ohio State University
PVP 200200232
PVP 200700369
Private company
Private company
PVP 200700409
Griffey et al., 2001
Griffey et al., 2005

† Cultivars listed as “private company” were provided by private companies without
information of their origin.
‡ Plant Variety Protection (PVP) number given
§ NR = Not Registered. Cultivars listed as NR have known origin, and these origins are
indicated.

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Table 1.2. Field location information by year, soil type, fertilizer & herbicide application and plot size for the trials
Trial Field Name
Year

Location
Information

Soil Type

Fertilizer and Herbicide

Plot size

Pre-plant Fertilizer: None
Spring Fertilizer: 89kg 46-0-0; 41kg N
Herbicide: 15 ml Harmony Extra + NIS
Pre-plant Fertilizer: None
Spring Fertilizer: 89kg 46-0-0; 41kg N
Herbicide: 15 ml Harmony Extra + NIS
Pre-plant Fertilizer: 45kg 6-24-24
Spring Fertilizer: 89kg 46-0-0; 41kg N
Herbicide: 15 ml Harmony Extra + NIS
Pre-plant Fertilizer: 90kg 7-12-28
Spring Fertilizer: 89kg 46-0-0; 41kg N
Herbicide: 15 ml Harmony Extra + NIS
Pre-plant Fertilizer: 135kg 9-23-30
Spring Fertilizer: 89kg 46-0-0; 41kg N
Herbicide: 15 ml Harmony Extra + NIS
Pre-plant Fertilizer: 101kg 7-12-27
Spring Fertilizer: 89kg 46-0-0; 41kg N
Herbicide: 15 ml Harmony Extra + NIS

4 rows,
1.5 m length,
2.1 m wide
6 rows,
3.6 m length,
1.1 m wide
6 rows,
3.6 m length,
1.1 m wide
6 rows,
3.6 m length,
1.1 m wide
6 rows,
3.6 m length,
1.1 m wide
6 rows,
3.6 m length,
1.1 m wide

2009

Agronomy
Farm

Ingham County
East Lansing, MI

Capac Loam,
0-3 percent slopes

2009

Clarksville

Ionia County
Clarksville, MI

Lapeer Sandy Loam,
2-6 percent slopes

2010

Agronomy
Farm

Ingham County
East Lansing, MI

Capac Loam,
0-3 percent slopes

2010

Saginaw

Saginaw County
Richville, MI

2011

Lenawee

Lenawee County
Britton, MI

Tappan-Londo
Loam,
0-2 percent slopes
Silty Clay Loam,
0-3 percent slopes

2011

Saginaw

Saginaw County
Richivlle, MI

!
!

Tappan-Londo
Loam,
0-2 percent slopes

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40!

Table 2.1. Number of days between planting date to PM, flowering date and PM date for
different locations in 2010 and 2011
Year
2010
2011

Location
Agronomy
Saginaw
Lenawee
Saginaw

Planting to
PM
251.3
270.0
271.3
278.1

Red
Flowering to
PM
34.6
32.4
30.4
30.6

White
Planting to
Flowering to
PM
PM
252.3
34.6
270.8
32.3
271.9
30.4
279.1
31.2

Table 2.2. Absorbance of a series of diluted standard solutions (p-nitrophenol in 1% tri-sodium
phosphate) at 400 nm and corresponding EmM value
Dilution fold
ΔE400†
50
2.937
100
1.460
150
0.964
300
0.709
600
0.231
Average
† ΔE400 = Absorbance (reaction) – Absorbance (blank)

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EmM
14.69
14.60
14.18
13.83
13.86
14.23

Table 2.3. The α-amylase activities for 24 wheat varieties at PM-3 in the absence of PHS were
ranked from smallest to the largest; the FN values were ranked from largest to the smallest
Wheat Type
Test
Rank
1
2
3
4
5
6
7
8
9
10
11
12

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White
α-amylase
FN test
activity test
MSU E5024
MSU E5024
Crystal
MSU E2043
MSU E2043
Jupiter
Envoy
Envoy
Ambassador
Coral
AgriProW1062
Ambassador
Coral
Crystal
Jupiter
Lowell
MSU D8006
MSU D8006
Lowell
Aubrey
Aubrey
AgriProW1062
Caledonia
Caledonia

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Red
α-amylase
FN test
activity test
Pioneer 25R62 Pioneer 25R62
Red Ruby
R055
OH04-264-58
Red Ruby
R055
Hyland Emmit
Arena
Tribute
MCIA Oasis
OH04-264-58
Roane
Roane
R045
Pioneer 25R47
Pioneer 25R47
R045
Tribute
Arena
Hopewell
Hopewell
Hyland Emmit
MCIA Oasis

Figure 2.1. The α-amylase activity (CU/g) at three days before PM (PM-3), PM and three days after PM (PM+3) for 24 Michigan
varieties, using grain frozen immediately after harvesting. LSD values are included at each time point (p<0.05).
(For interpretation of the references to color in this and all other figures, the reader is referred to the electronic version of this thesis.)

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Figure 2.2. Falling Number values at three days before PM (PM-3), PM and three days after PM (PM+3) for 24 Michigan varieties,
using grain frozen immediately after harvesting. LSD values are included at each time point (p<0.05).
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44!

Figure 2.3. The α-amylase activity (CU/g) at three days before PM (PM-3), PM and three days after PM (PM+3) for 24 Michigan
varieties, using grain frozen immediately after harvesting. LSD values are included at each time point (p<0.05). Red color represents
red wheat lines, and white color represents white wheat lines.

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Figure 2.4. Falling Number values (s) at three days before PM (PM-3), PM and three days after PM (PM+3) for 24 Michigan varieties,
using grain frozen immediately after harvesting. LSD values are included at each time point (p<0.05). Red color represents red wheat
lines, and white color represents white wheat lines.

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46!

400

Falling Number (s)

300

200

y = -397.46x + 374.21

100

R² = 0.5743
0

0

0.2

0.4

0.6

0.8

α-Amylase Activity (CU/g)

Figure 2.5. Correlation of α-amylase activity to FN for 24 wheat varieties.

400

Falling Number (s)

300

200

y = -353.14x + 365.39

100

R² = 0.5216
0

0

0.2

0.4

0.6

0.8

α-Amylase Activity (CU/g)

Figure 2.6. Correlation of α-amylase activity to FN for 12 red wheat varieties.
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47!

400

Falling Number (s)

300

200

y = -494.91x + 395.6

100

0

R² = 0.6881

0

0.125

0.25

0.375

0.5

α-Amylase Activity (CU/g)

Figure 2.7. Correlation of α-amylase activity to FN for 12 white wheat varieties.

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48!

LITERATURE CITED

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LITERATURE CITED

American Association of Cereal Chemists, International. 2002. Approved Method of AACCI,
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Abdulhussain, S. and G.M. Paulsen. 1989. Role of proteinaceous alpha-amylase enzymeinhibitors in preharvest sprouting of wheat grain. Journal of Agricultural and Food
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Afshari, R.T., S. Badri and A. Abbasi. 2011. Effects of gibberellin and abscisic acid on
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embryo of bread wheat cultivar 'RL4137'. Iranian Journal of Field Crop Science. 41:781789.
Barbeau, W.E., C.A. Griffey and Z.H. Yan. 2006. Evidence that minor sprout damage can lead to
significant reductions in gluten strength of winter wheats. Cereal Chemistry. 83:306-310.
Bewley J.D. and M. Black. 1994. Seeds: Physiology of Development and Germination. 2nd
edition. Plenum Press. pp: 5-350.
Campbell, K.A.G., H.N. Lafever, P. Finney, R.L. Gooding, L. Herald, A. Johnston, P. Lipps, and
R. Minyo. 2001. Registration of ‘Hopewell’ wheat. Crop Science. 41:1638–1639.
Clarke, J.M. 1983. Time of physiological maturity and post-physiological maturity drying rates
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Cornford, C.A., M. Black and J. Chapman. 1987. Sensitivity of developing wheat grains to
gibberellin and production of alpha-amylase during grain development and maturation.
Proceedings of fourth international symposium on pre-harvest sprouting in cereals.
Westview Press. pp:283–292.
Daussant, J. and H.A. Renard. 1987. Development of different alpha-amylase isozymes, having
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Daussant, J. and M. Renard. 1976. Immunochemical identification of alpha-amylases in
developing and germinating wheat seeds. Cereal Research Communications 4:201-212.
Falcinelli, M. and G.M.B. Giannoni. 1985. Physiological maturity and visual spike color in four
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Flintham, J.E. 2000. Different genetic components control coat-imposed and embryo-imposed
dormancy in wheat. Seed Science Research. 10:43-50.

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Gale, M.D. and C.C. Ainsworth. 1984. The relationship between alpha-amylase species found in
developing and germinating wheat grain. Biochemical Genetics. 22:1031-1036.
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physiological problem affecting bread-making quality of UK wheat. Aspects of Appl.
Biol. 15:115-124.
Gimbi, D.M. and N. Kitabatake. 2002. Changes in alpha- and beta-amylase activities during seed
germinaiton of African finger millet. International Journal of Food Sciences and
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Brann, E.G. Rucker, H.D. Behl, M.E. Vaughn, W.L. Sisson, T.R. Randall, R.A. Corbin,
J.C. Kenner, D.W. Dunaway, R.M. Pitman, A.E. Smid, H.E. Bockelman, C. Gaines, D.L.
Long, D.V. McVey, S.E. Cambron, and L. Witcher. 2005. Registration of ‘Tribute’
wheat. Crop Science. 45:419–420.
Griffey, C.A., T.M. Starling, A.M. Price, W.L. Sisson, M.K. Das, T.H. Pridgen, M.E. Vaughn,
W.L. Rohrer, and D.E. Brann. 2001. Registration of ‘Roane’ wheat. Crop Science.
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Groos, C., G. Gay, M.R. Perretant, L. Gervais, M. Bernard, F. Dedryver and D. Charmet. 2002.
Study of the relationship between pre-harvest sprouting and grain color by quantitative
trait loci analysis in a whitexred grain bread-wheat cross. Theor. Appl. Genet. 104:39-47.
Hagberg, S. 1960. A rapid method for determining alpha-amylase activity. Cereal Chemistry.
37:218-222.
Hanft, J.M. and R.D. Wych. 1982. Visual indicators of physiological maturity of hard red spring
wheat. Crop Science. 22:584-588.
Hill, R.D., S.M. Gubbels, L. Boros, M.J. Sumner and A.W. Macgregor. 1995. Location of alphaamylase/subtilisin inhibitor during kernel development and germination. Canadian
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Hucl, P. 1994. Repeatability of a simplified method for determining sprouting resistance in
wheat. Plant Varieties and Seeds. 7:79-84.
Hughes, K.R., C.A. Griffey, D.J. Parrish, W.E. Barbeau, E. Souza and W.E. Thomason. 2010.
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Humphreys, D.G. and J. Noll. 2002. Methods for characterization of preharvest sprouting
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Kruger, J.E. 1972. Changes in amylases of hard red spring wheat during growth and maturation.
Cereal Chemistry. 49:379-479.
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cereals. pp:61-84.
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Ward. 2010a. Registration of ‘Coral’ wheat. J. Plant Registrations. 4:205–214.
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Ward. 2010b. Registration of ‘Ambassador’ wheat. J. Plant Registrations 4:215–223.
Lunn, G.D., B.J. Major, P.S. Kettlewell and R.K. Scott. 2001. Mechanisms leading to excess
alpha-amylase activity in wheat (Triticum aestivum, L.) grain in the UK. Journal of
Cereal Science. 33:313-329.
Marchylo, B.A., L.J. Lacroix and J.E. Kruger. 1980. Alpha-amylase isoenzymes in canadian
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Mares, D.J. and K. Mrva. 2008. Late-maturity alpha-amylase: Low falling number in wheat in
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Number in wheat. Journal of Food Science. 43:652-653.
McCleary, B.V. and H. Sheehan. 1987. Measurement of cereal alpha-amylase: a new assay
procedure. Journal of Cereal Science. 6:237-251.
Metzger, R.J. and B.A. Silbaugh. 1970. Location of genes for seed coat color in hexaploid wheat,
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Moot, D.J. and D. Every. 1990. A comparison of bread baking, Falling Number, alpha-amylase
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Cereal Science. 11:225-234.
Mundy, J. 1984. Hormonal regulation of alpha-amylase inhibitor synthesis in germinating barley.
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Paterson, A.H. and M.E. Sorrells. 1990. Inheritance of grain dormancy in white-kernelled wheat.
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Sorrells, M.E., D. Benscher, and W.J. Cox. 2004. Registration of ‘Caledonia’ wheat. Crop
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Walker-Simmons, M. 1988. Enhancement of Aba Responsiveness in Wheat Embryos by HighTemperature. Plant Cell and Environment 11:769-775.
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Weselake, R.J., A.W. Macgregor, and R.D. Hill. 1985. Endogenous alpha-amylase inhibitor in
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Xiao, S.H., Yan, C.S. and S. Tang. 2005. Wheat Pre-harvesting Research. China Agriculture
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Zadoks, J. C., T. T. Chang and C.F. Konzak. 1974. Decimal code for growth stages of cereals.
Weed Research. 14:415-421.

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CHAPTER III
EVALUATION OF ΑLPHA-AMYLASE ACTIVITY AND FALLING NUMBER FOR
SOFT WHITE AND SOFT RED WHEAT VARIETIES ADAPTED TO MICHIGAN
FOLLOWING MULTIPLE PHS INDUCTION TREATMENTS

ABSTRACT
Pre-Harvest Sprouting (PHS) on wheat is a major threat to Michigan, which is the
primary soft wheat production area in Great Lakes. The objectives of this study were to evaluate
adapted wheat cultivars for α-amylase activity and the corresponding FN values around
physiological maturity (PM) time following artificial PHS induction treatments, determine the
effect of after ripening period on PHS severity, and compare the impact of two misting
procedures (severe and moderate) on tested wheat varieties. Twenty-four soft winter wheat
genotypes (12 red and 12 white) with varying levels of susceptibility to PHS were planted in αlattice design in two locations from 2009 to 2010. Samples were freeze-dried and evaluated for
α-amylase activity and FN values. ‘Lowell’ and ‘Jupiter’ showed significant differences at PM
for either α-amylase activity or FN values, compared between moderate misting and non-misting
treatment. In 2010 trial, genetic differences among 24 cultivars existed for both responses at PM
in all treatments. Five days of after-ripening period (ARP) has significant effect on dormancy
breaking for some genotypes. Generally, red wheat is more resistant to PHS than white wheat.
‘Jupiter’, ‘Lowell’ and ‘Caledonia’ were three main white wheat varieties susceptible to PHS.
White wheat varieties ‘MSU E5024’ and AgriPro ‘W1062’ showed PHS tolerance in this study.
Red wheat ‘Hopewell’ and ‘Red Ruby’ exhibited PHS susceptibility after severe misting.

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INTRODUCTION
Wheat is grown more widely than any other commercial crop and holds the greatest
world trade (FAO, 2002). Globally, it is the second major human food crop after rice. One key
motivation of the Michigan’s wheat business is the dominant position for Michigan in soft wheat
production in Great Lakes area. The other is that food industries that rely on soft wheat,
®

®

including the headquarters of such major international cereal companies as Kellogg’s , Post
®

and Jiffy , located in Michigan.
Pre-harvest sprouting (PHS) is the precocious germination of the grain prior to harvest in
the field. Higher than average rainfall and cool temperature after grain maturation (i.e., after
having reached physiological maturity) is the major cause of PHS. Pre-harvest sprouting in
wheat results in the activation of enzymes, including α-amylase, which degrades starch, the
major component of the endosperm (flour) of the grain. Such degradation results in poor grain
quality, including reduced test weight, alkaline water retention capacity, as well as altered grain
functionality (Sorrells et al., 1989; Barnard and Purchase 1998). Products made from PHS
degraded flour will often be porous, sticky, misshapen, have poor loaf volume and be off-color
(Ranhotra et al., 1977). Generally, the losses due to PHS are multifold, including loss of the
grain itself due to rejection by millers and food companies, economic losses to the wheat
business, and loss of wheat availability in future years because of the farmer avoidance of risk of
more losses due to PHS.
Susceptibility to PHS is dependent on both genotype and environmental conditions such
as temperature and moisture (Nielsen et al., 1984; Barnard and Smith, 2009). High temperatures
before physiological maturity (PM) reduced sprouting tolerance (Nielsen et al., 1984). In general,
cool temperature and moist conditions after PM are inclined to break dormancy and lead to PHS.
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55!

Monitored natural rainfall and artificial misting treatment have been widely used in PHS
research (Mares 1993; Humphreys and Noll, 2002; Hughes et al., 2010). Multiple wetting and
drying cycles help water to penetrate seeds quickly (Thomason et al., 2009). However, there is
no standard protocol to induce PHS after harvest in artificial environment.
Seed dormancy is considered the main factor to generate resistance to wheat PHS. Two
types of dormancy, seed coat-imposed dormancy and embryo dormancy, are present in wheat
(Paterson and Sorrells, 1990; Flintham, 2000). Physical restriction, gas exchange interference,
and the presence of inhibitors are major mechanisms to explain the seed coat effect contributing
dormancy (Bewley and Black, 1994). The embryo dormancy is not affected by surrounding
tissues. In cereals and other seeds, it is well established through physiological and genetic studies
that abscisic acid (ABA) plays an important role in the induction and maintenance of dormancy
(Gubler et al., 2005). During seed development, ABA content is low during the grain filling
stages, peaks around mid-maturation, and then declines gradually during seed drying stage (after
maturity) (Bewley and Black, 1994). Gibberellic acid (GA), which activates the production of αamylase in aleurone tissues in cereal grains, has antagonistic effect to ABA on dormancy (Gubler
et al., 2005).
Some morphology traits were also found to protect the grain from water. The presence of
awns keeps more water on the heads and increased the sprouting probabilities, compared to the
awnless lines (King and Richards, 1984). The existence and thickness of epicuticular waxes also
impact the water uptake rate (King and von Wettstein-Knowles, 2000).
After ripening period (ARP) is an essential process for seed to start germination. After
ripening period refers to a period of time that the seeds broke dormancy in dry conditions after
reaching PM and this effect depends on time length, moisture, temperature and oxygen level
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(Bewley and Black, 1994). The effect of breaking dormancy by six weeks ARP storage could be
equivalent to the effect of 100 mol of GA, but ARP and GA break seed dormancy in different
ways (Tavakkol-Afshari and Hucl, 2002). Abscisic acid decreased rapidly by hydration but the
GA biosynthetic pathway was activated and GA accumulated during ARP storage, both of which
are also effected by environment (Jacobsen et al., 2002). After ripening period has no effect on
expression of ABA biosynthesis genes, but promotes expression of an ABA catabolism gene, a
GA biosynthetic gene, and a GA catabolic gene following imbibition. In ARP, positive
correlation of the seed dormancy and phenolic contents suggests its dormancy control effect in
cereals (Weider et al., 1996). Cultivar and the ARP environment both affect the speed of losing
seed dormancy during ARP, and high temperature during ARP was found to be a positive factor
to accelerate the loss process (Hagemann and Ciha, 1987; Romagosa et al., 1999). Due to rapid
loss of seed dormancy in tested wheat varieties, the aim of breeding for gradual loss of dormancy
during after-ripening varieties to reduce PHS was proposed (Tavakkol-Afshari and Hucl, 2002).
The objective of this study was to evaluate the PHS resistance and susceptibility of the 24
wheat varieties adapted to Michigan, using α-amylase activity and Falling Number (FN) tests,
following multiple PHS inductions such as artificial misting and added ARP treatment. The first
misting experiment was conducted at three days before PM, at PM and three days after PM, to
identify the relationship between maturity and PHS severity. Subsequently, ARP treatment and
two different misting treatments were used to detect the effect of ARP on the 24 genotypes and
the genetic variation on different misting categories. These results could provide some insight on
line selection in breeding for PHS resistance and optimization of artificial PHS induction
procedures for soft winter wheat breeding in Michigan.

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MATERIAL AND METHODS
Plant Materials, Filed Design and Plot Sampling
In the fall of 2009 and 2010, 12 soft white and 12 soft red wheat varieties were grown in
a three-replication α-lattice design in two locations in Michigan each year. The details of field
design and planting has been described in material and methods part in chapter 2 of this thesis.
Physiological maturity (PM) was visually assessed within each season according to
chlorophyll loss. In 2010, approximately 200 spikes with stem (30-50 cm) were sampled at three
days prior to PM (PM-3), at PM and three days after PM (PM+3) from each plot. In 2011,
approximately 600 spikes with stem were randomly sampled at PM from each plot.
Post-harvest Treatment
In 2010, samples were divided into two subsamples, one of which was treated with
misting in the greenhouse to induce sprouting, while the other group was kept in the greenhouse
at ambient temperature without misting. The plants in each group were arranged in plastic tubes
(5.3 cm diameter and 25 cm deep), which were placed in racks so that stems were upright in
tubes (to help mimic the natural architecture of the plant in the field). The subsample to be
misted was placed on the greenhouse bench under a misting system made up of nozzles attached
to the pipelines (1.4 m interval) hanging above the bench and the time controller (Plug-In Digital
Timer, Model 15079, General Electric). The misting system was set to operate for 45 min every
6 h and the whole process lasts for 48 h, which is comparable to another recent study on PHS
(Humphreys and Noll, 2002).
In 2011, five days after-ripening storage and additional misting treatments were added to
the 2010 treatments. The details of the treatment arrangement are described in Table 3.1. Misting
treatment I was performed with 1.4 m interval nozzles, controlled by 6-zone misting controller
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(1626D, Phytochronics Inc., St. Louis, Mo), with the cycle of 45 min per 6 h, lasts for 48h.
Misting treatment II was performed with 35-inch interval nozzles, controlled by another
controller (Sterling, Superior Controls Co. Inc., Valencia, CA) in a separate room, with the cycle
of 20 s per 2 min, lasts for 72 h. Relative humidity and temperature were recorded (Watchdog
A150 Temp/RH Logger, Spectrum Technologies Inc., Plainfield, IL).
For both years, after misting treatment, all samples were trimmed to remove the stems
and the remaining spikes were maintained the metabolism level at -20 °C until the samples were
freeze-dried.
Sample processing, α-amylase and Falling Number measurements
After freeze-drying, cleaning and threshing, whole flour was obtained from 40 g of each
sample using a UDY Laboratory Mill with a 0.5 mm sieve. Alpha-amylase activity and FN tests
(AACCI Method 56-81.03) were conducted using the appropriate weight of each flour sample,
calculated based on its moisture level according to a Near Infrared Reflectance Spectroscopy
(NIRS) assessment. Alpha-amylase activity was assessed using the Ceralpha Method (AACCI
Method 22-02.01).
Statistical Analysis
Analyses of variance (ANOVA) and least square (LS) means of α-amylase activity and FN
values were performed using the PROC MIXED procedure of SAS for Windows, version 9.2
(Cary, NC). Bonferroni adjustment was used when the effect was not significant in ANOVA
table. Fisher’s least significant difference (LSD) was calculated for each specific treatment
category in red and white wheat. Pairwise comparisons were performed by Tukey’s Honestly
Significant Difference (HSD) test at a significance level of 0.05.

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RESULTS
PHS Severity vs. Maturity Categories: PM-3, PM, PM+3
The analysis of the misted and non-misted (ambient) treatment for 2010 data showed that
the α-amylase activity and FN values of the 24 genotypes fluctuated across the three maturity
time points (PM-3, PM, PM+3) and there no clear trends were observed (Figure 3.1, 3.2, 3.3 &
3.4). Fisher’s Least Significant Differences (LSD) were calculated at each time point, at p<0.05
level. Although significant differences existed among cultivars at each of the three time points,
the range of α-amylase activity and FN values were quite narrow in comparison with
immediately frozen samples (Figure 2.1 & 2.2 in Chapter 2).
The ANOVA analysis indicated that genotype has significant effect on both α-amylase
activity and FN test, which suggested that one part of the variance came from the genotypic
effects (p<0.05). In contrast, the effect of genotype × maturity × misting treatment interaction is
not significant, suggesting that this three way interactive effect may not contribute the variation
of observations (p<0.05). There were no significant effects for year, location, replication and
block in the ANOVA analysis (p<0.05). Due to our preplanned experimental hypothesis, it is
necessary to use Bonferroni adjustment, in the following pairwise comparisons between misted
and non-misted treatment for 24 genotypes at each maturity time (PM-3, PM and PM+3).
The 2010 results showed that only white wheat variety ‘Lowell’ at PM showed a
significant difference on α-amylase activity between misting and non-misting treatments (Figure
3.5), and no other significances were found in FN test results or other maturity time points.
However, the α-amylase activity of ‘Lowell’ at PM after misting treatment was still above the
comparable commercially acceptable FN values (250) for sound soft wheat (Figure 2.1 & 2.2 in
Chapter 2).
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Moderate Misting Treatments at PM in 2010 and 2011
In 2011, only PM was selected as the time for sampling, and five days ARP combined
with moderate misting or severe misting treatments were followed after sampling. The moderate
misting treatment without ARP was equivalent to the misting treatment that used in 2010 study.
The data at PM in 2010, and the data of treatment at moderate misting without ARP in 2011
were combined together for analysis. For both α-amylase activity and FN test, the effect of entry
× treatment interaction is significant, suggesting that genotypes do not respond uniformly to the
different misting treatments.
To conservatively estimate the differences between the misted and non-misted treatments,
Tukey’s Honestly Significant Difference (HSD) was used in the pairwise comparisons. The
results showed that there is no significant difference found in α-amylase activity results.
However, two white genotypes, ‘Lowell’ and ‘Jupiter’ were found to have significant differences
in FN between misting and non-misting treatments (Figure 3.6). Even so, the FN results
following misting were all above 250, which is the commercial acceptable line for sound soft
wheat.
PHS Severity, ARP and Misting Treatments: Within Genotype Comparisons
From the ANOVA test, the effects of genotype, treatment (misting and ARP), and
genotype × treatment interaction were significant (p < 0.05) for both α-amylase activity and FN
test. The effects of location, year, replication and blocks were not significant (p <0.05).
Significant differences were found within each genotype, between the following categories:
severe misting effect, moderate misting effect and ARP effect, using Tukey’s HSD method
(Table 3.2, 3.3 & 3.4).

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In table 3.2, when inspecting the α-amylase activity results of the severe misting
treatment, it was clear that 10 out of 12 white wheat genotypes were sensitive to the severe
misting with ARP, and seven out of 12 were sensitive to the severe misting without ARP. No red
wheat genotypes showed significant differences in the severe misting and non-misting
comparison for α-amylase activity test. For FN test, all 12 white wheat genotypes have
significant differences under both severe misting with ARP and without ARP categories. The red
wheat cultivars, ‘Red Ruby’, ‘Tribute’ and ‘R055’ showed significant differences between
severe misting and non-misting, under both with ARP and without ARP categories. Pioneer
‘25R47’, Pioneer ‘25R62’, ‘Roane’ and ‘R045’ were only susceptible to the severe misting with
ARP.
In table 3.3, in the moderate misting effect comparison for α-amylase activity test,
‘Lowell’ and ‘Jupiter’ showed significant differences between misting and non-misting, only
when they were treated with ARP storage. For FN test, these two white wheat genotypes were
significantly different compared to non-misting, under both severe misting with ARP and
without ARP treatments. ‘Aubrey’ was susceptible to moderate misting when it was treated with
ARP storage.
In table 3.4, the ARP effect comparison for α-amylase activity test, ‘Lowell’ and ‘Jupiter’
showed significant differences between with ARP and without ARP categories, for both misting
treatments. The sensitivity to ARP for ‘Ambassador’, ‘Aubrey’, ‘Caledonia’, ‘Coral’ and ‘MSU
D8006’ were only observed after severe misting. For FN test, only ‘Lowell’ was sensitive to
ARP in both misting categories. Four white genotypes (‘Aubrey’, ‘Caledonia’, ‘MSU E5024’,
AgriPro ‘W1062’) and two red genotypes (Pioneer ‘25R62’ and ‘R045’) showed significant
differences between with ARP and without ARP categories, following sever misting treatment.
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However, there was no significant difference between non-misting with ARP and non-misting
without ARP, for any of the 24 wheat genotypes.
PHS Severity, ARP and Misting Treatments: Within Treatment Comparisons
The comparisons were also made within each treatment, in order to identify the relative
position for 24 genotypes for both α-amylase activity and FN values (Figure 3.7 – 3.18). Least
significant differences (LSDs) were provided at each treatment for α-amylase activity and FN
tests, and significant differences were found between genotypes for both white and red lines, at
all six treatment categories: severe misting with ARP, severe misting without ARP, moderate
misting with ARP, moderate misting without ARP, non-misting with ARP, non-misting without
ARP.
For α-amylase activity test, following severe misting with ARP and severe misting
without ARP, results varied below 0.50 CU/g for all the red genotypes, but the results of white
genotypes ranged widely from 0.58 to 17.61 CU/g and 0.28 to 6.40 CU/g. The results for
moderate misting (with ARP and without ARP), showed less variation for both red and white
genotypes, and the majority are below 0.50 CU/g. The α-amylase activities of 24 wheat lines in
the two control groups were consistently low below 0.25 CU/g.
For FN test under severe misting, the red genotypes ranged from 164 to 325, some of
which were below the commercial acceptable value (250). The FN values of majority of white
lines were around 62, which is the lowest theoretical FN value. A few white genotypes showed
PHS tolerance with higher FN values, around 150. For moderate misting, the FN values of all red
genotypes were above 250 and the white genotypes were varied from 200 to 300. The average
FN values for both white and red varieties were higher without ARP treatment than with ARP

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treatment. Similar to the results in α-amylase activity test, two control groups showed high FN
values (>300), for both red and white genotypes.
Genotype Ranking
Alpha-amylase activity and FN values of 12 white and 12 red wheat genotypes were
ranked by percentile (percentage of scores in the frequency distribution) in each treatment
category separately (Table 3.5 & 3.6). The top three resistant genotypes, which showed lowest αamylase activity and highest FN, were in the top three rows of the table, and three most
susceptible genotypes was in the three bottom rows. For each ARP class (with ARP or without
ARP) and wheat color class, the times of genotype in the resistant group and susceptible group
were counted to give a new rank and remarks were given if there was only one hit in these two
groups (Table 3.7 – 3.10).
For the white wheat, the results showed that ‘MSU E5024’ and AgriPro ‘W1062’ were
consistently the two most PHS tolerant lines in both misting categories, irrespective of ARP.
‘Jupiter’ was one of the most PHS susceptible white wheat for both treatments with or without
ARP storage. ‘Lowell’ was another most susceptible genotype in the misting experiment with
ARP storage. Several other genotypes performed differently depending on the ARP / misting
treatments employed. ‘Envoy’ and ‘Crystal’ showed good resistance following ARP treatment.
However, ‘Envoy’ did not perform well in the without ARP group. ‘Caledonia’ and ‘Aubrey’
showed good resistance to the misting at PM, but the former did badly in the misting experiment
after ARP storage and the latter did badly in the misting without ARP.
Hyland ‘Emmit’ showed consistent resistance to PHS for both ARP treatments. ‘OH04264-58’ and ‘Arena’ were also resistant red wheat lines in this study. MCIA ‘Oasis’, ‘Roane’,
‘Tribute’ and two Pioneer varieties varied in the resistant and susceptible group. Although the
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red wheat were more resistant to PHS than white wheat overall, ‘Hopewell’ and ‘Red Ruby’
consistently grouped within the PHS susceptible red genotypes.
Scatter Plot of the Experimental Data between α-Amylase Activity and FN Values
Using three years data points from all experiments, α-amylase activity and FN values
were plotted to check their regression (Figure 3.19). It is clear that the correlation between αamylase activity and FN values across the whole data range was not linear. Falling Number value
did not correlate with α-amylase activity well (r2 = 0.5816) when FN in the range of 200-300.
The change of FN values was quite small and reached the minimum value (62), when α-amylase
activity was over 3.0 CU/g. When the variation of α-amylase activity was relative small in the
low range (0 to 0.4 CU/g), the FN values varied largely, which could be 150 to over 400.
DISCUSSION
In the summer of 2010 and 2011, there was no severe moisture condition in the field
during sampling to induce PHS. No visual evidence of sprouting and the general low α-amylase
activity and high FN values in the non-misting control groups validated the sound grain quality
of our samples.
In 2010, the fluctuation of α-amylase activity and FN results between PM-3 and PM+3
for the misting and non-misting suggested that the maturity stage had little effect on the misting
consequences. In the absence of PHS study, the converging trend of the α-amylase activity and
FN results after PM was observed (Chapter 2). It is possible that the effect of non-misting control
for two days was equivalent to the three days after PM in the field, on α-amylase activity level.
There was no significant difference found between misting and non-misting in FN test
and one white wheat genotype, ‘Lowell’, had significantly higher α-amylase activity after
misting treatment at PM, in 2010 study. For 2010 – 2011 experiment, which focused on PM,
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only white wheat cultivars, ‘Lowell’ and ‘Jupiter’, exhibited lower FN values after misting.
Although Tukey’s HSD test detected significant difference for these comparisons, the mean
values after misting for both α-amylase activity and FN value were still tend to be acceptable
(>250). All red and white genotypes were selected based on the variation on PHS response from
historical data, variation of response to the misting was expected. The results that only two lines
had general high FN after misting, even significantly lower than non-misting, suggested that the
artificial misting induction used in 2010 (moderate misting) might not be effective to the
majority of genotypes in our specific environment. The results also suggested that genotype,
environment and genotype × environment affected the PHS response. Since MSU previously
conducted visual sprout count evaluations in the same greenhouse using the same misting system
and during the same time of year, we knew that sprouting could occur using these facilities
during the summer. In addition, large variation in FN was observed in the comparable artificial
misting study on hard wheat (Humphreys and Noll, 2002). Industry reported that PHS can occur
within hours in the field, but perhaps the same responses were not as rapid in the greenhouse and
either a longer duration of wetting was needed, or more time was needed for the plant to respond
to the wetting prior to freezing. The general lack of significant differences between the misted
and ambient treatments within genotypes could have several different causes, such as specific
temperature conditions in the field prior to harvest or in the greenhouse post-harvest that
promoted higher levels of dormancy, drought condition before seed maturity, and insufficient
relative humidity levels in the misting system. Previous studies suggested that the cool
temperature during maturation led to higher levels of grain dormancy (Reddy et al., 1985).
Drought during grain filling stage has been shown to increase seed dormancy and non-dormant
plants can be induced to show a dormant phenotype (Biddulph et al., 2005). Thomason et al.
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(2009) pointed out that cool temperatures prior to PM, combined with dry conditions, might
result in higher dormancy of grain, and they also indicated that multiple wetting cycle help water
penetrating out layers of grain, which provided better misting effect. While other related research
(Paterson and Sorrells, 1990; Humphreys and Noll, 2002; Hughes et al., 2010) used air drying
following misting, our use of freezing samples to stop metabolic changes soon after misting may
have cut short the time for the grain to respond to the misting treatment. After ripening period
was indicated to break seed dormancy, by ABA degradation and activation GA synthesis
(Tavakkol-Afshari and Hucl, 2002). Short-term drying storage prior misting treatment was
reported in several PHS researches (Paterson and Sorrells, 1990; Singh et al., 2008), which could
be considered as equivalent to an ARP factor.
Significant difference among 24 genotypes to four different misting treatments for both
α-amylase activity and FN values indicated genotypic variation in the response to the PHS
induction. In two control treatments, significant differences were also observed in 24 genotypes,
which were in agreement with the previous results of α-amylase activity and FN values at PM+3
time points, in the absence of PHS (Bradford and Nonogaki, 2007).
Both severe misting and moderate misting successfully induced significant changes for
α-amylase activity and FN values in the 24 varieties. More varieties, especially the red wheat
genotypes, showing significant difference after severe misting in the FN test, than α-amylase
activity test, indicated that FN might be more easily to differentiate the changes in the PHS
experiment for the red wheat. ‘Lowell’ and ‘Jupiter’ showed consistently significant difference
even after moderate misting suggested that these two lines were susceptible to PHS.
Several genotypes receiving ARP treatment before misting, showed significantly higher
α-amylase activity and lower FN values, compared to the group without ARP storage. It
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suggested that the dormancy of these wheat varieties were more easily broken, compared to other
varieties, which showed no significant differences after ARP treatment. The dormancy reduction
after ARP might be caused by the ABA degradation and GA precursor synthesis (TavakkolAfshari and Hucl, 2002). However, the ARP effect on dormancy was different between severe
and moderate misting, even for the same genotype, which indicated that breaking dormancy was
affected by both environment and ARP storage. ‘Lowell’ and ‘Jupiter’ showed consistently
significant ARP effect for both severe and moderate misting, which implied that these two white
wheat varieties were susceptible to the dormancy release factors, such as ARP and weathering.
The summary of the percentile rank for white and red genotypes, in terms of the misting
responses, indicated two white wheat varieties, ‘MSU E5024’ and AgriPro ‘W1062’, are tolerant
to PHS. ‘Jupiter’, ‘Lowell’, ‘MSU E2043’ were the lines which are very susceptible to PHS.
‘Envoy’ and ‘Crystal’ are moderately tolerant to PHS if they received ARP treatment, and
‘Caledonia’ and ‘Aubrey’ were moderately tolerant to PHS if they were directly weathered at
PM. However, ‘Caledonia’ was susceptible to PHS after ARP storage, which validated the ARP
effect on the dormancy reduction. The wheat varieties, such as AgriPro ‘W1062’ and ‘Aubrey’,
showed susceptibility at PM and tolerance after ARP, suggested the dormancy might be obtained
in the beginning term of ARP storage. For the red wheat, ‘Hopewell’, ‘Red Ruby’, ‘R055’
showed significantly reduced FN after misting, suggested these red varieties are not tolerant to
the long term misting. However, the overall of red varieties showed significantly higher αamylase activity and lower FN values than white wheat, in both severe misting and moderate
misting group, which proved that red wheat were generally more resistant to PHS than white
wheat. In this study, Hyland ‘Emmit’, ‘Arena’ and ‘OH04-264-58’ showed PHS tolerance
consistently, suggested that those lines could be good source of PHS resistant for white wheat.
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There were no obvious effects of the awn on the PHS tolerance in our study. We were not
able to prove the previous conclusion that the awnless wheat was more resistant to sprouting than
awned wheat (King and Richards, 1984). From the pedigree analysis, two PHS tolerant white
wheat varieties, ‘MSU E5024’ and AgriPro ‘W1062’ shared the same pedigree, Pioneer ‘25W33’.
The PHS susceptible wheat lines, ‘Lowell’, ‘Caledonia’ and ‘Jupiter’ all have genetic similarity
from each other or ‘Genesee’. The PHS resistant or susceptible genes might be brought to the
progenies from the original pedigrees by genetic introgression.
The viviparous-1 (VP1) gene mutants in maize (Zea mays) have a defect in ABA
biosynthesis or signaling, indicating a role for vp1 in preventing precocious germination
(McCarty 1995). Wheat homologs (TaVP1) of the maize VP1 gene are also associated with
dormancy and PHS (Chang et al., 2010a; Chang et al., 2010b; Chang et al., 2011). VP1 gene was
genotyped in ‘MSU E5024’, which suggested that the PHS tolerance might result from the VP1
gene action (Lewis et al., 2011).
The scatter plots showed the similar results that significant changes in FN test could
occur without increase in α-amylase activity when the latter in low range (< 0.25 CU/g) (Souza
et al., 2011). The decline of FN may be due to swelling of the starch granules through hydration
or it may be result from the effect of other enzymes, such as proteases or lipases, which degraded
other large molecules in the grain (proteins and fats) to reduce the resistance of the plunger
movement. However, our result was contradictory to the conclusion that FN correlated well with
α-amylase activity in the range of FN 200 - 300 (Souza et al, 2011).
SUMMARY
Over years, locations and genotypes, significantly genetic differences were observed
around physiological maturity (PM) time, especially three days prior to PM (PM+3), for α!
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amylase activity and FN values in 24 Michigan adapted soft white and soft red winter wheat.
Pre-harvest sprouting (PHS) resistant, tolerant and susceptible soft white and soft red wheat
genotypes have been identified through this study. However, repeated experiments in multiple
years and locations are necessary to validate the evaluation results. The rank of base α-amylase
activity identified in either red or white wheat, in the absence of PHS, did not match the rank of
α-amylase activity under the artificial misting PHS induction treatments, except for the top and
bottom ones, ‘MSU E5024’ and ‘Caledonia’. This suggested that the enzyme accumulation level
was affected by both genotypic and environment factors. The α-amylase activity test and FN test
showed different power in analyzing PHS samples. For breeding purposes, using α-amylase
activity test is better than FN test, especially for white wheat, since it could not only provide the
direct enzyme activity information, but also having the unlimited range when the sample FN
results in low range (<150). However, FN test is more convenient and faster than α-amylase
activity test, and provided good differentiation in mid-high range (200-400). The average
temperature record for the severe misting, moderate misting and non-misting was 31.65°C,
32.25°C, 37.75°C, which indicated that the two misting environment provided lower temperature
conditions than non-misting control. The average relative humidity (RH) record for those three
environments were 80.4%, 76.9%, 45.4%, and the close RH between severe misting and
moderate misting implied that the frequency of watering might play an important role in
breaking seed dormancy. The results suggested that severe misting plus ARP treatment is the
effective approach to induce PHS for Michigan white and red wheat in greenhouse condition.
The genotyping of ‘MSU E5024’ has proved the existence of vp1 gene and it would be interested
genotyping its genetic similar line AgriPro ‘W1062’ and Pioneer ‘25W33’. Designing double
haploid population of these genotypes is expected to map the PHS related gene.
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Table 3.1. Post-harvesting treatment design of trial 2011
Treatment Code
A

After Ripening Period (ARP)
5 days

Misting Cycle
I: 20 seconds / 2 minutes, 72 hours total

B
C
D
E
F

None
5 days
None
5 days
None

I: 20 seconds / 2 minutes, 72 hours total
II: 45 minutes / 6 hours, 48 hours total
II: 45 minutes / 6 hours, 48 hours total
None
None

Table 3.2 Genotypes were shown in each treatment category when the pair wise comparisons
between severe misting (20 s per 2 min, last for 72 h) and non-misting for α-amylase activity and
FN tests were significant (p<0.05)
Test
Treatment
Category
White
Wheat
Genotypes

Red
Wheat
Genotypes

!
!

α-Amylase activity test
FN test
Severe misting / Non-misting
With ARP
Without ARP
With ARP
Without ARP
Ambassador
Ambassador
Ambassador
Aubrey
Aubrey
Aubrey
Caledonia
Caledonia
Caledonia
Coral
Coral
Coral
Coral
Crystal
Crystal
Crystal
Crystal
Lowell
Lowell
Lowell
Lowell
MSU D8006
MSU D8006
MSU D8006
MSU D8006
MSU E2043
MSU E2043
MSU E2043
MSU E2043
Jupiter
Jupiter
Jupiter
Jupiter
Envoy
Envoy
Envoy
Envoy
MSU E5024
MSU E5024
AgriPro W1062 AgriPro W1062
Pioneer 25R47
Pioneer 25R62 Pioneer 25R62
Red Ruby
Red Ruby
Roane
Tribute
Tribute
R055
R055
R045

!

71!

Table 3.3 Genotypes were shown in each treatment category when the pair wise comparisons
between moderate misting (45 min per 6 h, last for 48 h) and non-misting for α-amylase activity
and FN tests were significant (p<0.05)
Test
Treatment
Category
White
Wheat
Genotypes

α-Amylase activity test
FN test
Moderate misting / Non-misting
With ARP
Without ARP
With ARP
Without ARP
Aubrey
Lowell
Lowell
Lowell
Jupiter
Jupiter
Jupiter

Table 3.4 Genotypes were shown in each treatment category when the pair wise comparisons
between the group with ARP treatment and without ARP treatment for α-amylase activity and
FN tests were significant (p<0.05)
Test
Treatment
Category

White
Wheat
Genotypes

α-Amylase activity test
ARP effect for ARP effect for
severe misting
moderate
misting
Ambassador
Aubrey
Caledonia
Coral
Lowell
Lowell
Envoy
MSU D8006
Jupiter
Jupiter

Aubrey
Caledonia
Lowell

MSU E5024
AgriPro W1062
Pioneer 25R62
R045

Red Wheat
Genotypes
!
!

FN test
ARP effect for ARP effect for
severe misting
moderate
misting

!

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Lowell

Table 3.5. The α-amylase activities and FN values for 12 white wheat varieties at PM for misting and ARP treatment, were ranked
from smallest to the largest; the FN values were ranked from largest to the smallest
Test
Treatment

α-Amylase activity test

FN test

α-Amylase activity test

FN test

Severe
misting
w/ ARP

Moderate
misting
w/ ARP

Severe
misting
w/ ARP

Moderate
misting
w/ ARP

Severe
misting
w/o ARP

Moderate
misting
w/o ARP

Severe
misting
w/o ARP

Moderate
misting
w/o ARP

1

MSU E5024

MSU E5024

MSU E5024

MSU E2043

Aubrey

Ambassador

Aubrey

MSU D8006

2

Envoy

Crystal

Envoy

Crystal

AgriPro
W1062

MSU E5024

AgriPro
W1062

MSU E5024

3

AgriPro
W1062

AgriPro
W1062

AgriPro
W1062

MSU E5024

Caledonia

AgriPro
W1062

Caledonia

Envoy

4

Crystal

Envoy

MSU D8006

MSU D8006

Ambassador

MSU D8006

Ambassador

Crystal

5

Ambassador

MSU E2043

Crystal

MSU E5024

Envoy

MSU E5024

Coral

6

MSU E2043

Aubrey

Aubrey

Envoy
AgriPro
W1062

Lowell

Lowell

Crystal

Ambassador

7

MSU D8006

MSU D8006

Ambassador

Coral

Coral

Caledonia

Lowell

Caledonia

8

Aubrey

Ambassador

Caledonia

Ambassador

Crystal

Coral

MSU D8006

Lowell

9

Coral

Coral

MSU E2043

Aubrey

MSU D8006

Crystal

MSU E2043

Aubrey

10

Jupiter

Caledonia

Coral

Caledonia

Envoy

Aubrey

Envoy

MSU E2043

11

Lowell

Lowell

Jupiter

Lowell

Jupiter

MSU E2043

Coral

AgriPro
W1062

12

Caledonia

Jupiter

Lowell

Jupiter

MSU E2043

Jupiter

Jupiter

Jupiter

Rank

!
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73!

Table 3.6. The α-amylase activities and FN values for 12 red wheat varieties at PM for misting and ARP treatment, were ranked from
smallest to the largest; the FN values were ranked from largest to the smallest
Test
Treatment
Rank
1
2
3

α-Amylase activity test
Severe
Moderate
misting
misting
w/ ARP
w/ ARP
OH
MCIA Oasis
04-264-58
OH
Pioneer
04-264-58
25R62
Hyland
Pioneer
Emmit
25R47

FN test
Severe
Moderate
misting
misting
w/ ARP
w/ ARP
Hyland
Hyland
Emmit
Emmit
Roane

Roane

MCIA Oasis

Tribute

α-Amylase activity test
Severe
Moderate
misting
misting
w/o ARP
w/o ARP
OH
Arena
04-264-58
Hyland
Pioneer
Emmit
25R62
Pioneer
Arena
25R62
Pioneer
Pioneer
25R47
25R47
OH
Hyland
04-264-58
Emmit

4

Arena

R045

R045

5

Arena

MCIA Oasis

OH
04-264-58

Pioneer
25R62

6

Roane

Red Ruby

Arena

Red Ruby

R045

MCIA Oasis

7

R055

Hyland
Emmit

Hopewell

OH
04-264-58

MCIA Oasis

R045

8

Pioneer
25R47

Tribute

R055

Arena

Hopewell

9

Hopewell

R045

Pioneer
25R47

Pioneer
25R47

10

Tribute

R055

Tribute

11

Red Ruby

Roane

12

!
!

R045

Pioneer
25R62

Hopewell

FN test
Severe
Moderate
misting
misting
w/o ARP
w/o ARP
R045

Tribute

Hyland
Emmit

Hyland
Emmit
Pioneer
25R62

Roane
MCIA Oasis

Arena

OH
04-264-58

Red Ruby

Hopewell

MCIA Oasis

Roane

Tribute

Arena

R055

R055

R055

Hopewell

R055

Hopewell

Pioneer
25R62

MCIA Oasis

Tribute

R055

Tribute

Red Ruby

Red Ruby

!

Pioneer
25R47
OH
04-264-58
Pioneer
25R62

Hopewell

Red Ruby

Roane

Red Ruby

Pioneer
25R47

74!

Roane

R045

Table 3.7. The summary of hit times and remarks for the genotypes listed in the top-three group
of the percentile rank for white wheat in table 3.5
ARP

W/

W/O

Rank
1
2
3
3
4
6

Genotype
MSU E5024
AgriPro W1062
Envoy
Crystal
MSU E2043

Hit Times
4
3
2
2
1

Remark

Moderate Misting

1
2
2
2
5
5
5

AgriPro W1062
MSU E5024
Caledonia
Aubrey
Ambassador
Envoy
MSU D8006

3
2
2
2
1
1
1

Moderate Misting
Moderate Misting
Moderate Misting

Table 3.8. The summary of hit times and remarks for the genotypes listed in the top-three group
of the percentile rank for red wheat in table 3.6
ARP

W/

W/O

!
!

Rank
1
2
2
2
5
5
5

Genotype
Hyland Emmit
OH04-264-58
MCIA Oasis
Roane
Pioneer 25R62
Tribute
Pioneer 25R47

Hit Times
3
2
2
2
1
1
1

Moderate Misting
Moderate Misting
Moderate Misting

1
2
2
2
5
5
5

Hyland Emmit
Pioneer 25R62
Arena
R045
OH04-264-58
Roane
Tribute

3
3
2
1
1
1
1

Severe Misting
Moderate Misting
Severe Misting
Moderate Misting

!

75!

Remark

Table 3.9. The summary of hit times and remarks for the genotypes listed in the bottom-three
group of the percentile rank for white wheat in table 3.5
ARP
W/

W/O

Rank
1
1
3
4

Genotype
Jupiter
Lowell
Caledonia
Coral

Hit Times
4
4
3
1

Remark

Severe Misting

1
2
3
4
4
4

Jupiter
MSU E2043
Envoy
Coral
AgriPro W1062
Aubery

4
3
2
1
1
1

Severe Misting
Moderate Misting
Moderate Misting

Table 3.10. The summary of hit times and remarks for the genotypes listed in the bottom-three
group of the percentile rank for red wheat in table 3.6
ARP

W/

W/O

!
!

Rank
1
1
1
1
1
6
6

Genotype
Hopewell
Pioneer 25R62
Red Ruby
Roane
R055
Tribute
MCIA Oasis

Hit Times
2
2
2
2
2
1
1

Severe Misting
Moderate Misting

1
2
2
2
5
5

Red Ruby
R055
Hopewell
Tribute
Pioneer 25R47
Roane

3
3
2
2
1
1

Moderate Misting
Moderate Misting

!

76!

Remark

Table 3.11. Pedigree and awn information for white wheat
Color

Variety

Awn

Pedigree

MSU E5024

Awned

D6234 / Pioneer Brand 25W33

Agri Pro W1062

Awnless

Pioneer Brand 25W33/Caledonia

Ambassador

Awnless

Pioneer Brand 2737W/MSU Line D1148

Crystal

Awned

Pioneer Brand 2737W/MSU Line D1148

MSU E2043

Awned

MSU Line DC076/ Pioneer Brand 2552

Envoy

Awned

MSU Line DC076/ Pioneer Brand 2552

Caledonia

Awnless

Lowell

Awnless

Jupiter

Awned

Caledonia / NY88024-117

MSU D8006

Awned

Pioneer Brand 2555/Lowell

Coral

Awnless

MSU Line D3913/MSU Line D0331

Aubrey

Awnless

N/A (Private Company Line)

White

!
!

Ross Selection /3/( NY5207aB-2B-34 ) Burt // Genesee / CI
12658 /4/ Genesee
Genesee / Winoka /3/ Suweon92 / Brevor // 5* Genesee /4/ Talbot
/ CI8487 /3/ Genesee *4 // Norin 10 / Brevor

!

77!

Shared Genetic
Source
Pioneer Brand
25W33
Pioneer Brand
2737W,
MSU Line D1148
Pioneer Brand
2552, MSU Line
DC076

Genesee,
Caledonia,
Lowell

Table 3.12. Pedigree and awn information for red wheat
Color

Variety

Awn

Pedigree

Hyland Emmit

Awnless

Pioneer Brand 2510/Marilee

Arena

Awned

NASW84-345/Coker9835//OH419/OH389

OH04-264-58

Awned

OH645/HOPEWELL

Hopewell

Awnless

Logan / Hart // 32270A / Rousalka /3/ TN1685 / IA22 // 6767 /
216-6-3

Roane

Awned

VA-71-54-147/Coker-68-15//IN-635309-C-1-18-2-3-2

Pioneer Brand 25R47

Awned

WBE2190B1/WBA416H2(Houser/MO9545//W4034D/Augusta)//
Pioneer Brand 2552

Red Ruby

Awned

Pioneer 2552/Pioneer 2737W

MCIA Oasis

Awnless

IL85-3132-1/Irena//OH449/VA86-54-290

Tribute

Awned

VA92-51-39/AL870365

Pioneer Brand 25R62

Awned

R045

Awned

N/A (Private Company Line)

R055

Awnless

N/A (Private Company Line)

Red

!
!

Shared Genetic
Source

WBN0686C/WBJ0249B1

!

78!

Hopewell

Pioneer Brand
2552

Figure 3.1. The α-amylase activity (CU/g) at three days before PM (PM-3), PM and three days after PM (PM+3) for 24 MI cultivars
with artificial misting treatment immediately after harvesting in 2010.

!
!

!

79!

Figure 3.2. The Falling Number values (s) at three days before PM (PM-3), PM and three days after PM (PM+3) for 24 MI cultivars
with artificial misting treatment immediately after harvesting in 2010.

!
!

!

80!

Figure 3.3. The α-amylase activity (CU/g) at three days before PM (PM-3), PM and three days after PM (PM+3) for 24 MI cultivars
with non-misting treatment (ambient control) immediately after harvesting in 2010.

!
!

!

81!

Figure 3.4. The Falling Number values (s) at three days before PM (PM-3), PM and three days after PM (PM+3) for 24 MI cultivars
with non-misting treatment (ambient control) immediately after harvesting in 2010.

!
!

!

82!

.

Figure 3.5. The α-amylase activity (CU/g) of genotype ‘Lowell’ at PM for misting and nonmisting test in 2010. The letter indicated the significantly different group, at p<0.05 criteria.

Figure 3.6. The Falling Number values (s) of genotype ‘Lowell’ and ‘Jupiter’ at PM for misting
and non-misting test in 2010 and 2011. The letter indicated the significantly different group, at
p<0.05 criteria.

!
!

!

83!

Figure 3.7. The α-amylase activity (CU/g) for 24 MI cultivars with severe misting treatment (20 s per 2 min for 72 h), after five days
ARP storage in 2011. Left side showed red cultivars and right side showed white cultivars. In each part, the cultivars are ordered and
LSDs are provided (p<0.05).

!
!

!

84!

Figure 3.8. The α-amylase activity (CU/g) for 24 MI cultivars with severe misting treatment (20 s per 2 min for 72 h), immediately
after harvesting in 2011. Left side showed red cultivars and right side showed white cultivars. In each part, the cultivars are ordered
and LSDs are provided (p<0.05).

!
!

!

85!

Figure 3.9. The α-amylase activity (CU/g) for 24 MI cultivars with moderate misting treatment (45 min per 6 h for 48 h), after five
days ARP storage in 2011. Left side showed red cultivars and right side showed white cultivars. In each part, the cultivars are ordered
and LSDs are provided (p<0.05).

!
!

!

86!

Figure 3.10. The α-amylase activity (CU/g) for 24 MI cultivars with moderate misting treatment (45 min per 6 h for 48 h),
immediately after harvesting in 2011. Left side showed red cultivars and right side showed white cultivars. In each part, the cultivars
are ordered and LSDs are provided (p<0.05).

!
!

!

87!

Figure 3.11. The α-amylase activity (CU/g) for 24 MI cultivars with non-misting treatment (ambient control), after five days ARP
storage in 2011. Left side showed red cultivars and right side showed white cultivars. In each part, the cultivars are ordered and LSDs
are provided (p<0.05).

!
!

!

88!

Figure 3.12. The α-amylase activity (CU/g) for 24 MI cultivars with non-misting treatment (ambient control), immediately after
harvesting in 2011. Left side showed red cultivars and right side showed white cultivars. In each part, the cultivars are ordered and
LSDs are provided (p<0.05).

!
!

!

89!

Figure 3.13. The Falling Number (FN) values (s) for 24 MI cultivars with severe misting treatment (20 s per 2 min for 72 h), after five
days ARP storage in 2011. Left side showed red cultivars and right side showed white cultivars. In each part, the cultivars are ordered
and LSDs are provided (p<0.05). The horizontal line at 250 indicated the commercial acceptance FN for soft wheat.

!
!

!

90!

Figure 3.14. The Falling Number (FN) values (s) for 24 MI cultivars with severe misting treatment (20 s per 2 min for 72 h),
immediately after harvesting in 2011. Left side showed red cultivars and right side showed white cultivars. In each part, the cultivars
are ordered and LSDs are provided (p<0.05). The horizontal line at 250 indicated the commercial acceptance FN for soft wheat.

!
!

!

91!

Figure 3.15. The Falling Number (FN) values (s) for 24 MI cultivars with moderate misting treatment (45 min per 6 h for 48 h), after
five days ARP storage in 2011. Left side showed red cultivars and right side showed white cultivars. In each part, the cultivars are
ordered and LSDs are provided (p<0.05). The horizontal line at 250 indicated the commercial acceptance FN for soft wheat.

!
!

!

92!

Figure 3.16. The Falling Number (FN) values (s) for 24 MI cultivars with moderate misting treatment (45 min per 6 h for 48 h),
immediately after harvesting in 2011. Left side showed red cultivars and right side showed white cultivars. In each part, the cultivars
are ordered and LSDs are provided (p<0.05). The horizontal line at 250 indicated the commercial acceptance FN for soft wheat.

!
!

!

93!

Figure 3.17. The Falling Number (FN) values (s) for 24 MI cultivars with non-misting treatment (ambient control), after five days
ARP storage in 2011. Left side showed red cultivars and right side showed white cultivars. In each part, the cultivars are ordered and
LSDs are provided (p<0.05). The horizontal line at 250 indicated the commercial acceptance FN for soft wheat.

!
!

!

94!

Figure 3.18. The Falling Number (FN) values (s) for 24 MI cultivars with non-misting treatment (ambient control), immediately after
harvesting in 2011. Left side showed red cultivars and right side showed white cultivars. In each part, the cultivars are ordered and
LSDs are provided (p<0.05). The horizontal line at 250 indicated the commercial acceptance FN for soft wheat.

!
!

!

95!

Figure 3.19. Scatter plot of α-amylase activity (CU/g) and Falling Number values (s) for all the data points collected in 2009-2011
studies.

!
!

!

96!

LITERATURE CITED

!
!

!

97!

LITERATURE CITED

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