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The ability of plants to produce an incredible diversity of small molecules and complex polymers has made them an integral component of lives and societies. We have long harnessed the diversity of chemicals found in plants as food, commodities, and medicine. While plants innately produce millions of useful chemicals, biotechnology has allowed us to further tailor plants to our needs. An integral component of plant anabolism is the attachment of sugars to various chemicals, whether it be to small molecules or complex glycans. This unique specialty of plants creates novel opportunities to expand the ways in which we can improve their properties. In this work, we utilized the innate metabolism of plants to generate a variety of glycosides and glycans. First, we optimized the production of a glycoside, glucoraphanin, to permit expanding the portfolio of plants that produce this beneficial compound. Second, we developed transgenic crops capable of converting glucoraphanin from an inactive form to a chemopreventive form following cooking, thereby improving the nutritional quality of cooked foods. Last, we demonstrated the ability of plants to economically produce milk oligosaccharides, complex glycans that are normally produced only by mammals. Together, these results demonstrate our ability to engineer plants for improved nutritional quality and the production of novel compounds.

Communication is an essential component to all living organisms. In plants, the additional cell wall surrounding each cell adds a layer of complexity not observed in animals. To overcome the literal wall separating cells, plants have evolved specialized pores to connect adjacent cells. Plasmodesmata (PD) allow plants to have a continuous cytoplasm between cells. Although plasmodesmata may appear simple and lack regulation, their structural components and their regulatory machinery is complex and not well understood. Organelle-to-nucleus-to-plasmodesmata signaling (ONPS) have been worked as a leading model for a possible regulatory mechanism. Many of the details of organelle-to-nucleus retrograde signaling pathways have been elucidated in yeast, mammalian and plant model systems. Understanding mechanisms of chloroplast-to-nucleus signaling will help elucidate the functions of retrograde signaling in all organisms including bacteria and apicomplexans. Our previous work with mutants lacking the chloroplast RNA helicase ISE2 indicates that chloroplasts are important regulators of plant intercellular communication and trafficking mediated by pores in the plant cell walls called plasmodesmata. Loss of ISE2 has suggested defects in glucosinolates and in this dissertation we show how glucosinolates can regulate intercellular trafficking via plasmodesmata. I uncover the potential pathway of how the changes in the chloroplast by the loss of ISE2 modulate nuclear signaling and ultimately disrupts the biosynthesis of glucosinolates. I find that plants overexpressing ISE2 results in global physiological defects that can partially be described as auxin defects. However, they are a more complex phenomenon. Further, I reveal that the addition of glucosinolates alone to plants results in an increase in intercellular trafficking in a dose-dependent manner.

Beauveria is a genus of entomopathogenic fungi that exhibits traits suitable for the development of commercial biopesticide products to control various arthropod pests, as well as plant pathogens. The literature describes thousands of strains of Beauveria, largely limited to strains of Beauveria bassiana that have been isolated and screened for their ability to control pests around the world. Some of these pests, such as diamondback moth (DBM), the two-spotted spider mite, green peach aphid and housefly, are highly resistant to chemical insecticides. This study was conducted to find at least one New Zealand derived Beauveria isolate able to control DBM. The first question of this study was, “do other Beauveria species have the potential to control DBM”? Thus, 14 isolates from four Beauveria species were assessed for their control of DBM under in vitro conditions. Before the assessment, all 14 isolates were taxonomically identified using both morphological and DNA-based methods. The in vitro bioassays identified three isolates of B. bassiana (Mo1, CTL20, and CTA20) and B. pseudobassiana (FRhp, FW Mana, and I12 Damo) that successfully killed 50% of the DBM larvae (median lethal dose LD50) at a low dosage while isolates from two other species could not achieve LD50 at a higher rate. This is the first report that details B. pseudobassiana as a potential species for DBM control. Combining two or more fungal isolates may improve the efficacy of a biological control agent. The second question examined was, “do combinations of Beauveria isolates result in a synergistic or an antagonistic interaction”? Beauveria isolates were grouped according to their previously assessed virulence towards DBM. Under in vitro conditions, combining the low virulent isolates together resulted in a higher and faster DBM mortality rate than when these isolates were applied separately. Combining the three species of Beauveria together resulted in antagonistic interactions between the fungi, with lower mortality rates than when these isolates were applied separately. Diet can sometimes affect the susceptibility of an insect to entomopathogenic fungi. The third question of this study was “is there any difference in the susceptibility of DBM larvae to fungal infection by selected Beauveria isolates when raised on different brassicas”? DBM larvae were fed on four different brassicas with predicted high (broccoli and cabbage) and low (cauliflower and radish) glucosinolate levels. Glucosinolates have been implicated in the resistance of brassica hosts to plant pathogens. In vitro results showed that DBM larvae fed on broccoli and cabbage were more susceptible to infection by B. pseudobassiana I12 Damo than larvae raised on cauliflower and radish. Conversely, DBM larvae raised on cauliflower and radish were more susceptible to infection by B. bassiana CTL20 than larvae fed on broccoli and cabbage. Secondary metabolites produced by Beauveria spp. have previously been found to have insecticidal properties and can kill a wide range of insects, including DBM. The fourth question investigated was, “was there evidence that an isolate of Beauveria spp. produces toxins that kill DBM larvae directly”? B. pseudobassiana FW Mana was selected as it caused high DBM mortality in previous bioassays but exhibited less sporulation from DBM cadavers compared to the other isolates within the study. The filtered supernatant of B. pseudobassiana FW Mana was used for assays along with toxins from two other less-lethal isolates of Beauveria. As expected, the supernatant from B. pseudobassiana FW Mana caused significantly higher mortality than the supernatant from the other two isolates assessed. Overall, this study found several Beauveria isolates that have the potential to be developed and commercialised as biopesticides to control DBM.

Canola co-products have a high content of fiber and glucosinolates. Fiber reduces nutrient utilization in pigs, whereas glucosinolates are degraded to toxic products, which interfere with liver, kidney and thyroid functions. Negative effects of fiber can potentially be alleviated by fiber-degrading enzymes, whereas negative effects of glucosinolates in pigs can potentially be alleviated through reduction in hindgut pH. However, there is a lack of information on effects of supplemental fiberdegrading enzymes on digestion and fermentation characteristics of canola coproducts for pigs. Also, there is limited information on effects of reducing hindgut pH on toxicity of glucosinolates in pigs. Four experiments were conducted to fill these gaps in knowledge. The first experiment investigated effects of supplementing canola co-products with fiber-degrading enzymes on porcine in vitro digestion and fermentation of canola co-products. Supplemental fiber-degrading enzymes increased in vitro digestibility of canola co-products. The second experiment investigated effects of increasing levels of cold-pressed canola cake (CPCC) in diets for pigs from 0 to 40% on growth performance, organ weights, blood thyroid hormone levels. Growth performance, metabolic activity in liver and thyroid functions were negatively affected by dietary inclusion of CPCC at 40%. The third experiment investigated effects of reducing hindgut pH through dietary inclusion of high-amylose cornstarch (HA-starch) on the fore-mentioned response criteria and cecal concentration of glucosinolate degradation products in pigs fed diets that contained 40% CPCC. Dietary CPCC increased thyroid gland weight of pigs fed HA-starch-free diet, but not of pigs fed HA-starch-containing diet. Inclusion of HA-starch in CPCC-based diets increased isothiocyanate production in cecal digesta of pigs. However, nitriles were undetected in cecal digesta of pigs fed CPCC-based diets. Thus, the fourth experiment was conducted to determine effects of reducing pH on composition of glucosinolate degradation products in canola co-products using porcine in vitro fermentation technique. Reduction in fermentation medium pH from 6.2 to 5.2 increased production of indole- 3-acetonitriles. In conclusion, the results demonstrated that fiber-degrading enzymes can be supplemented to canola co-products-based diets for pigs to improve efficiency of nutrient utilization and that toxicity of canola glucosinolates can be alleviated through reduction in pH of hindgut of pigs.

Carinata Brassica carinata and camelina Camelina sativa are being genetically modified to improve their composition for biofuel production and other agricultural applications. After oil extraction, the residual de-oiled meals (40%) in protein. However, their inclusion in animal feeds is limited to 10% because of high levels of antinutritional factors (ANFs), mainly glucosinolates (GLS), sinapine, and crude fiber that limit diet intake, nutrient utilization, and lower thyroxine production, resulting in reduced growth. The nutritional value of carinata and camelina seed meals to fish were assessed by subjecting cold pressed (CP) carinata and camelina meals to extrusion (EX), solvent extraction (SE) and aerobic conversion (AC) or sequential process combinations to determine which process(es) yielded improved composition. Carinata meals generally yielded more crude protein and lower fiber. The primary protein increase for both meal types was due to oil removal and AC. Fiber increased with AC, but that step reduced GLS by at least 70%. Palatability was generally improved by SE. Apparent digestibility coefficients (ADCs) for protein were generally higher in carinata than camelina meals for both Rainbow Trout Oncorhynchus mykiss (RBT) and Hybrid Striped (Sunshine) Bass Morone chrysops ♀ x M. saxatilis ♂ (HSB). Overall protein ADCs were higher in HSB than in RBT. The use of AC on protein ADC was more effective in camelina meals. Two experiments were conducted to determine the maximum GLS concentration from cold-pressed carinata meal (CPCM) containing 61.2 μmoles of GLS and 6.07 mg of sinapine/g of meal that could be tolerated in HSB diets. In experiment 1, there was no effect of up to 2.71 μmoles of GLS and 0.181 mg of sinapine/g of diet on diet palatability, thyroxines production, deiodinase enzyme activity, and consequently HSB growth. In experiment 2, concentrations of ≥5.58 μmoles of GLS and ≥0.54 mg of sinapine/g of diet reduced feed consumption, utilization, and growth. Reduction in palatability due to GLS and sinapine from CPCM may not enable fish to consume enough GLS and / or GLS breakdown products to impair thyroid function. GLS were not lethal to HSB. Based on meal composition, protein ADCs, palatability and GLS tolerance, solvent extracted aerobically converted carinata meal, followed by a single wash (ACCM), was chosen for growth performance trials. In the RBT growth trial, we determined how much fish meal (FM) could be replaced with ACCM. We replaced 25, 50 and 75% of FM in the reference diet containing 20% FM as the sole animal meal, composing diets of 44% crude protein and 17% crude lipid. Diets were balanced for nutrients and fiber. After a 56-day growth period, replacement of more than 25% FM by ACCM resulted in reduced growth partly due to reduced feed consumption. Condition factor (Fulton’s K) decreased with increased FM replacement. Feed conversion ratio (FCR) had an inverse relationship with diet consumption. Results of this study showed that more than 25% of FM cannot be replaced by ACCM in low FM/animal (20%) diets of RBT; improved utilization of ACCM by RBT may occur with more animal meal inclusion or additional processing of ACCM to improve the feeding value. In the HSB growth trial, we determined the maximum inclusion of ACCM or double-washed carinata meal, without AC (WCM) in low (20%) animal diets. We included ACCM at 10 and 30%; and WCM at only 30% of the diets. All diets contained ~44% crude protein 12% crude lipid, and were balanced for fiber. After a 106-day growth period, we observed that HSB fed 30% WCM had a similar weight gain to HSB fed the FM reference diet and 30% ACCM but better than HSB fed 10% ACCM. High (30%) amounts of ACCM or WCM improved feed consumption. FCR of WCM was better than that of ACCM. HSB fed 30% WCM had smaller livers and higher condition factors than HSB fed other treatment diets. Survival (>99%) was similar among treatments. Hematocrit (Hk) and hemoglobin (Hb) contents of HSB were improved by ACCM but not WCM. These results showed that the second wash in WCM improved feed utilization more than ACCM. However, the extra wash step in WCM likely reduced the iron content of WCM resulting in lower Hk and Hb. At the end of the RBT growth trial, trypsin activity, protein ADCs, amino acid ADCs and avaialability were measured to account for observed differences in protein utlization and thus growth. Trypsin activity and protein ADCs were not altered with increasing ACCM in diets. Replacement of more than 25% of FM reduced apparent digestibility of arginine, histidine, isoleucine, leucine, phenylalanine, threonine, valine, and tyrosine. However, the above essential amino acids (EAAs) were not decreased in serum due to FM replacement with ACCM, although serum from RBT fed 75% ACCM contained lower lysine concentrations. ACCM diets had lower EAA peak concentrations and a slower release of EAAs in serum. Cumulative total EAAs in serum also decreased with ACCM inclusion. The pattern of total EAAs in serum for most sampling intervals best associated with muscle EAAs for the reference and 10 and 15% ACCM. Ratios of EAAs to lysine showed that tryptophan was the most limiting EAA. However, isoleucine, leucine, methionine, and phenylalanine were also inadequate for muscle synthesis for the first 9-12 hours after force-feeding. Optimal time for muscle synthesis was 36 or more hours because all EAAs were adequate except for isoleucine in the muscle of RBT fed the 10% ACCM diet. Following the HSB growth trial, trypsin activity, protein ADCs, amino acid ADCs and avaialability were measured to explain the observed differences in protein utilization and thus growth. Inclusion of up to 30% processed CM did not alter trypsin activity or protein ADCs. However, 30% ACCM reduced isoleucine, leucine, phenylalanine, threonine and valine ADCs. Feeding high ACCM (30%) reduced serum arginine and leucine. All inclusions of ACCM or WCM increased serum methionine. High inclusions of ACCM or WCM (30%) increased serum tryptophan and valine. The reference and 30% WCM diets resulted in the highest total essential amino acids (EAAs) in serum but the release of total EAAs in serum of HSB fed 30% WCM was elevated continuously over a longer period. High inclusions of ACCM or WCM (30%) increased muscle histidine but resulted in lower leucine and phenylalanine. Only 30% WCM increased muscle lysine and valine. However, all inclusion levels of ACCM or WCM increased muscle methionine. High inclusions of ACCM or WCM (30%) in diets resulted in more available total EAAs over a longer period. Muscle EAA to lysine ratios showed that only histidine concentrations were adequate for muscle synthesis over the 36-hour period.

Land plants respond to multiple abiotic stresses, including drought, through the signaling cascade induced by the phytohormone abscisic acid (ABA). Endogenous production, or externally applied ABA has a major function of eliciting guard cell closure, ultimately lowering transpiration and increasing drought tolerance in plants. Accordingly, the mechanisms of action in which ABA facilitates this response have been popular targets for agricultural research and applications. However, ABA has a multitude of responses aside from stomatal closure that are important for plant's survival to abiotic stress. In response to limited water availability, the phytohormone is known to maintain primary root growth while decreasing shoot growth, increase osmolyte accumulation, inhibit seed germination, and is involved in substantial crosstalk with other phytohormones. These responses are dependent on a core ABA signaling pathway comprised of three components: the ABA receptors known as the PYRABACTIN RESISTANT/PYRABACTIN RESISTANT-LIKE/REGULATORY COMPONENT OF ABA RECEPTORs (PYR/PYL/RCARs), the clade A protein phosphatase 2Cs (PP2Cs), and the sucrose nonfermenting related subfamily 2 (SnRK2s).The ABA receptors are the first interactors with ABA within the pathway, and cluster into three clades (I, II, III) based on sequence identity. Additionally, these clades can be further grouped based on oligomeric preference; clades I and II are preferentially monomeric, while clade III is preferentially dimeric. While some research has been done on non-redundant functions of the PYL proteins, many of the ABA responses remain uncharacterized with respect to the differential contributions of the different receptors. Additionally, most of the published ABA-receptor agonists are either direct ABA analogs displaying pan-agonist activity, or are primarily active only on the dimeric subgroup of the receptors. It would thus be potentially beneficial to develop agonists that show preferential activation of the monomeric receptors, allowing for temporal activation and subsequent analysis of their biological relevance.In order to identify compounds with novel selectivities, preferably on the monomeric receptors, I used high-throughput virtual screening to evaluate compounds unbiased to previous, active scaffolds. This resulted in a series of chemically similar hits which showed potent activity on the monomeric receptors, and translated to some in vivo responses. This potent, monomeric-specific scaffold was optimized using structure-aided design, improving the in vivo responses. Using this probe molecule I provide data that suggest that monomeric and dimeric ABA receptors may differentially control metabolomic and transcriptional responses, but are adequate in seed germination inhibition and primary root elongation.

Pieris rapae is an insect pest the feeds exclusively on species of the Brassicaceae. It is a serious economic pest in commercial horticulture as larvae chew large holes in leaves, can destroy developing heads of broccoli, cauliflower, and cabbage, and otherwise contaminate or stain produce, rendering it unmarketable. P. rapae is notoriously difficult to manage in organic production systems, and organic brassica growers traditionally rely upon floating row covers or biopesticides to prevent feeding damage. Natural pest resistance is important when making cultivar recommendations for production systems with limited pest management options, and our research evaluates natural and induced resistance in broccoli (Brassica oleracea var. italica) against P. rapae with a focus on the roles that glucosinolates (GSLs) play in resistance. GSLs are constitutive and inducible secondary metabolites in members of the Brassicaceae and are toxic to generalist insect pests. However, the relationship between GSL and P. rapae, a brassica specialist, is complex and not well understood. A controlled greenhouse study evaluated the effects of P. rapae herbivory on GSL induction and subsequent larval growth and performance on two broccoli cultivars (‘Beneforte’ and ‘Green Magic’) that varied in concentration of two GSLs, glucobrassicin and neoglucobrassicin. Our results show that neoglucobrassicin and glucobrassicin concentrations did not significantly increase as a result of herbivory, but the overall growth and performance of P. rapae was influenced by cultivar. Larvae consumed less total leaf area and weighed less as pupae when feeding on ‘Beneforte’, which also had a significantly higher glucobrassicin concentration, indicating that cultivar may be important in resistance against P. rapae. A field study performed at two certified organic farms in Minnesota in 2015 and 2016 evaluated six broccoli cultivars (‘Belstar,’ ‘Fiesta,’ ‘Green Magic,’ ‘Marathon,’ ‘Packman,’ and ‘Thompson’) for natural resistance to P. rapae and for yield performance. The cultivars in this study did not significantly differ in susceptibility to P. rapae infestation, and neoglucobrassicin and glucobrassicin concentrations did not explain P. rapae egg and larval abundance. We conclude that cultivar selection should not be based on natural resistance to pests in this case but rather based on cultivar performance. ‘Belstar,’ ‘Fiesta’, ‘Green Magic,’ and ‘Packman’ were top performers and produced a consistent and high quality product, and therefore we would recommended these cultivars as acceptable selections for organic systems in Minnesota.

Due to the phase-out of methyl bromide, there is a need for alternative, nonchemical fumigation treatments in strawberry production. Anaerobic soil disinfestation and biofumigation are two non-chemical methods for controlling soilborne plant pathogens of strawberry. This study was designed to observe strawberry fruit nutrition and soil volatiles of a strawberry field being treated with biofumigation treatments, anaerobic soil disinfestation treatments, and a combination of the two alternative methods. A trial was conducted with 11 pre-plant soil-incorporated treatments arranged in a randomized complete block design with 6 rows (blocks). Biofumigation treatments consisted of deactivated mustard meal, deoiled mustard meal, mustard pellets, and Biofence mustard seed meal. Other treatments included dried molasses as a carbon source for an anaerobic treatment and a Basamid® chemical treatment. Additional combination treatments of deactivated mustard meal combined with molasses, deoiled mustard meal combined with molasses, and molasses combined with soybean meal (to lower amendment Carbon:Nitrogen ratio) were also applied, as well as an untreated control. Soil samples were taken at designated times post irrigation application in order to measure sinigrin and allyl isothiocyanate simultaneously. Harvested fruit were counted, weighed, and graded into marketable and non-marketable categories, and were then analyzed for sugars (fructose, glucose, and sucrose), organic acids (malic and citric), and mineral content (B, Na, Mg, P, S, K, Ca, Fe, Cu, Mn, and Zn). The combination of deoiled mustard meal and molasses can provide a comparable marketable yield as the chemical treatment Basamid®. Fruit sugar and organic acid content did not consistently differ among most treatments. Likewise, there were no consistent patterns of differences among treatments in mineral content of either fruit or leaf tissues. In general, the alternative methods of biofumigation and soil anaerobic disinfestation produced fruit of equal quality to that produced using the Basamid® chemical treatment. Future work will evaluate pathogen and soil nutrient dynamics affecting productivity in these alternative soil disinfestation treatments.

Abstract: Canada is the top producer of Brassica oilseeds [B. napus L. (n = 19, AC genome)] in the world. Genetic diversity has declined in this crop in the recent years due to use of only superior and genetically narrow gene pool in breeding. Presence of adequate genetic diversity is important for further improvement of this crop through breeding. Genetically distinct germplasm of B. napus or its allied species can be used to broaden genetic diversity in Canadian B. napus canola. However, limited efforts have been made to utilize genetic diversity of the progenitor species B. rapa (n = 10, A genome) and B. oleracea (n = 9, C genome) in the breeding of this crop as interspecific cross often introduces undesirable traits in the breeding program. This M.Sc. thesis research was undertaken to develop genetically distinct B. napus lines through interspecific crosses between B. napus canola and B. rapa. For this, three genetically distinct B. rapa lines were used. The F1’s of B. napus × B. rapa interspecific crosses were self-pollinated for F2 as well as backcrossed to the B. napus parent for BC1F1 progenies. Pedigree breeding was applied where selection for plant fertility and glucosinolate content was done in each generation. SSR marker analysis of the F4 plants revealed that the three populations derived from B. napus × B. rapa crosses are genetically distinct from each other as well as from the B. napus parent; thus, the advanced generation populations derived from the progeny of these plants expected to carry allelic diversity of the B. rapa parents. Plant fertility and glucosinolates content in many of the F7 and BC1F4 families reached close to the B. napus parent. Flow cytometric analysis of F6 and BC1F3 families for nuclear DNA content indicated that many families are euploid B. napus type. Findings from this thesis research suggest that genetically distinct, fertile, euploid B. napus canola lines can be developed from both F2 and BC1F1 of the B. napus × B. rapa interspecific crosses.

Glucosinolates (GLSs) are sulfur-containing secondary metabolites produced by broccoli (Brassica oleracea subsp. italica) and other cruciferous vegetables. GSLs exist for use in plant defense, but are gaining research interest for their role in cancer prevention. Glucoraphanin (GR) is a particular glucosinolate found in broccoli that has great health benefit potential. Seleno-methylselenocysteine (SeMSC) is another compound unique to broccoli when it has been exposed to selenium during plant growth, and is also of interest for its chemopreventive potential. To understand the relationship between root fertilization of sulfur and selenium on GR and SeMSC concentrations in a production environment, we exposed a low-GR ("Green Magic") and a high-GR ("Beneforte") broccoli to sulfur (0 - 34 kg.ha��) and selenium (0 - 3.36 kg.ha��) fertilization treatments in the field. GR and SeMSC concentrations depended upon cultivar, treatment, and environmental factors."Beneforte" consistently delivered the highest GR concentration, and "Green Magic" the lowest, and "Beneforte" GR concentrations were less affected by the presence of Se treatments than "Green Magic". "Beneforte" also accumulated higher concentrations of SeMSC overall than "Green Magic". A colder, wetter spring in 2013 led to reduced sulfur uptake and lower concentrations of GR overall, while a warmer, drier climate during later Se applications increased Se uptake and subsequent SeMSC concentrations. Contrasting, a warmer, dryer spring the following year gave way to increased sulfur uptake and greater GR concentrations, and wetter, cooler conditions during Se applications negatively impacted SeMSC concentrations overall. To assess the efficacy of foliar Se fertilization as an alternative to root application, the same two varieties were grown in a greenhouse and were subjected to one of four sodium selenate treatments (0 - 93.74 mg Se.plant�). Again, Se significantly affected the concentration of GR in "Green Magic", but not in "Beneforte". Overall, a weak relationship between GR and SeMSC concentrations give a promising outlook to the ability to maximize GR and SeMSC for ultimate benefit upon consumption of broccoli. This is especially true in "Beneforte", where GR concentrations remain relatively stable in the presence of Se, but still allow Se uptake and SeMSC formation.

Studies were conducted to evaluate nutritional characteristics of three oilseed meals. Camelina and carinata meals were either cold-pressed or hexane-extracted and manufactured by 6 different processing conditions within extraction method. Flaxseed meals were either cold-pressed or solvent extracted and their processing condition unknown. Results are intended to provide helpful information for both processors producing meals and producers desiring to use these meals in livestock diets. The first set of studies was designed to determine a modification to the Tilley and Terry in vitro procedure that would generate adequate material for multiple analyses on residue of oilseed meals without compromising IVDMD. The modified procedure was then used to evaluate OM, CP and fat disappearance of camelina, carinata and flaxseed meals during a 48-h incubation. Cold-pressed and hexane extracted camelina and carinata meals from a single processing condition were selected together with the flaxseed meals for further in vitro analysis of DM and CP disappearance, glucosinolate concentrations and VFA production, and in situ degradation kinetics of DM and CP portions. During the 48 h incubation, differences in OM and crude fat disappearance due to oilseed (P < 0.01), extraction method (P < 0.01), and an oilseed by extraction method interaction (P < 0.01) were observed. No difference (P = 0.2) in the CP disappearance due to extraction method or oilseed by extraction method interaction was observed. During the 96 h incubation, hexane extracted meals (45.3%) had a greater (P= 0.03) portion of DM that was potentially degradable in the rumen than cold-pressed meals (39.3%). Results from both 48-h and 96 h incubations generally suggest that CP in carinata meals is more ruminally available than either camelina or flaxseed. With the exception of Gluconapin (GNA), we observed no significant effect (P > 0.05) of extraction method on glucosinolate concentration of carinata meals. Additionally, extraction method had no effect on glucosinolate concentrations in the supernatant of either cold-pressed or solvent extracted camelina meal. Flaxseed meals produced the greatest concentration of propionate (12.3 mM) with camelina meals producing the greatest amount of valerate (1.2 mM; P = 0.01) during the 96 h incubation period.

Broccoli sprouts are a rich source of glucosinolates, a unique group of phytochemicals which may play a role in preventing multiple types of cancer. In order to exert physiologic activity, glucosinolates must be converted to their bioactive form, known as isothiocyanates, by the heat-labile enzyme myrosinase. This study had two objectives: First, to determine how heating broccoli sprouts affects glucosinolate content and subsequent isothiocyanate formation in vitro. Secondly, to understand how these results translate in vivo, by studying the metabolism of glucosinolates and isothiocyanates in mice fed diets containing thermally processed broccoli sprout powders (BSPs). Fresh broccoli sprouts received one of three processing treatments: (1) freeze-dried raw, (2) steamed and freeze-dried, or (3) heated at 60°C and freeze-dried. The sprouts were powdered, and then analyzed for differences in glucosinolate content and conversion to isothiocyanates. Three different mouse diets were prepared, each incorporating one of the BSPs at 4%, and fed for 7 days (n=10 mice/group). An additional group was fed a diet containing the purified isothiocyanate sulforaphane. 24 hours prior to sacrifice, all mice were exposed dorsally to ultraviolet light. Ultra performance liquid chromatography–mass spectrometry was utilized to quantify metabolites of the two predominant isothiocyanates, sulforaphane and erucin, in the plasma, liver, skin, lungs, kidneys, and bladder. Additionally, the activity of myeloperoxidase, a biomarker for inflammation, was measured in the skin. Steamed BSP had the greatest concentration of glucosinolates, followed by raw and 60°C treated BSPs. However, isothiocyanate formation from the raw and 60°C treated BSPs was approximately 5 and 23-fold greater, respectively, than the steamed BSPs, indicating that heating intensity has disparate effects on isothiocyanate formation. Mice fed the steamed BSP diet had the lowest concentrations of isothiocyanate metabolites in plasma and all tissue sites, while consumption of the 60°C-treated BSP diet generally resulted in the highest concentrations. Particularly high levels of isothiocyanate metabolites were detected in the bladder. Interestingly, we observed the isothiocyanates sulforaphane and erucin interconvert in vivo, with erucin being the vastly favored form at several tissue sites, even in mice fed the sulforaphane diet. Dietary administration of the BSPs did not reduce myeloperoxidase activity compared to the control group. In conclusion, in accordance with other studies, we show steaming severely hinders glucosinolate conversion to isothiocyanates upon consumption. Feeding broccoli sprouts treated at 60°C resulted in significantly greater concentrations of isothiocyanates at many organ sites. Lastly, once converted, chemopreventive isothiocyanates were distributed systemically to all tissue types analyzed.

Glucosinolates and their hydrolysis products, found in plants of the order Brassicales, are a widely studied group of secondary chemicals known for their defensive properties against insect herbivores. However, the specific effects of individual glucosinolates as well as their hydrolysis products are still largely unknown. Arabidopsis thaliana (Col-0) genetic lines with mutations that modify the type of glucosinolate, such as myb28myb29, which lacks aliphatic glucosinolates, and the line cyp79B2cyp79B3 that does not produce indolyl glucosinolates, make it possible to test the specific effects of these different glucosinolates on herbivores. Likewise, the effects of different glucosinolate hydrolysis products can be evaluated using genetically modified lines of Col-0 (which naturally produces isothiocyanates), including the nitrile-producing line 35S:ESP and the double knockout tgg1tgg2. In no-choice experiments with the crucifer specialist insect Pieris rapae, differences in hydrolysis products had no significant effect on insect fitness-related traits, while differences in glucosinolate products did significantly affect pupae weight as well as days to pupation. Pieris rapae larvae spent significantly less time on plants lacking camalexin, and the pupae reared on plants lacking aliphatic glucosinolates weighed significantly less than those reared on the other genetic lines. In contrast, the generalist insect Spodoptera exigua was significantly affected in all fitness-related traits measured, preferring those lines lacking hydrolysis products (tgg1tgg2) as well as those lacking either aliphatic or indolyl glucosinolates (myb28myb29 and cyp79B2cyp79B3, respectively). Spodoptera exigua larvae spent significantly less time on plants lacking aliphatic glucosinolates, while the pupae and adults weighed more when reared on those lines lacking indolyl glucosinolates. Results from feeding choice trials showed that Pieris rapae had no particular preference for any of the genotypes. In contrast, Spodoptera exigua had a significant feeding preference for the double mutant tgg1tgg2, which virtually lacks hydrolysis products. Overall, this study provides evidence that variation in glucosinolate profiles and hydrolysis products can influence insect performance as well as feeding choices, although the responses are dependent on the insect species.

Wild radish ( L.), a facultative winter annual is a troublesome weed in small grain crops of the Southeastern United States. Besides being a weed, it may also be used as a cover crop for weed management due to its production of glucosinolates. Studies were conducted to evaluate the biology and ecology of wild radish as well as its glucosinolate production and its weed management possibilities. Wild radish emerging in fall months formed a rosette of leaves which aided its winter survival. Plants emerging from December through March that did not form a rosette had minimal survival. Wild radish life cycle ranged from 43.5 to 230.5 days. Plants emerging in the fall had greater biomass and seed production compared to ones emerging in summer months. Developmental phases most influenced by the emergence date were emergence to bolting and bolting to flowering. Phenological development phases, except for flowering to silique production, were dependent on both temperature and photoperiod. Wild radish seeds at maturation exhibited lower germination compared to seeds after-ripened in the field for 3 to 6 months. Temperatures of 5 to 15 C were required for germination. Germination was greater at fluctuating temperatures compared to constant temperatures. Burial of seeds (10-cm depth) decreased germination with red light increasing germination at 6 mo after retrieval from soil, indicating a phytochrome effect. Five glucosinolates - glucoerucin, glucotropaeolin, glucoraphenin, glucobrassicin, and gluconasturtin were present in plant parts ranging from cotyledon to flowering stage. The highest concentration of total glucosinolates was present at the flowering stage. Glucoraphenin and glucoerucin were the predominant glucosinolates among different accessions of wild radish. Accessions from Florida and Mississippi had the greatest total glucosinolate concentration. Glucosinolates that hydrolyze to form isothiocyanates, which inhibit seed germination, comprised greater than 80% of the total glucosinolates. Wild radish cover crop alone provided good early season but not season long weed control. Wild radish in conjunction with half rates of atrazine plus S-metolachlor provided season-long control of Florida pusley, large crabgrass, spreading dayflower, and ivyleaf morningglory without affecting sweet corn yields, ultimately resulting in less herbicides required for acceptable weed control.

Garlic mustard (GM), Alliaria petiolata, a European biennial herb invasive in North America, produces secondary metabolites that may contribute to its invasive success by allelopathically inhibiting growth of native plants. I tested this hypothesis by determining the levels, and stability, of these compounds in field soils. I also explored differences in the phytochemical profiles of GM and closely related North American species, and biogeographic differences in allelopathic effects on North American and European arbuscular mycorrhizal fungi (AMF). Furthermore, I exposed a North American annual herb, pale jewelweed (Impatiens pallida), and its AMF, to GM extracts in order to determine how GM metabolites interact to inhibit growth throughout the formation of mycorrhizal symbioses. Additionally, I determined whether or not allelopathic inhibition remained significant when resource competition was also occurring, by exposing jewelweed plants grown at various densities to GM extracts. Also, I assessed allelopathic effects on AMF diversity by performing terminal restriction fragment length polymorphism analysis on AMF DNA isolated from field soils collected either in or outside of GM populations. Finally, I explored how three methods commonly used to kill GM influenced the health of jewelweed plants later planted in the same soil. Secondary metabolites produced by GM decompose too quickly to account for long term inhibition, suggesting that degradates of these compounds are bioactive. None of the compounds produced by GM were found in four North American species, and North American AMF were more sensitive to allelopathic effects than European AMF. I found strong inhibition of seed germination and growth of uncolonized plants, but AMF appeared to protect colonized jewelweed plants from any allelopathic effects. Allelopathic effects were not as significant as effects of resource competition on jewelweed plant size. AMF diversity was lower in sites with GM than without. During simulated restorations, jewelweed plants were larger and more colonized by AMF in soils with the least amount of dead GM tissue remaining. GM can be allelopathic, but effects may not be strong enough to drive its invasive success. Instead, allelopathic effects may be more important in maintaining an invasion and must be addressed during restoration of invaded sites.

The effects of packaging treatments, post harvest cooling delay and storage duration on color, texture, ascorbic acid content, weight loss and glucosinolate retention in crown-cut heads of broccoli were studied. Oxygen and CO2 levels inside shrink wrap packages were also monitored. Shrink wrap packaging had a significant positive effect on hue angle (p