Expression of ZmGA20ox cDNA alters plant morphology and increases biomass production of switchgrass (Panicum virgatum L.)

Switchgrass (Panicum virgatum L.) is considered a model herbaceous energy crop for the USA, for its adaptation to marginal land, low rainfall and nutrient‐deficient soils; however, its low biomass yield is one of several constraints, and this might be rectified by modulating plant growth regulator levels. In this study, we have determined whether the expression of the Zea mays gibberellin 20‐oxidase (ZmGA20ox) cDNA in switchgrass will improve biomass production. The ZmGA20ox gene was placed under the control of constitutive CaMV35S promoter with a strong TMV omega enhancer, and introduced into switchgrass via Agrobacterium‐mediated transformation. The transgene integration and expression levels of ZmGA20ox in T0 plants were analysed using Southern blot and qRT‐PCR. Under glasshouse conditions, selected transgenic plants exhibited longer leaves, internodes and tillers, which resulted in twofold increased biomass. These phenotypic alterations correlated with the levels of transgene expression and the particular gibberellin content. Expression of ZmGA20ox also affected the expression of genes coding for key enzymes in lignin biosynthesis. Our results suggest that the employment of ectopic ZmGA20ox and selection for natural variants with high level expression of endogenous GA20ox are appropriate approaches to increase biomass production of switchgrass and other monocot biofuel crops.     

Transgenic miR156 switchgrass in the field: growth,recalcitrance and rust susceptibility

Sustainable utilization of lignocellulosic perennial grass feedstocks will be enabled by high biomass production and optimized cell wall chemistry for efficient conversion into biofuels. MicroRNAs are regulatory elements that modulate the expression of genes involved in various biological functions in plants, including growth and development. In greenhouse studies, overexpressing a microRNA (miR156) gene in switchgrass had dramatic effects on plant architecture and flowering, which appeared to be driven by transgene expression levels. High expressing lines were extremely dwarfed, whereas low and moderate‐expressing lines had higher biomass yields, improved sugar release and delayed flowering. Four lines with moderate or low miR156 overexpression from the prior greenhouse study were selected for a field experiment to assess the relationship between miR156 expression and biomass production over three years. We also analysed important bioenergy feedstock traits such as flowering, disease resistance, cell wall chemistry and biofuel production. Phenotypes of the transgenic lines were inconsistent between the greenhouse and the field as well as among different field growing seasons. One low expressing transgenic line consistently produced more biomass (25%–56%) than the control across all three seasons, which translated to the production of 30% more biofuel per plant during the final season. The other three transgenic lines produced less biomass than the control by the final season, and the two lines with moderate expression levels also exhibited altered disease susceptibilities. Results of this study emphasize the importance of performing multiyear field studies for plants with altered regulatory transgenes that target plant growth and development.     

半干旱黄土丘陵沟壑区柳枝稷（Panicum virgatum）的生物质形成

为深入认识半干旱黄土丘陵沟壑区引种能源植物柳枝稷生物质生产的开发潜力及其约束机制,调查了农田、植丛尺度上早熟和晚熟柳枝稷年度生命周期内生物量累积、分株建成动态,以及土壤水分供求平衡过程。研究发现,植丛尺度早熟柳枝稷抽穗比例近100％,分株生殖发生大小阈值依赖基本丧失,高度大小分布近似正态,种群内光资源竞争强度明显弱化,与晚熟类型形成明显差异。农田尺度晚熟柳枝稷生物质产量可以达到15t／hm^2,高出早熟类型近1倍,但是其立地80～400cm土层的含水量稳定在7％以下,土壤干旱已经发生,早期干旱胁迫导致的生长停滞,以及生长中后期的成片倒伏现象在两年的观测周期内连续出现。早熟柳枝稷立地则形成相对稳定的白草、柳枝稷复合优势植被结构,深层土壤水分含量稳定在10％以上,实现了跨年度的土壤水分供求平衡。植丛尺度的生物质形成在一定程度上取决于分株生殖发生的大小依赖程度和分株间竞争关系格局,基于植丛尺度普遍的生殖发生和明显弱化的光资源竞争,早熟柳枝稷表现出更为高效的生物质形成机制。农田尺度晚熟柳枝稷尽管在雨热同步期的降水资源利用效率上存在明显比较优势,但是在降水资源利用分配策略和效应上,早熟柳枝稷表现出了综合的生态适宜性优势。保证雨热同步期降水资源利用和保蓄的平衡,是半干旱黄土丘陵沟壑区生物质生产应该遵循的基本原则之一。     

Characterization of culturable bacterial endophytes of switchgrass (Panicum virgatum L.) and their capacity to influence plant growth

Switchgrass (Panicum virgatum L.) is a perennial warm season grass that is native to the plains of North America and is widely grown as a forage, bioenergy or groundcover crop. Despite its importance, a bottleneck in switchgrass production is poor seedling vigor, which as a perennial crop represents an important time for management. Herein, data identify a suite of culturable bacterial microflora extracted from switchgrass, and show their capability to influence host plant growth and development. A total of 307 bacterial isolates were cultured and isolated from surface sterilized switchgrass biomass and sequence identified into 76 strains (subspecies classification), 36 species and 5 phyla. Approximately 58% of bacterial strains, when reintroduced into surface‐sterilized switchgrass seeds, were documented to increase lamina length (cm from base to tip after 60 days growth) relative to uninoculated controls. Ecologically, Phylum Firmicutes was the most abundant bacterial classification and encompassed 75% of all isolates. Although the culturable bacterial community studies herein represent an unknown and assumedly minor proportion of the total microbiome, by focusing on culturable bacteria, we delineate functional feedback between the presence of isolated bacteria and switchgrass seedling growth.     

Switchgrass (Panicum virgatum L.) has ability to induce germination of Orobanche cumana

Orobanche cumana Wallr. (sunflower broomrape) is a devastating root parasitic weed, causing enormous crop losses worldwide. The question was whether or not switchgrass has the potential to be a ‘trap crop’ for O. cumana control. To answer this question, the field experiments, pot experiments, and laboratory experiments were conducted in this study. The ability of nine switchgrass (Panicum virgatum L.) cultivars to induce O. cumana seeds germination was tested. Results indicated that root extracts shoot extracts, rhizosphere soil, and root exudates from switchgrass induced O. cumana germination. Ability to induce germination varied significantly among growing stages, with the earlier part of growing season (grown for 2 weeks) generally inducing the highest O. cumana germination rates. The methanol was a more suitable solvent than distilled water for extracting germination stimulants from switchgrass plants. Ten-fold dilutions of the extracts generally induced higher germination rates than either undiluted or 100-fold dilutions. The germination rates of O. cumana seeds in shoot extracts treatments were positively correlated with those in the root extracts (R² = 0.6397; p < 0.01) and negatively correlated with those in the rhizosphere soil treatments ( R² = 0.4433; p < 0.05). In conclusion, it is believed that switchgrass is a potential trap crop for the control of root parasitic weed O. cumana.     

Effects of ecotypes and morphotypes in feedstock composition of switchgrass (Panicum virgatum L.)

Switchgrass (Panicum virgatum L.) is a C4 grass with high biomass yield potential and is now a model species for the Bioenergy Feedstock Development Program. Two distinct ecotypes (e.g., upland and lowland) and a range of plant morphotypes (e.g., leafy and stemmy) have been observed in switchgrass. The objective of this study was to determine the influence of ecotype and morphotype on biomass feedstock quality. Leaf and stem tissues of leafy and stemmy morphotypes from both lowland and upland ecotypes were analyzed for key feedstock traits. The leaf : stem ratio of leafy morphotype was more than 40% higher than the stemmy morphotype in both upland and lowland ecotypes. Therefore, the stemmy morphotype has significant advantages over leafy morphotype during harvesting, storage, transportation and finally the feedstock quality. Remarkable differences in feedstock quality and mineral composition were observed in switchgrass genotypes with distinct ecotypic origins and variable plant morphotypes. Lignin, hemicelluloses and cellulose concentrations were higher in stems than in the leaves, while ash content was notably high in leaves. A higher concentration of potassium was found in the stems compared to the leaves. In contrast, calcium was higher and magnesium was generally higher in the leaves compared to stems. The upland genotypes demonstrated considerably higher lignin (14.4%) compared with lowland genotypes (12.4%), while hemicellulose was higher in lowland compared with upland. The stemmy type demonstrated slightly higher lignin compared with leafy types (P < 0.1). Differences between the ecotypes and morphotypes for key quality traits demonstrated the potential for improving feedstock composition of switchgrass through selection in breeding programs.     

Targeted mutagenesis in tetraploid switchgrass (Panicum virgatum L.) using CRISPR/Cas9

The CRISPR/Cas9 system has become a powerful tool for targeted mutagenesis. Switchgrass (Panicum virgatum L.) is a high yielding perennial grass species that has been designated as a model biomass crop by the U.S. Department of Energy. The self‐infertility and high ploidy level make it difficult to study gene function or improve germplasm. To overcome these constraints, we explored the feasibility of using CRISPR/Cas9 for targeted mutagenesis in a tetraploid cultivar ‘Alamo’ switchgrass. We first developed a transient assay by which a non‐functional green‐fluorescent protein gene containing a 1‐bp frameshift insertion in its 5′ coding region was successfully mutated by a Cas9/sgRNA complex resulting in its restored function. Agrobacterium‐mediated stable transformation of embryogenic calli derived from mature caryopses averaged a 3.0% transformation efficiency targeting the genes of teosinte branched 1(tb1)a and b and phosphoglycerate mutase (PGM). With a single construct containing two sgRNAs targeting different regions of tb1a and tb1b genes, primary transformants (T0) containing CRISPR/Cas9‐induced mutations were obtained at frequencies of 95.5% (tb1a) and 11% (tb1b), respectively, with T0 mutants exhibiting increased tiller production. Meanwhile, a mutation frequency of 13.7% was obtained for the PGM gene with a CRISPR/Cas9 construct containing a single sgRNA. Among the PGM T0 mutants, six are heterozygous and one is homozygous for a 1‐bp deletion in the target region with no apparent phenotypical alterations. We show that CRISPR/Cas9 system can generate targeted mutagenesis effectively and obtain targeted homozygous mutants in T0 generation in switchgrass, circumventing the need of inbreeding.     

Physiological and growth responses of switchgrass (Panicum virgatum L.) in native stands under passive air temperature manipulation

In the central Great Plains of North America, climate change predictions include increases in mean annual temperature of 1.5–5.5 °C by 2100. Ecosystem responses to increased temperatures are likely to be regulated by dominant plant species, such as the potential biofuel species Panicum virgatum (switchgrass) in the tallgrass prairie. To describe the potential physiological and whole‐plant responses of this species to future changes in air temperatures, we used louvered open‐sided chambers (louvered OSC; 1 × 1 m, adjustable height) to passively alter canopy temperature in native stands of P. virgatum growing in tallgrass prairie at varying topographic positions (upland/lowland). The altered temperature treatment decreased daily mean temperatures by 1 °C and maximum temperatures by 4 °C in May and June, lowered daytime stomatal conductance and transpiration, decreased tiller density, increased specific leaf area, and delayed flowering. Among topographic contrasts, aboveground biomass, flowering tiller density, and tiller weight were greater in lowland sites compared to upland sites, with no temperature treatment interactions. Differences in biomass production responded more to topography than the altered temperature treatment, as soil water status varied considerably between topographic positions. These results indicate that while water availability as a function of topography was a strong driver of plant biomass, many leaf‐level physiological processes were responsive to the small decreases in daily mean and maximum temperature, irrespective of landscape position. The varying responses of leaf‐level gas exchange and whole‐plant growth of P. virgatum in native stands to altered air temperature or topographic position illustrate that accurately forecasting yields for P. virgatum in mixed communities will require greater integration of physiological responses to simulated climate change (increased temperature) and resource availability over natural environmental gradients (soil moisture).     

Genic microsatellite markers derived from EST sequences of switchgrass (Panicum virgatum L.)

Switchgrass is a large, North American, perennial grass that is being evaluated as a potential energy crop. There is a need to assess genetic diversity in stored accessions and in remaining native stands to assist breeding and conservation efforts. Marker development will also be necessary for genetic linkage mapping. Toward this end, 32 switchgrass genic di‐, tri‐ and tetranucleotide repeat microsatellites were identified from expressed sequence tags (ESTs). These microsatellites were used to screen individuals from two different named cultivars. The markers displayed a high level of polymorphism consistent with the tetraploid, allogamous behaviour of the cultivars tested.     

Gateway-compatible vectors for high-throughput gene functional analysis in switchgrass (Panicum virgatum L.) and other monocot species

Switchgrass (Panicum virgatum L.) is a C4 perennial grass and has been identified as a potential bioenergy crop for cellulosic ethanol because of its rapid growth rate, nutrient use efficiency and widespread distribution throughout North America. The improvement of bioenergy feedstocks is needed to make cellulosic ethanol economically feasible, and genetic engineering of switchgrass is a promising approach towards this goal. A crucial component of creating transgenic switchgrass is having the capability of transforming the explants with DNA sequences of interest using vector constructs. However, there are limited options with the monocot plant vectors currently available. With this in mind, a versatile set of Gateway-compatible destination vectors (termed pANIC) was constructed to be used in monocot plants for transgenic crop improvement. The pANIC vectors can be used for transgene overexpression or RNAi-mediated gene suppression. The pANIC vector set includes vectors that can be utilized for particle bombardment or Agrobacterium-mediated transformation. All the vectors contain (i) a Gateway cassette for overexpression or silencing of the target sequence, (ii) a plant selection cassette and (iii) a visual reporter cassette. The pANIC vector set was functionally validated in switchgrass and rice and allows for high-throughput screening of sequences of interest in other monocot species as well.     

黄土丘陵半干旱区引种禾草柳枝稷的生物量与水分利用效率

加强优良引种禾草植物的生态适应性研究对促进我国黄土高原半干旱区草地建设和草业科学发展具有十分重要的意义。比较研究了引种禾草柳枝稷(Panicumvirgatum)在黄土丘陵半干旱区不同立地条件下地上生物量的大小和季节累积差异及其水分利用特征。2001~2002年川地柳枝稷草地地上生物量达13000~16000kg/hm2,山地梯田和坡地为2300~2650kg/hm2。不同立地条件柳枝稷返青后的生物量累积过程呈二次或三次多项式。柳枝稷的绝对生长速率(AGR)在整个生长季内呈双峰曲线变化,川地柳枝稷草地的现存量和总量AGR最大值分别为158.93和169.83kg/(hm2·d),梯田分别为27.31和38.25kg/(hm2·d),坡地为37.0和36.69kg/(hm2·d)。坡地柳枝稷生物量较大值和AGR最大值出现时间最早。不同立地柳枝稷生物量相对生长速率(RGR)在整个生长季内呈双峰曲线变化,均以返青后的20d内最大,平均AGR以川地最大,坡地和梯田相近,但坡地两峰值高于梯田。不同立地柳枝稷草地土壤水分主要利用层次为0~2m,月平均含水量顺序为梯田>坡地>川地。川地柳枝稷叶片和整体生物量水分利用效率均最高,梯田整体生物量水分利用效率大于坡地,但二者叶片水分利用效率相近。川地和山地地表下5cm生育期平均地温均为17.60℃,但4~5月份川地地温高出山地1.2~2.8℃,川地4~10月份平均气温较山地高1.5℃,这些差异影响不同立地条件柳枝稷草地水分利用和生长进程。     

Genetic Structure of Remnant Populations and Cultivars of Switchgrass (Panicum virgatum) in the Context of Prairie Conservation and Restoration

Switchgrass (Panicum virgatum L.) is a dominant, perennial C₄ grass of North American tallgrass prairies with cultivars that are widely used in grassland restoration, pastures, and landscaping. However, these cultivars may be genetically dissimilar to small, remnant populations, raising concerns about altered genetic composition of native populations through gene flow. To address this issue on a local scale in Ohio and Illinois, we used microsatellite markers to characterize genetic diversity and differentiation of 10 remnant prairie populations (5 in each state) and 8 common cultivars. The bulk of genetic variation was found to reside within rather than among wild populations, consistent with the outcrossing breeding system of switchgrass. Genetic diversity was similar among the remnant populations despite large differences in area (approximately 2–2,590 ha), highlighting the importance of small native populations as reservoirs of variation and potential seed sources for prairie restoration. Cultivars generally had similar levels of variation to the wild populations, but we found clear genetic dissimilarity between wild and cultivated gene pools (especially for Kanlow, but also Trailblazer, Blackwell, Dacotah, Summer, and Sunburst cultivars). This suggests that using cultivars in local prairie restoration efforts may alter the genetic composition of wild populations. Whether such changes are deemed as negative depends on the cultivar under consideration and specific conservation goals for preserving native switchgrass populations. Patterns of genetic variation in remnant prairie populations and potential cultivar sources can be used to develop guidelines for restoration as well as future planting of cultivars for biofuels.     

Soil microbial community structure and enzymatic activity responses to nitrogen management and landscape positions in switchgrass (Panicum virgatum L.)

Switchgrass (Panicum virgatum L.) is usually grown on marginal land for biofuel system, in which nitrogen (N) is an essential management practice, and landscape position is a key topographical factor in impacting the production. However, limited information is available regarding how the N application and landscape positions affect soil microbial communities and enzyme activities under switchgrass. Thus, the specific objective of this study was to evaluate the responses of N rate (high, 112 kg N/ha; medium, 56 kg N/ha; and low, 0 kg N/ha) and landscape positions (shoulder and footslope) on soil biological health under switchgrass field. Data showed that N addition significantly influenced carbon and N fractions. The hot water‐soluble organic carbon (HWC) and nitrogen (HWN) fractions were significantly higher at footslope position than the shoulder position. The amount of total phospholipid fatty acid (PLFA), total bacterial, actinomycetes, gram‐negative and gram‐positive bacteria, total fungi, arbuscular mycorrhizal (AM) fungi, and saprophytes PLFAs were highest with medium and high N rates and footslope position. The N addition increased total PLFAs in N fertilizer treatments, viz. medium (5,946 ng PLFA‐C/g soil) and high N rates (5,871 ng PLFA‐C/g soil). Microbial biomass carbon and nitrogen and enzyme activities (urease, β‐glucosidase, acid phosphatase and arylsulfatase) were significantly enhanced by N fertilization (medium and high N rates) compared to control (low N rates) under footslope position. The urease activity under medium (36.3 µmol N‐NH₄⁺ g^?1 soil hr^?1) and high N rates (31.4 µmol N‐NH₄⁺ g^?1 soil hr^?1) was 42.9% and 23.6% higher than low N rates, respectively. This study suggests that the application of medium N rate in footslope position to switchgrass can enhance the soil biological properties and hence can protect the environment from the excessive use of N fertilizer.     

Enhancement of switchgrass (Panicum virgatum L.) biomass production under drought conditions by the ectomycorrhizal fungus Sebacina vermifera

Experiments were conducted to examine the effects of cocultivating the important bioenergy crop switchgrass with the ectomycorrhizal fungus Sebacina vermifera under severe drought conditions. Plants cocultivated with the fungus produced significantly higher biomass and had a higher macronutrient content than uninoculated control plants under both adequately watered and drought conditions.     

Transgenic switchgrass (Panicum virgatum L.) targeted for reduced recalcitrance to bioconversion: a 2‐year comparative analysis of field‐grown lines modified for target gene or genetic element expression

Transgenic Panicum virgatum L. silencing (KD) or overexpressing (OE) specific genes or a small RNA (GAUT4‐KD, miRNA156‐OE, MYB4‐OE, COMT‐KD and FPGS‐KD) was grown in the field and aerial tissue analysed for biofuel production traits. Clones representing independent transgenic lines were established and senesced tissue was sampled after year 1 and 2 growth cycles. Biomass was analysed for wall sugars, recalcitrance to enzymatic digestibility and biofuel production using separate hydrolysis and fermentation. No correlation was found between plant carbohydrate content and biofuel production pointing to overriding structural and compositional elements that influence recalcitrance. Biomass yields were greater for all lines in the second year as plants establish in the field and standard amounts of biomass analysed from each line had more glucan, xylan and less ethanol (g/g basis) in the second‐ versus the first‐year samples, pointing to a broad increase in tissue recalcitrance after regrowth from the perennial root. However, biomass from second‐year growth of transgenics targeted for wall modification, GAUT4‐KD, MYB4‐OE, COMT‐KD and FPGS‐KD, had increased carbohydrate and ethanol yields (up to 12% and 21%, respectively) compared with control samples. The parental plant lines were found to have a significant impact on recalcitrance which can be exploited in future strategies. This summarizes progress towards generating next‐generation bio‐feedstocks with improved properties for microbial and enzymatic deconstruction, while providing a comprehensive quantitative analysis for the bioconversion of multiple plant lines in five transgenic strategies.