University of Florida Thesis Or Dissertation Formatting

THE EFFECTS OF SOIL TYPE AND NITROGEN RATES ON GLUCOSINOLATE AND OIL PRODUCTION IN BRASSICA CARNATA

THEODOR LINARES STANSLY

A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE

UNIVERSITY OF FLORIDA

2016

To my parents, Philip and Silvia Stansly, I thank you from the bottom of my heart. Not all of the important lessons needed for a successful career can be taught in schools or universities. They must also come from those who have persevered through life’s obstacles without losing hope, inspiration, and spirit. I am grateful for everything you have done and continue to do throughout these years.

ACKNOWLEDGMENTS

Many thanks to Pete C. Andersen for allowing me the opportunity to pursue my passion for science and providing the steppingstones to a career as a professional researcher. I also want to thank David Wright, Jim Marois, Sheeja George, and Ramdeo

(Andy) Seapaul and the rest of the Brassica carinata research team at the UF North

Florida Research and Education Center for all your support and assistance in the development of my project. Mosbah M. Kushad from the University of Illinois and

Steven H. Miller at the University of South Florida for assisting in various aspects of data acquisition of the samples.

TABLE OF CONTENTS

page

ACKNOWLEDGMENTS ...... 4

LIST OF TABLES ...... 7

LIST OF FIGURES ...... 8

LIST OF ABBREVIATIONS ...... 9

ABSTRACT ...... 10

CHAPTER

1 LITERATURE REVIEW ...... 12

Climate Change and its Origins ...... 12 Renewable Fuel Standard ...... 13 Long Chain Fatty Acids ...... 14 Brassica carinata: The “New” Competitor ...... 16 The Glucosinolate-Myrosinase Defense System ...... 19

2 THE EFFECT OF SOIL TYPE ON GLUCOSINOLATE PRODUCTION IN BRASSICA CARINATA DURING VARIOUS STAGES OF ITS GROWTH CYCLE ...... 25

Introduction ...... 25 Materials and Methods ...... 27 Greenhouse and Irrigation ...... 27 Soil ...... 27 Tissue Sampling ...... 28 Glucosinolate Extraction and Purification ...... 28 Glucosinolate Quantification ...... 29 Glucosinolate Profiles ...... 30 Experimental Design and Statistics ...... 31 Results ...... 31 Roots ...... 31 Stem ...... 32 Leaves ...... 32 Seed ...... 33 Discussion ...... 33

3 THE INFLUENCE OF NITROGEN RATES ON OIL AND GLUCOSINOLATES IN SEEDS OF BRASSICA CARINATA ...... 39

Introduction ...... 39

Materials and Methods ...... 40 Soil ...... 40 Irrigation and Fertilization ...... 41 Glucosinolate and Fatty Acid Profiles ...... 42 Experimental Design and Statistics ...... 42 Results ...... 43 Discussion ...... 44

4 COMPARING TWO ETHIOPIAN MUSTARD GENOTYPES GROWN IN THREE TYPES OF AGRICULTURALLY MANAGED SOILS ...... 47

Introduction ...... 47 Materials and Methods ...... 48 Seed and Soil ...... 48 Irrigation and Fertilization ...... 49 Glucosinolate and Fatty Acid Profiles ...... 50 Experimental Design and Statistics ...... 50 Results ...... 51 Discussion ...... 51

5 CONCLUSION ...... 55

Volatile Identification and Quantification ...... 55 Myrosinase Assays ...... 55 Prospecting for Carinata’s Future ...... 57

LIST OF REFERENCES ...... 59

BIOGRAPHICAL SKETCH ...... 65

LIST OF TABLES

Table page

2-1 The pH, nutrients (ppm), organic matter (%) and electrical conductivity (EC) of the three soil types for potting B. carinata in a greenhouse...... 36

2-2 The Hoagland’s nutrient solution at varying nitrogen rates...... 36

2-3 Dry biomass of root, leaf, stem, seed of Brassica carinata grown in three soil types...... 36

2-4 Total Glucosinolate and oil content in the seeds of conventionally grown carinata using near infrared spectroscopy...... 37

LIST OF FIGURES

Figure page

1-1 Concept map for producing biodiesel via transesterification...... 22

1-2 Flow chart for the conversion of triglycerides into biojet or biodiesel via catalytic hydrothermolysis, hydroprocessing, and fractionation...... 22

1-3 Comparison of long chain fatty acid profiles from various oilseed crops as percent (%) of total oil. Source: ...... 23

1-4 Diagram of possible outcomes of glucosinolate and its derivative (isothiocyanate) after soil incorporation...... 24

2-1 Total glucosinolate concentration of the roots, stem, and leaves in Brassica carinata at different stages in its life cycle...... 38

3-1 Total biomass accumulation at various rates of nitrogen using modified versions of Hoagland solutions with standard error ...... 46

3-2 Distribution of oil, protein, total glucosinolate concentration, erucic acid, long and very long chain fatty acid in the seed, ...... 46

4-1 Percentage of seeds germination and establishment...... 53

4-2 Correlation of combined total glucosinolate and oil in the seeds of two B. carinata genotypes (AAC-A110 and ARG-137)...... 53

4-3 Distribution of total glucosinolate and oil in the seeds of B. carinata between genotype AAC-A110 and ARG-137 ...... 54

LIST OF ABBREVIATIONS

ASTM American Society for Testing and Materials

EISA Energy Independence and Security Act

EPAct Energy Policy Act (2005)

GHG Greenhouse gas

GLS Glucosinolate

HEFA Hydroprocessed Ester and Fatty Acids

HSD Honest Significant Difference

HPLC High Performance Liquid Chromatography

ITC Isothiocyanate

LCFA Long-chain fatty acid

NFREC North Florida Research and Education Center

RFS Renewable Fuel Standards

UF/IFAS University of Florida / Institute of Food and Agricultural Sciences

USDA-NRCS United States Department of Agriculture – Natural Resource Conservation Service

Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science

THE EFFECTS OF SOIL TYPE AND NITROGEN RATES ON GLUCOSINOLATE AND OIL PRODUCTION IN BRASSICA CARNATA

Theodor Linares Stansly

May 2016

Chair: Pete C. Andersen Major: Horticultural Sciences

Brassica carinata is an oilseed crop that can withstand many environmental stresses and capable of producing seeds with an ideal fatty acid profile well suited for the biofuel industry with recent research suggesting that B. carinata can be successfully grown in the southeastern U.S. However, biotic and abiotic stresses due to climate variability, effects of soil type, and different cropping systems will affect the availability of nutrients and therefore alter the oil and glucosinolate (GLS) concentration in the shoots, roots, and seeds. In addition, high concentration of GLS in seed meal can be toxic at high doses and reduce the quality for use as animal feed. In this study, we investigated how differently managed soils (i.e. pasture, conventional, and sand) and nitrogen fertilization rates affect oil and GLS concentration of roots, shoots, and seeds of B. carinata genotypes. Total GLS was quantified at four different growth stages by colorimetric means and GLS profiles analyzed using HPLC. Pasture soils significantly increased total dry biomass accumulation while sand reduced total GLS in plant tissues.

We also found a strong negative correlation between oil and GLS concentration (r = -

0.91, p < 0.01) and percent oil and protein (r = -0.96, p < 0.01) in the seeds when varying nitrogen fertilization rates. Indole GLS was mostly found in root tissue with

sinigrin (an allyl-GSL) making up the majority (>90%) of the GLS found in the shoots and seed.

CHAPTER 1 LITERATURE REVIEW

Climate Change and its Origins

The Department of Defense released the 2014 Climate Change Adaptation

Roadmap where Chuck Hagel (Secretary of Defense) identifies climate change as a

‘threat multiplier’; predicting a profound effect on the world stability including increased extreme weather events, rising sea-levels, food and water shortages, infectious diseases, and terrorism (Department of Defence, 2014). The concerns over these rapid changes in global climate are intensifying throughout the world. In fact, last December, world leaders gathered in Paris to negotiate a legally-binding deal that culminated with

195 countries agreeing to do their part in reducing greenhouse gas (GHG) emissions and keeping the average global temperature from increasing more than 2°C. However, this goes into effect only after 55 countries have deposited their instruments of ratification (European Commission, 2016).

Almost 200 years ago, a greenhouse-like effect was first proposed by Joseph

Fourier who also developed the Fourier Transform and Series to study periodic phenomena. This concept was confirmed 50 years later through carbon dioxide and water vapor experimentation by John Tyndall (Kandel, 2012). Thereafter, Svente

Arrhenius published the first version of Earth’s energy budget in 1896. In this study titled On the Influence of Carbonic Acid in the Air upon the Temperature of the Earth, he calculated the total amount of radiation energy being absorbed and released. Although, this model was able to accurately explain geological history, there were some anomalies in his data when he approached present times. This led him to an important question in which he asks “Is it probable that such great variations in the quantity of

carbonic acid as our theory requires have occurred in relatively short geological times?”

During this time, the British controlled the coal industry and built the infrastructures in the Middle East to support the industrial revolution (Barak, 2015). This excessive amount of coal helped professors like Arvid Hogbom to state that the discrepancies within Arrhenius’ model may be explained by the sudden increase in the burning of fossil fuel (Arrhenius, 1896).

Renewable Fuel Standard

Ninety-five percent of the scientific community are endorsing pro-action policy to combat global warming and climate change (Liu et al., 2015). The overall consensus is to shift from fossil fuel as the sole source of energy to more renewable options. This includes extracting energy from nuclear, wind, solar and fuel derived from algae and plants-based sources. Using plants as a feedstock for energy is a method that recycles carbon back into plant biomass after it has been consumed for fuel in a cyclical manner which can takes months rather than then the millions of years it took to sequester the carbon as oil, coal, or natural gas (Murphy & Kendall, 2015).

According to the United States Department of Agriculture Economic Research

Service (USDA-ERS), about 7.1 percent (13.8 billion gallons) of the total transport fuel consumption is biofuel with the overwhelming majority of it being ethanol (“U.S.

Bioenergy Statistics,” 2016). The introduction of the Renewable Fuel Standard (RFS1) created under the Energy Policy Act of 2005 (EPAct, P.L. 109-58) mandated all fuel companies to blend at least 7.5 billion gallons of renewable fuels into gasoline by 2012.

This was later expanded under the Energy Independence and Security Act of 2007

(EISA, P.L. 110-140) that greatly increased the renewable fuel production capacity to 36 billion gallon by 2022 (Schnepf & Yacobucci, 2013). In addition, EISA also included a

provision that included the production of biodiesel in addition to ethanol. The expanded

RFS (or RFS2) also divided the production of these biofuels into four categories:

1. Total renewable fuels – 36 billion gallons by 2022 with a 20% GHG emission reduction*.

2. Advanced biofuels – 21 billion gallons by 2022 with a 50% GHG emissions reduction and produced by non-corn feedstocks, including ethanol.

3. Cellulosic and agricultural waste-based biofuel – 16 billion gallons by 2022 with a 60% reduction in GHG emissions and derived using cellulose, hemicelluloses, and lignin.

4. Biomass-based diesel – biodiesel and renewable diesel

* Relative to conventional fuels to qualify (Schnepf & Yacobucci, 2013)

The sources for renewable fuel allowed as feedstock must fit into five categories that include crop residues, forest materials, separate food and yard waste, perennial grasses, and secondary annual crops which must have minimal or no change in land use (Schnepf & Yacobucci, 2013). The assumptions on land use and management vary between the Department of Defense, U.S. Environmental Agency, and the U.S.

Department of Agriculture (Keeler et al., 2013). Regardless, these policies set forth by the Federal Government and will undoubtedly bolster the biofuel industry and expand the research and infrastructure needed to become a competitor in world markets and ultimately mitigate the advancement of climate change.

Long Chain Fatty Acids

The American Society for Testing and Materials (ASTM) are responsible for laying out the standards and specifications for the different types of fuels and blends.

Biodiesel is distinct from renewable diesel and defined under the standard of ASTM

D6751 as derived from vegetable oils or animal fats and made up of long chain fatty

acids (LCFA) and VLCFA (Kumar & Sharma, 2015). Essentially, they are converted into biodiesel using transesterification when triglyceride oils reacted with an alcohol by a strong base catalyst separating them into a mixture of biodiesel and glycerol (see Figure

1-1).

On the other hand, renewable or “green” diesel is defined as the product of using thermal depolymerization, but can also include hydrotreating or hydroprocessing for removing contaminates and fractionation to separate the different products (see Figure

1-2). Thermal depolymerization breaks down long fatty acids into shorter molecules using heat and pressure producing a mixture of long and short-chain hydrocarbons with a maximum length of 18 carbon atoms. In this case, plants that consistently produce longer-chained fatty acids are desired since the end result is a higher proportion of 18 carbon-chained molecules producing a product with very similar characteristics to petroleum-derived diesel (Yoon, 2011).

Plant oil are composed of triglycerides that vary in carbon chain length and in number of double bonds. Some of the most sought after in the biofuel industry are

LCFA and the VLCFA which contain aliphatic tails with either 13-19 carbon atoms or more than 20 carbon atoms, respectively. The fatty acids vary in composition and abundance among plant species (see Figure 1-3). This is important because the initial composition of fatty acids in the seed will affect the cold flow properties and cetane number (Ramos et al., 2009). Another important VLCFA is erucic acid (C22:1) which is naturally found in the seed oil of plants in the family Brassicaceae. Seed oil that contains both low (<2%) and high (>50%) erucic acid content have been extensively bred for commercial use. The former targeted for human consumption since erucic acid

has been recognized as hazardous and the latter as an industrial important feedstock

(Guan et al, 2014; Yan et al., 2015). VLCFA are ideal for the production of bioethanol, biodiesel (including renewable diesel), and biolubricant (Yu et al., 2014).

A performance study comparing three renewable biofuel feedstock of camelina

(Camelina sativa L.), carinata (Brassica carinata), and pennycress (Thlaspi arvense L.) concluded that carinata had petroleum-like engine performance and combustion characteristics with an overall reduction in carbon monoxide emissions (Drenth et al.,

2014). The history of carinata has made it a very resilient plant that can out produce other Brassica species when grown in marginal lands (Cardone et al., 2003). Carinata is a crop that can grow with minimal inputs and still able to produce high amounts of oil with a particular fatty acid profile conducive to the biodiesel and renewable diesel industry.

Brassica carinata: The “New” Competitor

Brassica carinata A. Braun is a plant of many names, but it is commonly known as Abyssinian mustard, Ethiopian mustard, or just carinata. It is a member of the

Brassicaceae family (formerly Cruciferae) in the order Brassicales. Carinata is an amphidiploid (BBCC, 2n=34) thought to have originated in the Ethiopian highlands plateau by the interspecific hybridization of two other diploid Brassica species followed by chromosome doubling (Guo et al., 2012). One of the parent species is B. nigra or black mustard (BB, 2n=16) which has been historically used to make spice from the seed. The other is B. oleracea (CC, 2n=18) which is commonly eaten throughout the world and include food crops like broccoli, cauliflower, cabbage, kale, collard greens,

Brussel sprouts, and kohlrabi. Carinata spread from the plateau to the surrounding regions around the Mediterranean coast and out into parts of Asia and Europe before

being introduced to Canada and Australia during the 1980s. In North America, it has typically been farmed as a cool season summer crop in the Northern Plains of the

United States and the western Canadian prairies as a summer crop, but new research suggests that carinata can be grown in southeastern U.S. as a winter crop (Bliss et al.,

2014).

B. carinata is a tall herbaceous annual with a high level of branching and an extensive tap root system (Barro & Martin, 1999). It has a high water use efficiency and therefore can tolerate high levels of heat stress, water deficiency, and some salinity stress (Ashraf & Sharif, 1998). The seed pod contains up to 20 large seeds and a significant amount of lignin making it the most shatter-resistant species compared to other oil seed crops in the genus Brassica which include B. juncea (brown mustard), B. napus (rape), and B. rapa (field mustard) (Barro & Martin, 1999).

A 2013 study from Canada on nitrogen fertilization rates with carinata resulted in a decrease of plant densities, nitrogen use efficiency, and oil concentration in the seeds when applying at high rates (200 kg N ha-1). They also found that as the nitrogen rate increased so did protein content in the seeds (Johnson et al., 2013). In fact, this negative correlation between oil and protein accumulation in seeds has been observed in other studies with carinata and canola (Brassica napus) (Hocking et al., 1997; Pan et al., 2012; Rathke et al., 2005). Johnson et al. (2013) was unable to maximize seed production even at these high rates and hypothesized that carinata may require more nitrogen than previously thought. Treatments that did not receive any nitrogen showed a high level of variability for seed production which ranged from low (507 kg ha-1) to high

(2050 kg ha-1) amounts. Fertilization recommendations will be specific to the genotype and the location in which carinata is grown (Pan et al., 2011).

The inverse relationship between oil and protein production in seeds of Brassicas may be due to their competition for carbon. The nitrogen and carbon ratio in protein is much high than in oil allowing protein synthesis to occur more often at the expense of fatty acids (Rathke et al., 2005). Carinata is consistently shown to be highly responsive to nitrogen fertilization as long as it is not stressed for water. In periods where carinata is water-stressed, there is rapid loss in nitrogen usage and only 50 kg N ha-1 is needed to maximum seed yields (Pan et al., 2011). This can be observed as carinata adapts to drought by physiological changes that include reduction in leaf area and elongation of the leaves and roots (Rana & Chaudhary, 2013). Drought stress and high ambient temperatures (30°C) will increase the time it takes for carinata to germinate, but also decrease the germination rate of the seeds (Patanè & Tringali, 2011).

Sulfur concentration also contribute to protein accumulation in the tissue of

Brassica oleracea var. Capitata L. and peaked at the application rate of 80 kg ha-1

(Verma & Nawange, 2015). They achieved maximum growth, yield, and quality when simultaneously applying 150 kg ha-1 of nitrogen and 60 kg ha-1 of sulfur, possibly displaying an interaction. Namvar et al. (2015) used similar rates which was sufficient for maximum growth and development and seed yield in rapeseed plants. In general, carinata should follow similar nutrient management as canola. Ideally, it should be grown in soils containing a pH between 5.5 to 6.8 and applied at least 91 kg N ha-1, 45

-1 -1 -1 kg P2O5 ha , 91 45 kg K2O ha , and 28 kg S ha (Bliss et al., 2014).

The Glucosinolate-Myrosinase Defense System

The pungent sensation that one feels around the nasal cavities from eating mustard seeds, horseradish, wasabi (Eutrema japonicum) and other Brassica vegetables are quite different from the “spiciness” of capsaicin found in hot peppers.

Instead, these short lived sulfur-rich molecules are activated when glucosinolates (GLS) become hydrolyzed by endogenous thioglucosidase enzyme called myrosinase. These activated compounds commonly referred to as mustard oil or isothiocyanate (ITC) and serve many roles, but the primary role is to mediate the interaction between the biotic environment and the overarching needs of the plant (Kliebenstein, Kroymann, &

Mitchell-Olds, 2005). Their toxic nature helps the plant to defend itself against predation and disease, but the unique chemical characteristic of certain volatile ITCs can attract specialist feeders (Halkier & Gershenzon, 2006). For many years humans have benefited from ITC as a spice in the culinary arts, or as a biological fumigant or pesticide to reduce pest pressure in cropping systems, and recently as a cancer- preventing agent.

More than 120 types of GLS have been found almost exclusively from the order

Brassicales. They all share the same core structure consisting of a β-thio-glucose moiety and a sulfonated oxime linked to a variable R group (Fahey et al., 2001). These compounds are derived from amino acid homologues and are generally categorized into three main groups depending on its structure and parent amino acids of origin. The aliphatic GLS are derived from alanine, leucine, isoleucine, methionine, or valine. The aromatic GLS are derived from phenylalanine and tyrosine while the indole GLS from tryptophan (Halkier & Gershenzon, 2006).

Biosynthesis of aliphatic and aromatic GLS undergoes three phases of development. First, side chains are elongated by the insertion of methylene group and reconfigured to produce the core structure. Genetic studies have found the Arabidopsis greatly increased the tryptophan-metabolizing gene in the CYP gene family which catalyzes the reaction from the amino acid into an aldoxime and then to thiohydroximic acids. It is thought that an S-glucosyltransferase then converts the thiohydroximic acid into a GLS. Finally, there are additional modifications to the R group which determine the direction and the activity of the hydrolysis product (Jensen et al., 2014; Nissum et al., 2008).

Although, many of these compounds are produced throughout the entire plant, some are induced to produce higher quantities when the plant detects danger. For example, a review found that up to a 20-fold increase in indolic GLS, and to a lesser extent aromatic and aliphatic GLS, can occur after an attack by any herbivore or wounding (Textor & Gershenzon, 2008). There are some indications that carbon availability may be controlling the production of aliphatic glucosinolates. The accumulation of GLS hydrolysis products is controlled by the plant through the isolation of GLS and myrosinase into separate cellular compartments (Textor & Gershenzon,

2008). When the plant suffers cell rupture, myrosinase is released into the environment at which it then catalyzes the reaction from GLS to ITC.

The conversion of GLS to ITC mostly occurs when the myrosinase enzyme (β- thioglucoside glucohydrolases) catalyzes the hydrolysis reaction of GLS into isothiocyantes (ITC), thiocyanates, epithionitriles, and nitriles (Andreasson et al., 2001;

Textor & Gershenzon, 2008). The distribution and composition of GLS depend on

environmental conditions, tissues, growth stages, biotic and abiotic stresses, and among Brassica species. Carinata leaves have shown increases in GLS concentration acropetally and was generally higher in concentration during early stages of development with an allylglucosinolate (sinigrin) comprising between 80-90% of the total

GLS in the leaves and indole GLS in the roots (Glover et al., 1988). Bhandari et al.

(2015) evaluated at 9 Brassica crops and found that seeds had the highest and shoots the lowest concentration of aliphatic GLS which include sinigrin. Roots exclusively contained gluconasturtiin (aromatic GLS), in addition to glucobrassicin (indole GLS) which could also be found in the shoots. Indole GLS is also less stable then aliphatic

GLS and may not require myrosinase for the conversion to its more toxic form (Bhandari et al., 2015; Halkier & Gershenzon, 2006).

Many mustard species and varieties are being used in cropping systems as cover crops and as biofumigation agents for the reduction of pests and diseases.

However, once a GLS-containing plant is incorporated into the soil (Figure 1-4), the retention of the noxious products is generally short-lived and dependent on factors such as soil type and ecology, water content, and temperature. GLS and ITC are both prone to microbial degradation and mineralization in the soil (Mazzola & Zhao, 2010). The chemical nature of GLS cause it to become easily leached compared to ITC which binds strongly to nucleophilic groups increasing sorption to organic matter in the soil (Gimsing

& Kirkegaard, 2008). Furthermore, studies using Brassica napus suggested that the introduction of glucosinolate and its hydrolyzation products into the soil significantly alters the microbial and fungal population long after the active compounds have dissipated (Cohen et al., 2005).

Figure 1-1. Concept map for producing biodiesel via transesterification. Source: www.enerfish.eu

Figure 1-2. Flow chart for the conversion of triglycerides into biojet or biodiesel via catalytic hydrothermolysis, hydroprocessing, and fractionation. Source: Readi fuels (www.readifuels.com)

Figure 1-3. Comparison of long chain fatty acid profiles from various oilseed crops as percent (%) of total oil. Source: (Drenth et al., 2015)

Figure 1-4. Diagram of possible outcomes of glucosinolate and its derivative (isothiocyanate) after soil incorporation. Source: (Gimsing & Kirkegaard, 2009)

CHAPTER 2 THE EFFECT OF SOIL TYPE ON GLUCOSINOLATE PRODUCTION IN BRASSICA CARINATA DURING VARIOUS STAGES OF ITS GROWTH CYCLE

Introduction

The demand for hydroprocessed esters and fatty acids (HEFA) fuel as a source for renewable energy is on the rise. These fuels are comprised primarily of hydrocarbons rather than alcohols and esters that are associated with ethanol-based biofuel. HEFA fuel are commonly known as ‘drop-in’ fuel since there is no distinguishable differences to that of petroleum-based diesel and can therefore be put directly into use with minimal refining requirements (Brown & Radich, 2015). Currently,

50% blends have been approved for use in aviation with the possibility of using 100% biodiesel in the future. Carinata, has been bred to contain a large amount of very long- chain fatty acids (VLCFA). The characteristics of VLCFA make for efficient HEFA fuel production and therefore demanded by the biodiesel industry. Canada and Northern

Plains in the United States had been the traditional locations for carinata farms during the summer months. However, recent research is now being conducted to incorporate carinata as part of a winter crop rotation into the Southeastern United States with minimal changes to the existing infrastructure (Bliss et al., 2014).

Although, there are many studies on the various oilseed crops including those in the genus Brassica, little research have been published concerning carinata alone. This includes glucosinolate (GLS) production throughout different organs of the plant and also at various stages in its life cycle. GLS are secondary compounds that contain sulfur and nitrogen atoms with over 120 types already described (Textor & Gershenzon,

2008). After a cell rupture event, GLS are typically hydrolyzed by the enzyme myrosinase into what may be commonly called mustard oil which is comprised of toxic

compounds including ITC, thiocyanates, epithionitriles, and nitriles (Andreasson et al.,

2001; Textor & Gershenzon, 2008). All Brassica species use this glucosinolate- myrosinase defense as method to deter herbivory and pest attack by other organisms above and below the soil surface. The concentration and type of GLS will vary from plant to plant depending on the genetics, agronomic practices and climatic conditions.

Generally, the production of glucosinolates increases when grown in the tropics then when it is grown in the temperate regions (Tripathi & Mishra, 2007). Although, it is useful as a defensive mechanism, studies have shown that there exists a negative correlation between oil and protein accumulation in seeds with carinata and canola

(Hocking et al., 1997; Pan et al., 2012; Rathke et al., 2005).

My hypothesis is that the partitioning and concentration of glucosinolate will differ with plant organ as B. carinata completes its various life cycles. Also, the GLS concentration and oil being produced by B. carinata will be affected by the soils that vary in management history. My objective is to test soil management histories with the allocation of glucosinolates in plant tissue during each stage of plant development. We propose that the roots will have a higher concentration and different profile of GLS when grown in environments that differ soil quality and structure found with different management history. Plant tissue (roots, stems, leaves, and seeds) will be harvested and glucosinolate and oil content will be analyzed at different stages of plant development for possible difference in their production and profiles. They will be grown in a semi-controlled environment using soils that are representative to North Florida farming and pasture lands to observe the effects of GLS and oil production in the seeds.

This will be valuable information that can be used for future field trials and increase the success of carinata oil production in the southeastern United States.

Materials and Methods

Greenhouse and Irrigation

Brassica carinata variety AAC A110 was used with the seeds having been acquired from Agrisoma Biosciences Inc. (Saskatoon, SK SKN 0W9, Canada). Plants were grown in a greenhouse equipped with evaporative cooling units between the months of February and July. Tree pots with dimensions 19.7 cm x 31.75 cm and total volume of 0.00765 m3 were used for all experiments (Product # TP816, Stuewe and

Sons, Tangent, OR 97389 USA). Four 20 mm holes were punched into the soil for uniform placement of seeds and soil kept moist until seedlings establishment. Two weeks later, potted plants were thinned to one plant per pot and irrigated 3 times a day at a rate about 1.05 milliliter/second. For the first few weeks after seedling establishment, they were given 1.5 total min of water per day (94.5ml) with an increasing rate of 1 minute per event every month until the seeds were harvested.

Soil

The three soil treatments (Table 2-1) used in this study were selected to reflect three different agricultural management practices. Soil, designated as “pasture”, was collected in Quincy, Florida (Latitude 30.54988°, Longitude -84.59672°) where bahia grass (Paspalum notatum Flugge) had been grown for over 30 years. This soil was classified by the United States Department of Agriculture Natural Resource

Conservation Service (USDA-NRCS, websoilsurvey.sc.egov.usda.gov) as Orangeburg loamy sand and contained 2.0% organic matter. The “conventional” soil had an extensive land use history and collected on a farming site at a similar location (Latitude

30.54555°, Longitude -84.59966°) and classified as Orangeburg-Norfolk complex containing 0.5% organic matter. The soil labeled as “sand” is of construction quality, contains no organic matter, and used as a control medium. All soil types had a similar pH ranging from 6.0-6.4, but varied in nutrient content. To negate the effects of nutrient differences, all plants were fertilized through irrigation (i.e. fertigation) with 100% of the standard Hoagland nutrient solution (Table 2-2)

Tissue Sampling

At least 10 grams of tissue sample were collected at each of the 4 stages of plant development (i.e. juvenile, bolting, flowering, and mature). Most of the plants (80%) had to be in the same stage before collecting leaf, stem, and root tissue samples for GLS quantification and profile analysis. Initially, leaves and stem from the 3rd, 4th, and 5th leaf axial were collected during the juvenile stage and sampling location moved upwards at every subsequent sampling time in order to acquire leaves of similar maturity. Root tissue was collected by rinsing all the sand and debris from the root mass using water on top of a sifting screen. All sampled tissue was snap frozen in liquid nitrogen and stored at -80°C freezer until they were lyophilized (freeze-dried). They were then weighed, ground to a fine powder, placed into their respective labeled container, and stored at 4ºC until glucosinolates extraction. The rest of the plant biomass not used for glucosinolates extraction was placed in paper bags, dried in an oven at 48°C, and weighed for biomass estimation. Seeds were harvested at maturity, dried at 48°C, stored in plastic container, and ground just before extraction.

Glucosinolate Extraction and Purification

Total glucosinolate extraction followed a modified version of Gallaher et al.

(2012). First, 200 mg of dried and ground plant tissue was placed into glass test tubes

and heated on a heating block for 10 min at 80°C. Next, 3 ml of 80% boiling methanol was added and then vortexed every 5-10 minutes while continuing to heat at 80°C water in a water bath. Samples were centrifuged for 10 minutes at 5000 rpm and supernatant was transferred into a clean glass test tube. The pellet was resuspended and the process was repeated except for 15 minutes of heating and pooling the supernatant. A modified version of Widharna (2012) was followed for glucosinolate isolation and purification. Each ion exchange column was filled with 200 mg of Sephadex A-25 resin and saturated with water. The resin was allowed to swell while mixing to remove air bubbles. Two repetitions (x2) of 2 ml of 0.5 M sodium hydroxide (0.5M NaOH) was passed through the column followed by 4 ml of double-distilled water (dd-H2O) to remove any excess NaOH. To this, 2 ml of 0.5M pyridine acetate solution (x2) was added to derivative the column to an acetate form. The column was rinsed again by adding 4 ml of dd-H2O (x2) leaving meniscus of water on top of column for sample application. The total extract was then added into prepared column and rinsed again using 4 ml of dd-H2O (x2). Finally, the column was washed using 2 mL 30 % formic acid

(x2), followed by 2 mL dd-H2O (x2) and discarding the eluate. The glucosinolate was eluted using 2 ml of 0.3 M potassium sulfate. Part of the eluate was used immediately for spectrophotometric quantification while the rest was frozen at -20ºC.

Glucosinolate Quantification

A modified version of Kumar (2004) was used for glucosinolate quantification.

One ml of eluted glucosinolate sample solution was pipetted into glass test tubes (2 repetitions per sample), then 300 μL of 0.002 M sodium tetrachloropalladate solution

(2mM NaPdCl4) was transferred into each test tube every 30 seconds. This corrects for the time needed to rinse or retrieve a new cuvette and recalibrate the

spectrophotometer between each set of samples. After 30 min, absorbance values was measured at 405 nm while keeping a 30 second per observation pace. A standard curve was calculated using the range of absorbance values and a series of sinigrin concentrations (Sinigrin Hydrate (TLC), ≥99.0%, Sigma-Aldrich) ranging from 10-220

µM sinigrin.

Glucosinolate Profiles

Tissue samples were sent to the Plant Science Laboratory (University of Illinois,

Urbana, IL 61801) for glucosinolate profile analysis using high-pressure liquid chromatography (HPLC). Following a modified version of Kushad (1999), a 1 mL fraction of the supernatant was combined with 150 µL of 0.5 M barium acetate, vortexed for 5 s, and layered on a 1 cm DEAE-Sephadex A-25 column. Glucosinolates were desulfated on the column with 10 units of arylsulfatase type H-1 from H. pomatia

(Sigma, St. Louis, MO) suspended in 500 µL of dd-H2O. The columns were capped for

18 h. Desulfated glucosinolates were eluted off the column with 2 mL of dd-H2O and filtered through a 0.2 µm Acrodisc filter (Pall Gelman Laboratory, Ann Arbor, MI). A 20

µL fraction of the water filtrate was injected into a Hitachi HPLC system (Hitachi Ltd.,

Tokyo, Japan) equipped with a UV detector set at a 229 nm wavelength, a refrigerated autosampler set at 5 °C, a column heater set at 32 °C, and a Lichrosphere Hibar RP-18 column (Merck, Darmstadt, Germany). A gradient of 0-20% acetonitrile at 0.8 mL/min for 53 min was used to elute the desulfoglucosinolates off the column. The type and amount of glucosinolates were estimated based on retention times and response factors developed for desulfoglucosinolates in a standardized and certified rapeseed reference material (BCR 367) by the Commission of the European Community Bureau of

References (Brussels, Belgium). Sinigrin concentration was estimated by comparison of

HPLC retention time with that of sinigrin standard (Sigma) after on-column sulfatase treatment as described above. Seed tissues samples were analyzed for GLS profile as well as moisture content, percent oil, protein, and fatty acid profiles using near infrared spectroscopy (Agrisoma Biosciences Inc, 110 Gymnasium Pl, Saskatoon, SK S7N 0W9,

Canada).

Experimental Design and Statistics

This experiment was set up with a randomized complete block design with 5 blocks and 60 total observations. One factor was soil type (conventional, pasture, and sand) and the other is growth stage of development with 4 levels (juvenile, bolting, flowering, and agronomic maturity). A two-way analysis of variance (ANOVA) was used followed by Tukey’s studentized range test with honest significant difference (HSD) to determine significant differences in between soil type, biomass weight, GLS concentrations, and oil content in seeds using R Studio (version 0.98.109) statistical software.

Results

Roots

Root biomass was highest (up to a 40% increase) when grown in pasture soil, but no significant root growth occurred after the flowering stage (Table 2-3). The average root to shoot ratio decreased as carinata matured and ranged from 1.61 during the juvenile stage, 0.91 during the bolting stage, 0.66 during the flowering stage and

0.29 once the seeds were mature. Absorbance analysis revealed that GLS concentration in the roots increased from 16 to 55 µmol g-1 of tissue as the plant matured. It was also evident that GLS concentrations was less when carinata was grown in sandy soils compared to conventional or pasture soils. HPLC results showed

that mostly indole GLS were found in root tissue samples that included glucoraphanin, glucobrassicin, neoglucobrassicin, and 4-methoxyglucobrassicin with traces of sinigrin, progroitrin, and epiprogroitrin. Taking GLS concentration and biomass accumulation into account the estimated amount of total GLS in the plant was highest during the flowering stage and in pasture soils (3,809 µmols).

Stem

Pasture soil significantly increased stem biomass production followed by sand and then conventional soil. Although, there were no differences in the GLS concentration between soil types, there was a continuous increase in the GLS concentration of the stem as the plant continued through its development and peaked with an average of 51.9 µmols g-1 of tissue at the mature stage. Carinata grown in pasture soil also produced the most GLS (3,243 µmol) by the time it matured when compared to other soils. The most abundant GLS found in stems from conventional soils was sinigrin and comprised 70%-90% of the GLS extracted with glucoraphanin, gluconapin, epiprogoitrin and progroitrin accounting for the remainder

Leaves

Carinata increased its leaf biomass accumulation when grown in pasture soils followed by conventional and sand. There was also a marked reduction in GLS concentration when carinata left the vegetative stage (juvenile and bolting, 78.0 µmol g-1 tissue) and entered into its reproductive stage (38.5 µmol g-1 tissue).This led to no changes in total GLS accumulated in the leaves between the bolting stage and the flowering stage. HPLC and GS analysis shows that sinigrin made up almost all of the

GLS found in leaves with some traces of glucoraphanin and glucobrassicin.

Seed

There was no statistical difference between biomass accumulation (ranging from

11.6 to 13.5 grams per plant) and GLS concentration (ranging from 159.8 to 162.5 µmol g-1) between soil types. Therefore, there were also no differences in total GLS accumulated in the seeds. In addition, NIR analysis (Table 2-4) on seeds showed that there were no differences in percent protein and fatty acid content (including VLCFA) as well as GLS concentration between the different soil types. There was a wide range of

GLS identified in the seed profile with sinigrin being at least 90% of the total and progroitrin and epiprogroitrin equally making up another 5%. In addition to glucosinolates, there was also no difference between oil content among the different treatments (p>0.05). Seeds contained 21-27% oil with about 50 g kg-1 of LCFA and

VLCHA and a moisture content of less than 5%.

Discussion

There is a significant increase in total biomass accumulation when carinata is grown in pasture soil compared to conventionally managed soil or sand. This increase occurred in the roots, leaves and stem, but not among seed weights. The ecological, soil fertility, and microbial diversity differences between intensively-managed agricultural land to those that are natural or organically managed could be responsible for this outcome. A review comparing various farming systems found that a diverse farming system will increase biodiversity, soil quality, and crop resilience and resistant to climate change compared to conventional farming (Kremen & Miles, 2012). An indication of this is observed between the dry weights of carinata grown in conventional soil and in sand having no discernible difference. Land that has an intensive land use history tends to negatively impact the soil health or quality and therefore lead to a less robust

agricultural environment. Both, abiotic and biotic factors may be contributing to these differences. For example, an increase in soil quality that includes organic matter as well as contact with a more diverse population of microorganisms can increase decomposition rates, the availability of nutrients, and water moisture allowing a more stable growing condition (Bowles et al., 2014).

Soil ecology may play a significant role on the production of glucosinolates and other secondary metabolites in field trials where the plants are constantly exposed to the environment, but there was not enough statistical evidence in this experiment to support this hypothesis (p=0.06). The presence of nematodes and other potentially infectious diseases-causing organisms can stimulate carinata to increase GLS concentrations where it is needed (Textor & Gershenzon, 2008). In the future, studies that capture some of the more volatile forms of GLS would be useful to add to total GLS being produced and possible lead to the identification of GLS in which parasitic nematodes and other pests are particular sensitive. However, studies have found that the release of volatiles depend more on differences between Brassica species than the composition of glucosinolate or type of damage (van Dam et al., 2012). The breeding program to identify carinata accessions which can readily resist the attack of pests through the manufacturing of specific GLS profiles either in the roots while it is growing or after it is incorporated into the soil as an amendment strategy will be extremely useful for Florida’s higher pest pressures. This will also help farmers increase the soil quality as well as to suppress certain soil-borne diseases when GLS is used as a biofumigation agent.

There is evidence to suggest that B. carinata may be reallocating GLS to the seeds as it matures. This is consistent from the discovery of GLS transporter genes in

Arabidopsis thought to transfer GLS from various organs to the seed (Lu et al., 2014).

During the early stages of carinata development, the leaves have consistently contained a high GLS concentration (80 µmol g-1 tissue) until the flowering stage where it then was reduced by 50% (Figure 2-1). This would allow the plant to reduce the chances of herbivory due to the high toxicity of GLS hydrolysis products. The stem maintained a constant GLS concentration (of about 10 µmol g-1 tissue) throughout the vegetative state and flowering stage. At plant maturity there was a sudden spike of GLS concentration (over 70 µmol g-1 tissue). This sudden spike would occur in the absence of leaves or seeds once carinata has reached agronomic maturity and the seeds are no longer being supplied water, nutrients, and GLS. In 2003, a similar study on GLS concentration and stages of development was conducted in Denmark between four

Brassica species (Bellostas et al., 2004). They found that roots contributed the most for total GLS production during the vegetative or “leaf” stage (40 µmol g DM-1) and a decline of GLS in the stem and leaves thereafter. In the current study, roots of carinata didn’t reach that limit until after the bolting stage.

Carinata is a resilient oilseed crop that can grow on marginal lands and produce oil with the fatty acid profile suited for the biofuel industry. Already, crops are being grown in the Canadian Prairies and Northern United States and now being targeted for the Southeastern U.S. as part of a winter cropping system. The purpose of this study was to evaluate how well carinata can grow on these marginal soils and what effect does it have on glucosinolate (GLS) and oil production.

Table 2-1. The pH, nutrients (ppm), organic matter (%) and electrical conductivity (EC) of the three soil types for potting B. carinata in a greenhouse. Soil pH BV OM EC N P K Mg Ca Cu Mn Zn Pasture 6.4 N/A 2.00 0.12 0.10 72 125 192 753 0.58 23.57 6.22 Conventional 6.0 7.66 0.47 0.15 0.03 209 25 40 215 0.09 23.96 1.69 Sand 6.1 7.88 0.00 0.15 0.01 42 0 11 82 0.02 1.90 3.11

Table 2-2. The Hoagland’s nutrient solution at varying nitrogen rates. Weight (mg L-1) Chemical 0%-N 33%-N 66%-N 100%-N

Ca(NO3)2 * 4H2O 0 234.1 468.1 709.4

CaCl2 324.1 216.7 110.2 0

KNO3 0 92.6 185.2 281.7 KCl 153.0 102.4 52.0 0

MgSO4 * 7H2O 274.3 274.3 274.3 274.3

KH2PO4 75.9 75.9 75.9 75.9 FeNa ; EDTA 56.5 56.5 56.5 56.5 NaCl 9.83 9.83 9.83 9.83

MnCl2 * 4H2O 3.30 3.30 3.30 3.30

H3BO3 3.1 1.67 1.67 1.67

CuSO4 * 5H2O 3.09 3.09 3.09 3.09

ZnSO4 * 7H2O 0.48 0.48 0.48 0.48

(NH4)6MO7O24 * 4H2O 0.15 0.15 0.15 0.15

Table 2-3. Dry biomass of root, leaf, stem, seed of Brassica carinata grown in three soil types. Dry Biomass Weights (grams) Tissue Pasture Conventional Sand Roots 45.0a 30.4b 27.7b Leaves 20.6a 17.4ab 16.3b Stem 62.5a 49.9b 52.2ab Seed 11.6a 13.5a 13.3a Total 108.4a 84.5b 82.6b Weights with similar subscripts letter do not differ (Tukey’s HSD P > 0.05)

Table 2-4. Total Glucosinolate and oil content in the seeds of conventionally grown carinata using near infrared spectroscopy. OIL GLS C221 PROTEIN SATS C160 C180 C181 C182 C183 C201 MONO POLY LCFA VLCFA H2O (%) (µmol/g) (%) (%) (%) (%) (%) (%) (%) (%) (%) (%) (%) (%) (%) (%) 24.6 158.9 30.6 44.6 7.4 3.8 1.3 21.3 23.9 10.3 4.4 51.6 41.0 50.0 50.0 5.0 26.6 152.5 32.7 45.5 6.7 3.9 1.0 16.4 22.7 11.9 5.1 52.9 40.1 53.2 46.8 4.6 26.7 166.4 33.7 44.0 6.9 3.6 1.1 18.1 21.8 11.3 5.3 52.9 39.8 46.9 53.1 4.8 25.2 180.0 25.1 43.7 9.1 3.6 1.7 21.8 28.3 7.9 2.7 51.1 40.8 44.6 55.4 4.2 24.5 169.2 29.6 45.6 7.6 3.7 1.2 18.0 24.2 10.4 4.2 52.6 40.1 50.7 49.3 4.7 21.4 189.6 26.6 45.8 8.9 3.5 1.5 20.6 26.9 8.7 2.6 51.6 40.6 42.8 57.2 4.8 26.9 161.1 36.2 46.8 6.5 3.8 1.1 17.1 21.2 12.0 4.2 53.3 39.5 49.7 50.3 4.9 24.2 169.1 30.1 45.0 7.0 3.6 1.1 17.9 23.1 11.6 5.7 51.6 41.0 50.5 49.5 5.0 27.0 153.4 34.2 45.0 6.8 3.7 1.2 19.0 22.0 11.2 4.5 52.9 39.7 48.3 51.7 4.9 23.8 189.8 33.8 45.6 7.2 3.6 1.2 18.2 22.7 10.8 4.8 51.8 40.6 48.1 51.9 5.0 24.1 171.4 33.3 47.3 7.3 3.7 1.2 16.3 22.9 11.6 3.1 52.0 40.5 49.6 50.4 4.8 22.4 160.2 27.0 46.0 8.2 3.8 1.4 18.7 26.6 9.0 3.4 50.6 41.8 50.1 49.9 4.6

Changes in total GLS Concentration in B. carinata with Stages of Development

Roots Stem Leaves

90 )

/g 80

70 mol 60 50 40 30 20 Concentration (µ Concentration 10

GLS GLS 0 Juvenile Bolting Flowering Mature Stages of Development

Figure 2-1. Total glucosinolate concentration of the roots, stem, and leaves in Brassica carinata at different stages in its life cycle.

CHAPTER 3 THE INFLUENCE OF NITROGEN RATES ON OIL AND GLUCOSINOLATES IN SEEDS OF BRASSICA CARINATA

Introduction

In 2014, the United States Department of Defense has stated that climate change is the single biggest threat to our national security and that this ‘threat multiplier’ must be dealt with immediately. One plan to reduce emissions as well as conflict is to shift from petroleum-based fuels to more renewable sources of energy. This will help capture and reuse the carbon in a cyclic manner and therefore help mitigate the enormous release of greenhouse gas emissions (GHG) into Earth’s atmosphere.

Brassica carinata also known as Ethiopian mustard or carinata is an oilseed crop that is now being examined as a potential winter crop in the Southeastern United States. The fatty acid profile in the seeds of carinata are made up of very long chain fatty acids

(VLCFA) and well suited for use as biodiesel and biojet fuel.

Carinata is mostly grown during the summer months in the Canadian prairies and more recently in the US Northern plains. Currently, there is research to integrate carinata into the Southeastern US as a winter crop with minimal to no changes to current infrastructure (Bliss et al., 2014). Growing carinata between the fall and spring cropping seasons will help farmers increase their lands production value and likely the yield to subsequent crops by providing an additional ecological service which can include increasing organic matter and reduction in pests and diseases. Previous carinata research has mainly focused the ecological studies and breeding efforts on producing high yields in northern climates, but little is known for northern Florida.

Additional studies are needed to understand the best farming and soil management practices to increase the resilience of carinata in this region.

In this study, we will be growing carinata in soil collected from locations where there has been an intensive farming practice and compare that to soil collected from pasture land predominated with bahia grass for over 30 years. We will also vary the nitrogen fertilization rates while grown in the different soils. A study the on the effects of soil type found that having a higher clay content will increase the uptake of nitrogen

(Razzaghi et al., 2012). Differences in soil quality can affect the uptake of water and nutrients. My hypothesis is that carinata will grow significantly better if grown in soil collected from bahia grass (Paspalum notatum Flugge) pastures since it will contain better water holding capacity, increased soil quality, and a more complex microbial diversity then ‘conventional’ soil.

Materials and Methods

The Brassica carinata seeds (var. AAC A110) were acquired from Agrisoma

Biosciences Inc. (Saskatoon, SK SKN 0W9, Canada). This particular variety is considered the industry standard for the production of biodiesel. These plants were grown in a greenhouse using pots with 0.00765m3 of total volume and of dimensions

19.7cm x 31.75cm (Product # TP816, Stuewe and Sons, Tangent, OR 97389 USA).

Although, these soils were collected from similar location and of similar classification, they do vary in the nutrient and organic matter composition (Table 3-1). Except for phosphorous, pasture soils contain high levels if micronutrients and macronutrients and organic matter (2.0%) when compared to conventional soil (0.5%).

Soil

Tree pots with dimensions 19.7cm x 31.75cm and total volume of 0.00765 m3 were used for all experiments (Product # TP816, Stuewe and Sons, Tangent, OR 97389

USA). Four 20 mm holes were punched into the soil for uniform placement of seeds.

Two different soils were used as the growing medium to simulate the agroecosystems found around the Southeastern region of the United States. Soil from pastureland, designated as ‘pasture’, was collected on NFREC land (Latitude 30.54988°, Longitude -

84.59672°) where bahia grass (Paspalum notatum Flugge) has predominated for a minimum of 30 years. It is classified as Orangeburg loamy sand by the USDA Natural

Resource Conservation Service (2014). Only the top 15 cm of soil was collected to capture the area of the rhizosphere. It was then separated from the bahia grass root masses by carefully shaking it free and discarding large plant tissues. No sifting was done to preserve the soil structure. The “conventional” soil was also collected on

NFREC property and at a similar location (Latitude 30.54555°, Longitude -84.59966°) but has an intensive land use history. This includes the continuous tilling, spraying of herbicides and pesticides, and fertilization for the multitude of crops grown in the area over the years. The USDA-NRCS has classified this soil as Orangeburg-Norfolk complex (http://websoilsurvey.sc.egov.usda.gov).

Irrigation and Fertilization

Initially, four seeds were sown 25 ml deep and soil kept moist until seedlings establishment. Thereafter, potted plants were thinned to two plants per pot and irrigated

3 times a day at a rate of 1.05 ml s-1. Liquid fertilization during irrigation periods (i.e. fertigation) included 4 different nitrogen rates as treatment that include 0, 33, 66, and

100% strength of Hoagland’s nutrient solution (Table 3-2.). The entire seedling stand were supplied with the full nutrients 2 weeks after seedling establishment (98 mg N day-

1) before treatments were imposed. For the first month after start of treatments, they were given 1.5 total min of water and fertilization per day (94.5 ml) with an increasing

rate of 1 min per event (3 per day) every month until the seeds were harvested 4 months after planting.

Glucosinolate and Fatty Acid Profiles

Near infrared spectroscopy was performed by Agrisoma and used to determine total glucosinolate and oil content in the seeds as well as its fatty acid profile. Agrisoma carinata NIR seed screening was done on a FOSS XDS™ Rapid Content Analyzer. The instrument is fitted with an autosampling unit. Approximately 2-3g of seed (clean, dried to <8% moisture) is placed in a ringcup, tracking code is recorded, and sample spectra is collected at 0.5 nm increments, over the range of 400-2499.5 nm. Values for oil were determined using NMR on an Oxford MARAN Ultra benchtop NMR system using fresh carinata oil as a tuning sample, and various check lines to determine oil content. Fatty- acid values (and derived statistics, such as %Sats and %LCFA) were obtained using

Gas Chromatography of the Fatty-Acid Methyl Esters (FAMEs) using the protocol described by Taylor et al. (1992). Glucosinolate values were provided by the Canadian

Grains Commission.

Experimental Design and Statistics

This experiment was set up as a randomized control trial containing 2 factors, soil type and nitrogen rate. For soil type there are two levels labeled as “conventional” and “pasture”. There was four nitrogen rates labeled as 0, 33, 66, and 100% strength of the Hoagland’s nutrient solution. A two-way analysis of variance (ANOVA) was used followed by Tukey’s honest significant difference (HSD) to determine significant differences in between soil type and biomass weight, seed GLS and oil concentrations, and fatty acid profiles using R Studio (version 0.98.109) statistical software. The NIR data set was subjected to math treatment to refine the spectra and the WinISI software

package was used to determine the spectral regions most predictive of the measured parameters. We selected a number of points providing >99.99% correlation with the experimental values, and used these to develop the predictive equation.

Results

The biomass accumulated, including seed weights, between carinata grown in conventional soil and pasture soil were not significantly different (p>0.05, see Figure 3-

1). However, there were differences between nitrogen rates. Dry biomass was 50 g for

0% treatments. Both the 33% and the 100% N treatments had no difference producing

240 g and 290 g, respectively. Treatments that received 66% N had the highest biomass accumulation (over 350 g). This trend was similar to seed production in which the 66% N treatment achieved a 30% increase in seed production compared to the full nutrient treatment counterpart.

Near infrared spectroscopy revealed a continuous decrease in oil concentrations in the seeds of carinata with increasing rates of nitrogen. The amount of oil decreased from about 48% to 30% when the complete (100% N-rate) Hoagland nutrient solution was applied. This was the opposite in regards to crude protein (CP) and glucosinolate

(GLS) concentrations where there was a positive relationship. CP concentrations increased from 26 g kg-1 seed at the 0% N-rate to over 44g kg-1 of seed at the 100% N- rate. GLS concentrations ranged from less than 80 µmol g-1 of seed to over 140 µmol g-

1 (Figure 3-2). A Pearson correlation between nitrogen rate, percent oil, protein, and glucosinolate concentration in the seeds revealed a strong negative correlation between the concentration of oil and total glucosinolates (r= -0.91, p < 0.01) and the concentration of oil and protein (r = -0.96, p < 0.01) in the seeds when running nitrogen

deficiency trials. The estimated total nitrogen applied per plant was 1.38, 20.1, 38.8, and

58.0 g for the 0, 33, 66, and 100% N-rate, respectively.

Discussion

The negative correlation between oil and protein content has been seen in other studies and may be due to the competition for carbon skeletons between fatty acid and amino acid biosynthetic pathways (Johnson et al., 2013; Rathke et al., 2005). The decrease in oil concentration as the nitrogen rate increased produced significantly more oil since the higher nitrogen rates produced significantly more seeds. Carinata has consistently been shown to be highly responsive to nitrogen fertilization as long as it is not stressed for water (Pan et al., 2011). This can be observed as carinata adapts to drought by physiological changes that include reduction in leaf area and elongation of the leaves and roots (Rana & Chaudhary, 2013). Drought stress and high ambient temperatures (30°C) will increase the time it takes for carinata to germinate, but also decrease the germination rate of the seeds (Patanè & Tringali, 2011).

From this experiment, it is evident that nitrogen can impact the production of glucosinolate (GLS), protein, and oil in the seeds of Brassica carinata. Although, not quite statistically significant (p-value = 0.0545), there was a continuous decrease of erucic acid (C22:1) as the amount of nitrogen increased. This may be important since longer-chained fatty acids are desired to increase the proportion of 18 carbon-chained molecules in the final product that can closely resemble the characteristics to petroleum-derived diesel (Yoon, 2011). In field trials, studies suggest that the addition of plant growth-promoting rhizobacteria can increase oleic acid (C18:1) and linoleic acid

(C18:2) when compared with uninoculated control (Nosheen et al., 2013). Although, there were no significant differences between the soil types, there was a trend of

increasing biomass in pasture soils starting to occur. Expanding this experiment could find that pasture soils would be able to increase oil production and decrease GLS accumulation in the seeds given the same nitrogen fertilization rates.

Total Biomass vs Nitrogen Rates

Conventional Pasture

500.0

400.0

300.0

200.0

100.0 Dry Biomass (grams) Biomass Dry 0.0 0% 33% 66% 100% Nitrogen Rate (%)

Figure 3-1. Total biomass accumulation at various rates of nitrogen using modified versions of Hoagland solutions with standard error

Figure 3-2. Boxplots showing the distribution of oil (%), protein (%), total glucosinolate concentration (µmol/g), erucic acid (%), long-chain fatty acid (LCFA, %), and very long-chain fatty acid (VLCFA, %) in the seed,

CHAPTER 4 COMPARING TWO ETHIOPIAN MUSTARD GENOTYPES GROWN IN THREE TYPES OF AGRICULTURALLY MANAGED SOILS

Introduction

All members of the mustard family (Brassicaceae, formally Cruciferae) invest significant amount of energy for the production of glucosinolates (GLS). These are secondary compounds that are low in molecular weight, contain high levels of sulfur, and are derived from amino acids. They become ‘activated’ after a hydrolyzation event by a thioclucoside enzyme called myrosinase (E.C. 3.2.1.147). The product is commonly known as isothiocyanates (ITC) and also contain thiocyanates, nitriles, and epinitriles (Bones & Rossiter, 2006).

Once these compounds are activated they can become quite toxic. Farmers have often used Brassicas as a biofumigant to suppress and deter unwanted pests. However, there is some evidence to suggest that glucosinolates are interfering with oil production in the seeds. Elahi et al., found that over-expressing the BnLEC1 gene in (Brassica napus L.) can increase the production of oil in the seeds (7-16%) without altering the fatty acid profile or GLS concentrations. This gene was linked to fatty acid biosynthesis, sucrose metabolism, and glycolysis and when suppressed, there was a reduction in oil content (9-12%) and a dramatic increase of glucosinolates (Elahi, Duncan, & Stasolla,

2016). The negative correlation between glucosinolates and oil production must be investigated further to understand how GLS interferes with oil production.

Carinata (Brassica carinata A. Braun) is being introduced into the southeastern

United States as a feedstock for the biodiesel industry. They are already established in many parts of the Canadian prairies and the U.S. Northern Plains. However, the high temperatures, excessive rainfall, disease pressure, and low soil quality in northern and

central Florida will undoubtedly require new breeding efforts and protocols specific to the region. Carinata genotypes that express high levels of GLS may be more suited for the increase in pests. In this study we observe whether two B. carinata accessions that vary in seed glucosinolate accumulation have the capacity to produce the same amount of oil if they are subjected to a variety of soils gathered from intensive farming areas, pasture land, or sandy environments. My hypothesis is that the composition of these soils will influence nitrogen availability and leaching and therefore oil and GSL production. In addition, the genotype that accumulate more GLS in the seeds will be more capable of germinating and maintaining a rigorous growing behavior, but at the cost of oil productivity.

Materials and Methods

Seed and Soil

The Brassica carinata seeds were acquired from Agrisoma Biosciences Inc.

(Saskatoon, SK SKN 0W9, Canada). The accession AAC A110 is widely used around northern latitudes and considered the standard. In addition, accession (ARG-137-5.106) will be used since it has been determined to produce significantly more glucosinolates in the seeds (140 μM) than the industry standard (80 μM) and possibly a heightened interaction with the different soil types. These soils were selected to simulate three different ecosystems that include pasture land, heavily agriculture or conventionally managed land, and sand. They were all collected from Gadsden County, Florida in and around the property of the University of Florida extension office of North Florida

Research and Education Center (NFREC) located in the city of Quincy.

Soil designated as ‘pasture’ was collected on NFREC land (Latitude 30.54988°,

Longitude -84.59672°) where bahia grass (Paspalum notatum Flugge) has been

predominating for a minimum of 30 years. This soil composition is classified as

Orangeburg loamy sand by the USDA Natural Resource Conservation Service (2014).

Only the top 15 cm was collected to capture the majority of the organic matter and microbial diversity located within the rhizosphere. Debris in the rhizosphere was carefully separated from the soil by carefully shaking it free without sifting to preserve the soil structure. The ‘conventional’ soil was also collected on NFREC property and at a similar location (Latitude 30.54555°, Longitude -84.59966°) but has an intensive land use history. This includes the continuous tilling, spraying of herbicides and pesticides, and fertilization for the multitude of crops grown in the area over many years. The

USDA Natural Resource and Conservation Service have classified this soil as

Orangeburg-Norfolk complex. The “sand” is of construction quality and contains low levels of clay.

Although, the conventional and pasture soil come from a similar location classification, they do vary in the composition. The UF/IFAS Soil Testing Lab has indicated that all soils have similar pH ranging from 6.0 to 6.4. Pasture soils have high levels of nutrients when compared to conventional soil, except for phosphorous. As expected, sandy soil has no detectable organic matter. Conventional soil has a 0.47 % organic matter content compared to the pasture soil that has 2 %. There is some trace amount of nitrogen in the soils, but this will be supplemented with liquid fertilization during irrigation. (i.e. fertigation).

Irrigation and Fertilization

Tree pots with dimensions 19.7 cm x 31.75 cm and total volume of 0.00765 m3 were used for all experiments (Product # TP816, Stuewe and Sons, Tangent, OR 97389

USA). Four 20 mm holes were punched into the soil for uniform placement of seeds.

Pots were kept moist until seed germination and seedling establishment. After 1 week, the number of seeds were reduced to one per pot and then irrigated 3 times a day at a rate 1.05 ml/s. For the first month each pot received about 1.5 total minutes (94.5ml) of water per day with 100% Hoagland solution. Every month thereafter there was an increase of one minute for every event (3 times a day) until the seeds were harvested.

Glucosinolate and Fatty Acid Profiles

Gas Chromatography of the Fatty-Acid Methyl Esters (FAMEs) using the protocol described by Taylor et al. (1992). Glucosinolate values were provided by the Canadian

Grains Commission.

Experimental Design and Statistics

This trial was set up as a randomized complete block design with 6 blocks.

Factors include soil type (i.e. pasture, conventional, and sand) and carinata genotypes with two levels (AAC-A110 as “low” and ARG-137 as “high”). A 2-way ANOVA was performed with a Tukey’s studentized range test (HSD) to determine significant differences in between genotype and soil type, seedling establishment, GLS and oil

concentration in seeds between treatments by applying R Statistics. The NIR data set was subjected to math treatment to refine the spectra and the WinISI software package was used to determine the spectral regions most predictive of the measured parameters. We selected a number of points providing >99.99% correlation with the experimental values, and used these to develop the predictive equation.

Results

The carinata with the ARG-137 GLS genotype had a significant higher rate of seedling establishment in pasture and conventional soil. All (100%) of the seeds germinated successfully when they were planted in conventional soil compared to just over 54% for the AAC A110 GLS genotype (Figure 4-1). The total seed weight for the

ARG-137 GLS carinata remained the same throughout every soil treatment (ranged from 7.4-9.4 g pot-1) and was significantly lower than the AAC A110 GLS genotype

(ranging from 14.7 to 22.3 g pot-1). Near infrared spectroscopy revealed that the GSL genotype consistently produced significantly more oil and less GLS than the ARG-137

GLS genotype. The largest increase was observed in the pasture soil treatment where the AAC A110 GLS genotype had a 26.2% increase in oil accumulation and a 28.9% decrease in total glucosinolates in the seeds compared to the ARG-137 GLS genotype.

The amount of oil and GSL (Figure 4-2.) in the seeds inversely correlated (r = -.875).

Discussion

All Brassica plants produce glucosinolates (GLS) which help mitigate the interactions it has with the environment. A large majority of GLS are ‘activated’ after cell rupture by the enzyme myrosinase. The activated compound can be quite toxic and can be used as a biofumigant to deter and suppress diseases. This may be the reason why the seed germination and seedling establishment was more successful with AGR-137

who initially had twice the amount of GLS in the seeds. This study also showed that carinata GSL and oil distribution can vary greatly when grown in different soils (see

Figure 4-3).

The total GLS concentrations were quite high and could have been induced by factors such as heat stress or pests. An infestation of aphid and diamondback moths were found in over 70% of the plants and persisted over a period of two weeks until it was fully controlled. Glucosinolate are produced constitutively, but can also be up regulated when the plant senses the presence of pests or herbivory. Thereafter, the

GLS can be subsequently transferred to the seeds during maturation and lead to an overall increase of GLS in the data. Another trial with regular pesticide spraying would be appropriate as well as an alternate GLS quantification method to verify results.

Germination and Seedling Establisment within Three Different Soil Treatments 100 SH 90 80 SL 70 60 CH 50 40 CL 30

Germination Rate (%) Rate Germination 20 PH 10 PL 0 SH SL CH CL PH PL Treatments: Sand (S-), Conventional (C-), and Pasture (P-) GSL Production: High (-H; 140μM/g) and Low (-L; 85μM/g)

Figure 4-1. This bar graph shows the percentage of seeds that germinated and made it to seed establishment. The first letter signifies the soil treatment and the second letter identifies whether it is the high (H, ARG-137) or the low (L, AAC-A110) GLS producing genotype with their corresponding standard error (SE).

Total Glucosinolate and Percent Oil in the Seeds of Brassica carianta 240.0

220.0

200.0 R² = 0.7661 180.0

Glucosinolates Glucosinolates 160.0

(umol/g of tissue) of (umol/g 140.0 Total 120.0 15.0 20.0 25.0 30.0 35.0 Percent Oil in the Seeds (%)

Figure 4-2. Correlation of combined total glucosinolate and oil (%) in the seeds of two B. carinata genotypes (AAC-A110 and ARG-137).

Figure 4-3. Distribution of total glucosinolate and oil in the seeds of B. carinata between genotype AAC-A110 and ARG-137

CHAPTER 5 CONCLUSION

Volatile Identification and Quantification

Much of the research on plant glucosinolates may not be assessing certain unstable glucosinolate compounds that are easily volatilized from various parts of the plant and released in the air or soil. Particularly in the soil, the fate of GLS hydrolysis products vary from being bound in the soil matrix to degradation by the surrounding microbiota (Hanschen et al., 2015). They found that the stability of GLS hydrolysis products was largely dependent on its structure, the amount of water in the soil, and microbial population since autoclaving reduced the hydrolysis of GLS. Indolic GLS make up the majority of GLS being released from Brassica roots and are chemically unstable.

They can hydrolyze spontaneously without the need for myrosinase and still serve as a effective defense compound (Textor & Gershenzon, 2008).

Myrosinase Assays

Myrosinase activity is a useful assay to assess the conversion rate of GLS into its respective hydrolysis product as it is amended into the soil or when it is used as animal feed. In general, Brassica seed meal contains a high number of myrosinase enzymes which has been shown to be detrimental to the health of animals in high doses by reducing kidney and liver function, reduce palatability, and interfere with iodine uptake and availability (Tripathi & Mishra, 2007). A reduction in the amount of myrosinase isoforms in the seeds via precision or traditional breeding methods will be important to increase the value of carinata as a sustainable crop for use as animal feed.

In addition to the complexity involved with glucosinolates (GLS) production and distribution, there are also myrosinase with many isoforms that vary in levels of

specificity. A review by Nakano et al. (2014) found that a unique rod-shaped endoplasmic reticulum (ER) structure was found only in plants from the order

Brassicales which serves as a repository for β-glucosidases and found almost exclusively and ubiquitously in seedlings or mature roots. Nakano et al. (2014) illustrated two distinct myrosinase-glucosinolate delivery systems. They found that mustard oil bombs occur when myrosinase and the GLS accumulate in the vacuole of separate cells and are united during cell rupture or they can be found in the same cell, but segregated in subcellular compartments. These compartments are divided into the myrosinase-containing ER body and the vacuole where the GLS molecules are stored.

In this system, the myrosinase or the GLS can be translocated into the cytoplasm individually or at the same time (Nakano et al., 2014).

There is an increase in myrosinase activity in the soil after Brassica napus crop indicating that there may be an increase in rhizosphere bacteria with the ability to hydrolyze GLS (Borek et al., 1996). The allelopathic effects of GLS and its persistence in the soil can be detrimental to crops that are planted after carinata. Identifying and selecting microbes (i.e. microbial and fungal populations) that can breakdown GLS can help to further the investigation of GLS persistence in a variety of soil types and environments. There are some indications that certain bacteria in the soil may be influencing growth, seed yield, and oil quality of carinata at different nitrogen fertilization rates (Nosheen et al., 2013). Identifying the populations that can coexist in the rhizosphere of carinata would help build the resilience during stressful periods. DNA sequencing that uses the hypervariable region of the 16S rDNA of bacteria and 18S

rDNA of fungi can be used to build a profile for an ideal ecological environment in various soils and conditions.

Prospecting for Carinata’s Future

Canola oil can contain seed oil from a number of different Brassica species that contain low erucic acid concentration and therefore deemed safe for human consumption. Growing plants that can be used as food or fuel can create some tension between these two industries that are competing for land crushing facilities and other infrastructure and government subsidies. Focusing breeding efforts to produce seeds with specific fatty acid profiles are important to the success of carinata (Almeyehu and

Becker, 2001). A study using infrared spectroscopy suggests that the color of the seed may make a difference in regards to oil. It found that there is a reduced amount of oil and VLCFA profile in carinata seeds that are brown then those that are yellow (Pro et al., 2014).

The seed meal has also been found to contain a high amount of quality proteins and polyphenols which can be used for livestock feed (Das Purkayastha et al., 2013).

However, without the reduction of glucosinolate or myrosinases in the seed, value of carinata seed meal for livestock feed is diminished. On the contrary, increasing the release of specific GLS or myrosinase isoforms in the roots can help deter nematodes and other pests or increase the presence of beneficial microbial and fungal populations would be beneficial to farmers with high levels of pest pressure. Intensive breading programs are underway in pursuit of carinata resistant to white mold (Sclerotinia sclerotiorum), leaf spot (Alterneria spp.), turnip mosaic virus, and insects such as silverleaf whitefly (Bemisia argentifolii), diamondback moth (Plutella xylostella) and aphids (Bliss et al., 2014; Fahey et al., 2001; Kliebenstein et al., 2005).

In 2012, the Canadian Government alongside the National Research Council tested carinata-derived biojet fuel in a modified Falcon 20 twin-engine. This was achieved using the Resonance® Brand of carinata developed by Agrisoma Bioscience

Incorporated. These varieties are bred specifically for the biofuel industry and suited for the Northern climate, soil, and ecology. With great effort and some luck, the successful integration of carinata into the Southeastern U.S. will not only help increase farmer revenue, but ultimately reduce greenhouse gas emissions and help mitigate the change in global temperatures.

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BIOGRAPHICAL SKETCH

Theodor Linares Stansly was born in Bryan, Texas to Philip Anzolut and Silvia

Linares Stansly. He had lived in Venezuela, Ecuador, and Spain, and raised in Labelle,

Florida. His many years of working on fruit and vegetable crops with their respective diseases had fueled his interest in agriculture and advances in molecular biology.

Theodor transferred from Florida Gulf Coast University and then graduated from the

University of Florida with a Bachelor of Science degree majoring in horticultural sciences and specializing in plant molecular and cellular biology. He then began his

Master of Science degree in the Fall of 2013 in the Horticultural Sciences Department at the North Florida Research and Education Center located in Quincy, FL. Upon graduation, Theodor plans to continue experience in agriculture by pursuing a Doctor of

Philosophy degree.