Alleviating Heat Stress in Spring Canola (Brassica napus L.) with Foliar
Boron Treatment
by
Laxhman Ramsahoi
A Thesis
Presented to
The University of Guelph
In partial fulfilment of requirements
for the degree of
Master of Science
Guelph, Ontario, Canada
© Laxhman Ramsahoi, October, 2013
ABSTRACT
ALLEVIATING HEAT STRESS IN SPRING CANOLA (Brassica napus L.) WITH
FOLIAR BORON TREATMENT
Laxhman Ramsahoi
University of Guelph, 2013
Advisor:
Professor H.J. Earl
This thesis is an investigation of the effects of foliar boron (B) application on alleviating
heat stress in spring canola (Brassica napus L.). Canola is a cool season crop and as such it
is sensitive to high temperature stress during flowering. Heat stress during this period
results in yield loss due to pod abortions. Field and controlled environment experiments
were conducted to investigate the benefits of foliar B applications and to determine if B
application could alleviate the effects of heat stress. Field results indicated that benefits of
B application are inconsistent, but may be more likely when the growing season is very
warm and dry. In controlled environment experiments, plants were exposed to three
temperature regimes at three development stages for three durations. Based on this a heat
stress protocol that produced a 25% reduction in viable pod numbers was selected to be
used for testing B effects. Using this protocol, B application was shown to significantly
reduce pod abortions when compared with the treatments without B. B application
reduced pod abortion in the presence of heat stress.
ACKNOWLEDGEMENTS
I would like to offer my sincerest gratitude to my supervisor, Dr. Hugh Earl, for his
support and guidance throughout this work. One simply could not wish for a better, friendlier or
more helpful supervisor. I also acknowledge the extensive and critical reviews of this work
provided by members of my advisory committee, Dr. Bill Deen and Dr. Barry Shelp.
I am very grateful to the friendly, helpful and cheerful members and co-workers in the
Oilseeds physiology laboratory, Godfrey Chu and Li Guo, whose help was invaluable. Also to
the numerous summer students, Shuang Li, Kalista Ramsahoi, John Patterson, Hanna Smith and
Meagan Griffiths, without whom I might never have completed all my measurements. I also
appreciate the support of Dietmar Scholz with the controlled environment facilities. The
financial assistance that made possible this research was provided by the Ontario Canola
Growers Association and the Ontario Ministry of Agriculture and Food.
I thank my wife, Veronica and children for supporting me throughout all my studies at
the University and most importantly, to the “Lord Thy God” for seeing me through this study.
iii
TABLE OF CONTENTS
CHAPTER 1:
GENERAL INTRODUCTION AND PRELIMINARY TRIALS
1.1 General Introduction
...................................................................................
2
1.2 Preliminary Trials
...................................................................................
6
1.3 Hypotheses
...................................................................................
7
1.4 Research Objectives
...................................................................................
8
CHAPTER 2:
LITERATURE REVIEW
2.1 Canola: Canada’s Major Oilseed Crop
............................................................
2.2 Canola’s Susceptibility to High Temperature Stress
2.3 The Benefits of Boron Applications
CHAPTER 3:
14
....................................
15
............................................................
21
FIELD EXPERIMENTS
3.1 Research Objectives
....................................................................................
24
3.2 Materials and Methods
....................................................................................
24
3.2.1 Small plot field trials
........................................................................
24
3.2.1.1 Summer 2009 ........................................................................
24
3.2.1.2 Summer 2010 ........................................................................
27
3.2.1.3 Summer 2011 ........................................................................
28
3.2.1.4 Summer 2012 ........................................................................
28
3.2.2 Field protocols ....................................................................................
30
3.2.2.1 Canopy Reflectance Measurements ....................................
30
3.2.2.2 Protocol for Hand-harvesting and Pod Counts ...................
32
3.2.3 Quality analyses ..................................................................................
33
3.2.3.1 Green and Brown Seeds Determination ..............................
33
iv
3.2.3.2 Oil and Protein Analysis ......................................................
35
3.2.3.3 Procedures for FFA Analysis ................................................
35
..............................................................................................................
37
3.4 Discussion
........................................................................................................
58
CHAPTER 4:
CONTROLLED ENVIRONMENT EXPERIMENTS
3.3 Results
4.1 Research Objectives
......................................................................................
64
4.2 Materials and Methods
......................................................................................
64
4.2.1 Plant culture systems and stress treatments
.....................................
64
4.2.1.1 Experiment I ..........................................................................
65
4.2.1.2 Experiment II ..........................................................................
68
4.2.2 Measurements ....................................................................................
4.2.2.1 Protocol for Hand-harvesting and Pod Counts ....................
4.2.3 Procedures for FFA analysis
70
70
..............................................................
71
...............................................................................................................
73
4.4 Discussion ............................................................................................................
85
CHAPTER 5:
CONCLUSIONS
...............................................................
89
REFERENCES
...................................................................................................
91
4.3 Results
APPENDIX
.............................................................................................................
v
105
LIST OF TABLES
Table 1.1:
Spring canola yield response to foliar B applications in 2007 ………………… 10
Table 1.2:
Spring canola yield response to foliar B applications in 2008 …........................ 12
Table 3.1:
A summary of years and locations where field experiments were conducted in
Ontario and the outcome at each experimental site ......................................... 42
Table 3.2:
Effect of foliar B treatments on canopy colour, as measured by canopy reflectance
spectroscopy measurements for seven location-years ……............................... 47
Table 3.3:
Spring canola yield response to foliar B applications in 2009 ..........................
Table 3.4:
Spring canola yield response to foliar B applications in 2010 ........................... 50
Table 3.5:
Spring canola yield response to foliar B applications in 2011 ..........................
51
Table 3.6:
Spring canola yield response to foliar B applications in 2012 ..........................
52
Table 3.7:
Summary of the effects of foliar B treatments on yield of spring canola for all
location-years and means across location for 6 years ...................................... 53
Table 3.8:
Summary of location-years with yields, yield effects of foliar B applications on
spring canola and the available soil B as per soil test analysis ......................... 54
Table 3.9:
Parameters measured from hand harvested samples for all location-years .…..
49
55
Table 3.10: Parameters measured on the hand harvested samples for location-years with a
positive response to foliar B treatment ........................................................... 56
Table 3.11: Parameters measured on hand harvested samples for location-years with a negative
response to foliar B treatments ......................................................................... 57
Table 3.12: Effect of foliar B treatments on seed free fatty acid levels for all location-years
............................................................................................................................. 58
Table 4.1:
Temperature main effects and temperature x foliar B interactive effects on main
raceme pod counts and main raceme and whole plant seed yields ...................... 81
Table 4.2:
Contrasts between foliar B treatments and control under heat stress and control
conditions, for main raceme pod counts and main raceme and whole plant seed
.............................................................................................................................. 82
vi
LIST OF FIGURES
Figure 1.1: Growing season weather data for Elora, Ontario, 2007 .....................................
9
Figure 1.2: Growing season weather data for Elora, Ontario, 2008 ..................................... 11
Figure 3.1: The hand-held boom with flat fan nozzle sprayer, pressurized with CO2, used for
treatment applications ………………………………………………………… 30
Figure 3.2: The reflectance spectrometer (Unispec DC, PP Systems, MA, USA) used to
measure canopy reflectance in the field. ……………………………...……… 32
Figure 3.3: Instruments used for crushing seeds and a strip of 100 crushed canola seeds … 35
Figure 3.4: Growing season weather data for Elora, Ontario, 2009 .................................... 43
Figure 3.5: Growing season weather data for Elora, Ontario, 2010 .................................... 44
Figure 3.6: Growing season weather data for Elora, Ontario, 2011 .................................... 45
Figure 3.7: Growing season weather data for Elora, Ontario, 2012 .................................... 46
Figure 3.8: Canopy reflectance spectroscopy measurements normalized to reflectance at
850 nm for small plot spring canola trials at Meaford ON in 2009 ................... 48
Figure 4.1:
Spring canola plants at 3 days after first flower in the growth cabinet for heat
stress treatments ................................................................................................. 73
Figure 4.2:
Aborted pods and viable pods on main racemes, as affected by time of initiation
of heat stress and duration of heat stress ............................................................ 77
Figure 4.3:
Treatment effects on pod counts for the 28/20°C temperature regime at first
flower for 3 different durations .......................................................................... 78
Figure 4.4:
Morphological symptoms on spring canola bud, opened flower and mature pod as
affected by heat stress .................................................................................... ... 79
Figure 4.5:
Morphology of entire canola plants and main raceme for the greenhouse check
and 28/20°C stress temperature regime ............................................................. 80
Figure 4.6:
Aborted pods and viable pods on main racemes, as affected by temperature
regime and foliar B application .......................................................................... 83
Figure 4.7:
Flowering main racemes of spring canola plants in non-stressed environment and
stressed conditions ............................................................................................. 84
vii
LIST OF ABBREVIATIONS
7FF
Seven Days After First Flower
14FF
Fourteen Days After First Flower
ABA
Abscisic Acid
APFB
Alpine Plant Food Boron
B
Boron
CGC
Canadian Grain Commission
FF
First Flower
FFA
Free Fatty Acids
FID
Flame Ionization Detector
GC
Gas Chromatography
HPLC
High Performance Liquid Chromatography
IAA
Indole Acetic Acid
IUPAC
International Union of Pure and Applied Chemistry
LAR
Leaf Area Ratio
LWR
Leaf Weight Ratio
NDI
Normalized Difference Index
NIR
Near Infrared Reflectance
OCGA
Ontario Canola Growers Association
PAR
Photosynthetically Active Radiation
PPFD
Photosynthetic Photon Flux Density
SLW
Specific Leaf Weight
WCOCA
Western Canola Oilseeds Crushers Association
viii
CHAPTER 1
GENERAL INTRODUCTION AND PRELIMINARY TRIALS
1
1.1
General Introduction.
Canola (Brassica napus L.) acreage in Canada has steadily expanded to meet the growing
demand for canola oil for food uses and for use in the biofuel industry, and canola now ranks as
the most valuable field crop in Canada (Statistics Canada 2009, 2012). “Canola” refers to a
particular class of rapeseed, a species within the much larger Brassicaceae (former Cruciferae)
family which includes mustard, turnip, cauliflower, cabbage, rutabaga, radish and broccoli
(Robbelen et al. 1989, Statistics Canada 2009).
Through domestication, Brassica plant parts have been modified by humans in almost
every conceivable way including the root, stem, leaf, terminal and axillary buds and seeds
(Robbelen et al. 1989). The rapeseed branch of this plant family (Brassicaceae) was bred to
maximize production of the high-oil seeds that are used to produce vegetable oil (Al-Shehbaz
2011). These Brassica species of plants supply up to one third of the world’s food oil supply (AlShehbaz 2011).
Canola is very much Canadian; the very name given is the abbreviation of “Canadian Oil
Low Acid” developed and registered by the Western Canada Oilseeds Crushers Association
(WCOCA) (Adolphe 1974). Canola was adopted as the name to be used for the rapeseed
cultivars referred to as “double low”, indicating their low glucosinolate (β-D-thioglucoside-Nhydroxysulfates ) (less than 30 µmol g-1 of air-dried oil-free meal) and erucic acid (C22H42O2 )
(less than 2% in the oil) (Statistics Canada 2009).
2
In a global perspective, Canada ranked second in the world behind China in canola
production, with 14 million tonnes in 2011(Statistics Canada 2012). Canada is the world’s
largest exporter of canola oil; approximately 80% of oil produced in 2010 was exported and this
represented over 50% of the world’s total canola oil exports, with the United States being the
major importer (Statistics Canada 2009, 2012).
Since it has the ability to germinate and grow at low temperatures, canola is one of the
few oilseed crops well suited for the cooler production regions of Canada, China, India, Pakistan,
Northern Europe and the US (Robbelen et al. 1989). However, canola is also susceptible to high
temperature and associated abiotic stresses. With the increasing interest in canola and the
expanding demand for its products, cultivation has expanded to much warmer regions, making
the crop’s exposure to heat stress and resulting yield losses more common.
Boron (B) is an essential nutrient for normal plant growth (Warrington 1923), and B
availability in soil and irrigation water is an important determinant of agricultural productivity
(Tanaka and Fujiwara 2007). This micronutrient is required in particular by dicotyledonous
plants during both vegetative and reproductive growth (Gupta 1993). Boron is a member of the
subgroup III of metalloids and has intermediate chemical properties of both metals and nonmetals (Marschner 1995). The term ‘boron’ originated from the Arabic word buraq and the
Persian word burah, meaning borax (Na2B4O7 • 10H2O), which is the most common natural form
of B. The IUPAC name for borax is sodium decaborate tetrahydrate (Anon. 2012). Borax also is
known as sodium borate, sodium tetraborate or disodium tetraborate (Anon. 2012). In the soil, B
exists primarily as boric acid (H3BO3), which can be easily leached under high rainfall
conditions (Shorrocks 1997, Yan et al. 2006). The chemical forms of B normally taken up by
3
plants are undissociated boric acid (H3BO3 or B(OH)3 and borate (H2BO3-) (Camacho-Cristóbal
et al. 2007, 2008).
Tanaka and Fujiwara (2007) list three different mechanisms by which uptake of boric
acid may occur: passive diffusion across the lipid bilayer, facilitated transport by a major
intrinsic protein (MIP) channel, or a high-affinity transport system. The later mechanism is
energy-dependent and is induced in response to low B supply. In vascular plants B moves from
the roots via the transpiration stream and accumulates in growing points of leaves and stems
(Raven 1980, Shelp et al. 1995, Dannel et al. 2000, Stangoulis et al. 2001, Stangoulis et al.
2010). However, B can also be transported via phloem to both reproductive and vegetative
tissues (Shelp et al. 1995, Matoh and Ochiai 2005), although this capacity varies among species
(Brown and Shelp 1997).
Boron is essential for many plant functions such as; maintaining a balance between sugar
and starch, translocation of sugar and carbohydrates, pollination and seed reproduction, normal
cell division, nitrogen metabolism and protein formation, and cell wall formation. It plays an
important role in the proper function of cell membranes and the transport of K+ to guard cells for
internal water balance control system (Pilbeam and Kirkby 1983, Blevins and Lukaszewski
1998, Blaser-Grill et al. 1989, Goldbach et al. 2001, Yu et al. 2003, Camacho-Cristóbal et al.
2005 and Camacho-Cristóbal and Gonzalez-Fontes 2007.
Deficiency of B is evidenced by various symptoms including: darkening of tissues;
hypertrophy followed by death of shoot apices; curled and brittle leaves and short stubby roots;
distorted, hollow and cracked stems; and the abortion or dropping of flowers and fruits
4
(Whittington 1956, Blevins and Lukaszewski 1998, Anon 2003, Camacho-Cristóbal et al. 2008).
However, the range between deficient and toxic levels of boron is very narrow (Pilbeam and
Kirkby 1983, Camacho-Cristóbal et al. 2008). The typical visible symptom of B toxicity is leaf
burn – chlorotic and/or necrotic patches, often located at the margins and tips of older leaves
(Bergmann 1992, Bennett 1993,).
B deficiency has been reported in more than 80 countries and for 132 crops over the past
years (Shorrocks 1997), and globally has been identified as the second most important
micronutrient constraint in crops after zinc (Zn). B deficiency has been reported to cause
considerable yield reduction in cotton (Gossypium hirsutum L.), rice ( Oryza sativa L.),
maize/corn ( Zea mays L.), wheat ( Triticum aestivum L.), soybean ( Glycine max L.), groundnut
( Arachis hypogaea L.), oilseed rape/canola ( Brassica napus or B. rapa L.), alfalfa (Medicago
sativa L.) and orchards crops (Arora et al. 1985, Patil et al. 1987, Ali & Monoranjan 1989,
Dwivedi et al. 1990, Sinha et al. 1991, Niaz et al. 2002, 2007, Rashid et al. 2005, Zia et al.
2006). Rashid (2006) estimated a substantial potential net economic benefit from the use of B
fertilizers in B-deficient crops.
5
1.2
Preliminary Trials.
Foliar boron (B) is sometimes applied to canola during early flowering but prior
experiments have provided evidence that the benefits of this practice may be rare, especially if
the growing season is cool with adequate rainfall.
A series of field experiments entitled “Best Management Practices for Ontario Spring
Canola” were conducted by the OCGA (Ontario Canola Growers Association), OMAF (Ontario
Ministry of Agriculture and Food) and the University of Guelph in 2007 and 2008. These
experiments investigated the benefits of several fungicide, insecticide and foliar B fertilization
treatments, used as stand-alone treatments or in combination. These small-plot field experiments
were conducted at each of two locations in Ontario (Elora and Meaford) for 2007 and 2008, with
a subset of the treatments also tested in farm-scale strip trials at nine other locations across
Ontario in 2008.
Foliar treatments included the insecticide Matador 120 EC (lambda cyhalothrin; Syngenta
Canada Inc.) at 83 ml ha-1, the fungicide Proline 480 SC (prothioconazole; Bayer Crop Science)
at 350 ml ha-1 and the surfactant Agral 90 (nonyenoxy polyethoxy ethanol 90%; Syngenta) at
0.125% v/v. For liquid B (0-0-0-13.3; Alpine Plant Foods Corp.) the application rate was
3.4 L ha-1, which provided 0.45 kg ha-1 actual B. Another boron source (borax) at a low rate (0-00-15 at 2.3 kg ha-1) was also tested, as well as a high rate of borax (4.6 kg ha-1) (0.69 kg ha-1
actual B).
6
In 2007, a hot year (Figure 1.1), both Elora and Meaford locations indicated a yield
advantage close to 0.5 Mg ha-1 for foliar B applications at early flowering (P<0.10) (Table 1.1).
In 2008, a cooler year (Figure 1.2), there was no yield response to foliar B at these same two
locations (Table1.2). However, multi-location strip trials conducted by Brian Hall of OMAFRA
indicated a small but statistically significant (P< 0.1) benefit to foliar B in 2008 (B. Hall,
personal communication).
1.3
Hypotheses.
Based on these results, the hypothesis was developed that application of foliar B results
in a yield benefit to canola only under conditions of high temperature stress. Given the
documented importance of B for pollination and seed set (Pilbeam and Kirkby 1983, Blevins and
Lukaszewski 1998, Blaser-Grill et al. 1989, Goldbach et al. 2001, Yu et al. 2003, CamachoCristóbal et al. 2005 and Camacho-Cristóbal and Gonzalez-Fontes 2007), it was further
hypothesized that the yield benefit of foliar B under heat stress is associated with reduced flower
abortions and / or increased pod retention.
To test this hypothesis, we conducted a series of field experiments over four years (2009
– 2012), to add to the existing dataset (2007, 2008) described above. We measured the effects of
foliar B applications on both yield and pod abortions, with the prediction that the benefits of
foliar B would be more pronounced when the crop experienced heat stress. To test this
hypothesis more rigorously, we also conducted a controlled environment study to measure
directly if foliar B application could reduce pod losses associated with high temperature stress.
7
1.4
Research Objectives
1. To evaluate the potential yield benefits of foliar B applications to spring canola
under typical Ontario growing conditions, in replicated multi-location field trials.
2. To test the hypotheses that foliar B applications can increase yields under heat
stress in the field, by reducing pod abortions or by increasing seed size or number
of seeds per pod.
3. To develop a reproductive stage heat stress protocol, in controlled environment
experiments, that produces significant yield reductions in spring canola with heat
stress symptoms similar to those observed under field conditions.
4. To test the hypothesis that foliar B applications can alleviate the heat stress effects
induced by the protocol identified under Objective 3, under controlled
environment conditions.
8
45
450
Elora 2007
400
35
350
30
300
25
250
20
200
15
150
10
100
5
50
0
0
29-Aug
14-Aug
30-Jul
15-Jul
30-Jun
15-Jun
31-May
16-May
-50
1-May
-5
Figure 1.1. Growing season weather data for Elora ON, 2007.
Daily high and low temperatures are shown by closed and open circles, respectively. Cumulative
precipitation beginning May 1 is shown as the solid line. The dashed horizontal line represents
the temperature of 29.5°C, identified by Morrison and Stewart (2002) as a critical temperature
for heat stress effects in canola during the flowering period.
9
Cumulative precipitation (mm)
Temperature (°C)
40
Table 1.1. Spring canola yield response to foliar B applications in 2007.
Yield results for two locations (Elora and Meaford) and the mean response for these two
locations and contrasts between -B (Check, Lance, Matador and Lance + Matador) treatments
and +B (Boron, Lance + Boron, Matador + Boron and Lance + Matador + Boron) treatments (H.
Earl, unpublished data).
Treatment
Elora
Location
Meaford
Mean
______Yield (Mg ha-1)______
2.50
3.33
2.92
3.00
3.78
3.41
2.54
3.34
2.94
2.64
3.68
3.15
Check
Boron
Lance (Fungicide)
Lance + Boron
Matador (Insecticide)
Matador + Boron
Lance + Matador
Lance + Matador +
Boron
3.12
3.27
2.99
3.48
3.67
3.72
3.30
3.47
3.35
CV%
P-value
LSD 10%
2.86
10.5
0.02
0.31
3.61
9.5
0.43
NS
3.23
9.9
0.06
0.29
P-value
2.79
2.94
0.18
3.47
3.68
0.08
3.13
3.31
0.05
Contrast
- Boron (4 treatments)
+ Boron (4 treatments)
10
45
450
Elora 2008
400
35
350
30
300
25
250
20
200
15
150
10
100
5
50
0
0
29-Aug
14-Aug
30-Jul
15-Jul
30-Jun
15-Jun
31-May
16-May
-50
1-May
-5
Cumulative precipitation (mm)
Temperature (°C)
40
Figure 1.2. Growing season weather data for Elora ON, 2008.
Daily high and low temperatures are shown by closed and open circles, respectively. Cumulative
precipitation beginning May 1 is shown as the solid line. The dashed horizontal line represents
the temperature of 29.5°C, identified by Morrison and Stewart (2002) as a critical temperature
for heat stress effects in canola during the flowering period.
11
Table 1.2. Spring canola yield response to foliar B applications in 2008.
Yield results for two locations (Elora and Meaford) and the mean response for these two
locations and contrasts for -B (Check and Adjuvant only) treatments and +B (Liquid Boron +
Adjuvant, Low Borax + Adjuvant and High Borax + Adjuvant) treatments (H. Earl, unpublished
data).
Treatment
Elora
Location
Meaford
Mean
______Yield (Mg ha -1 )______
Check
Adjuvant only
3.24
3.19
4.83
4.78
4.04
3.99
Liquid Boron + Adjuvant (0.32 kg B ha -1 )
3.15
4.82
3.99
Borax + Adjuvant (0.32 kg B ha -1 )
3.48
4.90
4.19
Borax + Adjuvant (0.64 kg B ha -1 )
CV%
P-value
LSD 10%
3.26
6.7
0.31
NS
4.77
4.4
0.92
NS
4.02
5.3
0.13
NS
P-value
3.22
3.30
0.99
4.81
4.83
0.80
4.02
4.07
0.39
Contrast
- Boron (2 treatments)
+ Boron (3 treatments)
12
CHAPTER 2
LITERATURE REVIEW
13
2.1
Canola: Canada’s major oilseed crop
Canola is currently one of the most productive and important oilseed crops grown
worldwide (Aksouh et al. 2006) and also Canada’s major oilseed crop, totaling $5.6 billion in
farm receipts and bypassing wheat to become the most valuable field crop in Canada in 2010
(Statistics Canada 2012). In 2011, the Canola Council of Canada reported that Canadian-grown
canola contributed $15.4 billion in economic activity to the Canadian economy.
Canola is a readily marketed high value crop that augments the range of exports and
products, satisfies domestic needs for vegetable oil, displaces imports and extends field crop
rotations (Adolphe 1974, Angadi et al. 2000, OCGA 2003). In recent years, there has been an
increase in the demand for oil high in mono-unsaturated and poly-unsaturated fats, including
canola oil (Assad et al. 2002). In 2006, approximately 50% of all vegetable oil consumed by
Canadians was of sourced from canola (Statistics Canada 2009).
Increase in the world’s human population has increased the demand for food, including
vegetable oils such as canola oil, as well as protein sources for animal feed (Statistics Canada
2009). Recently there has been increasing demand for canola oil as a feedstock for biodiesel
production and other industrial uses (Statistics Canada 2009).
Canola can play a major role in supplying these demands, not only for Canada, but also
for the wider world. Changing government policies, world grain markets, the increasing
awareness of farmers of the benefits of good crop rotations, and the availability of suitable
14
seeding and harvesting technologies have encouraged farmers to diversify their cropping systems
to include canola (Angadi et al. 2000).
The land area under canola (and prior to canola, rapeseed) production has increased
steadily from 143 000 ha in 1956 to 6 806 100 ha in 2010 (Statistics Canada, 2009; 2012).
Survey results indicated that Canadian farmers seeded a record 8.7 million ha of canola in 2012,
up 13% or 1 000 000 ha from the previous record set in 2011. This was the sixth consecutive
annual record in canola area at the national level (Statistics Canada 2012). The acreage of canola
in non-traditional growing areas is increasing (Statistics Canada 2009, 2012).
2.2
Canola’s susceptibility to high temperature stress.
The increase in demand for canola and canola products has encouraged the expansion of
the canola growing area into warmer regions. Canola is adapted to cooler temperatures and is
susceptible to high temperature stress, particularly during reproductive development (Morrison
1993, Brandt and McGregor 1997, Angadi et al. 2000, Aksouh et al. 2001, Morrison and Stewart
2002). Aksouh et al. (2001) also stated that susceptibility is seen even in short episodes of heat
stress during seed development and that the effects are manifested as reductions in both yield and
quality.
15
High ambient temperatures during the reproductive development of cool-season field
crops (including canola) can significantly reduce economic yield (Morrison 1993). Morrison and
Stewart (2002) identified 29.5°C as a critical temperature for canola during flowering. Canola
seed yield was reduced in proportion to heat stress units accumulated during flowering, using
29.5°C as the stress base temperature. Temperatures above 32oC can occur on an average of
about 7 days in a given growing season in the Canadian semiarid prairie (McCaig 1997) and
temperatures this high can cause substantial yield losses in Brassica species (Angadi et al. 2000,
Morrison and Stewart 2002, Faraji et al. 2008). In Sothern Ontario, these temperatures occur
frequently during the summer which can be a major limitation for canola production, but heat
stress varies from year to year. Weather data recorded at the Elora Research Station, Ponsonby
ON, during the canola growing season (May 1 to August 31) for 2007 to 2012 indicated 18, 2, 1,
20, 11 and 22 days with temperatures exceeding 29.5°C, respectively (Environment Canada
2012).
Canola plants subjected to high temperatures (“heat blast”) often display increased
abscission of flowers, fruits and even leaves, due to an increased concentration of abscisic acid
(ABA) (Nilsen and Orcutt 1996). Jones (1992) reported that high temperatures reduce plant
biomass by decreasing photosynthesis, while also increasing transpiration (E) and stomatal
conductance (Gs). Crafts-Brandner and Slavucci (2000) showed that high temperature reduces
photosynthesis by contributing to Rubisco deactivation in Gossypium spp. and N. tabacum. The
Canadian Grain Commission reported that high temperatures during the growing season (field
heat stress) can lead to high FFA (free fatty acids) levels in canola seeds, sometimes higher than
1% of total oil (CGC 2012).The presence of FFA in oil suggests that the seeds have been stressed
(CGC 2012).
16
Aksouh et al. (2006) reported that high temperatures during seed maturation
proportionately reduces the percentage of linoleic (18:2) and linolenic (18:3) acids and increases
that of oleic acid (18:1). High temperatures also increase saturated fatty acids such as palmitic
(16:0) and stearic (18:0). These results are similar to earlier reports of rapeseed and other species
exposed to high temperature (Canvin 1965, Green 1986, Gibson and Mullen 1996). This
information was taken as evidence that oleic and linoleic desaturase activities are restricted by
high temperatures.
Qaderi et al. (2006) reported that canola plants grown under high temperatures have
shorter and thinner stems with smaller, thicker leaves, resulting in less leaf area per unit plant
biomass and ultimately less total plant biomass. Yield losses in canola are mainly due to abortion
of the pods or siliques. McGregor (1981) indicated that pod abortion increases in the warmer part
of the growing season and described abortion as “the failure of the pods to elongate, increase in
girth and become seed bearing”. Total seed yield is determined by the number of pods, seeds per
pod and seed weight; of these the number of pods is influenced most by the environment and
thus has the greatest influence on yield (Olsson 1960, Morrison and Stewart 2002). The
environment (high temperatures) can limit the number of seed-bearing pods that may be retained
to maturity, which affects seed yield.
Temperature and water stress are the two most common environmental factors that limit
crop productivity worldwide (Boyer 1982, Hall 1992). Potential oil yields are reduced frequently
by heat and water stresses (Mailer and Cornish 1987, Pritchard et al.2000, Si Ping et al. 2003).
17
High temperatures also accelerate the rate of plant development, thereby reducing the length of
the growing period and reducing the yield potential in T. aestivum L. (Entz and Fowler 1991).
Yield reductions due to high temperatures are very evident when comparing yields
obtained from canola crops grown in cooler areas with those grown in warmer areas (Akouh
2001, 2006). Mahan et al. (1995) reported that canola is under temperature stress for much of its
growing season. High temperature stress can reduce both the source (photosynthesis) and sink
(seed establishment) for photo assimilates (Hall 1992, Paulsen 1994, Mendham and Salisbury
1995). Heat stress can affect the crop during the vegetative stage but the reproductive stage is
most susceptible (Hall 1992, Paulsen 1994, Andagi et al. 2000, 2003). High temperatures during
flowering also cause a reduction in fertility and seed yield in other crops such as wheat (Saini et
al. 1983), maize (Herrero and Johnson 1980, Schoper et al. 1987, Mitchell and Petolino 1988,
Carlson 1990), cotton (Kittock et al. 1988), cowpea (Vigna uniguiculata L. (Walp.)) (Ahmed and
Hall 1993), flax (Linum usitatissimum L.) (Gusta et al. 1997), tomato (Solanum lycopersicum L.)
(Peet et al. 1998, Sato et al. 2002), common bean (Phaseolus vulgaris L.) (Shonnard and Gepts
1994), and pea (Pisum sativum L.) (Guilioni et al. 1997). The range of species adversely
affected by high temperature stress during the reproductive stage suggests that some common
mechanisms may be involved in heat-induced reduction of seed production (Young et al 2003).
Mendham and Salisbury (1995) reported that the effects of heat stress on the seed yield
potential of Brassica crops depends on the developmental stage at which the stress occurs (i.e.,
prior to vs. during flowering) and the duration of the stress. Richards and Thurling (1978), Hall
(1992) and Young et al. (2003) also identified the flowering stage as the most susceptible stage
18
for heat stress damage, probably due to effects on pollen development, anthesis and fertilization
(asynchrony of stamen and gynoecium development). Morrison and Stewart (2002) observed that
flowering of canola is inhibited as the temperature rises above 27ºC, whereas Polowick and
Sawhney (1988) demonstrated that sterile pods and pod abortion occurred at 32ºC. Morrison
(1993) reported completely sterile canola flowers at 27 / 17ºC day / night. Rao et al. (1992)
working with mustard - Brassica juncea L., and Guilioni et al. (1997) and Wang et al. (2006)
working with pea reported that high temperatures accelerate the rate of plant development and
cause abortion of flowers with concomitant loss in seed yield. Heat stress during flowering of
canola can prematurely end flowering, resulting in limited seed set (Faraji, et al. 2008).
Gan et al. (2004) reported on experiments in which canola plants were exposed to three
different temperature regimes (35/18ºC day/night (high), 28/18ºC (moderate) and 20/18ºC
(control)) in growth chambers. Heat stress resulted in a significant reduction in the number of
pods produced on both the main and other racemes; on average the plants at high temperatures
produced 25% of the pod numbers of the control and those at moderate temperatures produced
more than double the number of pods of the plants at high temperatures. The number of seeds per
pod and average seed size was also affected at high and moderate temperatures, both being
reduced by 25% and 22% respectively, compared to the controls. They also reported that total
seed yield per plant was affected by temperature stress, where the plants at moderate and high
temperatures produced yields 14% and 39% lower than the control, respectively.
Andagi et al. (2000, 2003), however, found in growth chamber experiments that the
earlier the heat stress occurred, the greater was the potential for recovery through increased
19
production of pods and branches post-stress. In that work the plants were grown under day/night
temperatures of 20/15°C until early flowering or early pod development, subjected to high
temperature stress of 28/15°C or 35/15°C for seven days, then allowed to recover at 20/15°C. All
of the plants began to recover from stress by continuing to flower after being returned to the
control temperature.
Young et al. (2003) demonstrated that exposure of canola plants to high temperatures at
early flowering reduces whole plant dry matter by 14%, and seed weight per plant by 53%.
Flowers either developed into seedless, parthenocarpic fruit or aborted on the stem in their high
temperature stress (35/15°C) when compared with the controls (23/18°C). They also reported
that under heat stress, pollen viability and germinability were slightly reduced; however, the
plants compensated for lack of fruit and seed production by increasing the number of lateral
inflorescences produced.
McGregor (1981) reported that a loss of reproductive growth early in flowering due to
high temperatures results in the production of branches and/or more flowers on the inflorescence
and/or more seeds per pod and/or increasing seed weight. The response of plants and sensitivity
of reproductive organs to heat stress determines the source-sink relationships within a plant (Hall
1992).
20
2.3
The potential benefits of boron applications.
Farmers in southern Ontario often apply foliar B to their spring canola but the benefits of
this practice are not well documented. Foliar B application has been shown to affect a wide
diversity of processes in vascular plants, such as root elongation, IAA oxidase activity, sugar
translocation, carbohydrate metabolism, nucleic acid synthesis, and pollen tube growth (Blevins
and Lukaszewski, 1998), activity of plasmalemma-bound enzymes regulating ion fluxes (BlaserGrill et al. 1989, Goldbach et al. 2001), levels of exoskeletal proteins in maize (Yu et al. 2003),
accumulation of phenolics and polyamines (Camacho-Cristóbal et al. 2005), and nitrogen
metabolism (Camacho-Cristóbal and Gonzalez-Fontes 2007).
Several researchers have demonstrated that canola has a higher requirement for B than do
cereals (Grant and Bailey 1993). Previous research with other species indicates that B has a
significant role in reproductive development (Thomson and Batjer 1950, Montgomery 1951
(Trifolium hybridum L.), Gauch and Dugger 1954, Gupta 1993, Assad et al. 2003), although the
specific physiological basis of this is still uncertain. To date, it is believed that one of the primary
roles of B is in cell wall structure and function (Herrera-Rodríquez et al. 2010). Others reported
that there is a direct involvement of B in cross-linking of cell wall rhamnogalacturonan II (RGII)
in pectin assembly showing that boron is essential for cell wall structure and function (Loomis
and Durst 1992, Matoh et al. 1993, Ishii and Matsunaga 1996, O’Neill et al. 1996 and more
recently Kobayashi et al. 2004.
Boron deficiency alters the structures of cell walls, leads to oxidative damage, and may
also result in redox imbalance because of the impaired cell wall structure (Kobayashi et al.
2004). Some evidences show that boron is involved in protecting the plasma membrane from
21
oxidative damage caused by toxic oxygen species (Roldan et al. 1992, Ferrol et al. 1993 and
Cakmak and Rӧmheld 1997. Similarly, B has been found to play a role in protecting thylakoid
membranes from oxidative damage (El-Shintinawy 1999). Yamauchi et al. (1986a, b) suggested
that B contributes to calcium metabolism in the cell walls of tomato. Boron also plays a vital role
in root elongation (Dugger 1983, Ali and Jarvis 1988, Marschner 1995. Dell and Huang (1997)
reported that root growth is more sensitive to boron deficiency than is shoot growth.
Other researchers have demonstrated that B is essential to development of both male and
female reproductive organs in canola (Dell and Huang 1997, Assad et al. 2002). Gupta (1993)
also indicated that B is an important factor in the fertilization process of flowers, and B-deficient
plants with may grow normally but seed yield may be severely reduced. Vaughan (1977) and
Blevins and Lukaszewski (1998) reported that B is vital for pollen tube growth and elongation
after pollination. Boron also affects fertilization by increasing the pollen producing capacity of
the anthers and pollen grain viability (Agarwala et al. 1981). Smith and Johnson (1969) (T.
repens L.) and Eriksson (1979) (T. pretense L.) reported that a foliar B application tends to make
clover flowers more attractive to pollinators by increasing the amount and altering the
composition of sugars in the nectar, thereby boosting pollination. The application B increases
canola grain yield by decreasing the number of sterile florets and improving pod development
(Nuttal et al. 1987, Porter 1993). Application of B to canola has also been found to significantly
increase grain oil content, especially when there is a significant effect on grain yield (Pageau et
al. 1999).
22
CHAPTER 3
FIELD EXPERIMENTS
23
3.1 Research Objectives
These experiments were conducted to: i) evaluate the potential benefits of foliar B
applications to spring canola under Ontario growing conditions, in replicated multi-location field
trials, and ii) to test the hypothesis that foliar B applications can increase yields under heat stress
in the field, by reducing pod abortions or by increasing seed size or number of seeds per pod.
3.2 Materials and Methods.
3.2.1 Small plot field trials.
3.2.1.1 Summer 2009
Each field experiment was conducted using a Randomized Complete Block Design
(RCBD) with four replications and five treatments. Treatments were all foliar applications onto
the crop during early flowering (approximately 20% bloom), and consisted of:
1. Control - (no treatment)
2. Foliar B, low rate - 3.4 Lha-1 liquid B (0-0-0-13.3) providing 0.45 kg ha-1 actual B plus
0.25 L ha-1 adjuvant. Spray solution contained 17 mL liquid B plus 1.25 mL adjuvant per
L water.
3. Foliar B, high rate - 6.8 L ha-1 liquid B (0-0-0-13.3) providing 0.90 kg ha-1 actual B plus
0.25 L ha-1 adjuvant. Spray solution contained 34 mL liquid B plus 1.25 mL adjuvant per
L water.
4. Foliar B, high rate - 4.6 kg ha-1 borax (0-0-0-15) providing 0.68 kg ha-1 actual B plus 0.25
L ha-1 adjuvant. Spray solution contained 46 g Borax plus 1.25 mL adjuvant per L water.
5. Adjuvant only - 0.25 L ha-1 or 1.25 mL adjuvant per L water.
24
The adjuvant used was Agral 90 (a.i nonylenoxy polyethoxy ethanol 90%, Syngenta Crop
Protection Canada Inc. Guelph, ON).
These trials were conducted at the Elora Research Station (Elora, Ontario) (43°39’0”N
80°25’0”W, 376 m elev.), Grand Valley (43° 54’43”N 80° 15’50” W, 220 m elev.), Shelburne
(44° 0’ 4” N 80° 0’ 12”W, 254 m elev.) and Meaford (44° 29’15”N 80° 34’ 13”W, 357 m elev.).
The individual plots were 1.42 m x 5 m (7.1 m2); with 3 m between replications (blocks)
and an extra planter tire width (13 cm) between plots to permit access for treatment applications
using a hand-held boom applicator. A single glufosinate-tolerant cultivar was used, InVigor 5030
(Bayer Crop Science, Calgary AB). Seeds were planted with a small-plot cone seeder (Hege
Company, Waldenburg, Germany) with 7” (18 cm) row spacing and 7 rows at a rate of 120 seeds
m-2. Seeds were pretreated with Helix Xtra seed treatment ( a.i. thiamethoxam 20.7%, Syngenta
Crop Protection Canada, Inc.), at a rate of 1.5 mL per 100 g of seeds, for early season control of
flea beetles, seed-borne blackleg, seed-borne Alternaria and the seedling disease complex
(damping-off, seedling blight, seed rot and root rot) caused by Pythium spp., Fusarium spp. and
Rhizoctonia spp. Plots were originally planted 7 m long, then trimmed to 5 m after emergence, so
as to ensure a uniform plant stand over the plot length. All plots received N, P and K fertilizers
according to soil test-based recommendations (OMAFRA 2002), and a soil test for B was also
conducted via the hot water extraction method (A & L Laboratories, London ON). Soil samples
were taken using a soil probe and from two depths (0-15 cm) and (15-30 cm) approximately 3
weeks prior to planting. For detailed soil analysis see appendix.
25
At Elora, pre-plant fertilizer application supplied 50 kg ha-1 actual P2O5 and K2O (0-2020), 100 kg ha-1 actual N (as ammonium nitrate - 34-0-0), and 20 kg ha-1 actual S plus an
additional 18 kg ha-1 N as ammonium sulphate (21-0-0-23). At other locations grower
cooperators fertilized the fields according to their standard practice for canola production.
Weed control was via pre-plant incorporated herbicide Rival (a.i. trifluralin 500 g L-1,
Nufarm Agriculture Inc. AB) and then by a post-emergent herbicide, Liberty (a.i. glufosinateammonium 18%, Bayer Crop Science) at the label recommended rate, applied prior to bolting.
All plots received Matador 120 EC insecticide (a.i. lambda-cyhalothrin 120 g L-1, Syngenta Crop
Protection Canada Inc.) using label recommended rates, applied at the same time that foliar B
treatments were applied (within one week of first flower, generally at 20% bloom).
Foliar B treatments were applied in the form of liquid B containing 10% actual B from
Na2B8O13 4H2 (Alpine Plant Foods Corp, New Hamburg, Ontario) or Borax (15% actual B from
Na2B4O7 110H2O; Plant Products Co. Ltd., Brampton, Ontario). All applications were made using
a hand-held boom with flat fan nozzles, pressurized with CO2 and calibrated to deliver a spray
volume of 200 L ha-1. The boom was held approximately 30 cm from the top of the main
racemes during application (Figure 3.1).
Canopy reflectance spectroscopy measurements were taken one week after foliar B
applications (see Section 3.2.2.1). One week prior to combine harvesting, a hand harvested subsample was taken, for pod count data (see Section 3.2.2.2).
26
Plant heights were measured just prior to harvesting of the entire plots by randomly
selecting a typical canola plant and measuring the distance from soil level to the tip of the main
raceme. Plots were harvested with a small plot combine (Wintersteiger, Laval QC). Seeds were
then placed in forced air driers at 60°C, for 3 days. The dried seeds were cleaned with a seed
cleaner (model ASC-3, Agriculex Inc., Guelph ON) to remove stem and pod remnants. Brown
seed counts and FFA content were determined for the bulk harvests from each plot (see sections
3.2.3.1 and 3.2.3.3). Analysis of variance and treatment contrasts were conducted using the
PROC GLM procedure in SAS (Version 9.2, SAS Institute Inc. Cary NC) to identify significant
treatment effects. The assumption of normal distribution of residual values was tested using the
PROC UNIVARIATE procedure in SAS (Shapiro-Wilks test for normality) and by examining
plots of residual vs. predicted values for each response variable. Treatment effects and planned
contrasts between groups of treatments were declared significant at P<0.1 since we expected a
priori that treatment effects would be small.
3.2.1.2 Summer 2010
Trials were conducted using the same experimental design and procedures as in 2009;
however, an additional treatment was included, which was:
Foliar B, low rate - 2.3 kg ha-1 borax (0-0-0-15) providing 0.34 kg ha-1 actual B plus 0.25 L ha-1
adjuvant. Spray solution contained 23 g borax plus 1.25 mL adjuvant per L water.
The same adjuvant was used as in 2009, Agral 90. Trial locations were the same as in 2009, but
in different fields.
27
3.2.1.3 Summer 2011
Trials were conducted using the same experimental design as in 2009 and 2010, but with
nine replications and the number of treatments was reduced to the following:
1. Control - (no treatment)
2. Foliar B, high rate - 6.8 L ha-1 liquid B (0-0-0-13.3) providing 0.90 kg ha-1 actual B plus
0.25 L ha-1 adjuvant. Spray solution contained 34 mL liquid B plus 1.25 mL adjuvant per
L water.
3. Adjuvant only - 0.25 L ha-1 or 1.25 mL adjuvant per L water.
The same adjuvant was used as in previous years, Agral 90.
Trial locations were the same as in 2009 and 2010, except for Grand Valley. The Grand
Valley location was replaced by a trial in Dundalk, Ontario (44° 5’ 22” N 80° 29’ 41”W, 526 m
elev.).
3.2.1.4 Summer 2012
Experimental trials were conducted using the same experimental design as in 2011, but
with four replications. The seeding rate was reduced to 100 seeds m-2.
These trials were also conducted on three of the same locations common to 2009, 2010
and 2011, but the Dundalk location was no longer available so that location was replaced by a
site in Arthur, Ontario (43° 82’ 33” N 80° 38’ 42”W, 250 m elev.). In 2012 a different
glufosinate-tolerant cultivar was used, InVigor 5440 (Bayer Crop Science), replacing InVigor
5030.
28
Figure 3.1. The hand-held boom with flat fan nozzle sprayer, pressurized with CO2, used for
treatment applications.
29
3.2.2 Field protocols
3.2.2.1 Canopy Reflectance Measurements.
Canopy reflectance measurements were taken using a Unispec-DC Spectral Analysis
System (PP Systems International Inc. Haverhill, MA. (Figure 3.2) on all plots at full flower
(approximately one week after B application), to detect possible effects of B on flowering. An
upward facing probe measured the incoming light and a second probe pointing down at the
vegetation at a 45° angle measured the reflected light simultaneously. Four scans were taken per
plot; this was done for all years except 2012. We calculated a normalized difference index based
on the yellow (560 nm) and near infrared (860 nm) wavebands (NDI560/850) as a measure of
canopy flower cover:
NDI560/850 = (R850-R560) / (R850+R560),
Where Rλ is fractional canopy reflectance of incident solar radiation in the 3-nm waveband
centered on λ.
30
Figure 3.2. The reflectance spectrometer (Unispec DC, PP Systems, MA, USA) used to measure canopy
reflectance in the field one week after B applications.
31
3.2.2.2 Protocol for Hand-harvesting and Pod Counts.
Samples were taken at about “swathing time” (approximately one week before direct
combining) so as to avoid shattering, and were taken from checks and foliar B treatments only.
Ten plants were removed from plots by cutting them below the lowest pod-bearing branch. Five
contiguous plants were taken from the second and sixth row, avoiding the first and last 1m of
each row. Sample locations were selected to avoid sampling unusually small, diseased or insector bird-damaged plants.
The main raceme was located for each plant, by starting at the bottom of the plant and
following the thickest stem section upwards. The number of set pods on the main raceme was
counted starting from the bottom going upwards. The number of aborted pods (including pod
scars and aborted flowers at the top of the raceme) was then counted on the main raceme.
Finally, the main racemes were detached from the rest of the plant and all ten main racemes from
one plot were placed in the same bag, which was then placed in a drier for subsequent hand
threshing.
Once the main racemes were processed the number of viable pods and aborted pods on
the other branches were counted, and then all branches were placed together in one bag for
drying and subsequent threshing. After hand threshing the seeds were cleaned with a column
blower (CB-1, Agriculex Inc. Guelph). Total seed weight and 1000-seed weight were determined
for all hand-harvested main raceme and branch samples.
32
Pod count and seed number data from the hand harvests were converted to a ground area
basis (pods m-2, seeds m-2). This was done by taking the count from each 10-plant sample, and
multiplying by the ratio of the bulk (machine) harvest dry seed weight (g m-2) to the hand-harvest
sample dry seed weight for that plot (g).
3.2.3 Quality analyses
3.2.3.1 Green and Brown Seeds Determination.
Canola seeds from the machine harvested samples were counted using a 100-well seed
counting paddle. The seeds were collected into the wells, and then taped onto a strip of masking
tape, which was inverted and placed on a flat surface and crushed with a roller (Figure 3.3).
Crushed seeds were individually inspected for colour variations from the normal yellow. The
percentages of green and brown seeds were recorded. This process was repeated again for each
sample, so as to get two crush strips per plot.
33
Figure 3.3. The tools used for crushing seeds (left) and a strip of 100 crushed canola seeds
(right).
34
3.2.3.2 Oil and Protein Analysis
Seeds from the machine harvest were analyzed for percent oil and percent protein using a
Technicon Infra Alyzer 450-NIR, Grain Analyzer, (Technicon Ireland Ltd.), calibrated with
standards provided by the Canadian Grains Commission.
3.2.3.3 Procedures for FFA Analysis
Free fatty acids were determined for the machine-harvested seed according to the method
of May and Hume (1993). For each sample 20 g of canola seeds were oven dried at 80°C for at
least 8 hours to facilitate grinding. After cooling, approximately 10 g of seeds were ground with
a coffee grinder. 400 mg of the ground seeds were placed on a 10-mL test tube and 1mL of
solution I (internal standard containing 1g heptadecanoic acid (C-17) (Fisher Scientific; Fair
Lawn, New Jersey, USA) dissolved in 1L of HPLC grade heptane) and 4 mL of solution II
(containing 40 parts isopropyl alcohol plus 1 part 1NH2SO4) were added. The test tube was
shaken vigorously using a vortex and let stand for 15 mins., after which 2 mL of heptane and 3
mL of deionised distilled water were added. The tube was shaken again then let stand a few
minutes to allow for phase separation. After the phase separation, a sample from the top layer
was withdrawn and placed in a GC auto sampler vial for analysis.
For FFA analysis a Hewlett Packard (HP) 6890 Series Gas Chromatograph (GC)
(Agilent, Wilmington, DE, USA) was used, equipped with a flame ionization detector (FID). The
column was a J&W 125-3212 DB-FFAP supplied by Chromatographic Specialities (Brockville
ON, Canada). The column was 15 m in length, 530 µm in diameter, with a1-µm film thickness.
The GC, inlet and detector temperatures were 270 and 330°C, respectively. The pressure on the
35
column was 153 kPa and the oven temperature program started at 220°C with a ramp rate of 1°C
min-1 to 225°C. The temperature was held at 225°C for 3.5 mins., followed by a ramp rate of 5°C
min-1 to 230°C. The oven stayed at 230°C for 1.5 mins. and was then cooled back to the starting
temperature. Total run time was 12 mins. The carrier gas was helium. Injection volume of the
sample was 2.0 µL, and a split injection (10:1) was used.
36
3.3 Results
Field trials were conducted over a period of 4 years at five locations in Ontario; however,
data were not obtained for all the sites in all years as some sites failed to produce acceptable data
due to various reasons (see Table 3.1).
The 2009 experimental year was a cool one (with reference to the 29.5°C temperature
identified by Morrison and Stewart (2002) as a critical temperature for heat stress in canola
during flowering) with adequate moisture evenly distributed throughout the growing season as
can be seen for Elora, ON (Figure 3.4), whereas the preliminary seasons at Elora (2007 and
2008) were warmer (Figure 1.1 and 1.2). The growing season in 2010 was warmer than 2009
with evenly distributed moisture during the middle part of the growing season at Elora (Figure
3.5). The growing season in 2011 was similar to 2010 in terms of the number of days above
29.5°C; however, there was a very hot and dry period between June 30 and July 30 in that year
(Figure 3.6). The 2012 growing season at Elora was hotter and drier than previous years, with a
long hot and dry period (from June 10 to July 25) and a lower cumulative precipitation (Figure
3.7).
Canopy reflectance spectroscopy measurements were taken at all sites on all plots at full
flower (approximately one week after B application) to detect possible effects of B on flowering.
These measurements showed no treatment effects at the Elora or Shelburne sites in 2009. At the
Meaford site, the check and adjuvant only treatments were significantly more reflective in the
yellow to orange region of the spectrum than were the B-containing treatments (Table 3.2). This
is consistent with a greater flower density in the check and adjuvant only treatments (Figure 3.8).
37
Reflectance measurements for the 2010 and 2011 growing seasons showed no significant effects
when contrasting the +B and – B treatments. No reflectance measurements were made in 2012.
Foliar B application did not significantly affect yield at either the Elora or the Shelburne
sites in 2009 (Table 3.3). Canola yields at the Meaford site were the highest that have ever been
recorded in small plot trials in Ontario. At that location, foliar B significantly suppressed yields,
as evidenced by a contrast between the three treatments containing B and the two treatments
without B (Table 3.3). The overall effect of B applications in 2009 was negative averaged across
sites, but this was entirely due to the strong and statistically significant negative response for
Meaford (- 6.7%) (Table 3.7).
In 2010, all of the field trials failed except Elora (Table 3.1). Table 3.4 indicates a
significant positive yield response to the foliar B application compared with the treatments
without B, in both individual treatment effects and contrasts between B treatments as a group and
treatments without B as a group. The average yield benefit from foliar B was 6.3% (Table 3.7).
However, contrasts between the low rate B treatments and the treatments without B, or between
high and low rates of B were not significant (Table 3.4).
Only three of the four locations produced any statistically acceptable data for the 2011
season (Table 3.1). There were no significant effects of foliar B on yield at Elora or Shelburne.
However, at a low yielding site at Dundalk, there was a significant yield response to foliar B and
a contrast between the B treatment and the two treatments without B was also significant (Table
3.5). The yield benefit from B in this case was 5.2% (Table 3.7).
38
Two experimental sites (Shelburne and Arthur) in 2012 produced no data (Table 3.1). Of
the other two sites (Elora and Meaford), only the latter showed a significant increase in yields
with foliar B treatment (Table 3.6) with an overall B effect of 5.4% (Table 3.7).
Table 3.8 shows the soil B levels determined by testing of soil samples collected prior to
planting. We found no correlation between soil B test results and the responsiveness of canola
yields to foliar B (analysis not shown).
Averaged across all nine location-years that produced data, there were few significant
effects of B treatment on yield components (Table 3.9). There was a small but significant
increase in the number of seeds per pod on main racemes, and a significant reduction in the
number of aborted pods on the branches. (Table 3.9).
However, considering only the three location-years with a positive response to foliar B
applications (Elora 2010, Dundalk 2011 and Meaford 2012; Table 3.7) there was a significant
increase in the number of viable pods on the main raceme with B application (Table 3.10). This
was associated with in increased number of seeds on the main raceme. Boron application also
decreased the number of aborted pods on the other branches. Overall, in addition to increasing
seed yield, B treatment tended to alter the yield distribution; there was an increase in the fraction
of viable pods, seed numbers and seed weight located on the main raceme, as opposed to the
other branches (Table 3.10). The fraction of aborted pods on the main raceme (aborted pods
39
divided by aborted + viable pods) was also decreased by B treatment, although the effect was not
quite statistically significant (P = 0.11) (Table 3.10).
On the other hand, for the single location where B treatments resulted in a negative yield
response (Meaford 2009 :Table 3.7), there was a significant decrease in the number of seeds on
the main raceme, and the fraction of total viable pods located on the main raceme also decreased
(Table 3.11). Numerically, the number of pods on the main raceme was reduced by B treatment
(although this effect was not quite statistically significant: P = 0.11). There was also a similar
trend towards a reduced number of aborted pods on the main raceme (P = 0.11) (Table 3.11). For
pod count results for individual location-years, see the Appendix.
Quality analyses for percent green and brown seed showed no significant treatment
effects (not shown), and free fatty acids levels also showed no significant treatment effects in
2009. In 2010, at the only location harvested, a contrast between B and no B treatments showed a
significant decrease in FFA levels for treatments containing B. In 2011, Elora and Shelburne
showed no B effect on FFA levels, but at Dundalk there was a significant decrease in FFA levels
for B-containing treatments relative to treatments containing no B. In 2012, there was no
treatment effect on seed FFA levels (Table 3.12).
40
Table 3.1. A summary of years and locations where field experiments were conducted in Ontario and the outcome at each
experimental site.
41
Figure 3.4. Growing season weather data for Elora ON, 2009.
Daily high and low temperatures are shown by closed and open circles, respectively. Cumulative
precipitation beginning May 1 is shown as the solid line. The dashed horizontal line represents
the temperature of 29.5°C, identified by Morrison and Stewart (2002) as a critical temperature
for heat stress effects in canola during the flowering period.
42
Figure 3.5. Growing season weather data for Elora ON, 2010.
Daily high and low temperatures are shown by closed and open circles, respectively. Cumulative
precipitation beginning May 1 is shown as the solid line. The dashed horizontal line represents
the temperature of 29.5°C, identified by Morrison and Stewart (2002) as a critical temperature
for heat stress effects in canola during the flowering period.
43
Figure 3.6. Growing season weather data for Elora ON, 2011.
Daily high and low temperatures are shown by closed and open circles, respectively. Cumulative
precipitation beginning May 1 is shown as the solid line. The dashed horizontal line represents
the temperature of 29.5°C, identified by Morrison and Stewart (2002) as a critical temperature
for heat stress effects in canola during the flowering period.
44
Figure 3.7. Growing season weather data for Elora ON, 2012.
Daily high and low temperatures are shown by closed and open circles, respectively. Cumulative
precipitation beginning May 1 is shown as the solid line. The dashed horizontal line represents
the temperature of 29.5°C, identified by Morrison and Stewart (2002) as a critical temperature
for heat stress effects in canola during the flowering period.
45
Table 3.2. Effect of foliar B treatments on canopy colour, as measured by canopy reflectance
spectroscopy measurements for seven location years.
The canopy “yellowness”, measured as a normalized difference index based on reflectance at
560 nm and 850 nm was used as an indication of flower cover. Measurements were made near
peak flower (about 1 week after foliar B treatments were applied).
(1)
The Normalized Difference Index was calculated using the following formula: NDI560/850 = (R850-R560)
/ (R850+R560).
46
Figure 3.8. Canopy reflectance spectroscopy measurements normalized to reflectance at 850 nm
for small plot spring canola trials at Meaford ON in 2009.
Each line is a mean of four scans per plot over four replications and scans were taken one week
after B applications.
47
Table 3.3. Spring canola yield response to foliar B applications in 2009.
Yield results for three locations (Elora, Shelburne and Meaford) and contrasts between
treatments without B (Check and Adjuvant only) and treatments containing B (Low liquid B +
Adjuvant, High liquid B + Adjuvant and Low Borax + Adjuvant), for these locations.
48
Table 3.4. Spring canola yield response to foliar B applications in 2010.
Yield results for Elora, ON and contrasts between treatments without B (Check and Adjuvant
only) and treatments containing B (Low liquid B + Adjuvant, High liquid B + Adjuvant, Low
Borax + Adjuvant and High Borax + Adjuvant), High B rate treatments (High liquid B +
Adjuvant and High Borax + Adjuvant) and Check, Low B treatments (Low liquid B + Adjuvant
and Low Borax + Adjuvant) and Check, and Low B treatments and High B treatment for this
location in 2010.
49
Table 3.5. Spring canola yield response to foliar B applications in 2011.
Yield results for three locations (Elora, Dundalk and Shelburne) and contrasts between
treatments without B (Check and Adjuvant only) and treatments containing B (High liquid B +
Adjuvant) for these locations in 2011.
50
Table 3.6. Spring canola yield response to foliar B applications for two locations (Elora and
Meaford) in 2012.
51
Table 3.7. Summary of the effects of foliar B treatment on yield of spring canola for 13 locationyears and means across locations for 6 years. Percentage yield increases/ decreases are shown in
bold when the B effect was significant at P<0.1.
52
Table 3.8. Summary of 13 location-years with yield effects of foliar B treatment on spring
canola and the available soil B as per soil test analysis.
For allocations prior to planting.
Year
Location Check
Foliar B
2007
2007
2008
2008
2009
2009
2009
2010
2011
2011
2011
2012
2012
__Yield (Mg ha -1)__
Elora
2.79
2.94
Meaford
3.47
3.68
Elora
3.21
3.29
Meaford
4.81
4.83
Elora
2.95
2.82
Meaford
7.91
7.38
Shelburne 3.26
3.24
Elora
2.54
2.70
3.32
3.31
Elora
Dundalk
1.93
2.03
Shelburne 2.28
2.30
Elora
1.69
1.69
Meaford
3.87
4.08
53
Effect (%) Soil B (ppm)
+5.3
+6.1
+2.4
+0.4
- 4.4
- 6.1
- 0.6
+ 6.3
- 0.3
+ 5.2
+ 0.1
0.0
+ 5.4
1.00
0.90
0.48
NA
1.10
0.80
1.15
1.45
0.41
0.49
0.54
0.75
0.45
Table 3.9. The parameters measured from hand harvested samples for all location-years (9
locations).
54
Table 3.10. The parameters measured on the hand harvested samples for location-years with a
positive response to B treatment.
Three locations (Elora 2010, Dundalk 2011 and Meaford 2012).
55
Table 3.11. The parameters measured on hand harvested samples for location-years with a
negative response to foliar B treatments.
One location (Meaford 2009).
56
Table 3.12. Effect of foliar B treatment on seed free fatty acid levels for all location-years (9
locations).
57
3.4 Discussion
The effect of foliar B on spring canola was not consistent throughout this study, but was
evident in most seasons, though not at all of the locations for these seasons. Soil B content as
determined by a soil test was not found to be predictive of the yield response to foliar B. Past
research has found varying levels of yield response to both soil-applied and foliar-applied B in
spring canola. Pageau et al. (1999) reported that in experiments conducted in Northern Quebec
soil-applied B at rates of 0.5, 1.0 and 2.0 kg ha-1 resulted in yield increases of 19, 26 and 31%,
respectively on boron deficient sites. In Blackville SC, Porter (1993) found that growing canola
crops on a Plinthic Paleudult soil, plants receiving foliar B had a 6.5% higher yield than those
receiving no B over the combined two years of study.
The growing conditions over the course of these field experiments were highly variable.
In 2007, a hot year, spring canola yields were increased by foliar B treatments by 5.7% when
averaged across both locations. In 2008, a much cooler year, the mean effect across both
locations was very small (1.2% increase) and not statistically significant. The 2009 season was a
canola-friendly year with minimal heat stress and adequate moisture. It was under these highyielding conditions that a negative response to foliar B was observed at Meaford, with no yield
response at the other two, lower-yielding locations. In contrast, 2010 was much warmer (but
with well distributed precipitation throughout the growing season); under these conditions a
significant yield increase with foliar B was observed at Elora. In 2011, a hotter year with
relatively poor distribution of precipitation, foliar B application had a positive effect on yield, but
only at the lowest-yielding location (Dundalk). Elora was higher yielding in 2011 than in 2010,
and was only responsive to foliar B in the lower-yielding year. In 2012, an extremely hot and
58
dry year at Elora, there was no response to foliar B; however, that trial was very low-yielding
due to water stress, with a very high CV for yield (19.8%). At Meaford in the same year, where
temperatures were also very hot but soil water was less limiting and yields were higher, there
was a positive response to foliar B.
Although no interaction between temperature and foliar B can be conclusively inferred
from these field trials (since temperature differences between the location-years are confounded
with any other differences that existed between the trials), in general the results support the
hypothesis that foliar B is more beneficial when yields are limited by high temperatures. No
benefit was seen in either of the cool years (2008, 2009), but responses were observed at least at
some sites in the warmer years (2007, 2009, 2010, 2011, 2012). Aksouh et al. (2001) found that
canola is susceptible to even short episodes of heat stress during seed development and that the
effects are manifested as reductions in both yield and quality.
The physiological basis of the foliar B effect is not completely clear, but some specific
trends were noticeable in the hand-harvested samples. Averaged across all nine location-years
where yield components were measured, foliar B was found to slightly reduce pod abortions on
the branches, and slightly increase seed number per pod on the main raceme. When considering
only those three location-years where a significant positive response to foliar B was observed,
the treatment still reduced the number of pod abortions on the branches. However, foliar B also
significantly improved all aspects of yield formation on the main raceme; the number of pods
and number of seeds on the main raceme increased with foliar B treatment, while pod and seed
numbers on the branches were not affected. Based on these observations, we speculate that
59
foliar B, applied during the early stages of main raceme flowering, tends to increase the number
of pods that are successfully set and filled in the main raceme. This appeared to result more
from an increase in main raceme flower production than from a reduction in main raceme pod
abortions, since the number of viable pods increased more than the number of aborted pods
declined. This increase in pod set also results in an overall shift in reproductive effort away from
the branches and towards the main raceme; while foliar B did decrease the number of pod
abortions on the branches, this did not result in an increase in pod numbers or seed yield on the
branches. In fact there was a (non-significant) reduction in pod numbers on the branches, but
this was smaller than the increase in pod numbers on the main raceme, so total crop yield
increased. The fraction of total pod numbers on the main raceme, fraction of total seed numbers
on the main raceme, and fraction of total seed weight on the main raceme all increased.
The single case where a significant yield reduction was observed in response to foliar B
(Meaford 2009) deserves special consideration. In this extremely high-yielding trial, main
raceme pod numbers were more than three times the typical number. Since flowering occurs
sequentially from the bottom to the top of the main raceme, in such long racemes the
developmental delay between the bottom and top of the raceme may have been similar to the
normal delay between the main raceme and the branches. In this scenario, foliar B application
reduced main raceme pod set, resulting in a reduced yield. That is, pod set on the laterdeveloping part of the main raceme may have been reduced by foliar B treatment in the same
manner as observed for the branches at other locations. The canopy reflectance data from this
60
site seem to confirm this, as they indicated a reduced flower density in the upper canopy of plots
that received B, consistent with arrested flower production on main racemes.
Taken together, these results suggest a model whereby foliar B exerts its effect by
favouring allocation of resources to regions of the plant that are currently flowering and setting
pods, to the possible detriment of flowering positions that would otherwise be establishing pods
shortly afterwards. In resource-limited scenarios, this could result in a yield benefit by reducing
allocation of resource to extraneous reproductive effort that would not ultimately produce mature
seed-bearing pods. In non-resource limited environments such as the Meaford 2009 trial, where
the crop could support extremely high pod loads, the resulting reduction in total potential pod
numbers would be detrimental to yield.
Our data also suggest that the benefits of foliar B are more likely to be observed under
conditions of heat stress. The sensitivity of reproductive development in canola to heat stress is
well documented. Morrison (1993) showed that high temperature stress causes sterility in
canola, reducing fertilization and resulting in parthenocarpy and reduced yields. Brandt and
McGregor (1997) reported that the yield of canola on summer fallow in Saskatchewan is closely
related to temperature during flowering and early seed development. Morrison and Stewart
(1993) showed that temperatures above 27°C can also lead to floral sterility and yield loss in
canola in controlled environment studies. Faraji et al. (2008) demonstrated that lower
temperatures result in greater seed yield in canola grown in Iran.
61
Previous research also implicates B fertility in reproductive success of canola. For
example, Nuttal et al. (1987) concluded from field studies done in Saskatchewan that B enhances
yield by decreasing the number of sterile florets and improving pod development, similar to our
observations. Also, Dell and Huang (1997) reported that in the field setting, sexual reproduction
is often more sensitive to low soil B than is vegetative growth, and seed yield reductions can
occur without symptoms being expressed during prior vegetative growth in canola. Assad et al.,
(2002) demonstrated in controlled environment studies that the reproductive stage of canola is
more sensitive than the vegetative stage to low B supply.
In conclusion, we found that foliar B application during early flowering sometimes
resulted in enhanced yield of spring canola, and that when those yield increases occurred they
were associated with increased pod numbers on the main raceme, but not on the branches. The
data also suggested that these beneficial effects of foliar B are more likely to be observed for
crops exposed to heat stress. This hypothesized interaction between heat stress and foliar B is
further investigated in the controlled environment study described in Chapter 4.
62
CHAPTER 4
CONTROLLED ENVIRONMENT EXPERIMENTS
63
4.1 Research Objectives
The results of the field trials described in Chapter 3 suggested that there may be an
interaction between foliar B application and temperature in spring canola, such that a yield
benefit from the B application is only observed when the crop is exposed to heat stress.
However, data in support of this idea were circumstantial, in that effects of temperature per se
could not be separated from any of the other factors that differentiated the various field locationyears from one another. To more rigorously test the hypothesized interaction between
temperature and foliar B application, we conducted a series of controlled environment
experiments with the following objectives:
1. To identify a reproductive stage heat stress protocol that produces significant yield
reductions in spring canola, with heat stress symptoms similar to those observed under field
conditions.
2. To test the hypothesis that foliar B applications can mitigate the effects of heat stress
on pod set in spring canola.
4.2 Materials and Methods
4.2.1 Plant Culture Systems and Stress Treatments.
64
4.2.1.1 Experiment I
A controlled environment experiment was conducted in the greenhouses and growth
cabinets of the University of Guelph, Ontario, Canada in the winter of 2009. Spring canola plants
were grown in the greenhouse with target temperatures of 20/15°C day/night with a photoperiod
of 16 hours / 8 hours light/dark, programmed for shade cloth deployment to control solar heating
above 35°C. Day length was extended with overhead high pressure sodium and metal halide
lamps delivering an additional flux of photosynthetically active radiation (PAR) of
approximately 600 µm m-2s-1 at the top of the plants during the experiment. A single LibertyLink cultivar was used as in the field trials, InVigor 5030 (Bayer Crop Science). Seeds were
pretreated as in the field trials with Helix X Tra seed treatment (a.i. thiamethoxam 20.7%,
Syngenta Crop Protection Canada, Inc.), at a rate of 1.5 ml 100 g-1 of seeds. Seeds were planted
approximately five seeds per pot in batches of seventy and were gradually thinned out leaving
one plant per pot.
Five-litre pots were filled with potting soil (Sunshine LA4- Mix, SunGro Inc. Vancouver,
Canada). Pots were arranged randomly on the bench, but positioned so as to maintain a uniform
density. Plants were watered daily and rotated to minimize bench position effects. Fertilizer was
applied daily with irrigation water, at a rate of 0.5 g L-1 of All Purpose 20:20:20 fertilizer (Plant
Products Co. Ltd., Brampton, Canada). Plants were kept well-watered at all time to prevent water
stress. Planting was staggered so that plants to be moved to the growth cabinets for heat stress
treatment would be at the various pre-defined developmental stages all at the same time (first
flower, 7 days after first flower or 14 days after first flower).
65
At the first flower stage (Figure 4.1), plants were moved into controlled environment
growth chambers (model Conviron PGW 36, Controlled Environment Limited, Winnipeg,
Canada) with light provided by a combination of Cool White and GroLite florescent tubes and
40-W clear incandescent bulbs (Sylvania, Arrow Rd, Guelph, ON Canada). The photosynthetic
photon flux density (PPFD) was maintained at 600 µmolm-2s-1, measured at the top of the plants.
Transfers from the greenhouse to the growth cabinets were made always at 10:00 am.
The treatments consisted of:
a) Exposure to one of three different growth cabinet temperatures (day/night):
1. 20/15°C ( Control)
2. 28/20°C
3. 32/20°C
b) Exposures beginning at one of three different developmental stages:
1. First flower (FF)
2. 7 days after first flower (7FF)
3. 14 days after first flower (14FF)
c) Exposure for one of three different durations:
1. 3 days
2. 7 days
3. 14 days.
There were therefore 27 growth cabinet treatment combinations, with six plants per treatment.
Each temperature setting was applied only once, with the nine starting time x exposure time
combinations applied to six plants (subsamples) within that treatment. The three temperature
treatments were applied sequentially, using the same growth cabinet. Thus, the experiment was
66
unreplicated, in the sense that each heat stress protocol was tested only once, sequentially – this
was a design compromise for this preliminary experiment, necessitated by the limited number of
available growth cabinets. Within a temperature, the experiment was treated as a completely
randomized design, with six replications of each exposure timing / duration treatment. This
permitted the detection of significant differences between the various exposure timing / duration
combinations within a temperature treatment, and between them and the associated greenhouse
check.
Plants were returned to the green house after the exposure treatment period, where they
remained for further growth and seed setting. In addition, there was a “greenhouse control”
treatment for each temperature treatment – six plants that were never moved from the greenhouse
to the growth cabinet. For each temperature treatment then, 60 plants were needed (six plants for
each of the nine exposure time x exposure duration combination, plus six for the greenhouse
control). The most uniform 60 plants were selected from among the 70 available, and the
remaining 10 plants were discarded.
Final data collection included seed weight per plant, and seed number per plant. These
parameters were measured separately for a) the main raceme and b) the rest of the plant. For
main racemes only, number of pods and number of aborted pods were also determined. Brown
seed percent and seed FFA content were measured for each “main raceme” and “rest of plant”
sample; because seed quantity was insufficient to perform these measurements for main racemes
of individual plants, the samples were instead bulked samples across the six plants. For each
temperature treatment, an analysis of variance was conducted using the PROC GLM procedure in
67
SAS to identify the significant effects of exposure time and exposure duration on the response
variables, treating each plant as an independent experimental unit.
4.2.1.2 Experiment II
This experiment was conducted using a control treatment (no stress) and a suitable heat
stress protocol, identified from Experiment I, and was also conducted in the greenhouses and
growth cabinets of the University of Guelph, Ontario, Canada in the winter of 2010. The design
was a split plot with six replications of the main plot factor (temperature; replicated in time), and
six sub-plot factors (foliar treatments).
Based on the results obtained in Experiment I, a single exposure duration x temperature
regime x developmental stage combination was selected. Plants were grown in the greenhouse as
described for Experiment I. Three days after first flower, plants were treated with one of six
different foliar treatments:
1.
Control - (no treatment)
2.
Liquid B, low rate - 3.4 L ha-1 liquid B (0-0-0-13.3) providing 0.45 kg ha-1 actual
B plus 0.25 L ha-1 adjuvant. Spray solution contained 17 mL liquid B plus 1.25
mL adjuvant per L water.
3.
Liquid B, high rate - 6.8 L ha-1 liquid B (0-0-0-13.3) providing 0.90 kg ha-1 actual
B plus 0.25 L ha-1 adjuvant. Spray solution contained 34 mL liquid B plus 1.25
mL adjuvant per L water.
68
4.
Borax, low rate - 2.3 kg ha-1 borax (0-0-0-15) providing 0.34 kg ha-1 actual B plus
0.25 L ha-1 adjuvant. Spray solution contained 23 g borax plus 1.25 mL adjuvant
per L water.
5.
Borax, high rate - 4.6 kg ha-1 borax (0-0-0-15) providing 0.68 kg ha-1 actual B
plus 0.25 L ha-1 adjuvant. Spray solution contained 46 g borax plus 1.25 mL
adjuvant per L water.
6.
Adjuvant only - 0.25 L ha-1 or 1.25 mL adjuvant per L water.
These treatments were identical to those used in the 2010 field experiments. They were
applied to emulate field application at a volume of 200 L ha-1, by arranging the plants on the
floor and using the same hand-held boom sprayer (Figure 5). The liquid boron was a
commercially available formulation (Alpine Plant Foods, New Hamburg, ON) The adjuvant was
Agral 90, (Norac Concepts Inc. Guelph, ON).
Four days after the foliar treatments were applied (7 days after first flower), half of the
plants from each foliar treatment were transferred to a controlled environment cabinet set at
20/15°C day / night temperature (Control), and the other half were transferred to another
controlled environment cabinet set at 28/20°C day / night temperature (Stress). The cabinets
were the same model used in Experiment I. Temperature treatments were randomly assigned to
the two growth cabinets with each replication of the experiment.
69
There were two plants per foliar treatment in each temperature regime. After 14 days in
the growth cabinets, the plants were returned to the greenhouse to grow to maturity, maintaining
the spatial arrangement of the split plot design. The entire experiment was repeated six times, to
provide six true replicates of the main plot (temperature stress) factor.
At maturity the main racemes were removed, and the viable and aborted pods
were counted. Seed weights were recorded for both main racemes and other branches. Measured
parameters were the same as for Experiment 1. An analysis of variance was conducted using the
Proc Glm procedure in SAS (Version 9.2, SAS Institute Inc. Cary NC), considering the
experiment as a split plot design with six replications. The temperature regime was the main plot
factor and the foliar treatment was the sub plot factor.
4.2.2 Measurements
4.2.2.1 Protocol for Hand-harvesting and Pod counts.
Plants were harvested from the pots by cutting them below the lowest pod bearing
branch. The main raceme was located for each plant, by starting at the bottom of the plant and
following the thickest stem section upwards. The number of set pods on the main raceme was
counted starting from the bottom going upwards. The number of aborted pods (including pod
scars and aborted flowers at the top of the raceme) were then counted on the main raceme and
recorded. Finally, the main racemes were detached from the rest of the plant and these were
placed in individual bags, which were then placed in a forced air drier at 60°C until they reached
constant weight, for subsequent hand threshing.
70
4.2.3 Procedures for FFA analysis
This procedure remained unchanged from the field experiments, except that similar
treatments were bulked across replications so as to have enough seed for the analysis.
71
Figure 4.1. Spring canola plants at 3 days after first flower in the growth cabinet for heat stress
treatments.
72
4.3 Results
In Experiment I, the 32/20°C and 28/20°C temperature regimes and exposure durations
produced statistically significant (P<0.1) effects on numbers of viable pods, aborted pods and
total pods (sum of viable and aborted pods), when compared with the greenhouse check. In
contrast, the 20/15°C temperature regime did not affect total pod numbers, and had only minor
effects on numbers of viable pods and the aborted pods (Figure 4.2).
The 32/20°C temperature regime produced very high pod abortions in all nine
starting time x duration treatments when compared with the greenhouse check, and also
significantly fewer viable pods (Figure 4.2 top). These effects were strongest when the
temperature stress was applied early during flowering, and became less marked (but still
significant) if the onset of the stress was delayed until 14 d after first flower. For the stress
treatments beginning at first flower or 7 d after first flower, the longer duration stresses (7 or 14
d) reduced viable pod numbers more strongly than did the 3-d stress duration. In general, plants
exposed to the 32/20°C stress treatment were taller and viney looking with distorted flowers and
pods (Figures 4.4 and 4.5).
When the 28/20°C stress protocol was tested, the greenhouse check had lower pod
numbers than the greenhouse check for the 32/20°C experiment. However, relative to its
corresponding greenhouse check, the 28/20°C temperature regime affected pod counts in much
the same manner as the 32/20°C regime, although the magnitude of the effects was somewhat
less. There were stronger effects when temperature exposure was applied early during flowering,
and they became less marked (but still significant) if the heat exposure was delayed. For the
73
stress treatments beginning at first flower the 7- or 14-d exposure reduced viable pod numbers
more than did the 3-d stress duration (Figure 4.2 middle).
Based on the results of Experiment I, the treatment identified to be used as the heat stress
protocol in Experiment II was the 28/20°C temperature regime beginning seven days after first
flower and lasting for fourteen days (indicated by the grey oval on Figure 4.2 middle).
Figure 4.3 shows how treatment effects on the main raceme yield components (number of
viable pods on main raceme, seeds per pod, and average seed weight) combined to significantly
reduce main raceme seed yield for the 28/20°C temperature treatments. For the treatment chosen
for Experiment II, pod number and seed weight were more strongly reduced than seeds per pod.
In Experiment II, plants were very highly branched, with approximately 87% of the total
seed yield borne on the branches and only 13% on the main racemes (Table 4.1). Effects of
temperature and foliar B treatments on whole-plant seed yield were minor, but there were strong
effects on the yield of main racemes. Averaged across foliar treatments, the heat stress
significantly (P = 0.01) reduced the number of viable pods on the main raceme, and significantly
(P = 0.08) increased the number of aborted pods on the main raceme and the percentage pod
abortion on the main raceme (P = 0.05). Significant temperature x foliar treatment interactions
were observed for both the number of aborted pods on the main raceme (P = 0.007) and percent
pod abortion on the main raceme (P = 0.02). In both cases, these interactions occurred because
the foliar treatments had no significant effect in the absence of the heat stress. Under heat stress
conditions, both of these parameters were significantly higher in the control treatments (i.e., the
74
check and adjuvant only treatments) than in any of the treatments receiving foliar B. There were
no significant differences among the four foliar B treatments (Table 4.1).
Because of the similarity among B treatments, we constructed contrasts to compare these
as a group to the control treatments. Under control temperature conditions, contrasts revealed no
significant effects of the foliar B treatments on any of the measured parameters (Table 4.2).
Under heat stress, the foliar B treatments strongly reduced the number of aborted pods and the
percent pod abortion on the main raceme, when compared to either the check, the adjuvant-only
treatment, or the mean of those two control treatments (P < 0.001 in all cases). Comparing the
foliar B treatments to the two control treatments, there was a significant (16.2%; P = 0.03)
increase in main raceme seed weight, which seemed to arise primarily from an increase in the
number of viable pods (13.5%; P = 0.08) (Table 4.2). The interactive effect of temperature and
foliar B treatment on main raceme pod counts is also shown in Figure 4.6. Figure 4.7 shows the
morphological characteristics of main racemes from the two temperature treatments, both with
and without B application.
The FFA levels were very low in both experiments. For Experiment I, mean FFA content
was 0.06% with a range of 0.02 – 0.14%. In terms of temperature regime, the 32/20°C had a
treatment mean of 0.08% with a range of 0.02-0.14%, the 28/20°C a mean of 0.06% and range of
0.03-0.08%, and for the 20/15°C a mean of 0.03% with a range of 0.02-0.07%.
75
In Experiment II, the mean FFA content was 0.30%, with a range of 0.28-0.31%. In terms
of the temperature regimes the treatment means were 0.29% and 0.30% for the 20/15°C and
28/20°C regimes, respectively.
76
200
Initiation of Treatments (32/20°C)
180
first flower
160
first flower + 7 d
AB
140
A
120
100
A
C
C
C
C
C
D
80
first flower + 14 d
aborted pods
BC
A
viable pods
60
B
BCD
CD
40
BC
DE
20
EF
F
0
Check
3
G
G
7
14
3
7
14
3
7
14
200
Initiation of Treatments (28/20°C)
Pods per plant (main raceme)
180
first flower
160
first flower + 14 d
first flower + 7 d
140
120
100
A
80
B
E
B
BC
BC
CD
DE
CD
DE
60
A
AB
40
AB
BC
C
7
14
C
20
D
Check
3
BC
7
14
D
0
200
BC
7
14
3
3
Initiation of Treatments (20/15°C)
180
first flower
160
first flower + 7 d
first flower + 14 d
140
120
100
80
60
Figure 4.2. Aborted
pods and viable (seed
bearing) pods on main
racemes, as affected by
time of initiation of heat
stress and duration of
heat stress.
The temperature
regimes were 32/20°C
(top), 28/20°C (middle)
and 20/15°C (bottom).
The oval in the middle
panel indicates the
treatment selected for
future experiments.
Within each panel,
treatments followed by
the same letter do not
differ for aborted pods
(top letters) or viable
pods (bottom letters) on
the main raceme,
according to a protected
LSD test (P<0.1).
B
AB
AB
A
A
AB
AB
AB
AB
AB
AB
AB
AB
A
AB
A
AB
A
3
7
14
3
7
14
AB
B
40
20
0
Check
3
7
14
Duration of stress exposure (d)
77
Number of viable pods
Viable pods on main raceme
70
60
50
40
30
20
10
0
A
AB
AB
BC
C
BC
C
BC
D
D
Treatments (28/20°C)
Figure 4.3. Treatment effects at
the 28/20°C temperature regime at
first flower for 3 days, 7 days or 14
days (FF3, FF7 or FF14,
respectively); beginning at 7 days
after flower for 3 days, 7 days or
14 days (7F3, 7F7 or 7F14,
respectively) or beginning at 14
days after first flower for 3 days, 7
days or 14 days (14F3, 14F7 or
14F14, respectively). The oval
indicates the treatment selected for
future experiments. Total seed
weight on the main raceme is the
product of viable pods by seeds per
pod by average seed weight on the
main raceme. Within each panel,
treatments followed by the same
letter do not differ for aborted pods
(top letters) or viable pods (bottom
letters) on the main raceme,
according to a protected LSD test
(P<0.1).
X
Number of seeds per pod
Seeds per pod on main raceme
20
18
16
14
12
10
8
6
4
2
0
A
A
A
A
A
A
A
A
A
B
Treatments (28/20°C)
X
Seed weight (mg)
Average seed weight on main raceme
4.5
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
A
AB
AB
AB
B
AB
BC
CD
DE
E
Treatments (28/20°C)
=
Seed yield (mg)
Total seed weight on main raceme
4500
4000
3500
3000
2500
2000
1500
1000
500
0
A
AB
ABC
ABC
BC
DC
C
DE
E
E
Treatments (28/20°C)
78
Figure 4.4. Morphological symptoms on spring canola bud (top), opened flower (middle) and
mature pod (bottom). Non heat stress symptoms on bud, flower and pod (20/15°C) (left) and heat
stress symptoms (32/20°C) (right).
79
Figure 4.5. Morphology of entire canola plants and main raceme for the greenhouse check (top)
and 32/20°C stress temperature regime (bottom). Temperature stress was initiated at 7 days after
first flower and lasted for 14 days. Control plants had moderate height and well developed main
racemes and branches, with viable pods along their whole lengths. Plants exposed to temperature
stress were taller and more slender with a viney appearance; on the main racemes, pod
developing before, during and after the stress treatments are easily distinguished.
80
Table 4.1. Temperature main effects and temperature x foliar B interactive effects on pod
characteristics of the main raceme and seed yields of the main raceme and whole plant.
Temperature
Treatment
Control
Stress
Mean
Mean
__________Main Raceme________
Viable Aborted
Pod
Seed
Pods
Pods Abortion Weight
(g)
(%)
Other Branches
Seed
Weight
(g)
Total
Seed
Weight
(g)
49.2
46.0
P-value 0.01
14.9
20.6
0.08
25.83
31.44
0.05
2.96
2.59
0.01
19.06
17.51
0.17
22.02
20.10
0.24
51.2
42.5
46.2
54.4
51.5
49.3
42.7
47.7
46.5
49.8
47.8
41.8
13.6
12.9
16.7
15.5
14.3
16.2
28.0
16.7
17.6
18.8
18.1
24.3
23.34
26.97
29.96
22.88
25.04
26.76
40.04
26.96
28.98
26.51
28.11
38.02
2.99
3.01
2.90
3.17
2.89
2.80
2.39
2.74
2.45
2.64
3.04
2.30
18.24
18.52
17.55
19.27
20.48
20.32
17.45
17.18
17.77
18.21
17.35
17.12
21.23
21.53
20.45
22.44
23.37
23.12
19.84
19.92
20.22
20.85
20.38
19.42
P-value (Temperature x treatment)
0.44
LSD 10% NS
0.007
3.84
0.02
7.38
0.41
NS
0.38
NS
0.08
2.79
Control
Control
Control
Control
Control
Control
Stress
Stress
Stress
Stress
Stress
Stress
Check
Low Liquid B
High Liquid B
Low Borax
High Borax
Adjuvant only
Check
Low Liquid B
High Liquid B
Low Borax
High Borax
Adjuvant only
81
Table 4.2. Contrasts between foliar B treatments and control treatments (check, adjuvant only,
mean of check + adjuvant only) under heat stress and control conditions, on main raceme pod
counts and main raceme and whole plant seed yields.
Contrasts
Control
B vs no B
__________Main Raceme________
Other Branches
Viable Aborted
Pod
Seed
Seed
Pods
Pods
Abortion Weight
Weight
(%)
(g)
(g)
Means
B (4 treatments)
No B (2 treatments)
P value
B vs Check
B (4 treatments)
Check (1 treatment)
P value
B (4 treatments)
Adj only (1 treatment)
P value
Check vs Adj only Check (1 treatment)
Adj only (1 treatment)
P value
B vs Adj only
Stress
B vs no B
B (4 treatments)
No B (2 treatments)
P value
B vs Check
B (4 treatments)
Check (1 treatment)
P value
B (4 treatments)
Adj only (1 treatment)
P value
Check vs Adj only Check (1 treatment)
Adj only (1 treatment)
P value
B vs Adj only
Total
Seed
Weight
(g)
48.6
50.3
0.62
48.6
51.2
0.54
48.6
49.3
0.87
51.2
49.3
0.73
14.9
14.9
0.99
14.9
13.6
0.53
14.9
16.2
0.52
13.6
16.2
0.32
26.21
25.05
0.64
26.21
23.34
0.38
26.21
26.76
0.86
23.34
26.76
0.40
2.99
2.89
0.55
2.99
2.99
0.98
2.99
2.80
0.37
2.99
2.80
0.49
18.96
19.28
0.69
18.96
18.24
0.49
18.96
20.32
0.19
18.24
20.32
0.12
21.95
22.17
0.79
21.95
21.23
0.51
21.95
23.12
0.28
21.23
23.12
0.17
47.9
42.2
0.08
47.9
42.7
0.21
47.9
41.8
0.14
42.7
41.8
0.86
17.8
26.1
<.0001
17.8
28.0
<.0001
17.8
24.3
0.002
28.0
24.3
0.15
27.64
39.03
<.0001
27.64
40.04
0.0003
27.64
38.02
0.002
40.04
38.02
0.62
2.72
2.34
0.03
2.72
2.39
0.13
2.72
2.30
0.06
2.39
2.30
0.74
17.63
17.29
0.67
17.63
17.45
0.86
17.63
17.12
0.63
17.45
17.12
0.80
20.34
19.63
0.40
20.34
19.84
0.64
20.34
19.42
0.40
19.84
19.42
0.76
82
Figure 4.6. Aborted pods and viable (seed bearing) pods on main racemes, as affected by
temperature regime and foliar B application. Within a temperature regime, treatments followed
by the same letter do not differ for aborted pods (top letters) or viable pods (bottom letters) on
the main raceme.
83
Figure 4.7. Flowering main racemes of spring canola plants in non-stressed and heat-stressed
environments, 20/15°C (top left) and 28/20°C (top right) respectively. Mature main racemes
following treatments (bottom left to right): heat stressed; heat stressed + Boron; non-stressed;
non-stressed + Boron (20/15°C and 28/20°C respectively).
84
4.4 Discussion
In Experiment I, the 32/20°C regimes produced significant treatment effects, however,
they were considered too severe since a lot of pods were aborted and/or misshapen in a manner
not normally seen in the field. This temperature combination almost eliminated viable pods on
the main raceme when exposure began at first flower. Heat stress symptoms were also evident on
the flowers (Figure 4.4), where the pistil was elongated before the flower opened; also the stigma
was receptive before pollen was available and the mature pods were parthenocarpic and sterile.
This agrees with work published by Gan et al. (2004). Nilsen and Orcutt (1996) also reported
that canola plants subjected to high temperatures displayed increased abscission of flowers, fruits
and even leaves; these effects were very evident also in the current study under the 32/20°C
regime. The total number of pod positions was high, but a lot more aborted. These plants were
also highly branched, with total pod counts exceeding 2000 per plant (data not shown). Likely,
the extensive abortion of the main raceme pods increased the post-stress source: sink ratio such
that the plant diverted photosynthate to production of additional fruiting positions on the lateral
branches. This study agrees with work done by Hall (1992), Paulsen (1994) and Mendham and
Salisbury (1995) who stated that high temperature stress can affect the source: sink ratio for
photassimilates in canola. In the field, canola crops that are unsuccessful in establishing an
adequate pod load will often produce additional branches and racemes late in the season.
The 28/20°C temperature regimes in Experiment I produced less severe but statistically
significant reductions in viable pod numbers compared with the greenhouse check (Figure 4.2).
85
Figure 4.3 shows the effect of timing and duration of exposure to the 28/20°C regime on main
raceme seed yield and its components. The magnitude of the effect was dependent on the
duration of the stress treatment and the developmental stage at which it was initiated. This agrees
with the finding of Mendham and Salisbury (1995) that the effects of heat stress on the seed
yield potential of Brassica crops depend on the developmental stage at which the stress occurs
and the duration of the stress. The specific regime chosen for Experiment II (14-d duration
beginning 7 d after first flower) showed significant but moderate treatment effects with a 25%
decrease in viable pod numbers on the main raceme.
In Experiment II, pod counts showed that the heat stress treatments reduced the number
of viable pods and increased the number of aborted pods compared to the control temperature
regime (Figure 4.6), although these effects were not as pronounced as observed in Experiment I
(Figure 4.2). The application of foliar B, however, reduced the magnitude of this effect in the
presence of heat stress. Indeed, there was a very strong temperature x foliar B interaction, with
main raceme pod counts only responding to B applications in the presence of stress. In the
absence of heat stress foliar B had no significant effect. Applying the adjuvant alone did not
significantly affect pod numbers under heat stress, indicating that the observed effects of the
foliar B treatment were due to the B component itself. These results strongly support our
hypothesis that foliar B provides a significant benefit only when the crop is under heat stress
during the early reproductive stage.
The precise physiological basis of this interaction cannot be deduced from the
experiments. Boron has many important roles in the reproductive physiology of plants, as
86
evident in the literature. Some possible roles of B include sugar transport (which could be
implicated in source-sink relations), pollen viability, cell wall synthesis, lignification, cell wall
structural integrity, carbohydrate metabolism, ribose nucleic acid (RNA) metabolism,
respiration, indole acetic acid (IAA) metabolism, phenol metabolism (Parr & Loughman, 1983;
Welch, 1995; Young et al., 2003; Ahmad et al., 2009).
In Experiment II, seed yield measured at the whole plant level was not significantly
affected by any of the treatments. However, these plants were grown in the greenhouse at
relatively low densities (16 m-2, approximately 25% of a normal field population), and thus were
very large and highly branched compared to those grown in the field experiments described in
Chapter 3; this extensive branching resulted in an abnormal distribution of seed production
between the main raceme and the branches. For the controlled environment experiment
approximately 87% of the total seed yield was borne on the branches with only 13% on the main
racemes; thus factors affecting main raceme seed yield specifically would have only a minor
effect on total plant yield. Our heat stress treatments were specifically designed to affect main
raceme pod set, by virtue of their timing relative to main raceme development. In this controlled
environment experiment, considering only the main racemes, under heat stress the B treatment
increased yield by 16% (Table 4.2, contrast between the four B and the two non-B treatments),
whereas yield on the branches was unaffected. In the field, plants bore much more of their total
yield on the main racemes; averaged across all location-years for which we had yield component
data, we found that 42% of the seed yield was produced on the main racemes (Chapter 3).
Probably more important than the distinction between main racemes and branches is the fact that
plants grown in the field at normal densities have a shorter flowering period and so overall
87
reproductive development is more synchronized across the different parts of the plant (since
branches flower later than the main raceme). Thus, a transient stress occurring at a particularly
sensitive stage has a larger likelihood of affecting overall yield.
Based on the evidence provided in the controlled environment experiments, it is apparent
that foliar B applications to spring canola during early flowering can reduce pod abortion in the
presence of heat stress, but only for those pod positions that are at a particular phase of
reproductive development when the stress occurs. Considering the 16% yield benefit from foliar
B on main racemes under heat stress in the controlled environment, and the large percentage of
seeds borne on the main racemes in the field (42%), the 5 to 6% yield benefit seen from foliar B
in the field is well within the range that could be explained by the temperature x foliar B
interaction we observed in the controlled environment experiment. These results support our
hypothesis that foliar B application increases yield of spring canola by alleviating the effects of
heat stress.
88
CHAPTER 5
CONCLUSIONS
This study was undertaken to evaluate the potential benefits of foliar B application to
spring canola under typical Ontario growing conditions, and to understand the physiological
basis of those effects (if any) at the whole plant and whole crop levels. Field trials included
thirteen location-years in Ontario from 2007 until 2012. A significant yield benefit was seen on
four occasions, slight (non-significant) yield increase or no effect was seen on eight occasions,
and there was one example of significant yield suppression from foliar B at a very high yielding
site in a cooler growing season.
Where foliar B resulted in a significant yield benefit in the field, we observed increased
pod numbers and seed yield on the main raceme, a higher fraction of total plant pod numbers and
total seed weight being borne on the main raceme, and a reduction in the number of pod
abortions on the other branches. In the single case where foliar B reduced yield (Meaford 2009),
there was a reduction in the number of seeds and fraction of pods borne on the main raceme; also
there were fewer aborted pods but also a reduced number of total flower positions. Based on
these observations, we suggest that foliar B may induce the crop to prioritize retention / filling of
the pods that are being established when the B is applied, to the detriment of flowering positions
that might otherwise be established later (either on the branches or higher on the main raceme).
In a resource-limited environment where the crop would typically establish many more flowers
than it can convert to viable pods, this reallocation of resources may be beneficial to yield. In an
89
extremely high-yielding environment like Meaford 2009, the loss of those future potential pod
positions would be detrimental to yield.
A significant yield benefit from foliar B application was observed only in seasons when
there was a significant heat stress, which supported the hypothesis that foliar B exerts its
beneficial effect by reducing the effects of heat stress. This idea was further investigated in a
controlled environment experiment where the effects of foliar B were measured both in the
presence and the absence of heat stress. We found a strong temperature x foliar B interaction on
main raceme pod counts and seed yield. Foliar B had no effect under control (non-stressful)
temperature conditions, but significantly increased main raceme pod counts and yield when
plants were exposed to heat stress during flowering and pod set. In all cases during the
controlled environment experiments, the number of viable pods was the primary yield
component affected by heat stress.
These results support the hypothesis that foliar boron application can help mitigate heat
stress effects in spring canola specifically by reducing heat-induced pod abortions. As a whole,
foliar B applications can alleviate the effects of heat stress in spring canola by decreasing pod
abortions and increasing yields in very warm growing seasons. Additional work should be
undertaken to determine the precise physiological mechanisms by which foliar B exerts these
beneficial effects under high temperatures.
90
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103
APPENDIX
104
A 1. The parameters measured for hand harvested samples taken from the 2009 trials.
Elora
No Boron
1973
Parameter
M ain raceme pods (m-2 )
Boron
1633
P-value LSD 10%
0.09
0.11
266
12.1
21.2
42.1
3.16
2895
235
12.5
22.2
35.9
3.28
2426
0.59
0.86
0.43
0.59
0.16
0.20
NS
NS
NS
NS
NS
NS
Pods , total (m-2 )
1074
27.6
16.3
43.6
2.98
4867
1049
29.9
19.0
45.7
3.08
4060
0.88
0.39
0.28
0.55
0.24
0.07
NS
NS
NS
NS
NS
0.15
Seeds , total (1000s m-2 )
Fraction of pods on main raceme
Fraction of s eeds on main raceme
Fraction of s eed weight on main raceme
85.8
0.42
0.49
0.50
81.6
0.40
0.44
0.45
0.52
0.57
0.17
0.14
NS
NS
NS
NS
M ain raceme pods (m-2 )
5896
5370
0.11
NS
M ain raceme aborted pods (m-2 )
Percent pod abortion on main raceme (%)
Seeds per pod, main raceme
837
12.2
23.9
140.9
3.39
4974
693
11.3
23.6
125.4
3.46
5220
0.11
0.44
0.71
0.02
0.32
0.63
NS
NS
NS
0.02
NS
NS
Pods , total (m-2 )
3377
41.0
20.9
103.2
3.39
10870
3388
38.7
19.2
99.2
3.34
10589
0.98
0.40
0.29
0.55
0.71
0.71
NS
NS
NS
NS
NS
NS
Seeds , total (1000s m-2 )
Fraction of pods on main raceme
Fraction of s eeds on main raceme
Fraction of s eed weight on main raceme
244.1
0.55
0.58
0.58
224.7
0.50
0.56
0.57
0.07
0.06
0.26
0.56
0.13
0.13
NS
NS
Shelburne
2046
2021
0.90
NS
261
11.5
21.1
42.8
3.21
3129
263
11.5
21.3
42.5
3.26
2853
0.96
0.94
0.90
0.92
0.50
0.25
NS
NS
NS
NS
NS
NS
Pods , total (m-2 )
1428
31.0
17.3
53.2
3.05
5175
1351
32.3
17.8
49.9
3.12
4875
0.65
0.62
0.72
0.43
0.25
0.21
NS
NS
NS
NS
NS
NS
Seeds , total (1000s m-2 )
Fraction of pods on main raceme
Fraction of s eeds on main raceme
Fraction of s eed weight on main raceme
96.0
0.40
0.44
0.45
92.5
0.41
0.46
0.47
0.42
0.65
0.73
0.73
NS
NS
NS
NS
M ain raceme aborted pods (m-2 )
Percent pod abortion on main raceme (%)
Seeds per pod, main raceme
Seeds on main raceme (1000s m-2 )
main raceme 1000-s eed weight (g)
Other branches pods (m-2 )
Other branches aborted pods (m-2 )
Percent pod abortion on other branches (%)
Seeds per pod, other branches
Seeds on other branches (1000s m-2 )
Other branches 1000-s eed weight (g)
Meaford
Seeds on main raceme (1000s m-2 )
main raceme 1000-s eed weight (g)
Other branches pods (m-2 )
Other branches aborted pods (m-2 )
Percent pod abortion on other branches (%)
Seeds per pod, other branches
Seeds on other branches (1000s m-2 )
Other branches 1000-s eed weight (g)
M ain raceme pods (m-2 )
(m-2 )
M ain raceme aborted pods
Percent pod abortion on main raceme (%)
Seeds per pod, main raceme
Seeds on main raceme (1000s m-2 )
main raceme 1000-s eed weight (g)
Other branches pods (m-2 )
Other branches aborted pods (m-2 )
Percent pod abortion on other branches (%)
Seeds per pod, other branches
Seeds on other branches (1000s m-2 )
Other branches 1000-s eed weight (g)
105
A 2. The parameters measured for hand harvested samples taken from the 2010 trials.
Elora
Parameter
-2
Main raceme pods (m )
Main raceme aborted pods (m-2)
Percent pod abortion on main raceme (%)
Seeds per pod, main raceme
-2
Seeds on main raceme (1000s m )
main raceme 1000-seed weight (g)
Other branches pods (m-2)
Other branches aborted pods (m-2)
Percent pod abortion on other branches (%)
Seeds per pod, other branches
Seeds on other branches (1000s m-2)
Other branches 1000-seed weight (g)
-2
Pods, total (m )
-2
Seeds, total (1000s m )
Fraction of pods on main raceme
Fraction of seeds on main raceme
Fraction of seed weight on main raceme
106
No Boron
1421
Boron
1606
P-value LSD 10%
0.14
NS
258
15.4
19.8
28.3
3.04
2451
239
12.7
21.6
34.8
3.11
2449
0.67
0.27
0.25
0.09
0.59
0.99
NS
NS
NS
0.11
NS
NS
943
27.1
20.2
49.6
2.99
789
24.3
21.1
51.6
2.76
0.29
0.42
0.32
0.56
0.07
NS
NS
NS
NS
0.08
3871
4055
0.38
NS
77.9
0.37
0.36
0.37
86.4
0.39
0.40
0.43
0.16
0.20
0.16
0.03
NS
NS
NS
0.04
A 3. The parameters measured for hand harvested sample taken from 2011 trials.
E lora
Parameter
M ain raceme pods (m-2 )
No B oron
2329
B oron
2011
M ain raceme aborted pods (m-2 )
Percent pod abortion on main raceme (%)
Seeds per pod, main raceme
Seeds on main raceme (1000s m-2 )
main raceme 1000-s eed weight (g)
Other branches pods (m-2 )
453
16.7
19.6
43.4
3.00
3817
309
13.0
19.8
39.9
2.98
3939
0.07
0.02
0.91
0.52
0.78
0.78
Other branches aborted pods (m-2 )
Percent pod abortion on other branches (%)
Seeds per pod, other branches
Seeds on other branches (1000s m-2 )
Other branches 1000-s eed weight (g)
Pods , total (m-2 )
1499
29.2
35.7
127.7
2.79
6146
1390
26.6
35.6
135.5
2.73
5950
0.52
0.40
0.96
0.49
0.38
0.65
Seeds , total (1000s m-2 )
Fraction of pods on main raceme
Fraction of s eeds on main raceme
Fraction of s eed weight on main raceme
Dundalk
M ain raceme pods (m-2 )
171.1
0.38
0.26
0.42
175.3
0.34
0.23
0.39
0.53
0.51
0.48
0.51
1607
1889
0.19
M ain raceme aborted pods (m-2 )
Percent pod abortion on main raceme (%)
Seeds per pod, main raceme
Seeds on main raceme (1000s m-2 )
main raceme 1000-s eed weight (g)
Other branches pods (m-2 )
603
26.5
16.9
27.1
3.63
1759
495
20.8
16.3
30.2
3.57
1874
0.23
0.003
0.57
0.24
0.51
0.58
Other branches aborted pods (m-2 )
Percent pod abortion on other branches (%)
Seeds per pod, other branches
Seeds on other branches (1000s m-2 )
Other branches 1000-s eed weight (g)
Pods , total (m-2 )
992
36.8
29.2
51.3
3.14
3365
852
32.4
26.6
50.0
3.17
3763
0.24
0.28
0.23
0.78
0.84
0.11
0.61
0.59
0.37
0.44
P-value
0.39
Seeds , total (1000s m-2 )
Fraction of pods on main raceme
Fraction of s eeds on main raceme
Fraction of s eed weight on main raceme
Shelburne
M ain raceme pods (m-2 )
78.4
0.48
0.35
0.55
80.2
0.51
0.39
0.58
1254
1324
0.51
M ain raceme aborted pods (m-2 )
Percent pod abortion on main raceme (%)
Seeds per pod, main raceme
Seeds on main raceme (1000s m-2 )
main raceme 1000-s eed weight (g)
Other branches pods (m-2 )
451
26.6
18.0
22.2
4.32
2113
259
16.1
19.6
25.9
4.17
1756
0.01
0.001
0.19
0.02
0.03
0.14
Other branches aborted pods (m-2 )
Percent pod abortion on other branches (%)
Seeds per pod, other branches
Seeds on other branches (1000s m-2 )
Other branches 1000-s eed weight (g)
Pods , total (m-2 )
930
29.6
27.3
54.7
4.08
3366
839
32.4
30.5
52.4
3.92
3081
0.58
0.11
0.18
0.56
0.11
0.31
76.9
0.39
0.30
0.47
78.3
0.44
0.33
0.51
0.67
0.11
0.20
0.20
Seeds , total (1000s m-2 )
Fraction of pods on main raceme
Fraction of s eeds on main raceme
Fraction of s eed weight on main raceme
107
A 4. The parameters measured for hand harvested sample taken from 2012 trials.
Elora
No Boron
Boron
P-value
Main raceme pods (m-2)
Parameter
1909
2068
0.65
Main raceme aborted pods (m-2)
340
496
0.23
Percent pod abortion on main raceme (%)
15.0
19.4
0.22
Seeds per pod, main raceme
19.4
19.4
0.98
(1000s m-2)
36.8
40.0
0.56
main raceme 1000-seed weight (g)
2.78
2.73
0.09
1220
1400
0.38
470
596
0.21
Percent pod abortion on other branches (%)
27.6
29.3
0.24
Seeds per pod, other branches
16.0
11.8
0.33
branches (1000s m-2)
19.5
17.1
0.73
Other branches 1000-seed weight (g)
2.69
2.63
0.43
3129
3467
0.51
Seeds, total (1000s m )
56.3
57.1
0.92
Fraction of pods on main raceme
0.61
0.60
0.66
Fraction of seeds on main raceme
0.65
0.73
0.51
0.66
0.73
0.51
1566
1765
0.74
214
193
0.69
Percent pod abortion on main raceme (%)
11.9
10.2
0.09
Seeds per pod, main raceme
Seeds on main raceme
Other branches pods (m
Other branches aborted
Seeds on other
-2)
pods (m-2
)
-2
Pods, total (m )
-2
Fraction of seed weight on main raceme
Meaford
Main raceme pods (m-2)
Main raceme aborted pods (m
-2)
23.6
27.1
0.26
(1000s m-2)
36.7
47.2
0.46
main raceme 1000-seed weight (g)
3.56
3.54
0.78
Other branches pods (m )
3408
3005
0.29
Other branches aborted pods (m-2)
986
893
0.73
Percent pod abortion on other branches (%)
22.0
22.4
0.95
Seeds per pod, other branches
Seeds on main raceme
-2
21.3
21.7
0.83
branches (1000s m-2)
72.8
65.5
0.57
Other branches 1000-seed weight (g)
3.08
3.24
0.08
4974
4771
0.51
109.4
112.7
0.40
Fraction of pods on main raceme
0.32
0.36
0.69
Fraction of seeds on main raceme
0.34
0.40
0.55
Fraction of seed weight on main raceme
0.37
0.42
0.65
Seeds on other
-2
Pods, total (m )
-2
Seeds, total (1000s m )
108
A 5. Soil test analyses for location-years 2009 to 2012.
Year Location Sample Depth OM
2009
Elora
Meaford
Shelburne
(cm)
(%)
0 - 30
0 - 15
15 - 30
0 - 15
15 - 30
3.3
4.5
3.0
4.4
3.2
Phosphourous
K
B
Cu
Fe
Mn
Zn
Mg
Ca
Na pH
CEC
Base Saturation
S
Al
Nitrate -N
Bicarb Bray
(%)
(ppm)
(ppm) (ppm) (ppm) (ppm) (ppm) (ppm)
(meq/100g) K Mg Ca Na (ppm) (ppm) (ppm) (ppm) (ppm) (ppm) (ppm)
12
40
19
27
9
21
99
35
53
18
64
134
52
90
49
320
190
160
160
155
2870
1320
1210
3760
3810
7
8
9
8
9
7.7
6.4
6.6
7.8
7.9
17.2
11
8.8
20.4
20.5
1.0 16 83
3.1 14 60
1.5 15 69
1.1 6.5 92
0.6 6.3 93
0
0
0
0
0
1.1
0.9
0.7
1.5
0.8
1.7
1.2
0.9
2.5
2.0
67
83
74
80
64
116
62
87
54
64
3.6
5.2
2.7
5.1
2.4
9
14
11
11
8
727
1047
1104
547
412
19
17
12
13
8
Year Location Sample Depth OM
Phosphourous
K
Mg
Ca
Na pH
CEC
Base Saturation
B
Cu
Fe
Mn
Zn
S
Al Nitrate -N
Bicarb Bray
(%)
(%) (ppm) (ppm) (ppm) (ppm) (ppm) (ppm)
(meq/100g) K Mg Ca Na (ppm) (ppm) (ppm) (ppm) (ppm) (ppm) (ppm)
(ppm)
(cm)
2010
Elora
0 - 15
15 - 30
5.8
4.7
11
10
Year
Location
Sample Depth
(cm)
2011
Dundalk
0 - 15
15-30
0 - 15
15-30
0 - 15
15-30
Elora
Shelburne
Meaford
64
51
520
460
2670
2630
22
18
17.9
17.2
7.4
7.6
Soil moisture Ammonium N Nitrate N
(%)
(mg/kg)
(mg/kg)
19.6
16.08
22.46
14.49
19.83
17.69
5.7
6.13
6.23
2.44
5.7
1.39
OM
Phosphorus
K
(cm)
(%)
Bicarb Bray
(ppm) (ppm)
0 - 15
15 - 30
0 - 15
15 - 30
4.2
3.0
4.1
2.7
Year Location Sample Depth
2012 Elora
16
12
24
12
37
27
47
17
79
47
9.24
6.69
13
5.83
17.6
11.4
Mg
Ca
0.9 24.2
0.8 22.3
330
265
165
130
2590
2530
1130
940
109
0.5
0.5
1.8
1.1
66
61
3.2
3.0
79
61
4.3
3.4
9
8
736
787
19
14
B
(ppm)
P, Extractable
(mg/L soil)
Mg, Extractable
(mg/L soil)
K, Extractable
(mg/L soil)
pH
S
(%)
0.62
0.36
0.53
0.29
0.61
0.46
35
9
6.6
1.9
11
9.7
320
330
510
430
130
130
97
60
95
94
77
67
7.8
7.8
7.6
7.8
7.9
8.0
<0.01
<0.01
0.04
<0.01
<0.01
<0.01
Na
pH
2
3
5
1
CEC
Base Saturation
(%)
(meq/100g) K Mg Ca
(ppm) (ppm) (ppm) (ppm)
57
35
151
71
74
77
7.8
8.0
6.7
6.9
15.9
15.0
8.6
7.2
0.9
0.6
4.5
2.5
17
15
16
15
82
85
65
66
B
S
Al
K/Mg
ratio
Na (ppm) (ppm) (ppm)
0.1
0.1
0.3
0.1
0.8
0.7
0.6
0.3
8
5
17
8
726
579
953
1356
0.05
0.04
0.28
0.17