Rhizophagus irregularis and biochar can synergistically improve the physiological characteristics of saline-alkali resistance of switchgrass.

Inoculation of arbuscular mycorrhizal fungi (AMF) or biochar (BC) application can improve photosynthesis and promote plant growth under saline-alkali stress. However, little is known about the effects of the two combined on growth and physiological characteristics of switchgrass under saline-alkali stress. This study examined the effects of four treatments: (1) no AMF inoculation and no biochar addition (control), (2) biochar (BC) alone, (3) AMF (Rhizophagus irregularis, Ri) alone, and (4) the combination of both (BC+Ri) on the plant biomass, antioxidant enzymes, chlorophyll, and photosynthetic parameters of switchgrass under saline-alkali stress. The results showed that the above-ground, belowground and total biomass of switchgrass in the BC+Ri treatment group was significantly higher (+136.7%, 120.2% and 132.4%, respectively) than in other treatments compared with Control. BC+Ri treatment significantly increased plant leaves' relative chlorophyll content, antioxidant enzyme activity, and photosynthesis parameters. It is worth noting that the transpiration rate, stomatal conductance, net photosynthetic rate, PSII efficiency and other photosynthetic-related indexes of the BC+Ri treatment group were the highest (38% to 54% higher than other treatments). The fitting results of light response and CO2 response curves showed that the light saturation point, light compensation point, maximum carboxylation rate and maximum electron transfer rate of switchgrass in the Ri+BC treatment group were the highest. In conclusion, biochar combined with Ri has potential beneficial effects on promoting switchgrass growth under saline-alkali stress and improving the activity of antioxidant enzymes and photosynthetic characteristics of plants.

Improved node culture methods for rapid vegetative propagation of switchgrass (Panicum virgatum L.).

BACKGROUND: Switchgrass (Panicum virgatum L.) is an important bioenergy and forage crop. The outcrossing nature of switchgrass makes it infeasible to maintain a genotype through sexual propagation. Current asexual propagation protocols in switchgrass have various limitations. An easy and highly-efficient vegetative propagation method is needed to propagate large natural collections of switchgrass genotypes for genome-wide association studies (GWAS). RESULTS: Micropropagation by node culture was found to be a rapid method for vegetative propagation of switchgrass. Bacterial and fungal contamination during node culture is a major cause for cultural failure. Adding the biocide, Plant Preservative Mixture (PPM, 0.2%), and the fungicide, Benomyl (5 mg/l), in the incubation solution after surface sterilization and in the culture medium significantly decreased bacterial and fungal contamination. In addition, "shoot trimming" before subculture had a positive effect on shoot multiplication for most genotypes tested. Using the optimized node culture procedure, we successfully propagated 330 genotypes from a switchgrass GWAS panel in three separate experiments. Large variations in shoot induction efficiency and shoot growth were observed among genotypes. Separately, we developed an in planta node culture method by stimulating the growth of aerial axillary buds into shoots directly on the parent plants, through which rooted plants can be generated within 6 weeks. By circumventing the tissue culture step and avoiding application of exterior hormones, the in planta node culture method is labor- and cost-efficient, easy to master, and has a high success rate. Plants generated by the in planta node culture method are similar to seedlings and can be used directly for various experiments. CONCLUSIONS: In this study, we optimized a switchgrass node culture protocol by minimizing bacterial and fungal contamination and increasing shoot multiplication. With this improved protocol, we successfully propagated three quarters of the genotypes in a diverse switchgrass GWAS panel. Furthermore, we established a novel and high-throughput in planta node culture method. Together, these methods provide better options for researchers to accelerate vegetative propagation of switchgrass.

Sulfur supply reduces barium toxicity in Tanzania guinea grass (Panicum maximum) by inducing antioxidant enzymes and proline metabolism.

Sulfur (S) can play essential roles in protecting plants against abiotic stress, including heavy metal toxicity. However, the effect of this nutrient on plants exposed to barium (Ba) is still unknown. This study was designed to evaluate the S supply on oxidative stress and the antioxidant system of Tanzania guinea grass under exposure to Ba, grown in a nutrient solution under greenhouse conditions. It was studied the influence of S/Ba combinations in nutrient solution on oxidative stress indicators (hydrogen peroxide, malondialdehyde, and proline) and antioxidant enzyme activities (superoxide dismutase, catalase, ascorbate peroxidase, guaiacol peroxidase, and glutathione reductase). The treatments consisted in thirteen S/Ba combinations in the nutrient solution (0.1/0.0; 0.1/5.0; 0.1/20.0; 1.0/2.5; 1.0/10.0; 1.9/0.0 - control; 1.9/5.0; 1.9/20.0; 2.8/2.5; 2.8/10.0; 3.7/0.0; 3.7/5.0 and 3.7/20.0 mM of S and Ba, respectively). The plants were grown for two growth periods, which consisted of fourteen days of S supply and the eight days of Ba exposure each one. The severe S deficiency decreased the superoxide dismutase activity, regardless of Ba exposure in recently expanded leaves and culms plus sheaths. However, supplemental S supply (above 1.9 mM S, which corresponds to S supply adequate to plant growth) it improved the superoxide dismutase activity in these tissues under high Ba concentrations. Conversely, the severe S deficiency increased the activities of catalase, ascorbate peroxidase, and glutathione reductase in grass leaves slightly, without Ba exposure influence. It was observed that the supplemental S supply also induced the guaiacol peroxidase activity and proline production in culms plus sheaths under high Ba rates, showing values until 2.5 and 3.1 folds higher than the control treatment, respectively. In plants under exposure to 20.0 mM Ba, the supplemental S supply decreased the malondialdehyde content in culms plus sheaths in 17% compared to 1.9 mM S. These results indicate that supplemental S supply can mitigate Ba toxicity in Tanzania guinea grass, mainly by improving superoxide dismutase and guaiacol peroxidase activities, and proline metabolism.

Botanical and chemical fungicides in the treatment of commercial seeds of Brachiaria brizantha and Panicum maximum.

Brazil is the largest producer, consumer, and exporter of forage seeds in the world. However, the high incidence of pathogens threatens the seed production system, resulting in decreases in production and quality. Currently, the emphasis has been placed on the use of alternative products as a way of maintaining productivity and environmental sustainability. Thus, the objective of this study was to evaluate the mycoflora associated with the commercial seeds of Brachiaria brizantha and Panicum maximum treated with chemical and alternative fungicide. To test with chemical and alternative treatments, seeds were subjected to fluazinam + thiophanate-methyl, fludioxonil + metalaxyl-M, noni essential oil, rosemary pepper essential oil, sulfuric acid. There was also an untreated control. The most efficient products in the control of incident fungi were, in decreasing order, fluazinam + thiophanate-methyl, fludioxonil + metalaxyl-M, noni essential oil, rosemary pepper essential oil, and sulfuric acid. Fungicides mainly controlled the genera Alternaria, Aspergillus, Bipolaris, Chaetomium, Cladosporium, Colletotrichum, Curvularia, Fusarium, Penicillium, Phoma and Rhizopus. Essential oils showed positive effects in controlling Bipolaris, Curvularia, and Fusarium. Sulfuric acid reduced the incidence of Bipolaris, Colletotrichum, Curvularia, Fusarium, Phoma, and Rhizopus. Seed treatment is one of the promising strategies for obtaining seeds of a healthy quality. As a result, the fungicides and botanical components listed above were promising in reducing fungi associated with forage seeds and can be used for seed treatments and in the development of new products with a natural base through essential oils.

Nanoparticle titanium dioxide affects the growth and microRNA expression of switchgrass (Panicum virgatum).

Nanoparticle TiO2 is a common chemical used in daily life. As increasing usage of TiO2, it is becoming a potentially dangerous contaminant to the environment. However, the impact of TiO2 is not well understood. In this paper, switchgrass was employed to investigate the impacts of nanoparticle TiO2 on plant growth and development as well as the potential impact on the expression of microRNAs (miRNAs). TiO2 significantly affected switchgrass seed generation as well as plant growth and development in a dose-dependent manner. Particularly, TiO2 significantly inhibited root development. miRNA expressions were also significantly altered. Nanoparticle TiO2 may regulate plant development through controlling the expression of certain miRNAs. Among the 16 tested miRNAs, the expression of some miRNAs, such as miR390 and miR399 was increased with increasing TiO2 concentrations; the expression of some miRNAs, such as miR169 was decreased with increasing TiO2 concentrations; the other miRNAs show different expression patterns.

Sulfamethoxazole sorption by cattail and switchgrass roots.

Sorption to roots is one of several mechanisms by which plant-assisted attenuation of antibiotics can be achieved. The objectives of this study were to (1) evaluate the sorption of sulfamethoxazole (SMX) by cattail and switchgrass roots, (2) determine the kinetics of SMX sorption by cattail and switchgrass roots, and (3) characterize the temperature-dependency of SMX sorption. A batch sorption experiment was conducted to measure SMX sorption by roots of the two plant species using five initial antibiotic concentrations (2.5, 5, 10, 15, and 20 µg L-1) and eight sampling times (0, 0.5, 1, 2, 4, 8, 12, and 24 h). Another batch experiment was conducted at three temperatures (5, 15, and 25 °C) to determine the effect of temperature on sorption kinetics. SMX sorption followed pseudo-second-order kinetics. The pseudo-second-order rate constant (k2) decreased with increasing temperature for both plant species. The rate constant followed the order: 5 °C = 15 °C > 25 °C for cattail and 5 °C > 15 °C = 25 °C for switchgrass. Results from this study show that switchgrass roots are more effective than cattail roots in the removal of SMX. Therefore, the use of switchgrass in systems designed for phytoremediation of contaminants might also provide an efficient removal of some antibiotics.

Transcriptome-based analyses of phosphite-mediated suppression of rust pathogens Puccinia emaculata and Phakopsora pachyrhizi and functional characterization of selected fungal target genes.

Phosphite (Phi) is used commercially to manage diseases mainly caused by oomycetes, primarily due to its low cost compared with other fungicides and its persistent control of oomycetous pathogens. We explored the use of Phi in controlling the fungal pathogens Puccinia emaculata and Phakopsora pachyrhizi, the causal agents of switchgrass rust and Asian soybean rust, respectively. Phi primes host defenses and efficiently inhibits the growth of P. emaculata, P. pachyrhizi and several other fungal pathogens tested. To understand these Phi-mediated effects, a detailed molecular analysis was undertaken in both the host and the pathogen. Transcriptomic studies in switchgrass revealed that Phi activates plant defense signaling as early as 1 h after application by increasing the expression of several cytoplasmic and membrane receptor-like kinases and defense-related genes within 24 h of application. Unlike in oomycetes, RNA sequencing of P. emaculata and P. pachyrhizi did not exhibit Phi-mediated retardation of cell wall biosynthesis. The genes with reduced expression in either or both rust fungi belonged to functional categories such as ribosomal protein, actin, RNA-dependent RNA polymerase, and aldehyde dehydrogenase. A few P. emaculata genes that had reduced expression upon Phi treatment were further characterized. Application of double-stranded RNAs specific to P. emaculata genes encoding glutamate N-acetyltransferase and cystathionine gamma-synthase to switchgrass leaves resulted in reduced disease severity upon P. emaculata inoculation, suggesting their role in pathogen survival and/or pathogenesis.

Effects of water availability and UV radiation on silicon accumulation in the C4 crop proso millet.

Proso millet (Panicum miliaceum L.) is an annual thermophilic, drought-resistant, short-season C4 grass that is suitable for growing under the present changing meteorological conditions. However, water shortage can reduce plant production and hinder mineral nutrition in plants, including silicon, which is of crucial importance for grasses. Ultraviolet radiation can mitigate the impacts of water shortage, and therefore we examined the effects of moderate water shortage and reduced ultraviolet radiation on different leaf traits, including leaf levels of silicon and other elements, and plant biomass production. Moderate water shortage and ambient ultraviolet radiation did not affect the contents of photosynthetic pigments, while they reduced stomata density. Water shortage significantly decreased leaf light reflectance in the ultraviolet and violet regions. Leaf silicon, calcium, phosphorus, and sulphur levels were significantly lower with reduced water availability and significantly higher with reduced ultraviolet radiation. Leaf silicon levels ranged from 1.5% to 2.5% of leaf dry mass, with leaf calcium levels from 0.3% to 0.6%. Except for chlorine and potassium, the levels of these elements in the leaves were significantly positively related (p ≤ 0.05) to soil moisture levels. Water availability, but not ultraviolet radiation, significantly reduced living leaf biomass.

Partial replacement of nitrate by ammonium increases photosynthesis and reduces oxidative stress in tanzania guinea grass exposed to cadmium.

In order to grow and effectively uptake and accumulate cadmium (Cd), plants used for phytoextraction have to cope with toxicity, which may be influenced by the supply of nitrate (NO3-) and ammonium (NH4+). Thus, we evaluated the effect of these nitrogen forms on the photosynthetic and antioxidant enzyme activities of Panicum maximum cv. Tanzania (tanzania guinea grass) under Cd stress. Plants were grown in nutrient solution under greenhouse conditions and subjected to a 3 × 3 factorial experiment. They were supplied with three NO3-/NH4+ ratios (100/0, 70/30 and 50/50) and exposed to three Cd rates (0.0, 0.5 and 1.0 mmol L-1), being arranged in a randomized complete block design with three replications. Gas exchange parameters, oxidative stress indicators, proline concentration and antioxidant enzyme activities were studied. Exposure to Cd reduced photosynthesis by causing stomatal closure and impairing electron transport. However, the simultaneous supply of NO3- and NH4+, particularly at a 50/50 ratio, restored gas exchange and improved the function of photosystem II, increasing the photosynthetic capacity of the grass. Plants grown with 50/50 showed reduced lipid peroxidation along with increased proline synthesis. Moreover, this NO3-/NH4+ ratio increased the tolerance of tanzania guinea grass to Cd by inducing high superoxide dismutase and glutathione reductase activities in shoots and roots, respectively, maintaining cellular homeostasis and reducing oxidative stress. The negative effects of Cd on photosynthesis and on the balance between oxidants and antioxidants are attenuated by the partial replacement of NO3- by NH4+ in the nutrient solution.

Nitrate and ammonium proportion plays a key role in copper phytoextraction, improving the antioxidant defense in Tanzania guinea grass.

Various nitrate and ammonium proportions (NO3-/NH4+) in the growth media can increase metal phytoextraction compared to supplying solely NO3-. However, there are no studies showing these effects in plants under copper (Cu) contamination as well as their consequences in plant stress tolerance. The objective was to evaluate the effect of NO3-/NH4+ proportions in Cu phytoextraction by Panicum maximum cv. Tanzania and its consequence in the oxidative stress, photosynthesis, and antioxidant system under Cu stress. The experiment was carried out in a randomized complete block design, by using a 3 × 4 factorial with six replications. Three NO3-/NH4+ proportions (100/0, 70/30, and 50/50) were combined with four Cu rates (0.3, 250, 500, and 1000 µmol L-1) in the nutrient solution. It was found that the largest Cu accumulation in the shoots occurred at the first harvest of the plants supplied with 70/30 NO3-/NH4+ and Cu 1000 µmol L-1. Such plants also displayed high concentrations of proline in the shoots as well as high superoxide dismutase activity in the roots. Malondialdehyde concentration was high in the plant parts at the Cu rate of 1000 µmol L-1. Hence, transpiration rates, stomatal conductance, quantum efficiency of photosystem II, electron transport rate, and net photosynthesis were all low at the Cu rate of 1000 µmol L-1. Catalase, guaiacol peroxidase, ascorbate peroxidase, and glutathione reductase activities in the roots were high when plants were exposed to Cu 1000 µmol L-1. In conclusion, the combination of NO3- with NH4+ increases copper phytoextraction that causes oxidative stress, but also favors the antioxidant system of Tanzania guinea grass in attempt to tolerate such stress.

Ozone phytotoxicity to Panicum maximum and Cenchrus ciliaris at Indo-Gangetic plains: an assessment of antioxidative defense and growth responses.

Two common tropical grassland species, Panicum maximum Jacq. (Guinea grass) and Cenchrus ciliaris (Buffel grass) of Indo-Gangetic plains were assessed for their responses under future level of O3 (ambient +30 ppb) using open top chambers. Plants were assessed for foliar injuries, pigments, growth, biomass accumulation, histochemical localization of reactive oxygen species (ROS), antioxidant defense system and ROS scavenging activities at two stages. Foliar injuries were noticed at an early stage in P. maximum compared to C. ciliaris. Significant reductions were observed in total chlorophyll, growth and total biomass in both species. Significant increases in contents of melondialdehyde and ascorbic acid in P. maximum while total phenolics and thiols in C. ciliaris were found. Histochemical analysis showed more production of superoxide radicals and hydrogen peroxide in leaf tissues of P. maximum compared to C. ciliaris. It can be concluded that higher level of primary antioxidants (total phenolics and thiols) along with superoxide dismutase and ascorbate peroxidase scavenged O3 effectively in C. ciliaris causing less reduction of biomass which is used as a feed for cattles. In P. maximum, more photosynthates were allocated for defense, leading to higher reduction in total biomass compared to C. ciliaris. The leaf area ratio was higher in P. maximum compared to C. ciliaris under elevated O3. The study further suggests higher susceptibility of P. maximum compared to C. ciliaris under future level of O3 exposure.

Overexpression of gene encoding the key enzyme involved in proline-biosynthesis (PuP5CS) to improve salt tolerance in switchgrass (Panicum virgatum L.).

KEY MESSAGE: Genetic improvement through overexpressing PuP5CS in switchgrass is feasible for enhancing plant salt stress tolerance. Switchgrass (Panicum virgatum L.) has developed into a dedicated bioenergy crop. To improve the biomass production of switchgrass grown on different types of soil, abiotic stress tolerance traits are considered for its genetic improvement. Proline accumulation is a widespread response when plants are subjected to abiotic stresses such as drought, cold and salinity. In plants, P5CS gene encodes the key regulatory enzyme that plays a crucial role in proline biosynthesis. Here, we introduced the PuP5CS gene (from Puccinellia chinampoensis) into switchgrass by Agrobacterium-mediated transformation. Transgenic lines overexpressing the PuP5CS gene showed phenotypic advantages, in leaf width, internode diameter, internode length, tiller numbers and precocious flowering under normal conditions, and the transgenic lines displayed better regenerative capacity in forming more tillers after harvest. Moreover, the PuP5CS gene enhanced the salt tolerance of transgenic switchgrass by altering a wide range of physiological responses. In accordance with the physiological results, histological analysis of cross sections through the leaf blade showed that the areas of bulliform cells and bundle sheath cells were significantly increased in PuP5CS-overexpressing leaves. The expression levels of ROS scavenging-associated genes in transgenic plants were higher than in control plants under salt stress. The results show that genetic improvement through overexpressing PuP5CS in switchgrass is feasible for enhancing plant stress tolerance.

Transcriptome analysis of Cd-treated switchgrass root revealed novel transcripts and the importance of HSF/HSP network in switchgrass Cd tolerance.

KEY MESSAGE: Transcriptome analysis of Cd-treated switchgrass roots not only revealed novel switchgrass transcripts and gene structures but also highlighted the indispensable role of HSF/HSP network in switchgrass Cd tolerance. Switchgrass (Panicum virgatum L.), a C4 perennial tall grass, can be used for revegetation of Cd-contaminated soil. In the present study, a comparative transcriptome analysis of Cd-treated switchgrass roots was conducted. The result revealed a total of 462 novel transcripts and refined gene structures of 2337 transcripts. KEGG pathway and Gene Ontology analyses of the differentially expressed genes (DEGs) suggested that activation of redox homeostasis and oxidation-related metabolic processes were the primary response to Cd stress in switchgrass roots. In particular, 21 out of 23 differentially expressed shock transcription factor genes (HSFs), and 22 out of 23 differentially expressed heat shock protein genes (HSPs) had increased expression levels after Cd treatment. Furthermore, over-expressing one HSP-encoding gene in Arabidopsis significantly improved plant Cd tolerance. The result highlighted the activation of the redox homeostasis and the involvement of the HSF/HSP network in re-establishing normal protein conformation and thus cellular homeostasis in switchgrass upon Cd stress. These DEGs, especially those of the HSF/HSP network, could be used as candidate genes for further functional studies toward improved plant Cd tolerance in switchgrass and related species.

Enhanced Cold Tolerance and Tillering in Switchgrass (Panicum virgatum L.) by Heterologous Expression of Osa-miR393a.

The microRNA393 (miR393) family is one of the conserved miRNA families in the plant kingdom. MiR393 was reported to regulate rice tillering and abiotic stress resistance positively through an auxin signaling pathway. However, little is known about the function of miR393 in switchgrass (Panicum virgatum L.), an important bioenergy C4 grass plant. We tested the expression level of miR393 and its four putative target genes (PvAFB1, PvAFB2, PvAFB3 and PvTIR1) in switchgrass, and found that these genes all responded to cold stress and exogenous 1-naphthaleneacetic acid (NAA) treatment. To investigate the function of miR393 in switchgrass, we enhanced miR393 expression by introducing an Osa-miR393a gene into switchgrass. The results showed that cold tolerance of the transgenic T0 and T1 generation plants was highly improved. Cold tolerance-related genes PvCOR47, PvICE1 and PvRAV1 were negatively regulated by exogenous NAA, and the expression of these genes was significantly higher in transgenic plants than in wild-type plants. The transgenic T1 seedlings were more tolerant to exogenous NAA treatment, accumulating less H2O2 after cold treatments. It was also observed that the miR393/target module regulates cold tolerance responses in Arabidopsis. In addition, transgenic plants overexpressing miR393 had significantly more tillers and higher biomass yield per plant in greenhouse and field tests. Forage quality analyses revealed that the soluble sugar contents of transgenic plants were increased markedly. Overall, the results suggested that overexpression of miR393 improved cold tolerance and tillering of switchgrass through regulation of auxin signaling transduction.

Allelopathic effects of exogenous phenylalanine: a comparison of four monocot species.

MAIN CONCLUSION: Exogenous phenylalanine stunted annual ryegrass but not switchgrass or winter grain rye, with deuterium incorporation up to 3% from phenyalanine-d 8 . Toxicity to duckweed varied with illumination intensity and glucose uptake. Isotopic labeling of biomolecules through biosynthesis from deuterated precursors has successfully been employed for both structural studies and metabolic analysis. Phenylalanine is the precursor of many products synthesized by plants, including the monolignols used for synthesis of lignin. Possible allelochemical effects of phenylalanine have not been reported, although its deamination product cinnamic acid is known to have deleterious effects on root elongation and growth of several plant species. The effects of phenylalanine and its deuterated analog phenylalanine-d 8 added to growth media were studied for annual ryegrass (Lolium multiflorum), winter grain rye (Secale cereale), and switchgrass (Panicum virgatum) cultivated under hydroponic conditions. Growth of annual ryegrass was inhibited by phenylalanine while switchgrass and rye were not significantly affected. Growth was less affected by deuterated phenylalanine-d 8 than by its protiated counterpart, which may be a typical deuterium kinetic isotope effect resulting in slower enzymatic reaction rates. Deuterium incorporation levels of 2-3% were achieved in biomass of switchgrass and annual ryegrass. Both protiated and deuterated phenylalanine were moderately toxic (IC25 values 0.6 and 0.8 mM, respectively) to duckweed (Lemna minor) grown using a 12 h diurnal cycle under photoautotrophic conditions. A significant increase in toxicity, greater for the deuterated form, was noted when duckweed was grown under higher intensity, full spectrum illumination with a metal halide lamp compared to fluorescent plant growth lamps emitting in the blue and red spectral regions. Supplementation with glucose increased toxicity of phenylalanine consistent with synergy between hexose and amino acid uptake that has been reported for duckweed.

Expression in grasses of multiple transgenes for degradation of munitions compounds on live-fire training ranges.

The deposition of toxic munitions compounds, such as hexahydro-1, 3, 5-trinitro-1, 3, 5-triazine (RDX), on soils around targets in live-fire training ranges is an important source of groundwater contamination. Plants take up RDX but do not significantly degrade it. Reported here is the transformation of two perennial grass species, switchgrass (Panicum virgatum) and creeping bentgrass (Agrostis stolonifera), with the genes for degradation of RDX. These species possess a number of agronomic traits making them well equipped for the uptake and removal of RDX from root zone leachates. Transformation vectors were constructed with xplA and xplB, which confer the ability to degrade RDX, and nfsI, which encodes a nitroreductase for the detoxification of the co-contaminating explosive 2, 4, 6-trinitrotoluene (TNT). The vectors were transformed into the grass species using Agrobacterium tumefaciens infection. All transformed grass lines showing high transgene expression levels removed significantly more RDX from hydroponic solutions and retained significantly less RDX in their leaf tissues than wild-type plants. Soil columns planted with the best-performing switchgrass line were able to prevent leaching of RDX through a 0.5-m root zone. These plants represent a promising plant biotechnology to sustainably remove RDX from training range soil, thus preventing contamination of groundwater.

Toxicity of coal fly ash (CFA) and toxicological response of switchgrass in mycorrhiza-mediated CFA-soil admixtures.

Increasing support for the use of Coal fly ash (CFA) in agriculture has necessitated a better understanding of the effects of the CFA in various cropping schemes. Experiments were conducted to assess mutagenic response of a mutant strain of Salmonella enterica serovar Typhimurium (TA100) to varying concentrations of CFA-water extracts, determine oxidative stress in switchgrass (Panicum virgatum L.) at varying levels of CFA-soil admixtures, and evaluate mycorrhiza-mediated modulation of oxidative stress responses of CFA-grown switchgrass. The TA100 exposed to 0%, 5%, 10%, 15%, 20% and 25% (w/v) CFA-water extracts elicited significant (p < 0.05) mutagenic responses at 20% and 25% extract levels but not below the 15% level. In greenhouse pot experiment, CFA-soil admixtures at 7.5% and 15% (w/w) significantly (p < 0.05) decreased the activities of superoxide dismutase (SOD) by 19.1% and 28.3% respectively, compared to control soil (0% w/w CFA/soil). Under the same conditions, activities of glutathione peroxidase (GPx) decreased by 75.9% and 66.9%. In contrast to the antioxidant enzyme activities, levels of malondialdehyde (MDA) an indicator of lipid peroxidation increased significantly (p < 0.05) by 30.49% and 38.38%. Inoculation of 7.5% and 15% CFA-soil admixtures with arbuscular mycorrhizal fungi (AMF), Rhizophaga clarus enhanced the activities of both SOD and GPx in the switchgrass, while it significantly (p < 0.05) reduced the levels of MDA. The study demonstrated that incorporation of CFA (at concentrations considered to be non-mutagenic against TA100) as soil amendment produced concentration-dependent oxidative stress responses in switchgrass; however, inoculation of the CFA-soil admixtures with AMF significantly modulated the oxidative stress responses.

Availability and toxicity of cadmium to forage grasses grown in contaminated soil.

It is important to know the mechanisms for forage development, especially those related to the tolerance of potentially toxic elements, when considering their use in phytoremediation in heavy metal contaminated areas. In this study, we evaluated plant growth, concentration, and the availability of cadmium (Cd) for forage grasses (Panicum maximum Jacq. cv. Aruana and cv. Tanzânia; Brachiaria decumbens cv. Basilisk; Brachiaria brizantha cv. Xaraés and cv. Marandu) cultivated in Cd contaminated soils. The experiments were performed under greenhouse conditions over a 90-day evaluation period, and the Cd rates were 2, 4, and 12 mg/kg of soil. The relative growth rate of the forage grasses decreased as Cd rates increased, and the following descending order of susceptibility was observed: Marandu > Xaraés > Aruana > Tanzânia > Basilisk, with regard to phytotoxicity in these plants. The forage Cd concentration increased in line with increases in the Cd rates. Cd contents extracted by Mehlich-1 and by diethylenetriaminepentaacetic acid presented high positive correlation with forage relative growth. The forage plants did not block Cd entry into the food chain because they were not capable of limiting Cd absorption.

Inside out: high-efficiency plant regeneration and Agrobacterium-mediated transformation of upland and lowland switchgrass cultivars.

KEY MESSAGE: Selection of pre-embryogenic callus from a core structure from mature seed-derived callus is the key for high-efficiency plant regeneration and transformation of switchgrass different cultivars. Switchgrass (Panicum virgatum L.) has been identified as a dedicated biofuel crop. For its trait improvement through biotechnological approaches, we have developed a highly efficient plant regeneration and genetic transformation protocol for both lowland and upland cultivars. We identified and separated a pre-embryogenic "core" structure from the seed-derived callus, which often leads to development of highly regenerative type II calluses. From the type II callus, plant regeneration rate of lowland cultivars Alamo and Performer reaches 95%, and upland cultivars Blackwell and Dacotah, 50 and 76%, respectively. The type II callus was also amenable for Agrobacterium-mediated transformation. Transformation efficiency of 72.8% was achieved for lowland cultivar Alamo, and 8.0% for upland cultivar Dacotah. PCR, Southern blot and GUS staining assays were performed to verify the transgenic events. High regenerative callus lines could be established in 3 months, and transgenic plants could be obtained in 2 months after Agrobacterium infection. To our knowledge, this is the first report on successful plant regeneration and recovery of transgenic plants from upland switchgrass cultivars by Agrobacterium-mediated transformation. The method presented here could be helpful in breaking through the bottleneck of regeneration and transformation of lowland and upland switchgrass cultivars and probably other recalcitrant grass crops.

Induced Phytoextraction of Lead Through Chemical Manipulation of Switchgrass and Corn; Role of Iron Supplement.

The effects of combined chemical application of benomyl, ethylenedianinetetraacetate (EDTA), and iron (Fe) (foliar and root) on lead (Pb) phytoextraction by switchgrass (Panicum virgatum) and corn (Zea mays) was examined. Switchgrass was grown in Pb-contaminated urban topsoil with the following treatments: (C) Control, (B) benomyl, (E) EDTA, (F) foliar-Fe, (BE) benomyl + EDTA, (BF) benomyl + foliar-Fe, (FE) foliar-Fe + EDTA, (BFE) benomyl + foliar-Fe + EDTA. Corn was grown in sand-culture supplemented with Pb (500 mg kg(-1)) with the following treatments: (C) control, (B) benomyl, (E) EDTA, (F) root-Fe, (BE) benomyl + EDTA, (BF) benomyl + root-Fe, (FE) root-iron + EDTA, and, (BFE) benomyl + root-Fe + EDTA. All treatments were replicated three times and pots were arranged in a completely randomized design. Plants were analyzed for element concentration (Fe, Zn, P, and Pb) using either inductively coupled plasma (argon) atomic emission spectroscopy (ICP-AES) or graphite furnace atomic absorption spectrometer. Iron supplementation (foliar and root) affected Pb-translocation in plants. Foliar-Fe treatment increased translocation ratio of Pb (TF-Pb) significantly compared to other treatments with the exception of plants treated with benomyl and BF. Root-Fe treatment in combination with EDTA (FE) increased TF-Pb significantly compared to other treatments. Phytoextraction was improved by the combined chemical application; plants treated with BFE treatment increased Pb-total-phytoextraction by 424% compared to Control plants.