GENETIC VARmBiLETYOF ”AVAILABLE” METmonmE ‘ f , TOTAL PROTEIN 3:350:51; GGAWW AND 6THER TRANS fNTETRAPLOiD POTATOES Thesis for the Denge of'Ph;D... ‘ MICHIGANSTATE‘UN-IVERSITY * ROBERT'LUESCH'ERTZ ' ’ Am LIBRARY AA111111111111111111111111 . ”113323“ 3 1293 10262 2416 ——— —.—.— V" This is to certify that the thesis entitled Genetic variability of "available" methionine, total protein, specific gravity and other traits in tetraploid potatoes presented by l Robert Luescher has been accepted towards fulfillment of the requirements for Ph.D. degreein Crop and Soil Sciences 7 watt-(m DACWZ: A? 72. 0-7639 g“ 2/5 ABSTRACT GENETIC VARIABILITY OF ”AVAILABLE" METHIONINE. TOTAL PROTEIN. SPECIFIC GRAVITY AND OTHER TRAITS IN TETRAPLOID POTATOES By Robert Luescher Parental cultivars of potatoes (Solanum tubegosum L. and §. tuberosum - g. stgloniferum hybrids) and 320 segre- gating offspring representing 8 crosses were analysed in duplicate for total protein, ”available” methionine and specific gravity. Significant differences were found for all three traits among parents and families. Heritability. mainly in the nar- row sense. varied from 79 to 100% for "available” methio- nine. from 10 to 28% for total protein and from 35 to 99% for specific gravity. Total protein in the offspring varied from 7.5 to 18.8 mg/lOO mg dry matter. "available" methionine varied from 0.9 to 2.2 mg/l6 mg N and total dry matter ranged from 12.7 to 2&% of the fresh weight. ”Available” methionine. total protein and specific Robert Luescher gravity were all positively correlated with chip color (r- 0.1h. 0.13 and O.hh respectively). No definite relationship between "available" methio- nine content and rest period could be observed. From the 320 segregating offspring. 16 clones were selected according to their total protein and ”available" methionine content. These samples were analysed for protein and nonprotein nitrogen. free methionine and free cysteine. The BV of the total protein and the nonprotein nitrogen fraction was assessed by means of a microbiological method using Strgptococcus zygoggngs. In this study free methionine (mg/l6 mg N) ranged from, 6.3“ to 0.97% and was highly correlated with ”available" methionine (rs 0.96). Free methionine provided between 12 and 62% of all methionine present in the total protein. Free methionine and "available" methionine seemed to be independent of the total protein level. No measurable amounts of free cysteine could be de- tected. The BV of the total protein was negatively correla- ted with the total protein level (r= -0.55). The BV of the nonprotein N fraction was very dependent upon % nonprotein N and the total protein level (r= -0.96 and -O.69 respectively). Tuberin, tuberinin, globulin II, prolamin and glutelin were isolated from the dry matter of three advanced seedlings by means of conventional extracting procedure. Tuberin. tuberinin and prolamin of the cultivars 58 and 322-6 contained similar amounts of methionine and cystine, Robert Luescher whereas the sum of methionine and cystine of the same proteins of 709 were considerably lower. Electrophoretic analyses showed that tuberin is com- posed of at least three major protein bandso The relative. quantity of these bands varied between genotypes and could thus cause some variation in the content of the sulfur con- taining amino acids. The bands of tuberinin were almost identical to the bands of tuberin. but in different pro- portions. Selection criteria to develop a potato high in the sulfur amino acids were discussed. I'lll'lllll'lull’ll’lfl'l‘ III! GENETIC VARIABILITY OF ”AVAILABLE” METHIONINE. TOTAL PROTEIN. SPECIFIC GRAVITY AND OTHER TRAITS IN TETRAPLOID POTATOES By Robert Luescher 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 1972 ACKNOWLEDGMENTS The author expresses his gratitude to Dr. N. R. Thomp- son for making his stay at the Michigan State University a learning experience. His encouragement towards independent research and his guidance during the preparation of his dissertation are greatly appreciated. My gratitude goes to the members of the committee: Dr. 0. Mickelsen, Dr. C. L. Bedford. Dr. D. Penner and Dr. N. R. Thompson for discussions. comments and criticism. and a liberal use of their time. The author expresses thanks to Dr. C. M. Harrison for his help in the revision of the draft. Gratitude is expressed to Dr. D. Penner for providing laboratory facility and guidance so essential for this work. I would like to thank Mr. R. Kitchen who was a great help with the technical aspects of planting and harvesting. ii TABLE OF CONTENTS LIST OF TABLES . . . . LIST OF FIGURES . . . LIST OF DEFINITIONS . INTRODUCTION . . . . . REVIEW OF LITERATURE . Nitrogen containing Amino acid composition and nutritive value of the various nitrogen containing con- stituents . . . . Environmental influences on the compo- sition of the nitrogen containing con- Btituents Of the pOtatO e e e e e e e e e The role of methionine in the potato Relations among specific gravity. total nitrogen and quality factors of potatoes. constituents of Toxic constituents of potatoes. . . potatoes. Page vii iix 10 12 1a 15 PART I. INHERITANCE OF ”AVAILABLE" METHIONINE. TOTAL PROTEIN AND SPECIFIC GRAVITY IN TETRAPLOID POTATOES Introduction . . . . . Materials and Methods Results and Discussion Summary. . . . . . . . iii 17 17 20 29 Page PART II. CAUSE OF VARIATION IN "AVAILABLE” METHIONINE IN POTATO PROTEIN Introduction . . . . . . . . . . . . . . . . . . ”31- Materials and Methods . . . . . . . . . . . . . 32 Results and Discussion . . . . . . . . . . . . . 33 Summary. . . . . . . . . . . . . . . . . . . . . 39 PART III. ELECTROPHORESIS AND ANALYSIS OF THE SULFUR AMINO ACIDS OF VARIOUS ' POTATO PROTEINS' Introduction . . . . . . . . . . . . . . . . . . 41 Materials and Methods . . . . . . . . . . . . . #1 Results and Discussion .-. . . . . . . . . . . . an Summary . . . . . . . . . . . . . . . . . . . . 52 SUMMARY AND CONCLUSIONS . . . . . . . . . . . . 54 REFERENCES 0 O O C O C C C C C O O O O O O O O O 56 APPENDIX A. Data of 320 offspring representing 8 crosses . . . . . . . . . . . . . . . . . 61. Be Data Of parental Onltivars e e e e e e e e e 69 iv Table 1. 2. 9. LIST OF TABLES Net protein production of corn. wheat and pOtatoes in the Us Se A. e e e e e 0 Influence of fertilizer level on the flat prOtein content 0 e e e e e e e e e 0 Composition of variance and covariance components in half-sib families in a tetraploid............... Parent and offspring family means for "available” methionine, total protein and specific gravity . . . . . . . . . . Narrow sense heritability estimates for "available” methionine. total protein and specific gravity . . . . . . . . . . Means and ranges of chip color. rest period and total dry matter of 320 seedlings re- presenting 8 crosses . . . . . . . . . . Correlations among ”available" methionine. total protein. specific gravity, fresh weight, total dry matter, rest period and chip color (data from 320 seedlings) . . Means of two analyses for total protein, "available" methionine. methionine, free methionine, nonprotein N, BV of nonprotein N, BV of total protein and contribution of free methionine to methionine of total protein . . . . . . . . . . . . . . . . . Correlations among total protein. ”avai- lable” methionine. methionine, cystine. nonprotein N. free methionine. BV of total protein and RV of nonprotein N (data from 16 seedlings analysed in 2 replications eaCh)e e e e e e e e e e e o Page 12 19 21 25 2'7 28 35 36 Table 10. 11. 12. Distribution of total nitrogen in 60 g dry matter of 3 cultivars . . . . . Methionine and cystine contents of tuberin. tuberinin. prolamin and the nonprotein N fraction of 3 cultivars..............o. Upper limits in sulfur amino acid contents of potatoes grown with low and high nitrogen fertilization . . . . . vi Page “5 #5 51 LIST OF FIGURES Figure 1. Relationship between levels of nitrogen fertilization and yield of 2 potato varieties e e e e e e e e e e e e e e 2. Offspring: Mid-parent regression for "available" methionine e e e e e e e 3. Offspring: Mid-parent regression for tOtal prOtein e e e e e e e e e e e e 4. Offspring: Mid-parent regression for SPOOifiC graVIty e e e e e e e e e e 5. Regression of ”available” methionine on free methionine e e e e e e e e e 6. Scheme for fractionation of potato prOteins e e e e e e e e e e e e e e 7. Densitgmetrical readings of the gels Of 5 e e e e e e e e e e e e e e e e 8. Densitometrical readings of the gels Of 322-6 0 e e e e e e e e e e e e e 9. Densitometrical readings of the gels Of 709 e e e e e e e e e e e e e e 0 vii Page 11 22 22 23 3a “3 47 48 49 “7‘1 _‘ “(J-l"... ‘5." ” ‘l £Ih€ LIST OF DEFINITIONS "available” methionine: methionine determined microbio- logically in an enzymatic digest of the total protein. b: regression coefficient. BV: biological value of protein. Cov: covariance. cystine: cystine determined microbiologically in an acid hydrolysate of the total protein. EAA - Index: essential amino acid index. free cysteine: cysteine determined microbiologically in the nonprotein nitrogen fraction. free methionine: methionine determined microbiologically in the nonprotein nitrogen fraction. methionine: methionine determined microbiologically in an acid hydrolysate of the total protein. NPU: net protein utilization. P: probability of error. r: correlation coefficient. total protein: (Kjeldahl N of potato flour) x 6.25. V: variance. viii INTRODUCTION Plant geneticists have bred plants to alter specific characters such as color. size. shape. disease resistance and yield. Biologists studying plants and animals have shown that specific proteins are characteristic of certain families (Dalby and Lillevik. 1969: Boulter and Thruman. 1968). The nutritional implications of this type of work are obvious, namely. that quantitative variation in protein occurs with genetic variation. and can be further enhanced by making suitable crosses. With the increasing world population and the ensuing food shortage which threatens to affect even the economically developed countries. scientists have begun to look seriously at many hitherto unexplored areas for protein rich foods. Emphasis has been put on plant sources as primary providers of both protein and calories. Algae and leaves are being examined as possible pro- tein sources for humans. Although such food can be used in animal feeds or make interesting academic studies. accepta- bility is an important factor in human nutrition. Most of these unusual sources of food need either to be processed suitably or require tremendous effort to educate peeple to accept these unusual foods. 2 Protein deficiency is a major nutritional and health problem in the world today. Kwashiorkor. a protein deficien- cy disease. is common in many developing countries. Since f population growth may increase more rapidly than food supply. the problem of protein deficiency is likely to become even more acute. This problem can be averted only by a combination of population control. improved agricultural practices. food preservation and economical development. The available animal protein is insufficient to balance the world diet. Two thirds of the protein for present world consumption comes from cereals (Borgstrom. 1967). Cereals. however. do not provide balanced proteins. In addition. the primary aim of past breeding practices was based on the mis- conception that quantity alone can feed the world. Most of the cereals first cultivated contained 12 to 15% protein. In contrast. the present high yielding soft wheat varieties contain 10% or less of protein. If the dietary protein is significantly less than 8% of the calories provided by human milk. it is not possible to feed an infant enough food to meet the protein requirement. If proteins of lower quality than those of human milk are fed. the intake should be proportionally higher. but efforts should be made to provide the infant with the highest possi- ble quality of protein. Future agricultural research must not overlook the improvement of the nutritional quality of food crops. For centuries the potato (Solgnum tuberosum L.) has been a reliable food source for man and animal. The ”Incas" built a civilization around the potato and the Irish. before the blight catastrophes of the 18A0's. exis- ted on potatoes with a small amount of animal protein. Al- though the potato is considered primarily starch. it can supply more than 1000 lbs of protein per acre. In addition. the nutritive value of its protein is far superior to that of corn and wheat. Kofranyi and Jekat (1967) reported the daily protein requirement for man to be an average of 0.55g per kg body weight when potato was the only source of protein. That is almost equal to the nutritional value of whole egg protein and better than beef. tuna. whole milk. wheat flour. corn. rice. soybean and kidney bean protein. In its fresh state. the potato has only an average of 2% total protein. but can range from 1.5% up to 4.0%. How- ever. on a dry weight basis the total protein content of po- tatoes is not different from that of wheat and can amount to more than 17%. One hectare of land under potato cultivation can supply the protein requirement for 9.5 people. while the protein of wheat from the same land can satisfy only 6.3 people (Borgstrom. 1969). The figures in Table 1 clearly demonstrate that the potato can easily outyield the two main crops of the U. S. A. . Recent calcultaions of the composition of average na- tional diets. based on PAC information. concluded that lysine rarely appears to be the limiting amino acid in characteristic regional diets: it is usually the sulphur-containing amino u acids (Auret gt,gl.. 1968). Miller and Donoso (1963) re- ported similar results after feeding regional diets to rats. Table 1: Net protein production of corn. wheat and potatoes in the U0 Se Ac Corn++* Wheat+++ Potatoes Yield+ tons/ha #.5 2.1 2h.h Dry matter tons/ha 3.3 1.8 b.9 % protein content++ 8.5 12 2 NPU++ 50 60 70 Net protein++ kg/ha 193 161 340 +production Yearbook 1970 ++Kuppuswamy 23,31.. I958 +++yield of grain yield (tons/ha) x %protein x NPU kg net protein/ha = - 10 Amino acid analysis and studies with animal and human adults fully agree that the sulfur containing amino acids are the first limiting in potato protein (Schuhpan. 1958: Rios gt,§;.. 1972: Kiss and Metzfox. 1972). Kiss and Metzfox (1972) proved in their study with human beings that the pro- tein value of dehydrated potato flakes can be improved by adding methionine to the diet. To obtain a potato diet higher in methionine. various theoretical approaches may be taken. These include genetic selection of potato tubers having a higher methionine content. addition of purified methionine in the industrial processing of dehydrated po- tato flakes or education of consumers in usage of desirable food combinations. The attempt to improve the biological value of potato protein by increasing its methionine content may result in an additional benefit. Methionine appears to be a precur- sor of some flavor compounds in potatoes (Gumbmann and Burr. 196“). By increasing the methionine content and thus possibly. increasing the flavor. it may be possible to enhance the ”taste appeal" of this food. This could become an important consideration in determing whether consumers will purchase and use potatoes in their daily diet. Recent studies indicate that in some plant tissues methionine is a natural precursor of the plant hormone ethy- lene (Lieberman gt,gl.. 1966: Burg and Clagett. 1967). Poabst gt_§;. (1968) showed that potato tubers contain endo- genous ethylene. Since gibberellic acid is known to stimulate potato sprouting. and ethylene and gibberellic acid interact with each other. the possibility that methionine could indi- rectly be involved in controlling the rest period should not be excluded in future studies. The object of this research was to investigate the ge- netic variability of methionine in potato protein. To faciliate 6 eventual breeding work in this area. it is necessary to have more information about the components responsible for such a genetic variation. Attention will also be given to possible side effects of methionine content on agronomic factors and quality factors of the potato. REVIEW OF LITERATURE N t 0 en ontaini const tuents of ot toes Nitrogen has been found bound in free amino acids and amides. in protein soluble in various extracting solutions. in an insoluble protein residue and in trace quantities of numerous constituents. These include nucleic acids. alka- loids. choline. enzymes and some vitamins. The proteins present in the potatoes are: Tuberinin: (Albumin) is soluble in water. It is heteroge- neous in composition and the role ascribed to it is mainly enzymic. Tubgrin: (Globulin) is insoluble or sparingly soluble in water. but its solubility is greatly enhanced by the addition of neutral salts like sodium chloride. In the potato. the quantitative distribution of tuberinin and tuberin is pH dependent. At a pH of 6. tuberinin is present in small amounts whereas at a pH of 3 more tuberinin can be ob- tained at the expense of tuberin (Jirgensons. 19h6). Jir- gensons postulated that tuberin when exposed to an acid en- vironment is converted to tuberinin plus a very insoluble casein-like protein. This conversion was reversible in an alkaline environment. Tuberin can easily be extracted with 2% NaCl solution. Its molecular weight is estimated to be between 295.000 and 330.000 (Hoelzl and Bancher. 1961). Tuberin and tuberinin are present in a dissolved form in the cell sap of the potato. If once the cell wall is ruptured. these two proteins can easily be extracted with water (Hoelzl and Bancher. 1961). At a pH of 6.8 both proteins have a negative isoelec- tric point (Groot gt,§l.. 1947). Together they account for 30 to 60% of the total protein. Protein cgystals: have been observed in the protoplasm of the outer layers of potato cells. Depending upon varieties. these crystals can also be synthesized in other tissues of the potato tuber (Hoelzl and Bancher. 1959). These crystals. hardly exceeding the size of l cubicpn. are assumed to be of a globular protein type and represent only a small fraction of the total protein. Prolgmins: are soluble in 70 vol.% ethanol. Glutelins: are soluble in a 0.2% NaCl solution made up in 60 vol.% ethanol. Sclereoproteins: are found in the residue which can not be extracted with the above mentioned extracting solutions. This fraction can be 10% of the total protein. The nonprotein nitrggen fraction is composed mainly of free amino acids and amides. The principle amides are asparagine and glutamine. The free amino acids and the amides represent the amino acid pool of the plant and are involved in many different physiological activities of the plant. Together they can contain between 35 and 65% of the total nitrogen. Amino acid composition and nutritive value of the various n tr n onta n constitu nts Lindner £3.31. (1960) measured the relative amounts of different proteins in the potato and found that of the total protein. tuberin accounted for 76.4%. globulin II 1.4%. tuberinin (albumin) #%. prolamin 1.8%. glutelin 5.5%. and the insoluble residue 11%. The essential amino acids in the nonprotein nitrogen fraction are present at a much lower level than in the protein fractions. Frequently no free tryptophan or no free cysteine could be detected (Woodward and Talley. 1953). The difficul- ties involved in the proper recovery of cysteine may partially explain these variations. Furthermore one has to consider that the potato tuber is a metabolically active unit. even when stored at low temperature. Therefore the chemical composition of the tuber may vary if analysed at different physiological stages. The nonprotein nitrogen alone cannot promote growth of weanling rats (Slack. 1948: Chick and Cutting. 1943). The same investigators have shown that the nitrogen of the intact potato supports growth at least as well as tuberin alone. The complementary nutritive effect between the tuberin and the nonprotein nitrogen fraction of the press juice could not be explained in terms of their amino acid contents. A paper by Rose gt_gl. (1948) contains a description of ex- periments in which growth of weanling rats was definitely stimulated by the addition of 2% glutamic acid to a diet in which the nitrogen. adequate in amount. was supplied as a mixture of the ten ”essential” amino acids. It may be pos- sible that some nutritional significance is attached to the relatively large amounts of glutamine present in the potato tuber. The insoluble scleroproteins are found mainly in the skin and outer cortex. When these layers were removed. the apparent digestibility was raised from 74 to 79% (Chick and Slack. 1949) resulting in better growth of the rats. In nitrogen balance studies. using human adults as test individuals. potato protein proved to have the best nu- tritive value of all analysed plant proteins (wheat flour. corn. rice. algae. soybean and kidney bean protein). Jekat and Kofranyi (1970) demonstrated by means of human bio-assays 10 complementary effects of potato protein with egg. soybean. algae. rice. corn. beans and wheat protein. The lowest ever reported minimal protein requirement per kg body weight was obtained with a mixture of 36% whole egg protein and 64% potato protein (Kofranyi and Jekat. 1967). y In other studies using a similar technique as Jekat and Kofranyi (1970). potato diets were supplemented with leucine. phenylalanine and methionine. In comparison with the regular potato diet. only the human adults whose diets were supplemented with methionine showed significantly im- proved utilization of the potato protein. This is another proof that methionine is the first limiting amino acid in potato protein (Kies and Metzfox. 1972). Environmental influences on thp composition of thg nitrogen containing ponstituents of the potato One of the most important factors in this respect is the level of soil fertility and nutrient availability. This factor influences the nonprotein nitrogen fraction of the potato. because this chemically mobile part is actively involved in the metabolic processes of the plant. 11 Figure 1: Relationship between levels of nitrogen fertili- zation and yield of 2 potato varieties+. g ’4'.“ :} u,o Bona a 3.6 .‘.a‘.01ympip. a 3 2 .§ ' P . so kg‘/ ha 5 2'8 K . 100 kg / ha 2’ 2.4 'U 0 so 100 150 200 kg N fertilizer /’ha + Schuhpan. 1970 Table 2 demonstrates the detrimental effect of exces- sive nitrogen fertilization on potato protein quality. How- ever. attention must be given to other factors which are in- fluenced by fertilization. Figure 1 shows the dependence of total dry matter production upon nitrogen fertilization and Table 2 reflects the response of applied nitrogen fertilizer to net protein content. 12 Table 2: Influence of fertilizer level on the net protein content"' level of N fertilizer *BV net protein content++ kg/ha relative/ha O 76 100 50 82 144 100 74 184 200 71 201 + BV. determined on the growing pig %N x 6.25 x true absorption x BV 1656 ++ net protein content = +++ Brune. 1968/69 Most of these experiments used Just one potato Variety. How- ever. the extent of response in amino acid composition as influenced by nitrogen fertilizer may be dependent upon the genotype. Schuhpan (1970). for instance. reported that when lZOkg/ha nitrogen fertilizer was applied. the variety Bona had an EAA-Index of 64 and the variety Olympia 86. In addi- tion. the EAA-Index of the variety Olympia decreased only slightly with increasing amounts of nitrogen. whereas that of Bona dropped from a high of 95 to a low of 60. The role of methionine n th ot to Methionine as a precursor is involved in the synthesis 13 of ethylene and flavor compounds. Burton (1957) reviewed earlier work on the effects of ethylene and carbon dioxide on sprouting. Brief treat- ments with ethylene at intervals stimulated sprouting. while treatments of longer duration suppressed it. Since conditions leading to ethylene accumulation in storage would also lead to carbon dioxide accumulation. Burton suggested that sprout suppression attributed to high CO2 was. in fact. caused by ethylene. His own work suggested that carbon dioxide stimu- lated sprouting when ethylene was removed. Poabst pp pi. (1968) proved chemically that potato tubers contain endo- genous ethylene. and treatment with gibberellic acid increased the ethylene content. This result conflicts with Burton's hypothesis. since gibberellic acid stimulates potato sprouting. It may be that the influence of gibberellic acid is strong enough to overcome any ethylene produced as a result of its application. Ethylene has been recognized as an endogenously pro- duced hormone which initiates fruit ripening and regulates many aspects of plant growth. It has been reported to be pro- duced nonenzymically from methionine and its analogues. me- diated either by a Cu++ -ascorbate -H202 (Lieberman pi pi.. (1965) or by a FMN -light system (Yang pp gi.. 1966: Yang pp_pi.. 1967). The findings that methionine stimulates ethy- lene production and is readily converted to ethylene in fruit and vegetative tissues (Lieberman pp,gi.. 1966: Burg and Clagett. 1967) indicate that this amino acid is a natural 14 precursor of ethylene in plant tissues. The importance of sulfur-containing compounds to fla- vor lies in their extremely low odor thresholds. From all sulfur-containing volatiles produced during the cooking process. methylmercaptan and dimethyl-disulfide make up 90% of the mixture. with ethyl-mercaptan and methyl-sulfide making up most of the remainder (Gumbmann and Burr. 1964). Although the pathways and intermediates of the sulfur metabolism in plants and animals represent an area largely unexplored. there is sufficient information available to account for the appearance of these compounds in food pro- ducts. Both. primary and secondary mechanisms may be regarded as responsible for the production of volatile compounds du- ring cooking. For example. the breakdown of the sulfur amino acids is thought to be the primary source for simple organic sulfur compounds. Pathways through which methionine. cystine and cysteine could be converted into flavor compounds are re- ported by Neukom (1967). Relations gmopg specific grgvityI total nitrogen and quality factops of potatoes Certain aspects of the quality of fried potato products are apparently associated with high starch content. Thus po- tatoes of high specific gravity. in general. give french fries that are crisper than those prepared from tubers of low specific 15 gravity (Kirkpatrick p1,51.. 1956). In selecting potatoes for processing into chips. it is important that tubers of high specific gravity or dry matter content are chosen. Smith (1951) showed that for every increase in specific gravity of 0.005. there is approximately an increase of one percent in yield of chips. Pope pp_gi. (1971) found an in- verse relation between specific gravity and potato chip oil content and yellowness of potato chips. Houghland (1966) observed that in potatoes having 13.3% total solids. about 56.5% of this dry matter consisted of starch. At'a total solids content of 32.2%. however. he showed that starch content to be 79.0% of the dry matter. On a fresh basis there was an increase of only 1% in the non-starch fraction of the tuber. and an increase of 18.9% in the solids or an average of 0.053% non-starch for each 1% increase in total solids between these extreme. Thus. any effort to increase the protein content of potatoes by breeding for an increase in dry matter content would show little pro- mise of success. Fitzpatrick pp,gi..(l969) confirmed these findings. Tpxic constitugnts of potatops gpotpgsp inhibitors: The crystallization of two thermolabile (trypsin inhibitors from the potato was described by Sohonie and Ambe (1955). The potato may contain a number of other 16 protease inhibitors which. because of incomplete characte- rization may or may not be identical. These include also a potent inhibitor of chymotrypsin (Ryan and Balls. 1962). The molecular weight of this inhibitor is approximately 22.000. The inhibitor was devoid of carbohydrate. It rather unexpectedly contained no cystine but rather 4 residues of cysteic acid and l residue of methionine sulfoxide. If the latter should be true. then. unlike most of the other pro- tease inhibitors which have been characterized. the chymo- trypsin inhibitor is apparently devoid of disulfide bridges (Balls and Ryan. 1962. 1963). Ryan (1966) points out that _ the chymotrypsin inhibitor of the potato is quickly destroyed by heating in the intact potato even through the purified in- hibitor is quite stable. Cho n ste s i b to : The only cholinesterase inhibitor identified is the glycoside. solanine. which is present in highest concentration in the sprouts and skin (especially when green) of the potato. Although human fatalities due to the consumption of green potatoes have been reported from time to time (Hanson. 1925: Hams and Cockburn. 1918). proof that solanine was "the” causative agent is.largely indirect. However. it is significant that solanine is not destroyed by cooking (Baker pp gi.. 1955). and the poisoning of livestock has sometimes been observed even with cooked potatoes (Kings- bury. 196“) e PART I INHERITANCE OF "AVAILABLE” METHIONINE. TOTAL PROTEIN AND SPECIFIG GRAVITY IN TETRAPLOID POTATOES INHERITANCE OF ”AVAILABLE” METHIONINE. TOTAL PROTEIN AND SPECIFIC GRAVITY IN TETRAPLOID POTATOES This portion of research was undertaken to study the inheritance pattern of methionine. total protein and spe- cific gravity. Also. the possible relationships among these three traits and potato chip color. fresh weight. total dry matter and rest period were investigated. MATERIALS AND METHODS Five thousand segregating progenies (1 tuber each) representing 8 crosses and the parental clones were planted on the Montcalm Experimental Farm in 1971. Plant spacings were 45 cm and row spacings 86.4 cm. The soil. a uniform Montcalm sandy loam. was fertilized with 185 kg N. 110 kg P205 and 110 kg K20 per hectare. At harvest. 7 hills of each parent and 40 cultivars from each cross were randomly selected and stored at room temperature until analysed. To determine the specific gravity. the potatoes from each hill were weighed in air and in water. From each first year seedling 3 tubers were taken and cut longitudinally into halves. From one half of each of 17 18 three tubers. 5 slices were taken for chipping. From each of the other three halves. 7 to 10 two mm thick slices were removed and immediately frozen on a layer of dry ice. After freeze drying the samples were ground in a Wiley mill through a sixty mesh screen. Most of the peel was removed in this process. From each parent. 7 hills were chosen at random for ana- lysis. Two 3 tuber samples were taken from each hill and pre- pared as outlined above. The freeze dried samples were used for the analysis of total protein and "available” methionine (Luescher and Thompe son. 1972). For chipping. the potato slices were rinsed in cold water and fried at 190 C until water evaporation ceased. The potato chip color was estimated using the standard color chart of the National Potato Chip Institute. After chipping the remaining potatoes were stored at 4.5 C and checked monthly for sprouts. When half of the tu- bers of a seedling showed visible sprouts (2 mm). it was con- sidered to have broken the rest period. The female parents in population I originated from a breeding program in which S. stoloniferum was backcrossed several times with S. tuberosum L. and the progenies selected for specific gravity. All the other parental cultivars were strictly of the S. tuberosum type. 4 The following variance and covariance components (Table 3) were estimated for ”available” methionine. total 19 Table : Composition of variance and covariance components of half-sib families in a tetraploid+. Source of variance Estimate Composition Amo half-sib family v 1/ 0'2 + 1/ «’- nIoffspring) means 0 4 A 36 D ++ Within half-sib families V 3/0'2 +35/ 402-10240‘2 Among mid-parents VMP 1/20“: + 1/2 6123*1/20'3'*1/2d% Family (offspring) mean: Cov l/Zdfi + 1/6 0'; mid-parents covariance where Oi = additive genetic variance 0%. ‘2. «F = interaction variances of 2. 3. and 4 alleles Heritability mainly in the narrow sense was calculated in two ways: 02 + 4/ 1. 4vO/(vo-1-vw) = A 36‘2” ;A*¢g+°‘%*°‘§+ as OF34-2/'1d2 2. 2 Cov/(ZVMP-HIE) = A 6 D 1* I)" '1'" F+ E ++ in this variance estimate the environmental variance is included because no replications were available from the first year seedlings + Kempthorne. 1955 20 protein and specific gravity and interpreted genetically assuming autotetraploid inheritance and absence of in- breeding in the ancestries of the parental clones (Kempt- horne. 1955). In both cases the numerator contains some variance due to the interactions of 2 alleles which is analogous to dominance deviations in the diploid case. The variance of half-sib family means contains the least of this interaction variance. Falconer (1960) points out that the half-sib cor- relation and the regression of offspring on the male parent give the most reliable estimation of heritability. RESULTS AND DISCUSSION There is limited information on the genetic variabi- lity of methionine in potatoes. From the total phenotypic variance in the parental nursery the variance between the two 3 tuber samples approached zero (P=O.89). This indi- cates that one 3 tuber sample per hill (first year seedling) is a representative sample for methionine analysis. In population 11 the offspring family means of methio- nine were somewhat higher (but not significant) than those in population I (Figure 2. Table 4). In population II. total protein averaged 9% higher than in population I. This diffe- rence and the positive correlation between total protein 21 uaoo.a .ooo.a nnao.~ osoo.a oo.~H nn.~a Ho.oH oo.aH Ne.H we.” HH.H o~.H m a unnaxam-eoa oooo.~ ooo.a memo.” memo.a oa.na oa.ma on.oa oe.a~ an.H on.a mo.H a~.~ a o -Haaxen-moe some.“ - - peso.” am.~a - - sa.aa ~n.a - : o~.H o ~n-ooaxon-woa o~ao.~ one.” onno.a oaoo.a mm.~H ma.aa no.nH ae.eH om.H nm.a ma.H e~.H n ooexan-eoa Ha cane.” woo.” ammo.” anoo.a em.HH am.aa no.n~ oa.sH ma.a on.a me.a mH.H a ooaxoe-a~n ammo.~ moo.” mmao.~ odaa.a HH.HH ~o.~H no.na oo.a mm.H ma.a mo.d n:.a n ooaxmoaa~n ease.” nae.” anao.a oaao.a H¢.~H aa.ma no.mH on.m~ on.H mu.” me.H oH.H a moaxmnuamn neao.a. ~mo.u ammo.” omoa.a ~n.aa mm.~a no.na ou.o~ mn.H m~.~ ma.a ao.a a ooaxo -own H mcflammmo assuming: . b 0 92:93.3 $8.393: b o unauamuuo 05.233: b O honescmmouo mmucemm couumaaaom muu>mum owuuoeam :«ovohm Hmpoa mausoacvoz mmouu .h»«>ahw advance» was fineness ago MEOOH\wSV :aououn Hope» .Az wsofi\uev ocdcodguos :ounsaua>m: you memes hawemh waduAmumo can acoumm .: oHpmn OFFSPRING OFFSPRING 1.7 1.6 1.5 1.4 14 13 12 11 O 22 Figure 2 : Offspring : Mid-parent regression for "available” methionine Population II : b2 8 0.90 r2 = 0099 7 o 5 o '3 . 8o 4. . Population I : b = 1.17 efl' 1 4'1 2 r1 = 0.93 I I i I r I 1.1 1.2 1.3 1.4 1.5 1.6 ”available" methionine. mg met /'16mg N MID - PARENTS Figure 3 : Offspring : Mid-parent regression for total protein ‘ Population II . b2 = 0.37 r2 = 0.99 :1 5 ’07/ . (D . ‘.",a’ 8 . C) .afl"’ (L A . ‘,%; Population I . bl = 0.33 1: r1 = 0.93 I r I I I I I 10 11 12 13 14 15 16 total protein. mg Nx6.25 / 100mg dry matter MID - PARENTS OFFSPRING 1.085 1.085 1.075 1.070 1.065 23 Figure 4 : Offspring : Mid-parent regression for specific gravity '1 ’3 I e 1 2 A.“ J an .1 1 50 q q d 1 8 b30014“ : 7° 0 r=0078 1 L "1‘V'V‘fiV'VfVV'tfrf'T'T‘iffi O 1.065 1.070 1.075 1.080 1.085 1.090 specific gravity MID - PARENTS 24 and ”available” methionine (r-0.16. Pm macaw a: a as on we no H.an a ao.~ am.H mm.H mm.oa .>¢ as am an o.en as.” Ha.H ma.~ o.oH m -a mm mm on A.em ao.~ Ho.~ m~.~ m.oa as-“ an am as m.om no.H ow.a om.a e.HH a~-m 0: mm Hm o.m: um.d mm.n Hm.H o.HH qun an o on o we a u.oo e ma.H am.H ao.~ oo.mH .>< .an ow Ho a.no oa.a ma.~ mm.” a.n~ aaa-a we on an m.mo w~.H 0:.H mn.H ~.mH nnum mm as on “.mn mn.a an.a oo.a n.0a Nmum an as ea n.nm oe.a . oo.H ao.a m.aH an-” ma on an o oo o m.ma o mn.o ca.” oo.H an.o .>< DH 05 HOH Hemn Hweo Hnofl :0..." o.m m lm RN so as n.~n Ha.o mn.a mH.H o.m ~m-~ NH om mm o.ae an.o nn.~ Ha.o m.o~ o:-~ 0H mm mm 3.3: mm.o mm.a «H.H ~.m omua as o as n on a o.mn n mw.o 3H.H mo.~ aw.oa .>< H: as on o.~n Hm.o oH.H a.” m.ma an-w on as we m.on oa.o mH.~ ma.o n.o~ m~-o am we ww w.~o mo.H HH.H :~.H m.wa m :w on as do a.no ao.o nH.H mo.H a.o~ 0H-m . z Hmuou z saopoua hooves has :aououq Have» no on“: 2 z wso0fi\ws use: weod\ws z m5wa\ws z meoa\ms msooa\ms :oazpes op onwc0usvos caououncoc :«ououasoc or“: cancofinuoe . .oz ovum «0 soausnuuucoom no >m ho >m z saovouACoz :ougpoe ooh» usacownpos :oanmaflm><. campoua Haney ocoau .cdouoan Havoc ho ocncounp :os op ocdcoqguoe noun no couusnauucoo one came noun Have» no >m .z :«ouonmco: mo >m .z :«opouacos .osdsoanuoa oouu .ocdsoanvos .ocacouzpos :oanma :am>m: .caovoun Have» new commence at» no name: .m:mammw 36 T ble : Correlations among total protein. ”available" me- thionine. methionine. cystine. nonprotein N. free methionine. BV of total protein and BV of nonpro- tein N (data from 16 seedlings analysed in 2 re- plications each. total protein 1.00 (%of dry matter) "available" methionine -0.11 1.00 (mg met/16 mg N) methionine -0.49a 0.86b 1.00 (mg met/l6 mg N) cystine -0.08 -0.39 -0.20 1.00 (ms eye/16 ms N) free N 0.63” 0.26 -0.09 -0.03 1.00 (% of total N) free methionine b b (mg free met/' 0.09 0.96 0.76 -0.45 0.30 1.00 1 mg free N) av of total -0.5sa 0.40 0.62” 0.00 -0.u1 0.24 1.00 protein BV of non- -0.69” -o.15 0.21 -0.02 -o.96” -o.24 0.60a protein N c+ = s o it it I” 3» £13 3- '6 :3 3 '< m 1"” 5' d- 0 O O h' ora Ho en 't 5 l" O :3 2 B 'U 1"” 3 O (D d' ’1 :3 O‘ H' d- O O (b H :3 5' d- 8. z o l-' s a 3- 2 0 :3 I (D a: significant at P: 0.05 b: significant at P: 0.01 37 Neuberger and Sanger (1942) compared heat coagulation. precipitation with trichloroacetic acid and filtering through a membrane with an average pore size of 7 mp . All three methods gave comparable results. * Only traces of free cysteine could be detected in the 16 analysed cultivars. The regression of ”available" methionine (mg met/16 mg N) on free methionine (mg free met/16 mg nonprotein N)«amounted to 0.76 and accounted for 93% of the total variation in "available” methionine (Figure 5). Although total protein was highly correlated with nonprotein N. it had no influence on free methionine (Table 8). ' In the low total protein - low methionine group. free methionine provided 18% of all the methionine present in the total protein. whereas in the high protein - high methionine group. free methionine accounted for 57% (Table 8). From the nutritional point of view an increase in methio- nine or cystine or both would be desirable. In the clones analysed however. cystine was present exclusively in the protein fractions. The various proteins could theoretically differ in their quantity and in their cystine content and thus cause variation in overall cystine content (Part III of this thesis). Variation in methionine is composed of variation in free methionine and in methionine present in the proteins. The latter could be explained as for cystine. According to this study. however. free methionine is responsible for 93% 38 of the variation in ”available" methionine. Free methionine ranged from 0.34 to 2.07 mg/16 mg nonprotein N. Whether or not there exists an upper limit for free methionine could not be appraised. It must be re- membered. however. that these potato cultivars received 185 kg of N fertilizer per hectare during their growth. Mulder and Bakema (1956) reported that free methionine dropped from 1.9% when fertilized with 33 kg N/ha to 1.0% when fertilized with 150 kg N/ha. If this observation is taken into account. the highest free methionine content of 2.07% is remarkable where 185 kg N/ha were applied. The fact that growing conditions have a great influence on free methionine is a major drawback. If the genotype x environment interactions are reasonably small. the geneti- cal gain in free methionine would be actual. A potato high in free methionine must be cooked and processed carefully to avoid a loss of nonprotein nitrogen in cooking or rinsing water. The high free methionine con- tent could enhance the formation of flavor compounds. S. zypogenes has an absolute requirement for exogenous leucine. methionine. tryptophan. arginine. histidine. iso- 1eucine. valine and glutamic acid. Thus the amount of these amino acids determines the EV obtained by this microbiolo- gical assay. The BV of the nonprotein nitrogen was very dependent upon the level of the nonprotein nitrogen and total protein (r= -O.96 and -O.69 respectively). Even the EV of the total 39 protein was negatively correlated (Table 9) with these two factors: however. the regression coefficients were not significant. At the 0.01% level. no group means of the EV results could be separated with Duncan's Multiple Range Test (Table 8). High and low totalfprotein cultivars were found with a high ”available” methionine content. The high "available" methionine content was proportional to the amount of non- protein nitrogen. SUMMARY Sixteen cultivars selected for their total protein and ”available" methionine contents were studied. Free methionine ranged from 0.34 to 2.07 mg/16 mg nonprotein N and was highly correlated with "available” methionine (r= 0.96). Free methionine contributed from 12 to 62% of all methionine present in the total protein. Free methionine and ”available" methionine were independent of the total protein content. No measurable amounts of free cysteine could be de- tected. Advantages and disadvantages of a potato high in ”available” methionine are discussed. The BV of the total protein determined by microbio- logical assay was negatively correlated with total protein 40 content and positively with methionine. However. the group means could not be separated with Duncan's Multiple Range TeSt at P: 0.010 PART III ELECTROPHORESIS AND ANALYSIS OF THE SULFUR AMINO ACIDS OF VARIOUS POTATO PROTEINS ELECTROPHORESIS AND ANALYSES OF THE SULFUR AMINO ACIDS OF VARIOUS POTATP PROTEINS Variation in amino acid composition can be due to variation in the nonprotein fraction and/or variation in the protein fractions of the total protein in potatoes. In Part II of this thesis variability in "available" me- thionine could be explained mainly by the variability in free methionine. Lindner pp pi. (1960) isolated five different proteins and analysed three of them for methionine but not for cystine. Only limited information is available about the uniformity of these extracted proteins. This research was designed to investigate the vari- ability of methionine and cystine in the classical proteins of three potato cultivars. MATERIALS AND METHODS Random samples. 4 kg each. of the experimental culti- vars Nos. 58. 709 and 322-6 were used for this study. All three cultivars have a completely different pedigree: Num- ber 58 is an inbred Merrimack. 322-6 originates from a breeding program in which S. stoloniferum was backcrossed several times with S. tub rosum. and 709 is S. tuberosum. 41 42 All three cultivars were grown on a Montcalm sandy loam and fertilized with 220 kg N. 150 kg P205 and 110 kg K20 per hectare. The tubers were cut into 2 mm thick slices and immediately frozen on a layer of dry ice. After freeze drying the samples were ground in a Wiley mill to pass through a sixty mesh screen. Most of the peel was removed in this process. Methionine. cystine and total protein were determined according to Luescher and Thompson (1972). Free methionine and nonprotein nitrogen were assayed as outlined in Part II of this thesis. To isolate the various proteins. the procedure out- lined by Lindner pp,gi. (1957) was applied (Figure 6). The potato flour together with the extracting solutions were in all cases blended for four minutes at room tempera- ture. The cellophane bags1 were dialized against 100 times its volume of distilled water for 48 hours at 4 C. During dialysis the water was changed 4-5 times. After the first complete isolation. tuberin. tuberinin. globulin II and prolamin were dissolved in their corresponding extracting solutions followed by centrifugation. dialysis. centrifugation and freeze drying. In addition tuberin was washed 4 times with distilled water. Electrophoretic separations of the proteins were per- formed according to Davis (1964). Instead of preparing a sample gel. the sample was mixed with a sucrose solution 1 Number 27/100 was obtained from Union Carbide 3 I“ :q4393dv usuaau onoeuu .mamhamav unnumCLoasn zazammaze szun madame madame oaoouu *.Ho> ow :« ave; w~.o mucosa ouoouu .mdmhdsuv as con and: cowpomuvxm ufimfiuuow acuacChomsm ZHEEOmL ugvdeafim Osvm . en. zzozxz: A 2 HH zaqanoqu .59 o... ... .313 . segue euoouu .: a 3 .u 33.3 V Cofiunwzumhucoo .HOSUMHa cm Newhhv A « souvezzamugcec munhasav Ho mwmhamfiv museum moeav n .Hozooae R.Ho> on .uaosdeae as con gear coaooeaoxe «condemn «cevmcuodsm vcmumcuodsm oaufimom A .c«3 on .u oow.oa . consensuaupeoo coaumusumm now ou as A .caa mg .m oo:.o« v couumusuuupcoo aomw. as V Co 533.3 no.3» n A .82 no" as con fit. 532.528 «superheazm oscwmom A .c«3 mm .u oom.oa . coaoeesuauoceo mead» n mouaa hog omzdz n.~ mannamucoo soap Isaom Hoaz ma as con gawk coapomhpxo macaw cannon sedan ouoouh u ow A smog..4d dd accusaq v usumvoun oumvoa no coupscowuomum Lou osozou . 44 and an aliquot portion was layered on top of the spacer gel. The gels were stained for 15 minutes with Coomassie blue. 0.125 g in a solution of 5 parts methanol. 5 parts distilled water and 1 part acetic acid respectively. For destaining the gels were soaked in a solution containing 5% methanol and 7% acetic acid at 50 C for 2h hours. The destaining solution was changed twice. Protein extracts from the potato flour were prepared in the following manner: a sample containing 50 mg of to- tal protein was extracted with h ml of a 2% NaCl solution containing 500 mg NaHSOB per 100 ml. then centrifuged (10.000g, 10 min.) and the supernatant was saved. The super- natant of a second extraction with 4 m1 of the same solution was added to the first one and the volume was made up to 8 m1. For the electrophoretic analysis the extract was diluted 1:1 with a 40% sucrose solution. From this mixture 10 pl portions were analysed. One mg each of tuberinin and tuberin was dissolved in 1 ml of a 2% NaCl solution and then diluted 1:1 with a 40% sucrose solution. RESULTS AND DISCUSSION Tuberin and the nonprotein nitrogen fraction contained 86 to 92% of the total nitrogen. The amount of nonprotein “5 Table 10: Distribution of total nitrogen in 60 g dry matter of 3 cultivars. 58 322-6 mg % mg % mg % Protein fractions Tuberin 2059 28.8 1769 33.5 2082 35.4 Globulin II 41 0.6 20 0.“ 16 0.3 Tuberinin 77 1.1 60 1.2 56 1.0 Prolamin #2 0.6 32 0.6 #9 0.8 AGlutelin 6 0.1 5 0.1 6 0.1 unknown nitro- 60 0.8 32 0.6 128 2.2 gen compounds Residue #82 6.7 267 5.1 579 9.8 Total 2767 38.7 2189 41.0 2916 “9.6 Nonprotein N #379 61.3 3093 58.6 2968 50.h Total protein 71h6 100.0 5282 100.0 5884 100.0 nonprotein N ob- tained by heat 31.6 “5.6 55.6 coagulation Table 11: Methionine and cystine contents of tuberin, tu- berinin. prolamin and the nonprotein N fraction of 3 cultivars. 58 709 322-6 met cys+ total met+ total met+ cys+ total Tuberin 2.9 1.2 u.1 2.0 1.4 3.4 2.7 1.1 3.8 Tuberinin 2.0 1.0 3.0 1.5 1.5 3.0 2.0 1.2 3.2 Prolamin 0.3 3.7 0.0 0.3 3.4 0.3 3.9 “.2 Nonprotein 0.8 trace 0.8 0.7 trace 0.? 0.3 trace 0.3 N fraction * amino acids are expressed in mg/16 mg N; means of two Aindependent analyses #6 nitrogen obtained by heat coagulation and by the extraction procedure agreed reasonably well considering the large number of nitrogen analyses involved in the extraction pro- cedure (Table 10). Ammonium sulfate (at 80% saturation) did not precipitate all nitrogen present in larger molecules of the 2% NaCl solution. In Table 10 this residue left after precipitation with (NHu)2 SO“ was called "unknown nitrogen compounds”. The four major nitrogen containing constituents, tu- berin, tuberinin. prolamin and the nonprotein fraction were analysed for methionine and cystine (Table 11). Tuberin and tuberinin of the cultivars 58 and 322-6 contained about twice as much methionine as cystine. Cultivar 709 had consi- derably less methionine in both the tuberin and tuberinin fraction. 0n the other hand both of these protein fractions contained more cystine than 58 and 322-6. In all three cul- tivars the sum of methionine and cystine was smaller in tuberinin than in tuberin suggesting that these two proteins differ more than by an atom of hydrogen as indicated by Jirgensons (1906). Tuberin, the main protein fraction, con- tained amounts of sulfur amino acids similar to milk but only about 75% of the methionine and 50% of the cystine pre- sent in the protein of whole egg (Block. 1951). Some variabi- lity can be expected in the amino acid composition of tube- rin, however, to what extent was not determined from this study. Prolamin is an excellent source of cystine. However, 1+7 Figure 2: Densitometrical readings of the gels of 58 O 1 '5 whole flgur >. 3:} m B C 5 0.9 -o E; A 0.3 r . r' o 1 2 3 4 running distance. cm 1'5 tuberinin >. +3 ”.3 5 009 -o +3 9. <3 0.3 o 1 2 3 4 running distance, cm (:> tuberin (:> >. :3 009 U) s 0) -o +3 0-3 D. O 0 1 2 3 4 running distance. cm A.B,C 1 major protein bands 48 Figure 8 : Densitometrical readings of the gels of 322-6 0 1'5 whole flgur >. .p ”4 m g 0.9 'o . A 4.: 87 0.3 0 1 2 3 running distance. cm 1.5 9 tuberinin >. -———-'-— +3 ‘3 g 009 .5 A .5 p. c> 0.3 o 1 2 3 running distance. cm 3 0.9 tuberin 0H 0) c 0 vs .3 D. c: 0 1 2 running distance. cm A,B.C : major protein bands 4:- 49 Figure 2 s Densitometrical readings of the gels of 709 0 G9 1'5 whole flour Opt. density >. .p g 0.9 A Q) ' B '9 C .3 o. c: 0.3 0 1 2 3 4 running distance. cm >. tuberin 33 0.9 m c 0 -o ' B a 0.3 A C c: y H 0 1 2 3 4 running distance, cm A.B.C : major protein bands 50 the sum of cystine and methionine was not greater than in tuberin. The recordings of the densitometer of the gels are presented in the Figures 7, 8 and 9. The gels of the whole potato flour of 322-6 and 58 had 3 major bands (A. B. and C). In 709 band C was greatest, although the other two bands were present as can be seen from the tuberin fraction. Most of the bands in all three cultivars could be found in tu- berin as well as in tuberinin. However their relative quanti- ties seemed to differ. For instance band A was most abundant in the tuberinin of all three cultivars whereas bands B and C predominated in the whole flour and in the tuberin fractions. Not only was the sulfur amino acid composition of tu- berin, tuberinin and prolamin similar for the cultivars 58 and 322-6, but also the densitometrical recordings of their gels had some similarities. A few major bands make up most of the proteins defined in the classical terms. The relative portions of these bands can vary from genotype to genotype. The fact that the same proteins of the three cultivars differed in methionine and cystine leads to the conclusion that the protein bands must vary in the amounts of sulfur amino acids. In a breeding program for increased sulfur containing amino acids, production practices must be considered. Depending upon the growing practices two different approaches have to be chosen. The deciding factor in this respect is the extent of applied nitrogen fertilizer. High 51 Tab e 121 Upper limits in sulfur amino acidrcontents of potatoes grown with low and high nitrogen_ fertilization* Growing conditions No or only limi- Intensive ni- ted nitrogen trogen ferti- fertilization lization Tuberin Nonprotein Tuberin Nonprotein fraction fraction % N of total N 80 20 no 60 % met 3 1.0 3 2-5 % cys 1.5 - 1.5 -' % met of total 2.6 2.? protein % eye of total 1.2 0.6 protein cys + met of 3.8 3-3 total protein % of the sulfur 63% 55% amino acids pre- sent in the whole egg protein + The methionine and cystine values are the highest values obtained from analysis of 700 segregating cultivars for "available" methionine. 52 rates of nitrogen cause an increase in the nonprotein ni- trogen fraction at the expense of the protein fractions (Mulder and Bakema, 1956). In areas where little or no nitrogen fertilizer is applied (e.g. in developing countries) a potato with a high tuberin content should be sought. The ratio of protein ni- trogen /'nonprotein nitrogen would be the selection cri- teria in this case. The composition in sulfur amino acids which could be expected is presented in Table 3. In this country, another approach must be taken. Ni- trogen fertilization of 100 to 200 kg per hectare is common. Thus it would be difficult to find potato clones in which ' total proteins would contain 80% tuberin. Thus the ideal potato to select should be high in free methionine and tu- berin and should account for 40 to 50% of the total protein. The possible presence of free cysteine should be kept in mind. Here. methionine content would be the most important selection criteria but in a final evaluation cystine and free cysteine should be considered. SUMMARY From the dry matter of three cultivars. tuberin, tu- berinin, globulin II. prolamin and glutelin were isolated. 0f the total nitrogen, tuberin accounted for 29 to 35%. the nonprotein nitrogen fraction for 50 to 61%. the residue for 53 5 to 10% and all the other fractions for less than 2.5%. Tuberin. tuberinin and prolamin of the cultivars 58 and 322-6 contained similar amounts of methionine and cystine. whereas the sums of methionine and cystine of the corres- ponding proteins of 709 were considerably lower. The electrophoretic separation showed that tuberin is composed of at least 3 major bands. The proportioncof these 3 bands was dependent upon the genotype and could possibly be responsible for the differences in the content of the sulfur amino acids. Tuberinin contained primarily the same protein bands as tuberin. however. their relative portions were different. Different section criteria applicable to breeding a potato high in the sulfur amino acids are discussed. «SUMMARY AND CONCLUSIONS SUMMARY AND CONCLUSIONS In this study the high heritability of the amino acid methionine was established. With this information it should be possible to breed a potato variety containing two or more mg met/16 mg N. Seventy g of such a potato protein would provide 1.4 g methionine or more than the daily minimal requirement of 1.1 g for a human adult. Based on this study one hectare of land planted with 26,000 hills of the potato clone 7-34 would produce 11,200 kg of dry matter which would contain 1,900 kg of total pro- tein. The methionine content of this clone was 1.6 mg/16 mg N. This example demonstrates the tremendous potential of the po- tato to produce large quantities of proteins as well as car- bohydrates. Human nutritionists and food technologists should give more attention to the potato as a source of protein. It is known that methionine is a precursor of flavor compounds. It would be very worthwhile to know whether the formation of these flavor compounds is proportional to the content of methionine in potatoes. This knowledge would help the potato breeder to direct his future research. This study indicated that the mostly abundant protein. the tuberin. is composed of at least 3 separate proteins. It would be of practical interest to know whether these proteins contain different amounts of sulfur containing amino acids. This information would help the plant breeder to obtain the 54 55 highest overall content in sulfur amino acids. 3. 4. 7. From the data presented it can be inferred that: "Available"‘ methionine is highly heritable and is in- dependent upon the level of total protein. Total protein and specific gravity are moderately he- ritable. No negative correlations could be observed between "avai- lable” methionine and rest period. fresh weight. speci- fic gravity. total protein, chip color and total dry matter production. Ninty-three percent of the variation in "available" me- thionine was due to variation in free methionine. Tuberin is composed of at least three major protein bands. The relative presence of these bands seems to vary from genotype to genotype and could therefore cause some va- riation in the content of the sulfur amino acids. Tuberinin contained almost the same bands as tuberin, but in different proportions. The cultural practices dictate the selection criteria to be applied in order to breed a potato high in sulfur amino acids. LITERATURE CITED LITERATURE CITED Auret, M.. J. Perisse. F. Sizaret and M. Cresta. 1968. Nutrition News Letter 6, Nr. 4: 1. Baker, Le Ce. Le He Lampitt and Le Ce ”eradithe 1955c Solanine. glycoside of the potato. III. An improved method of extraction and determination. 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DATA OF 320 OFFSPRING REPRESENTING 8 CROSSES Leggnd: total protein chip color rest period fresh weight specific gravity total dry matter . mg ( N x 6.25 )/100mg dry matter "available” methionine: mg ”av.” met/16mg N : according to the color chart of the National Potato Chip Institute 1 10 O‘MNNH very very rest rest rest rest rest rest bright dark p. broken in December' p. broken in January p. broken in February p. broken in March p. broken in April p. broken in May : total weight of one hill. in g : ( wt. in air )/( wt. in air-wt. in water : calculated from specific garavity and fresh weight, in g Cross Clone Total "av."me- Chip Rest Fresh Spec. Total prot. thionine color p. wt. grav. d.m. 1 2 13.67 1.77 5.5 1 420 1.077 84 1 5 9.48 1.40 3.5 l 970 1.072 187 1 6 10.82 1.40 3.5 3 1070 1.075 214 l 7 11.56 1.47 5.5 3 1065 1.081 226 1 8 8.70 0.90 5.5 l 1060 1.076 214 1 9 13.67 1.17 5.5 2 465 1.069 87 1 10 12.29 1.80 6.5 1 855 1.069 160 1 11 13.45 1.65 9.0 l 595 1.062 102 l 12 10.96 1.60 7.0 l 1115 1.070 211 l 14 12.14 1.45 1.5 1 1180 1.078 243 1 15 7.69 1.40 5.5 1 930 1.075 186 61 Cross Clone Total ”av."me- Chip Rest Fresh Spec. Total prot. thionine color wt. grav. d.m. 1 16 11.23 0.95 5.5 3 645, 1.066 116 1 17 9.42 1.37 6.0 1 380 1.070 72 1 18 12.19 1. 7 5.0 2 1215 1.085 269 1 19 10.17 1.02 5.5 1 680 1.079 142 1 20 10.16 1.52 9.5 2 945 1.074 187 1 21 10.25 0.95 7.5 1 970 1.066 175 1 22 14.29 1.27 7.5 2 1180 1.063 205 1 2 11.20 1.25 9.5 2 395 1.082 85 1 2 9.68 1.35 7.5 3 350 1.077 71 1 25 8.91 1.10 5.5 1 1515 1.063 263 1 26 12.79 1.55 7.5 2 1370 1.083 297 1 29 14.75 1.60 7.5 2 805 1.073 157 1 30 12.33 1.75 5.0 1 600 1.081 128 1 31 13. 2 1.35 2.5 2 460 1.082 99 1 32 10.70 1.80 .0 2 1115 1.082 239 1 34 9.16 1.02 7.5 1 570 1.075 114 1 36 16.91 1.15 7.5 1 520 1.072 100 1 37 12.82 1.22 7.5 2 1315 1.069 245 1 38 14.79 1.90 5.0 1 1560 1.072 01 1 9 11.60 1.07 7.5 2 2140 1.070 04 1 0 10.76 0.90 5.0 3 640 1.076 129 1 41 8.85 1.27 7.5 2 465 1.081 99 l 42 10.01 1.20 7.5 2 805 1.073 157 1 :2 9.94 l. 0 7.5 2 545 1.079 113 1 11.56 1.15 7.5 3 350 1.077 71 1 46 10.16 1.30 7.5 2 1395 1.077 284 1 47 9.82 1.35 7.5 3 530 1.071 101 1 48 17.57 1.37 3.5 1 660 1.073 129 1 49 10.64 1. 7 3.5 2 1520 1.086 339 2 26 14.23 1.52 1.5 2 690 1.087 156 2 31 12.51 1.15 4.5 1 835 1.084 183 2 32 12.38 1.37 3.5 3 790 1.082 170 2 36 11.81 1.57 5.5 2 1095 1.079 228 2 37 11.71 1.60 5.5 3 1660 1.078 342 2 38 15.53 1.42 5.5 2 1720 1.068 317 2 39 12.13 1.30 1.5 4 540 1.091 126 2 42 14.45 1. 0 7.5 1 870 1.081 18 2 43 12.47 1.25 3.5 3 1365 1.079 28 2 50 12.32 1.42 5.5 3 640 1.076 129 2 51 11.31 1.13 5.5 3 565 1.076 114 2 53 12. 8 1.30 1.5 1 630 1.086 141 2 59 13.84 1.30 3.5 1 530 1.093 126 2 60 10.18 1.30 3.5 1 1215 1.090 282 2 62 9.32 1.15 3.5 2 600 1.091 140 2 64 10. 1.17 3.5 1 1260 1.082 270 2 65 15.52 1.45 9.5 2 620 1.069 116 2 71 12.73 1.52 7.5 1 1430 1.071 273 2 72 13.60 1.17 5.5 2 685 1.087 154 2 73 13.00 1.70 3.5 2 955 1.085 211 63 Rest Cross Clone Total "av."me- Chip Fresh Spec. Total # # prot. thionine color p. wt. grav. "d.m. 2 78 14.01 1.50 1.5 1 1505 1.079 313 2 81 10.34 1.52 1.5 1 785 1.090 182 2 83 8.43 1.22 7.5 3 1360 1.084 298 2 8 13.17 1.42 9.5 1 1560 1.072 301 2 86 14.08 1.60 3.5 3 825 1.078 170 2 87 13.45 1.40 7.5 2 695 1.077 142 2 8 11.44 1.30 3.5 1 870 1.067 159 2 90 10.48 1.52 7.5 1 770 1.069 144 2 92 8.62 1.13 5.5 3 710 1.084 155 2 93 8.40 1.25 5.5 3 605 1.080 127 2 95 12.85 1.27 135 2 1095 1.079 228 2 97 15.00 1.40 3.5 1 935 1.07 187 2 98 9.42 1.45 9.5 l 775 1.05 120 2 104 13.25 1.50 5.5 2 500 1.075 100 2 105 17.31 1.40 1.5 2 630 1.068 116 2 109 13.77 1.20 . 3.5 1 1765 1.073 345 2 110 13.23 1.32 3.5 2 930 1.075 186 2 111 11.47 1.20 7.5 3 1340 1.072 259 2 112 12.97 1.40 3.5 2 585 1.073 114 2 113 12.25 1.35 1.5 2 415 1.078 86 3 1 11.85 1.75 2.0 2 1065 . 1.115 304 3 2 8.96 1.55 1.5 3 2330 1.079 485 3 3 13.51 1.70 2.5 5 1080 1.085 239 3 4 11.61 1.13 2.5 3 505 1.086 113 3 5 11.39 1.45 1.5 5 2180 1.082 468 3 6 16.59 1.65 3.5 4. 505 1.074 100 3 7 9.33 1.20 4.5 4 785 1.083 170 3 8 11.94 1.77 4.5 g 410 1.093 98 3 9 11.54 1.75 6.5 685 1.079 143 3 10 p 9.23 1.60 6.0 5 810 1.087 183 3 11 9.16 1.45 1.5 3 950 1.092 224 3 14 9.78 1.75 5.5 1 1450 1.078 299 3 15 11.08 1.55 3.5 4 1180 1.083 256 3 18 14.38 1.80 2.5 3 1030 1.084 226 3 19 10.11 1.85 5.5 5 1065 1.081 226 3 21 13.67 1.50 9.5 1 650 1.092 154 3 22 11.59 1.92 7.5 2 1 00 1.079 271 3 23 10.87 1.25 3.5 2 1 75 1.073 288 3 24 10.77 1.85 3.5 4 520 1.094 125 3 25 13.88 1.72 5.5 3 695 1.094 167 3 26 .10.55 1.65 1.5 2 1390 1.086 310 3 27 11.23 1.65 1.5 3 1660 1.085 367 3 28 10.99 1.65 9.5 3 1180 1.088 269 3 30 12.42 1.55 3.5 3 1450 1.082 311 3 31 13.13 1.65 1.5 1 870 1.087 196 3 32 9.72 1.42 3.5 3 .1000 1.092 236 3 34 11.71 1.97 1.5 2 1250 1.087 282 3 36 10.96 1.77 3.5 4 475 1.092 112 64 Cross Clone Total "av.”me- Chip Rest Fresh Spec. Total # prot. thionine color p. wt. grav. d.m. 3 37 9.99 1.25 5.5 2 840 1.084 184 3 38 12.46 1.80 1.5 '3 740 1.088 168 3 39 9.63 1.45 5.5 3 410 1.079 85 3 40 9.15 1.05 5.5 2 700 1.069 131 3 41 12.08 1.70 3.5 3 920 1.089 211 3 42 12.22 1.62 9.5 4 1220 1.080 257 3 43 9.04 1.55 7.5 4 965 1.084 211 3 45 9.38 1.35 5.5 3 1325 1.07? 270 3 46 11.63 1.50 5.5 3 810 1.080 170 3 47 7.53 1.50 1.5 4 325 1.102 84 3 48 10.01 1.75 2.0 3 790 1.090 183 3 50 9.29 1.37 3.5 3 1005 1.080 211 4 1 13.82 1.65 3.5 3 1495 1.087 337 4 141 10.79 1.47 7.5 5 1230 1.079 256 4 142 12.31 1.05 5.5 2 235 1.093 56 4 145 10.31 1.15 3.5 3 1470 1.077 300 4 147 11.62 1.35 5.5 3 545 1.069 -102 4 148 12.58 1.50 5.5 R 1320 1.084 226 4 149 13.45 1.75 3.5 0 1.073 86 4 154 11.81 1.35 7.5 3 1120 1.082 240 4 156 11.5 1. 5 5.5 2 645 1.084 141 4 159 11.1 1.35 5.5 650 1.083 141 4 160 12.57 1.32 3.5 3 380 1.086 85 4 161 11.11 1.10 5.5 2 575 1.075 115 4 162 12.45 1.30 7.5 3 815 1.079 170 4 163 11.98 1. 5 5.5 2 540 1.080 114 4 165 12.34 1.60 1.5 3 2340 1.083 507 4 166 11.36 1.45 5.5 2 30 1.081 198 4 168 12.11 1.47 5.5 2 30 1.075 86 4 170 11.84 1.17 9.5 1 635 1.067 116 4 180 13.80 1.35 3.5 2 570 1.096 140 4 181 7.50 1. 5 9.5 4 830 1.071 158 4 189 10.37 1.17 3.5 2 2020 1.086 451 4 191 11.76 1.20 7.5 3 2330 1.071 445 4 194 14.20 1. 2 5.5 5 690 1.087 156 4 197 15.17 1.82 1.5 5 1130 1.076 228 4 198 11.83 1.45 3.5 3 1615 1.080 340 4 297 11.77 1.55 1.5 4 590 1.092 139 4 314 12.46 1.45 5.5 $3 910 1.077 186 4 331 11.48 1.40 5.5 2 825 1.078 170 4 333 13.44 1.60 5.5 1 795 1.082 171 4 341 12.26 1.25 1.5 3 780 1.076 157 4 345 11.01 1. 5 5.5 2 785 1.083 170 4 349 10.61 1.45 3.5 3 960 1.091 225 4 357 8.04 1.25 9.5 4 910 1.070 172 4 365 11.25 1.55 3.5 5 770 1.077 157 4 367 12.02 1.60 5.5 3 970 1.078 200 4 370 12.45 1.57 9.5 2 740 1.065 132 65 Cross Clone Total "av.”me- Chip Rest Fresh Spec. Total prot. thionine color p. wt. grav. d.m. 4 383 11.50 1.65 5.5 3 520 1.083 113 4 387 12.47 1.20 5.5 2 1000 1.081 213 4 390 13.86 1.20 3.5 3 1290 1.079 269 4 394 9.52 1.35 5.5 3 1180 1.078 243 5 l 13.49 1.75 3.5 3 1490 1.072 288 5 2 12.44 1.74 1.5 3 1675 1.067 305 5 3 13.13 1.46 5.5 2 945 1.080 199 5 5 13.62 1.67 9.5 , 3 1050 1.065 187 5 6 12.94 1.85 9.5 2 910 1.064 160 5 7 14.28 1.80 2.0 3 1150 1.070 217 5 8 15.78 1.70 3.5 2 1430 1.067 261 5 9 13.60 1.67 3.5 3 400 1.081 85 5 10 16.43 1.31 7.5 2 550 1.068 101 5 11 12.85 1. 0 7.5 3 1355 1.067 247 5 12 12.36 1.88 1.5 2 900 1.071 172 5 1 12.95 1.46 5.5 2 930 1.075 186 5 1 10.53 1.97 9.5 4 1170 1.068 216 5 15 14.67 1.60 1.5 3 1015 1.074 ‘ 200 5 16 8.58 1.49 9.5 2 1315 1.078 271 5 17 13.16 1.32 5.5 2 1010 1.074 199 5 18 13.86 1.63 1.5 4 850 1.076 171 5 19 11.34 1.75 5.5 2 1575 1.071 301 5 20 12.65 1.29 1.5 3 1625 1.071 16 5 21 11.16 1.67 9.5 2 23 0 1.068 32 5 22 16.49 1.79 5.5 g 785 1.054 121 5 2 12.2 0.97 5.5: 535 1.081 114 5 2 15.2 1.38 7.5 5 1050 1.060 176 5 25 10.63 1. 1 9.5 3 770 1.069 144 5 26 11.59 1.22 1.5 4 385 1.085 85 5 27 10.90 1. 1.5 4 520 1.083 113 5 28 9.77 1.22 7.5 5 1680 1.077 343 5 29 13.95 1.67 1.5 3 990 1.076 200 5 30 13.86 1.52 1.5 4 1475 1.073 288 5 31 11.85 1.90 7.5 3 1170 1.068 216 5 32 15.16 1.76 1.5 4 545 1.069 102 5 33 15.23 1.77 5.5 4 910 1.071 174 5 34 13.68 1.50 5.5 l 990 1.076 200 5 35 12.89 1.54 7.5 3 1500 1.071 286 5 36 12.74 1.64 1.5 1 845 1.076 170 5 37 12.78 1.71 9.5 5 1230 1.074 243 5 38 8.84 1.54 7.5 5 2195 1.071 419 5 9 12.26 1.71 7.5 3 1010 1.080 212 5 0 12.05 1.45 7.5 2 1255 1.07 245 6 l 11.05 1.42 9.5 3 1000 1.06 176 6 2 9.23 1.25 9.5 5 1 20 1.065 235 6 3 11.69 1.35 3.5 5 1 0 1.070 276 6 4 11.97 1.30 7.5 2 1145 1.070 216 6 5 14.59 1.25 5.5 3 1000 1.075 200 Cross Clone Total "av.”me- Chip Rest Fresh Total prot. thionine color p. wt. d.m. 6 6 9.00‘ 1.09 7.5 .3 915 173 6 7 14. 56 1.43 1.5 4 590 115 6 8 18. 53 1.25 3.5 3 1000 174 6 9 14. 54 1.52 7.5 3 1165 ~ 205 6 10 12.39 1.15 7.5 5 1525 288 6 11 12. 36 1.35 3.5 3 1320 246 6 12 13. 97 1.35 7.5 3 2300 4 4 6 1- 12.6 1.65 9.5 2 735 1 6 1 15.8 1.45 3.5 4 1400 249 6 15 12.90 1.25 7.5 3 830 158 6 16 11.29 1.30 7.5 2 2070 355 6 17 13.02 1.13 5.5 2 1340 247 6 18 15. 92 1.40 3.5 3 920 174 6 19 11.86 1.36 7.5 1 2230 92 6 20 11. 67 1.27 7.5 3 2120 05 6 21 16.79 1. 0 7.5 3 560 102 6 22 8.57 1.36 9.5 3 1715 . 320 6 2 13.63 1.26 1.5 2 2010 375 6 2 1 .72 1.28 5.5 3 1240 245 6 25 11.53 0.99 9.5 4 1145 216 6 26 12.11 1.25 9.5 3 560 114 6 27 11.56 1.49 5.5 4 850 159 6 28 16. 31 1.09 7.5 3 535 88 6 29 11. 71 1.29 9.5 3 1520 277 6 30 11. 47 1.41 7.5 4 1970 376 6 31 11. 01 1.25 7.5 5 780 157 6 32 12.77 1. 2 7.5 5 1065 226 6 33 14.82 1.27 7.5 3 945 187 6 34 13.86 1.19 9.5 5 360 72 6 35 1 .52 1.37 5.5 4 850 171 6 36 12.68 1.13 7.5 3 595 115 6 37 13.52 1.35 9.5 2 1145 204 6 38 12.98 1.50 3.5 3 940 198 6 9 12.34 1.47 7.5 5 990 187 6 0 11.32 1.37 3.5 3 735 169 7 1 11.26 1.67 7.5 3 780 157 7 2 12.17 1.36 7.5 3 810 121 7 3 13.40 1.63 5.5 5 1425 235 7 4 12.86 1.79 9.5 4 1020 175 7 5 11. 56 1.29 7.5 3 1460 276 7 6 9-99 1-74 7-5 3 990 187 7 7 12.11 1.39 9.5 5 1800 344 7 8 11.06 1.65 9.5 4 1500 251 7 9 13.98 1.73 7.5 2 1095 190 7 10 10.52 1.51 3.5 3 1365 261 7 11 12.07 1.47 1.5 4 990 213 7 12 13.71 1.51 7.5 4 740 119 7 13 13.21 1.73 7.5 3 1010 186 Cross Clone Total "av."me- Chip Rest Fresh Spec. Total prot. thionine color wt. grav. d.m. 7 14 12.55 1.87 5.5 3 750 1.041 95 7 15 15.61 1.77 1.5 2 535 1.059 88 7 16 16.72 1.60 9.5 3 1435 1. 067 262 7 17 16.31 1.68 5.5 4 1010 1.063 175 7 18 12.53 1.42 7.5 5 1890 1.068 349 7 19 14.56 1.69 7.5 3 915 1. 064 161 7 20 11.93 1.64 9.5 a 660 1.056 105 7 21 12. 31 1.61 5.5 - 1315 1.065 234 7 22 13.11 1.61 7.5 4 1435 1.067 262 7 2 13.26 1.40 1.5 2 670 1.081 142 7 2 11. 54 1.58 7.5 2 14 5 1.071 274 7 25 12.72 1.70 7.5 2 1001.061 176 7 26 13.76 1.59 3.5 3 710 1.076 143 7 27 11.57 1.53 7.5 5 1120 1.067 204 7 28 10. 68 1.54 7.5 4 870 1.067 159 7 29 16.20 1.60 1.5 2 975 1.066 176 7 30 11.86 1.79 1.5 3 480 1.079 . 100 7 31 13.37 1.32 5.5 2 940 1.080 198 7 32 12.01 1. 5 9.5 5 810 1.066 146 7 33 13.62 1.60 1.5 3 1065 1.070 201 7 34 17. 59 1.60 5.5 1 2g40 1.066 422 7 35 10. 75 1.06 5.5 3 20 1.079 171 7 36 11. 69 1.32 9.5 5 745 1.064 131 7 37 11. 81 1.50 8.0 5 1315 1.065 234 7 38 10.23 1.70 1.5 6 1355 1.080 285 7 9 12.10 1.24 9.5 3 1080 1.048 153 7 0 12.27 1.55 1.5 2 1255 1. 077 256 8 1 10.81 1.65 5.5 3 1970 1.071 376 8 2 12.05 1.46 7.5 4 890 1. 066 160 8 2 12.14 1.34 5.5 3 1405 1. 073 274 8 16.60 1.47 5.5 2 1340 1. 059 221 8 5 13.78 1.28 3.5 3 1095 1. 068 202 8 6 11.76 1.65 5.5 2 1900 1.064 334 8 7 11.02 1.84 8.0 3 615 1. 069 115 8 8 12.63 1.72 4.5 5 1535 1. 077 313 8 9 13.56 1.20 4.5 2 2010 1. 066 362 8 10 13.41 1.27 2.5 3 900 1. 078 185 8 11 11. 81 1.51 4.5 5 1375 1. 062 236 8 12 14.46 1.41 5.5 3 810 L 073 158 8 13 11.49 1.24 8.5 3 1000 1. 064 176 8 14 12.52 1.36 6.5 6 1230 1. 060 206 8 15 11.98 1.24 4.5 3 1455 1.062 250 8 16 11.29 1.52 5.0 3 1050 1.071 200 8 17 10.18 1.22 8.5 6 1640 1.055 257 8 18 12.96 1.11 9.5 2 1365 1.054 211 8 19 9. 65 1.31 6.5 4 720 1.083 156 8 20 13. 86 1.49 7.0 5 610 1.061 103 68 Cross Clone Total ”av.”me- Chip Rest Fresh Spec. Total # # prot. thionine color p. wt. grav. d.m. 8 21 13.12 1.29 3.5 5 1325 1.064 233 8 22 11.79 1.78 .5 4 2135 1.066 395 8 23 13.83 1.37 5.5 3 18 0- 1.064 324 8 2 12.85 1.20 5.5 2 1810 1.071 346 8 25 11.14 1.35 5.5 3 1850 1.066 33 8 26 12.45 1. 0 5.5 4 1500 1.064 26 8 27 13.57 1.35 5.0 O 895 1.065 159 8 28 11.77 1. 6 3.0 2 1340 1.076 270 8 29 10.64 1.31 5.5 4 1240 1.069 231 8 30 12.13 1.27 5.5 4 715 1.075 143 8 31 9.68 1.44 5.5 5 600 1.081 128 8 32 11.77 1.35 5.5 3 820 1.072 158 8 33 10.13 1.61 5.5 5 2010 1.066 362 8 34 10.54 1.24 8.0 3 2340 1.059 386 8 35 7.77 1.20 5.5 3 1165 1.074 230 8 36 8.04 1. 7 7.5 5 1165 1.069 217 8 37 13.22 1.43 4.5 4 1105 1.068 204 8 38 15.16 1.33 6.5 3 1845 1.060 ‘309 8 9 11.69 1.71 6.5 3 1505 1.067 274 8 0 12.36 1.47 3.5 4 2240 1.074 442 B. DATA OF THE PARENTAL CULTIVARS Cultivar Hill Sample Total "av." me- Specific # # of hill prot. thionine gravity 320- 6 1 B 8.79 1.03 1.118 320- 6 2 A 9.67 1.09 320- 6 2 B 10.41 1.10 1.124 320- 6 3 B 13.15 0.95 1.103 320- 6 6 A 10.35 1.02 320- 6 6 B 10.00 1.13 1.099 320- 6 8 A 9.94 1.05 320- 6 8 B 9.41 0.98 1.117 320~ 6 9 A 13.03 1.19 321-38 2 A 12.01 1.07 321-38 5 A 15. 52 1.08 321-38 5 B 15.55 1.17 1.074 321-38 6 A 15.81 1.10 321-38 6 B 17.89 1.09 1.071 321-38 7 A 16.64 1.12 321-38 7 B 15.63 1.17 1.080 321-38 8 B 14. 46 1.14 1.094 321-70 1 A 12.17 1.22 321-70 1 B 13. 25 1.21 1.089 321—70 2 A 16.78 1.11 321-70 2 B 13. 74 1.10 1.094 321-70 3 A 12.56 1.14 321-70 3 B 14.17 1.20 1.096 321¥70 4 A 13 75 1.08 321-70 4 B 16.86 1.14 1.111 321-70 5 A 17.98 1.28 321-70 5 B 18.86 1.31 1.082 69 70 Cultivar H111 Sample Total ”av.” me- Specific # # of hill prot. thionine gravity 321-70 6 B 12.29 1.10 1.100 321-70 7 A 14.27 1.08 709 1 A 13.43 1.38 709 l B 1 .10 1.50 1.078 709 2 A 13.92 1.51 709 2 B 15.46 1.38 1.079 709 3 A 14.00 1.54 709 3 B 16.07 1.43 1.073 709 4 A 15.62 1.52 709 4 B 15.80 1.55 1.073 709 5 A 1 .19 1.40 709 5 B 1 .24 1.38 1.076 709 6 A 15.78 1.44 709 6 B 15.70 1.40 1.0765 709 7 A 15.10 1.43 709 7 B 16.02 1.50 1.077 706-34 2 A 12.74 1.17 706-34 5 A 13.32 1.17 706-34 5 B 1 .76 1.16 1.062 706-34 7 A 12.33 1.05 706-34 7 B 12.54 1.05 1.059 706-34 8 A 9.84 1.05 706-34 9 A 17.70 1.25 706-34 9 B 13.10 1.17 1.072 706-34 10 A 14.38 1.15 735- 1 1 A 10.22 1.04 735- 1 2 A 10058 1.14 735- 1 2 B 10.59 1.12 1.082 735- l 3 A 12.04 1.22 735- 1 g B 10.46 1.25 1.078 735- 1 A 10.41 1.07 735- l 5 A 9.80 1.23 735- 1 5 B 10.73 1.27 1.066 735- 1 6 A 11.08 1.13 735- 1 6 B 10.29 1.09 1.067 Cultivar Hill Sample Total ”av." me- Specific #‘ # of hill prot. thionine gravity 735- 1 7 B 11.17 1.00 1.071 711- 8 1 A 12.04 1.45 711' 8 1 B 16076 10““ 10068 711- 8 2 A 15.95 1.52 711- 8 3 A 1 .01 1.57 711- 8 3 B 1 .54 1.63 1.067 711- 8 4 A 17.42 1.49 711- 8 4 B 15.97 1.44 1.070 711- 8 5 A 18.47 1.44 711- 8 5 B 15.67 1.50 1.065 711- 8 6 A 13.21 1.54 711- 8 6 B 1 .88 1.45 1.069 711- 8 7 A 17.91 1.43 321-65+ 9.00 1.45 1.111 + mean values from the 1969 crop of the same field "I44444444