AN AGRONOEflC END PHYSEOLOGECAL mm; “123% OF GPAQUE . 2, AND NOW MAIZE {3; mass u Thesis for Degree of Pb. D. ‘I‘JflCHTGAN STATE UNEVERSETY ROBERT E. ‘WlTTERS 1970 .“h?”’ l..-. - u- . LIBRARY 1 mam um. ‘fH". 51“: This is to certify that the thesis entitled AN AGRONOMIC AND PHYSIOLOGICAL COMPARISON OF OPAQUE-Z AND NORMAL MAIZE (Egg m L.) presented by Robert E. Witters has been accepted towards fulfillment of the requirements for —Ph—D— deqree in sum—W ( \ _2' ///> ,,/ 7 , 4 (6C W/fig/zxd Major professor Date Q/x Q: 7 (.77 0-169 5. .A- «u. . . _r {e r ABSTRACT AN AGRONOMIC AND PHYSIOLOGICAL COMPARISON OF OPAQUE-Z AND NORMAL MAIZE (Zea mays L.) By Robert E. Witters Experiments were conducted on Opaque-2 and conven- tional yellow dent maize (normal maize) under field and laboratory conditions. The near-isogenic single-cross hybrids, opaque-2 (W64A-op-2 X MS-14-0p-2) and normal maize (W64A-TRf X M-14-TRf) were used to study yield and components of yield during the 1968 growing season. A Split-split plot design was used to analyze the two hybrids grown at four plant population rates and three levels of supplemental nitrogen. Field experiments showed that the opaque-2 hybrid averaged five bushels per acre* more than its normal counterpart. The Opaque-2 hybrid had a yield high of 109 bushels per acre which was obtained at a plant population rate of 31,500 and 125 pounds of nitrogen. The normal *All calculations are based on one bushel of maize weighing 56 pounds at 15.5 percent moisture. Robert E. Witters hybrid yielded a high of 86 to 87 bushels per acre at a plant population rate of 26,600 and 31,500 with 125 pounds of nitrogen. The normal hybrid had a higher shelling per- cent at all levels of nitrogen and all population rates. The opaque-2 hybrid was generally higher in percent mois- ture at harvest; this may be attributed to looser arrange- ment of storage starch, higher sugar content, and more viscous protein storage forms. The normal hybrid had higher kernel weight, but lower total kernels per ear row, total kernels per ear, and total kernel weight per ear than the opaque-2 hybrid. Germination tests in warm soil showed that opaque-2 and normal maize germinate almost 100 percent under near ideal conditions whereas tests in cold soil showed that normal maize germinated twice as well as opaque-2 maize (70.6 vs 30.3 percent). Germination of the two maize types under cold test conditions in a sterile media indicated that soil and seed-borne microorganisms play a greater role than low temperature in the failure of seeds to germinate. The effects of low temperature on germination were more evident in Opaque-2 than in normal maize. Several opaque-2 and normal maize types were analyzed in the laboratory. A modification of the Lowry colorimetric method for protein determination was shown to Robert E. Witters be highly correlated with the standard micro-Kjeldahl method. Normal maize had a slightly higher correlation with the micro-Kjeldahl protein than the opaque-2 maize. Regression lines were calculated for use in the conversion of the colorimetric absorbance values to percent protein by the micro-Kjeldahl method. Greater precision in conversion was found by using separate lines for Opaque-2 and normal ‘maize. A modified TNBS (2,4,6-trinitrobenzenesulfonic acid) method was used to determine the lysine content in whole ground meal of opaque-2 and normal maize. The method was found to be a reliable method for lysine analysis in maize. Lysine analyses of maize meal indicated that Opaque- 2 hybrids contained from 20 to 100 percent more total lysine than the normal maize. The combination of opaque-2 inbred lines used to produce the hybrid influenced the lysine content of the kernel. Maternal effects on the endosperm composition appeared to influence the lysine content in the kernel. The effect of one, two, or three Opaque-2 gene doses in the endosperm tissue resulted in relatively small differences in total kernel lysine content. This indicates that a normal type plant could be used as a female parent plant to develop better kernel character- istics in Opaque-2 maize. Robert E. Witters Lysine analysis in the extracted alkali-soluble proteins showed that an average of 72.6 percent of the total kernel lysine of opaque-2 single-cross hybrids was found in the alkali-soluble protein fraction compared to 72.1 percent for normal single-cross hybrids and 80.5 percent in three-way hybrids. Three general patterns of protein distributions (based on lysine content) were found from analyses of five ammonium sulfate fractions of the alkali-soluble proteins of opaque-2 and normal maize. The percent lysine distri- butions for the 5, 10, 20, 30 and 80 percent ammonium sulfate fractions were: type I - 19.0, 45.8, 17.8, 3.8 and 11.9: type II - 52.1, 22.4, 12.6, 3.0 and 10.1; type III - 54.1, 11.2, 19.0, 4.3 and 10.6. The number of hybrids represented by each lysine distribution was: type I - one, type II - five, and type III - seven. The type I distribution was representative of only one hybrid, W64A-op-2 X MS-l4-op-2 (BC-3). The near-isogenic normal counterpart and the BC-5 opaque-2 hybrid of the same pedigree were characterized by type II lysine distribu- tions. The near-isogenic hybrids W64A X Oh-43 and W64A- op-2 X Oh-43-op-2 had type III lysine distributions in their alkali-soluble proteins and both contained relatively Robert E. Witters low levels of total kernel lysine. Most three-way hybrids also had the type III lysine distribution. ACKNOWLEDGEMENTS The author wishes to express his appreciation to Dr. Elmer C. Rossman for his help and guidance throughout this investigation. Thanks are also due to Dr. C. M. Harrison for his appraisal of the manuscript and for serving as a guidance committee member; to Dr. Donald Penner for his contribu- tion of time and equipment toward completion of the chem- ical analyses; to Drs. H. D. Foth, T. J. Johnston and C. J. Pollard who served as guidance committee members. The advice and constructive criticisms offered by my fellow graduate students is also appreciated. The author is deeply grateful to his wife Joan, without whose devotion and work this manuscript would not be possible and to my three daughters Debra, Teresa and Roberta for their patience during this course of study. The research was supported in part by funds from the National Defence Education Act, Title IV. A grant from DeKalb Ag Research was used for a portion of the Operating budget and is acknowledged.with appreciation. ii AN AGRONOMIC AND PHYSIOLOGICAL COMPARISON OF OPAQUE-Z AND NORMAL MAIZE (Zea mays L.) By . Vi Robert E. Witters A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of CrOp and Soil Sciences 1970 TABLE OF CONTENTS LIST OF TABLES . LIST OF FIGURES. INTRODUCTION . LITERATURE REVIEW. MATERIALS AND METHODS. RESULTS AND DISCUSSION . . . . . . . . Agronomic Field Studies . Laboratory Studies. SUMMARY. LITERATURE CITED . iii Page iv vii 14 26 26 78 100 105 Table LIST OF TABLES Results of protein fractionation from defatted corn endosperm (10 g) of +, op-2 and fl-2 by the modified Mendel-Osborne method. . . . . . . . . . . . . . Average yields for two maize hybrids (normal and opaque-2) at four plant populations and three nitrogen levels, East Lansing, 1968. . . . . Analysis of variance for yield of two corn hybrids at four plant populations and three nitrogen levels, East Lansing, 1968. Average shelling percent for two maize hybrids (normal and opaque-2) at four plant populations and three nitrogen levels, East Lansing, 1968. Analysis of variance for shelling percent of two corn hybrids at four plant pOpula- tions and three nitrogen levels, East Lansing, 1968. . . . . Average percent moisture for two maize hybrids (normal and Opaque-2) at four plant populations and three nitrogen levels, East Lansing, 1968. Analysis of variance for percent moisture of two corn hybrids at four plant popula— tions and three nitrogen levels, East Lansing, 1968. . . . . . . . . . iv Page 27 28 36 37 42 43 Table 10. ll. 12. l3. 14. 15. 16. Average weight per 100 kernels for two maize hybrids (normal and opaque-2) at four plant populations and three nitro- gen levels, East Lansing, 1968. Analysis of variance for weight per 100 kernels of two corn hybrids at four plant populations and three nitrogen levels, East Lansing, 1968. Average number of kernel rows per ear for two maize hybrids (normal and opaque-2) at four plant populations and three nitro- gen levels, East Lansing, 1968. Analysis of variance for number of kernel rows per ear of two corn hybrids at four plant populations and three nitrogen levels, East Lansing, 1968. Average number of kernels per ear row for two maize hybrids (normal and opaque-2) at four plant populations and three nitrogen levels, East Lansing, 1968. Analysis of variance for number of kernels per ear row of two corn hybrids at four plant populations and three nitrogen levels, East Lansing, 1968. Average total kernel number per ear for two maize hybrids (normal and opaque-2) at four plant populations and three nitrogen levels, East Lansing, 1968. . . Analysis of variance for total kernel number per ear of two corn hybrids at four plant populations and three nitrogen levels, East Lansing, 1968. Average total kernel weight per ear for two hybrids (normal and opaque-2) at four plant populations and three nitrogen levels, East Lansing, 1968. . Page 47 48 52 53 57 58 62 63 66 Table Page 17. Analysis of variance for total kernel weight per ear of two corn hybrids at four plant populations and three nitrogen levels, East Lansing, 1968. . . . . . . . . . . 67 18. Comparison of soil warm test germination percent in opaque-2 vs normal maize. . . . . . 71 19. Comparison of cold test germination percent in soil of opaque-2 and normal maize. . . . . . . . . . . . . . . . . . . . . 73 20. Comparison of cold test germination percent in sterile media of opaque-2 and normal maize. . . . . . . . . . . . . . . . . . 75 21. Comparison of two colorimetric absorbance values and the mean absorbance with the percent protein determined by the micro- Kjeldahl method. . . . . . . . . . . . . . . . 81 22. Lysine analyses of ground meal and alkali- soluble protein fractions in maize. . . . . . . 87 23. Comparison of percent lysine distributions in the ammonium sulfate fractionations of the alkali-soluble proteins in opaque-2 and normal maize. . . . . . . . . . . . . . . . 95 vi Figure LIST OF FIGURES Standard curve used for TNP-L-lysine analysis based on absorbance at 346 mp and 420 mp wavelengths. Hybrid main effect for yield of opaque-2 and normal maize. . . . . . . . . . Population main effect for yield at plant populations of (1) 16, 900; (2) 21, 300; (3) 26, 600; and (4) 31, 500. . Hybrid X nitrogen interaction for yield of opaque-2 and normal maize grown at supplemental nitrogen rates of (1) 0 lbs; (2) 125 lbs; and (3) 250 lbs per acre. Hybrid X population interaction for yield of opaque- -2 and normal maize at plant populations of (l) 16, 900; (2) 21, 800; (3) 26, 600; and (4) 31, 500. . Nitrogen X population interaction for yield at 3 supplemental nitrogen rates of (l) 0 lbs; (2) 125 lbs; (3) 250 lbs, and 4 plant populations of (1) 16,900; (2) 21,800; (3) 26,600; and (4) 31,500. Hybrid main effect for shelling percent in opaque-2 and normal maize. POpulation main effect for shelling percent at populations of (1) 16, 900; (2) 21, 800; (3) 26, 600; and (4) 31, 500. . . . . vii Page 25 29 29 30 3O 31 38 38 Figure Page 9. Hybrid X nitrogen interaction for shell- ing percent in Opaque-2 and normal maize grown at supplemental nitrogen rates of (l) 0 lbs; (2) 125 lbs; and (3) 250 lbs. . . . 39 10. Hybrid X population interaction for shelling percent in opaque-2 and normal maize grown at plant populations of (l) 16, 900; (2) 21, 800; (3) 26, 600; and (4) 31,500.. . . . . . . . . . . 39 11. Hybrid main effect for percent moisture for opaque-2 and normal maize. . . . . . . . . 44 12. Hybrid X nitrogen interaction for percent moisture at harvest in opaque-2 and normal maize at supplemental nitrogen rates of (l) 0 lbs; (2) 125 lbs; and (3) 250 lbs. . . . . . . . . . 45 13. Hybrid X population interaction for percent moisture at harvest in opaque-2 and normal maize grown at plant popula- tions of (1) 16,900; (2) 21,800; (3) 26,600; and (4) 31,500. . . . . . . . . . . 45 14. Hybrid main effect for weight per 100 kernels of opaque-2 and normal maize. . . . . . 49 15. Hybrid X nitrogen interaction for weight/ 100 kernels of opaque-2 and normal maize at supplemental nitrogen rates of (l) 0 lbs; (2) 125 lbs; and (3) 250 lbs. . . . . . . . . . 50 16. Hybrid X population interaction for weight/100 kernels of opaque-2 and normal maize at plant populations of (1) 16,900; (2) 21,800; (3) 26,600; and (4) 31,500. . . . . 50 17. Population main effect for number of kernel rows/ear in opaque-2 and normal maize O O O O O O O O O O O O 0 I O O O O O O O 54 viii Figure Page 18. Hybrid X nitrogen interaction for number of kernel rows per ear in opaque-2 and normal maize grown at supplemental nitrogen rates of (l) 0 lbs; (2) 125 lbs; and (3) 250 lbs. . . . . . . . . . 55 19. Hybrid X population interaction for number of kernel rows per ear in opaque-2 and normal maize grown at plant popula- tions of (1) 16,900; (2) 21,800; (3) 26,600; and (4) 31,500. . . . . . . . . . . 55 20. Hybrid main effect for number kernels/ear row in opaque-2 and normal maize. . . . . . . . 59 21. Population main effect for number of kernels/ear row in opaque-2 and normal maize. . . . . . . . . . . . . . . . . . . . . 59 22. Hybrid X nitrogen interaction for number of kernels/ear row in opaque-2 and normal 'maize grown at supplemental nitrogen rates of (l) 0 lbs; (2) 125 lbs; and (3) 250 lbs. . . 60 23. Hybrid X population interaction for number of kernels/ear row in opaque-2 and normal maize grown at plant populations of (l) 16, 900; (2) 21, 800; (3) 26, 600; and (4) 31,500.. . . . . . . . . . . . 60 24. Hybrid main effect for total kernels/ear in opaque-2 and normal maize. . . . . . . . . . 64 25. Population main effect for total kernels/ ear row in opaque-2 and normal maize. . . . . . 64 26. Hybrid X nitrogen interaction for total kernels/ear in opaque-2 and normal maize grown at supplemental nitrogen rates of (l) 0 lbs; (2) 125 lbs; and (3) 250 lbs. . . . 65 ix Figure Page 27. Hybrid X population interaction for total kernels/ear in opaque-2 and normal maize grown at plant popula- tions of (1) 16,900; (2) 21,800; (3) 26,600; and (4) 31,500. . . . . . . . . . . 65 28. Hybrid main effect for total kernel weight/ear in Opaque-2 and normal maize. . . . . . . . . . . . . . . . . . . . . 68 29. Population main effect for total kernel weight/ear in opaque-2 and normal maize. . . . 68 30. Hybrid X nitrogen interaction for total kernel weight/ear in opaque-2 and normal maize grown at supplemental nitrogen rates of (l) 0 lbs; (2) 125 lbs; and (3) 250 lbs. . . 69 31. Hybrid X pOpulation interaction for total kernel weight/ear in opaque-2 and normal maize grown at plant population rates of (1) 16,900; (2) 21,800; (3) 26,600; and (4) 31,500. . . . . . . . . . . 69 32. Regression analysis of total kernel protein by the modified.Lowry colori- metric and micro-Kjeldahl methods in opaque-2, normal, and in both maize types combined. . . . . . . . . . . . . . . . . 84 33. Representative lysine distribution patterns based on the percent lysine in each of the five ammonium sulfate fractions of the alkali-soluble proteins in opaque-2 and normal maize. . . . . . . . . . . . . . . . . . 99 INTRODUCTION The opaque-2 and floury-Z mutant genes in maize (Egg m§y§_L.) have been recognized since the mid-nineteen- thirties. However, the discovery that the mutant genes alter the protein composition and amino acid pattern in ‘maize, was not made until 1963 at Purdue University. Nutritionists have known since the early 1900's that normal maize protein is of poor quality because it is nearly devoid of two essential amino acids, lysine and tryptophan. Since the major effect of the two mutant genes is to shift the kernel protein storage pattern from nutritionally poor zein to nutritionally balanced glutelin, the kernel protein is present in a dietary balanced condition. Thus, these improvements in protein quality over normal maize should provide a more complete feed that would require less sup- plementation. This would be especially important in those areas and countries where maize is the major feed used. Although there are great nutritional benefits created by the incorporation of the Opaque-2 and floury-2 genes into normal maize types, observation from breeding programs 1 2 indicated that some of the kernel characteristics of opaque-2 maize might present some problems in commercial production. The objectives of this study were to study various agronomic and physiological aspects of high-lysine and normal dent maize. Included are (1) field studies of yield and components of yield; (2) germination tests; (3) evalua- tion of a colorimetric method of protein determination; and (4) analysis of lysine content in whole kernel protein and alkali-soluble protein. LITERATURE REVIEW A new era of investigations has been opened for plant breeders, plant physiologists and nutritionists since Mertz, Bates and Nelson discovered that opaque-2 (op-2) (Mertz gt a}, 1964) and floury-2 (fl-2) (Nelson g£_al, 1965) genes alter the amino acid pattern of maize protein (Bres- sani and Elias, 1969). The recessive mutant opaque-2 gene was first described by Singleton and Jones in 1935 (Emerson gt 31, 1935). Mumm reported the recessive mutant floury-Z gene in the mid-nineteen thirties (Emerson gt El: 1935). In normal maize, the amino acids lysine and tryp- tOphan respectively, were found to be the first and second nutritionally limiting amino acids in humans (Osborne and Mendel, 1914; Rose, 1957; Sauberlich, 1953b). Analyses of opaque-2 maize endosperm showed the lysine content was 69 percent more than normal maize (Mertz, 1964) and the amount of tryptophan was more than doubled (Nelson £3 a1, 1965). The floury-2 maize endosperm.was shown to contain similar proportions of lysine and tryptophan as found in opaque-2 but in addition increased.methionine, the third nutritionally 3 4 limiting amino acid, by 50 percent (Nelson gt a1, 1965). Osborne and Mendel (1914) showed that normal maize endosperm contained approximately 80 to 85 percent and the embryo 15 to 20 percent of the total kernel protein. Tests showed that normal endOSperm protein consisted of 16 to 26 percent acid-soluble (albumins + globulins), 41 to 60 per- cent alcohol-soluble (zein), and 17 to 31 percent alkali- soluble (glutelin) fraction (Concon, 1966). Protein analyses of W64A normal inbred, W64A-op-2, W64A-fl-2, and W64A-op-2-fl-2 were as follows (Jimenez, 1966): Table 1. Results of protein fractionation from defatted corn endOSperm (10 gm) of +, op-2 and fl-2 by the modified.Mendel-Osborne method. (Percent- age of the soluble protein in the endosperm.) Fraction + 0-2 f1-2 0-2:f1-2 Albumin (water- 3.7 14.7 14.6 11.0 soluble) Globulin (salt- 1.7 4.4 4.4 6.1 soluble) Zein (alcohol- 54.2 25.4 28.7 18.7 soluble) Glutelin (alkali- 40.4 55.5 52.3 64.2 soluble) In all four genotypes most of the lysine present in the endosperm was contributed by the glutelins (Jimenez, 1966). Also, the glutelins in the three mutant genotypes were higher concentration than in normal maize. The 5 concentration of lysine was higher in the glutelin fraction in opaque-2 and floury-Z than in normal glutelin (Jimenez, 1966). Analyses of opaque-2 and normal maize by Jimenez (1961) showed that zein contained very little lysine. He showed that normal zein contributed more to the total kernel lysine content because normal maize contained about twice as much zein as opaque-2 maize. The glutelin protein frac- tion has been shown to contain greater quantities of lysine and tryptOphan than all other extractable protein fractions combined (Sauberlich, 1953a; Lloyd and.Mertz, 1958). Thus, to obtain high kernel lysine content, selection for non- zein protein appeared to be the best criterion, a method (Frey 25 El: 1949b) found to be successful in selection for tryptophan content in maize. A survey of some normal inbred lines showed some with a whole kernel lysine content equal to and higher than some Opaque-2 or floury-2 lines (Paez gt_al, 1969c). High protein lines of normal maize usually were nutritionally inferior to normal maize on a per unit basis because of the protein increase being derived from a larger zein fraction (Frey §£_al, 1949b; Frey, 1951; Sauberlich £5 al, 1953a, 1953b). Field Study Performance of near-isogenic lines of opaque-2 and normal maize in Illinois trials by Alexander g£_al, 1968, was as follows: In 1966 trials with BC2 (second backcross) material, 5 of 7 opaque hybrids yielded significantly less than their normal counterparts. The 1967 trials showed 7 opaque hybrids equal to or higher than their near-isogenic normal hybrids although differences were not statistically significant. Colyer and Kroth (1968) developed response curves for nitrogen effect on maize yields in 1965 (grown under good summer moisture conditions) on two Missouri silt loam soils. Their results indicated a steady increase in yield up to about 150 pounds N/A and a slight decrease above that rate on both soil types. In 1969 trials at Michigan State University, Lucas (1969b) found the following results from nitrogen applica- tion on Mich. 500-2X (single cross) hybrid grown on sandy loam soil: N (actual) Yield (Bu/A) 0 103 100 lbs May 156 100 lbs May + 100 lbs June 155 Stevenson and Baldwin (1969) reported increases in maize yields from spring applied nitrogen over applications 7 at any other time on a clay loam. They also found yield responses in the order of anhydrous ammonia, urea and ammonium nitrate as nitrogen sources. The effect of population on yield was also studied by Colyer and Kroth (1968) on two Missouri silt loam soils. In 1965 they found yield reSponse curves to pOpulation effects with maximum yields obtained at between 16,000- 18,000 plants per acre. Yield response to four pOpulation rates of maize (Lucas, 1969a) grown on loamy sand at East Lansing, Michigan was as follows: population yield (Bu/A) 20,000 145 24,000 156 28,000 162 32,000 162 Maize yield was found to be increased by tassel removal at or near tassel emergence and the increases were larger at higher populations (Hunter gt 31, 1969). Their results also showed a decrease in yield when leaf tissue was removed with the tassel although these effects were smaller at higher pOpulations. Some effects of male-sterile cytoplasm and pollen fertility restorer genes on yield performances of hybrid maize are listed below: 8 l. Stringfield (1958) showed Texas-type male sterile cytoplasm with Rf restorer genes was usually more productive in adapted genetic background plants. 2. Everett (1960) showed a non-significant reduction in yield of 6.3 percent in a restored-sterile three-way cross hybrid. 3. Josephson and Knicer (1962) found no difference between yields of nine restored-sterile hybrids and their normal counterparts. Paez §£_§l (1969b) found significant differences in percent moisture at harvest between opaque¥2 and floury-2 vs normal maize. Opaque-2 exceeded normal lines by 1.9 percent moisture and floury-2 by 1.6 percent moisture. Similar differences were found in Illinois tests of single- cross (2X) opaque-2 varieties and their normal counter- parts (Alexander, 1968). Opaque-2 and floury-Z lines were significantly lower (3.5 gm and 2.3 gm resPectively) than normal maize in Missouri tests for weight per 100 kernels (Paez g£_§l, 1969b). Alexander (1968) likewise found single-cross opaque-2 varieties to be significantly lower in kernel weight. Germination Standard warm test germination results may fail to predict field stand accurately because of its failure to detect weaknesses in seeds (Delouche and Caldwell, 1960). Attempts by other workers to predict field performance more accurately have included seed behavior before or during germination (Moore, 1962), or germination of seed under unfavorable environmental conditions in cold test (Isely, 1957). Investigators (Hooks and Zuber, 1963; Ho and Melhus, 1940) found soil microorganisms had an adverse effect on seed germination and vigorous seedling growth. Micro- organisms were found to have adverse affects on corn at times including when the corn was planted in cold wet soil (Menon and Williams, 1957). With the advent of early maize planting dates, cold wet soils play a larger role in determining final plant population. Thus the cold test germination was found to be a more likely indicator of field germination than standard warm tests (Neptune and Rossman, 1953). Pinnell (1949) found high correlations between cold test germination results and field performance in inbred lines, single-cross and double-cross hybrids. Cold test germination studies reported by Alexander 10 (1968) indicated that on the average, inbred Opaque-2 lines germinated only one-half as well as their normal counter- parts. However some Opaque-2 lines were found to germinate nearly as well as the normal. Protein Analysis The Kjeldahl method of protein determination is used extensively but shortcomings such as requirements of expen- sive permanent equipment, use of large quantities of strong acid or alkali and use of excessive heat have definitely limited its usefulness (Zeleny, 1941). The sensitive colorimetric method of protein estima- tion using the Folin-Ciocalteu phenol reagent has been used widely with many different proteins (Bailey, 1967). The 'measurement of protein with copper and the Folin-Ciocalteu reagent has the advantage of requiring no digestion, is 10 to 20 times more sensitive than the ultraviolet absorption at 280 mp, is less liable to disturbance of turbidities, is several times more sensitive than the ninhydrin reaction, and is 100 times more sensitive than the biuret reaction (Lowry g£_§1, 1951). Lowry (1951) originally developed the colorimetric method of protein analysis using a determination wavelength of 750 mp and protein sources of crystalline trypsin, ll crystalline chymotrypsin, crystalline serum albumin, cytro- chrome Q, crystalline zinc insulin, gelatin and L-tyrosine. Chow and Goldstein (1960) showed that any peptide bond will yield some color in the Lowry method but that certain amino acid sequences, not necessarily containing aromatic amino acids, were more chromogenic than others and thus accounted for the majority of color developed. Jennings (1961) found very good correlation between Kjeldahl protein (total N X 6.25) and Lowry protein in bar- ley. He also found the colorimetric method to be as repro- ducible as the Kjeldahl method for protein determination. Alkali-Soluble Protein Extraction Breeding and selection for protein quality in maize requires analysis of the constituent proteins of the corn kernel. Results of a number of analyses have indicated large analystical variations in apparent percentages of different protein fractions. These variations could be due to alterations in extraction procedures (Nagy £3 a1, 1941). Jones and Csonka (1928) extracted glutelins (alkali- soluble protein) from whole ground maize with 0.2 percent sodium hydroxide after previous removal of salt-soluble and alcohol-soluble proteins (zein). Bressani and Mertz (1958) 12 removed 90 to 95 percent of the total endosperm proteins with a c0pper extractant. Glutelins were removed from the total extract after first removing the alcohol-soluble proteins. L-Lysine Analysis With the advent of high-lysine maize a quick, reliable lysine analysis became even more needed. The colorimetric procedure using the reagent l-fluoro-2, 4-dinitrobenzene (FDNB) has been used extensively to determine free é-amino groups of proteins to study their functional role in the biological activity of proteins (Ikenaka, 1959). Carpenter (1960) developed a method involving the use of FDNB to determine the available lysine measured as (-dinitr0pheny11ysine, in protein foodstuffs. High correlation was observed between the available lysine content and the biological value of the protein (Boyne gt; 2;, 1961). Due to the laborious and time consuming nature of the FDNB method, an alternate method utilizing the reagent 2,4,6-trinitrobenzenesu1fonic acid (TNBS) which specifically reacts with primary amino groups (Okuymma and Satake, 1960; Kotaki and Satake, 1964) appeared to be a simpler method of measuring available lysine in proteins (Kakade and Liener, 1969). 13 A TNBS method was developed by Habeeb (1966) to determine amino groups of proteins and Haynes gt a; (1967) used the TNBS method to study the role amino acids play in the activity of certain protease inhibitors. Habeebs method did not differentiate between é-amino and N-terminal amino groups in proteins. Kakade and Liener (1960) developed a TNBS method of analysis specific for available lysine in certain protein foodstuffs. In foodstuffs, some of the N-terminal-amino groups react with carbohydrate, thus rendering them unavailable for effective utilization by animals (Blom gt 31, 1967). Thus the blockage of the pheny- lation reaction by the £-amino—carbohydrate complex, result- ed in a measure of nutritionally available lysine (Kakade and Liener, 1969). The compound é-TNP-L-lysine was shown to have an absorption spectrum.with two peaks with maximum absorption at a wavelength of 346 mp and a smaller peak at 420 mp (Okuyama and Satake, 1960). MATERIALS AND METHODS Yield Studies Near-isogenic opaque-2l and normal maize hybrids (W64A-op-2 X MS-14-op-2 and W64A-TRf X M-14-TRf) were grown on the Crop Science Farm at East Lansing, Michigan in 1968. The maize was grown on Conover silt loam soil previously planted to maize. A split-split plot design was used to evaluate yield and yield components of the two hybrids planted at four populations and three nitrogen rates, rep- licated four times. The maize was planted by hand in 36 inch rows, May 18, 1968 and 280 pounds of 10-20-20 fertil- izer banded to the side and below the seed. To insure correct plant populations in the 30 foot, one-row plots, the normal seed was planted 1.5 times and the Opaque-2 seed planted approximately 2.5 times more than the desired final plant populations. The plots were thinned to desired plants per plot when the maize was approximately six to eight inches high. Final plant populations were 16,900, 21,800, 26,000 and 31,500 plants per acre. Nitrogen [KNH4)2NO31 1The opaque-2 hybrid was released in 1969 for certified seed production as Michigan 510-2XHL. 14 15 was applied at the rates of 0, 125 (applied July 7), and 250 pounds (125 on July 7 and 125 pounds on July 20) of actual nitrogen per acre with a hand applicator. Cultural practices included post-emergence herbicide (Atrazine) and two cultivations. A11 normal maize plots were detasseled to prevent outcrossing on the opaque-2. The plots were irrigated twice (about three inches total) during the growing season. All plots were hand harvested on October 12, 1968. Mois- ture samples were taken by slicing a one inch section from the center of 10 randomly chosen ears from each plot and dried at 105 C for 72 hours. Components of yield were calculated from a random sample of 10 ears from each plot. After ear and kernel measurements for field components, the 10 ears were machine shelled for shelling percent. Analyses of all yield and components of yield data were computed by STAT series AOV routines. Germination Several Opaque-2 and normal maize hybrids were germinated in order to test their viability and to a certain extent, their vigor. Germination methods used were warm test, cold test with a soil mixture, and cold test with l6 sterile non-soil conditions. The warm test germination was conducted by placing one-fourth inch of moist field-greenhouse soil mixture on previously water soaked folded newspaper. One hundred seeds for each of four replications were placed in equi- distant cross rows (approximately one inch apart) on the soil and covered with a thin layer of soil. The neWSpaper was rolled, fastened, and placed on end in humidity chambers at 25 C at near 100 percent relative humidity. After six days the rolls were removed and percent germination deter- mined by counting all seeds with normal roots and shoots, each three inches or greater in length. Preparation of neWSpaper and placement of seed for the cold test germination in soil was similar to the warm tests. Newspaper rolls containing soil and seed were placed in a cool room at 5 C for five days, then they were removed to 25 C chambers for six days to complete germination as in the warm test. Cold test germination was also conducted with sterile non-soil conditions. Glass petri-dishes (15 X 100 mm) containing two layers of water saturated blotter paper were autoclaved at 120 C (16 psi) for two hours. Sixty seeds of each maize sample were surface sterilized with a five percent laundry bleach solution for 10 minutes l7 (stirring of seeds was necessary to allow complete surface contact with the bleach). The samples were thoroughly rinsed twice with sterile water. The 60 seeds of each sample were divided into three petri-dishes (20 seeds per dish) along with approximately five ml of sterile water (enough water to saturate the blotter paper with no excess). The petri-dishes were placed in a dark box in a cool temperature room (5 C) for five days. Then the petri- dishes (in the box) were moved to a warm room (25 C) for six days. Percent germination was determined by counting only seedlings with normal roots and shoots one inch or greater in length. Protein Analysis A modification of the colorimetric nitrogen determin- ation method by Jennings (1961) was used to evaluate its use for determination of kernel protein content in maize. A modified biuret reagent used for initial extraction was prepared as follows: 15 ml of 10 N potassium hydroxide and 2.5 gm of potassium sodium tartrate (A.R.) was dissolved in approximately 900 ml of distilled water; 30 m1 of a four percent copper sulfate pentahydrate solution was added slowly with continuous stirring. Final volume was brought 18 to one liter with distilled water. All maize samples were ground in a Wiley mill through a 40 mesh screen. Five hundred mg samples of the ground ‘meal were placed in 125 ml Erlenmeyer flasks. A meal sample was placed into a water jacketed semi-micro blending jar (Eberbach Corp.) on a Waring blender (model 1042) after the addition of 25 ml biuret reagent and two m1 carbon tetrachloride. The mixture was blended at high speed for three minutes, then poured into the original flask. The blender jar was washed twice with an automatic pipette syringe using a total of 25 m1 additional biuret reagent. The bottle was stOppered with a cork and placed in a constant temperature (40 C) water bath shaker (Research Specialties Co.) for 1.0 hour. Upon completion of the reaction, portions of the biuret reagent extracts were centrifuged in 50‘m1 centrifuge tubes at 2500 X g for 10 minutes. Reagents for the treatment of the biuret extract were as follows: Reagent A. Two percent sodium carbonate in 0.1 N sodium hydroxide. Reagent B. 0.5 percent copper sulfate pentahydrate in one percent potassium sodium tartrate solution. A few drops of dilute sodium 19 hydroxide solution were necessary to clear the solution. Reagent C. Mixed 50 m1 of reagent A with one m1 of reagent B. Discarded after one day. Reagent D. Diluted Folin-Ciocalteu reagent (obtained commercially) to 1.0 N in acid. See Folin-Ciocalteu (1927) for preparation of this reagent. V A Hamilton automatic diSpensing syringe was used to transfer 0.2 ml of the centrifuged extracts (two replica- tions) into 15 X 150 mm test tubes. Ten m1 of reagent C was added and thoroughly mixed. After at least 15 minutes, 1.0 ml reagent D was added while vigorously stirring the reaction mixture on a test tube mixer (Scientific Products No. 58220). Immediate and thorough blending of the Folin- Ciocalteu reagent is critical. Sixty minutes was allowed for full color development before reading absorbance on a Spectronic 20 colorimeter with a .20 ml flow-through cuvette attachment, at a wavelength of 760 mp. A total of 16 samples, seven normal and nine opaque-2, were colorimetrically analyzed.with two replica- tions (run on different days) for each sample. The samples were also analyzed by the micro-Kjeldahl method on a mois- ture free basis. Correlation and regression analysis was 20 then performed on data from the colorimetric vs the micro- Kjeldahl analyses. Alkali-Soluble Protein Extraction The glutelin fraction was extracted from 10 gm of meal previously ground through a 40 mesh screen. The meal was defatted in 125 m1 Erlenmeyer flasks with hexane- dioxane (1:2 v/w) on a Burrell wrist-action shaker for one hour. The solvent was poured off and the samples evacuated to dryness. The alkali-soluble fraction was extracted in 250 ml centrifuge bottles with 0.1 N sodium hydroxide (5:2, v/w) for 4.5 hours on a Burrell wrist-action shaker at 23 C. The extracts were centrifuged at 7000 X g for 20 minutes and the supernatant poured off. The meal was then washed with 0.1 N sodium hydroxide (1:1, v/w) and centrifuged. After a second similar wash and centrifuga- tion, the extracts were stored at 2 C. TNP-L-Lysine Analysis Lysine analysis on whole meal was performed on material previously ground through a’40 mesh screen and stored in plastic bags at 0 C. Four replications of one mg of meal were accurately weighed and placed in 16 X 150 mm test tubes. One ml of borate buffer (pH 8.5) was added to each tube. Three m1 of concentrated hydrochloric acid (HCl) 21 were added to one replicate to form a blank solution and all tubes were then placed in a darkened constant temperature (40 C) water shaker bath for 10 minutes with moderate shaking. One ml of 1.0 percent 2,4,6-trinitrobenzene- sulfonic acid (TNBS) solution was added to all tubes and the reaction allowed to continue with shaking for two hours at 40 C. At the conclusion of the shake time, three m1 of con- centrated HCl was added to the remaining tubes to stOp the phenylation reaction. Glass shell vials (21 X 70 mm) were inverted over the reaction tubes. The tubes were auto- claved at 120 C (15-17 psi) for one hour. When removed from the autoclave the tubes were placed in a refrigerator at 2 C and allowed to cool to approximately room.tempera- ture. Five ml of distilled water was added to each cooled tube. Two extractions with 10 ml ethyl ether were made to remove TNP-N-terminal amino acids or peptides and picric acid which form during the course of the reaction. Insol- uble particulate matter was filtered out with Whatman No. 42 acid washed, filter paper. Residual ether was removed by placing the tubes in hot water for approximately 10 minutes. The aqueous solution was read at a wavelength of 346 mp on a Beckman DU Spectrophotometer against the blank solution. 22 Alkali-Soluble Protein Fractionation The extracted alkali-soluble protein was fractionated with ammonium sulfate [KNH4)ZSO4] into five components. A quantity of ammonium.sulfate solution (saturated at 23 C) equal to five percent of the extract volume of 15 ml was placed with the extract into 50 ml centrifuge tubes. The tubes were placed on a Burrell wrist-action shaker for 20 minutes, removed and Spun at 3000 X g for 20 minutes. The supernatant was poured into an ammonium sulfate solution 10 percent of its volume, then shaken and Spun as previously described. Repetition of this procedure was used to produce fractions of 10, 20, 30 and 80 percent ammonium sulfate. The precipitated material in each fractionation was resolubilized in 15 ml of borate buffer (pH 8.5) with shak- ing. All fractions were stored at 2 C. Lysine Analysis of Alkali-Soluble Protein Alkali-soluble protein extract aliquots of 0.25 ml were pipetted into 16 X 150 mm test tubes and one m1 borate buffer (pH 8.5) added to each tube (one HCl blank and three reaction replications). The tubes were then placed in a constant temperature (40 C) water bath shaker. Subsequent treatment was the same as for lysine analysis of whole meal samples, except 4.75 ml water was added to the cooled 23 hydrolysate instead of five ml in order to bring the final reaction mixture volume to 10 ml and no filtration was necessary. Lysine Analysis of Fractionated Alkali-Soluble Protein Aliquots of one ml were used to analyze the lysine content of the buffered ammonium sulfate fractions of the alkali-soluble protein. Three reaction tubes and one HCl blank were treated as previously stated for the alkali- soluble protein analyses. TNP-L-Lysine Standard Curve é-TNP-L-lysine HCl-HZO (Nutritional Biochemicals Corp.) was used to produce a standard curve (figure 1) for calculation of quantities of L-lysine in meal and extracts. To obtain analogous conditions for production of the standard curve as used for meal and extract analysis, the following procedure was used: Ten mg e-TNP-L-lysine HCl-HZO was added to one ml distilled.water and one m1 borate buffer (pH 8.5) in a test tube and allowed to stand at 40 C for one hour. After three ml HCl was added to the reaction mixture, a shell vial was placed over the tube and the contents autoclaved at 120 C (15-17 psi) for one hour. The solution was then cooled to room temperature. After 24 the addition of five ml distilled water the solution was twice extracted with 10 m1 ethyl ether. Excess ether was volatilized off with a hot water bath. Aliquots were taken such that final quantities of 50, 100, 150, 200 and 250 pg TNP-L-lysine were contained in 10 ml distilled water. Absorbance of é-TNP-L-lysine was measured at wavelengths of 346 mp and 420 mp on a Beckman DU Spectrophotometer. 25 O 346 mp Absorbance (346 mp and 420 mp) 0 50 100 * 150 200 250 300 pg {-TNP-L-lysine Figure 1. Standard curve used for TNP-L-lysine analysis based on absorbance at 346 mp and 420 mp wavelengths. RESULTS AND DISCUSSION Agronomic Field Studies Yield Analysis The opaque-2 maize hybrid averaged 82.3 bushels per acre2 and the normal hybrid averaged 77.3 bushels per acre over the entire experiment. This difference of about five bushels per acre was highly significant (figure 2, table 2). Generally higher yield for the opaque-2 hybrid was unexpected in view of previous results (Alexander, 1968; Rossman, 1969) in which the normal hybrids averaged zero to 15 percent higher yield than opaque-2. Detasselinginjury to the normal hybrid could account for a portion of the difference here. The population main effect was highly significant indicating that as population rates increased, average yield of both hybrids increased, 68.4, 79.8, 84.0, 87.1 bushels for populations of 16,900, 21,800, 26,600 and 31,500 reapectively (figure 3, table 2). Only at the population 2All calculations are based on one bushel of maize weighing 56 pounds at 15.5 percent moisture. 26 an.o . a x a - so can an.o . a x a - nc.nma ~.~o o.o. e.n~ ~-osvoao 0.x" o.oo o.n~ dunno: z.on~ z-n- z-o anus»: .2'7 A-vuouu ouuuoauv neauuouounu cououuuz u punch: Aa-ao-ca-m= x ~-ao-¢4osa n.~m o.am o.~o o.ooa o.o~ o.an ~.~o o.oa n.~. ~.oo o.~a ~.n. a.sa n.oo n.oo n.oa a.ao ~-o=rqao Auau.s~-: x uuu-¢< z-on~ z-n~a z-o .o>< z-on~ z-n- z-o .o>< z-cn~ z-n- z-o .o>< x-on~ z-n~a z-o con.~n coo.o~ oou.a~ coo.o~ noun»: coda-anaoa sauna .nom~ .wcuocoq uoou .-o>o~ conouuac noun» use occauodsnon ocean unou u. Anuoarono 1a. nuluonv ovuunas snail 03» now endow» ououo>< .N oaaoh 28 Table 3. Analysis of variance for yield of two corn hybrids at four plant pOpulations and three nitrogen levels, East Lansing, 1968. Degrees Approximate of Mean Significance Source Freedom Square of F Statistic Replications 3 201.71 .510 Nitrogen 2 839.45 .094 Error R X N 6 233.70 Population 3 1613.81 .002 Error R X P 9 139.54 Nitrogen X 6 163.40 .074 Population Error R X N X P 18 69.16 Hybrid Hybrid 1 580.71 .001 Hybrid X Nitrogen 2 69.55 .218 Hybrid X Population 3 68.65 .213 Hybrid X Nitrogen 6 111.60 .037 X POpulation Error 36 43.71 29 Amos. n my .oon.~m Ase was ”80.8 Ame moom.am Ame Moos.sa AHV mo maoaumaaaoa ucmam um UHOH% How uoommm cams aowumasaom .m mhdwflm coaumasaom ucmHm s m N . a Z . ; -......-_ .A.ce J on aloe/nq u; p131; cm :8. a B .onama Hmahoc paw unmoumao mo vHOHA Mom uommmm came panama .N ouawfih shuns: Nuno .IL A]- z . . E .IIIIJ. T H Laos 4_on aloe/nq u: p131; 30 90 0) . m 0 op-2 \ 80 9 B / \n N O .5 a 3 70 m oa >‘ 60 l 2 3 Nitrogen Figure 4. Hybrid X nitrogen interaction for yield of Opaque-2 and normal maize grown at supplemental nitrogen rates of (l) 0 lbs; (2) 125 lbs; and (3) 250 lbs per acre (F = .094). \O O \l 0 Yield In bu/acre oo <3 \1 2: O‘ O l 2 3 4 Plant Population Figure 5. Hybrid X population interaction for yield of Opaque-2 and normal maize at plant populations 0f (1) 16,900; (2) 21,800; (3) 26,000; and (4)3l,500. (F = .213) 3] 100 P O itro-2 90 . 0 II a U / 8 / NitrO-3 \ O 3 '2 80 P n -H ___ ———o 3 . \ . .3 Nitro-l >-' 70 b 8 o 60 _ l~ II: If ‘4: L__._. l 2 3 4 Plant POpulation Figure 6. Nitrogen X population interaction for yield at 3 supplemental nitrogen rates: (1) 0 lbs, (2), 125 lbs, (3) 250 lbs, and 4 plant populations: (1) 16,900, (2) 21,800, (3) 26,600, and (4) 31,500. (F = .074). 32 rates of 16,900 and 31,500 were yields produced which were significantly different between the two hybrids. The hybrid X nitrogen (figure 4) and hybrid X population (figure 5) interactions show that on the average, the Opaque-2 hybrid yielded more than the normal at all levels of nitrogen and all population levels (table 2). Failure of the yield curves to break and turn down at 26,600 and 31,500 at these relatively low yield levels, less than 100 bushels per acre, was unusual. Most hybrid X pOpulation studies at these yield levels show lower yields at populations of 20,000 and above (Rossman and Cook, 1966). The nitrogen X pOpulation interaction (figure 6) shows that on the average, the 125 pound rate of supple- mental nitrogen (nitro-2) produced the highest yields at all levels of population. The highest yield, 109.0, for the opaque hybrid was produced with a pOpulation rate of 31,500 and 125 pounds of nitrogen. The highest yields for the normal hybrid were 86 and 87 bushels, produced at a popula- tion of 26,600 with 125 pounds of nitrogen, and 31,500 with either 125 or 250 pounds of nitrogen. There was no significant difference (LSD) between the two hybrids at any one level of nitrogen (table 2) even at the 125 pound nitrogen rate where highest yields were obtained (88.6 for opaque-2 and 80.9 for normal). No 33 significance (LSD) was found at any one pOpulation level (table 2) including the highest yielding population (31,500 plants per acre) where yields were 83.2 bushels per acre (normal) and 91.0 bushels per acre (opaque-2). Greatest differences in yields were obtained at the high- est level of plant population (31,500 plants per acre) but significance (LSD) was found between only the yields at the zero pounds nitrogen rate and the 125 pOunds nitrogen rate. The amount of outcrossing on opaque-2 from normal pollen was estimated at less than one percent. Most comparisons of near-isogenic Opaque-2 vs normal hybrids with reSpect to yield have indicated that normal maize out-yields the opaque-2 hybrids, although some exceptions have been reported, (Alexander, 1968). While the opaque-2 averaged 82.3 bushels per acre the normal averaged 77.3 bushels per acre in this experiment, under similar cultural practices in the same field the single- cross hybrid, Michigan 500-2X (2X = single-cross), yielded 139.2 bushels per acre (Rossman g£_al, 1968). Under the conditions of this experiment, neither the opaque-2 nor normal hybrids could be considered commercially competitive hybrids. However, it was necessary during the growing season to walk between the rows many times for purposes of detasseling and hand pollination. Therefore compaction of 34 the soil between the rows of maize may have lowered aera- tion in the root zone thus reducing yield in both hybrids. The normal hybrid in this experiment contained the Texas source of male sterility with restorer genes present. Past results have indicated that variable results may be expected in hybrids containing male sterility and restorer genes, but generally non-significant results have been obtained (Stringfield, 1958; Josephson and Knicer, 1962). Removal of tassel alone enhanced yields, removal of tassel plus one leaf tended to give yields equivalent to complete plants, and removal of the tassel plus two leaves decreased yields from five to seven percent in studies by Hunter e_t g (1969) . This experiment encompassed all three combinations of tassel removal with perhaps more removal of tassel plus two leaves than tassel alone. Therefore it is possible that there was some reduction in yield from detasseling the normal hybrid. The effect of population indicated that these two hybrids maintained their yield under stress from high populations, but their yields were not exceptionally high having been irrigated and grown during a relatively good maize growing season in Michigan. Previous data showed that nitrogen levels of 100 to 35 150 pounds of nitrogen often resulted in higher yields than either lower or higher nitrogen levels (Lucas, 1969b). Similar results occurred in this experiment for each hybrid and for all population levels. Shelling Percent The hybrid main effect indicated that the normal hybrid had a highly significant greater (85.5 vs 80.5 percent) shelling percent than opaque-2 (figure 7). The population main effect (figure 8) showed that over the entire experiment, as population rates increased, the shelling percentage of both the opaque-2 and normal hybrids also increased (82.4, 82.9, 83.1 and 83.7 percent). The interactions between hybrids X nitrogen level (figure 9) and hybrid X pOpulation level (figure 10) showed that the normal hybrid had a greater shelling per- cent at all levels of nitrogen and all population rates. A highly significant increase in shelling percent was shown at all nitrogen and all pOpulation levels for normal maize over Opaque-2 maize (table 4). Results of the shelling percent experiment are consistent with previous comparisons of opaque-2 and normal maize. The kernel of opaque-2 had a lower weight per 100 kernels and a lower Specific gravity than normal maize 3&6 m.ow m.nm owouo>< sees»: no. moo. - z x m - emu see. u A x m - no.9mu o.aa m.om ~.~o ~-sseseo e.nm n.nm s.na uuauoz z-om~ z-m- z-o sauna: Aucoouoa mafiaaonm owouo>uv newuouuoucw cumouuuz x vuupzx Am-eo-sA-mz x ~-eo-< z-ow~ z-m~H z-o .s>< z-on~ z-n- z-o .s>< z-on~ z-n- z-o .s>< z-om~ z-n- z-o oom.~n oos.e~ oom.- ooo.ee sauna: gang—act ufiddm .wooH .wcfimccq umam .aHO>OH cowouuac wounu use occuuquaon panda uaou ua Auuonvuao vac Huauocv spawn»; «name can you unbound mcuuaosa owauo>< .e vanes 37 Table 5. Analysis of variance for shelling percent of two corn hybrids at four plant populations and three nitrogen levels, East Lansing, 1968. Degrees Approximate of Mean Significance Source Freedom Square of F Statistic W Replications 3 1.341 .681 Nitrogen 2 4.119 .275 Error R X N 6 2.555 Population 3 7.203 .016 Error R X P 9 1.215 Nitrogen X Population 6 1.630 .025 Error R X N X P 18 .508 Hybrid 1 596.605 .0005 Hybrid X Nitrogen 2 7.920 .008 Hybrid X POpulation 3 3.786 .064 Hybrid X Nitrogen X 6 2.387 .158 Population Error 36 1.432 38 Acne. n so .oom.em Ase use mooe.e~ Ame moom.H~ Amy mooe.ea Ase mo acowumanmon um unnamed waHHHmsm How uommmo dams coaumfinaom .w muawfih cowumanaom ucmHm s m N H A file -lls file .7]- Hm mm quaazaa Enlllaqs on $8. a B .ouwma Hashes paw Nuoawmno a“, ucmuhoa wawaamsm now assume sees sees»: sense: «:90 .n madman E H ills -lls N a s nuaozaa BUIIIBHS Shelling Percent 39 90 85 ° ° ° N ONO 80 \o op-Z 75 fi -. ‘ — 1 2 3 Nitrogen Figure 9. Hybrid X nitrogen interaction for shelling per- cent in opaque-2 and normal maize grown at supplemental nitrogen rates of (l) 0 lbs; (2) 125 lbs; and (3) 250 lbs. (F = .008) Shelling Percent Figure 10. 90 85 ° o/° ° N 0 o__. o op-2 80 o/ 75 W l 2 3 4 Plant Population Hybrid X population interactiOn for Shelling percent in opaque-2 and normal maize grown at plant populations of (1) 16,900; (2) 21,800; (3) 26,600; and (4) 31,500. (F = .064) 4O (Wichser, 1966; Alexander, 1969; Paez g£_§l, 1969b). The lower density of opaque-2 appears to be the result of a smaller proportion of endOSperm in the kernel near or complete absence of horny endosperm and much more loosely arranged starch in the endOSperm (Watson, 1966). Kernels of most Opaque-2 types have a shrunken appearance when dried at maturity. The shrunken appearance is due to the more loosely arranged endosperm material. Percent Moisture at Harvest The percent moisture at harvest averaged higher in the opaque-2 hybrid than in the normal over the entire experiment (37.6 vs 36.3 percent, figure 11). The hybrid X nitrogen interaction (figure 12) shows an exception to this trend in that the normal hybrid had a higher percent moisture than the opaque-2 hybrid with zero pounds nitro- gen. At 125 pounds nitrogen and 250 pounds nitrogen, the opaque-2 hybrid had a significantly higher percent moisture than the normal hybrid. The Opaque-2 hybrid had a higher percent moisture than the normal hybrid at all population rates (figure 13). There was no significant difference between the hybrids at any one population level (table 6). Opaque-2 maize has, in previous experiments, been 41 shown to have a higher moisture content at harvest than its normal counterpart (Alexander, 1968). The alteration of the endosperm storage pattern in opaque-2 probably accounts for the higher moisture content. For example, Watson (1966) reported that the sugar content of Opaque-2 maize was 2.6 percent compared to 1.9 percent in normal maize. The Storage form of starch in Opaque-2 has been shown to be more loosely arranged and horny endosperm has been lack- ing (Wichser, 1966). The loosely arranged starch in the endOSperm would tend to hold more water than the compacted starch which forms the horny endosperm. Also, the storage protein shifted from zein to non-zein forms (Concon, 1966). Duvick (1961) Speculated that zein was stored in tight granules in the endOSperm tissue. Wolf 93 a; (1967), using electron microscopy and biochemical analysis, found Duvick's speculation was true and that glutelins, globu- linS, and albumins are viscous in nature. She found that opaque-2 maize had fewer endOSperm protein granules than did normal. It appears that, as was found with starch, the tight globular granules of zein would retain less water than the more viscous protein forms. 42 owmuo>< seeps: o.nm n.on n.0m a.nm o.nn s.mm .o>< zlonm Zumwa oom.~n .moo~ .wCAmcau umam .mdo>oa cowouuac moucu one accuuaannoa usage know us Amnonwuno van Huauocv «wanna; oana can you ouaunaoa unbouon owuuo>< o.mn Zuo n.0n .o>< ~.on n.5n o.¢m w.nn N.om ~.mn 0.5m w.mm 2-0mw znmmfi Zuo .o>< ooe.o~ cbwuadnnom ucaam ~.mn m.nm n.5m m.mm $.Qn 0.0m zuomu zumwa 2-0 oom.- Amununaoa 0.9m .o>< os.~ a z x = - no.amu os.~ u z x a - mo.nmu e.mn A.an n.sn ~-o=eseo n.0m ~.nm s.en asauoz z-on~ z-n- z-o sens»: accouoa owuuo>av abuuuououan unwound: x pawns: A~-eo-sa-mz x ~-eo-¢sto e.en e.sn a.en ~-oseseo Aume-sD-z x uma-< papa»: .wooa H.m~ o.¢~ m.c~ m.mN .o>< 2-0mm znnmfi Zoo 00m.am .wcuaCuA uaom .uao>u~ :uwouuua no. o~.u I z x I n nmg ee.~ . e x m - no. and ~.N~ ~.N~ o.- «toavuno o.n~ o.m~ o.n~ unluoz z-cn~ z-m- z-o sens»: Andean»: cod you uzwuoa ammuo>¢v acauuquoucu cumouuuz K vaunzz 3-8-3-? x «-8433 o.- m.a~ H.N~ ¢.N~ n.- ~.~N s.- N.- m.- ~.m~ h.- m.~u Nuoscqao Amma-s~-x x «me-< z-OnN z-n- 2-0 .o>< zlomw Zumma 2-0 .o>< zuonu zunuu 2-0 ooe.e~ oom.- ooe.e~ sees»: seeuaaaeoe segue oousu van acouuodsnoa unaua know u- Autosvuao can Aaauocv coaunzs unaua can you awash»: oo~ Mon unwav: ow-uo>< .m o~AOH Table 9. Analysis of variance for weight per 100 kernels of two corn hybrids at four plant populations and three nitrogen levels, East Lansing, 1968. Source Replications Nitrogen Error R X N Population Error R X P Nitrogen X Population Error R X N X P Hybrid Hybrid X Nitrogen Hybrid X Population Hybrid X Nitrogen X Population Error Degrees of Freedom 3 2 36 Mean Square M 115. 13. 10. .603 .030 .339 .058 .375 .920 .012 194 938 395 .959 .499 Approximate Significance of F Statistic .853 .084 .988 .511 .0005 .001 .001 .699 26 24 22 Weight/100 kernels (gm) 20 49 LILY—I:— Figure 14. N op-2 Hybrid Hybrid main effect for weight per 100 kernels of opaque-2 and normal maize. (F = .0005) Weight/100 kernels (gm) Figure 15. Weight/100 kernels (gm) Figure 16. 50 27.5 D 25.0 \ n— _—n N 22.5 o 2 O _ 0/ Op 20.0 1 2 3 Nitrogen Hybrid X nitrogen interaction for weight/100 kernels of opaque-2 and normal maize at supple- mental nitrogen rates of (l) 0 lbs; (2) 125 lbs; (3) 250 lbs. 27.5 25.0 ° N /u/ - "/0. O 22.5 \,_ fie . \O op-z 20.0 1 2 3 4 Plant Population Hybrid X population interaction for weight/100 kernels of Opaque-2 and normal maize at plant populations of (1) 16,900; (2) 21,800; (3) 26,600; and (4) 31,500. 51 Row Number/Ear The population main effect was highly Significant for row number per ear. Number of rows per ear decreased as pOpulation level increased (16,900 = 16.0; 21,800 = 15.4; 26,600 = 15.0; 31,500 = 14.8, figure 17). The hybrid X pOpulation interaction was highly significant (figure 19). The opaque-2 hybrid averaged lower on kernel row number at low populations and averaged higher at the high populations than the normal hybrid. No clear explan- ation is apparent for this interaction. It would appear that if lack of population stress was conducive to a greater number of kernel rows for one hybrid it would be conducive for both hybrids. There were no significant differences between hybrids at any of the three nitrogen rates or at any of the four pOpulation rates tested (table 10). Kernels/Ear Row The hybrid main effect showed that the Opaque-2 maize had a highly significant greater number of kernels per ear row than normal maize (35.9 vs 29.0, figure 20). The population main effect was highly significant and Showed that as pOpulation rate increased the number of £522 ammuw>< saunas .mooH .weeecsu seem .sus>su csmouusc amuse use ~.mH 0.0H ~.¢~ ~.mH ZuOmN zunua 00n.Hn «.ma 0.0A zuo n.0H .o>< o.n~ H.na 0.0a a.ma ~.m~ n.0a 0.0a o.n~ zuonN zunua 2:0 .u>< 000.0N ceauoasnom ucoum oe.a . z x a - no.ama 2; .. e x a - 3.80 d2 92 «.2 «-2625 “.3 DD «.2 28oz 2-an .32 2... Sue": Anne pom axon gonna: mo Nunez: owINo>nv neuuoeuouau unwound: x wanna: o.nH ~.na m.m~ w.na 2-0mm zunNH 000.~N 0.m~ o.n~ 2-0 «.mu n.m~ 0.n~ n.na 0.0a N.n~ c.0u 0.o~ .o>< zocnu Ionuu 2-0 ooo.o~ accuuaannoa ucaan know us Auuoauaao one auahocv uuwunms onaqa can you ado his anon ducks: «0 yonasc awauo>< 3-8-3%: 0. «lo-.320 «nonvoao Cup-3-x x mes-<33 guano: seas»: .oH wanna 53 Table 11. Analysis of variance for number of kernel rows per ear of two corn hybrids at four plant populations and three nitrogen levels, East Lansing, 1968. Degrees of Mean Source Freedom Square Approximate Significance of F Statistic Replications 3 .946 Nitrogen 2 .155 Error R X N 6 2.438 POpulation 3 6.494 Error R X P 9 .551 Nitrogen X Population 6 .325 Error R X N X P 18 .935 Hybrid ' 1 1.330 Hybrid X Nitrogen 2 1.647 Hybrid X Population 3 7.751 Hybrid X Nitrogen X 6 1.412 Population Error 36 1.493 .766 .939 .002 .902 .352 .343 .004 .475 54 16 . ______ Number of kernel rows per ear I II I Figure 17. I j 2 3 4 Plant Population Population main effect for number of kernel rows/ear in opaque-2 and normal maize. (F = . 002) 55 H 16.0 M U \ g. 3 15.5 0 ° ° N H. C) a . , o — 3 1500 /o p '44 o 0 c3 z 14.5 1 2 3 Nitrogen Figure 18. Hybrid X nitrogen interaction for number of kernel rows per ear in Opaque-2 and normal maize grown at supplemental nitrogen rates of (l) 0 lbs; (2) 125 lbs; and (3) 250 lbs. (F = .343) H 17 q; n o \ m g 16 \\\\\\\\\ '3: O O l: 15 ° ° op-2 °. ...————————-° N E 14 l 2 3 4 Plant Population Figure 19. Hybrid X population interaction for number of kernel rows per ear in opaque-2 and normal maize grown at plant populations of (1) 16,900; (2) 21,800; (3) 26,600; and (4) 31,500. (F - .004) 56 kernels per ear row decreased (16,900 = 35.2; 21,800 = 33.8; 26,600 = 31.8; 31,500 = 21.1, figure 21). The hybrid X nitrogen interaction was not signifi- cant (figure 22), but at each of the three nitrogen rates, the opaque-2 hybrid had a highly significant greater number of kernels per ear row than the normal hybrid (table 12). The hybrid X population interaction indicated the opaque-2 maize had a significantly greater number of kernels per ear row than its normal counterpart at all populations (figure 23, table 12). Total Kernels/Ear The opaque-2 hybrid averaged 544.1 kernels per ear compared to the normal hybrid average of 451.5 kernels per ear (figure 24). The population main effect was highly significant. Number of kernels per ear decreased as popula- tion increased (16,900 = 562.1; 21,800 = 521.1; 26,600 = 475.7; 31,500 = 431.4, figure 25). The hybrid X nitrogen interaction was not signif- icant (figure 26). At each of the three nitrogen rates the Opaque-2 hybrid had a Significantly greater number of kernels per ear than the normal hybrid (table 14). The 125 pound nitrogen rate produced the highest average yields for both hybrids and also produced the greatest number of 537 0.0N m-mN m.c~ .o>< 210mm owmuo>< cues»: 00n.am n.~m n.¢~ zuo .mooa .wcumcmq umam .m~o>oH cowouua: mouSu $5.4 n z x x - mo.owa m~.s u e x x - no.0mo c.en H.em A.sn N-oseseo m.m~ e.s~ ~.m~ usauoz z-omw z-nwe z-o sfiuasx A3ou moo you oaocuox mo Mensa: owouo>¢v coHuomuouGH cowouuwz x pawn»: A~-eo-sH-mz x -eo-< z-omu z-m- z-o .s>< z-om~ z-nNH z-o .s>< z-onu z-msn z-o ooo.e~ oom.s~ ooe.ea uses»: cauumaaaom ucuam 0cm oceaumanaoa ocean uaou ucfimnosvuno one amahosv moans»; ouaqa can now no» you non nauflhox mo hogan: omeko>< .Na canes 58 Table 13. Analysis of variance for number of kernels per ear row of two corn hybrids at four plant populations and three nitrogen levels, East Lansing, 1968. Degrees Approximate of Mean Significance -“ Source Freedom Square of F Statistic ‘ Replications 3 8.83 .663 Nitrogen 2 31.10 .222 Error R X N 6 15.91 POpulation 3 172.32 .0005 Error R X P 9 10.10 Nitrogen X Population 6 6.95 .899 Error R X N X P 18 19.70 Hybrid 1 1141.26 .0005 Hybrid X Nitrogen 2 1.91 .843 Hybrid X Population 3 32.88 .046 Hybrid X Nitrogen X 6 10.52 .476 POpulation Error 36 11.14 59 Amoco. u_mv .mnaoa Hmahoa 0am Nuodvmao a“ son um0\maochox mo Hogan: How uommmo came aoaumanaom aowumanmom ocean e m N .HN seamen mm on mm 00 no; lea/SISuJax go °oN Amoco. n so .ONHQB Hmahoa can Nuonvmao a“ Son Hem \maoahox Hogans Mom . assume sees seesaw .o~ seamen sees»: N-eo z In to no: Jae/staulax go 'ON C In on N 00 No. of kernels/ear row Figure 22. No. of kernels/ear row Figure 23. 60 40 o 35 o/ \0 Op-2 30 a/.\' N 25 H— ‘ ‘ 1 2 3 Nitrogen Hybrid X nitrogen interaction for number of kernels/ear row in Opaque-2 and normal maize grown at supplemental nitrogen rates of (1) 0 lbs; (2) 125 lbs; and (3) 250 lbs. (F = .843) 40 °\° o 35 \ e n\\\\\\\\\ o op-2 30 '\ 25 “\° N l 2. 3 4 Plant Population Hybrid X pOpulation interaction for number of kernels/ear row in opaque-2 and normal maize grown at plant populations of (1) 16,900; (2) 21,800; (3) 26,600; and (4) 31,500. (F = .046) 61 kernels per ear, indicating a greater total develOpment of the individual ears. The hybrid X population interaction was highly significant. As pOpulation rate increased the total number of kernels per ear decreased, but the trend was more pro- nounced in the normal hybrid than in opaque-2 (figure 27). Comparison of the two hybrids at individual plant popula- tion rates (table 14) showed that significant differences were obtained only at 26,600 and 31,500 pOpulations. Total Kernel Weight/Ear The hybrid.main effect showed the total kernel weight per ear of opaque-2 maize was significantly greater than that of the normal hybrid (120.4 vs 109.1 gm” figure 28). The population main effect was highly significant. As population increased, total kernel weight per ear de- creased (16,900 = 130.0; 21,800 = 120.5; 26,600 = 109.5; and 31,500 = 98.9 gm, figure 29). The hybrid X nitrogen interaction was not signif- icant (figure 30). Opaque-2 maize averaged greater total kernel weight per ear than normal at all three nitrogen rates but the differences were not significant at any one level of nitrogen. The hybrid X population interaction was significant 62 nn.on I z x z a and mm.on I m x x u amd e.¢sn s.emn ~.nmn ~-oauuao ~.ans n.mcs «.mne duauoz z-on~ z-n- z-o cans»: Auno non humans Hocuox Aouou ownuo>av noduoauuuaH cowouuaz x caunhz H.¢vn N.~¢¢ N.¢o¢ «.mom o.hnc N.aon «.mmn o.wwm ¢.m¢m ¢.mnm n.mon m.@om m.an¢ n.0nm H.Qan ~.ooc m.~on n.~on a.oo¢ a.o~¢ m.non o.om¢ m.oon n.nhq .o>< zuonm Zunma 2-0 .o>< Zuonu ZunNH 2-0 .o>< z-on~ Zunmm ammuo>< sauna: com um coo om com on :ouuoaaoom unaam n.¢~n e.ow¢ Zno h.mom e.¢nn .o>< a~-ao-sa-mx x ~-ao-<¢ozv o.aon a.~mm a.¢mm ~-oavaao Amma-¢u-x x «ma-u~ cowouuac wanna use acouuoaaaon ocuaa know an Amuosvono van anhocv ovaqun onuqa otu Mom hue hon Russia geckos Hauou owquo>< .ca manoh 63 Table 15. Analysis of variance for total kernel number per ear of two corn hybrids at four plant popula- tions and three nitrogen levels, East Lansing, 1968. Degrees Approximate of Mean Significance Source Freedom Square of F Statistic Replications 3 4,299.43 .636 Nitrogen 2 6,054.60 .473 Error R X N 6 7,120.38 POpulation 3 76,241.73 .0005 Error R X P 9 3,395.05 Nitrogen X Population 6 1,586.88 .846 Error R X N X P 18 3,649.62 Hybrid 1 205,882.26 .0005 Hybrid X Nitrogen 2 649.92 .811 .Hybrid X POpulation 3 26,853.85 .0005 Hybrid X Nitrogen X 6 2,927.17 .473 Population Error 36 3,083.51 Amoco..n mv .onwma Hmahoc pow Nuosumoo SH Bow Hmo\mfioauox Hmuou Mom uoommm cams cowumasoom .mN ouawam couumasoom unmam o m N a . [E fill 4 I I 64 _ A as om¢ com O In In coo lea/stauaaa 19301 Amoco. u my . .onama Hmauoa pom Nuosvmno a“ Hmo\maoaumx amuou you uoommo Gama wanna: vaunsm Numo z .qN «gamma 1 o In \‘r com 0 In Ln com lea/SIauaax 19:01 65 H600 - <3 G) \ o g 550 P o/ \o Op-2 '33 500 E ,2 II a N '3 450 D n / 4.3 O F'400 - f a a n l 2 3 Nitrogen Figure 26. Hybrid X nitrogen interaction for total kernels/ ear in Opaque-2 and normal maize grown at supplemental nitrogen rates of (l) 0 lbs; (2) 125 lbs; and (3) 250 lbs. (F = .811) 600 P g 550 b u\.o/° £2 \\\\\\\\\ \\\\\\\\\ 35.00 » H ° ° 013’2 0 {3450 - .2 3400 L E3 \n N 350 - *f L _I ‘ 1 2 3 4 Plant Population Figure 27. Hybrid X pOpulation interaction for total kernels/ear in opaque-2 and normal maize grown at plant populations of (1) 16,900; (2) 21,800; (3) 26,600; and (4) 31,500. (F = .0005) 66 «.0NH o.¢o~ ~.oo~ “.mo . 0>< owmuo>¢ sauna: .mooa .mcamcwa ummm H.00a n.wo~ ~.oo~ o.mw n.ooH ~.mo 2-0mm Zumma z-o con.~m .m~o>oa cowouuwc oops» «.mwa m.m- s.oNH m.-~ m.mo n.no n.5m ¢.¢m .o>< ZnOmN z-m~H zuo ooo.o~ ceauuasoom can mcoHuoaooon ucaao poem um m.w~a H.~H~ .o>< ucaaa a~-o=vuao no. ma.o~ . z x m - on; ma.o~ . a x = - no.ama n.o~a ~.n- a.aaa ~-oacuao a.hoa «.moa o.oaa auauoz z-om~ z-n~a 2-0 sauna: Anna non unwwma Hocuox Houou owoum>uv coauoououca cowouuwz x manna: n.0ma m.m~a o.waa «.6Ha 2-0mm Zunma oom.- w.oaa o.nNu zuo n.a- c.0ma .o>< Aw-ao-¢2-mz x ~-ao-< .oa manna 67 Table 17. Analysis of variance for total kernel weight per ear of two corn hybrids at four plant popula- tions and three nitrogen levels, East Lansing, 1968. Degrees Approximate of Mean Significance Source Freedom Square of F Statistic Replications 3 355.89 .592 Nitrogen 2 43.55 .920 Error R X N 6 518.09 Population 3 4352.43 .0005 Error R X P 9 209.46 Nitrogen X POpulation 6 108.21 .806 Error R X N X P 18 219.72 Hybrid 1 3090.33 .0005 Hybrid X Nitrogen 2 111.72 .573 Hybrid X Population 3 848.43 .011 Hybrid X Nitrogen X 6 122.44 .714 Population Error 36 197.82 68 Amoco. u av 825. n B 63% .ouw a Hmauoa mam guano: pom Numacmoo nosumno.aH Moo H3 Hocuox Hmuou aw Hmo\u3 Hochox Houou . N How uommwo\:wme aowumasaom .om onswwm How uoommm cams vaunxm mm mhswwm coflumasaom unmam pwunzm e m N H Tao z Hull— IIHUIIDIQ Al- om -fil- 42: L Tu 0 w 3 e m. T.— 2: a u m .oaa m a .l T. A . oHH WW .m. m m I, ONH w, a e oma m” 1 a a oma omH 69 130 E‘s H ' °“‘r--. @120 o/ o op-Z fl 3110 - °\ C ”\D a: N .2 H _3100r O . ‘ ‘ h ‘" 1 2 3 Nitrogen Figure 30. Hybrid X nitrogen interaction for total kernel weight/ear in opaque-2 and normal maize grown at supplemental nitrogen rates of (l) 0 lbs; (2) 125 lbs; and (3) 250 lbs. (F = .573) H o :3 120 \ o ‘t a g, 1:110 E o op-Z 3 100 H n \n N CU l5 90 H H ‘ _f L 1 2 3 4 Plant Population - Figure 31. Hybrid X population interaction for total kernel weight/ear-in Opaque-2 and normal maize grown at plant population rates of (1) 16,900; (2) 21,800; (3) 26,600; and (4) 31,500. (F'= .0005) 70 (figure 31). There was a trend toward lower total kernel weight for both hybrids as pOpulation increased, but the differences between the two were significant only at the 26,600 population (table 16). The components of yield analyses revealed some factors which help to explain the higher average yields obtained in the opaque-2 hybrid compared to the normal. The opaque-2 maize deve10ped larger individual ears than the normal, as reflected by the component, total kernel weight per ear. This value is obtained by multiplying the weight per 100 kernels X total kernels per ear. The individual opaque-2 ears contained enough total kernels per ear to more than offset the higher test weight found in the normal hybrid, thus resulting in overall higher yields. Germination Study Warm test germination of single-cross hybrids showed the overall germination percent of the normal hybrids was 98.6 percent compared to 96.0 percent for Opaque-2 (table 18). Fungicide treated seeds in this test had essentially no advantage over untreated ones (97.4 vs 97.1 percent). Results of the warm test germination showed that under near ideal conditions almost perfect germination can be expected from either normal or Opaque-2 maize whether 71 Table 18. Comparison of soil warm test germination percent in opaque-2 vs normal maize. Captan Percent Pedigree Treated Germination M-14 X W64A No 98.5 Yes 97.5 W64 X M-l4 No 99.3 Yes 98.0 M-l4-TRf X W64A-TRf Yes 99.8 W64A-op-2 X MS-l4-op-2 Yes 97.8 (medium flats) W64A-op-2 X MS-14-op-2 Yes 97.5 (medium.rounds) W64A-0p—2 X MS-l4-0p-2 No 96.3 MS-l4-op-2 X W64A-op-2 Yes 93.8 (medium flats) MS-l4—op-2 X W64A-op-2 Yes 95.3 (medium rounds) MS-14-op-2 X W64A-0p-2 No 95.8 Overall Normal 98.6 Overall Opaque-2 96.0 Overall Treated 97.4 Overall Untreated 97.1 72 treated with fungicide or not. Cold test germination with a field soil mixture show- ed normal hybrids had a 70.6 percent overall germination compared to 30.3 percent for opaque-2 hybrids (table 19). Fungicide treated seed averaged 50.7 percent compared to 34.0 percent for untreated seed for all hybrids. Treated opaque-2 seed germinated 49.0 percent compared to 18.1 per- cent for untreated opaque-2 seed. Germination percent for normal treated seed was 78.4 compared to 58.8 percent for untreated seed. The added stress in the form of low initial tempera- ture and soil microorganisms greatly lowered the germina- tion percent in the cold test in soil when compared to warm test germination. Alexander (1969) showed that cold test germination reduced percent germination in opaque-2 hybrids to about one-half that of normal hybrids. His results did not stipulate whether the seeds were fungicide treated or not. It is obvious that treatment with fungicide is of great value for increasing germination for both types of seed, Opaque-2 and normal. All counts in these two tests were based on seeds which had developed normal roots and shoots of a minimum of three inches each, after six days in warm temperature. Therefore, not only viability but also vigor of the seeds 73 Table 19. Comparison of cold test germination percent in soil of opaque-2 and normal maize. Captan Percent Pedigree Treated Germination W64A X Mr14 Yes 78.5 No 47.0 M-l4 X W64A Yes 93.0 No 70.5 W64A X Sib Yes 63.8 W64A-op-2 X MS-14-op-2 Yes 36.0 (medium flats) W64A-op-2 X MS-14-op-2 Yes 41.3 (medium rounds) W64A-op-2 X MS-14-op-2 No 21.0 MS-l4-op-2 X W64A-op-2 Yes 61.0 (medium.f1ats) MS-14-op-2 X W64A-op-2 Yes 36.3 (medium rounds) MS-14-op-2 X W64A-op-2 No 21.8 MS-l4-op-2 X Sib Yes 26.5 No 9.8 W64A-op-2 X Sib No 19.8 Opaque-2 Overall 30.3 Treated 40.2 Untreated 18.1 Normal Overall. 70.6 Treated 78.4 Untreated 58.8 Overall Treated 50.7 Overall Untreated 34.0 74 was tested. Results indicate that opaque-2 maize should be planted at higher planting rates per acre to obtain plant populations equivalent to those of normal maize. Cold test germinations in a sterile media were per- formed to further characterize germination of opaque-2 maize in comparison to normal (table 20). The object of this test was to eliminate effects of soil and seed surface micro- organisms, thus separating the effect of low temperature from the effects of microorganisms. The procedures of cold test germination in sterile media reported by other invest- igators were found to be inadequate in this test. The commonly used method of placing bleach sterilized seeds into a sterile glass petri-plate containing a filter paper, with 10 m1 sterilized water containing antibiotic (Penner, 1969) was unsuccessful. The amount of water that surrounded the seeds apparently affected metabolism (specifically reSpira- tion), enough that the swelled seeds germinated very poorly (0-20 percent in each petri-plate) and some that did germinate produced atypical roots and shoots. Another method of germinating seeds in sterile condi- tions (Chilton and Isely, 1961) involved the placement of bleach sterilized seeds into sterilized glass petri-plates which contained no filter paper. The closed petri-plates were then placed into a plastic bag which contained two 75 Table 20. Comparison of cold test germination percent in sterile media of opaque-2 and normal maize. Captan Percent Pedigree Treated Germination w W64A X M-l4 (1967 Seed) No 98.3 M-14 X W64A (1967 Seed) No 95.0 M-l4-TRf X W64A-TRf (1968 Seed) Yes 100.0 W64A X Oh-43 (1968 Seed) Yes 96.6 W64A (1968 Seed) No 95.0 M-14 (1968 Seed) No 96.6 W64A-op-2 X MS-l4-op-2 (1967 Seed) Yes 46.6 (medium flats) W64A-0p-2 X MS-14-0p-2 (1967 Seed) Yes 60.0 (medium,round) W64A-op-2 X MS-l4-op-2 (1967 Seed) No 58.3 MS-l4-op-2 X W64A-op-2 (1967 Seed) Yes 85.0 (medium flats) MS-l4-op-2 X W64A-0p—2 (1967 Seed) No 80.0 W64A—op-2 X MS-l4-op-2 (1968 Seed) Yes 93.3 W64A-op-2 X MS-l4-op-2 (1968 Seed) No 96.6 W64A-op-2 (1967 Seed) No 25.0 W64A-op-2 (1968 Seed) No 93.3 MS-14-op-2 (1968 Seed) No 90.0 Overall Opaque-2 72.8 Overall Normal 96.9 1967 Seed (Opaque-2) 68.0 1967 Seed (Normal) 97.8 Average 1967 Seed 82.9 1968 Seed (Opaque-2) 93.3 1968 Seed (Normal) 96.1 Average 1968 Seed 94.7 Overall Treated 80.3 Overall Untreated 82.8 76 layers of saturated blotter paper. The humidity within this plastic bag was supposed to be adequate for germina- tion. When this technique was used the seeds swelled during the cold temperature treatment but when placed in warm temperature conditions the moisture supply was inade- quate for good germination. Consequently, most seeds began to dry out although a few seeds in some petri-plates devel- oped very short roots and shoots. The method reported in this experiment appeared to be very adequate. The method provides for adequate sterilization of materials and moisture supply without creating a thick film of water on the outside of the seeds which would interfere with gas exchange. Average germina- tion percent of Opaque-2 maize was 72.8 compared to 96.9 for normal. There were essentially no differences due to seed treatment. Untreated seed had a 82.8 percent germina- tion compared to 80.3 percent for treated seed. This test included seed produced in 1967 and 1968. Data indicate that percent germination of normal maize was essentially the same for seed from both years (97.8 for 1967 vs 96.1 percent for 1968). However, 1967 opaque-2 seed had a germination percent of 68.0 compared to 93.3 for 1968 seed indicating that the age of the seed greatly affected via- bility. The cold test germinations indicated that 77 approximately half the effect of lower germination rates in 1967 opaque-2 maize, resulted from cold temperature effects and half from effects of microorganisms. Also, the opaque-2 seed was much weaker than normal in the tests where stress from cold temperature and soil was placed on the seed during the germination process. Pavlov (1963) stated that although zein was synthe- sized late in seed deve10pment, it was the first protein fraction to be utilized in germination. His results showed that zein was used at a much higher rate than glutelin especially in the first few days of germination. It appeared from his results that zein was the most readily available source of nitrogen for initial embryo development. Pavlov's data relates to this study of germination in Opaque-2 and normal maize. The storage protein in opaque-2 material has been shown to shift from about 55 percent zein in normal maize to about 25 percent zein in opaque-2 maize (Concon, 1966). The shift in zein storage pattern in Opaque-2 maize may explain the partial reduction in percent germination which was not due to the effects of microorganisms. Since zein content is lower in opaque-2, its breakdown during early germination processes with cold temperature stress, may result in an inadequate supply of nitrogen to the embryo axis. Even though the 78 nitrogen present in the embryo would seem to provide adequate nitrogen for growth, it appeared that endOSperm protein played a role in initial embryo development. Laboratory Studies Colorimetric Protein Analysis The standard Kjeldahl method of protein analysis utilizes expensive equipment, strong acids and high temp- eratures and for this reason is not desirable in many laboratories. Consequently, a rapid colorimetric method of protein determination may be preferred in laboratories where many routine protein analyses must be made. The "Lowry" colorimetric method of protein deter- mination has wide use with many protein types as a rapid and accurate form of analysis. The Lowry method was modified by Jennings (1961) for use with barley. However, no information was available concerning the use of the modified Lowry technique with maize. Since previous analysis of opaque-2 maize showed that basic amino acids were present in greater proportion than in normal maize (Bates, 1966) it was decided not to use a colorimetric procedure in which color reaction was based on the basic amino acids (MacKenzie and Perrier, 1969). The Lowry 79 method utilizes the Folin-Ciocalteu phenol reagent which reacts with certain, as yet unknown, amino acid sequences causing the majority of the color development. Several modifications of the method by Jennings (1961) were necessary to produce high correlations between the colorimetric procedure and the micro-Kjeldahl method for protein determination. Mixing the meal sample with 25 m1 biuret reagent and stirring in ahigh-speed micro- blender for three minutes, produced more reproducible results than omitting the blending at the beginning of the extraction period. Using a Hamilton type syringe for measuring 0.2 m1 of the biuret extract instead of 0.1 ml as suggested by Jennings (1961), produced greater color development which was more reproducible between determina- tions. It is necessary to follow a fairly close time schedule throughout the Lowry procedure. Some aspects of the method required greater adherence to a precise time schedule than others. For example, after placing 0.2 ml of the extract into the test tubes, 10 m1 of reagent C was added with stirring, followed by a 15 minute wait before adding reagent D. A time schedule was necessary such that the 15 minute interval was rather precise between individ- ual test tubes. Best results were obtained when reagent D 80 (Folin's reagent) was placed in a darkened 25 ml burette and 1.0 m1 of the reagent added while the reaction mixture was being stirred vigorously. The reaction between the phenol reagent and the protein extract occurred very quickly (Lowry £5 91, 1951), consequently immediate and thorough mixing was very important for obtaining best results. Jennings (1961) suggested that 30 minutes be allowed for color development after the addition of the Folin phenol reagent (reagent D). After a time study on stable color development it was determined that a period of at least 60 minutes was necessary for the most reproducible results with'maize. A comparison of values obtained from the colorimetric method and micro-Kjeldahl method of protein determination is presented in table 21. Two absorbance values (absorb- ance l and 2) were obtained from analyses on the same sample run one or two days apart. Each absorbance value, (e.g. absorbance-l) was the mean of two determinations made on a single replication of the sample extracted. Correlations between the two absorbance values were calculated to estimate the reproducibility of the analysis from day to day batches. Correlations between the two ab- sorbance values were highly significant (r = .987 for normal maize, r = .961 for opaque-2 maize and r = .984 for 81 .N Hones: cowumoaaamu vaHm xx .H Hones: comumoHHomu wHon *l HNa. moo. amm. Amuv couumcuaumumc mo .uumoo Hamum>o coo. «mm. amm. Auv .uumoo couumamuuou Hamum>o Ham. mam. New. . Amuv coaumcaaumume mo .uumoo mmm. cam. was. Any .uumoo aouumamuuou aw.m Ham. man. man. ~-ao- oocmnHomnm ofiuuosauoHoo 03u mo acmfiumasoo .HN manma 82 the two groups combined) and indicated that absorbance values obtained for a sample run on one day were highly reproducible with those run on the same sample the next day. Correlations between colorimetric absorbance means and the micro-Kjeldahl protein values were slightly higher for normal maize than for opaque-2 maize. Correlation coefficients (r) were: (1) .962 for normal maize, (2) .989 for Opaque-2 maize and (3) .960 for the combined normal plus opaque-2 types. All correlation coefficients were highly significant and showed that there was a high level of agreement in protein determinations made with these two procedures. The coefficient of determination, r2, is the percent of the total variability accounted for by the relationship determined with the correlation coefficient. These values were 92.5 percent, 88.1 percent, and 92.5 percent for the normal maize samples, for opaque-2 samples, and for both groups combined, respectively. The correlation coefficients and coefficients of determinations showed that the Lowry method.was a very good alternative method for maize protein determination. The correlation between methods was slightly lower for opaque-2 samples. This is illustrated further in the 83 regression analysis (figure 32). Comparison of regression lines for opaque-2 and normal types showed that in the mid—range, 10 to 12 percent protein, the difference between the two lines was very small. Utilizing these regression lines to convert absorbance values from the colorimetric technique to equivalent micro-Kjeldahl values, the :1 estimates would differ only 0.2 to 0.3 percent protein at ‘ most. If the protein content of the maize sample was less than 10 percent or greater than 12 percent, the error of conversion could be as much as one percent protein. This conversion error would be reduced considerably if the "overall" regression line was used for normal samples, but only a slight reduction in error would result for opaque-2 samples. Absorbance values determined by the modified Lowry method should be converted to micro-Kjeldahl protein per- centages by using the separate regression lines for opaque-2 and normal maize. The colorimetric determination of maize protein described in this experiment should allow the processing of approximately 40 previously ground meal samples per day. Since the Lowry method is fairly simple and rapid, it should be a good analytical tool for routine protein determinations on maize samples. 84 .AHHmuo>ov cmuanfioo moaxu oNHmE nuon aw mam .Hmsuoa .Nuosvmmo a“ muonuma Hamuaofixuouofia cum oahuoEHuoaoo musoq umamapofi.osu up caououa Hocuox Hmuou mo mammamam aoammmuwmm .Nm madman aaououm ucouumm an ca mu ea ma «a HH ou a m i 1 1 4 I I d J . m. x_uwo~o. + «mesa. n w "~-o:smao x Gamma. + 50000. n m "umauoz . a. x ENS. + ~28. u m "Sears m . m a. 9 w a moa nu 9 0 m a 0. L No 85 TNP-L-Lysine Analysis A modification of the 2,4,6-trinitrobenzenesulfonic acid (TNBS) method (Kakade and Liener, 1969) was used to determine the lysine content of maize meal samples, extract- ed alkali-soluble proteins, and salt fractionations [(NH4)ZSO4] of the alkali-soluble proteins. The TNBS method for lysine analysis has been used successfully with certain foodstuffs (Kakade and Liener, 1969) but there was no reported data concerning its use with maize. Satake 25 a1 (1960) showed that the optimum pH for the TNP-lation with TNBS was from pH 7.5 to 8.5 at 40 C. Kakade and Liener (1969) used a reaction mixture buffered with a four percent sodium bicarbonate (NaHCOB) solution (pH 8.5). I found that a four percent NaHCO3 solution had a pH of about 7.0 and was unstable over time. Therefore, I used a borate buffer (pH 8.5). Removal of insoluble particulate matter by filtra- tion was suggested by Kakade and Liener (1969). I found that the material through which the hydrolysate was filtered affected the absorbance values. Whatman No. 42, acid washed filter paper was the best material for filtration. Degradation rate of the TNP-L-lysine product over time was not the same from sample to sample. Absorbance values were more reproducible when determined immediately 86 after the excess ether was volatilized from the samples. Analyses of ground meal (whole kernel) samples showed that single-cross Opaque-2 hybrids averaged 55.9 percent more lysine than the normal hybrids (5.2 vs 3.4 gm lysine per 100 gm protein, table 22). Three-way hybrids contained 41.2 percent more lysine than the normal single- cross hybrids (4.8 vs 3.4 gm lysine per 100 gm protein). (W64A X MS-14-0p-2) with one endOSperm gene for Opaque-2 (+, +, 0p-2), contained 5.7 gm lysine per 100 gm protein compared to 6.2 gm per 100 gm protein for the reciprocal hybrid (MS-14-op-2 X W64A) with two doses of opaque-2 in the endosperm (+, 0p-2, op-Z). Individual single-cross opaque-2 hybrids ranged in lysine content from 4.0 to 6.6 gm per 100 gm protein. Both single-cross normal hybrids, W64ArTRf X Mr14-TRf and W64A X Oh-43, contained 3.4 gm lysine per 100 gm protein. The hybrid W64A-op-2 X MS-l4-op-2, produced from inbreds backcrossed (BC-3) three times to Opaque-2, had 6.6 gm lysine per 100 gm protein. The same hybrid produced from inbreds backcrossed five times (BC-5) had 6.3 gm lysine per 100 gm protein. The reciprocal cross MS—14-0p-2 X W64A-0p-2, had a lysine content of 4.3 gm per 100 gm protein, suggesting that the 2N endOSperm contribution from the female parent might have a large effect on protein 87 5.0a H.0 0.0 ~-ao-~-um-mm-0oa-o x Am-ao-0u-0z x ~-ao-<0030 0.m0 0.0 o.0 N-ao-mm-0ou-u x A~-ao-su-0z x ~-ao-<0030 0.0a 0.0 0.0 ~-ao-m003 x Am-ao-0u-mz x N-ao-<0030 ~.a0 0.0 m.m N-ao-am-ma-z x A~-ao-0a-02 x ~-ao-<0030 H.00 a.m 0.0 ~-ao-u0-3 x Am-ao-0a-0z x ~-ao-<0030 5.0a N.0 5.0 ~-ao.+.+ u sumamoeam .~-ao-0a-02 x <003 a.a0 N.0 «.0 +.~-ao.~-ao u sumamoucm .<003 x N-ao-0a-0z a.ma 0.0 0.0 ~-ao-<003 x N-ao-0u-0z 0.0a 0.0 0.0 N-ao-ms-ao x ~-ao-<00z 0.0a 0.~ 0.0 00-00 x <003 0.m0 0.0 0.0 A0-om0 N-ao-0a-mz x ~-ao-<003 a.~a 0.0 0.0 Am-om0 ~-ao-0a-mz x ~-ao-¢003 0.a0 m.N 0.0 uaa-0a-z x 0m9-<003 Gowuomum Auamuoum Acamuoum mmuwwnom maanaom . Hams aw oou\amv aw 00H\amv -uamaaa cu maumuoua maasaom H00: «Nam: cu msaqu Houoe :HmeH< CH ozoucoo oawmka mo udmoumm ucmucoo mcwmmq .mNHmE EH mcowuomum cflmuoum oHLDHOmuHmeHm mom Hmma unsoum mo mmm%amcm mawmhd .NN mHQmH 88 storage in the kernel. Lysine content of three-way hybrids ranged from 3.9 to 5.8 gm per 100 gm protein. Two of the three-way hybrids, (W64A-op-2 X MS-l4-op-2) X C105-HP-op—2 and (W64A-op-2 X MS-l4-op-2) X C105-HP-op-2-f1-2, were near- isogenic hybrids except that one of them contained the “' double mutant opaque-2 and floury-Z genes. The lysine contents were 5.8 gm per 100 gm protein for the double mutant and 4.9 gm per 100 gm protein for its Opaque-2 counterpart. Nelson (1966) showed that the lysine content of the double mutant form of an inbred line contained a lower lysine content than its Opaque-2 counterpart. Since the double mutant form of the three-way hybrid in this experiment contained a greater amount of lysine than the opaque-2 hybrid, further studies should be made to deter- mine the effects of double mutant genes in other hybrids. The double mutant kernels have been shown to have greater kernel weight than opaque-2 maize and could therefore be economically important. Analyses of total kernel lysine content in Opaque-2 and normal maize hybrids indicated the following: 89 There was a wide range of lysine content, 6.6 gm to 4.0 gm per 100 gm protein, among opaque-2 hybrids. From a comparison of reciprocal crosses there was evidence that the female parent influenced the total kernel lysine content in opaque-2 maize (6.6 gm vs 4.3 gm per 100 gm protein). There was no significant difference in total kernel lysine content (6.6 gm vs 6.3 gm per 100 gm protein) between a single-cross produced with BC-3 and with BC-5 Opaque-2 inbred parents. There were relatively small differences in total kernel lysine content for endosperms containing one, two and three doses of opaque-2 genes 5.7 gm for one dose (Op-2, +, +), 6.2 gm for two doses (Op-2, op-2, +), and 6.6 gm for three doses (op-2, 0p-2, op-2) . If one endosperm gene dosage is sufficient to increase the kernel lysine content to 5.7 gm per 100 gm protein in hybrids many of the poor kernel characteristics associated with kernels with three opaque-2 dosages could be more easily overcome. Except for the double mutant (op-2, fl-2) the 90 three-way opaque-2 hybrids averaged lower in total kernel lysine content than the best single-cross hybrids (4.9 gm vs 6.5 gm per 100 gm protein). Results of the TNP-lysine analyses indicated quanti- tative values for lysine which were equivalent to or slight- ly higher than previously reported for maize. Alexander (1968) reported that opaque-2 hybrids (single-cross and three-way hybrids) averaged about 3.8 gm lysine per 100 gm protein and normal hybrids (single-crosses) averaged about 2.5 gm lysine per 100 gm protein. Paez g£_§1’(l969a) reported lysine values of .49, .52 and .38 percent (percent lysine on a whole kernel basis) for three opaque-2 inbred lines and values of .52 and .58 percent (based on the whole kernel) for two single-cross opaque-2 hybrids (an average of 4.5 gm per 100 gm protein based on 12 percent protein content of the kernel). He reported three normal inbreds with lysine contents of .28, .29 and .26 percent (based on the whole kernel). The average lysine content of the opaque-2 hybrids reported herein was 5.2 gm per 100 gm protein and the average for the normal hybrids was 3.4 gm lysine per 100 gm protein. The highest lysine value obtained in this experiment (6.6 gm lysine per 100 gm protein) was 1.5 gm 91 to 2.0 gm per 100 gm protein higher than previously report- ed data (Alexander, 1968; Paez £2 al, 1969a). Alexander (1968) reported that opaque-2 maize con- tained 52 percent more lysine than normal hybrids and Paez gt al (1969a) reported an average of 64 percent more lysine in opaque-2 maize. The TNBS method of lysine anal- ysis showed that on the average, opaque-2 maize contained 53 percent more lysine than normal maize. This indicates that the relationship between the lysine values for opaque-2 and normal maize were in good agreement with previously reported data. It is believed that the TNBS technique for deter- mination of lysine in whole ground meal of maize is reli- able and fairly rapid (15 to 20 ground samples per day). The method should be of value for routine analyses of lysine content in maize. Further characterization of the protein storage pattern in opaque-2 and normal maize was provided by lysine analysis of the extracted alkali-soluble proteins (table 22). Single—cross opaque-2 hybrids contained from 3.0 gm to 4.8 gm of lysine per 100 gm protein in the alkali- soluble protein fraction. This represented a range of 67.7 to 79.1 percent of the total lysine in the kernel with an 92 average of 72.6 percent. Jimenez (1966) reported that 78.6 percent of the lysine in opaque-2 maize endosperm.was con- tained in the alkali-soluble protein fraction. )(Jimenez' data represents percent lysine in alkali-soluble protein compared to the total lysine content in endosperm material from inbred lines W64A and W64A-op-2.) Since both endo- sperm and germ protein in opaque-2 maize have been shown to contain nutritionally sufficient amounts of lysine (Wichser, 1966) the proportion of lysine in the alkali- soluble proteins should be nearly equivalent, whether the values were based on germ, endOSperm or whole kernel protein. Consequently, the values reported by Jimenez (1966) are in good agreement with the values reported here. Normal single-cross hybrids contained 2.3 gm to 2.6 gm lysine per 100 gm protein in the alkali-soluble protein fraction. The lysine content of this fraction represented 67.6 to 76.5 percent of the total kernel lysine, an average of 72.1 percent. The alkali-soluble proteins in normal maize endosperm contained 80.0 percent of the total lysine in data reported by Jimenez (1966). Normal maize endosperm has been shown to contain about 80 percent of the total kernel protein, but it was nutritionally deficient in lysine (Wichser, 1966). Wichser found that the germ contained about 20 percent of 93 the total kernel protein and that it contained a nutrition- ally sufficient quantity of lysine. Since Jimenez (1966) reported that 80 percent of the lysine in normal maize endosperm was in the alkali-soluble protein, it was expected that a greater pr0portion than 72.6 percent of the lysine would be found in the alkali-soluble proteins of the whole kernel. Several factors could influence the values of lysine content in the alkali-soluble proteins of normal maize. The material used by Jimenez was from an inbred line (W64A) and the material used in this report were single-cross hybrids (W64A-TRf X M-l4-TRf and.W64A X Oh-43). The differences in lysine content could have been due to different extraction procedures as mentioned by Nagy 25.2l (1941). Cultural treatments such as nitrogen and differences in environment conditions could also affect the protein storage pattern in maize. The three-way Opaque-2 hybrids had lysine contents in the alkali-soluble protein ranging from 3.4 to 4.4 gm per 100 gm protein in the whole kernel. Lysine content of the alkali-soluble proteins ranged from 70.8 to 89.8 percent (average 80.5 percent) of the whole kernel protein. A further breakdown of the alkali-soluble proteins by ammonium sulfate fractionation provided further insight into the effects of the Opaque-2 gene on kernel storage 94 proteins. Since different proteins have been shown to pre- cipitate from solution at different salt concentrations (Mahler and Cordes, 1968), lysine analysis of each of the five fractions indicated that variability existed in the alkali-soluble protein storage pattern. Five ammonium sulfate precipitated protein fractions were obtained by using salt concentrations of 5, 10, 20, 30 and 80 percent. The percent lysine found in each frac- tion based on the total amount of lysine in all five fractions for any given sample is listed in table 23. Three general forms of protein distribution (based on lysine analysis) were found in the opaque-2 and normal hy- brids (figure 33). The type I lysine distribution pattern contained 19 percent of the lysine in fraction one, 45.8 percent in two, 17.8 percent in three, 3.8 percent in four and 11.9 percent in fraction five. The type I dis- tribution form was found in only one hybrid [(W64A-op-2 X MS-l4-op-2 (BC-3)]. The average of the five hybrids which had a type II lysine distribution contained 52.1 percent in fraction one, 22.4 percent in two, 12.6 percent in three, 3.0 percent in four, and 10.1 percent in fraction five. The average lysine distribution of the seven hybrids 95 0.0 0.0 0.00 0.00 0.00 000 0-00-0-00-00-000-0 000-00-00-02 00-00030 0.00 0.0 0.00 0.00 0.00 000 0-00-00-000-0 000-00-00-02 00-00030 0.0 0.0 0.00 0.0 0.00 000 0-00-0003 000-00-00-02 -00-00030 0.00 0.0 0.00 0.0 0.00 000 0-00-00-3 000-00-00-02 x 0-00-00030 0.00 0.0 0.00 0.00 0.00 000 0-00-0003 0 0-00-00-0: 0.0 0.0 0.00 0.00 0.00 000 0-00-00-00 0 0-00-0003 0.00 0.0 0.00 0.0 0.00 000 00-00 x 0003 0.00 0.0 0.00 0.00 0.00 00 0-00-00-00-: 000-00-00-02 x 0-00-00030 0.00 0.0 0.00 0.00 0.00 00 0-00 .+ .+ - 000000000 .0-00-00-00 x 0003 0.00 0.0 0.00 0.00 0.00 00 + .0-00 .0-00 000000000 .0002 x 0-00-00-02 0.00 0.0 0.00 0.00 0.00 00 00-000 0-00-00-0: 0 0.00.0003 0.0 0.0 0.0 0.00 0.00 00 000.00.: 0 000-0003 0.00 0.0 0.00 0.00 0.00 0 00-000 0-00-00-0: 0 0-00-0003 monks mouwauom 0000 0000 0000 0000 000000000000 000 000 000 00 0000000 .mcoHuowum pooHAEOU 050 CH oc0mha 050 no comma ao0uomum £00m o0 mc0mza unmouom .oNHmB Huauoa paw N-oavmmo c0 mGHmuoua man9000-Hamxao onu mo muowum uncauomum mummaam abacoeam 0:0 :« muoguanfiuumau oGHmha unmouoa mo a0000wmsoo .00 00000 96 represented by the type III pattern contained 54.1 percent in fraction one, 11.2 in two, 19.0 in three, 4.3 in four, and 10.6 percent in fraction five. Comparison of the two near-isogenic hybrids used in the agronomic field studies, W64A-TRf X M-l4-TRf and W64A-0p-2 X MS-14-op-2 (BC-3), indicated a major shift in alkali-soluble protein storage forms between fractions one and two. The normal hybrid contained 71.2 percent of the lysine in fraction one compared to 19.0 for Opaque-2. The lysine content found in fraction two was 13.8 percent in the normal hybrid and 45.8 percent for opaque-2. This represented from 65 to 85 percent of the total lysine and indicated a radical difference in protein storage forms between these two hybrids. Further backcrossing (BC-3 vs BC-5) of the two inbred parents of the opaque-2 hybrid, resulted in an alkali-soluble protein distribution of type II, the same as characterized the normal hybrid. Even though these two Opaque-2 hybrids (BC-3 and BC-S) con- tained about the same total kernel lysine content (6.6 vs 6.3 gm per 100 gm protein) the type of protein found in the alkali-soluble fraction was very different in their salt solubility characteristics. The shift in protein storage pattern may have been the result of further selection in backcrossing the inbred lines or some 97 differences in cultural practices. However they were grown in the same field under similar conditions during the 1968 growing season. The reciprocal single-cross opaque-2 hybrid [MS-l4- op-2 (BC-5) X W64A-op-2] had a lysine distribution (type III) in the alkali-soluble proteins which was different from either of the other two opaque-2 hybrids with similar pedigrees. This change in protein distribution was pri- marily in fractions two and three and indicates that reciprocal female parents in a hybrid may affect the type of alkali-soluble proteins which are stored in the kernel. The near-isogenic hybrids W64A X Oh-43 and W64A- 0p-2 X Oh-43-op-2 had alkali-soluble protein distributions which were nearly alike (type 111) when based on the lysine content of each fraction. Both of these hybrids had some- what lower lysine contents in the whole kernel analysis. The two hybrids W64A X MS-l4-0p-2 and MS-l4-0p-2 X W64A, reSpectively, contained one and two gene doses of the opaque-2 gene in their endosperm tissue. Both of these hybrids were represented by the type II lysine distribution in the alkali-soluble proteins. The three-way hybrid (W64A X MS-14-op-2) X M-l3-HP- op-2 had a lysine distribution of type II but the other four three-way hybrids had type III distributions. 98 The result of the analysis of lysine distribution in alkali-soluble proteins in opaque-2 and normal hybrids was not extensive enough to give a clear picture of the influence the Opaque-2 gene had on the storage protein form in the alkali-soluble proteins. Much more research will be necessary to establish correct genetic-protein distribution relationships. It is conceivable that analyses of the protein fractions of maize in conjunction with kernel lysine analyses could provide more Specific answers to plant breeders concerning the influence from the Opaque-2 or floury-2 genes, the female parent, and gene dosage on the protein quality of maize. 99 H00 1 um: e n .1 S .w. t mo 0 n e C r e P Nm . o O p b Ir u so No we m m we no we we moaned" >BBosHQB mfiwmmnm O / h HH 7.x. HHH O \ O O \ O we we mo mo wwmsnm uu. wmvflmmmSnmnH