Mini-synplastomes for plastid genetic engineering.

In the age of synthetic biology, plastid engineering requires a nimble platform to introduce novel synthetic circuits in plants. While effective for integrating relatively small constructs into the plastome, plastid engineering via homologous recombination of transgenes is over 30 years old. Here we show the design-build-test of a novel synthetic genome structure that does not disturb the native plastome: the 'mini-synplastome'. The mini-synplastome was inspired by dinoflagellate plastome organization, which is comprised of numerous minicircles residing in the plastid instead of a single organellar genome molecule. The first mini-synplastome in plants was developed in vitro to meet the following criteria: (i) episomal replication in plastids; (ii) facile cloning; (iii) predictable transgene expression in plastids; (iv) non-integration of vector sequences into the endogenous plastome; and (v) autonomous persistence in the plant over generations in the absence of exogenous selection pressure. Mini-synplastomes are anticipated to revolutionize chloroplast biotechnology, enable facile marker-free plastid engineering, and provide an unparalleled platform for one-step metabolic engineering in plants.

Climbing plants: attachment adaptations and bioinspired innovations.

Climbing plants have unique adaptations to enable them to compete for sunlight, for which they invest minimal resources for vertical growth. Indeed, their stems bear relatively little weight, as they traverse their host substrates skyward. Climbers possess high tensile strength and flexibility, which allows them to utilize natural and manmade structures for support and growth. The climbing strategies of plants have intrigued scientists for centuries, yet our understanding about biochemical adaptations and their molecular undergirding is still in the early stages of research. Nonetheless, recent discoveries are promising, not only from a basic knowledge perspective, but also for bioinspired product development. Several adaptations, including nanoparticle and adhesive production will be reviewed, as well as practical translation of these adaptations to commercial applications. We will review the botanical literature on the modes of adaptation to climb, as well as specialized organs-and cellular innovations. Finally, recent molecular and biochemical data will be reviewed to assess the future needs and new directions for potential practical products that may be bioinspired by climbing plants.

Improved tissue culture conditions for the emerging C4 model Panicum hallii.

BACKGROUND: Panicum hallii Vasey (Hall's panicgrass) is a compact, perennial C4 grass in the family Poaceae, which has potential to enable bioenergy research for switchgrass (Panicum virgatum L.). Unlike P. hallii, switchgrass has a large genome, allopolyploidy, self-incompatibility, a long life cycle, and large stature-all suboptimal traits for rapid genetics research. Herein we improved tissue culture methodologies for two inbred P. hallii populations: FIL2 and HAL2, to enable further development of P. hallii as a model C4 plant. RESULTS: The optimal seed-derived callus induction medium was determined to be Murashige and Skoog (MS) medium supplemented with 40 mg L-1 L-cysteine, 300 mg L-1 L-proline, 3% sucrose, 1 g L-1 casein hydrolysate, 3 mg L-1 2,4-dichlorophenoxyacetic acid (2,4-D), and 45 µg L-1 6-benzylaminopurine (BAP), which resulted in callus induction of 51 ± 29% for FIL2 and 81 ± 19% for HAL2. The optimal inflorescence-derived callus induction was observed on MP medium (MS medium supplemented with 2 g L-1 L-proline, 3% maltose, 5 mg L-1 2,4-D, and 500 µg L-1 BAP), resulting in callus induction of 100 ± 0.0% for FIL2 and 84 ± 2.4% for HAL2. Shoot regeneration rates of 11.5 ± 0.8 shoots/gram for FIL2 and 11.3 ± 0.6 shoots/gram for HAL2 were achieved using seed-induced callus, whereas shoot regeneration rates of 26.2 ± 2.6 shoots/gram for FIL2 and 29.3 ± 3.6 shoots/gram for HAL2 were achieved from inflorescence-induced callus. Further, cell suspension cultures of P. hallii were established from seed-derived callus, providing faster generation of callus tissue compared with culture using solidified media (1.41-fold increase for FIL2 and 3.00-fold increase for HAL2). CONCLUSIONS: Aside from abbreviated tissue culture times from callus induction to plant regeneration for HAL2, we noted no apparent differences between FIL2 and HAL2 populations in tissue culture performance. For both populations, the cell suspension cultures outperformed tissue cultures on solidified media. Using the methods developed in this work, P. hallii callus was induced from seeds immediately after harvest in a shorter time and with higher frequencies than switchgrass. For clonal propagation, P. hallii callus was established from R1 inflorescences, similar to switchgrass, which further strengthens the potential of this plant as a C4 model for genetic studies. The rapid cycling (seed-to-seed time) and ease of culture, further demonstrate the potential utility of P. hallii as a C4 model plant.

Pollen-mediated gene flow from transgenic to non-transgenic switchgrass (Panicum virgatum L.) in the field.

BACKGROUND: Switchgrass is C4 perennial grass species that is being developed as a cellulosic bioenergy feedstock. It is wind-pollinated and considered to be an obligate outcrosser. Genetic engineering has been used to alter cell walls for more facile bioprocessing and biofuel yield. Gene flow from transgenic cultivars would likely be of regulatory concern. In this study we investigated pollen-mediated gene flow from transgenic to nontransgenic switchgrass in a 3-year field experiment performed in Oliver Springs, Tennessee, U.S.A. using a modified Nelder wheel design. The planted area (0.6 ha) contained sexually compatible pollen source and pollen receptor switchgrass plants. One hundred clonal switchgrass 'Alamo' plants transgenic for an orange-fluorescent protein (OFP) and hygromycin resistance were used as the pollen source; whole plants, including pollen, were orange-fluorescent. To assess pollen movement, pollen traps were placed at 10 m intervals from the pollen-source plot in the four cardinal directions extending to 20 m, 30 m, 30 m, and 100 m to the north, south, west, and east, respectively. To assess pollination rates, nontransgenic 'Alamo 2' switchgrass clones were planted in pairs adjacent to pollen traps. RESULTS: In the eastward direction there was a 98% decrease in OFP pollen grains from 10 to 100 m from the pollen-source plot (Poisson regression, F1,8 = 288.38, P < 0.0001). At the end of the second and third year, 1,820 F1 seeds were collected from pollen recipient-plots of which 962 (52.9%) germinated and analyzed for their transgenic status. Transgenic progeny production detected in each pollen-recipient plot decreased with increased distance from the edge of the transgenic plot (Poisson regression, F1,15 = 12.98, P < 0.003). The frequency of transgenic progeny detected in the eastward plots (the direction of the prevailing wind) ranged from 79.2% at 10 m to 9.3% at 100 m. CONCLUSIONS: In these experiments we found transgenic pollen movement and hybridization rates to be inversely associated with distance. However, these data suggest pollen-mediated gene flow is likely to occur up to, at least, 100 m. This study gives baseline data useful to determine isolation distances and other management practices should transgenic switchgrass be grown commercially in relevant environments.

The performance of pathogenic bacterial phytosensing transgenic tobacco in the field.

Phytosensors are useful for rapid-on-the-plant detection of contaminants and agents that cause plant stress. Previously, we produced a series of plant pathogen-inducible synthetic promoters fused to an orange fluorescent protein (OFP) reporter gene and transformed them into tobacco and Arabidopsis thaliana plants; in these transgenic lines, an OFP signal is expressed commensurate with the presence of plant pathogens. We report here the results of 2 years of field experiments using a subset of these bacterial phytosensing tobacco plants. Time-course analysis of field-grown phytosensors showed that a subset of plants responded predictably to treatments with Pseudomonas phytopathogens. There was a twofold induction in the OFP fluorescence driven by two distinct salicylic acid-responsive synthetic promoters, 4 × PR1 and 4 × SARE. Most notably, transgenic plants containing 4 × PR1 displayed the earliest and highest OFP induction at 48 and 72 h postinoculation (h p.i.) upon inoculation with two phytopathogens Pseudomonas syringae pv. tomato and P. syringae pv. tabaci, respectively. These results demonstrate transgenic tobacco harbouring a synthetic inducible promoter-driven OFP could be used to facilitate monitoring and early-warning reporting of phytopathogen infections in agricultural fields.

Origin of a novel regulatory module by duplication and degeneration of an ancient plant transcription factor.

It is commonly believed that gene duplications provide the raw material for morphological evolution. Both the number of genes and size of gene families have increased during the diversification of land plants. Several small proteins that regulate transcription factors have recently been identified in plants, including the LITTLE ZIPPER (ZPR) proteins. ZPRs are post-translational negative regulators, via heterodimerization, of class III Homeodomain Leucine Zipper (C3HDZ) proteins that play a key role in directing plant form and growth. We show that ZPR genes originated as a duplication of a C3HDZ transcription factor paralog in the common ancestor of euphyllophytes (ferns and seed plants). The ZPRs evolved by degenerative mutations resulting in loss all of the C3HDZ functional domains, except the leucine zipper that modulates dimerization. ZPRs represent a novel regulatory module of the C3HDZ network unique to the euphyllophyte lineage, and their origin correlates to a period of rapid morphological changes and increased complexity in land plants. The origin of the ZPRs illustrates the significance of gene duplications in creating developmental complexity during land plant evolution that likely led to morphological evolution.

Bacterial pathogen phytosensing in transgenic tobacco and Arabidopsis plants.

Plants are subject to attack by a wide range of phytopathogens. Current pathogen detection methods and technologies are largely constrained to those occurring post-symptomatically. Recent efforts were made to generate plant sentinels (phytosensors) that can be used for sensing and reporting pathogen contamination in crops. Engineered phytosensors indicating the presence of plant pathogens as early-warning sentinels potentially have tremendous utility as wide-area detectors. We previously showed that synthetic promoters containing pathogen and/or defence signalling inducible cis-acting regulatory elements (RE) fused to a fluorescent protein (FP) reporter could detect phytopathogenic bacteria in a transient phytosensing system. Here, we further advanced this phytosensing system by developing stable transgenic tobacco and Arabidopsis plants containing candidate constructs. The inducibility of each synthetic promoter was examined in response to biotic (bacterial pathogens) or chemical (plant signal molecules salicylic acid, ethylene and methyl jasmonate) treatments using stably transgenic plants. The treated plants were visualized using epifluorescence microscopy and quantified using spectrofluorometry for FP synthesis upon induction. Time-course analyses of FP synthesis showed that both transgenic tobacco and Arabidopsis plants were capable to respond in predictable ways to pathogen and chemical treatments. These results provide insights into the potential applications of transgenic plants as phytosensors and the implementation of emerging technologies for monitoring plant disease outbreaks in agricultural fields.

Immunoparesis and monoclonal gammopathy of undetermined significance are disassociated in advanced age.

Immunoparesis and a skewed serum free light chain (FLC) ratio are indicators of immune dysfunction predictive of progression from monoclonal gammopathy of undetermined significance (MGUS) to multiple myeloma (MM). Previous studies have reported increased prevalence of MGUS by age, but no study has examined the relationship between immunoparesis and abnormal FLC ratios in the elderly. We screened 453 older adults (median age, 80 years; range, 65-96) to characterize the patterns of immunoparesis and abnormal FLC ratio in relation to MGUS. We defined MGUS in 4.4% of the subjects; the prevalence was 12.5% among individuals of >90 years. In MGUS (vs. non-MGUS) cases, immunoparesis and abnormal FLC ratios were detected in 70.0% (vs. 49.0%; P = 0.07) and 50.0% (vs. 12.9%; P = 0.0001), respectively. Based on small numbers, MGUS patients with abnormal FLC ratio were borderline (P = 0.07) more likely to have immunoparesis. Overall, the prevalence of immunoparesis varied in a nonlinear fashion, with lowest frequencies in the youngest and oldest groups. Our observed disassociation between MGUS prevalence and impaired immunoglobulin production suggests that separate mechanisms are involved in the development of MGUS and immunoparesis in advanced age. These findings emphasize the need for molecularly defined methods to characterize myeloma precursor states and better predict progression to MM.

Monitoring the Environmental Impact of TiO2 Nanoparticles Using a Plant-Based Sensor Network.

The increased manufacturing of nanoparticles for use in cosmetics, foods, and clothing necessitates the need for an effective system to monitor and evaluate the potential environmental impact of these nanoparticles. The goal of this research was to develop a plant-based sensor network for characterizing, monitoring, and understanding the environmental impact of TiO2 nanoparticles. The network consisted of potted Arabidopsis thaliana with a surrounding water supply, which was monitored by cameras attached to a laptop computer running a machine learning algorithm. Using the proposed plant sensor network, we were able to examine the toxicity of TiO2 nanoparticles in two systems: algae and terrestrial plants. Increased terrestrial plant growth was observed upon introduction of the nanoparticles, whereas algal growth decreased significantly. The proposed system can be further automated for high-throughput screening of nanoparticle toxicity in the environment at multiple trophic levels. The proposed plant-based sensor network could be used for more accurate characterization of the environmental impact of nanomaterials.

Characterization of physicochemical properties of ivy nanoparticles for cosmetic application.

BACKGROUND: Naturally occurring nanoparticles isolated from English ivy (Hedera helix) have previously been proposed as an alternative to metallic nanoparticles as sunscreen fillers due to their effective UV extinction property, low toxicity and potential biodegradability. METHODS: This study focused on analyzing the physicochemical properties of the ivy nanoparticles, specifically, those parameters which are crucial for use as sunscreen fillers, such as pH, temperature, and UV irradiation. The visual transparency and cytotoxicity of ivy nanoparticles were also investigated comparing them with other metal oxide nanoparticles. RESULTS: Results from this study demonstrated that, after treatment at 100°C, there was a clear increase in the UV extinction spectra of the ivy nanoparticles caused by the partial decomposition. In addition, the UVA extinction spectra of the ivy nanoparticles gradually reduced slightly with the decrease of pH values in solvents. Prolonged UV irradiation indicated that the influence of UV light on the stability of the ivy nanoparticle was limited and time-independent. Compared to TiO2 and ZnO nanoparticles, ivy nanoparticles showed better visual transparency. Methylthiazol tetrazolium assay demonstrated that ivy nanoparticles exhibited lower cytotoxicity than the other two types of nanoparticles. Results also suggested that protein played an important role in modulating the three-dimensional structure of the ivy nanoparticles. CONCLUSIONS: Based on the results from this study it can be concluded that the ivy nanoparticles are able to maintain their UV protective capability at wide range of temperature and pH values, further demonstrating their potential as an alternative to replace currently available metal oxide nanoparticles in sunscreen applications.

Bio-synthesis of gold nanoparticles using English ivy (Hedera helix).

Gold nanoparticles (AuNPs) have drawn significant interest in recent years due to unique properties that make them advantageous in biomedical applications, including drug delivery and tissue engineering. In this paper, we have developed multiple methods for the synthesis of AuNPs using English ivy as the substrate. In the first method, we have used actively growing English ivy shoots to develop a sustainable system for the production of ivy nanoparticles. The second method was developed using the extract from the adventitious roots of English ivy. The nanoparticles formed using both methods were compared to determine the size distribution, morphology, and chemical structure of the nanoparticles. Characterization of the AuNPs was conducted using ultraviolet-visible (UV-Vis) spectroscopy, dynamic light scattering (DLS), scanning electron microscopy (SEM), and energy-dispersive X-ray spectroscopy (EDS). In addition to the structural differences between the AuNPs formed from the different methods, details of the methods in terms of yield, duration, and speed of AuNP formation are also discussed. Further, this paper will show that AuNPs formed using both methods demonstrated efficient uptake in mammalian cells, which provides the potential for biomedical applications. The two methods developed through this research for eco-friendly synthesis of AuNPs present an alternative to traditional chemical synthesis methods.

Targeting the proteasome with bortezomib in multiple myeloma: update on therapeutic benefit as an upfront single agent, induction regimen for stem-cell transplantation and as maintenance therapy.

Bortezomib is the first therapeutic inhibitor of the proteasome that has demonstrated a significant clinical response in patients with otherwise refractory or rapidly advancing disease. Bortezomib has received US Federal Drug Administration approval for the treatment of the hematologic malignancies such as multiple myeloma and mantle cell lymphoma. Herein, the use of bortezomib as an upfront therapy, as an induction regimen before stem-cell transplantation and as maintenance therapy in the treatment of multiple myeloma is discussed.

Nanoparticle biofabrication using English ivy (Hedera helix).

BACKGROUND: English ivy (Hedera helix) is well known for its adhesive properties and climbing ability. Essential to its ability to adhere to vertical surfaces is the secretion of a nanocomposite adhesive containing spherical nanoparticles, 60-85 nm in diameter, produced exclusively by root hairs present on adventitious roots. These organic nanoparticles have shown promise in biomedical and cosmetic applications, and represent a safer alternative to metal oxide nanoparticles currently available. RESULTS: It was discovered that the maximum adventitious root production was achieved by a 4 h application of 1 mg/ml indole-3 butyric acid (IBA) to juvenile English ivy shoot segments cultured in custom vessels. After incubation of the shoots under continuous light at 83 µmol/m2 s at 20°C for 2 weeks, the adventitious roots were harvested from the culture system and it was possible to isolate 90 mg of dry weight nanoparticles per 12 g of roots. The nanoparticle morphology was characterized by atomic force microscopy, and found to be similar to previous studies. CONCLUSIONS: An enhanced system for the production of English ivy adventitious roots and their nanoparticles by modifying GA7 Magenta boxes and identifying the optimal concentration of IBA for adventitious root growth was developed. This system is the first such platform for growing and harvesting organic nanoparticles from plants, and represents an important step in the development of plant-based nanomanufacturing. It is a significant improvement on the exploitation of plant systems for the formation of metallic nanoparticles, and represents a pathway for the generation of bulk ivy nanoparticles for translation into biomedical applications.

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

BACKGROUND: The ubiquitin protein is present in all eukaryotic cells and promoters from ubiquitin genes are good candidates to regulate the constitutive expression of transgenes in plants. Therefore, two switchgrass (Panicum virgatum L.) ubiquitin genes (PvUbi1 and PvUbi2) were cloned and characterized. Reporter constructs were produced containing the isolated 5' upstream regulatory regions of the coding sequences (i.e. PvUbi1 and PvUbi2 promoters) fused to the uidA coding region (GUS) and tested for transient and stable expression in a variety of plant species and tissues. RESULTS: PvUbi1 consists of 607 bp containing cis-acting regulatory elements, a 5' untranslated region (UTR) containing a 93 bp non-coding exon and a 1291 bp intron, and a 918 bp open reading frame (ORF) that encodes four tandem, head -to-tail ubiquitin monomer repeats followed by a 191 bp 3' UTR. PvUbi2 consists of 692 bp containing cis-acting regulatory elements, a 5' UTR containing a 97 bp non-coding exon and a 1072 bp intron, a 1146 bp ORF that encodes five tandem ubiquitin monomer repeats and a 183 bp 3' UTR. PvUbi1 and PvUbi2 were expressed in all examined switchgrass tissues as measured by qRT-PCR. Using biolistic bombardment, PvUbi1 and PvUbi2 promoters showed strong expression in switchgrass and rice callus, equaling or surpassing the expression levels of the CaMV 35S, 2x35S, ZmUbi1, and OsAct1 promoters. GUS staining following stable transformation in rice demonstrated that the PvUbi1 and PvUbi2 promoters drove expression in all examined tissues. When stably transformed into tobacco (Nicotiana tabacum), the PvUbi2+3 and PvUbi2+9 promoter fusion variants showed expression in vascular and reproductive tissues. CONCLUSIONS: The PvUbi1 and PvUbi2 promoters drive expression in switchgrass, rice and tobacco and are strong constitutive promoter candidates that will be useful in genetic transformation of monocots and dicots.

Phenotypic effects of changes in the FTVTxK region of an Arabidopsis secondary wall cellulose synthase compared with results from analogous mutations in other isoforms.

Understanding protein structure and function relationships in cellulose synthase (CesA), including divergent isomers, is an important goal. Here, we report results from mutant complementation assays that tested the ability of sequence variants of AtCesA7, a secondary wall CesA of Arabidopsis thaliana, to rescue the collapsed vessels, short stems, and low cellulose content of the irx3-1 AtCesA7 null mutant. We tested a catalytic null mutation and seven missense or small domain changes in and near the AtCesA7 FTVTSK motif, which lies near the catalytic domain and may, analogously to bacterial CesA, exist within a substrate "gating loop." A low-to-high gradient of rescue occurred, and even inactive AtCesA7 had a small positive effect on stem cellulose content but not stem elongation. Overall, secondary wall cellulose content and stem length were moderately correlated, but the results were consistent with threshold amounts of cellulose supporting particular developmental processes. Vibrational sum frequency generation microscopy allowed tissue-specific analysis of cellulose content in stem xylem and interfascicular fibers, revealing subtle differences between selected genotypes that correlated with the extent of rescue of the collapsing xylem phenotype. Similar tests on PpCesA5 from the moss Physcomitrium (formerly Physcomitrella) patens helped us to synergize the AtCesA7 results with prior results on AtCesA1 and PpCesA5. The cumulative results show that the FTVTxK region is important for the function of an angiosperm secondary wall CesA as well as widely divergent primary wall CesAs, while differences in complementation results between isomers may reflect functional differences that can be explored in further work.

Recent advances in use of fresh frozen plasma, cryoprecipitate, immunoglobulins, and clotting factors for transfusion support in patients with hematologic disease.

Hematologic diseases include a broad range of acquired and congenital disorders, many of which affect plasma proteins that control hemostasis and immune responses. Therapeutic interventions for these disorders include transfusion of plasma, cryoprecipitate, immunoglobulins, or convalescent plasma-containing therapeutic antibodies from patients recovering from infectious diseases, as well as concentrated pro- or anticoagulant factors. This review will focus on recent advances in the uses of plasma and its derivatives for patients with acquired and congenital hematologic disorders.

A Robust Method to Quantify Cell Wall Bound Phenolics in Plant Suspension Culture Cells Using Pyrolysis-Gas Chromatography/Mass Spectrometry.

The wide-scale production of renewable fuels from lignocellulosic feedstocks continues to be hampered by the natural recalcitrance of biomass. Therefore, there is a need to develop robust and reliable methods to characterize and quantify components that contribute to this recalcitrance. In this study, we utilized a method that incorporates pyrolysis with successive gas chromatography and mass spectrometry (Py-GC/MS) to assess lignification in cell suspension cultures. This method was compared with other standard techniques such as acid-catalyzed hydrolysis, acetyl bromide lignin determination, and nitrobenzene oxidation for quantification of cell wall bound phenolic compounds. We found that Py-GC/MS can be conducted with about 250 µg of tissue sample and provides biologically relevant data, which constitutes a substantial advantage when compared to the 50-300 mg of tissue needed for the other methods. We show that when combined with multivariate statistical analyses, Py-GC/MS can distinguish cell wall components of switchgrass (Panicum virgatum) suspension cultures before and after inducing lignification. The deposition of lignin precursors on uninduced cell walls included predominantly guaiacyl-based units, 71% ferulic acid, and 5.3% p-coumaric acid. Formation of the primary and partial secondary cell wall was supported by the respective ~15× and ~1.7× increases in syringyl-based and guaiacyl-based precursors, respectively, in the induced cells. Ferulic acid was decreased by half after induction. These results provide the proof-of-concept for quick and reliable cell wall compositional analyses using Py-GC/MS and could be targeted for either translational genomics or for fundamental studies focused on understanding the molecular and physiological mechanisms regulating plant cell wall production and biomass recalcitrance.

Comparative genome analysis of lignin biosynthesis gene families across the plant kingdom.

BACKGROUND: As a major component of plant cell wall, lignin plays important roles in mechanical support, water transport, and stress responses. As the main cause for the recalcitrance of plant cell wall, lignin modification has been a major task for bioenergy feedstock improvement. The study of the evolution and function of lignin biosynthesis genes thus has two-fold implications. First, the lignin biosynthesis pathway provides an excellent model to study the coordinative evolution of a biochemical pathway in plants. Second, understanding the function and evolution of lignin biosynthesis genes will guide us to develop better strategies for bioenergy feedstock improvement. RESULTS: We analyzed lignin biosynthesis genes from fourteen plant species and one symbiotic fungal species. Comprehensive comparative genome analysis was carried out to study the distribution, relatedness, and family expansion of the lignin biosynthesis genes across the plant kingdom. In addition, we also analyzed the comparative synteny map between rice and sorghum to study the evolution of lignin biosynthesis genes within the Poaceae family and the chromosome evolution between the two species. Comprehensive lignin biosynthesis gene expression analysis was performed in rice, poplar and Arabidopsis. The representative data from rice indicates that different fates of gene duplications exist for lignin biosynthesis genes. In addition, we also carried out the biomass composition analysis of nine Arabidopsis mutants with both MBMS analysis and traditional wet chemistry methods. The results were analyzed together with the genomics analysis. CONCLUSION: The research revealed that, among the species analyzed, the complete lignin biosynthesis pathway first appeared in moss; the pathway is absent in green algae. The expansion of lignin biosynthesis gene families correlates with substrate diversity. In addition, we found that the expansion of the gene families mostly occurred after the divergence of monocots and dicots, with the exception of the C4H gene family. Gene expression analysis revealed different fates of gene duplications, largely confirming plants are tolerant to gene dosage effects. The rapid expansion of lignin biosynthesis genes indicated that the translation of transgenic lignin modification strategies from model species to bioenergy feedstock might only be successful between the closely relevant species within the same family.

Embryogenic cell suspensions for high-capacity genetic transformation and regeneration of switchgrass (Panicum virgatum L.).

BACKGROUND: Switchgrass (Panicum virgatum L.), a North American prairie grassland species, is a potential lignocellulosic biofuel feedstock owing to its wide adaptability and biomass production. Production and genetic manipulation of switchgrass should be useful to improve its biomass composition and production for bioenergy applications. The goal of this project was to develop a high-throughput stable switchgrass transformation method using Agrobacterium tumefaciens with subsequent plant regeneration. RESULTS: Regenerable embryogenic cell suspension cultures were established from friable type II callus-derived inflorescences using two genotypes selected from the synthetic switchgrass variety 'Performer' tissue culture lines 32 and 605. The cell suspension cultures were composed of a heterogeneous fine mixture culture of single cells and aggregates. Agrobacterium tumefaciens strain GV3101 was optimum to transfer into cells the pANIC-10A vector with a hygromycin-selectable marker gene and a pporRFP orange fluorescent protein marker gene at an 85% transformation efficiency. Liquid cultures gave rise to embryogenic callus and then shoots, of which up to 94% formed roots. The resulting transgenic plants were phenotypically indistinguishable from the non-transgenic parent lines. CONCLUSION: The new cell suspension-based protocol enables high-throughput Agrobacterium-mediated transformation and regeneration of switchgrass in which plants are recovered within 6-7 months from culture establishment.

Statistical tools for transgene copy number estimation based on real-time PCR.

BACKGROUND: As compared with traditional transgene copy number detection technologies such as Southern blot analysis, real-time PCR provides a fast, inexpensive and high-throughput alternative. However, the real-time PCR based transgene copy number estimation tends to be ambiguous and subjective stemming from the lack of proper statistical analysis and data quality control to render a reliable estimation of copy number with a prediction value. Despite the recent progresses in statistical analysis of real-time PCR, few publications have integrated these advancements in real-time PCR based transgene copy number determination. RESULTS: Three experimental designs and four data quality control integrated statistical models are presented. For the first method, external calibration curves are established for the transgene based on serially-diluted templates. The Ct number from a control transgenic event and putative transgenic event are compared to derive the transgene copy number or zygosity estimation. Simple linear regression and two group T-test procedures were combined to model the data from this design. For the second experimental design, standard curves were generated for both an internal reference gene and the transgene, and the copy number of transgene was compared with that of internal reference gene. Multiple regression models and ANOVA models can be employed to analyze the data and perform quality control for this approach. In the third experimental design, transgene copy number is compared with reference gene without a standard curve, but rather, is based directly on fluorescence data. Two different multiple regression models were proposed to analyze the data based on two different approaches of amplification efficiency integration. Our results highlight the importance of proper statistical treatment and quality control integration in real-time PCR-based transgene copy number determination. CONCLUSION: These statistical methods allow the real-time PCR-based transgene copy number estimation to be more reliable and precise with a proper statistical estimation. Proper confidence intervals are necessary for unambiguous prediction of trangene copy number. The four different statistical methods are compared for their advantages and disadvantages. Moreover, the statistical methods can also be applied for other real-time PCR-based quantification assays including transfection efficiency analysis and pathogen quantification.