II gum; ngngngm: ["1 gum ! WW g __——.———— __ 83E ABSTRACT A STUDY OF PHYTATE VARIABILITY IN WHEAT By Walter David Worrall Phytic acid, a compound in seeds which chelates divalent metals, was measured in 30 soft winter wheat lines grown in five 10- cations throughout southern Michigan and 42 winter wheats from the U.S.D.A. world wheat collection. Environmental and genetic controls on phytate were examined as a prelude to the inclusion of selection pressure for phytate into a wheat breeding program. Sufficient gene- tic variability was found to warrant further study, however, environ- mental influences were quite strong. Further studies should be con- ducted to examine the total variability available for phytic acid. Nevertheless, decreases of the magnitude found in this study would serve to substantially increase dietary mineral availability in popu- lations whose primary food source is wheat. A STUDY OF PHYTATE VARIABILITY IN WHEAT By Walter David Worrall A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Crop and Soil Sciences 1975 ACKNOWLEDGEMENTS To Dr. Everett Everson, I wish to express my deepest gratitude for professional guidance and unwaivering friendship throughout my undergraduate and graduate studies. Sincere appreciation is expressed to Dr. Fred Elliott whose advice has helped me overcome many personal idiosyncrasies. In addition, I would like to thank Dr. Wayne Adams whose concern for the dissemination of knowledge has introduced me to many new facets of science. Finally, I wish to thank my wife, Julie, and my son, Davey, whose love, trust, understanding and friendship are the guiding forces in my life. ii INTRODUI LITERATl MATERIAT RESULTS DISCUS S CONCLUS BI BLIOG APPENDI APPEND‘.‘ TABLE OF CONTENTS Page INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 LITERATURE REVIEW . . . . . . . . . . . . . . . . . . . . . . . . . . 3 MATERIALS AND METHODS . . . . . . . . . . . . . . . . . . . . . . . . 11 RESULTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 DISCUSSION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 CONCLUSIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31 BIBLIOGRAPHY . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32 APPENDIX A Entry means of percent phytate for the 30 entries included in the 1974 advanced regional yield trial study. . . . . . . . . . . . . . . . . . . . . . . . . 35 APPENDIX B Intranursery analyses of variance with LSD values and coefficients of variation for the five nurseries of the advanced regional yield trial study in 1974. . . . . . 36 iii Table 10. Table 10. LIST OF TABLES Page Location means and ranges of percent phytate for 30 soft wheat lines grown in the 5 advanced regional yield trial nurseries for 1974. . . . . . . . . . . . . . . . . . . l4 Numberscfiflines significantly less than the highest line and their percentage of the total of 30 entries according to LSD tests on intranursery entry means for percent phytate . 14 Mean phytate content and chelation capacities of the 30 included in the 1974 advanced regional yield trial nurseries . . . . . . . . . . . . . . . . . . . . . . . ... . l7 Ranks of entry means for percent phytate in descending order for the 30 lines included in the 1974 advanced regional yield trial study . . . . . . . . . . . . . . . . . . l9 Internursery analysis of variance for percent phytate in 30 lines of soft wheat included in the 1974 advanced regional yield trial study . . . . . . . . . . . . . . . . . . 20 Separation of variance components from mean squares derived from the internursery analysis of variance . . . . . . 20 Analysis of variance of percent phytate of 42 lines from the U.S.D.A. world wheat collection grown in unreplicated headrows at two locations in 1975 . . . . . . . . 21 Identification and origin of 42 lines from the U.S.D.A. World Wheat Collection grown in unreplicated headrows in two locations in 1975 and analyzed for percent phytate from which chelation capacity was computed . . . . . . . . . . 22 Names or accession numbers and pedigrees of the thirty varieties and lines included in the 1974 advanced regional yield trials . . . . . . . . . . . . . . . . . . . . 24 Results of analyses of soil samples taken in October 1973 for five of the advanced regional yield trial nurseries . . . 28 iv LIST OF FIGURES Figure Page 1. Myo-inositol-l,2,3,4,5,6—hexakis (dihydrogen phosphate) . . . 3 2. Distribution of internursery entry means of percent phytate for the 30 lines in the advanced regional yield trials for 1974 . . . . . . . . . . . . . . . . . . . . l8 INTRODUCTION Phytate, myo-inositol-l,2,3,4,5,6-Hexakis(dihydrogen phosphate), is a compound found in plant seeds and functions as a phosphorus- store for germination and early seedling growth. It may also function in the initiation of seed dormancy by chelating the divalent metal cations which are often cofactors in the enzymatic reactions involved in germination. It is this property that makes phytate a nutritional liazard to populations whose major food source is unleavened wheat {Droducts. An enzyme capable of breaking down the phytate molecule 11as been identified and is produced by a variety of microorganisms :including bread yeasts. This enzyme is present in varying concentrations :in the digestive systems of many animals, but at inadequate concentrations :in the human gastrointestinal tract to facilitate substantial phytate lareakdown. Consequently, alternative methods of decreasing the clietary intake of phytate must be examined. One possible method might lae genetic manipulation of phytate levels in the wheat kernel if suffi- cient genetic variability could be identified. It was the purpose of 'this study to examine the genetic and environmental variation of a Ilumber of wheat genotypes grown in different locations and to determine the feasibility of genetically altering phytate content in wheat by ‘plant breeding. This was accomplished by analyzing thirty high performing inbred wheat lines grown in a variety of Michigan environments as well as lanalyzing a sample of varieties from the world wheat collection. Due to -1- the small amount of seed available, lines from the world collection were planted in unreplicated headrows in only two environments, whereas, the thirty Michigan lines were planted in larger test plots in five environments. Consequently, the bulk of this study is concerned with results from the five—environment, thirty—line analysis. LITERATURE REVIEW Posternak, in 1903 first isolated and described a phosphorus containing compound found almost exclusively in the outer layers of seeds (McCollum, 1957). Due to its plant origin, Posternak named this substance phytic acid and assigned it the formula C2H809P2. Since then various other structural formulae have been assigned to phytic acid including C6H24027P6’ (Neuberg, 1908) and C6H18024P6, (Anderson, 1914). X-ray crystallography and nuclear magnetic resonance have shown that the structure proposed by Anderson is the proper structure for naturally occurring phytic acid of plant origin, (Johnson and Tate, 1969), (Oberleas, 1971). Figure l. myo-Inositol-l,2,3,4,5,6-hexakis (dihydrogen phosphate) The occurrence of phytate in diverse species of plants has been shown by many authors. Wheat contains 53% of its phosphorus as phytate (Williams, 1970), corn - 37%, rice - 59%, (Oke, 1965). Recent work with cereals has shown that the highest concentrations of phytate are found in the aleurone layer of seeds. O'Dell, §£_§1, (1972), found that 87.1% of the phytate phosphorus in wheat was found in this seed fraction. 3 Nelson, g£_al, (1968), reported up to 77% of the total phosphorus present in wheat bran as phytate. This is in contrast to phytate- phosphorus concentrations of 12.9% and 2.2% for wheat germ and endo— sperm respectively, (O'Dell, g£_al,, 1972). Scanning electron micro- scopy has shown a close association of phytate with the aleurone grains, (Stevens, 1973; Jones, 1969). The above evidence indicates that phytate is a valuable storage form of phosphorus for seeds during germination when phosphorus serves as an energy source (Morton and Raison, 1963). In fact, phytate may have three additional physiological roles. 1. Experiments have shown that the phytate molecule is de- phosphorylated in a stepwise fashion throughout germination and during the earliest stages of seedling growth (Mihailovic, 33 al., 1965), indicating that it provides the growing plant with a time-released pool of inorganic phosphorus (Hall and Hodges, 1966), (Abernathy, 33 31,, 1973). 2. Phytate may also function in the onset of seed dormancy since the stepwise phosphorylation of the inositol molecule from inositol monophosphate to inositol hexaphosphate involves a drain on the ATP pool within the seed (Sobolev and Rodionova, 1966; Mandel and Biswas, 1970). Williams (1970), measured the concentration of ATP and found that it did not fall during the synthesis and phosphorylation of phytate during seed ripening. However, he did hypothesize an alternative mechanism through which phytate might facilitate seed dormancy. As shown in Figure 1, phytate has twelve replaceable hydrogen atoms. It is this characteristic which gives phytate the property of forming insoluble precipitates by chelating divalent metals such as calcium, iron, zinc and manganese in the order of stability Zn++, Mn++, Fe++, Ca++, (Vohra, 1965). These metals are often cofactors in enzymatic reactions within the seed. If, during ripening, they are tightly che- lated, then metabolic activities are reduced and dormancy may be ini- tiated (Williams, 1970). Williams recognized, however, that this is a secondary mechanism possibly supplementing hormonal control of dormancy. 3. Finally, following the stepwise dephosphorylation during germination, the remaining myo-inositol may function as a precursor for cell wall polysaccharide biosynthesis (Loewus, 1974). The physiological role of phytate is quite speculative and needs clarification. Shortly after isolation and identification, phytate became of interest to nutritionists. Early in the 1900's phytin, the calcium- magnesium salt of phytic acid, was produced commercially and sold both as a tonic and as a source of what was believed to be readily utilizable phosphorus (McCance and Widdowson, 1935). However, considerable time elapsed before its anti-nutritional characteristics were elucidated. In 1949, Mellanby conducted nutritional experiments with diets con- sisting of high extraction cereal flours. Mellanby found these diets had a strong ricketogenic effect on puppies due to phytic acid complexes with calcium. He also recognized that the ricketogenic effects of white flour were significantly less than the respective whole flour counter— parts. Later, chemical evaluations proved that lower extraction flours decreased the phytate concentration by as much as ninety percent (McCance and Widdowson, 1935). Hoff—Jorgensen, g£_§1, (1946), carried out animal and human studies which introduced an age factor into responses to dietary phytic acid. They first showed that puppies were less severely affected than older dogs by dietary phytic acid. Similar studies with human infants and older children followed. In both experi- ments, the results were similar; high levels of phytic acid decreased calcium availability more severely in older than in younger members of the same species. Recently, equivalent experiments were repeated with chickens to explain this differential age response. Older hens had a decreased activity of phytase, the enzyme responsible for phytate hydrolysis (Maddaiah, 1964). One possible reason why nutritional work with phytate came to a near standstill in the 1950's may have been a report by Walker, g£_§13 (1948). Walker and his coworkers designed an experiment in which four healthy European males were fed two experimental diets for periods ranging from four to nine weeks each. Following a control period, a diet consisting of whole wheat "war-bread" was fed for two to nine weeks followed by a 70% extraction white bread diet for a duration of one to nine additional weeks. Balance studies for calcium, magnesium and phosphorus were conducted. Walker's conclusions were that, l) phytate was present in the intestine as phytin, 2) phytin was hydrolyzed at such a point in the digestive tract as to allow the absorption of calcium, magnesium and phosphorus, and 3) over time, the human gut can adapt itself to the presence of phytate and maintain nutrient equilibria. The validity of these conclusions remains in question today although differences in opinion are not as great as they were then. McBean and Speckman (1974) claim that although very little is known about the interrelationships of different dietary components on mineral metabolism, the body can adapt to low levels of calcium availability both by decreasing renal excretion and by increasing absorptive capacity of the gut. A number of questions arise from such a statement: 1) For how low a level of mineral availability can the body be expected to compensate? 2) Since phytate seems to be the complexing agent in- volved, how much dietary phytic acid is tolerable? 3) If a differen- tial response to equivalent amounts of ingested phytate exists, when is the ability to adapt to high phytate intakes acquired, how long does it last and how is it manifested? 4) Is rickets the only nutritional malady upon which phytate exerts an effect or can other diseases be attributed to its ability to chelate metal ions? In the past fifteen years scientists have begun investigating some of these questions. Iron deficiency anemia was identified in Iran in 1961 (Prasad, g£_§1,). Over a period of two years Prasad and his coworkers studied eleven patients admitted to the Nemazee Hospital at the University of Shiraz, Iran for the following clinical features: short stature, hypogonadism, hepatosplenomegaly and iron deficiency anemia. Upon treatment with oral doses of iron, each patient promptly exhibited signs of recovery. Serum zinc was determined on only one patient and was found to be half the normal level. Prasad hypothesized that the same factors governing the absorption of iron also control the absorption of zinc. In fact, a number of the features which Prasad originally attributed to iron deficiency were probably caused by a de- ficiency in zinc. Additional evidence has since been added to this hypothesis by other workers in Iran (Reinhold, 1972; Reinhold, e£_§13, 1973). It is now generally agreed that sexual dysfunction and dwarfism are manifestations of zinc deficiency (Halsted, 1972). An interesting discovery by the researchers at Shiraz was the high incidence of geophagia, or clay eating, among the subjects they studied (Prasad, gt_§l,, 1961; Reinhold, 1972, Halsted, g£_§1,, 1972). Without exception, all subjects studied had, for an extended period of time, practiced geophagia. An American medical student living in a village in Iran in 1960 studied this phenomenon in 171 village residents (Greenwald, unpublished). He found no one in this village who had a history of prolonged geophagia without exhibiting mineral deficiency symptoms or deficiency symptoms without a history of geophagia (Prasad, .S£.§ln: 1961). Halsted, gt_§l, (1974) claimed that geophagia is a result of zinc deficiency; that is, individuals whose tissue zinc concentrations have fallen below a given level develop a physiological craving for zinc which drives them to eat dirt. However, Reinhold (1972) and Prasad, g£_§1, (1961), believe that geophagia, which is normally restricted to young children, is practiced because it relieves hunger and possibly because of the pleasing taste of clay. The latter explanation seems more reasonable in light of the work done by Byrd and Matrone (1965). This study provided chemical evidence to prove the theory that a triple- factor interaction exists between zinc, calcium and phytate. Other workers had noticed that mineral absorption was decreased in animals fed diets of high extraction flours and high levels of dietary calcium (O'Dell, 1969; Reinhold, g£_§1,, 1973; Oberleas and Prasad, 1969; Likuski and Forbes, 1965; Likuski and Forbes, 1964). Prasad attributed this to an interaction between phosphate and calcium, a fact indirectly correct since the phosphate content of high extraction flours can be traced to phytate. However, the clays typically eaten in Iran are highly calcareous (Reinhold, 1972), which adds credence to the theory of Reinhold and Prasad. A third factor, zinc, is also involved. "Since zinc is a trace element, it does not supply enough cations to form a significant precipitation of zinc-phytate. However, calcium cations increase the total cationic environment sufficiently to initiate a co- precipitation with zinc to form insoluble phytates. Therefore, zinc in the presence of calcium would not be as available for absorption and zinc deficiency would be aggravated," (Byrd and Matrone, 1965). It is possible that zinc deficiency is even more complex since addition of histidine to low zinc soybean meal chick rations has been shown to cause remission of some zinc deficiency symptoms (Dahmer, st 31., 1966). The mystery of complexing factors in the human diet is far from being solved. Evidence indicates that high phytic acid levels indirectly affect many physiological activities of the body. For instance, Atkinson, g£_§1,, 1972, found lower carboxypeptidase activity in zinc deficient rats indicating an indirect effect of phytate on protein metabolism. This could possibly explain the malnourished state of the subjects studied in Iran, many of whom exhibited symptoms similar to kwashiokor. Many approaches have been offered as solutions to the phytate problem. Phytase exists in insufficient quantities in the human gas- trointestinal tract to handle the levels of dietary phytic acid charac— teristics of many areas of the developing world. However, phytase is produced by a number of fungi and therefore can be produced commercially and utilized as a food additive. This, however, has little applicability to the developing world where food processing is almost nonexistent. Reinhold (1975) found that significant destruction of phytate in 10 unleavened bread products could be achieved if the dough was allowed to stand for at least two hours. This would not be an insurmountable cul- tural change for the people involved. Finally, genetic manipulation of phytate within the seed might be possible if sufficient genetic variation exists. This seems a viable alternative since it is believed that different classes of wheat have different phytate contents (Mattern, pers. comm.), however, genetic manipulation may cause repercussions in terms of the physiological functions of the seed. Consequently, the physiological and genetic parameters of phytate must be studied con- currently. MATERIALS AND METHODS The wheat breeding program at Michigan State University is set up such that all lines under initial consideration are grown in preliminary comparison nurseries and, after evaluation for yield and quality charac- teristics, the most elite materials entered in advanced regional yield trial nurseries. Samples for this study were taken from the latter only. Advanced regional yield trials were planted at five locations throughout southern Michigan. The sites chosen represent the hetero- geneous nature of the environments in which Michigan wheats are grown. The 1974 nurseries were located in Mbnroe, Lenawee, Ionia, Tuscola, and Berrien Counties and are designated M31, L41, SSl, T61 and B91, respectively. The thirty soft winter wheat entries included in each nursery were identical and consisted of 12 commercial varieties and 18 advanced selections from the Michigan State University wheat breeding project. Entries were grown as four-row plots, each row twelve feet long with twelve inches between rows. The nursery design was a 5 X 6 rectangular lattice with each entry replicated three times. Only the middle two rows of each plot were harvested, and after yield and test weight evaluations were made, a twenty gram subsample was taken from each plot for phytate analysis. In addition, 42 lines selected for winter hardiness characteristics from the U.S.D.A. world wheat collection were planted in unreplicated headrows in a completely randomized design in two locations. All samples were ground in a Udy Cyclone Sample Mill, stored for one week in the same room so that moisture levels were constant and then sealed in airtight glass containers until needed. 11 12 Oberleas (1971) has outlined a number of quantitative techniques for the determination of phytate and inositol phosphates. In order to analyze the large volume of material to be included in this study, a relatively rapid determination procedure was desirable. Consequently, the procedure decided upon was Oberleas' iron method. This technique makes use of the fact that phytate is soluble in 0.6% HCl. Extraction of phytic acid from the whole grain flour was accomplished by shaking 2.5 grams of flour in 1.2% HCl. Ten ml. of filtrate was diluted with deionized water to bring the acid concentration to 0.6% and ferric chloride added to facilitate the formation of the flocculent ferric— phytate precipitate. The precipitate was centrifuged at 4500 rpm, washed with a solution of 0.6% HCl and 4% Na2804, (sodium sulfate added to increase the firmness of the precipitate) and centrifuged again. The last step was repeated after which the precipitate was dissolved in 3 ml. of concentrated H2804 and brought to volume in 100 m1. volume- tric flasks. Iron concentrations were determined on a Perkin-Elmer Mbdel 303 Atomic Absorption Spectrophotometer. Finally, a conversion factor, based on the fact that iron binds to phytate in an ion-molecule ratio of 4 Fe:l phytate, was used to convert ppm iron into percent phytate. This ratio does not agree with the structure of the phytate molecule shown in Figure 1. This is because a phytate molecule not only chelates iron, but may also become bound to other phytate molecules; a stereochemical process which overcomes the steric hindrances of the inositol hexaphosphate molecule. Accuracy of the above procedure was determined daily by analyzing duplicate samples of a check variety from a commercial seed source. Also -13- a number of different concentrations of commercially prepared phytic acid were analyzed midway through the study to determine recovery rates which proved to be nearly 100%. Finally, all samples were replicated in the laboratory and reanalyzed if the values for percent phytate for the two replicates showed more than a 10% difference. Chelation capability will be defined in this thesis as the maximum amount of iron capable of being bound by the phytate in a 100 gram sample of flour. Since the molecular weight of phytate is 468.05 and the atomic weight of iron is 55.8, the phytate in 100 grams of flour is capable of binding 223.2 grams of iron (4 x 55.8) once the flour is ingested. Since our values for phytate are expressed as percentages and chelation capabilities based on 100 grams of whole grain flour, multiplication of the percent phytate by a constant (0.4769) equals the chelation capability. Analysis of variance was conducted within and between locations to determine the significance of entry'@3rietj)differences and genotype X environment interaction. Analysis as randomized blocks was considerably more efficient than analysis as rectangular lattices. Therefore, appropriate adjustments in analysis procedures were made. RESULTS Analysis of variance tables for individual nurseries may be seen in Appendix B. Table 1 shows that mean values of phytate for entries within nurseries ranged from a high of M31 of 1.240% to a low in 851 of 0.819% with an overall mean of 1.03% and a standard deviation of 0.1220. Table 1. Location means and ranges of percent phytate for 30 soft wheat lines grown in the 5 advanced regional yield trial nurseries for 1974. Nursery i High Line Low Line M31 1.045 1.240 0.842 L41 1.032 1.131 0.917 851 0.955 1.045 0.819 T61 1.002 1.131 0.898 B91 1.112 1.222 0.964 Table 2, which summarizes the results of LSD tests performed on each nursery, shows that at the 5% level of probability, a large number of the varieties in each nursery had phytate values significantly differ- This was also ent from the line with the highest percentage phytate. true at the 1% level of probability. Table 2. Numbers of lines significantly less than the highest line and their percentage of the total of 30 entries according to LSD tests on intranursery entry means for percent phytate. Locations M31 L41 $51 T61 B91 # of # of # of # of # of sig. % of sig. % of sig. % of sig. % of sig. % of lines total lines total lines total lines total lines total ‘LSD 05 11 37 14 47 8 27 4 13 10 33 LSD.01 2 7 8 27 3 10 l. 3 4 l3 l4 15 Internursery entry means and their respective chelation capabilities are presented in tabular form in Table 3 and graphically in Figure 2. The highest internursery entry mean was found to be 1.093% and the lowest 0.955% giving a range of 0.138%. Table 4 shows the rank for the means of individual entries over locations. Obviously, quite a bit of variability in rank exists, how- ever, entry 2 was found to be stable for low phytate at all five loca- tions and entries 6, 22 and 25 were relatively stable for high phytate at four of the five locations. Numerous other lines exhibited equivalent responses at three of the five nurseries. At first glance, the varia— bility in rank might be indicative of an entry X location interaction. This was not the case as the internursery analysis of variance in Table 5 shows. Differences among entry means were found to be highly signi— ficant, but their interaction with location showed no significance (P<.454). Separation of variance components, shown in Table 6, further illustrates this fact. Locations, replications and entries account for over ninety-eight percent of the total variance while interaction of location X entry accounts for an insignificant 1.15%. Correlation coefficients were calculated between phytate and yield and phytate and test weight but no significant correlation was found. Analysis of lines from the U.S.D.A. world wheat collection indi- cated a highly significant difference between environments, (P<;0005), as shown in Table 7, however, differences in entry mean values of phytate were less significant (P<;077) than differences between entry means in the regional yield trials. A slightly wider range in phytate values was observed in these lines as compared to the regional yield trials. This can be seen in Table 8 where the lowest line had a phytate value l6 (averaged over two locations) of 0.895% and the highest line 1.070% for a total range of 0.175%. This difference may be of no significance since a wider range would be expected with fewer observations per line and more lines. 17 Table 3. Mean phytate content and chelation capacities of the 30 lines included in the 1974 advanced regional yield trial nurseries.* Chelation Capacity Entry # % Phytate (mg. Fe/100g. Flour) 1 1.075 512.7 2 0.968 461.6 3 1.016 484.5 4 1.012 482.6 5 0.955 455.4 6 1.078 514.1 7 1.027 489.8 8 1.002 477.9 9 0.967 461.2 10 1.011 482.1 11 1.043 497.4 12 1.093 521.3 13 1.049 500.3 14 0.999 476.4 15 1.021 486.9 16 1.053 502.2 17 0.997 475.5 18 1.058 504.6 19 1.056 503.6 20 1.023 487.9 21 1.029 490.7 22 1.044 497.9 23 1.052 501.7 24 1.057 504.1 25 1.044 497.9 26 1.025 488.8 27 1.070 510.3 28 0.969 462.1 29 1.049 500.3 30 1.038 495.0 Standard error entry mean = 0.1220 *Mean of 3 replications/location and 5 locations Frequency 18 .95 .97 .99 |.O| LOB l.05 LO? |.O9 .96 .98 LOO l.02 I.O4 LOG LOB I.IO °/. Phytate Figure 2. Distribution of internursery entry means of percent phy- tate for the 30 lines in the advanced regional yield trial for 1974. Table 4. Variety Entry # \DmVOUI-L‘UJNH 19 Ranks of entry means for percent phy- tate in descending order for the 30 lines included in the 1974 advanced regional yield trial study. Nurseries fliflfllflllfil l 4 ll 27 15 27 28 25 25 29 23 2 19 19 27 20 15 17 15 25 30 30 22 14 21 3 11 8 6 19 15 29 18 11 7 7 27 21 30 ll 29 19 30 13 17 6 21 20 28 23 5 25 4 23 16 2 12 3 l7 6 9 26 5 24 1 8 13 29 26 26 17 22 27 12 4 12 14 16 2 14 24 23 23 8 28 26 7 2 l6 3 18 l 24 3 5 21 8 9 20 24 10 24 28 5 20 13 9 7 22 10 19 17 26 7 2 14 20 10 4 8 16 10 12 9 9 ll 18 13 21 22 4 17 15 1 18 28 16 14 29 30 22 6 l 10 13 25 3 6 18 12 20 Table 5. Internursery analysis of variance for percent phytate in 30 lines of soft wheat included in the 1974 advanced regional yield trial study. Sig. of Source 51f. _S_S_ Mi F Statistic Location 4 1.2144 0.3036 0.066 Reps within location 10 0.9735 0.0973 Entries 29 0.5303 0.0183 0.005 Locations X Entries 116 1.1432 0.0099 0.454 Residual Error 290 2.8172 0.0097 Total 449 6.6785 Table 6. Separation of variance components from mean squares derived from the internursery analysis of variance. Source Expected Mean Squares 2 2 2 Locations MSl = o + e10L + reoL Reps within location M82 = 02 + eofi E t 1 MS = 2 + 02 + 1 2 Locations X Entries M84 = 02 + IOEE Residual Error MS4,= 02 L = Locations (5) R = Replications (3) E = Entries (30) 2 - - 229 3 237 CL - M81 '- M32 - 0.00 (9. 0) re 2 a OR = M52 - M85 = 0.00292 (50.03%) e 0% = M83 - M84 0.00056 ( 9.59%) I]: 2 . OLE = M54 - M55 = 0.000067(l.15%) r Table 7. Source Locations Entries Error Total 21 Analysis of variance of percent phytate of 42 lines from the U.S.D.A. world wheat collection grown in unreplicated headrows at two locations in 1975. Significance _d§ Mean Square of F Statistic 1 0.0677 0.0005 41 0.0026 0.077 41 0.0016 83 22 Table 8. Identification and origin of 42 lines from the U.S.D.A. World Wheat Collection grown in unreplicated headrows in two locations in 1975 and analyzed for percent phytate from which chelation capacity was computed. Chelation I.D.# Origin % Phytate Capacity (mg. Fe/100 g.) C1012514 Texas 0.970 462.6 P1294987 Bulgaria 0.895 426.8 C1007166 China 0.913 435.4 P1285999 Poland 0.918 437.8 PI294992 Bulgaria 0.948 452.1 P1254839 Austria 0.965 460.2 C1011515 Nebraska 0.962 458.5 C1011724 Wisconsin 0.965 460.2 C1005086 China 0.979 466.9 P1167406 England 0.958 456.9 CIOll676 Kansas 0.953 454.5 C1011879 Wisconsin 1.005 479.3 P1285974 Poland 0.975 465.0 *SD072447 South Dakota 0.939 447.8 *SD072448 South Dakota 0.953 454.5 C1007l66 China 0.943 449.7 P1294987 Bulgaria 0.963 459.3 P1262661 U.S.S.R. 1.003 478.3 C1014486 Montana 0.973 464.0 C1006938 Canada 0.988 471.1 CIOl3670 Canada 1.018 485.5 C1008033 Montana 1.008 480.7 C1006914 U.S.S.R. 0.999 476.4 CIOl3547 Nebraska 1.030 491.2 PI262636 U.S.S.R. 1.020 486.4 C1014000 South Dakota 1.008 480.7 CI010722 China 1.013 483.1 **C1015327 1.054 502.7 C1012138 Minnesota 0.995 474.5 C1005149 Minnesota 0.980 467.4 P1167406 England 0.960 457.8 C1008034 Montana 0.970 462.6 C1005549 Montana 0.963 459.3 CIOll676 Kansas 1.002 477.9 P1254839 Austria 1.036 494.1 C1012138 Minnesota 0.973 464.0 PI278467 U.S.S.R. 0.997 475.5 P1285974 Poland 1.008 480.7 PI294937 Bulgaria 0.977 465.9 P1285999 Poland 0.983 468.8 P1267143 U.S.S.R. 0.958 456.9 P1262632 U.S.S.R. 1.070 510.3 Standard error of entry mean = 0.054 *C1 or PI numbers not available **Variety Sundance, origin unknown DISCUSSION The thirty entries included in the advanced regional yield trial nurseries are soft winter wheats proven to be of good pastry quality, high yielding and well adapted to Michigan environments. These lines vary in insect resistance, disease resistance, height, yield, test weight and winter hardiness. Since no selection pressure has been exerted for phytate content they are assumed to be quite variable for this trait also. However, it can be seen in Table 9 that there is a preponderance of genes from the varieties Genesee, Arthur and Redcoat in the pedigrees of many of these lines. Therefore, one cannot assume that they represent a randomly distributed sample population of all wheats for phytate even though they have never been selected for this character. As an additional check on variability for phytate levels, winter wheats of widely diver- gent genetic origin from the U.S.D.A. world wheat collection were also tested (see Table 8). Significant variability for phytate was found in the advanced regional yield trials and is evidenced by the fact that an overall average of 31% of the lines in each nursery were statistically significant at the .05 level. The inheritance of phytate was not examined but appears to be com— plex since in the absence of any genotype X environment interaction, only one genotype responded equally in all environments. Although the Possibilities for genetic manipulation of phytate are substantial, a more comprehensive survey of world wheat germplasm must be completed before conclusive statements may be made on the total amount of variability available. 23 24 Names or accession numbers and pedigrees of the thirty varie- ties and lines included in the 1974 advanced regional yield trials. Table 9. Variety or Entry Michigan Numbers Accession No. 1 Genesee 2 Yorkstar 3 Arrow 4 Ionia 5 Frederick 6 Tecumseh 7 Ticonderoga 8 Arthur 9 Arthur 71 10 Abe ll Oasis 12 Logan 13 A7170 14 B0272 15 30253 16 A9094 17 A9096 Pedigree Yorkwinl/Honor/Forward Genesee*3/3/Yorkwinl/Brevor/Norin 10 Avon sib./lHeines VII/Cornell Genesee*3/Redcoat Washington sel. lOl/Genesee//C.D. 6707 Minhardi/Wabash/S/Fultz sel./Hungarian/Z/ W38/3/Wabash/4/Fairfield/6/Redcoat sib./Wisc. C1012633/7/Vigo/4/Trumbull/2/Hope/Hussar/3/ Fulhio/Purkof (Purdue 427a1-1-3)*3/5/Kenya Farmer Genesee/4/82a1—2-4-7 (NY wheat-rye)/3/Genesee// Caldwell 8/ Cornell 595/5/Heines VII/6/ Genesee*Z/lBrevor/Norin 10/3/Avon sib. (NY 4848-2) Minhardi/Wabash/S/Fultz sel./Hungarian/Z/W38/ 3/Wabash/4/Fairfield/6/Redcoat sib./Wisc. C1012633/7/Vigo/4/Trumbull/2/H0pe/Hussar/3/ Fulhio/*Purkof/5/Kenya Farmer Arthur*5/3/Purdue 6028A2-15-9—2/2/Riley sib. *Z/Riley 67 Arthur*4/3/Purdue 6028A2-15—9-2/2/Riley*2/ Riley 67 Arthur 71/5/Arthur*3/3/Ribox/2/Riley*2/ Riley 67 (Purdue 6559 sel.)*2/4/Arthur*2/ 3/Ri1ey 67*2/2/Riley/Bu1garia 88 (P194407) Vermillion/Lucas Genesee*Z/Redcoat/lTalbot Genesee/Redcoat//Genesee*2/Redcoat/4/Norin lO/Brevor/lYorkwin/3/2*Genesee Genesee*Z/Redcoat/3/Suwon 92/Brevor//5*Genesee Asosan/3*Genesee German MI/3*Genesee 25 Table 9. Continued Variety or Entry Michigan Numbers Accession No. Pedigree 18 A7054 Genesee*2/Redcoat 19 A7055 Genesee*2/Redcoat 20 B0223 Purdue 5517-31/3/Suwon 92/Brevor//5* Genesee 21 A8175 Suwon 92/Burt//3*Genesee/4/Norin 10/Brevorl/ Yorkwin/3/2*Genesee 22 30215 Asosan/4*Genesee 23 B0261 Asosan/4*Genesee 24 B0216 Asosan/3*Genesee//Genesee/Redcoat 25 30270 Asosan/3*Genesee/4/Norin 10/Brevor//Yorkwin/ 3/2*Genesee 26 A8177 German MI/3*Genesee 27 30254 German MI/3*Genesee 28 B0255 German MI/3*Genesee 29 A9051 AC4835 (Durum Fly Resistance)/4*Genesee 30 B0240 AC4835 (Durum Fly Resistance)/5*Genesee 26 Although no significant genotype X environment interaction was identified, environment did play a role in determining phytate levels in wheat. This can be seen by the analysis of variance and separation of variance components for the regional yield trials and the analysis of variance of the U.S.D.A. lines. In both experiments macro- and micro- environments had an effect. The fact that variance due to replications in the regional yield trials accounted for about 50% of the total varia- bility is an indication of the differential genetic response to soil heterogeneity. The extent of this heterogeneity follows from analyses of soil samples shown in Table 10. Unfortunately, these were gross samples representative of soil conditions for entire nurseries and therefore do not supply the information needed to precisely determine the soil factors responsible for the large variation between replications. Future studies should include plot-by—plot soil analyses to overcome this difficulty. Macro— environmental effects are shown by the significance levels of the location main effects in the analysis of variance. The contribution of individual main effects is substantial, explaining in part the variability of entry ranks between nurseries seen in Table 4. Apparently main effects com- pound one another in a random fashion resulting in a variety of entry responses but not necessarily in a genotype X environment interaction. Lowering phytate a few tenths of a percent seemed of little con- sequence so a method was devised which brought this seemingly miniscule adjustment into proper perspective. Since the phytate determination procedure involved evaluation of the ability of the phytate in various lines of wheat to chelate iron, the method facilitated determination of the actual weight of iron, in milligrams chelated by 100 grams of whole grain 27 flour. This number was defined as an entry's chelation capacity. 0n the basis of the intranursery entry means mentioned previously, a differ— ence of 0.421% phytate exists (1.240% - 0.819%). Phytate chelates iron in a molecular ratio of 1:4 (phytate:iron). Consequently, 1.240 grams of phytate is capable of binding 591 milligrams of iron, whereas, 0.819 grams of phytate has the capability of chelating only 391 milligrams; a difference of 66%. This decrease in phytate also means a decrease in phosphorus within the seed. This is apparently accomplished without any adverse effect on germination or seedling vigor since a comparison of ranks based on phytate and yield shows no relationship. The lines lowest in phytate are not necessarily the lines lowest in yield; in fact, the variety Yorkstar which has the lowest phytate content over the five nurseries had the highest yield over these nurseries. Nutritionally, the effect of lowering the chelation capability of wheat 66% may prove important to populations whose primary food source is wheat. Nevertheless, it would appear from the data that even the lowest level of phytate in the wheats analyzed is capable of chelating the total daily dietary in- take of metal cations many times over. If this is so, what good would it do to lower phytate? There exists a multiplicity of answers to this question. Phytate is concentrated in the outer layers of the wheat kernel. In the rural areas of developing countries where phytate has the most significant nutritional impact, the crude milling practices employed fail to pulverize the bran layer into fine flour. This results in a number of phytate molecules, being closely associated with each other and probably partially bound together in the coarse bran fraction of the flour. Some of the binding sites may also have already been used to chelate metal ions in the intact seed. As the phytate enters the upper intestine, 28 Table 10. Results of analyses on soil samples taken in October 1973 for five of the advanced regional yield trial nurseries. _pH_ ._P:_ ._E: Ca* ‘Mg: Zn** Fe** M31 5.7 115 180 1309 125 11.6 40 L41 6.6 273 660 3200 540 15.2 132 851 6.8 30 154 4571 640 8.0 —- T61 7.2 128 324 5788 870 8.4 24 B91 6.4 121 348 3055 648 7.6 24 * Pounds per acre in upper 8" of soil **Parts per million in upper 8" of soil 29 the primary absorption region for metal ions, lower phytate levels obviously indicate a greater dilution effect within the lumen. The probability of a phytate molecule and a metal cation meeting in exactly the proper orientation to form a chelation product is, therefore, greatly reduced. When all these factors are considered, even a relatively small reduction in phytate would have a marked effect on mineral availability. The interrelationships of many physiological components of the seed must be studied before the nutritional impact of any one component may be fully understood. For example, research is presently underway to determine the feasibility of genetically decreasing the levels of certain protease inhibitors in seeds which have the effect of lowering the digestibility of plant proteins. However, if this is accomplished the results might be indirectly related to mineral avail- ability since phytate is closely associated with plant protein. The sooner bran proteins are hydrolyzed in the upper part of the gastrointes- tinal tract, the sooner free phytate will become available for chelation of metals. Since so little is understood about the physiological role of phytate within the seed it would be naive to attempt to genetically alter phytate levels without conducting concurrent studies correlating germination, dormancy and seedling vigor with various phytate levels. Before plant breeders undertake a detailed genetic analysis of phytate a new determination procedure must be made available. The iron method in current vogue is too time consuming to accommodate the large volume of material plant breeders must consider. A coloremetric quanti- fication procedure for phosphorus is available (Oberleas, 1971), which 30 improves the accuracy of phytate determinations but is more lengthy than the iron method. There is reason to believe, however, that a gel elec— trophoresis procedure will soon be published which will satisfy the re- quirements of both time and accuracy (Purvis, pers. comm.). CONCLUSIONS Genetic variability for phytate in wheat lines included in the advanced generation yield trials was identified and found to be of sufficient magnitude to warrant further investigation. Analyses of forty-two lines obtained from the U.S.D.A. small grain collection indicate that additional variability may be available. Environmental influences on phytate were also quite strong and should be investigated further. Obviously, detailed studies of soil components both in the field, greenhouse and possibly hydroponics are needed to elucidate these effects. Although phytate exerts little influence on the health of people eating highly diversified diets, it has profound effects on the nutritional well being of a considerable portion of the developing world's population. A decrease in phytate levels of the extent described in this study would be a big step toward bettering the diet of the world's poor. 31 10. 11. 12. 13. 14. 15. BIBLIOGRAPHY Abernathy, Rollin H., G. M. Paulsen and R. Ellis, Jr. 1973. Re— lationship Among Phytic Acid, Phosphorus and Zinc During Maturation of Winter Wheat. J. Agr. Food Chem. 21:282-284. Anderson, Robert J. 1914. Concerning Phytin in Oats. J. Biol. Chem. 17:171-190. Atkinson, J., P. Vohra and F. H. Kratzer. 1972. The Effect of Available Dietary Zinc on the Utilization of Protein by the Chick and Japanese Quail. Brit. J. Nut. 27:461-466. Byrd, Cynthia A. and G. 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APPENDICES 35 Appendix A: Entry means of percent phytate for the 30 entries included in the 1974 advanced regional yield trial study.* Table A1 Nurseries Entry # M31 L41 $51 T61 B91 1 1.240 1.088 0.983 0.932 1.115 2 0.977 0.965 0.894 0.956 1.014 3 0.983 1.127 0.933 0.978 1.055 4 0.997 1.031 0.957 1.005 1.065 5 0.842 0.917 0.926 1.007 1.096 6 1.158 1.058 0.996 1.060 1.105 7 1.051 0.961 0.936 1.019 1.151 8 1.091 0.965 0.929 0.898 1.129 9 0.900 1.017 0.819 1.007 1.112 10 1.127 1.004 0.932 0.915 1.090 11 1.133 0.975 1.015 0.962 1.113 12 1.224 1.050 1.023 0.991 1.152 13 1.083 0.969 1.014 0.962 1.222 14 1.087 1.050 0.874 0.934 1.064 15 1.033 0.999 0.891 1.018 1.169 16 1.061 1.039 0.961 1.094 1.117 17 0.980 0.995 0.914 1.042 1.053 18 0.977 1.084 1.025 1.002 1.171 19 1.023 1.131 0.902 1.093 1.157 20 0.997 1.081 0.995 0.970 1.080 21 1.079 0.988 0.887 1.085 1.105 22 1.059 1.064 1.006 0.969 1.130 23 1.015 1.087 0.893 1.043 1.213 24 1.055 1.006 0.987 1.090 1.151 25 1.045 1.063 0.978 1.023 1.141 26 1.066 1.024 0.977 0.970 1.092 27 1.135 1.026 0.962 1.131 1.105 28 0.953 1.031 0.969 0.904 0.964 29 0.995 1.084 1.045 1.021 1.118 30 0.980 1.107 1.012 0.981 1.124 *Means over three replications. 36 Appendix B: Intranursery analyses of variance of percent phytate with LSD and coefficients of variation for the 5 nurseries of the advanced regional yield trial studies in 1974. Table B1 Location M31: Source .9: SS MS Replications 2 0.10023 0.05011 Entries 29 0.64109 0.02211 Error 58 1.16478 0.02008 Total 89 1.90610 Least Significant Differences: P<.05 LSD = 0.2314 P<.01 LSD = 0.3008 Coefficient of Variation = 13.56% Table B2 Location L41: Source .df SS MS Replications 2 0.16694 0.08347 Entries 29 0.23937 0.00825 Error 58 0.29825 0.00514 Total 89 0.70456 Least Significant Differences: 0.1043 0.1356 P<.05 LSD P<.01 LSD Coefficient of Variation = 6.1838% Appendix B: Location 851: Source Replications Entries Error Total 37 Continued Table B3 d: 2 0 29 0 58 0. 89 0. Least Significant Differences: P<.05 LSD P<.01 LSD 0.1206 0.1567 Coefficient of Variation = 7.7346% Location T61: Source Replications Entries Error Total Table B4 ‘df 2 0 29 0. 58 0. 89 1. Least Significant Differences: P<.05 LSD P<.01 LSD 0.1768 0.2298 Coefficient of Variation = 10.8040% SS .15522 .25772 31611 72905 SS .33793 30969 67988 32750 MS 0.07761 0.00889 0.00545 MS 0.16897 0.01068 0.01172 38 Appendix B: Continued Table BS Location B91: Source .df SS MS Replications 2 0.21317 0.10658 Entries 29 0.22557 0.00778 Error 58 0.35814 0.00617 Total 89 0.79687 Least Significant Differences: P<.05 LSD P<.01 LSD .1252 .1627 Coefficient of Variation = 6.8908% "I11111111111111.11111“