MicroRNA expression analysis in the cellulosic biofuel crop switchgrass (Panicum virgatum) under abiotic stress

Switchgrass has increasingly been recognized as a dedicated biofuel crop for its broad adaptation to marginal lands and high biomass. However, little is known about the basic biology and the regulatory mechanisms of gene expression in switchgrass, particularly under stress conditions. In this study, we investigated the effect of salt and drought stress on switchgrass germination, growth and the expression of small regulatory RNAs. The results indicate that salt stress had a gradual but significant negative effect on switchgrass growth and development. The germination rate was significantly decreased from 82% for control to 36% under 1% NaCl treatment. However, drought stress had little effect on the germination rate but had a significant effect on the growth of switchgrass under the severest salinity stress. Both salt and drought stresses altered the expression pattern of miRNAs in a dose-dependent manner. However, each miRNA responded to drought stress in a different pattern. Salt and drought stress changed the expression level of miRNAs mainly from 0.9-fold up-regulation to 0.7-fold down-regulation. miRNAs were less sensitive to drought treatment than salinity treatment, as evidenced by the narrow fold change in expression levels. Although the range of change in expression level of miRNAs was similar under salt and drought stress, no miRNAs displayed significant change in expression level under all tested salt conditions. Two miRNAs, miR156 and miR162, showed significantly change in expression level under high drought stress. This suggests that miR156 and miR162 may attribute to the adaption of switchgrass to drought stress and are good candidates for improving switchgrass as a biofuel crop by transgenic technology.     

Energy crop and biotechnology for biofuel production

Selection of energy crops is the first priority for large-scale biofuel production in China.As a major topic, it was extensively discussed in the Second International Symposium on Bioenergy and Biotechnology, held from October 16-19(th), 2010 in Huazhong Agricultural University(HZAU), Wuhan, China, with more than one hundred registered participants(Figure 1).     

The yield and composition of switchgrass and coastal panic grass grown as a biofuel in southern England

Switchgrass (Panicum virgatum L.) and coastal panic grass (Panicum amarum A.S. Hitchc. & Chase) are perennial grasses indigenous to North America. Switchgrass has been shown to have good potential as a biofuel crop in both the US and Canada. In the study reported here, seven varieties of switchgrass and one panic grass were evaluated for 5 years under the temperate maritime conditions in Southern England. Both species had 0 or 60 kg N ha(-1) applied annually in spring as treatment. Yield was measured after flowering and when stems were dead in the winter. Yield increased annually for 4-5 years except for the variety Dacotah, and in the fifth year dead stem yields ranged from 8.82 to 13.97 t dm ha(-1). There was no response to N except for one variety in one year. Mineral concentration in biomass was higher at flowering than at dead stem harvest and delaying harvesting further provided more time for P, K and Cl to be leached but yield also declined.     

Genomics-assisted breeding for crop improvement

Genomics research is generating new tools, such as functional molecular markers and informatics, as well as new knowledge about statistics and inheritance phenomena that could increase the efficiency and precision of crop improvement. In particular, the elucidation of the fundamental mechanisms of heterosis and epigenetics, and their manipulation, has great potential. Eventually, knowledge of the relative values of alleles at all loci segregating in a population could allow the breeder to design a genotype in silico and to practice whole genome selection. High costs currently limit the implementation of genomics-assisted crop improvement, particularly for inbreeding and/or minor crops. Nevertheless, marker-assisted breeding and selection will gradually evolve into 'genomics-assisted breeding' for crop improvement.     

Harvest index: a review of its use in plant breeding and crop physiology

This review charts the use of the concept of harvest index in crop improvement and physiology, concentrating on the literature from the last 20 years. Evidence from abstract journals indicates that the term has been applied most to small grain cereal crops and pulses, in India, Western Europe and the USA, and that it has been less useful for maize and tuber crops. Standard methods of measuring harvest index, the associated problems of measurement and interpretation, and representative values for a range of world species are reviewed. The values for modern varieties of most intensively-cultivated grain crops fall within the range 0.4 to 0.6. Variation between varieties of the same species is illustrated by trends in the harvest indices of old, outclassed and recent varieties of temperate and mediterranean wheat and barley (compared under uniform conditions); this shows a progressive increase throughout the present century, although improvement has been much slower in Australia and Canada than in the UK. In most cases, the improvement in harvest index has been a consequence of increased grain population density coupled with stable individual grain weight. The high heritability of harvest index is explored by examining its (rather weak) response to variation in environmental factors (fertilisation, population density, application of growth regulators) in the absence of severe stress. A fuller perspective is gained by reviewing aspects of the harvest index of rice, maize and tropical pulses. With rice, attention must be paid to the fact that the adhering lemma and palea (not primarily part of economic yield) can make up 20% of grain weight; and there are important interactions among biomass, grain yield and season length. Maize differs from most small grain crops in that harvest index (in N. American varieties) was already high at the start of this century, and increases in yield potential have been largely the consequence of increased biomass production. The harvest index of many pulse species and varieties tends to be low because selection has been for some yield in all seasons. Extension of the harvest index concept to express the partitioning of mineral nutrients as well as dry matter (e.g. the nitrogen harvest index) has provided a range of responses whose implications for production and breeding remain to be explored. It is concluded that even though the principal cereal crops appear to be approaching the upper limit of harvest index, and future yield gains will have to be sought by increased biomass production, there will still be a need for the concept of harvest index as a tool in interpreting crop response to different environments and climatic change.     

Construction of a GFP-BAR plasmid and its use for switchgrass transformation

 A dual marker plasmid comprising the reporter gene sgfp (green fluorescent protein) and the selectable bar gene (Basta tolerance) was constructed by replacing the uidA (β-glucuronidase, GUS) gene in a uidA-bar construct with sgfp. A particle inflow gun was used to propel tungsten particles coated with this plasmid into immature inflorescence-derived embryogenic callus of switchgrass (Panicum virgatum L.). GFP was observed in leaf tissue and pollen of transgenic plants. Nearly 100 plants tolerant to Basta were obtained from the experiments, and Southern blot hybridization confirmed the presence of both the bar and sgfp genes. Plants regenerated from in vitro cultures of transgenic plants grew on medium with 10 mg l^–1 bialaphos. When the pH indicator chlorophenol red was in the medium, the transgenic plantlets changed the medium from red to yellow. Basta tolerance was observed in T₁ plants resulting from crosses between transgenic and nontransgenic control plants, indicating inheritance of the bar transgene. Received: 11 May 2000 / Revision received: 21 August 2000 / Accepted: 22 August 2000     

Euglena sp. as a suitable source of lipids for potential use as biofuel and sustainable wastewater treatment

Prolific algal growth in sewage ponds with high organic loads in the tropical regions can provide cost-effective and efficient wastewater treatment and biofuel production. This work examines the ability of Euglena sp. growing in wastewater ponds for biofuel production and treatment of wastewater. The algae were isolated from the sewage treatment plants and were tested for their nutrient removal capability. Compared to other algae, Euglena sp. showed faster growth rates with high biomass density at elevated concentrations of ammonium nitrogen (NH₄-N) and organic carbon (C). Profuse growth of these species was observed in untreated wastewaters with a mean specific growth rate (μ) of 0.28 day^?1 and biomass productivities of 132 mg ?L^?1? day^?1. The algae cultured within a short period of 8 days resulted in the 98 % removal of NH₄-N, 93 % of total nitrogen 85 % of ortho-phosphate, 66 % of total phosphate and 92 % total organic carbon. Euglenoids achieved a maximum lipid content of 24.6 % (w/w) with a biomass density of 1.24 g ?L^?1 (dry wt.). Fourier transform infrared spectra showed clear transitions in biochemical compositions with increased lipid/protein ratio at the end of the culture. Gas chromatography and mass spectrometry indicated the presence of high contents of palmitic, linolenic and linoleic acids (46, 23 and 22 %, respectively), adding to the biodiesel quality. Good lipid content (comprised quality fatty acids), efficient nutrient uptake and profuse biomass productivity make the Euglena sp. as a viable source for biofuel production in wastewaters.     

Improving efficiency of breeding for higher crop yield

Summary Exclusive selection for yield raises, the harvest index of self-pollinated crops with little or no gain in total bipmass. In addition to selection for yield, it is suggested that efficient breeding for higher yield requires simultaneous selection for yield's three major, genetically controlled physiological components. The following are needed: (1) a superior rate of biomass accumulation. (2) a superior rate of actual yield accumulation in order to acquire a high harvest index, and (3) a time to harvest maturity that is neither shorter nor longer than the duration of the growing season. That duration is provided by the environment, which is the fourth major determinant of yield. Simultaneous selection is required because genetically established interconnections among the three major physiological components cause: (a) a correlation between the harvest index and days to maturity that is usually negative; (b) a correlation between the harvest index and total biomass that is often negative, and (c) a correlation between biomass and days to maturity that is usually positive. All three physiological components and the correlations among them can be quantified by yield system analysis (YSA) of yield trials. An additive main effects and multiplicative interaction (AMMI) statistical analysis can separate and quantify the genotype × environment interaction (G × E) effect on yield and on each physiological component that is caused by each genotype and by the different environment of each yield trial. The use of yield trials to select parents which have the highest rates of accumulation of both biomass and yield, in addition to selecting for the G × E that is specifically adapted to the site can accelerate advance toward the highest potential yield at each geographical site. Higher yield for many sites will raise average regional yield. Higher yield for multiple regions and continents will raise average yield on a world-wide basis. Genetic and physiological bases for lack of indirect selection for biomass from exclusive selection for yield are explained.     

Making the most of 'omics' for crop breeding

Adoption of new breeding technologies is likely to underpin future gains in crop productivity. The rapid advances in 'omics' technologies provide an opportunity to generate new datasets for crop species. Integration of genome and functional omics data with genetic and phenotypic information is leading to the identification of genes and pathways responsible for important agronomic phenotypes. In addition, high-throughput genotyping technologies enable the screening of large germplasm collections to identify novel alleles from diverse sources, thus offering a major expansion in the variation available for breeding. In this review, we discuss these advances, which have opened the door to new techniques for construction and screening of breeding populations, to increase ultimately the efficiency of selection and accelerate the rates of genetic gain.     

The global potential for Agave as a biofuel feedstock

Large areas of the tropics and subtropics are too arid or degraded to support food crops, but Agave species may be suitable for biofuel production in these regions. We review the potential of Agave species as biofuel feedstocks in the context of ecophysiology, agronomy, and land availability for this genus globally. Reported dry biomass yields of Agave spp., when annualized, range from <1 to 34 Mg ha^?1 yr^?1 without irrigation, depending on species and location. Some of the most productive species have not yet been evaluated at a commercial scale. Approximately 0.6 Mha of land previously used to grow Agave for coarse fibers have fallen out of production, largely as a result of competition with synthetic fibers. Theoretically, this crop area alone could provide 6.1 billion L of ethanol if Agave were re‐established as a bioenergy feedstock without causing indirect land use change. Almost one‐fifth of the global land surface is semiarid, suggesting there may be large opportunities for expansion of Agave crops for feedstock, but more field trials are needed to determine tolerance boundaries for different Agave species.     

Microbial lipids as a source of biofuel

This review presents the main directions and experimental data aimed at searching for active producers of lipids among different species of microorganisms and ways to optimize the lipidogenesis process in the most promising stains. It was shown that enzymatic processes can be directed by maintaining the necessary cultivation conditions. The influence on the growth, development, and biochemical activity of the microbial medium composition and temperature and the aeration and oxidation reduction conditions was considered. Changes in these factors affected the biosynthetic activity of microorganisms and lipidogenic yeasts and the composition of synthesized lipids. The ability of lipidogenic yeasts, as well as the relatively rapid ability of changing the amount and composition of lipids by direct cultivation, leads to the conclusion that lipids obtained by microbial synthesis can be a source of commercial raw materials for biofuel.     

Selection, breeding and engineering of microalgae for bioenergy and biofuel production

Microalgal production technologies are seen as increasingly attractive for bioenergy production to improve fuel security and reduce CO(2) emissions. Photosynthetically derived fuels are a renewable, potentially carbon-neutral and scalable alternative reserve. Microalgae have particular promise because they can be produced on non-arable land and utilize saline and wastewater streams. Furthermore, emerging microalgal technologies can be used to produce a range of products such as biofuels, protein-rich animal feeds, chemical feedstocks (e.g. bioplastic precursors) and higher-value products. This review focuses on the selection, breeding and engineering of microalgae for improved biomass and biofuel conversion efficiencies.     

Molecular design of laccase cathode for direct electron transfer in a biofuel cell

In order to improve the direct electron transfer in enzymatic biofuel cells, a rational design of a laccase electrode is presented. Graphite electrodes were functionalized with 4-[2-aminoethyl] benzoic acid hydrochloride (AEBA). The benzoic acid moiety of AEBA interacts with the laccase T1 site as ligand with an association constant (K(A)) of 6.6×10(-6) M. The rational of this work was to orientate the covalent coupling of laccase molecule with the electrode surface through the T1 site and thus induce the direct electron transfer between the T1 site and the graphite electrode surface. Direct electron transfer of laccase was successfully achieved, and the semi-enzymatic fuel cell Zn-AEBA laccase showed a current density of 2977 μA cm(-2) and a power density of 1190 μW cm(-2) at 0.41 V. The molecular oriented laccase cathode showed 37% higher power density and 43% higher current density than randomly bound laccase cathode. Chronoaperometric measurements of the Zn-AEBA fuel cell showed functionality on 6 h. Thus, the orientation of the enzyme molecules improves the electron transfer and optimizes enzyme-based fuel cells efficiency.     

Bermuda grass as feedstock for biofuel production: a review

Bermuda grass is a promising feedstock for the production of fuel ethanol in the Southern United States. This paper presents a review of the significant amount of research on the conversion of Bermuda grass to ethanol and a brief discussion on the factors affecting the biomass production in the field. The biggest challenge of biomass conversion comes from the recalcitrance of lignocellulose. A variety of chemical, physico-chemical, and biological pretreatment methods have been investigated to improve the digestibility of Bermuda grass with encouraging results reported. The subsequent enzymatic hydrolysis and fermentation steps have also been extensively studied and effectively optimized. It is expected that the development of genetic engineering technologies for the grass and fermenting organisms has the potential to greatly improve the economic viability of Bermuda grass-based fuel ethanol production systems. Other energy applications of Bermuda grass include anaerobic digestion for biogas generation and pyrolysis for syngas production.     

Agave as a biofuel feedstock in Australia

The opportunity for commercial production of Agave in Australia stems from the substantial carbohydrate and fibre content of Agave, the nature of carbohydrates stored, the pre‐existence of an Agave ethanol‐producing industry in Mexico, demand for biofuel feedstocks, impressive water‐use efficiencies of plants with the CAM pathway, and legislation mandating the ethanol content of fuel in Australia, where an estimated 748 ML will be required for blending with petrol by 2010–2011, compared with about 440 ML in 2009. Agave has potential as a crop for areas of seasonally limited rainfall in Australia, a judgement based upon desktop analyses and agronomic experience of growing Agave in Australia before 1915. Development of a viable Australian Agave farming system requires production be located in suitable regions, efficient propagation methods, mechanized production, and viable business plans at grower, processor and marketing levels. Growers and processors agree that Agave will not be grown commercially until plants are grown, maintained and harvested in Australia, product is produced and tested, and yield and risks are evaluated. To this end, Agave were imported into Australia from Mexico and a tissue culture propagation technique developed. A trial crop of Agave tequilana was planted in northern Queensland and CO₂ exchange, nitrogen and water dynamics, carbohydrate content, and system inputs and outputs are being monitored. The experience will be used to fine‐tune the farming system, assess production costs and develop robust life‐cycle assessments. Processing of plants from trials will test harvesting and transport infrastructure and will provide material to processors for testing. Samples will be provided to potential producers of value‐added products. An Australian Agave industry should provide opportunities for stimulating agronomic, scientific and commercial exchange between Australia and Mexico. Successful integration of Agave into Australian agriculture will require a biofuels‐focussed breeding programme in collaboration with Mexican researchers.     

Endophytic fungi as a source of biofuel precursors

Endophytic fungi, isolated from a number of different species of tropical plants, were investigated for lipid biodiesel precursor production. The extracts produced from liquid cultures of these fungi were subjected to acidcatalyzed transesterification reactions with methanol producing methyl esters and then analyzed through chromatographic (GC-FID) and spectrometric techniques (MS, NMR 1H). The European Standard Method, EN 14103, was used for the quantification of methyl esters extracted from the fungi of the species and genera studied. Xylariaceous fungi exhibited the highest concentrations of methyl esters (91%), and hence may be a promising source for biofuel.     

Protoplast fusion for crop improvement and breeding in China

Protoplast fusion offers an opportunity for circumventing barriers to sexual reproduction and allows for gene transfer of nuclear and cytoplasmic genomes to enrich the gene pool of cultivated species. Moreover, protoplast fusion effectively generates novel germplasm for elite breeding of conventional crosses and promotes crop improvement in existing cultivars. Over the past few decades, protoplast fusion in China has considerably progressed not only for food crops but also for economic plants. In this review, we present and discuss the development of somatic hybrids in wheat, rapeseed, citrus, and cotton, some of which are already in use or have potential for future commercial use in China. For example, an elite salt- and drought-resistant line, Shangrong No. 3, has already been registered as a commercial wheat cultivar. Some other hybrids have been found to have disease resistance as well as modified fatty acids, high oil and protein contents, novel cytoplasmic male sterility, and numerous other desirable agronomic characteristics that are useful for further breeding.