Participatory Plant Breeding and Agroecology to Develop Intermediate Wheatgrass for Sustainable Grain Production

Participatory Plant Breeding and Agroecology to Develop Intermediate Wheatgrass for Sustainable Grain Production

Summary

Perennial crops that live for many years increase sustainability by holding soil, reducing nutrient runoff, limiting pesticide use, and boosting farmer incomes through a decrease in inputs. All commonly grown grain crops are annuals, but we are working to develop intermediate wheatgrass (IWG), which has potential to become the first widely grown perennial grain crop.

This project will develop improved IWG plants and determine the effect of nitrogen fertility on sustained grain yield in IWG.

We are conducting a participatory plant breeding program that includes growing IWG on college campuses, on an NGO research station in Kansas, and at a commercial nursery in Wisconsin in order to identify superior genotypes. Through cross-pollination, we are combining several important traits that we have discovered: large seed, shortness, and shatter resistance. To answer the key question of the role of soil nitrogen (N) in sustained yield, a range of N addition treatments have been applied to IWG stands on three on-farm fields and at research stations in Kansas and Minnesota. Plant tissue nitrogen and seed yield traits are being measured.

Results from the proposed research will be published in peer-reviewed journals, reported to the scientific community through seminars, published in the popular press (web and hard copy), and presented to several hundred yearly visitors to The Land Institute.

Objectives/Performance Targets

We expect the proposed project to contribute to the following outcomes:
• Increased knowledge of how to grow and breed IWG for use as a perennial grain crop. Through experimentation and interaction with members of the community of practice, we have been improving our methodology for growing and breeding IWG. We expect this trend to continue, which will be evidenced by accelerated progress in the breeding program and improved techniques for growing the crop.

• Increased optimism for the potential of IWG as a crop in the food and agriculture community. Optimism is essential to achieving expanded research, development, and planting of the crop. This optimism will be derived from clear progress in the breeding program and the discovery of reliable techniques for raising a productive crop.

• Increased scientific research with IWG throughout the north central United States. For a new crop to succeed, a diverse team of researchers must be assembled to work on the challenges as they arise. Interest from scientists in many fields is growing, and we soon hope to have a wide array of scientific collaborators.

• IWG is planted on a large acreage for commercial use, which reduces soil erosion on sloping lands. This is an intermediate term outcome. Commercial plantings depend upon the development of improved varieties, agronomic practices, processing techniques, and marketable products. We expect that even with sustained research funding, this outcome is at least 10 years away.

Short-term outcomes will include increased knowledge of how to grow and breed IWG, optimism for its potential as a crop within the food and agriculture community, and an increased number of scientists actively engaged in IWG research.

Intermediate outcomes will include widespread planting of IWG by farmers and commercial use of the grain for food. To evaluate, we will measure indicators of progress toward these outcomes.

In the long term, we anticipate that diverse perennial grain cropping systems could replace more than 50% of current annual crop acreage. Success in breeding, growing, processing, and marketing IWG will serve as a proof of concept for perennial grains in general. IWG will spark interest in and funding for many other perennial grains, including wheat, rice, sunflowers, dry beans, maize and sorghum. Wherever perennial grain crops are planted, soil erosion will be reduced below replacement levels, nitrate loss to ground and surface waters will be reduced by more than 90%, and herbicide contamination will be sharply reduced. Furthermore, reduced input costs will benefit farmers and rural communities economically.

Accomplishments/Milestones

Trials with nitrogen fertilizer rates ranging from zero to 200 lbs/acre were established at four locations. Biomass was most responsive on the farm with the loam soil. On the farm with sandy soil, water was limiting and therefore biomass was less responsive. But at both locations, 100 kg per ha appeared to be adequate for maximum biomass yield. On the organic farm, biomass yield responded differently, probably due to the very low availability of nitrogen provided in an organic form to the soil surface of a perennial plant.

Seed yield was variable across locations, but general trend indicated that in this year fertilization with 100 kg/ha was adequate to insure maximum seed yield potential (see figure).

We used controlled pollinations in the greenhouse to mate plants outstanding for seed size, seed yield, short stature, free threshing ability, earliness, and non-shattering. Seed of these plants were planted in small pots and transplanted to the field in fall 2011 to establish a new breeding nursery with about 14,000 individuals. We have kept track of both the male and female parents of every cross so that the performance of families as well as individuals can be used in making selections. See attached pedigree figure for a visual illustration of three generations of intermediate wheatgrass breeding.

This year we again performed an additional cycle of bulked mass selection for large, naked seed. We used a robotic seed size sorter to select the largest seeds out of 65,000 naked seeds that were harvested from the previous generation. The largest seeds were planted, and a new nursery containing 142 plants was established fall 2011.

Starch analysis. Determination of the total starch content was performed by using an enzymatic approach. Starch was hydrolyzed to glucose using ?-amylase and amyloglucosidase. Glucose was then measured by enzymatic oxidation forming gluconic acid and hydrogen peroxide which oxidizes p-hydroxybenzoic acid and 4-aminoantipyrine to a quinoneimine dye. Dye formation was monitored spectrophotometrically. Total starch content was determined to be 43.7 ± 0.9% (n=3), considerably lower than the starch contents in bread wheat (Triticum aestivum L.).

The determination of the amylopectin/amylose ratio was based on the formation of a complex between the lectin concanavalin A and amylopectin leading to a precipitation. Amylose and the precipitated amylopectin were separated and determined by enzymatic hydrolysis to glucose which was analyzed similar as described above. The amylose content was determined to be 22.95 ± 0.42% of the total starch content. Thus, the determined amylose/amylopectin ratio of ca. 23/77 is a common ratio for cereal grains.

Starch functionality was determined on the Rapid Visco Analyzer. The IWG had a much lower peak viscosity than did whole wheat pastry flour (1200 cps vs 3200 cps). This can be due to there being less starch in the flour as evidenced by the higher protein content in the IWG compared to the pastry flour. The lower peak viscosity indicates that it could be hard to have enough viscosity in a batter to hold gas during chemical leavening, and if one were to use IWG as the sole flour, it would probably be necessary to add a pregelatinized starch or gum to improve the baked volume.

Non-starch polysaccharides. Non starch polysaccharides were preparatively isolated using a heat stable ?-amylase, amyloglucosidase, and the protease alcalase. Water insoluble non-starch polysaccharides were separated by centrifugation and water soluble non-starch polysaccharides were precipitated in 80% ethanol. Both ash and residual protein content in the soluble and insoluble non-starch polysaccharide preparations will be analyzed. The neutral monosaccharide composition of the non-starch polysaccharides will be determined by acid hydrolysis, followed by reduction and acetylation of the liberated neutral monosaccharides. The formed alditol acetates will be analyzed by GC-FID. Uronic acids will be measured enzymatically after acidic hydrolysis of the polysaccharides.

Hydroxycinnamic acids. The contents of ester-linked monomeric hydroxycinnamic acids, i.e. ferulic acid (trans/cis), p-coumaric acid, and sinapic acid, were analyzed by RP-HPLC-UV after alkaline hydrolysis of the flours. Alkaline hydrolysis was performed for 18 h at room temperature using 2 M NaOH. Liberated hydroxycinnamic acids were extracted from the acidified hydrolysate using liquid/liquid extraction with diethyl ether. The combined organic phases were dried, the residue was re-dissolved in methanol/water, and monomeric hydroxycinnamic acid contents were analyzed using HPLC: trans-ferulic acid, 910 ± 133 ?g/g; cis-ferulic acid , identified, but below limit of quantification; trans-p-coumaric acid, 50 ± 13 ?g/g; trans-sinapic acid, 140 ± 25 ?g/g. The ferulic acid contents are higher than those usually found in bread wheat (roughly between 350 – 600 ?g/g).

Ferulic acid dimers and trimers were analyzed by RP-HPLC-MS. Alkaline hydrolysis and extraction was performed as described for the hydroxycinnamic acid monomers. Separation and detection, however, was performed by HPLC-MS. The following amounts of ferulic acid dimers (dFA) and trimers (tFA) were determined: 8-8cyclic-dFA, 86 ± 0 ?g/g; 8-8non-cylic-dFA, 17 ± 4 ?g/g; 8-8tetrahydrofuran-dFA, 6 ± 1 ?g/g; 8-5cyclic-dFA, 38 ± 2 ?g/g; 8-5non-cyclic-dFA, 14 ± 3 ?g/g; 8-5decarboxylated-dFA, not detected [sum 8-5-dFA, 52 ?g/g]; 5-5-dFA, 29 ± 6 ?g/g; 8-O-4-dFA 66 ± 5 ?g/g; 5-5/8-O-4-tFA 5 ± 3 ?g/g; 8-5non-cyclic/8-O-4-tFA, detected, but below limit of quantification; 8-5non-cyclic/5-5-tFA, detected, but below limit of quantification; 8-8cyclic/8-O-4-tFA, detected, but below limit of quantification, 8-O-4/8-O-4-tFA, not detectable. The diferulic acid contents are comparable to those found in bread wheat; however, the 22.9 composition is very different. While 8-5-coupled dimers usually dominate in wheat (and in most other cereals) the 8-8-coupled dimers, especially the 8-8cyclic-dimer, are dominant in the intermediate wheat grass. Surprisingly, most of the trimers were found below the limit of quantification. This is somewhat unusual given the high amounts of monomeric ferulic acid attached to the intermediate wheat grass cell wall polymers.

Protein. The intermediate wheatgrass had much higher protein levels than what is typically found in wheat flour. Protein content was analyzed using a nitrogen analyzer following the Dumas AOAC method. Using a protein conversion factor of 5.7, the IWG averaged 17.55% protein, whereas wheat pastry flour was measured at 10.7% protein. For reference, all-purpose flour is typically around 10% protein, bread flour about 12% and whole-wheat flour around 14% protein. Gluten, gliadin and glutenin fractions were extracted from IWG using a series of solvent extractions with water-saturated butan-1-ol, NaCl and aqueous ethanol and aqueous propan-1-ol. Gluten was extracted from whole wheat flour and white all-purpose flour following the traditional hand wash method. Gliadin and glutenin fractions were also extracted from whole wheat flour and white all-purpose flour following the mentioned series of solvent extractions. Protein fractions were analyzed by SDS-poly acrylamide gel electrophoresis using hand-cast 13% acrylamide gels. Compared to whole and white wheat flours, the IWG lacked high molecular weight (HMW) glutenins, which are responsible for the elasticity of a dough system. IWG gluten had mostly ? and ? gliadins and some low molecular weight (LMW) glutenins. Amino acid profiling of the IWG protein is underway, and results will be provided soon.

Baking tests. Standard AACC cookie, biscuit and bread baking tests were conducted with IWG being compared to equivalent flours for each product. For the cookie tests, IWG cookies had a similar spread but were slightly thicker, so the spread factor was lower than for all-purpose flour and whole wheat pastry flour. But structurally, IWG makes an acceptably shaped and set cookie. For biscuits, IWG had similar height and spread to whole wheat pastry flour and all-purpose flour. IWG performed very differently when making bread compared to whole wheat and white bread flour. For the same formula, the height in mm at the center of the loaf was 117 mm for bread flour, 50 mm for IWG and 65 mm for whole wheat flour. There was also much less of a crown formed. The gluten forming ability was also lacking, and it was almost impossible to sheet the dough with IWG as it would shred and not hold together.

Flavor Analysis. The flavor compounds in traditional yeast leaven bread made from whole grain hard wheat versus intermediate wheat grass (IWG) were characterized by gas chromatography-olfactometry (GC-O) and gas chromatography-mass spectrometry (GC-MS) techniques. Immediately after baking, the crust was removed, frozen with liquid nitrogen, and ground with a mortar and pestle. The ground sample was subsequently extracted with 1 L dichloromethane [spiked with both 20 ppb of 2-methyl-3-heptanone (internal standard) and butylated hydroxytoluene (BHT)] for 15 hours under a blanket of argon gas, at room temperature under stirred agitation. The dichloromethane layer was removed, and another 1 L of dichloromethane was added, with stirred agitation under argon, for another 3 hours, and also removed. The combined extracts were dried over sodium sulfate and concentrated by vigreux distillation to 200ml, then fractionated by a high vacuum distillation (Solvent Assisted Flavor Evaporation – SAFE). The volatile isolate was concentrated by vigreux distillation to a final volume of 1 mL. Qualitative GCO analysis of the flavor isolate indicated differences in the intensity of the ‘key’ flavor compounds 2-acetyl-1-pyrroline, methional, 2-ethyl-3,5-dimethylpyrazine, furaneol, acetyl formoin, 2-acetyl-2-thiazoline and 2-phenylethanol (identified by GC-MS) between the IWG and hard wheat samples. The differences noted in flavor development in the IWG (in comparison to the wheat sample) provide a basis to design IWG products to have a more traditional flavor of wheat-based bread.

Conclusion. The data gathered so far revealed some key characteristics and challenges that need to be addressed in order to use IWG in food applications. Additional characterization is needed so that breeders can modify key traits to enhance the quality of IWG.

IWG nurseries were established using clonally propagated plants at three college campuses. We tried to establish these plots in the spring to accelerate the program. Unfortunately, it was a stressful summer in much of the country and therefore establishment was therefore not as good as we hoped. However, we still hope to get meaningful data from the experiments next year.

• Intermediate wheatgrass biomass response to N at three locations IWG_N_Biomass
• Average seed yield of intermediate wheatgrass in response to N
• Full pedigrees of 14,000 plants in third generation of IWG breeding pedigree

Impacts and Contributions/Outcomes

As a result of SARE-funded research with IWG over the past several years, two breeding programs to develop IWG for use as a perennial grain have been initiated at universities. Additionally, work to develop food products from IWG grain will be expanding.

Intermediate wheatgrass has received attention in the popular press over the past year, most notably in the National Geographic Magazine: http://ngm.nationalgeographic.com/2011/04/big-idea/perennial-grains-text.

Collaborators: