Potential long-term (~7months) sub-lethal impacts of soil-applied low levels of Roundup herbicide formulation were investigated in a greenhouse environment using the vegetative clones of succulent non-crop plant model, Bryophyllum pinnatum (Lam.) Oken. An eleven day LC50 (concentration that killed 50% of the plants) was found to be 6.25% (~1.25mg glyphosate/mL and 1.25mg pelargonic acid/mL combined), and complete mortality occurred at 12.5%, of the field application rate (i.e., ~20mg glyphosate/mL and 20mg pelargonic acid/mL as active ingredients). While sub-lethal Roundup (1-5%) exposures led to hormesis-characterized by a significant increase in biomass and vegetative reproduction, higher concentrations (≥6.25%) were toxic. A significant interaction between Roundup concentrations and leaf biomass was found to influence the F1 plantlets' biomass. Biomass asymmetry generally increased with increasing Roundup concentrations, indicating that plants were more stressed at higher Roundup treatments but within the low-dose regime (≤5% of the as-supplied formulation). While leaf apex region demonstrated higher reproduction with lower biomass increase, leaf basal area showed lower reproduction with greater biomass increase, in plantlets. The results suggest long-term exposures to drifted low levels of Roundup in soil may promote biomass and reproduction in B. pinnatum.

Precise engineering of genomes holds great promise to advance our understanding of gene function and biotechnological applications. DNA double strand breaks are repaired via imprecise non-homologous end joining repair or via precise homology-directed repair processes. Therefore, we could harness the DSBs to engineer the genomes with a variety of genetic outcomes and with singlebase- level precision. The major barrier for genome engineering was the generation of site-specific DNA DSBs. Programmable DNA enzymes capable of making a complete and site-specific cut in the genome do not exist in nature. However, these enzymes can be made in in vitro as chimeric fusions of two modules, a DNA binding module and a DNA cleaving module. The DNA cleaving module can be programmed to bind to any user-defined sequence and the DNA cleaving module would generate DSBs in the target sequence. These enzymes called molecular scissors include zinc finger nucleases (ZFNs) and transcriptional activator like effector nucleases (TALENs). The programmability of these enzymes depends on protein engineering for DNA binding specificity which may be complicated, recourse intensive and suffer from reproducibility issues. Recently, clustered regularly interspaced palindromic repeats (CRISPR)/ CRISPR associated endonuclease 9 (Cas9) an adaptive immune system of bacterial and archaeal species has been developed for genome engineering applications. CRISPR/Cas9 is an RNA-guided DNA endonuclease and can be reprogrammed through the engineering of single guide RNA molecule (sgRNA). CRISPR/Cas9 activity has been shown across eukaryotic species including plants. Although the engineering of CRISPR/Cas9 is quite predictable and reproducible, there are many technological challenges and improvements that need to be made to achieve robust, specific, and efficient plant genome engineering. Here in this thesis, I developed a number of technologies to improve specificity, delivery and expression and heritability of CRISRP/Cas9-modification in planta. Moreover, I used these technologies to answer basic questions to understand the molecular underpinning of the interplay between splicing and abiotic stress. To improve Cas9 specificity, I designed and constructed a chimeric fusion between catalytically dead Cas9 (dCas9) and FOKI catalytic DNA cleaving domain (dCas9.FoKI). This synthetic chimeric fusion enzyme improved Cas9 specificity which enable precision genome engineering. Delivery of genome engineering reagents into plant cells is quite challenging, I developed a virus-based system to deliver sgRNAs into plants which facilitates plant genome engineering and could bypass the need for tissue culture in engineering plant genomes. To improve the expression of the CRISPR/Cas9 machinery in plant species, I developed a meiotically-driven expression of CRISPR/Cas9 which improved genome editing and heritability of editing in seed progeny, thereby facilitating robust genome engineering applications. To understand the molecular basis of the interplay between splicing stress and abiotic stress, I used the CRISPR/Cas9 machinery to engineer components of the U2snRNP complex coupled which chemical genomics to understand the splicing stress regulation in response to abiotic stress conditions. Finally, I harnessed the technological improvements and developments I have achieved with CRISPR/Cas9 system to develop a directed evolution platform for targeted trait engineering which expands and accelerates trait discovery and engineering of plant species resilient to climate change conditions.

Goatsrue is an introduced perennial plant that has proven to have great invasive potential, leading to its classification as a noxious weed in many states and at the federal level. This research focused on seed biology, herbicide control, and toxic dynamics of goatsrue. Physical dormancy of mature goatsrue seed was tested through scarification using sulfuric acid with exposures of up to 60 minutes resulting in 100% germination. Comparison of dormancy for 26-year-old and 6-month-old goatsrue seed indicated aged seeds had reduced dormancy levels compared to newly harvested seeds, but had similar viability. Goatsrue seedling emergence was inversely related to burial depth; emergence was greatest at 0.5 cm soil depth (93%), and no emergence occurred from 12 and 14 cm. Goatsrue seed density ranged from 14,832 seeds m-2 to 74,609 seeds m-2 in the soil seed bank of five goatsrue-infested areas. Viability and dormancy of seeds recovered from the soil seed bank survey ranged from 91 to 100% and 80 to 93%, respectively. Goatsrue was most sensitive to the ALS inhibitor herbicides chlorsulfuron and imazapyr in greenhouse trials. Field studies showed that plots treated with dicamba, chlorsulfuron, metsulfuron, aminopyralid, triclopyr, and picloram provided at least 93% control of goatsrue 12 months after treatment at two field sites and increased perennial grass cover at one site. All treatments at one site decreased seedling goatsrue cover 11 months after treatment. The concentration and pools (dry weight x concentration) of the toxin galegine, found in goatsrue, vary over plant tissues and phenological growth stages. Galegine concentration is significantly different among plant tissues; reproductive tissues have the highest levels of galegine (7 mg/g), followed by leaf (4 mg/g), and then stem (1 mg/g) tissues. Galegine pools or the total amount of galegine per stalk was lowest at the vegetative growth stage and increased until reaching a maximum at the immature pod stage, but decreased nearly in half at the mature seed stage. Average galegine concentration also peaked at the immature pod stage and decreased by half at the mature seed stage. Thus, goatsrue is most toxic in its phenological development at the immature pod stage.

Research trials performed in the greenhouse compared the tolerance and response of Hordeum jubatum and desirable pasture grass species to herbicides, salinity, and flooding. Desirable grass species used in this study included: `Fawn' tall fescue (Festuca arundinaceae), `Garrison' creeping foxtail (Alopecurus arundinaceus), `Palaton' reed canarygrass (Phalaris arundinacea), `Climax' timothy (Phleum pratense), `Alkar' tall wheatgrass (Thinopyrum ponticum), `Potomac' orchardgrass (Dactylis glomerata), and `Mustang' altai wildrye (Leymus angustus). Tolerance to herbicides, salinity, and flooding varied significantly among grass species. Herbicide tolerance was tested using four herbicides at five rates each. The herbicides used were imazapic (Plateau), propoxycarbazone (Olympus), sulfosulfuron (Outrider), and flucarbazone (Everest) at rates of 0, 10, 25, 50, 100, and 200 g ha-1. Foxtail barley was least tolerant of sulfosulfuron and propoxycarbazone. Tall fescue, creeping foxtail, and reed canarygrass were susceptible to all the herbicides tested. Timothy and foxtail barley were moderately tolerant while tall wheatgrass exhibited the greatest tolerance to flucarbazone. Orchardgrass was most tolerant to propoxycarbazone. Salinity tolerance was determined by exposing grasses to increasing electrical conductivity (EC) over time. Reed canarygrass and timothy were most susceptible to salinity. Orchardgrass, creeping foxtail, and tall fescue were moderately tolerant of salinity. Foxtail barley, altai wildrye, and tall wheatgrass exhibited the highest tolerances to salinity, and continued to persist at the highest EC levels tested. Flooding tolerance was determined by flooding grasses in 18 cm of water for 2, 4, 6, and 8 weeks. Grasses that were able to extend above the water surface survived, whereas plants that failed to extend beyond the water surface experienced higher mortality rates.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第17830号 / 農博第2015号 / 新制||農||1016(附属図書館) / 学位論文||H25||N4787(農学部図書室) / 30645 / 京都大学大学院農学研究科農学専攻 / (主査)教授 稲村 達也, 教授 冨永 達, 教授 奥本 裕 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DGAM

The resistance of Palmer amaranth (PA) and the tolerance (natural resistance) of pitted morningglory (PM) to glyphosate have made these species among the most common and troublesome weeds in the southeastern U.S. since the adoption of glyphosate-resistant (GR) crops. Populations of GR PA (R1 and R2) were identified in Mississippi. The inheritance of glyphosate resistance was examined in reciprocal crosses (RC) between glyphosate-resistant (R) and -susceptible (S) parents (Female-S × Male-R, S/R, and Female-R × Male-S, R/S), and second reciprocal crosses (2RC) (Female-S/R × Male-S/R, S/R//S/R, and Female-R/S × Male-R/S, R/S//R/S). Dose-response assays resulted in 17- to 4old resistance to glyphosate compared with S. Population S accumulated 325- and 8-times more shikimate at the highest glyphosate dose than in R1 and R2, respectively. cDNA sequence analysis of the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene indicated no target site mutation. Genomes of R1, R2, RC, and 2RC contained from 1- to 59old more copies of EPSPS gene than S; EPSPS was highly expressed in R1 and R/S, but was poorly expressed in S, S/R, and R2. EPSPS activity was lower in S and S/R than in R and R/S, glyphosate absent; all were inhibited by glyphosate. Western Blot analysis confirmed an increased EPSPS protein level to EPSPS copy number correlation. Thus, the level of resistance was decidedly influenced by the direction of the cross. R and S female plants were reproductively isolated and seed were still produced, suggesting that PA can produce seed both apomictically and sexually (facultative apomixis). This mode of reproduction determined the low copy number inheritance, as well as guaranteeing the GR trait stability in the R populations. Dose-response assays resulted in 2.6old variability in tolerance to glyphosate between the most tolerant (MT) and the least tolerant (LT) PM populations. The level of tolerance positively correlated with the time of exposure to GR-crop system. Less shikimate was recovered in MT as compared to LT. Levels of aminomethylphosphonic acid (AMPA) were not different between populations and sarcosine was not present in either populations. Consequently, metabolism of glyphosate to AMPA or sarcosine is not a common factor in explaining natural resistance levels.

S-metolachlor is an effective herbicide used to control/suppress annual grasses, nutsedges and several broadleaf weeds in sweetpotato. However, a decline in storage root quality and yield has been reported under certain environmental conditions. Information is limited on the effect of S-metolachlor application followed immediately by rainfall on sweetpotato growth and development under different temperatures, as well as the optimum application time. Therefore, the objectives of this study were to evaluate sweetpotato responses to interactive effects of S-metolachlor, temperature and rainfall, and to determine S-metolachlor optimum application time. A sunlit, controlled environment experiment was conducted to investigate sweetpotato response to S-metolachlor and rainfall immediately after application under different temperatures. Sweetpotato slips were transplanted into sandy soil filled pots. Treatment combinations included five levels of S-metolachlor, 0.00, 0.86, 1.72, 2.58 and 3.44 kg ha-1, two levels of rainfall, 0 and 38 mm and three temperatures, 25/17, 30/22 and 35/27 °C, day/night. After POST application of S-metolachlor and rainfall, all plants were transferred to sunlit growth chambers that were maintained at their respective temperatures and ambient CO2 concentration for 60 days. In another experiment, S-metolachlor application time was varied to investigate sweetpotato growth and development. Two levels of S-metolachlor 0.0 and 1.0 kg ha-1 and three application times 0, 5 and 10 days after transplanting (DAT) were used and plants were harvested five times, 5, 10, 15, 20 and 80 DAT to estimate plant growth and development. Shoot, root and total plant biomass yields declined with increasing concentration of S-metolachlor across temperatures. In addition, storage root yield and quality decline was S-metolachlor rate dependent and aggravated by rainfall immediately after herbicide treatment across temperatures. S-metolachlor was more injurious on most plant component parameters in the optimum and high temperatures where plant growth was vigorous than in the low temperatures. S-metolachlor application at 0 and 5 days affected sweetpotato growth, including storage roots, but delaying until 10 days minimized the injury. These results can be used to weigh the risk of crop injury against the weed control benefits of S-metolachlor when making management decisions, and to determine application time based on weather information.

Glyphosate-resistant (GR) horseweed has become a major problem in many row crop production systems in the United States. Horseweed is a winter annual weed common in no-till production systems. Fall-applied herbicides were compared with spring-applied treatments for the control of horseweed. In cotton, fall-applied trifloxysulfuron provided similar or greater control of horseweed when compared to spring-applied treatments of glyphosate + dicamba. Cotton yields with fall-applied trifloxysulfuron, clomazone, and flumioxazin were comparable to or better than spring-applied glyphosate + dicamba both years. Fall-applied cloransulam-methyl, flumetsulam, sulfentrazone, and the combination of chlorimuron-ethyl + metribuzin resulted in horseweed control and soybean yields comparable to spring-applied glyphosate + 2,4-D both years. Multiple-resistance to glyphosate and paraquat exists in a horseweed population from Mississippi. Herbicide rates of 0.066 kg ae/ha glyphosate and 0.078 kg ai/ha paraquat were required to reduce susceptible horseweed biomass 50%; whereas, rates of 0.78 kg/ha glyphosate and 0.67 kg/ha paraquat were required to reduce biomass of resistant horseweed to a similar intent. This is the first broadleaf weed species reported as exhibiting multiple-resistance to glyphosate and paraquat. The addition of metribuzin to paraquat improved control of paraquat-resistant horseweed. Paraquat at 0.84 kg/ha plus all rates of metribuzin controlled 15-cm tall horseweed at least 90% both years compared to 73% with 0.84 kg/ha paraquat alone. The addition of 1 and 2% methylated seed oil (MSO) to saflufenacil controlled horseweed 91 and 93%, respectively compared to 78% control with saflufenacil alone. The addition of saflufenacil to glyphosate improved control of GR horseweed from 50% to 100% at 21 d after treatment; control of horseweed with the combination of saflufenacil + glyphosate was additive. Saflufenacil did not affect absorption of glyphosate in glyphosate-susceptible horseweed; however, absorption increased in GR horseweed from 36 to 44% at 48 h after treatment with the addition of saflufenacil when compared to glyphosate alone treatments. Overall, the addition of saflufenacil reduced glyphosate translocation in horseweed at least 6%; however, due to the exceptional efficacy of saflufenacil on horseweed these reductions did not reduce control of GR horseweed.

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