Introduction

Renewable energy derived from recently living organisms, such as bioethanol, biodiesel, and biogas will play a major role in achieving the goal of energy-independency from fossil fuels, stimulating the rural economy, and alleviating the pressure of global warming [28]. Sorghum [Sorghum bicolor (L.) Moench] is a highly efficient photosynthetic C4 plant species that has been found to be a promising feedstock for bioethanol production [5]. Sugars derived from the juice, starch from grain, and cellulose and hemicellulosic polysaccharides from biomass of sorghum can be converted to ethanol. Sorghum has the highest water use efficiency among major crop plants and is extraordinarily tolerant to low soil fertility, important traits that are essential for survival and productivity in arid and semi-arid areas with limited irrigation capability. These traits make sorghum particularly advantageous as an alternative bioenergy feedstock because it can be grown profitably on marginal land and therefore, would not remove more fertile land from existing food and fiber production [14].

As a close relative to bioenergy perennial grasses such as switchgrass, sugarcane, and Miscanthus, sorghum has been proposed as a model plant for plant-based bioenergy research [5]. Recent progress in sorghum genomic studies has generated a series of important resources and tools that can be used to identify favorable genes or alleles for enhancement of resistance to abiotic stresses and for improvement of biofuel-related traits. For example, well-established genetic, physical, and cytological maps facilitate the mapping and identification of genes responsible for important agronomic traits. Furthermore, the construction of cDNA microarrays provides a platform for high throughput gene discovery [3, 17]. An important milestone was the recent completion of the genomic sequence for the sorghum inbred line, BTx623 (http://www.phytozome.net/sorghum). The identification of sorghum genes contributing to the “bioenergy quality” of sorghum biomass will undoubtedly be accelerated through utilization of these important genomic resources and biotechnological tools.

A number of plant traits have been demonstrated useful for production of bioenergy including brown midrib mutations for high conversion efficiency from biomass to oligosaccharides [15], high biomass yield, and better tolerance to abiotic stresses [5, 22]. A collection of sorghum natural and induced mutations (available at Plant Stress and Germplasm Development Unit, USDA-ARS at Lubbock, Texas) and a sorghum diversity panel provide useful resources for selecting agronomic traits for bioenergy production [6]. However, the number of lines is very limited for both populations. A systematically pedigreed mutagenized population will facilitate the selection of mutants with traits beneficial to bioenergy conversion efficiency or biomass production and elucidation of the function of the genes identified through cell wall genomics. Sorghum mutagenesis has been conducted in the past, however, the resulting lines were not preserved or are currently not available [11, 13]. Here, we report the generation of an ethyl methanesulfonate (EMS)-mutagenized sorghum mutant population, describe several useful mutant phenotypes, and discuss their potential utilization for bioenergy research. Results from our on-going phenotyping and genotyping studies indicate that this mutant population can serve as a useful genetic resource for both sorghum functional genomics and bioenergy research.

Methods

Line Purification and Mutagenesis

The sorghum [Sorghum bicolor (L.) Moench] inbred line BTx623, which has served as a parent for several mapping populations and the source for genome sequencing [1, 20, 26], was used to generate the pedigreed mutant population. BTx623 seeds were obtained from the National Germplasm Resources Information Network of USDA-ARS (http://www.ars-grin.gov/). Initial observations found that the seedlings from the original seeds showed minor variations in height and panicle size. However, no genetic heterogeneity was detected using ten publicly available SSR markers [12]. To ensure the homogeneity of the seeds used for mutagenesis, the original line was self-fertilized for six generations by single seed descent (SSD) to purify the line. At every generation, one plant that displayed the most typical characteristics of the original BTx623 was selected for further propagation. After six generations of selfing and purification, about 2 kg seeds were obtained for mutagenesis.

From the purified BTx623, batches of 100 g of dry seed (∼3,300 seeds) were soaked with agitation (16 h at 50 rpm on rotary shaker) in 200 ml of tap water containing EMS concentrations ranging from 0.1 to 0.3% (v/v). The treated seeds were subsequently thoroughly washed in about 400 ml of tap water for 5 h at ambient temperature, changing the wash water every 30 min. Then the mutagenized seeds were air-dried and prepared for planting.

Field Planting and Mutant Population Development

The air-dried seeds were planted at a density of 120,000 seeds per hectare. Before anthesis, each panicle was bagged with a 400 weight rain-proof paper pollination bag (Lawson Bags, Northfield, IL) to prevent cross pollination. After bagging, each bag was injected with 5 ml chlorpyrifos (Dow AgroSciences) at 0.5 ml/liter to control corn earworms that might hatch within the bag and destroy the seeds. Sorghum panicles were harvested manually and threshed individually. Each fertile panicle was planted as an M2 head row. Three panicles from each M2 head row were bagged before anthesis and only one fertile panicle was used to produce the M3 seeds. Duplicate leaf samples were collected from the same fertile plant for extracting DNA, and both the leaf samples and the panicle were barcoded. To avoid cross-contamination of leaf samples with dead pollen that could fall onto the leaves during pollen shedding, leaves were thoroughly rinsed with de-ionized water before sampling. The seeds from the barcoded plants were harvested and used to propagate the M3 generation. It should be noted that substantial mutant lines displayed diminished seed production during the M3 generation. Thus, ten panicles were bagged for each M3 head row and pooled as M4 seeds. The M4 seeds will be made available to the sorghum research community for forward and/or reverse genetic studies.

Field Observation and Phenotyping

Mutation phenotypes were systematically evaluated by visual observation in the M3 generation. Limited phenotyping was conducted at the M2 generation. Due to the large number of the mutants selected at M2 generation that displayed poor seed setting, systematic evaluation of mutant phenotypes was deferred to the M3 generation. Each M3 row was carefully inspected at least three times during the growing season (before flowering, after flowering, and when the majority of the plants reached physiological maturity). Distinguishable phenotypes were recorded and photographed with a digital camera.

Results

EMS Concentration and Mutation Rate for Development of Mutant Population

The fully sequenced sorghum inbred line BTx623 was used to generate the pedigree for the mutant population. To ensure obtaining an adequate mutation rate and sufficient number of mutagenized lines, a range of concentrations from 0.1 to 0.3% (v/v) EMS was used to generate the mutant population. Approximately 50,000 sorghum seeds were treated with EMS (∼10,000 seeds for each of the EMS concentration at 0.1%, 0.15%, 0.2%, 0.25%, and 0.3%). Over 10,000 fertile panicles were obtained from the M1 plants treated with various EMS concentrations (Table 1). Sorghum appeared to be very sensitive to EMS treatment. Even at 0.1% EMS, only about 40% of planted M1 seeds grew into viable plants that set M2 seeds. Only 41 fertile panicles were obtained from the 10,000 0.3% EMS-treated M1 plants; most of the M2 seeds from the panicles failed to germinate or developed into healthy plants in the next generation. 0.25% EMS treatment seemed to be the highest concentration that resulted in reasonable number of fertile panicles. The panicles from M1 plants treated with various EMS concentrations were threshed individually to develop the pedigreed mutant population.

Table 1 Number of M1 panicles that set seeds. About 10,000 mutagenized seeds were planted for each EMS concentration. Additional 500 lines treated with 0.25% EMS were developed in greenhouse
Full size table

Since the EMS concentrations used to generate this mutagenized sorghum population were relatively low compared with those used in other plants [7, 10, 21, 24], the M2 seeds from 0.25% EMS treated plants were used first to develop the pedigreed population. To maximize the utility of the limited M2 seeds, 50 (or all if less than 50) of the M2 seeds from each M1 panicle were planted as a head row. Three panicles from each row were bagged before anthesis. To prevent redundancy of mutations, only one fertile plant from each M2 head row were selected to produce M3 seeds. A total of 1,246 M3 lines were produced in the field. An additional 500 M3 lines were produced in the greenhouse. Each M3 family represented an independent mutant population derived from one original EMS-treated seed. In total, about 1,600 mutant lines were advanced to the M3 generation or had produced the pooled M4 seeds.

Mutants with Clear Phenotypes in the Field Observation

For a thorough characterization of mutant phenotypes, the lines within the mutagenized population will need to be screened under different conditions such as chemical stress (nutrition or salt), physiological stress (drought, high temperature, cold, and altered light intensity) in both greenhouse and field. Identification of obvious morphological alterations in the field is one of the easiest and least expensive approaches for mutant screening. Because of the high rate of sterility that still existed in the M2 generation, systematic evaluation of the mutation phenotypes of the pedigreed mutant population was deferred to the M3 generation. The advantage of screening mutants using M3 plants is that any observed mutant phenotype is most likely preserved in the pedigreed family since all progenies within an M3 family are derived from germlines of the same genotype. Throughout the growing season, the M3 families were repeatedly evaluated for phenotypes distinctive from the wild type BTx623. In the field, all M3 families were inspected closely for novel morphological traits compared to the wild type. Every M3 family segregated for at least one distinctive mutant phenotype and many lines segregated for multiple mutant phenotypes at an approximately 1:3 ratio (mutant phenotype to wild type). The observed mutant phenotypes can be viewed online (http://www.lbk.ars.usda.gov/psgd/sorghum/till/index.aspx). A number of mutants with traits suitable for bioenergy research are described in detail in the following sections.

Novel Sorghum brown midrib Mutants

Mutagenesis is largely a destructive process that eliminates, reduces, or modifies the function of specific gene products and/or metabolic pathways. However, mutant phenotypes that may play positive roles in crop improvement can be produced. A particular class of phenotypes that may be useful for improving bioenergy conversion efficiency is represented by the sorghum brown midrib (bmr) mutants, because of the decrease in the lignin content of sorghum biomass and the increase in sugar yield after enzymatic saccharification [15, 22]. Nineteen sorghum bmr mutants were previously isolated via chemical mutagenesis [13]. Including several spontaneous bmr mutants, a total of 28 bmr mutants are known in sorghum [15, 22]. We identified ten new independent bmr mutants from 768 M3 families that were used in a pilot TILLING (Targeting Induced Local Lesions IN Genome) study [25]. The new bmr mutants were named sequentially following the order of the previously known bmr mutants (Fig. 1a). Among the ten newly isolated bmr mutants, bmr34 and bmr35 were found to harbor mis-sense mutations in the gene encoding caffeic acid O-methytransferase (COMT), a key enzyme in lignin biosynthesis [2, 4, 23]. In bmr34, a transition mutation from G:C to A:T converted the proline (Pro) residue at 150 to leucine (Leu); in bmr35, the glycine (Gly) residue at 325 was converted to serine (Ser). Both mutations represent COMT alleles different from the previously identified bmr12, bmr18, and bmr26 alleles which contain non-sense mutations resulting in premature stop codons [2, 25]. No additional mutations were found in the COMT gene in the remaining bmr mutants. Some of these mutants may represent novel loci that regulate lignin biosynthesis.

Fig. 1
figure 1

Selected mutants with traits suitable for bioenergy research. a New sorghum brown midrib (bmr) mutants. Sorghum bmr mutants were isolated from M3 families. Only one bmr mutant was harvested from each family. The new bmr mutants were named sequentially following pre-existed sorghum bmr mutants. The bmr phenotype was further confirmed in the M4 plants. Top panel, one-month old wild type and bmr mutant plants. Bottom panel, leaf segment from the eighth (the youngest fully expanded) leaf. b An example of sorghum erect leaf (erl) mutant. c Examples of multiple tiller mutants. The photographs were taken on September 29, 2008 when the wild type plants had reached physiological maturity. The photograph in the middle is a multiple tiller mutant that has similar maturity to wild type. The photograph on the right is a multiple tiller mutant that matures late

Full size image

Erect Leaf Mutants

In general sorghum exhibits an open canopy with wide leaf angles that almost parallel to the ground. However, in other species such as maize, a major trait that has contributed to yield improvement and productivity is the introduction of acute leaf angles that allow maize to be planted at higher densities to capture more sunlight per unit land area [8]. Erect leaf (erl) mutants were also observed in our mutagenized sorghum population (Fig. 1b). Among the 1,600 M3 plots in the field, over 50 plots segregated for leaf angles that vary from the parent line. Although these erl mutants need to be confirmed under different environments and plant densities, they may prove to be useful for improving sorghum biomass and grain production based on the yield improvement achieved in hybrid maize with increased planting density since the advent of hybrid technology [8].

Multiple Tiller Mutants

Another class of mutants potentially useful for bioenergy research is multiple tiller mutants (mtl) (Fig. 1c). Wild type BTx623 plants usually develop one to three stalks under the environmental conditions at Lubbock, TX and using sub-surface drip irrigation and may also develop a few upper nodal tillers after the seeds on the main panicle reach physiological maturity. Over 60 M3 plots segregated for tillering mutants that produced from 6 to over 20 healthy tillers. These multiple tiller mutants may be a useful genetic resource for increasing biomass yield or improving the efficiency for converting biomass into cellulosic ethanol in sorghum breeding programs.

Late Flowering Mutants

Early flowering cultivars are required for grain production at certain growing zones but late flowering cultivars may be required for biomass production because extending the growing season may increase the biomass yield proportionally. Furthermore, if no grain is involved in the collection of biomass it may simplify the process of converting biomass into cellulosic ethanol. Many M3 plots segregated for moderately late flowering phenotypes. Extremely late flowering mutants as shown in Fig. 1c were rare. From the 1,600 M3 plots, nine M3 lines segregated for extreme late flowering phenotype. Some of the late flowering mutants showed an ideal phenotype for enhanced biomass production.

Discussion

The US government has set a target to replace 30% of the petroleum fuel used for transportation with renewable resources by 2030 [9]. Bioenergy derived from plant products will play a major role. The perennial C4 grass switchgrass and Miscanthus are considered superior potential feedstocks because of their highly efficient C4 photosynthesis, long growing season, and water use efficiency [5, 18]. As a C4 plant with excellent tolerance to drought and high temperature stresses, low soil fertility, and high water use efficiency, sorghum may serve as another potential feedstock for bioethanol production. A large collection of germplasm, well established genetic maps, completed genome sequence, and close evolutionary relationship to the other C4 biofuel feedstocks make sorghum an ideal model plant for bioenergy research [5]. Our pedigreed sorghum mutant population adds a useful genetic resource to help identify traits and the associated genes that are important for biomass yield and biomass conversion efficiency. In this population, each line (M3 family) was derived from a single M1 seed. The advantage of this pedigreed population is that it provides an immortal supply of seeds, allowing multiple screens for mutants carrying traits useful for improving agronomical performance and enhancing bioenergy yield and biomass conversion efficiency. Well-characterized mutants will also assist in determining the underlying biological processes associated with these desirable traits.

Biomass yield and biomass conversion efficiency are two critical factors for bioenergy feedstocks [8, 22]. Mutations that can potentially improve both traits were observed in the sorghum mutant population. The bmr mutants have been demonstrated in both maize and sorghum to produce higher sugar yield from enzymatic saccharification of stovers [22]. From a small-scale pilot TILLNG study, 10 bmr mutants were identified including two novel alleles of bmr12 (Fig. 1a). In 2008 field plots, over 50 independent bmr mutants were identified. These mutants are yet to be confirmed in the next generation. Some bmr mutants may represent different alleles of the previously known bmr mutants, i.e. bmr6 or bmr12, but others may represent new loci. Genetic classification or genetic complementation study are required to determine how many loci these bmr mutants may represent. Nevertheless, our observations demonstrate that the sorghum mutant population possesses valuable mutations likely to contribute to improved biomass conversion efficiency.

The cell wall is a major component of biomass and the conversion efficiency of cell wall to fermentable sugars is determined by its structure and composition. Plants devote ∼2,500 genes (∼10% of the genes in their genomes) to construction and dynamic rearrangement of their cell walls during growth [19, 27]. About 1,200 genes related to cell wall biogenesis and modifications have been identified within the maize genome through characterization of maize mutants and bioinformatic annotation (http://cellwall.genomics.purdue.edu). The sorghum mutant population and the completed genome sequence will help to identify genes responsible for sorghum cell wall biosynthesis and modification.

Compared with sorghum, a distinct characteristic of modern maize hybrids is acute leaf angles that allow high planting density to capture more sun radiation per unit land area [8]. Erect leaf mutants in rice have been shown to display increased biomass and grain yield [16]. The sorghum erl mutants isolated from our sorghum mutant population, coupled with multiple tillering traits, provide potential resources to improve biomass and grain yields of sorghum. Furthermore, high biomass yield traits may be stacked with traits that increase biomass conversion efficiency to develop ideal feedstocks for bioenergy (for example, crossing bmr mutants with mtl mutants and/or erl mutants to develop double or triple mutant plants).

In summary, a pedigreed mutant population in sorghum has been generated using EMS-mutagenesis. Phenotyping through visual observation has identified traits that are potentially useful in improvement of biomass production and conversion efficiency. This mutant population is likely also to contain traits that are not immediately visible but do affect biomass quality, digestibility or yield. In order to fully capture the utility of the sorghum mutant population, mutants will need to be screened under different conditions. In the next few years we intend to add additional mutant lines to this population. A number of the mutant lines identified from this population are currently being characterized by other researchers for their potential utility in bioenergy production. We believe this sorghum mutant population is a valuable resource for the scientific community. Multilateral collaborations screening this mutant population combined with the characterization of the various mutants will provide valuable information for the study and improvement of sorghum as a bioenergy crop. Interested researchers who wish to screen the sorghum mutant population can make arrangements through contacting the authors at zhanguo.xin@ars.usda.gov.