Medical glove durability during exposure to different solvent agents: an ex-vivo experimental study.

BACKGROUND: Medical professionals are constantly exposed to bodily fluids and sanitizing agents during routine medical procedures. Unbeknownst to many healthcare workers, however, the barrier integrity of medical gloves can be altered when exposed to these substances, potentially resulting in exposure to dangerous pathogens. METHODS: This experimental study was designed to test the hypothesis that the durability of both natural and synthetic solvent-exposed medical gloves will be lower than the durability of the gloves in air. The testing consisted of a sample of commercially available medical gloves exposed to 70% ethanol, phosphate buffered saline, and deionized water, aimed at simulating the environments in which medical gloves are commonly worn. Gloves were included in this study based on their performance in previous durability studies in air. Data were collected over a period of three months. The glove assessment device automatically detects pinhole-sized perforations in medical gloves, eliminating the need to visually inspect each glove. Relative durability was measured as the average number of sandpaper touches until glove puncture. RESULTS: Four out of five glove brands performed better when exposed to all three solvents than in air, which is likely due to slippage in the interface between the wet glove and the sandpaper. Sensicare Micro, a polyisoprene surgical glove, had the most consistent durability in all three solvents tested. A two-way ANOVA revealed that both glove brand (P = 0.0001), solvent (P = 0.0001), and their interaction (P = 0.0040, α = 0.05) significantly affected average glove durability. CONCLUSIONS: Glove durability did not remain consistent in 70% ethanol, phosphate buffered saline, deionized water, and air. These results make it clear that additional testing and labeling information would help healthcare workers select gloves for use in specific environments to ensure the best barrier protection against disease or toxins.

Carbon Monoxide Alleviates Salt-Induced Oxidative Damage in Sorghum bicolor by Inducing the Expression of Proline Biosynthesis and Antioxidant Genes.

Crop growth and yield are affected by salinity, which causes oxidative damage to plant cells. Plants respond to salinity by maintaining cellular osmotic balance, regulating ion transport, and enhancing the expression of stress-responsive genes, thereby inducing tolerance. As a byproduct of heme oxygenase (HO)-mediated degradation of heme, carbon monoxide (CO) regulates plant responses to salinity. This study investigated a CO-mediated salt stress tolerance mechanism in sorghum seedlings during germination. Sorghum seeds were germinated in the presence of 250 mM NaCl only, or in combination with a CO donor (1 and 1.5 µM hematin), HO inhibitor (5 and 10 µM zinc protoporphyrin IX; ZnPPIX), and hemoglobin (0.1 g/L Hb). Salt stress decreased the germination index (47.73%) and root length (74.31%), while hydrogen peroxide (H2O2) (193.5%), and proline (475%) contents increased. This increase correlated with induced HO (137.68%) activity and transcripts of ion-exchanger and antioxidant genes. Salt stress modified vascular bundle structure, increased metaxylem pit size (42.2%) and the Na+/K+ ratio (2.06) and altered primary and secondary metabolites. However, exogenous CO (1 µM hematin) increased the germination index (63.01%) and root length (150.59%), while H2O2 (21.94%) content decreased under salt stress. Carbon monoxide further increased proline (147.62%), restored the vascular bundle structure, decreased the metaxylem pit size (31.2%) and Na+/K+ ratio (1.46), and attenuated changes observed on primary and secondary metabolites under salt stress. Carbon monoxide increased HO activity (30.49%), protein content, and antioxidant gene transcripts. The alleviatory role of CO was abolished by Hb, whereas HO activity was slightly inhibited by ZnPPIX under salt stress. These results suggest that CO elicited salt stress tolerance by reducing oxidative damage through osmotic adjustment and by regulating the expression of HO1 and the ion exchanger and antioxidant transcripts.

Natural rubber - Increasing diversity of an irreplaceable renewable resource.

This paper discusses the importance of introducing domestic natural rubber production and presents the rediscovery of a rubber-producing species, Scorzonera tau-saghyz or "mountain gum", originally discovered in 1929 on the Karatau mountains in Kazakhstan. This plant could potentially also be cultivated in the U.S. In this exploratory work, roots (2-5 years old) were harvested on June 16, 2021 from wild strands in the Karatau mountains, Kumantas ridge, and Saraba, Kazakhstan, and processed at the Ohio State University. The rubber extraction method was based on an indigenous method in Kazakhstan to make natural chewing gum. Water extraction followed by purification yielded 16.2 wt% rubber from the dry roots, in comparison with 4-8 wt% from most rubber dandelion (Taraxacum kok-saghyz) plants, also a potential domestic rubber producing plant. High-resolution size exclusion chromatography was used to analyze rubber samples. The molecular weights and gel and oligomer contents were very similar to the rubber from Hevea brasiliensis, the current commercial source of natural rubber. More detailed investigations of this very interesting rubber-producing plant are in progress.

Effect of drought stress on natural rubber biosynthesis and quality in Taraxacum kok-saghyz roots.

Taraxacum kok-saghyz (TKS) is a potential source of natural rubber (NR) that can be grown in temperate regions with limited water availability. However, the effect of drought stress on NR production and properties in TKS isn't well studied. This study examined how different levels of drought stress (30, 60 and 90%) influenced the NR content, molecular weight (Mw), glass transition temperature (Tg), gene expression, and biochemical parameters in TKS roots. The results showed that drought stress didn't significantly change the NR content, but increased the Mw and the expression of CPT and SRPP genes, which are involved in NR biosynthesis. The NR from TKS roots (TNR) had a high Mw of 994,000 g/mol and a low Tg of below -60°C under normal irrigation, indicating its suitability for industrial applications. Drought stress also triggered the accumulation of proline, H2O2, MDA, and antioxidant enzymes (CAT, APX, GPX) in TKS roots significantly, indicating a drought tolerance mechanism. These findings suggest that TKS can produce high-quality NR under drought stress conditions and provide a sustainable alternative to conventional NR sources.

Durability Variation Among Medical Gloves Made from Existing and New Elastomers Poses a Risk to Public Health.

Despite being an essential line of defense in preventing the spread of diseases, medical glove durability is neither measured routinely nor has standard specifications. In this study, a new glove durability assessment device is used to objectively compare the durability of gloves made of a variety of elastomers from different manufacturers. Results are related to several mechanical tests, including stress relaxation, tensile and tear tests. Overall, natural latex gloves far outperformed those made of synthetic elastomers, and there is great disparity among the different brands of nitrile gloves, some of which do not meet nitrile glove performance requirements. The study includes prototype gloves made from guayule latex, a domestic source of alternative natural rubber latex, currently under commercial development. The guayule gloves outperformed all other gloves tested, including those made from Hevea latex, without posing allergy risks. Mechanical analysis demonstrated that the guayule gloves are as strong as the best alternatives, are softer and more elastic, have better tear strength, and have such low stress relaxation that they cause very little hand fatigue during use. Guayule latex can address the need for domestic production of gloves to resolve supply chain and quality issues and encourage a shift back to natural latex gloves, which will significantly diversify the natural rubber supply.

Characterization and potential valorization of industrial food processing wastes.

Valorization and utilization of industrial food processing waste as value added products, platform chemicals and biofuels, are needed to improve sustainability and reduce waste management costs. Various industrial food waste stream samples were characterized with respect to their physico-chemical characteristics and elemental composition. A subset of starchy food wastes and milk dust powder were evaluated in batch fermentation to acetone, a useful platform chemical. Production levels were similar to acetone produced from glucose but were achieved more quickly. Lactose concentration negatively affected fermentation and led to 50 % lower acetone concentration from milk dust powder than from starchy wastes. Uncooked starch waste can produce 20 % more acetone than cooked and modified starch waste. Fatty waste and mineral waste can be digested anaerobically generating biogas. Calorific value of soybean waste was 40 MJ/kg sufficiently high for biodiesel production. Low C/N ratios of wastewater and solids from food processing waste makes them unsuitable for anaerobic digestion but these waste types can be converted thermochemically to hydrochar and used as soil amendments. Low calorific content (10-15 MJ/kg) vegetable wastes also are not ideal for energy production, but are rich in flavonoids, antioxidants and pigments which can be extracted as valuable products. A model mapping food waste characteristics to best valorization pathway was developed to guide waste management and future cost and environmental impact analyses. These findings will help advance food industry knowledge and improve sustainable food production through valorized processing waste management.

Programmable chemical actuator control of soluble and membrane-bound enzymatic catalysis.

Enzyme catalyzed reactions can be started, stopped, and throttled by regulating ion concentration in solution. The flow of charge through solution is related to the flow of electrons through an electric circuit using electrochemistry. However, current electrochemical methods to vary ionic concentration are largely limited to the electrode surface, making it impractical to exploit ionic concentration as a control variable in traditional enzyme assays. This chapter presents an electrochemical cell, termed a programmable chemical actuator (PCA), that uses selective electrochemical REDOX reactions to regulate the concentration of ions that control enzyme catalyzed reaction kinetics. The PCA is a polypyrrole polymerized and equilibrated to impart Mg2+, Mn2+, or OH- ion selectivity to working electrodes. Duplex pulse amperometry demonstrated reversible control over the pH-dependent kinetics of alkaline phosphatase, a soluble homodimeric enzyme, during a spectrophotometric assay. PCAs were also used to extend electrochemical control to the membrane-bound enzyme complex rubber transferase by modulating Mg2+ and Mn2+ concentrations during a radiometric enzyme assay. PCAs demonstrated tunable control over rubber transferase activity and rubber molecular weight without dislodging either the elongating rubber molecule or irreversibly inhibiting the rubber transferase complex. This chapter presents methods to fabricate and operate PCAs to facilitate insights into the roles of ions in biochemical pathways and environmental responses in plants.

Sustainable Epoxidized Guayule Natural Rubber, Blends and Composites with Improved Oil Resistance and Greater Stiffness.

Production of petroleum-based synthetic rubbers (SRs) causes an enormous carbon footprint for the rubber industry. Carbon footprint would be reduced if natural rubber (NR) could take a larger market share and replace significant quantities of SR. However, some SRs have higher oil resistance than NRs, and, in applications where these properties are needed, chemically modified NR will be required. Epoxidation is a chemical modification of NR which partially converts unsaturated bonds on the backbone of NR to epoxy groups. In this research, epoxidized guayule natural rubber (EGNR)/guayule natural rubber (GNR) blends and GNR were used to make carbon black (CB) filled composites. The processability, mechanical properties, swelling behaviors and dynamic mechanical properties were characterized at various epoxide fractions. Composites made with EGNR/GNR had higher oil resistance, wet traction and stiffness than GNR composites, although tensile strength and elongation at break were reduced by epoxidation. EGNR is expected to lead to the development of new NR products with similar properties to SR, eroding SR markets and increasing the sustainability of the rubber industry.

Liquid Guayule Natural Rubber, a Sustainable Processing Aid, Enhances the Processability, Durability and Dynamic Mechanical Properties of Rubber Composites.

Petroleum-based oils are widely used as processing aids in rubber composites to improve processability but can adversely affect rubber composite performance and increase carbon footprint. In this research, liquid guayule natural rubber (LGNR), produced from guayule natural rubber, was used as a renewable processing aid to replace naphthenic oil (NO) in Hevea natural rubber, styrene-butadiene rubber (SBR) and guayule natural rubber (GNR) composites. The rheological properties, thermal stability, glass transition temperature, dynamic mechanical properties, aging, and ozone resistance of rubber composites with and without NO or LGNR were compared. Natural and synthetic rubber composites made with LGNR had similar processability to those made with NO, but had improved thermal stability, mechanical properties after aging, and ozone resistance. This was due to the strong LGNR-filler interaction and additional crosslinks formed between LGNR and the rubber matrices. The glass transition temperature of SBR composites was reduced by LGNR because of its increased molecular mobility. Thus, unlike NO, LGNR processing aid can simultaneously improve rubber composite durability, dynamic performance and renewability. The commercialization of LGNR has the potential to open a new sustainable processing-aid market.

Guayule Natural Rubber Latex and Bi2O3 Films for X-ray Attenuating Medical Gloves.

Existing natural latex radiation-attenuating gloves (RAGs) contain a high loading of radiation attenuation filler that reduces their mechanical properties to below Food and Drug Administration (FDA) medical glove requirements. RAGs are commonly formulated using Hevea natural rubber latex and lead-based fillers. The former can cause life-threatening allergic responses and the latter are known for their toxicity. In this work, a new lead-free RAG formulation based on circumallergenic guayule natural rubber latex (GNRL) and non-toxic radiation attenuation filler bismuth trioxide (Bi2O3) was developed. GNRL films with Bi2O3 loadings ranging from 0 to 300 PHR at different thicknesses were prepared. Radiation attenuation efficiencies (AE) at 60, 80, 100, and 120 kVp were determined and attenuation isocontour curves predicted film thickness and Bi2O3 loading required to meet or exceed the radiation attenuation requirements of ASTM D7866 and commercial RAGs. Optimal curing conditions for GNRL/Bi2O3 films with 150 PHR Bi2O3 were investigated by varying curing temperatures and time from 87 °C to 96 °C and 65 min to 90 min, respectively. In general, as the loading of the filler increased, the density of the films increased while the thickness decreased. GNRL/Bi2O3 films with 150 PHR Bi2O3 and 0.27 mm provided 5% more AE than RAG market average attenuation at the same thickness. The films with 150 PHR Bi2O3 cured under near-optimal conditions (90 °C/85 min, and 87 °C/65 min) met both the radiation attenuation standard (ASTM D7866) and the natural latex surgeon and examination glove standards (ASTM D3577 and D3578, respectively). Thus, gloves made using our formulations and protocols demonstrated potential to meet and surpass medical natural latex glove standards, offer a single product for both infection control and radiation protection instead of double-gloving, provide a greater degree of comfort to the user, and simultaneously reduce contact reactions and eliminate potential latex allergic reaction.

Adapting the Accelerated Solvent Extraction Method for Resin and Rubber Determination in Guayule Using the BÜCHI Speed Extractor.

Guayule (Parthenium argentatum Gray) is a promising alternative source to Hevea brasiliensis for the production of natural rubber, which can reach levels of 8-9% under industrialized farming conditions. The most common method for determining rubber concentration is by accelerated solvent extraction (ASE), a technique developed by the Dionex Corporation and almost exclusively performed with the Dionex ASE-200 or 350 systems. Herein, it is sought to apply and adapt the most common methods used in the literature for the Dionex system to another extraction platform, the BÜCHI Speed Extractor E-914. Results showed that using a sand sandwich method to confine the sample in the center and exploiting a larger cell volume (80 mL) for extraction prevents the occurrence of overpressure and problems with clogging. Under optimized conditions, the coefficient of variation was <15% for both resin quantification for samples containing 5.0-15.8% of resin and for rubber quantification for samples with 1.7-10.3% rubber content. The extraction time for resin (2 cycles of 5 min each) was smaller than for rubber (2 cycles of 20 min each). It would be interesting to carry out interlaboratory comparisons to standardize the method at an international level.

Inactivation of the Levansucrase Gene in Paenibacillus polymyxa DSM 365 Diminishes Exopolysaccharide Biosynthesis during 2,3-Butanediol Fermentation.

The formation of exopolysaccharides (EPSs) during 2,3-butanediol (2,3-BD) fermentation by Paenibacillus polymyxa increases medium viscosity, which in turn presents considerable technical and economic challenges to 2,3-BD downstream processing. To eliminate EPS production during 2,3-BD fermentation, we used homologous recombination to disable the EPS biosynthetic pathway in P. polymyxa The gene which encodes levansucrase, the major enzyme responsible for EPS biosynthesis in P. polymyxa, was successfully disrupted. The P. polymyxa levansucrase null mutant produced 2.5 ± 0.1 and 1.2 ± 0.2 g/liter EPS on sucrose and glucose, respectively, whereas the wild type produced 21.7 ± 2.5 and 3.1 ± 0.0 g/liter EPS on the same substrates, respectively. These levels of EPS translate to 8.7- and 2.6-fold decreases in EPS formation by the levansucrase null mutant on sucrose and glucose, respectively, relative to that by the wild type, with no significant reduction in 2,3-BD production. Inactivation of EPS biosynthesis led to a considerable increase in growth. On glucose and sucrose, the cell biomass of the levansucrase null mutant (8.1 ± 0.8 and 6.5 ± 0.3 g/liter, respectively) increased 1.4-fold compared to that of the wild type (6.0 ± 0.1 and 4.6 ± 0.3 g/liter, respectively) grown on the same substrates. Evaluation of the genetic stability of the levansucrase null mutant showed that it remained genetically stable over fifty generations, with no observable decrease in growth or 2,3-BD formation, with or without antibiotic supplementation. Hence, the P. polymyxa levansucrase null mutant has potential for use as an industrial biocatalyst for a cost-effective large-scale 2,3-BD fermentation process devoid of EPS-related challenges.IMPORTANCE Given the current barrage of attention and research investments toward the production of next-generation fuels and chemicals, of which 2,3-butanediol (2,3-BD) produced by nonpathogenic Paenibacillus species is perhaps one of the most vigorously pursued, tools for engineering Paenibacillus species are intensely sought after. Exopolysaccharide (EPS) production during 2,3-BD fermentation constitutes a problem during downstream processing. Specifically, EPS negatively impacts 2,3-BD separation from the fermentation broth, thereby increasing the overall cost of 2,3-BD production. The results presented here demonstrate that inactivation of the levansucrase gene in P. polymyxa leads to diminished EPS accumulation. Additionally, a new method for an EPS assay and a simple protocol employing protoplasts for enhanced transformation of P. polymyxa were developed. Overall, although our study shows that levan is not the only EPS produced by P. polymyxa, it represents a significant first step toward developing cost-effective 2,3-BD fermentation devoid of EPS-associated complications during downstream processing.

Narrowing the Gap for Bioplastic Use in Food Packaging: An Update.

Plastic production has outgrown most other man-made materials, with more than 90% being petroleum-based and nonbiodegradable. Packaging, primarily food packaging, consumes the most plastic and is the largest contributor to municipal solid waste. In addition, its dependence on crude oil feedstock makes the plastic industry unsustainable and renders plastic markets vulnerable to oil price volatility. Therefore, the development of bioalternatives to conventional plastics is now a priority of the food packaging industry. Bioplastics are polymers that are either biobased (fully or partially), or biodegradable, or both. This review aims to provide an insightful overview of the most recent research and development successes in bioplastic materials, focusing on food packaging applications. Bioplastics are compared to their conventional counterparts with respect to their mechanical, thermal, barrier, and processability properties. The gaps between bio- and conventional plastics in food packaging are elucidated. Potential avenues for improving bioplastic properties to broaden their food packaging applications are critically examined. Furthermore, two of the most controversial topics on bioplastic alternatives, sustainability assessment and their impact on the plastic waste management system, are discussed.

Natural rubber biosynthesis in plants, the rubber transferase complex, and metabolic engineering progress and prospects.

Natural rubber (NR) is a nonfungible and valuable biopolymer, used to manufacture ~50 000 rubber products, including tires and medical gloves. Current production of NR is derived entirely from the para rubber tree (Hevea brasiliensis). The increasing demand for NR, coupled with limitations and vulnerability of H. brasiliensis production systems, has induced increasing interest among scientists and companies in potential alternative NR crops. Genetic/metabolic pathway engineering approaches, to generate NR-enriched genotypes of alternative NR plants, are of great importance. However, although our knowledge of rubber biochemistry has significantly advanced, our current understanding of NR biosynthesis, the biosynthetic machinery and the molecular mechanisms involved remains incomplete. Two spatially separated metabolic pathways provide precursors for NR biosynthesis in plants and their genes and enzymes/complexes are quite well understood. In contrast, understanding of the proteins and genes involved in the final step(s)-the synthesis of the high molecular weight rubber polymer itself-is only now beginning to emerge. In this review, we provide a critical evaluation of recent research developments in NR biosynthesis, in vitro reconstitution, and the genetic and metabolic pathway engineering advances intended to improve NR content in plants, including H. brasiliensis, two other prospective alternative rubber crops, namely the rubber dandelion and guayule, and model species, such as lettuce. We describe a new model of the rubber transferase complex, which integrates these developments. In addition, we highlight the current challenges in NR biosynthesis research and future perspectives on metabolic pathway engineering of NR to speed alternative rubber crop commercial development.

Synergistic Mechanisms Underlie the Peroxide and Coagent Improvement of Natural-Rubber-Toughened Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) Mechanical Performance.

Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) is a promising bio-based and biodegradable thermoplastic with restricted industrial applications due to its brittleness and poor processability. Natural rubber (NR) has been used as a toughening agent, but further physical improvements are desired. In this study, rubber toughening efficiency was significantly improved through the synergistic use of a trifunctional acrylic coagent and an organic peroxide during reactive extrusion of PHBV and NR. The rheological, crystallization, thermal, morphological, and mechanical properties of PHBV/NR blends with 15% rubber loading were characterized. The peroxide and coagent synergistically crosslinked the rubber phase and grafted PHBV onto rubber backbones, leading to enhanced rubber modulus and cohesive strength as well as improved PHBV⁻rubber compatibility and blend homogeneity. Simultaneously, the peroxide⁻coagent treatment decreased PHBV crystallinity and crystal size and depressed peroxy-radical-caused PHBV degradation. The new PHBV/NR blends had a broader processing window, 75% better toughness (based on the notched impact strength data), and 100% better ductility (based on the tensile elongation data) than pristine PHBV. This new rubber-toughened PHBV material has balanced mechanical performance comparable to that of conventional thermoplastics and is suitable for a wide range of plastic applications.

Unusual subunits are directly involved in binding substrates for natural rubber biosynthesis in multiple plant species.

Rubber particles from rubber-producing plant species have many different species-specific proteins bound to their external monolayer biomembranes. To date, identification of those proteins directly involved in enzymatic catalysis of rubber polymerization has not been fully accomplished using solubilization, purification or reconstitution approaches. In an alternative approach, we use several tritiated photoaffinity-labeled benzophenone analogs of the allylic pyrophosphate substrates, required by rubber transferase (RT-ase) to initiate the synthesis of new rubber molecules, to identify the proteins involved in catalysis. Enzymatically-active rubber particles were purified from three phylogenetically-distant rubber producing species, Parthenium argentatum Gray, Hevea brasiliensis Muell. Arg, and Ficus elastica Roxb., each representing a different Superorder of the Dicotyledonae. Geranyl pyrophosphate with the benzophenone in the para position (Bz-GPP(p)) was the most active initiator of rubber biosynthesis in all three species. When rubber particles were exposed to ultra-violet radiation, 95% of RT-ase activity was eliminated in the presence of 50 µΜ Bz-GPP(p), compared to only 50% of activity in the absence of this analog. 3H-Bz-GPP(p) then was used to label and identify the proteins involved in substrate binding and these proteins were characterized electrophoretically. In all three species, three distinct proteins were labeled, one very large protein and two very small proteins, as follows: P. argentatum 287,000, 3,990, and 1,790 Da; H. brasiliensis 241,000, 3,650 and 1,600 Da; F. elastica 360,000, 3,900 and 1,800 Da. The isoelectric points of the P. argentatum proteins were 7.6 for the 287,000 Da, 10.4 for the 3,990 Da and 3.5 for the 1,790 Da proteins, and of the F. elastica proteins were 7.7 for the 360,000 Da, 6,0 for the 3,900 Da, and 11.0 for the 1,800 Da proteins. H. brasiliensis protein pI values were not determined. Additional analysis indicated that the three proteins are components of a membrane-bound complex and that the ratio of each small protein to the large one is 3:1, and the large protein exists as a dimer. Also, the large proteins are membrane bound whereas both small proteins are strongly associated with the large proteins, rather than to the rubber particle proteolipid membrane.

Analysis of the first Taraxacum kok-saghyz transcriptome reveals potential rubber yield related SNPs.

Taraxacum kok-saghyz (TK) is a potential alternative crop for natural rubber (NR) production, due to its high molecular weight rubber, short breeding cycle, and diverse environmental adaptation. However, improvements in rubber yield and agronomically relevant traits are still required before it can become a commercially-viable crop. An RNA-Seq based transcriptome was developed from a pool of roots from genotypes with high and low rubber yield. A total of 55,532 transcripts with lengths over 200 bp were de novo assembled. As many as 472 transcripts were significantly homologous to 49 out of 50 known plant putative rubber biosynthesis related genes. 158 transcripts were significantly differentially expressed between high rubber and low rubber genotypes. 21,036 SNPs were different in high and low rubber TK genotypes. Among these, 50 SNPs were found within 39 transcripts highly homologous to 49 publically-searched rubber biosynthesis related genes. 117 SNPs were located within 36 of the differentially expressed gene sequences. This comprehensive TK transcriptomic reference, and large set of SNPs including putative exonic markers associated with rubber related gene homologues and differentially expressed genes, provides a solid foundation for further genetic dissection of rubber related traits, comparative genomics and marker-assisted selection for the breeding of TK.

Improved axenic hydroponic whole plant propagation for rapid production of roots as transformation target tissue.

BACKGROUND: Plant roots are used as an efficient target tissue for plant transformation assays. In root propagable species transformed roots are able to regenerate into whole plants without the addition of exogenous hormones, thus avoiding somaclonal variation associated with many plant transformation protocols. Plants grown in soil or soilless solid medium have roots that tend to be extremely delicate and are difficult to sterilize in advance of plant transformation experiments. Axenic tissue culture plants grown on semi-solid media are slow to produce large amounts of biomass compared to plants grown in solution-based media. METHODS: Seeds were germinated and grown for 14 days on half-strength semi-solid Murashige and Skoog medium containing 1% sucrose. Seedlings were then transferred to Magenta™ GA7 vessels containing either liquid or semi-solid ½ MS medium with 0.25, 0.5, 1, 2 or 3% sucrose. In the hydroponics (liquid medium) treatments, expanded clay balls were used to anchor seedlings. Hydroponic vessels were fitted with a sterile air aeration hose and filled ¾ full (100 mL) with liquid ½ MS media. Liquid media were replaced after 7 days. All plants were grown under fluorescent lights for 14 days. RESULTS: We have developed an improved axenic hydroponic propagation system for producing large quantities of plant roots for use in transformation assays using Taraxacum kok-saghyz as a model for root propagable species. Plants grew significantly faster in liquid media than on solid media. Addition of sucrose from 0.25 to 2% was correlated with an increase in biomass accumulation in plants grown in liquid media. CONCLUSIONS: Our improved axenic hydroponic method yields sufficient quantities of roots for extensive plant transformation/molecular studies.

Chloroplast genome resources and molecular markers differentiate rubber dandelion species from weedy relatives.

BACKGROUND: Rubber dandelion (Taraxacum kok-saghyz, TK) is being developed as a domestic source of natural rubber to meet increasing global demand. However, the domestication of TK is complicated by its colocation with two weedy dandelion species, Taraxacum brevicorniculatum (TB) and the common dandelion (Taraxacum officinale, TO). TB is often present as a seed contaminant within TK accessions, while TO is a pandemic weed, which may have the potential to hybridize with TK. To discriminate these species at the molecular level, and facilitate gene flow studies between the potential rubber crop, TK, and its weedy relatives, we generated genomic and marker resources for these three dandelion species. RESULTS: Complete chloroplast genome sequences of TK (151,338 bp), TO (151,299 bp), and TB (151,282 bp) were obtained using the Illumina GAII and MiSeq platforms. Chloroplast sequences were analyzed and annotated for all the three species. Phylogenetic analysis within Asteraceae showed that TK has a closer genetic distance to TB than to TO and Taraxacum species were most closely related to lettuce (Lactuca sativa). By sequencing multiple genotypes for each species and testing variants using gel-based methods, four chloroplast Single Nucleotide Polymorphism (SNP) variants were found to be fixed between TK and TO in large populations, and between TB and TO. Additionally, Expressed Sequence Tag (EST) resources developed for TO and TK permitted the identification of five nuclear species-specific SNP markers. CONCLUSIONS: The availability of chloroplast genomes of these three dandelion species, as well as chloroplast and nuclear molecular markers, will provide a powerful genetic resource for germplasm differentiation and purification, and the study of potential gene flow among Taraxacum species.

Compartmentalized Metabolic Engineering for Artemisinin Biosynthesis and Effective Malaria Treatment by Oral Delivery of Plant Cells.

Artemisinin is highly effective against drug-resistant malarial parasites, which affects nearly half of the global population and kills >500 000 people each year. The primary cost of artemisinin is the very expensive process used to extract and purify the drug from Artemisia annua. Elimination of this apparently unnecessary step will make this potent antimalarial drug affordable to the global population living in endemic regions. Here we reported the oral delivery of a non-protein drug artemisinin biosynthesized (∼0.8 mg/g dry weight) at clinically meaningful levels in tobacco by engineering two metabolic pathways targeted to three different cellular compartments (chloroplast, nucleus, and mitochondria). The doubly transgenic lines showed a three-fold enhancement of isopentenyl pyrophosphate, and targeting AACPR, DBR2, and CYP71AV1 to chloroplasts resulted in higher expression and an efficient photo-oxidation of dihydroartemisinic acid to artemisinin. Partially purified extracts from the leaves of transgenic tobacco plants inhibited in vitro growth progression of Plasmodium falciparum-infected red blood cells. Oral feeding of whole intact plant cells bioencapsulating the artemisinin reduced the parasitemia levels in challenged mice in comparison with commercial drug. Such novel synergistic approaches should facilitate low-cost production and delivery of artemisinin and other drugs through metabolic engineering of edible plants.