Blake L. Joyce

Multiple Polyploidy Events in the Early Radiation of Nodulating and Nonnodulating Legumes

Unresolved questions about evolution of the large and diverse legume family include the timing of polyploidy (whole-genome duplication; WGDs) relative to the origin of the major lineages within the Fabaceae and to the origin of symbiotic nitrogen fixation. Previous work has established that a WGD affects most lineages in the Papilionoideae and occurred sometime after the divergence of the papilionoid and mimosoid clades, but the exact timing has been unknown. The history of WGD has also not been established for legume lineages outside the Papilionoideae. We investigated the presence and timing of WGDs in the legumes by querying thousands of phylogenetic trees constructed from transcriptome and genome data from 20 diverse legumes and 17 outgroup species. The timing of duplications in the gene trees indicates that the papilionoid WGD occurred in the common ancestor of all papilionoids. The earliest diverging lineages of the Papilionoideae include both nodulating taxa, such as the genistoids (e.g., lupin), dalbergioids (e.g., peanut), phaseoloids (e.g., beans), and galegoids (=Hologalegina, e.g., clovers), and clades with nonnodulating taxa including Xanthocercis and Cladrastis (evaluated in this study). We also found evidence for several independent WGDs near the base of other major legume lineages, including the Mimosoideae-Cassiinae-Caesalpinieae (MCC), Detarieae, and Cercideae clades. Nodulation is found in the MCC and papilionoid clades, both of which experienced ancestral WGDs. However, there are numerous nonnodulating lineages in both clades, making it unclear whether the phylogenetic distribution of nodulation is due to independent gains or a single origin followed by multiple losses.

Within-plant distribution and emission of sesquiterpenes from Copaifera officinalis

Copaifera officinalis, the diesel tree, is known for massive production of oleoresin, mainly composed of sesquiterpene hydrocarbons. In this study, composition of these sesquiterpenes and their concentrations in leaves, stems and roots of C. officinalis at two developmental stages, including the three-week old (TW) seedlings and two-year old (TY) trees, were determined. The leaves of TW seedlings and TY trees contained similar number of sesquiterpenes, which also had comparable concentrations. The stems of TW seedlings had higher concentrations of sesquiterpenes than those of TY trees. In contrast, the number of sesquiterpene species and their concentrations in the roots of TW seedlings were much lower than those in the roots of TY trees. Cluster analysis of sesquiterpenes estimated that there are at least four terpene synthase genes involved in the production of sesquiterpenes in C. officinalis. Because sesquiterpenes are highly volatile, emissions of sesquiterpenes from healthy and wounded TW seedlings were examined using headspace analysis. Whereas very low emission of sesquiterpenes was detected from undamaged plants, the physically injured seedlings emitted a large number of sesquiterpenes, the quality and the relative quantity of which were similar to those in leaves determined using organic extraction. The implications of our findings to the biosynthetic pathways leading to the production of sesquiterpenes as well as their biological roles in C. officinalis are discussed.

Stable Transformation of Ferns Using Spores as Targets: Pteris vittata and Ceratopteris thalictroides

Ferns are stably transformed by using spores as targets. Ferns (Pteridophyta) are very important members of the plant kingdom that lag behind other taxa with regards to our understanding of their genetics, genomics, and molecular biology. We report here, to our knowledge, the first instance of stable transformation of fern with recovery of transgenic sporophytes. Spores of the arsenic hyperaccumulating fern Pteris vittata and tetraploid ‘C-fern Express’ (Ceratopteris thalictroides) were stably transformed by Agrobacterium tumefaciens with constructs containing the P. vittata actin promoter driving a GUSPlus reporter gene. Reporter gene expression assays were performed on multiple tissues and growth stages of gametophytes and sporophytes. Southern-blot analysis confirmed stable transgene integration in recovered sporophytes and also confirmed that no plasmid from A. tumefaciens was present in the sporophyte tissues. We recovered seven independent transformants of P. vittata and four independent C. thalictroides transgenics. Inheritance analyses using β-glucuronidase (GUS) histochemical staining revealed that the GUS transgene was stably expressed in second generation C. thalictroides sporophytic tissues. In an independent experiment, the gusA gene that was driven by the 2× Cauliflower mosaic virus 35S promoter was bombarded into P. vittata spores using biolistics, in which putatively stable transgenic gametophytes were recovered. Transformation procedures required no tissue culture or selectable marker genes. However, we did attempt to use hygromycin selection, which was ineffective for recovering transgenic ferns. This simple stable transformation method should help facilitate functional genomics studies in ferns.

Switchgrass (Panicum virgatum L.) cell suspension cultures: Establishment, characterization, and application

Switchgrass (Panicum virgatum L.) is a warm-season perennial grass that has received considerable attention as a potential dedicated biofuel and bioproduct feedstock. Genetic improvement of switchgrass is needed for better cellulosic ethanol production, especially to improve cellulose-to-lignin ratios. Cell suspension cultures offer an in vitro system for mutant selection, mass propagation, gene transfer, and cell biology. Toward this end, switchgrass cell suspension cultures were initiated from embryogenic callus obtained from genotype Alamo 2. They have been established and characterized with different cell type morphologies: sandy, fine milky, and ultrafine cultures. Characterization includes histological analysis using scanning electron microscopy, and utility using protoplast isolation. A high protoplast isolation rate of up to 10(6) protoplasts/1.0g of cells was achieved for the fine milky culture, whereas only a few protoplasts were isolated for the sandy and ultrafine cultures. These results indicate that switchgrass cell suspension type sizably impacts the efficiency of protoplast isolation, suggesting its significance in other applications. The establishment of different switchgrass suspension culture cell types provides the opportunity to gain insights into the versatility of the system that would further augment switchgrass biology research.

Ethanol and High-Value Terpene Co-Production from Lignocellulosic Biomass of Cymbopogon flexuosus and Cymbopogon martinii

Cymbopogon flexuosus, lemongrass, and C. martinii, palmarosa, are perennial grasses grown to produce essential oils for the fragrance industry. The objectives of this study were (1) to evaluate biomass and oil yields as a function of nitrogen and sulfur fertilization, and (2) to characterize their utility for lignocellulosic ethanol compared to Panicum virgatum (switchgrass). Mean biomass yields were 12.83 Mg lemongrass ha-1 and 15.11 Mg palmarosa ha-1 during the second harvest year resulting in theoretical biofuel yields of 2541 and 2569 L ethanol ha-1 respectively compared to reported 1749–3691 L ethanol ha-1 for switchgrass. Pretreated lemongrass yielded 198 mL ethanol (g biomass)-1 and pretreated palmarosa yielded 170 mL ethanol (g biomass)-1. Additionally, lemongrass yielded 85.7 kg essential oil ha-1 and palmarosa yielded 67.0 kg ha-1 with an estimated value of USD

Investigation and Optimization of Biodiesel Chemistry for HCCI Combustion

857 and

Morphology and ploidy level determination of Pteris vittata callus during induction and regeneration

1005 ha-1. These data suggest that dual-use crops such as lemongrass and palmarosa may increase the economic viability of lignocellulosic biofuels.

An Improved Tissue Culture System for Embryogenic Callus Production and Plant Regeneration in Switchgrass (Panicum virgatum L.)

Over the past 5 years, ORNL has run 95 diesel range fuels in homogeneous charge compression ignition (HCCI), including 40 bio-diesels and associated diesel fuels in their blending. The bio-diesel blends varied in oxygen content, iodine number, cetane, boiling point distribution, chemical composition, and some contained nitrogen. All fuels were run in an HCCI engine at 1800 rpm, in the power range of 2.5 to 4.5 bar IMEP, using intake air heating for combustion phasing control, and at a compression ratio of 10.6. The engine response to fuel variables has been analyzed statistically. Generally, the engine responded well to fuels with lower nitrogen and oxygen, lower cetane, and lower aromatics. Because of the wide range of fuels combined in the model, it provides only a broad overview of the engine response. It is recommended that data be truncated and re-modeled to obtain finer resolution of engine response to particular fuel variables

Switchgrass (Panicum virgatum L.) polyubiquitin gene (PvUbi1 and PvUbi2) promoters for use in plant transformation

BackgroundMorphological and ploidy changes of the arsenic hyperaccumulator, Chinese brake fern (Pteris vittata) callus tissue are described here to provide insight into fern life cycle biology and for possible biotechnology applications. Pteris vittata callus was studied using transmission and scanning electron microscopy, and flow cytometry.ResultsCallus induction occurred both in light and dark culture conditions from prothallus tissues, whereas rhizoid formation occurred only in dark culture conditions. Callus tissues contained two types of cells: one actively dividing and the other containing a single large vacuole undergoing exocytosis. Sporophytes regenerated from callus asynchronously form clusters of cells in a manner apparently analogous to direct organogenesis. Extracellular matrices were observed in actively-growing callus and at the base of regenerating sporophytes. Callus tissue nuclei were found to be primarily diploid at induction and throughout maintenance of cultures indicating that callus cell fate is determined at induction, which closely follows apogamous sporophyte development. Presence of a dense extracellular matrix in conjunction with sporophyte development suggests a link between the suspensor-like activity of the embryonic foot during normal fern embryo development and the suspected functions of extracellular matrices in angiosperms.ConclusionsFurther investigation could lead to a better understanding of genes involved in P. vittata embryo development and apogamous sporophyte development. The methodology could be useful for in vitro propagation of rare and valuable fern germplasm.