Agrobacterium-mediated sorghum transformation

Agrobacterium tumefaciens was used to genetically transform sorghum. Immature embryos of a public (P898012) and a commercial line (PHI391) of sorghum were used as the target explants. The Agrobacterium strain used was LBA4404 carrying a `Super-binary' vector with a bar gene as a selectable marker for herbicide resistance in the plant cells. A series of parameter tests was used to establish a baseline for conditions to be used in stable transformation experiments. A number of different transformation conditions were tested and a total of 131 stably transformed events were produced from 6175 embryos in these two sorghum lines. Statistical analysis showed that the source of the embryos had a very significant impact on transformation efficiency, with field-grown embryos producing a higher transformation frequency than greenhouse-grown embryos. Southern blot analysis of DNA from leaf tissues of T₀ plants confirmed the integration of the T-DNA into the sorghum genome. Mendelian segregation in the T₁ generation was confirmed by herbicide resistance screening. This is the first report of successful use of Agrobacterium for production of stably transformed sorghum plants. The Agrobacterium method we used yields a higher frequency of stable transformation that other methods reported previously.     

Rapid and reproducible Agrobacterium-mediated transformation of sorghum

A rapid and reproducible Agrobacterium-mediated transformation protocol for sorghum has been developed. The protocol uses the nptII selectable marker gene with either of the aminoglycosides geneticin or paromomycin. A screen of various A. tumefaciens strains revealed that a novel C58 nopaline chromosomal background carrying the chrysanthopine disarmed Ti plasmid pTiKPSF₂, designated NTL₄/Chry5, was most efficient for gene transfer to sorghum immature embryos. A NTL₄/Chry5 transconjugant harboring the pPTN290 binary plasmid, which carries nptII and GUSPlus ^TM expression cassettes, was used in a series of stable transformation experiments with Tx430 and C2-97 sorghum genotypes and approximately 80% of these transformation experiments resulted in the recovery of at least one transgenic event. The transformation frequencies among the successful experiments ranged from 0.3 to 4.5%, with the average transformation frequency being approximately 1% for both genotypes. Over 97% of the transgenic events were successfully established in the greenhouse and were fully fertile. Co-expression of GUSPlus ^TM occurred in 89% of the transgenic T₀ events. Seed set for the primary transgenic plants ranged from 145 to 1400 seed/plant. Analysis of T₁ progeny demonstrated transmission of the transgenes in a simple Mendelian fashion in the majority of events.     

Photosynthetic response of sweet sorghum to drought and re‐watering at different growth stages

Sweet sorghum (Sorghum bicolor) is a C4 drought resistant species with a huge potential for bioenergy. Accentuated reductions in water availability for crop production and altered rainfall distribution patterns, however, will have direct impact on its physiological attributes, metabolic functions and plant growth. The objective of this study was to evaluate the effects of drought and re‐watering on the photosynthetic efficiency of sweet sorghum. Durable or short transient drought stress periods were imposed at early and late growth stages and compared with well‐watered plants. In spite of very similar drought levels at early and late growth stages (Ψ_soil = ?1.6 and ?1.7 MPa), the decrements in maximum quantum yield (?_Po) and performance index (PI) were about twice at late than at early growth stages. All the PI components, that is, density of active reaction centers (RCs), excitation energy trapping and conversion of excitation energy into electron flow followed a similar decreasing pattern. Upon re‐watering and regardless the duration and growth stage of the drought period, all the photosynthetic functions, and particularly those of photosystem II (PSII), fully recovered. Such effective self‐regulating functional activity by PSII photochemistry likely contributes to both high drought resistance and photosynthetic recovery capacity of sweet sorghum. At vegetative growth stages, the down regulation of the photochemistry seems to be the main photoprotective/regulative mechanisms, while at late growth stages, the accumulation of compatible solutes likely has a more preponderant role. The observed sugar concentration increments likely contributed to prevent permanent photo‐oxidative destruction of the PSII RCs of mature droughted sweet sorghum plants.     

Produced by a Strain of Unidentified Actinomycetes sp.

The possibility of using two kinds of sorghum as raw materials in consolidated bioprocessing bioethanol production using Flammulina velutipes was investigated. Enzymatic saccharification of sweet sorghum was not as high as in brown mid-rib (bmr) mutated sorghum, but the amount of ethanol production was higher. Ethanol production from bmr mutated sorghum significantly increased when saccharification enzymes were added to the culture.     

Legacy effects of individual crops affect N2O emissions accounting within crop rotations

Uruguay is pursuing renewable energy production pathways using feedstocks from its agricultural sector to supply transportation fuels, among them ethanol produced from commercial technologies that use sweet and grain sorghum. However, the environmental performance of the fuel is not known. We investigate the life cycle environmental and cost performance of these two major agricultural crops used to produce ethanol that have begun commercial production and are poised to grow to meet national energy targets for replacing gasoline. Using both attributional and consequential life cycle assessment (LCA) frameworks for system boundaries to quantify the carbon intensity, and engineering cost analysis to estimate the unit production cost of ethanol from grain and sweet sorghum, we determined abatement costs. We found 1) an accounting error in estimating N₂O emissions for a specific crop in multiple crop rotations when using Intergovernmental Panel on Climate Change(IPCC) Tier 1 methods within an attributional LCA framework, due to N legacy effects; 2) choice of baseline and crop identity in multiple crop rotations evaluated within the consequential LCA framework both affect the global warming intensity (GWI) of ethanol; and 3) although abatement costs for ethanol from grain sorghum are positive and from sweet sorghum they are negative, both grain and sweet sorghum pathways have a high potential for reducing transport fuel GWI by more than 50% relative to gasoline, and are within the ranges targeted by the US renewable transportation fuel policies.     

Trichoderma Harzianum WKY1: an indole acetic acid producer for growth improvement and anthracnose disease control in sorghum

The efficiency of two isolates of Trichoderma harzianum (WKY1 and WKY5) as bio-control agents against anthracnose disease in sorghum was investigated. In vitro, T. harzianum WKY1 isolate showed superiority in terms of inhibition of both mycelial growth and spore germination of Colletotrichum sublineolum, the causative agent of sorghum anthracnose, as well as induction of the sorghum seed germination over T. harzianum WKY5 isolate. The culture filtrate of the selected isolate (T. harzianum WKY1) was analysed using GC-MS system to determine their chemical constituents. Twenty-nine components with varied existence percentages were identified. Although T. harzianum WKY1 produced the phytohormone indole-3-acetic acid (IAA) on tryptophan free medium, a marked dependency on tryptophan for the production of IAA was noticed. Nutritional components were optimized for maximizing IAA production using the central composite design. The optimum levels were 1.06, 29.86 and 2.93?g?L^?1 from tryptophan, sucrose and NaNO₃, respectively, with a maximum IAA biosynthesis (138.9?µg?mL^?1) after five days of incubation. Production of IAA in the culture filtrate of T. harzianum WKY1 was qualitatively and quantitatively analysed by LC-MS system using a reference standard of IAA. Under greenhouse conditions, application of T. harzianum WKY1 and/or its filtrate reduced greatly the disease severity as well as improved the plant growth of sorghum. From the present data, we can recommend the application of T. harzianum WKY1 as a dual purpose bio-agent for biological control of anthracnose disease and plant growth promotion.     

Study of a single and mixed culture for the direct bio-conversion of sorghum carbohydrates to ethanol

Fusaium oxysporum F3 alone or in mixed culture with Saccharomyces cerevisiae 2541 fermented soluble and insoluble carbohydrates of sweet sorghum stalk directly to ethanol. Both microorganisms were first grown aerobically and fermented sorghum stalk to ethanol thereafter. During fermentation, insoluble carbohydrates were hydrolysed to soluble sugars by the celluloytic system of F. oxysporum. Ethanol yields as high as 24.4 and 33.5 g/100 g dry stalks were obtained by F. oxysporum and the mixed culture respectively, representing a theoretical yield enhancement of 11.6% and 53.6% respectively. The corresponding ethanol concentrations in the fermentation medium were 4.6% and 6.4% (w/v). These results clearly demonstrated that a large portion of insoluble carbohydrate from sorghum was converted by simultaneous saccharification and fermentation to ethanol, making the process promising for bioethanol production.     

Agronomic Considerations for Sweet Sorghum Biofuel Production in the South-Central USA

Sweet sorghum (Sorghum bicolor (L.) Moench) is currently recognized throughout the world as a highly promising biomass energy crop. Production systems and management practices for sweet sorghum have not been fully developed for the USA, although sporadic research efforts during recent decades have provided some insights into production of sweet sorghum primarily for fermentable sugar production. Field plot experiments were conducted at sites across Louisiana to assess biomass and sugar yield responses to N fertilizer, plant density, and selected cultivars. Although linear increases in stem biomass production and fermentable sugar yield were obtained with increasing N fertilizer rate under irrigated conditions, most of the increase was from the initial 45 kg N ha⁻¹ increment. Nitrogen fertilization increased stem biomass production but not fermentable sugar yield in some non-irrigated environments. Increased plant density contributed to fermentable sugar yield only under growth-limiting conditions, particularly under limited soil moisture. Location effects indicate that sweet sorghum may not be suitable for some sub-optimal cropland and pasture environments in Louisiana. During the primary growing season, cultivar did not affect fermentable sugar yields, although Dale was consistently high in sugar concentration during this period. Nitrogen fertilizer increased fermentable sugar yields only when moisture was not limiting. Overall results indicate that in environments where soil moisture limits plant growth, sugar yield responses are likely from increased plant density and not from increased N fertilization.     

Genome-wide patterns of genetic variation in sweet and grain sorghum (Sorghum bicolor)

Background

Sorghum (Sorghum bicolor) is globally produced as a source of food, feed, fiber and fuel. Grain and sweet sorghums differ in a number of important traits, including stem sugar and juice accumulation, plant height as well as grain and biomass production. The first whole genome sequence of a grain sorghum is available, but additional genome sequences are required to study genome-wide and intraspecific variation for dissecting the genetic basis of these important traits and for tailor-designed breeding of this important C₄ crop.

Results

We resequenced two sweet and one grain sorghum inbred lines, and identified a set of nearly 1,500 genes differentiating sweet and grain sorghum. These genes fall into ten major metabolic pathways involved in sugar and starch metabolisms, lignin and coumarin biosynthesis, nucleic acid metabolism, stress responses and DNA damage repair. In addition, we uncovered 1,057,018 SNPs, 99,948 indels of 1 to 10 bp in length and 16,487 presence/absence variations as well as 17,111 copy number variations. The majority of the large-effect SNPs, indels and presence/absence variations resided in the genes containing leucine rich repeats, PPR repeats and disease resistance R genes possessing diverse biological functions or under diversifying selection, but were absent in genes that are essential for life.

Conclusions

This is a first report of the identification of genome-wide patterns of genetic variation in sorghum. High-density SNP and indel markers reported here will be a valuable resource for future gene-phenotype studies and the molecular breeding of this important crop and related species.     

A Novel Wild-Type Saccharomyces cerevisiae Strain TSH1 in Scaling-Up of Solid-State Fermentation of Ethanol from Sweet Sorghum Stalks

The rising demand for bioethanol, the most common alternative to petroleum-derived fuel used worldwide, has encouraged a feedstock shift to non-food crops to reduce the competition for resources between food and energy production. Sweet sorghum has become one of the most promising non-food energy crops because of its high output and strong adaptive ability. However, the means by which sweet sorghum stalks can be cost-effectively utilized for ethanol fermentation in large-scale industrial production and commercialization remains unclear. In this study, we identified a novel Saccharomyces cerevisiae strain, TSH1, from the soil in which sweet sorghum stalks were stored. This strain exhibited excellent ethanol fermentative capacity and ability to withstand stressful solid-state fermentation conditions. Furthermore, we gradually scaled up from a 500-mL flask to a 127-m³ rotary-drum fermenter and eventually constructed a 550-m³ rotary-drum fermentation system to establish an efficient industrial fermentation platform based on TSH1. The batch fermentations were completed in less than 20 hours, with up to 96 tons of crushed sweet sorghum stalks in the 550-m³ fermenter reaching 88% of relative theoretical ethanol yield (RTEY). These results collectively demonstrate that ethanol solid-state fermentation technology can be a highly efficient and low-cost solution for utilizing sweet sorghum, providing a feasible and economical means of developing non-food bioethanol.     

Studies on fermentation of sorghum silage during storage,and its effect on milch animals

Three silages: sorghum alone (T₁); sorghum + 0.5% urea (T₂); and sorghum + wheat straw in the ratio of 4:1 (T₃), were assessed for their biochemical products and stability. The performance of milch cattle maintained on these forage diets was compared with wheat straw as a control (T₄). Animals of all four groups were given sufficient concentrate to satisfy their nutrient requirements. On the basis of fermentation products all three silages were classed as ‘good’ to ‘very good’. The palatability of these silages was relatively better than that of the control (T₄). Milk production appeared to be better with non-urea silages, but not significantly so.     

Optimized Agrobacterium-mediated sorghum transformation protocol and molecular data of transgenic sorghum plants

Agrobacterium-mediated sorghum transformation frequency has been enhanced significantly via medium optimization using immature embryos from sorghum variety TX430 as the target tissue. The new transformation protocol includes the addition of elevated copper sulfate and 6-benzylaminopurine in the resting and selection media. Using Agrobacterium strain LBA4404, the transformation frequency reached over 10% using either of two different selection marker genes, moPAT or PMI, and any of three different vectors in large-scale transformation experiments. With Agrobacterium strain AGL1, the transformation frequencies were as high as 33%. Using quantitative PCR analyses of 1,182 T₀ transgenic plants representing 675 independent transgenic events, data was collected for T-DNA copy number, intact or truncated T-DNA integration, and vector backbone integration into the sorghum genome. A comparison of the transformation frequencies and molecular data characterizing T-DNA integration patterns in the transgenic plants derived from LBA4404 versus AGL1 transformation revealed that twice as many transgenic high-quality events were generated when AGL1 was used compared to LBA4404. This is the first report providing molecular data for T-DNA integration patterns in a large number of independent transgenic plants in sorghum.     

A novel cost-effective technology to convert sucrose and homocelluloses in sweet sorghum stalks into ethanol

Background

Sweet sorghum is regarded as a very promising energy crop for ethanol production because it not only supplies grain and sugar, but also offers lignocellulosic resource. Cost-competitive ethanol production requires bioconversion of all carbohydrates in stalks including of both sucrose and lignocellulose hydrolyzed into fermentable sugars. However, it is still a main challenge to reduce ethanol production cost and improve feasibility of industrial application. An integration of the different operations within the whole process is a potential solution.

Results

An integrated process combined advanced solid-state fermentation technology (ASSF) and alkaline pretreatment was presented in this work. Soluble sugars in sweet sorghum stalks were firstly converted into ethanol by ASSF using crushed stalks directly. Then, the operation combining ethanol distillation and alkaline pretreatment was performed in one distillation-reactor simultaneously. The corresponding investigation indicated that the addition of alkali did not affect the ethanol recovery. The effect of three alkalis, NaOH, KOH and Ca(OH)₂ on pretreatment were investigated. The results indicated the delignification of lignocellulose by NaOH and KOH was more significant than that by Ca(OH)₂, and the highest removal of xylan was caused by NaOH. Moreover, an optimized alkali loading of 10% (w/w DM) NaOH was determined. Under this favorable pretreatment condition, enzymatic hydrolysis of sweet sorghum bagasse following pretreatment was investigated. 92.0% of glucan and 53.3% of xylan conversion were obtained at enzyme loading of 10 FPU/g glucan. The fermentation of hydrolyzed slurry was performed using an engineered stain, Zymomonas mobilis TSH-01. A mass balance of the overall process was calculated, and 91.9 kg was achieved from one tonne of fresh sweet sorghum stalk.

Conclusions

A low energy-consumption integrated technology for ethanol production from sweet sorghum stalks was presented in this work. Energy consumption for raw materials preparation and pretreatment were reduced or avoided in our process. Based on this technology, the recalcitrance of lignocellulose was destructed via a cost-efficient process and all sugars in sweet sorghum stalks lignocellulose were hydrolysed into fermentable sugars. Bioconversion of fermentable sugars released from sweet sorghum bagasse into different products except ethanol, such as butanol, biogas, and chemicals was feasible to operate under low energy-consumption conditions.

     

Piercing and vacuum infiltration of the mature embryo: a simplified method for <Emphasis Type="Italic">Agrobacterium-</Emphasis>mediated transformation of <Emphasis Type="Italic">indica</Emphasis> rice

Traditional transformation methods are complex and time consuming. It is generally difficult to transform indica rice varieties using traditional transformation methods due to their poor regeneration. In this contribution, a simple method was developed for the transformation of indica rice. In this method, the mature embryos of soaked seeds were pierced by a needle, and then soaked in the Agrobacterium inoculum under vacuum infiltration. The inoculated seeds germinated and grew to maturation (T ₀) under nonsterile conditions. The herbicide or antibiotic analysis and molecular analysis were conducted on T ₀ plants. The results showed that although the efficiency of transformation was about 6.0%, it was easier to transform indica rice using the proposed method, and the transformation process was significantly shortened. The success of transformation was further confirmed by the genetic and molecular analyses of T ₁ transformants.     

Regional water resource implications of bioethanol production in the Southeastern United States

The Energy Independence and Security Act (EISA) of 2007 mandates US production of 136 billion L of biofuel by 2022. This target implies an appropriation of regional primary production for dedicated feedstocks at scales that may dramatically affect water supply, exacerbate existing water quality challenges, and force undesirable environmental resource trade offs. Using a comparative life cycle approach, we assess energy balances and water resource implications for four dedicated ethanol feedstocks – corn, sugarcane, sweet sorghum, and southern pine – in two southeastern states, Florida and Georgia, which are a presumed epicenter for future biofuel production. Net energy benefit ratios for ethanol and coproducts range were 1.26 for corn, 1.94 for sweet sorghum, 2.51 for sugarcane, and 2.97 for southern pine. Corn also has high nitrogen (N) and water demand (11.2 kg GJ_net^?1 and 188 m³ GJ_net^?1, respectively) compared with other feedstocks, making it a poor choice for regional ethanol production. Southern pine, in contrast, has relatively low N demand (0.4 kg GJ_net^?1) and negligible irrigation needs. However, it has comparatively low gross productivity, which results in large land area per unit ethanol production (208 m² GJ_net^?1), and, by association, substantial indirect and incremental water use (51 m³ GJ_net^?1). Ultimately, all four feedstocks require substantial land (10.1, 3.1, 2.5, and 6.1 million ha for corn, sugarcane, sweet sorghum, and pine, respectively), annual N fertilization (3230, 574, 396, 109 million kg N) and annual total water (54 400, 20 840, 8840, and 14 970 million m³) resources when scaled up to meet EISA renewable fuel standards production goals. This production would, in turn, offset only 17.5% of regional gasoline consumption on a gross basis, and substantially less when evaluated on a net basis. Utilization of existing waste biomass sources may ameliorate these effects, but does not obviate the need for dedicated primary feedstock production. Careful scrutiny of environmental trade‐offs is necessary before embracing aggressive ethanol production mandates.     

Transgenic sorghum with improved digestibility of storage proteins obtained by <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation

Development of transgenic plants with modified seed storage protein composition and increased nutritive value is one of the most promising areas of genetic engineering. This task is especially important for sorghum—a unique drought tolerant cereal crop that is characterized, however, by a relatively poor nutritive value in comparison with other cereals. It is considered that one of the reasons of the low nutritive value of the sorghum grain is the resistance of one of its seed storage proteins, γ-kafirin, located in the outer layer of endosperm protein bodies, to protease digestion. Using Agrobacterium-mediated genetic transformation, we obtained transgenic sorghum plants (Sorghum bicolor (L.) Moench) harboring a genetic construct for RNAi silencing of the γ-kafirin gene. In the T₁ generation, the plants with almost floury or modified endosperm texture of kernels were found. In these kernels, the vitreous endosperm layer has been reduced and/or covered by a thin layer of floury endosperm. In vitro protein digestibility (IVPD) analysis showed that the amount of undigested protein in transgenic plants from the T₃ generation was reduced by 2.9–3.2 times, in comparison with the original non-transgenic line, and the digestibility index reached 85–88% (in comparison with 59% in the original line). In T₂ families, the plants combining high IVPD with vitreous endosperm type were found. In the electrophoretic spectra of endosperm proteins of transgenic plants with increased digestibility, the proportion of 20 kD protein that is encoded by the γ-kafirin gene, was significantly reduced, in comparison with the original non-transgenic line. HPLC analysis showed total amino acid content in two out of the three studied transgenic plants from the T₂ generation was reduced in comparison with the original non-transgenic line, while the lysine proportion increased by 1.6–1.7 times. The mechanisms conditioning improved digestibility of storage proteins in transgenic plants are discussed. The results of experiments demonstrate that it is feasible to develop sorghum lines combining high protein digestibility and vitreous endosperm that has a high breeding value.     

Ethanol production potential of sweet sorghum assessed using forage fiber analysis procedures

Sweet sorghum (Sorghum bicolor (L.) Moench) is widely recognized as a highly promising biomass energy crop with particular potential to complement sugarcane production in diversified cropping systems. Agronomic assessments have led to identification of four cultivars well suited for such sugarcane‐based production systems in southern Louisiana. Sweet sorghum biofuel production systems are currently being developed, and research producing large sample numbers requiring ethanol yield assessment is anticipated. Fiber analysis approaches developed for forage evaluation appear to be useful for screening such large numbers of samples for relative ethanol yield. Chemical composition, forage fiber characteristics, digestibility, and ethanol production of sweet sorghum bagasse from the four cultivars were assessed. Measures of detergent fiber, lignin, and digestibility were highly correlated with ethanol production (P < 0.01). The best linear regression models accounted for about 80% of the variation among cultivars in ethanol production. Bagasse from the cultivar Dale produced more ethanol per gram of material than any of the other cultivars. This superior ethanol production was apparently associated with less lignin in stems of Dale. Forage evaluation measures including detergent fiber analyses, in vitro digestibility, and an in vitro gas production technique successfully identified the cultivar superior in ethanol yield indicating their usefulness for screening sweet sorghum samples for potential ethanol production in research programs generating large sample numbers from evaluations of germ plasm or agronomic treatments. These screening procedures reduce time and expense of alternatives such as hexose sugar assessment for calculating theoretical ethanol yield.     

Molecular mapping and candidate gene identification of the Rf2 gene for pollen fertility restoration in sorghum [Sorghum bicolor (L.) Moench]

The A1 cytoplasmic–nuclear male sterility system in sorghum is used almost exclusively for the production of commercial hybrid seed and thus, the dominant genes that restore male fertility in F₁ hybrids are of critical importance to commercial seed production. The genetics of fertility restoration in sorghum can appear complex, being controlled by at least two major genes with additional modifiers and additional gene–environment interaction. To elucidate the molecular processes controlling fertility restoration and to develop a marker screening system for this important trait, two sorghum recombinant inbred line populations were created by crossing a restorer and a non-restoring inbred line, with fertility phenotypes evaluated in hybrid combination with three unique cytoplasmic male sterile lines. In both populations, a single major gene segregated for restoration which was localized to chromosome SBI-02 at approximately 0.5 cM from microsatellite marker, Xtxp304. In the two populations we observed that approximately 85 and 87% of the phenotypic variation in seed set was associated with the major Rf gene on SBI-02. Some evidence for modifier genes was also observed since a continuum of partial restored fertility was exhibited by lines in both RIL populations. With the prior report (Klein et al. in Theor Appl Genet 111:994–1012, 2005) of the cloning of the major fertility restoration gene Rf1 in sorghum, the major fertility restorer locus identified in this study was designated Rf2. A fine-mapping population was used to resolve the Rf2 locus to a 236,219-bp region of chromosome SBI-02, which spanned ~31 predicted open reading frames including a pentatricopeptide repeat (PPR) gene family member. The PPR gene displayed high homology with rice Rf1. Progress towards the development of a marker-assisted screen for fertility restoration is discussed.     

Simultaneous saccharification and fermentation of sweet sorghum carbohydrates to ethanol in a fed-batch process

Summary The simultaneous saccharification and fermentation (SSF) of sweet sorghum carbohydrates to ethanol by Fusarium oxysporum F3 alone or in mixed culture with Saccharomyces cerevisiae 2541 or Zymomonas mobilis CP4 in a fed-batch fermentation process was studied. While SSF was adequately carried out by the first microorganism the process achieved its maximum value by the mixed culture of the fungus and yeast. Under optimum conditions, ethanol yields and concentrations as high as 29.7 g of ethanol per 100 g of dry sorghum stalk and 7.5 % (w/v) respectively were obtained. These values together with the high yield of sorghum crop in Greece make this process promising and worthy of further investigation for the production of fuel bioethanol.