2005 Annual Report 1.What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? What does it matter? Despite the global acceptance of dry bean (Phaseolus vulgaris L.) as a food source and the nutritional superiority of the seeds of this crop compared to cereals, root crops, and other vegetables, beans do not supply inherent nutrients to the fullest potential to humans. To enhance the food value of beans, several constraints must be resolved, and these form the objectives for the work of this project: a. Determine why beans are less digestible than other crops to improve the bioavailability of nutrients, b. Determine the chemical changes that occur in seeds causing polymerization of macromolecules and how these compounds interact to cause after-darkening and seed hardening during storage, c. Research the hard-to-cook defect that leads to prolonged cooking time, d. Reduce flatulence in beans, e. Determine the positive and negative effects of seed coat pigments (flavonoid and polyphenolic compounds) in human diets, f. Determine the inheritance of food quality component traits, and discover molecular markers associated with the traits to increase selection efficiency, and g. Release new cultivars in all market classes with improved nutrient bioavailability. Approach: The determination of indigestible starch in beans includes digestion with enzymes, solubilization of the starch, and enzymatic degradation to glucose-the basic carbohydrate unit of starch. Enzymatic digestion is performed using the Total Dietary Fiber (TDF) assay. In this assay three sequential enzyme digestions are carried out using alpha-amylase, protease, and amyloglucosidase, where each enzyme has a specific function in the process of digestion. The indigestible starch remaining in TDF is determined through solubilization with 2M NaOH, and glucose content is calculated after digestion with amyloglucosidase. Using the digestion procedure mentioned, we will screen dry bean genotypes of diverse origin that mirror the adapted accessions grown in the U.S. to assess variability for TDF, resistant starch, and indigestible protein. The next step in the research is to develop unique populations where we can employ tools of molecular biology to locate (map) the genes controlling starch digestibility on specific chromosomes. DNA sequences located in close proximity to the genes involved in digestibility and other food quality traits can then be used as markers to assist the breeding efforts and, thus, increase the efficiency of the breeding process. Phytic acid, the major storage form of phosphorus (an important mineral for growth and development in plants) in dry bean seeds, reduces the bioavailability of zinc in human diets. Marginal zinc deficiency is a widespread problem, especially for people consuming vegetarian diets rich in food legumes to satisfy protein requirements. Because trace minerals, like zinc, are important not only for human nutrition but also for plant nutrition, plant breeding holds great promise for making a significant, sustainable, low-cost contribution to the reduction of micronutrient deficiencies in humans. Breeding for reduced levels of phytic acid along with increased zinc density will be a useful strategy to increase the amount of bioavailable zinc in diets. Pigments imparting color to bean seed coats belong to a class of simple phenols known as flavonoids. Flavonoids obtained commercially and also those isolated from various plant species are known to be effective free radical scavengers. Compounds that disrupt free radical formation are called antioxidants. Condensed tannins and hydrolyzable tannins of high molecular weight are also known to be effective antioxidants and have greater antioxidant activity than the monomer flavonoids. The establishment of the relationship between the Mendelian genes controlling bean seed coat color synthesis and the individual flavonoids and tannins produced may open new opportunities for bean breeders to develop new germplasm that provides industry with opportunities of commercial potential as nutraceutical foods. Additionally, the resolution of the genes responsible for flavonoid and tannin formation, along with the antioxidant activity of these compounds may enable breeders to select for a range of antioxidant potential and, thus, balance antioxidant activity with antinutritional effects. Dry bean has been used as the primary source of protein and calories for many populations lacking meat in the diet, particularly in the lesser developed countries of Africa and Latin America. However, heightened U.S. consumer awareness of the link between food consumption and health has led to the promotion of less saturated fat, cholesterol, sugar, and sodium (salt), in the diet, and a preference for complex carbohydrates such as fiber. Hence, dry bean has been promoted as a health food because of its superior nutritional quality, especially dietary fiber and low fat. The food guide pyramid re-designed by the USDA in 2005, recommends eating several cups a week of cooked dry beans under the vegetable group, which calls for 2 ½ cups of vegetables per day. Beans may also be categorized under the meat group if an individual, such as a vegetarian, does not receive adequate protein through meat consumption. Gastrointestinal discomfort from eating beans is often the result of the poor digestibility of bean carbohydrates and protein. As beans cook, the starch granules melt, hydrate, and swell. Swelling of the granules, called starch gelatinization, causes starch to become sticky and absorb more water. The degree of gelatinization that occurs as beans cook largely determines the completeness of digestion of starch. Dietary starch that does not digest is known as "resistant" starch. Resistant starch is a large contributor to gastrointestinal discomfort. The suppression of gelatinization may be caused by a limited supply of water to hydrate starch inside the cell because of insufficient space inside the cell associated with the physical strength of the cell wall. We will determine if cell walls crystallize during cooking by observing optical changes that occur with a light microscope. The loss of birefringence, as the optical changes that occur are called, under polarized light microscopy is used as an indicator of crystallization. 2.List the milestones (indicators of progress) from your Project Plan. Identification of cell structural factors: • Determine resistant starch (FY03/04) • Determine influence of mechanisms that entrap starch in cells (FY05/06) • Determine the effect of cell wall crystallization on cellular breakdown and cooking time (FY07/08) Determination of the genetic basis of food quality traits: • Determine the inheritance of starch digestibility (FY07/08) • Determine the inheritance of protein digestibility (FY07/08) • Determine the inheritance of phytic acid (FY04/05) • Identify major genes affecting culinary quality traits (FY04/05) • Identify markers associated with seed coat microstructural traits (FY04/05) • Determine the role of the seed coat microstructure in water imbibition and color loss due to leaching in black beans (FY05/06) Enhancement and release (germplasm): • Improved food quality (FY07/08) • Improved disease resistance (FY07/08) • Development of germplasm with shorter cooking times (FY07/08) Isolation, identification, and characterization seed coat flavonoids and determination of the relationship between seed coat color determining genes and the flavonoids they control: • Determine the antioxidant potential of flavonoids (FY04/05) • Isolate, identify, and characterize seed coat flavonoids from selected genetic stocks (FY07/08) • Determine the function of P and B genes 4a.What was the single most significant accomplishment this past year? Merlot small red dry bean was developed and released cooperatively by the USDA-ARS Sugarbeet and Bean Research Unit, East Lansing, Michigan and the Michigan Agricultural Experiment Station in 2005. Merlot is an upright, short-vine (Type IIA), full season cultivar with consistent and desirable canning quality, and the first small red commercial cultivar with resistance to bean rust disease. The release of Merlot improves the seed characteristics, canning quality, plant architecture, and disease resistance of the small red market class. The upright architecture helps to stabilize yield and provides the opportunity for growers to directly cut the variety, thereby preserving post-harvest quality and substantially reducing labor costs. The multiple disease resistance of Merlot reduces the need to use chemicals, reducing production costs and protecting the environment. The superior visual characteristics of both the dry and canned seed are expected to make Merlot highly marketable, and both bean shippers and processors have shown a great interest in Merlot. 4b.List other significant accomplishments, if any. Research was conducted to determine the inheritance of zinc and the relationship between zinc level and phytic acid concentration in navy beans. This research can be the foundation for a breeding program to increase zinc concentration in dry beans, and thereby aid the global problem of zinc deficiency in areas of the world where diets are largely based on grains and legumes. 4d.Progress report. The first two milestones listed above were completed. The phytic acid work was published in Crop Science. The third milestone, identifying genes affecting culinary traits, was substantially met. A paper describing the progress of this milestone will be published in the 2005 Annual Report of the Bean Improvement Cooperative and final results are expected by December 2005. The fourth milestone, identifying seed coat markers was substantially met. 5.Describe the major accomplishments over the life of the project, including their predicted or actual impact. i.) Ten dry bean cultivars in seven market (commercial) classes and three small red germplasm breeding lines were cooperative releases between the Michigan Agricultural Experiment Station and/or the Washington Agricultural Research Center and USDA-ARS Sugarbeet and Bean research Unit, East Lansing, Michigan.'Kodiak' and 'Burke' pinto beans, 'Beluga' alubia bean, 'Chinook' light red kidney bean, 'Glacier' great northern bean, 'Phantom' and 'Jaguar' black beans all have high yields, acceptable to excellent canning quality, and multiple disease resistance. The high yields of these varieties may increase profits for small farmers who generally rely on a good production to offset profit risk because of a reduced farm size compared to large scale operations. The multiple disease resistance of these cultivars reduces the need to use chemicals, thus reducing production costs and protecting the environment. 'LeBaron' small red bean is early maturing and very upright in growth habit. Its attractive vegetative appearance and uniform "dry down" and early maturity has attracted growers to plant 'LeBaron' as a second crop in a double-crop production system. 'Rojo Chiquito' Central American Red market class is the first small red with the dominant I gene form of resistance to bean common mosaic virus, and it has excellent canning quality with good firmness, and a very attractive bright red color after cooking. 'Rojo Chiquito' is becoming popular among Central American bean brokers and may represent a niche market for beans that are exported from the U.S. to this Region. The erect, upright germplasm lines, ARS-R93344, ARS-R93346, and ARS-R93349 all have excellent dry seed appearance and good to excellent canning quality. Impact: These germplasms represent a significant improvement in the adaptation of small red beans for the humid Great Lakes region of the U.S. Midwest and likely will be valuable as parental lines in bean breeding programs. Cultivar and germplasm enhancement and release are linked to National Program 301 Problem Areas I d-Expanding Germplasm evaluations and characterizations and II b-Genetic Improvement. ii.) We developed new and fundamental knowledge of cellular changes that occur during cooking of dry beans that lead to their ability to be digested. Heat induces cell walls to crystallize during the cooking process of dry beans. The cell wall crystallization forms a barrier that keeps water and digestive enzymes from solubilizing and breaking down starch. The indigestible starch passes through the gastrointestinal tract to the lower gut where microorganisms there metabolize it, thus, producing gas, which is subsequently expelled. Impact: This research provided the important knowledge base needed to develop strategies for improving dry bean digestibility. Studies on genes differentially expressed during cell wall crystallization will aid in identifying and manipulating the genes controlling crystallization. iii.) Research showed that both the genotype and processing method affect the digestibility of dry beans. There were significant differences among a select sample of 41 dry bean genetic stocks for total dietary fiber, indigestible starch, and indigestible protein. Canned beans contained less total dietary fiber, and indigestible starch and protein than beans cooked in a pot on a stovetop. Impact: This research provides plant breeders with the knowledge needed to develop selection strategies to lower the indigestible residue in cooked beans. The finding that canned beans contained less indigestible starch and protein than beans cooked in a pot on a stovetop may influence a larger consumption of canned beans; thus, leading to higher profits for industry and increased nutrition for consumers. iv.) We have determined that the flavonoids kaempferol 3-0-Beta-D glucoside and kaempferol 3-0-Beta-D-glucopyranoside-2 1-0-Beta-D xylopyranoside are the compounds responsible for yellow color in beans; the anthocyanins, delphinidin-3-o-glucoside, petunidin-3-0-glucoside, and malvidin-3-0-glucoside impart the black color to black beans; and the garnet red color of kidney beans is due primarily to proanthocyanidins which mask the yellow kaempferol flavonoids. Impact: This research was the breakthough needed to determine the function of genes in the biosynthetic pathway leading to the various flavonoid compounds involved in bean seed coat color. v.) An experiment was conducted in black bean to study water imbibition and its relationship to the thickness and continuity of the waxy cuticular layer of the seed coat. 'Shiny Crow' has a shiny seed coat and 'Raven' and 'Black Magic' both have opaque seed coats. Differences were detected between the three cultivars for thickness and continuity of the waxy epicuticular layer using scanning electron microscopy. In 'Shiny Crow' the epicuticular layer had a uniform thickness over the seed coat. The waxy epicuticular layers of 'Raven' and 'Black Magic' were of uneven thickness over the entire seed coat with areas varying in thickness by more than 1 micrometer. 'Black Magic' had the thickest epicuticular wax layer. Blanching in near boiling water (95ºC) caused a breakdown of the waxy epicuticular layer in all three genotypes. Data from the experiment indicated that breakdown of the waxy epicuticular layer occurred most rapidly in the thinnest regions, allowing water to penetrate the seed coat faster than in the thicker regions. Since water uptake is a key factor in leaching of pigments in thermally processed black beans, the thickness of the waxy epicuticular layer may control the magnitude of pigment loss after canning in black beans. This research on the effect of leaching on product quality of thermally processed black beans is linked to National Program 306, Problem Area I c-Factors and Processes That Affect Quality. vi.) An experiment was conducted to develop baseline data for cooking time on representative genotypes of several market classes that are of interest to growers who export beans to Central America and the Caribbean. Pinto beans were the fastest cooking among the seven market classes studied and small-red beans were the slowest cooking (110.3 min on average). There was a negative relationship (r = -0.84) between cooking time and water absorption. Since fast-cooking beans imbibed more water than slow-cooking ones, water absorption should be useful to predict cooking time in beans. Impact: Breeders should be able to select fast cooking genotypes from slow cooking ones. Selection based on the water absorption of a breeding line as an indirect estimation of its cooking time is rapid and saves resources. Research on the cookability and cooking rate of beans is linked to National Program 301, Problem Area I d-Expanding Germplasm Evaluations and characterizations. 6.What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end-user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? We provided new information to the scientific community and to bean grower and shipper industries on the importance of beans in a healthy diet and on important findings on the digestibility of beans and methods for improving the bioavailability of nutrients in this crop. New knowledge based on our research provided a basis to improve the health and nutrition of civil society. New cultivars of dry beans in several market classes were released to industry. These new cultivars have the potential of enhancing the profitability to growers and reducing costs for consumers. Cultivar releases in the small red market class have the potential to replace existing varieties grown in the humid Midwest, which will help stabilize production and grower income. In addition, disease resistance in the cultivars reduces the reliance on agricultural chemicals to control diseases and insect pests, thus, protecting the environment and reducing production costs. Several major consultations with the national media led to the publication of major articles publicizing our findings on flavonoids and their antioxidant potential and other health benefits obtained from including beans in the diet. New and improved cultivars have been released to industry and are currently being used by growers. New knowledge on digestibility and the health benefits from eating beans has been published in the national media and presented at health workshops. 7.List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: List your peer reviewed publications below). Bushey, S.M., Harris, L.S., Hosfield, G.L. 2004. Early generation tests for predicting color loss of black beans during thermal processing. Bean Improvement Cooperative Annual Report. 47:139-140. Review Publications Cichy, K.A., Forster, S., Grafton, K.F., Hosfield, G.L. 2005. Inheritance of seed zinc accumulation in navy bean. Crop Science. 45(3):864-870.