Zinc accumulation,photosynthetic gas exchange,and chlorophyll a fluorescence in Zn-stressed Miscanthus × giganteus plants

Photosynthetica - Accumulation and distribution of zinc within Miscanthus × giganteus plants grown on elevated Zn concentrations and their photosynthetic performance were investigated. High...     

Failure of androgenesis in Miscanthus × giganteus in vitro culture of cytologically unbalanced microspores

Miscanthus × giganteus is a popular energy crop, which due to its hybrid origin is only vegetatively reproduced. Asexual embryogenesis in anther and microspore culture leading to double haploids production could allow to regain the ability for sexual reproduction and to increase the biodiversity of the species. Therefore, the goal of this paper was to investigate the requirements of androgenesis in Miscanthus. The standard protocols used for monocotyledonous plants were applied with many modifications regarding the developmental stage of the explants at the time of culture initiation, stress treatment applied to panicles and isolated anthers as well as various chemical and physical parameters of in vitro culture conditions. Our results indicated that the induction of androgenesis in M. × giganteus is possible. However, the very low efficiency of the process and the lack of regeneration ability of the androgenic structures presently prevent the use of this technique.     

Changes in Phenolics Distribution After Chemical Pretreatment and Enzymatic Conversion of Miscanthus × giganteus Internode

In addition to lignin, grass cell walls are characterized by the presence of hydroxycinnamic acids that play a significant role in cross-linking polymers into a cohesive network, and pretreatments are required to overcome the recalcitrance of lignocelluloses prior to enzymatic bioconversion of polysaccharides. The effects of dilute acid and ammonium hydroxide pretreatments were studied on the chemical composition and enzymatic saccharification of Miscanthus internodes fragments. The hydroxycinnamic acid content was reduced after both pretreatments, while lignin got enriched in condensed linked structures. In addition, dilute acid pretreatment was effective in decreasing xylan content of Miscanthus, while ammonia treatment induced a marked swelling effect on the cell walls of parenchyma, vascular sclerenchyma, and epidermal sclerenchyma. The phenol distribution at the cell level was estimated using UV transmission microspectrophotometry. Internode cell walls displayed different UV spectra according to the cell type. However, the secondary cell walls had similar UV spectra after pretreatment, whereas spectra recorded at the cell corner region displayed variations according to cell type and pretreatment. Acid pretreatment was more efficient than ammonia to improve the conversion of polysaccharides by a Trichoderma cellulolytic cocktail. Although pretreatments achieved moderate saccharification yields, the secondary cell walls were altered at some pit regions of the vascular sclerenchyma whereas parenchyma appeared recalcitrant. Variations in the UV spectra of enzyme-digested cell walls suggest pretreatment-dependent heterogeneity of the phenolic distribution in the more recalcitrant cell walls.     

Genomic and small RNA sequencing of Miscanthus × giganteus shows the utility of sorghum as a reference genome sequence for Andropogoneae grasses

Background

Miscanthus × giganteus (Mxg) is a perennial grass that produces superior biomass yields in temperate environments. The essentially uncharacterized triploid genome (3n = 57, x = 19) of Mxg is likely critical for the rapid growth of this vegetatively propagated interspecific hybrid.

Results

A survey of the complex Mxg genome was conducted using 454 pyrosequencing of genomic DNA and Illumina sequencing-by-synthesis of small RNA. We found that the coding fraction of the Mxg genome has a high level of sequence identity to that of other grasses. Highly repetitive sequences representing the great majority of the Mxg genome were predicted using non-cognate assembly for de novo repeat detection. Twelve abundant families of repeat were observed, with those related to either transposons or centromeric repeats likely to comprise over 95% of the genome. Comparisons of abundant repeat sequences to a small RNA survey of three Mxg organs (leaf, rhizome, inflorescence) revealed that the majority of observed 24-nucleotide small RNAs are derived from these repetitive sequences. We show that high-copy-number repeats match more of the small RNA, even when the amount of the repeat sequence in the genome is accounted for.

Conclusions

We show that major repeats are present within the triploid Mxg genome and are actively producing small RNAs. We also confirm the hypothesized origins of Mxg, and suggest that while the repeat content of Mxg differs from sorghum, the sorghum genome is likely to be of utility in the assembly of a gene-space sequence of Mxg.     

Improving micropropagation of Mentha × piperita L. using a liquid culture system

In the current study, in vitro shoot proliferation and plant regeneration of Mentha × piperita L. (peppermint) cultivar ‘Black Mitcham’ was compared in semi-solid and liquid culture systems. Shoot tips from field-grown plants were used as explants to study shoot proliferation response on either Murashige and Skoog (MS) or Chee and Pool (C2D) medium containing varying levels of 6-benzylaminopurine (BAP), kinetin, and 6-γ,γ-dimethylallyl aminopurine (2iP). Differences in leaf ultrastructure and antioxidant capacity of greenhouse-grown and micropropagation-derived plants were studied to identify potential changes occurring during in vitro culture. Among the various media treatments tested, the maximum number of shoots was produced on the C2D medium with 4.0 μM BAP (40.7) followed by the MS medium with 4.0 μM BAP (32.2). Among the rooting treatments, shoots on the MS medium with 1.0 μM indole-3-butyric acid (IBA) produced the maximum number of roots (14.4). The number of shoots produced in Liquid Lab Rocker® (LLR) vessels containing liquid C2D medium with BAP (103.4) was significantly higher than that produced on semi-solid medium (40.7). No differences were observed in the leaf ultrastructure and antioxidant capacity of leaf extracts obtained from greenhouse-grown and micropropagation-derived plants. The study indicates that the liquid culture system under the described conditions can enhance peppermint micropropagation, with plant material being potentially valuable for use in herbal supplements and essential oil production.

     

Biomass Reducing Potential and Prospective Fungicide Control of a New Leaf Blight of Miscanthus × giganteus Caused by Leptosphaerulina chartarum

During a multistate survey (2009) of diseases of perennial grasses under consideration as biofuel feedstocks, a new leaf blight of Miscanthus?×?giganteus caused by Leptosphaerulina chartarum was observed on 100 % of the plants evaluated in research plots near Lexington, Kentucky. A greenhouse study was conducted to evaluate the effect of L. chartarum on M.?×?giganteus biomass and to identify effective foliar fungicides against the disease. Eleven broad-spectrum fungicides were tested with rates typically used in agronomic crops. Fungicides tested included active ingredients from five different chemical groups, demethylation inhibitors (prothioconazole, tebuconazole, cyproconazole, propiconazole, tetraconazole, flutriafol, and metconazole), quinone outside inhibitors (pyraclostrobin), succinate dehydrogenase inhibitors (boscalid), methyl benzimidazole carbamates (thiophanate-methyl), and chloronitriles (chlorothalonil). Infection by L. chartarum significantly lowered aboveground biomass by an average of 33 %. Application of cyproconazole, flutriafol, tebuconazole, or prothioconazole significantly reduced disease severity, with cyproconazole and flutriafol ranking best at an average of 29 % disease control. However, no fungicide treatment resulted in biomass loss abatement. Greater rates or a combination of fungicides might be needed to achieve adequate control.     

An improved lentiviral system for efficient expression and purification of β-defensins in mammalian cells

β-Defensins are a family of conserved small cationic antimicrobial peptides with different significant biological functions. The majority of mammalian β-defensins are expressed in epididymis, and many of them are predicted to have post-translational modifications. However, only a few of its members have been well studied due to the limitations of expressing and purifying bioactive proteins with correct post-translational modifications efficiently. Here we developed a novel Fc tagged lentiviral system and Fc tagged prokaryotic expression systems provided new options for β-defensins expression and purification. The novel lentiviral system contains a secretive signal peptide, an N-terminal IgG Fc tag, a green fluorescent protein (GFP), and a puromycin selection marker to facilitate efficient expression and fast purification of β-defensins by protein A magnetic or agarose beads. It also enables stable and large-scale expression of β-defensins with regular biological activities and post-translational modification. Purified β-defensins such as Bin1b and a novel human β-defensin hBD129 showed antimicrobial activity, immuno-regulatory activity, and expected post-translational phosphorylation, which were not found in Escherichia coli (E. coli) in expressed form. Furthermore, we successfully applied the novel system to identify mBin1b interacting proteins, explaining Bin1b in a better way. These results suggest that the novel lentiviral system is a powerful approach to produce correct post-translational processed β-defensins with bioactivities and is useful to identify their interacting proteins. This study has laid the foundation for future studies to characterize function and mechanism of novel β-defensins.     

Nitrogen Fertilization Does Significantly Increase Yields of Stands of Miscanthus × giganteus and Panicum virgatum in Multiyear Trials in Illinois

The C4 perennial grasses Miscanthus × giganteus and Panicum virgatum (switchgrass) are emerging bioenergy crops. They are attractive because they are productive and recycle nutrients to the overwintering belowground rhizomes, before the dry shoots are harvested. They are therefore expected to require minimal fertilizer inputs, a desirable trait for any crop. Until now, Europe has had the only long-term trials of M. × giganteus, and these have either shown no or a small effect of nitrogen fertilization, while trials of P. virgatum in the USA have shown a clear positive effect of N fertilization. This study exploited the first long-term trials of M. × giganteus in the USA, and first side-by-side comparison with P. virgatum, to test the hypothesis that N fertilization would only improve yields of the latter. A split-plot N fertility treatment (0, 67, 134, and 202 kg(N)?ha^?1) was added to >5-year-old replicated stands of the two crops at seven locations on contrasting soils in the US Midwest. Averaged across all locations, M. × giganteus yields increased significantly from 23.4 Mg ha^?1 with no N fertilization to 28.9 Mg ha^?1 (+25 %) at a N application rate of 202 kg ha^?1. P. virgatum also showed significant yield increases from 10.33 Mg ha^?1 at 0 kg(N)?ha^?1 to 13.6 Mg ha^?1 (+32 %) at 202 kg(N)?ha^?1. Both species therefore responded to N fertilization and to a similar extent. The increase per unit of added N was small compared to crops such as Zea mays and unlikely to be economically worthwhile. Nitrogen fertilization arrested most of the long-term yield decline that would otherwise have occurred in P. virgatum, but eliminated only about 40 % of the decline observed in M. × giganteus, suggesting additional causal factors for long-term yield decline in this crop. While the crops responded to nitrogen addition at some locations, they did not at others. Therefore a one-case-fits-all optimum fertilization rate cannot be prescribed.     

Abundance and Diversity of Ammonia-Oxidizing Prokaryotes in the Root–Rhizosphere Complex of Miscanthus × giganteus Grown in Heavy Metal-Contaminated Soils

Mine wastes have been considered as a source of heavy metal (HM) contamination in the environment and negatively impact many important ecosystem services provided by soils. Plants like Miscanthus, which tolerate high HM concentrations in soil, are often used for phytoremediation and provide the possibility to use these soils at least for the production of energy crops. However, it is not clear if plant growth at these sites is limited by the availability of nutrients, mainly nitrogen, as microbes in soil might be affected by the contaminant. Therefore, in this study, we investigated in a greenhouse experiment the response of ammonia-oxidizing microbes in the root–rhizosphere complex of Miscanthus × giganteus grown in soils with different levels of long-term arsenic (As) and lead (Pb) contamination. Quantitative PCR of the ammonia monooxigenease gene (amoA) was performed to assess the abundance of ammonia-oxidizing bacteria (AOB) and archaea (AOA) at two different points of plant growth. Furthermore, bulk soil samples before planting were analyzed. In addition, terminal restriction fragment length polymorphism (T-RFLP) analysis was used to investigate the diversity of archaeal amoA amplicons. Whereas high concentrations of As and Pb in soil (83 and 15?g/kg, respectively) resulted independent from plant growth in a clear reduction of AOA and AOB compared to the control soils with lower HM contents, in soils with contamination levels of 10?g/kg As and 0.2?g/kg Pb, only AOB were negatively affected in bulk soil samples. Diversity analysis of archaeal amoA genes revealed clear differences in T-RFLP patterns in response to the degree of HM contamination. Therefore, our results could clearly prove the different response patterns of AOA and AOB in HM-contaminated soils and the development of archaeal amoA phylotypes which are more tolerant towards HMs in soil samples from the areas that were impacted the most by mining waste, which could contribute to functional redundancy of ammonia-oxidizing microbes in soils and stability of nitrification pattern.     

Assessment of Aboveground and Belowground Vegetative Fragments as Propagules in the Bioenergy Crops Arundo donax and Miscanthus × giganteus

Giant miscanthus (Miscanthus × giganteus) and giant reed (Arundo donax) are leading bioenergy crops. Both exhibit many invasive characteristics, though only giant reed is known to be invasive. Despite this, neither produces viable seed, limiting movement to vegetative propagules. Therefore, to assess vegetative fragments as potential propagules, we quantified seasonal changes in culm node viability and performance in giant miscanthus and giant reed under greenhouse conditions. Giant miscanthus culms were collected in spring, summer, fall, and winter from established fields, while giant reed culms were collected in summer, fall, and winter from feral stands. Treatments at each timing consisted of whole culms and single-node culm fragments planted in soil or placed in standing water for an 8-week period. Giant miscanthus whole culms and fragments produced shoots and roots in both soil and standing water immediately following cutting from spring to summer, but failed to produce shoots and roots after fall and winter cutting dates. All rhizome fragments survived and generated shoots and roots after burial. By comparison, giant reed produced shoots and roots in both soil and standing water throughout the year, regardless of cutting date. With giant miscanthus, precautions should be taken when living culms or rhizome fragments are harvested and transported through riparian habitats during the summer months. By comparison, giant reed showed a remarkable increase in propagule generation and productivity throughout the year and, thus, escaped propagules present a far greater risk of unintentional establishment compared to giant miscanthus.     

Establishment of a micropropagation supporting technology for the Fraxinus mandshurica × Fraxinus sogdiana

In Vitro Cellular & Developmental Biology - Plant - The interspecific hybridization can take advantage of heterosis and combine the double parental traits most extensively, and is an effective...     

Root System Dynamics of Miscanthus × giganteus and Panicum virgatum in Response to Rainfed and Irrigated Conditions in California

Miscanthus (Miscanthus × giganteus) and switchgrass (Panicum virgatum) are large perennial grass bioenergy crops in the USA and Europe. Despite much research into their agronomic potential, few studies have examined in situ root growth dynamics under irrigation and soil water deficits, particularly as they relate to shoot performance. We grew miscanthus and switchgrass in outdoor mesocosms under irrigated and rainfed conditions and assessed the spatial distribution and abundance of roots using minirhizotron images and whole root system sampling. Despite surviving an extended period of drought, shoot and root biomass, root length density, numbers of culms, and culm height were reduced in both species under rainfed (dry) conditions. However, rainfed switchgrass far outperformed rainfed miscanthus in all shoot and root growth metrics. The rainfed (drought) treatment reduced switchgrass and miscanthus whole plant biomass by 83 and 98 %, culm production by 67 and 90 %, and root length density by 67 and 94 % compared to irrigated plants, respectively. Root nitrogen concentration was higher for miscanthus (3-fold) and switchgrass (4-fold) in the rainfed treatment compared to irrigated plants and did not significantly differ between species. Unlike miscanthus, switchgrass grew roots continuously into regions of available soil moisture as surface soil layers grew increasingly dry, indicating a drought avoidance strategy. Our study suggests that switchgrass is more likely to tolerate drought by mining deep wet soils, while miscanthus relies on shallow rhizome production to tolerate dry soils.     

An efficient micropropagation system for <Emphasis Type="Italic">Eclipta,alba</Emphasis>—A valuable medicinal herb

Summary An efficient rapid and large-scale in vitro clonal propagation of the valuable medicinal herb Eclipta alba (Asteraceae) by enhanced axillary shoot proliferation in cotyledonary node segments was designed. The medium type, various carbon sources, plant growth regulators, and coconut water markedly influenced in vitro propagation of Eclipta alba. An in vitro plantlet production system has been investigated on Murashige and Skoog (MS) medium with the synergistic combination of benzyladenine (4.4μM), kinetin (4.6μM), 2-isopentenyladenine (4.9μM), gibberellic acid (1.4μM), 5% coconut water, and 3% sucrose which promoted the maximum number of shoots as well as beneficial shoot length: Subculturing of cotyledonary node segments on a similar medium enabled continuous production of healthy shoots with similar frequency. Rooting was highest (94.3%) on full strength. MS medium containing 9.8 μM indolebutyric acid. Micropropagated plants established in garden soil, farmyard soil, and sand (2∶1∶1) were uniform and identical to the donor plant with respect to growth characteristics as well as floral features. These plants grew normally without showing any morphological variation.     

Greenhouse Gas Emissions, Nitrate Leaching, and Biomass Yields from Production of Miscanthus × giganteus in Illinois, USA

Understanding the effects of nitrogen (N) fertilization on Miscanthus × giganteus greenhouse gas emissions, nitrate leaching, and biomass production is an important consideration when using this grass as a biomass feedstock. The objective of this study was to determine the effect of three N fertilization rates (0, 60, and 120?kg?N?ha^?1 using urea as the N source) on nitrous oxide (N₂O) and carbon dioxide (CO₂) emissions, nitrogen leaching, and the biomass yields and N content of M. × giganteus planted in July 2008, and evaluated from 2009 through early 2011 in Urbana, Illinois, USA. While there was no biomass yield response to N fertilization rates in 2009 and 2010, the amount of N in the harvested biomass in 2010 was significantly greater at the 60 and 120?kg?N?ha^?1?N rates. There was no significant CO₂ emission response to N rates in 2009 or 2010. Similarly, N fertilization did not increase cumulative N₂O emissions in 2009, but cumulative N₂O emissions did increase in 2010 with N fertilization. During 2009, nitrate (NO ₃ ^? ) leaching at the 50-cm soil depth was not related to fertilization rate, but there was a significant increase in NO ₃ ^? leaching between the 0 and 120?kg?N?ha^?1 treatments in 2010 (8.9 and 28.9?kg?NO₃?CN?ha^?1?year^?1, respectively). Overall, N fertilization of M. × giganteus led to N₂O releases, increased fluxes of inorganic N (primarily NO ₃ ^? ) through the soil profile; and increased harvested N without a significant increase in biomass production.     

An efficient protoplast-to-plant system for the hybrid ornamental shrub,Weigela ×florida cv. Bristol Ruby (Caprifoliaceae)

Strategies were developed for the successful isolation of large numbers of highly viable protoplasts from the leaves, stems and roots of axenic plants of the hybrid ornamental shrubWeigela ×florida cv Bristol Ruby. Protoplasts, of all sources, were cultured on different media, leading to the establishment of sustained divisions, and coupled with the production of multi-celled (>50 cells) colonies. However, those colonies derived from mesophyll protoplasts only were capable of a further proliferation to the callus stage. Upon transfer to a regeneration medium consisting of MS salts and organics plus a range of concentrations of NAA and BAP, such calli underwent caulogenesis, with optimum responses for a medium with 1.0 mg l⁻¹ NAA and 1.0 mg l⁻¹ BAP. The protoplast-derived shoots thus obtained were multiplied on MS medium with 0.1 mg l⁻¹ IBA, 0.5 mg l⁻¹ BAP and 0.1 mg l⁻¹ GA₃. Individual shoots were subsequently rooted on a half-strength MS medium plus 3.0 mg l⁻¹ IBA, and complete protoplast-derived plants were finally transferred to the glasshouse for acclimatization.     

Correction to: Improving micropropagation of Mentha × piperita L. using a liquid culture system

In Vitro Cellular & Developmental Biology - Plant - This correction reflects Sadanand A. Dhekney’s updated e-mail address and affiliation.     

Comparative Biogeochemical Cycles of Bioenergy Crops Reveal Nitrogen-Fixation and Low Greenhouse Gas Emissions in a Miscanthus × giganteus Agro-Ecosystem

We evaluated the biogeochemical cycling and relative greenhouse gas (GHG) mitigation potential of proposed biofuel feedstock crops by modeling growth dynamics of Miscanthus × giganteus Greef et Deuter (miscanthus), Panicum virgatum L. (switchgrass), Zea mays L. (corn), and a mixed prairie community under identical field conditions. DAYCENT model simulations for miscanthus were parameterized with data from trial plots in Europe and Illinois, USA. Switchgrass, corn, and prairie ecosystems were simulated using parameters published in the literature. A previously unknown source of nitrogen (N) was necessary to balance the plant nutrient budget in miscanthus crops, leading us to hypothesize that miscanthus growth depends on N-fixation. We tested for nitrogenase activity by acetylene reduction of whole rhizomes and bacteria isolated from the rhizosphere and miscanthus tissue. Our results supported the hypothesis that biological N-fixation contributed to the N demand of miscanthus, a highly productive perennial grass. Corn agro-ecosystems emit 956 to 1899 g CO_2eq m⁻²y⁻¹ greater GHGs (including CO₂, N₂O, CH₄) to the atmosphere than the other biofuel crop alternatives because of greater N₂O emissions from fertilizer additions. Of the feedstock crops evaluated in this study, miscanthus would result in the greatest GHG reduction.