Introduction
Long-term Goals and Supporting Objectives
Our long-term goals focus on expanding canola acreage and increasing its production in
South Dakota and nearby areas. By developing locally adapted canola varieties with a combination of conventional and modern technologies, by optimizing production practices specifically for South Dakota and similar environments, and by educating potential canola growers about the benefits of growing canola, we will reach this goal. This project predominantly addresses the development of canola varieties adapted for South Dakota growing conditions and educating potential growers.
Supporting objectives include the following:

Beginning a breeding program to develop canola varieties that are able to consistently yield 2500 lbs/a with oil content of over 40% in most parts of South Dakota and similar environments.

Identifying and recommending to South Dakota growers the best currently available canola varieties with the best adaptation to South Dakota growing environments.

Educating South Dakota farmers on the adoption of canola in their crop rotation systems.
Body of Knowledge
Canola, a class of rapeseed ( Brassica ) oil crop, contains less than 2% of erucic acid in seed oil and less than 30 μmoles g
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of glucosinolates in oil-free meal. Since it offers one of the highest-quality oils available for human consumption, processors primarily extract its oil for cooking usage. Processors also find it suitable for biodiesel production and other industrial applications with increasing demand. Additionally, canola meal or whole canola seed provides a high-protein feed source for animals and the rapeseed/canola plant supplies cut hay and good grazing as animal feed.
Currently, over eighty percent of United States canola is grown in North Dakota (National
Agricultural Statistics Service), with Oklahoma and Minnesota following as the next two largest producers. In our effort to expand canola acreage, we look to South Dakota, which is climatically suitable for canola growth (Table 1) and has been growing canola in some northern counties, with an average annual acreage of about 3,600 between 1995 and 2002 (Grady, 2002).
Canola is a cool season crop. Its best growth occurs between 53°F and 86°F with maximum growth and development at 70-77°F. Of the three canola species, B. juncea and B. rapa appear to have a higher optimum temperature than B. napus . Canola seeds can germinate when soil temperature is above 38°F, and canola seedlings can tolerate temperatures as low as 24°F.
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Although northern South Dakota usually has the last spring frost in late May, with appropriate soil conditions, early sowing (early to mid April) is feasible in southern areas of the state. Like most Brassica species, canola has indeterminate inflorescence. This means its seed production is only limited by how much energy its photosynthesis and nutrient supplies can provide and by how long the favorable climate for flowering and pod filling can last. With Fall’s first killing frost usually occurring in late September to early October, South Dakota generally has a longer growing season than North Dakota or northern Minnesota and thus offers a higher yield potential for growing canola.
The lack of suitable and well-adapted varieties for the local climate poses the biggest challenge to growing canola in South Dakota. Varieties developed for North Dakota, Minnesota, or Canada usually have common characteristics of growth and development: a short vegetative growing stage (about 60 days) and a short reproductive stage (about 20 days). Because they bud quickly, they do not build up enough leaf area for photosynthesis to sustain maximum seed production under South Dakota conditions. Therefore, developing varieties with longer vegetative and reproductive stages will realize increased yield potential in South Dakota.
Heat stress during flowering and pod setting time is the key impediment for growing canola in South Dakota. Heat stress depends largely on genotypes and physiological conditions.
Morrison and Stewart (2002) reported that the threshold temperatures for heat stress that causes yield losses for all Brassica species was 85.1°F. Other reported critical temperatures for heat stress are from as low as 77-81°F to as high as 86-90°F in Canada or 95°F in Australia for B. napus (Canola Council of Canada). Although South Dakota weather is good for canola growth in summer, with daily highs frequently above 90°F in July and August, the northern varieties would not tolerate the severe heat stress that would occasionally occur during the flowering time in
South Dakota. The high summer temperatures usually shorten the flowering duration and often reduce number of pods per plant and seeds per pod. Fortunately, by overcoming these hurdles through genetic improvement, canola has an excellent potential to be a cash crop in South
Dakota and southern Minnesota.
Canola is a close relative of the model plant Arabidopsis. Therefore, we can readily apply molecular knowledge gained from Arabidopsis to canola improvement. As in canola, high temperatures trigger early flowering in Arabidopsis (Blázques et al., 2003). Physiological and molecular studies have revealed that high temperatures induced early flowering in Arabidopsis through a specific flowering pathway – the autonomous pathway. Mutants of FVE and FCA in this pathway are late flowering and insensitive to high temperature (Blázques et al., 2003;
Balasubramanian et al., 2006). Therefore, FVE and FCA genes are potential thermosensors that determine flowering time under higher temperatures. Manipulation of expression of these genes will reduce or eliminate the sensitivity of plants to high temperature.
Recent studies suggested that defects in anther development and pollen sterility in high temperatures could be the major cause of reduced seed/grain yield in many species including
Arabidopsis (Robertson et al., 2002; Zinn et al., 2010). In Arabidopsis, the pollen abortion was associated with suppressed expression of auxin synthesis genes and thus low auxin content.
Auxin is an important plant hormone. Exogenous application of auxin on heat-stressed (88-91°F)
Arabidopsis and barley inflorescence completely reversed male sterility caused by heat, resulting in normal grain and seed development (Sakata et al., 2010). Thus, maintaining high auxin level in the anther under high temperature may be the key to preventing yield loss caused by heat stress. In Arabidopsis, YUCCA is a gene in the auxin synthesis pathway whose expression in the anther was repressed by high temperature (Sakata et al., 2010).
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While increasing canola acreage in South Dakota suggests competition with soybean or wheat for acreage, canola has great economical potential as an alternative to soybean or wheat in the rotation system. It has a seed yield potential similar to or better than soybean or wheat but with higher cash value potential.
According to USDA’s data over
2008-2010, average prices in South
Dakota for soybean and wheat were $0.18 and $0.15 per pound, respectively. Although South
Dakota does not have a significant canola market, it is close to North
Dakota, where the average canola price over those three years was
$0.18 per pound. Soybean yield in
South Dakota is about 40 bushels
(2400 lbs) or $432 per acre.
Average wheat yield in South
Dakota is about 45 bushels (2,700 lbs) per acre with a current market value of about $405 per acre. In the late 90s, we conducted yield trials in Watertown and Webster, South
Dakota with four B. rapa and some of 59 B. napus varieties then available and obtained a three-year mean yield of 1552 lbs/a (Table 2).
Six open-pollinated and four hybrid varieties of B. napus were able to yield more than 2000 lb/a in a single year, with the highest single year yield of 2518 lbs/a (or
$453/a) from a hybrid in
Watertown. These data show how some of the varieties developed for the area to our north could perform equally well in South Dakota, and newly developed/recently released varieties will surely perform even better than those used in our 90s’ trial. In addition, canola usually
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contains twice as much oil as soybean (40% vs.
20%) and has a better quality/fatty acid composition as edible oil and feedstock to biodiesel than soy oil. Considering that the current market price of canola oil is about $0.23 per pound, it clearly has the competitive edge against soybean and wheat economically.
Canola can be of winter type or spring type. Winter-type varieties usually have higher yield than the spring-type because they have a longer vegetative growing stage to increase their leaf area, and the larger, stronger plants tolerate heat stress better during the reproductive stage.
Consequently, winter canola usually out-yields spring canola by about 30% or more but is only grown in the southern and eastern states where the winter is mild. At this time, growing winter canola in South Dakota with the available varieties is not practical; however, we will strive to develop new winter-hardy varieties that can survive South Dakota’s winter. In addition, we will use winter genotypes to improve spring canola’s tolerance to late frosts in spring. Crossing between spring and winter types can not only combine the merits from both types, but can also broaden the range of segregation in progenies and enhance genetic diversity (gene pool) of canola.
Objectives
Begin a breeding program that will develop canola varieties that are able to consistently yield
2500 lbs/a with oil content of over 40% in most parts of South Dakota and similar environments. This objective aims at converting the longer production season to higher yield in South Dakota. Realizing this objective will provide South Dakota growers with locally adapted varieties for maximum return and significantly expand canola acreage.
To identify and recommend to South Dakota growers the best currently available canola varieties with the best adaptation to South Dakota growing environments. This objective aims at increasing canola acreage in South Dakota before locally bred varieties are available.
To educate South Dakota farmers on the adoption of canola in their crop rotation systems.
This will be critical for expansion of canola acreage in South Dakota. We need to establish research programs and related demonstration trials to convince South Dakota farmers that canola is a value crop and inspire them with interest in growing the crop.
Rationale and Significance
Food use of canola oil is increasing due to its high health value. As a growing industry, canola oil-based biodiesel will help reduce US dependency on foreign petroleum-based fuels.
Currently, US production of canola only meets part of the ever-increasing domestic demand.
Increasing canola acreage is a way to reduce the canola shortage. From both agronomic and economic viewpoints, canola can be an excellent alternative to soybean and wheat in the crop rotation system. Since canola produces twice the oil per acre as soybean when its yield is similar, it has great potential as a cash crop that brings farmers higher profits than soybean and wheat.
The proposed research aims at increasing canola acreage and production in South Dakota and similar environments. We will develop varieties that are better suited to South Dakota growing conditions than currently available cultivars. Increasing heat tolerance during flowering and seed set will be our first priority. Molecular knowledge gained in the model species Arabidopsis, a close relative of canola, will be applied to aid our breeding efforts. While the breeding program is getting underway, we will test currently available canola varieties to determine which have the best adaptation to South Dakota growing environments and recommend these to growers. We
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will educate South Dakota growers on the economic and agronomic benefits of including canola in their cropping systems.
This project identifies and addresses major impediments to expansion of canola acreage and production in South Dakota and similar environments. It meets the research priorities of the
North Central Region Canola Research program. This project will increase crop diversity and thus sustainability of agricultural production in South Dakota.
Approach
The proposed activities
A multidisciplinary research team has been assembled to carry out the proposed research. Dr.
Yang Yen, a plant molecular biologist/geneticist, will take the leading role. He will coordinate all the project activities and be in charge of the molecular assay in the lab. Dr. Guo-Liang Jiang, an oilseed breeder, will lead the breeding efforts. Dr. Yajun Wu, a plant stress physiologist, will evaluate freezing and heat stress tolerance in canola. Ms. Kathleen Grady, an oilseed agronomist/breeder, will be in charge of the field trials and extension efforts. One research technician will be hired to assist the PI/Co-PIs in this project for field and lab work.
Germplasm collection and evaluation of introduced varieties/lines are the first critical steps for the genetic improvement program. Therefore, germplasm collected worldwide from germplasm banks and research institutions will be evaluated for superior materials to start our breeding program, which will be done with a combination of conventional variety development and biotechnology. Invitations will be sent to private seed companies for their participation in field evaluations. Collected germplasm and current varieties will be evaluated under field conditions for their performance in South Dakota. The germplasm will also be assayed in the lab for expression of genes that are critical for early vigor and tolerance to heat stress. Lines with desired traits will be used as parents for variety development. Breeding activities will involve inbred line development and hybrid variety development. The best current varieties will be recommended to growers for incorporation into their cropping systems.
In addition to conventional genetic improvement through breeding, we will take advantage of knowledge gained through Arabidopsis research. Relevant genes and pathways will be genetically engineered by overexpression or inhibition of the expressions to improve early vigor and to strengthen heat tolerance.
Methods to be used in carrying out the proposed project
Germplasm collection and evaluation:
Germplasm lines will be collected worldwide from germplasm banks and research institutions, including spring and winter types of all three species ( B. napus, B. rapa and B. juncea ), with emphasis on lines developed in major canola-growing regions. Since initial seed quantities of some accessions are likely to be small, a seed increase will be conducted the first year for all germplasm lines. Single row, non-replicated plots of each line will be sown at
Brookings, SD in early spring. If seeds are enough to perform replicated/multiple-row trials, a randomized complete block design (RCBD) will be employed. Accessions will be blocked according to species ( B . napus , B . rapa , B . juncea ) and rows of two current commercial varieties of each Brassica species will be included as checks at set intervals. Notes will be taken on agronomic traits such as days and Growing Degree Days (GDDs) to onset of flower, end flower, and maturity; cold tolerance (in case frost occurs), heat tolerance (in case heat stress occurs), plant height, lodging, seed shatter, and seed yield per plant/yield traits (number of pods per plant
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and number of seeds per pod/plant, 1000-grain weight). Harvested seed will be non-destructively analyzed for oil content and fatty acid composition via NMR and/or NIR. Some germplasm may be discarded based on these initial observations.
Also during the first year, a selection of currently available commercial and public springtype canola varieties will be evaluated for their performance in replicated trials at Brookings and an off-campus site in the northern part of the state. These trials will include varieties from all three Brassica species. Any lines/accessions with sufficient seed will also be incorporated into these tests. Experiment design will be a split-plot design with three replications. Whole plots will be Brassica species and sub-plots will be varieties. Sub-plot size will be approximately 5’ x
20’, with 7 inch row spacing. Traits to be measured include GDDs to onset of flower, end flower, and maturity; plant height, lodging, seed shatter, disease and insect damage, and seed yield. It is expected that differences in maturity dates will necessitate multiple harvest dates.
Harvested seed will be analyzed for oil content and oil fatty acid composition. These yield trials will be continued each year, incorporating advanced materials developed by the breeding program as they become available. After the initial three years of testing, the data will be summarized over years and locations and the best currently available commercial or public varieties/hybrids will be recommended to growers.
The second year, field evaluation of the collected germplasm increased in year one will be conducted at SDSU’s Brookings Campus and an off-campus site in the northern part of the state.
A split-plot design will be adopted, with Brassica species as main plots and lines/accessions as subplots. Approximately 100 lines will be evaluated per year in single-row plots replicated three times at each location with three to five of the best currently available varieties identified in the year one yield trials as checks. Certain laboratory/greenhouse/growth chamber evaluations will also be conducted on all lines. Traits to be evaluated include the following:
Germination-related traits such as germination percentage and germination rate, the number of days to 50% germination (These traits will be evaluated under controlled conditions in the lab under various temperature regimes.)
Growth-related traits such as tolerance to early frost and tolerance to late heat, leaf area index before budding (early vigor), GDDs to begin flower, end flower, and maturity
Yield-related traits such as number of pods per plant, number of seeds per pod and seed weight (g/1000 seeds), seed shatter, plant lodging, seed yield, disease and insect damage, oil content (%), oil fatty acid composition
Tolerance to early frost and late heat
Climate records will be kept near each field evaluation site. If frost (temperature <28°F) occurs after seedling emergence, notes will be taken on percent plant survival as soon as possible after the frost event. Heat tolerance will be evaluated in greenhouse/growth chamber studies.
Plants at different stages will be exposed to heat at 90°F for three hours daily for five days.
Flowering time and yield will be recorded and compared to that of unstressed controls. For effect of high temperatures on anther development, heat stress will be applied after plants flower.
Number of pods per plant and number of seeds per pod will be recorded and compared to unstressed controls.
Calculation of GDD
GDDs will be calculated by averaging the daily maximum and daily minimum temperatures and subtracting the assumed minimum base temperature for growth to occur. A minimum base
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temperature of 0°C (32°F) will be used for this study. Accumulated GDDs between planting and onset of flowering (at least one open floret on 50% or more plants), end of flowering, and maturity (10% of seed has changed color) will be recorded.
Estimation of leaf area index and growth rate
From one week after emergence until flowering, each line will be visually scored each week on a 1 to 5 scale for estimation of leaf area index. Plant growth rate will be estimated by measurement of leaf number and plant height of the main stem. Three plants per plot will be tagged one week after emergence. The total leaf number for each plant will be counted and height of the main stem will be recorded each week until the first flower opens.
Yield-related traits
At maturity, each plot will be scored on a 1-5 scale based on seed shatter and plant lodging.
Damage due to disease and/or insect pests will be estimated. Five to ten plants per plot will be randomly sampled (avoiding plot ends) at maturity for measurement of yield components
(pods/plant, seeds/pod, 1000 seed weight). The entire remaining plot area will be harvested and total seed weight added to that from the yield component sample for estimation of total plot yield. It is expected that differences in maturity dates will necessitate multiple harvest dates.
Harvested seed will be analyzed for oil content (NMR or NIR) and oil fatty acid composition
(GC).
Development of varieties:
Based on the field evaluation, materials with required traits will be used as parents.
Conventional breeding approaches will be predominantly employed. Single cross, three-way cross, and double cross will be made to combine desirable traits and/or genes and produce new segregates with improved performance and/or a combination of different merits. Single seed descent (SSD) and individual selection will be applied to handle the breeding populations.
Backcrossing will also be conducted in case to transfer simply inherited characters. To integrate the merits of different types, e.g. the winter-hardiness of winter type and the vigorous early development/growth of spring type, crosses between spring and winter types will be made. It can be expected that such sub-wide crosses will also produce a broad range of segregation in progenies and enhance genetic diversity (gene pool) of canola. Early-generation selection will focus on heat resistance, cold tolerance, vigorous and longer vegetative growth, more pods per plant and more seeds per pod. Yield and quality (oil and protein) will be determined in late generations. Superior inbred lines will be selected and used as the parents for hybrid variety development. Biotech engineering and molecular techniques will also be employed to facilitate and speed up the breeding progress, especially for heat tolerance and early growth vigor.
Superior inbred lines with good performance will be crossed with each other in a diallel and/or factorial mating design, depending upon the number of inbred lines used. Superior inbred lines with good combining ability will be used to produce hybrids for further trials. Hybrids will be evaluated in multiple environments (years and locations) for yield, quality (oil content, fatty acids, protein, etc.) and other characteristics (cold tolerance in seedling stages, heat resistance during flowering, and disease/pest resistance). The best hybrids will be released and recommended to farmers. To fully realize the yield potential of new varieties, cropping management and operation will also be investigated. The optimal growing practices will be recommended to farmers along with the release of hybrid varieties. Male-sterility will be used in
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hybrid development and production.
Molecular physiological analyses
We propose to clone the genes involved in flowering time control at high temperatures and in auxin synthesis in anthers and in freezing tolerance. Their expression will be studied in different varieties/lines to determine whether these genes are associated with heat tolerance. The genes selected for study include FT , FVE , FCA , YUCCA and CBF3 . FT is an integrator of different flowering pathways in Arabidopsis and other plants. Its expression level is directly correlated with flowering time. It encodes a protein called florigen that can move through phloem from leaves to inflorescence stem where it initiates floral development (flowering) (Jaeger and Wigge,
2007). FVE and FCA are two genes required for early flowering at high temperatures in
Arabidopsis (Blázques et al., 2003). YUCCA is a gene in auxin synthesis pathway in Arabidopsis, whose expression in the anther was repressed by high temperature (Sakata et al., 2010). CBF genes are well studied in Arabidopsis and other plants and play important roles in resistance to freezing stress (Thomashow, 2010). Overexpression of CBF genes in canola has improved tolerance to freezing and drought (https://www.msu.edu/~mftlab/).
FT genes in canola have been cloned and their sequences are available. Canola FT genes showed 85-87% nucleotide sequence identity to Arabidopsis FT genes (Wang et al., 2009).
Based on the high similarity of these two genomes, we will not expect difficulty to clone other genes. However, canola has a bigger genome than Arabidopsis due to triplication of many homologous regions (Wang et al., 2009). We will expect to identify several paralogues in canola for many genes in Arabidopsis. Gene expression study will help us to determine which paralogue is the key in the high temperature response/freezing tolerance.
The varieties that show good performance in the field evaluation will be selected for greenhouse/growth chamber studies for their tolerance to high temperature. Young plants at different stages will be exposed to heat at 90°F for three hours daily for five days. Flowering time and yield will be recorded and compared to unstressed controls. Leaf tissues will be harvested three days after treatment for quantification of FT , FVE and FCA transcript level using quantitative PCR. Correlation analysis will be computed between the transcript level and flowering time as well as yield. Expression level of the CBF genes in leaves of young seedlings will be studied to correlate with early-stage frost tolerance such as seedling survival rate and leaf electrolyte leakage.
For effect of high temperatures on anther development, heat stress will be applied after plants flower. Number of pods per plant and number of seeds per pod will be recorded and compared to unstressed controls. Developing young anthers will also be harvested after heat stress for quantification of YUCCA transcript level.
Once the key genes that are correlated with stress tolerance are identified, we plan to overexpress or down-regulate their expression through genetic engineering. Canola plants can be easily transformed via agrobacterium-mediated transformation. We plan to generate transgenic canola plants in year 3 and will have these transgenic plants tested in controlled environments by year 5 for their improved stress tolerance. Our ultimate goal is to facilitate the transfer of desired traits into local adapted germplasm through combined traditional crossing and molecular engineering.
Expected outcomes
- Identify about 10-20 lines for further work and hopefully a few varieties adapted to South
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Dakota for recommendation to growers
Develop superior inbred lines adapted to South Dakota and similar environments, and make new crosses with these lines for further development of hybrid varieties
Generate knowledge on physiological and molecular understanding of heat tolerance, flowering time control, and freezing tolerance in canola
Increase knowledge of producers about the performance and production practices of canola and how it may fit into traditional cropping systems
Produce extension/outreach materials and events, including factsheets, web-based content, field days, and meetings
Establish new internal collaborations among SDSU research and extension faculty
Means by which results will be analyzed, assessed or interpreted
To correctly analyze and interpret the results, we will statistically analyze the data using appropriate methods and means, depending on experimental designs. ANOVA and correlation will be predominantly computed, and the software SAS, AgroBase, and Excel will be used to process and analyze the data.
Uses for results or products
The germplasm will be shared with other breeders and researchers. The findings from molecular studies will be published in peer-reviewed journals. The genetic materials generated from the project will be made available to the public. Data from breeding field trials will be available upon request. Data from performance trials of commercial varieties will be published in reviewed extension publications and posted on extension-related websites such as eXtension and iGrow. Identified superior varieties will be recommended to potential growers. Results of this research will be presented at regional or national meetings or published as journal articles.
Timeline for multi-year projects
This proposed research is a multi-year project. Germplasm collection and evaluation (both field and lab) will be conducted every year. Breeding and extension efforts will start the second year. Optimization of production practices will start when the first variety from this program is put in yield trials. Here is the outline for the proposed work:
Research activities
Germplasm collection
Field trial
Greenhouse/growth chamber studies
Breeding-crossing
Plant transformation
Molecular assay/analysis
Year 1 Year 2 Year 3 Year 4 Year 5
X X X X X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
The following table outlines the procedure and time framework of variety development:
Year Crop season
1 Local nursery
Winter nursery (GH or off-location)
Population/material Activity and expected outcomes
Desired PIs Cross between selected PIs to produce F seeds.
1
F
1 s and desired PIs Plant F
1
seeds to produce F
2
seeds, and make new crosses.
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2
3
Local nursery
Winter nursery (GH or off-location)
Local nursery
F
F
F
2
3
4 s, F
1 s
pops, and F s, F
2:4 lines
2:3 plant rows, and F
1 s
Plant F
2 s and advance generation by SSD; select superior plants, and make new crosses and handle them similarly to previous crosses.
Advance generation by SSD, and bulk harvest plant rows.
Plant F
4 s and harvest individuals; evaluate
F
2:4
plant rows, select plants from the best rows
Plant F
4:5
plant rows and bulk harvest. Winter nursery (GH or off-location)
Local Nursery
F
4:5 plant rows
4 F
4:6 rows Evaluate F
4:6
lines and select the best lines and plants.
5
Winter nursery (GH or off-location)
Local nursery
F
4:7 lines, F rows/lines
6:7
Inbred lines and new F
1
hybrids
Increase seeds of best lines/plants selected in previous season, and make new crosses with superior lines
Evaluate inbred lines and F
1
hybrids; select superior lines and hybrids; make new crosses; and repeat the crosses and increase the parental lines for further trials
Outreach/Extension Activities
Field days/tours will be held each year at the sites of replicated yield trials to showcase canola as a potential cash crop.
Seminars/workshops will be held during the winter months featuring speakers on various aspects of canola production, how canola can fit into crop rotations, and economics and marketing issues.
Extension publications (printed and electronic) will detail research results.
Brochures and webinars will be produced for canola promotion and crop management.
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