The role of high lysine cereals in animal and human nutrition in Asia - S. K. Vasal

S. K. Vasal
The International Maize and Wheat Improvement Center
(CIMMYT)-Mexico

INTRODUCTION

Cereals play an important role in world agriculture. They contribute significantly to the global food pool in achieving food and nutritional security. Considering the area sown and annual production volume, they occupy an important position in the world economy and trade as food, feed and industrial grain crops. In 2000, the area harvested was roughly 675 million hectares which produced 2059.8 million tonnes with an average yield of 3049 kilograms per hectare (Table 1). As can be seen, wheat, rice and maize are of prime importance but area and production from other crops such as barley, sorghum, oats, rye and millet are also quite significant. It may be noted that maize has a great potential for yielding more per unit of land area than other cereals.

TABLE 1
World cereal statistics; area, yield and production in 2000

Source: FAO

In Asia, the area devoted to cereals was 301.8 million hectares with a production volume of 938.8 million tonnes (Table 2). This is almost 50 percent of total world cereal production. Rice is the most important crop in Asia occupying almost half of the cereal area, with a production of paddy rice touching 540 million tonnes. The other two important crops are wheat and maize, which rank second and third respectively. Other crops of importance with significant area are barley, sorghum and millets. Oats and Rrye are also grown but their area is quite small, less than one million hectares annually being sown to each crop.

TABLE 2
Asian cereal statistics; area, yield and production in 2000

Source: FAO

Some of the cereal crops, particularly rice, wheat and to some extent maize, sorghum and millet are consumed by humans as staple foods to meet energy and protein requirements. Feed use of cereals in Asia is more in some countries than others, but at least 158 million tonnes were used in 2000 for livestock (Table 3). Food and feed use of cereals will be greatly prioritized in future in view of projected world population growth of 80 million people every year. Unfortunately much of the increase in population will take place in the developing countries mostly concentrated in South Asia. It is expected that demand for food and meat products will increase dramatically in the next two decades. A demand driven livestock revolution is underway in Asia and it is very likely that demand for meat and other animal products may almost double by 2020. This in turn will increase demand of cereals for feeding livestock. The demand for some cereals such as maize will increase more rapidly, and will perhaps overtake demand for rice and wheat in the next two decades.

TABLE 3
Feed use of grains in Asia

Cereal proteins vary in protein content but in general are of poor quality because of a lack of balance in amino acid composition. Breeding for improved amino acid composition has been attempted in some crops and commercially exploitable high lysine varieties are now available, at least in maize. This paper will discuss development efforts in improving protein quality in different crops, as well as their future role in livestock and human nutrition.

PROTEIN RELATED NUTRITIONAL CHARACTERISTICS OF CEREALS GRAINS

The crude protein content varies in different crops (Table 4). Rice is quite low in protein (7 percent). Intermediate levels of 9-10 percent are encountered in maize, sorghum and barley. Wheat, oats and triticale exhibit a high protein content of 12 percent and more. In general high protein content is inversely correlated with yield.

TABLE 4
Protein and lysine content of cereal crops

In wheat and oats, however, high protein lines with good yielding ability are available. As far as protein quality is concerned, unfortunately, all cereals are deficient primarily in lysine with a secondary deficiency in threonine or tryptophan (Table 5).

TABLE 5
Limiting amino acids in cereal protein

The poor quality of proteins is attributed to a high concentration of prolamin storage protein fraction in cereals. This particular fraction is practically negligible or devoid of lysine. The high level of this fraction is the sole cause of poor protein quality in cereals. The prolamin contents of major cereals fall into three distinct classes or groups (Table 6). The high prolamin group constitutes 50-60 percent of protein, as is the case in maize and sorghum, intermediate 30-40 percent as in barley and wheat and the low prolamin group with only 5-10 percent as in rice and oats. The protein quality of cereals, like protein quantity, is inversely related to the protein content. Those groups of cereals such as rice and oats, which have low prolamin content, thus exhibit superior protein quality. It may be pointed out that prolamin is one of the four protein fractions which make up cereal protein. The other three fractions are albumins, globulins and glutelins and are soluble in water, saline solution and alkali solution, respectively. The prolamins being soluble in alcohol are rich in proline and glutamine, but are low in basic amino acids including lysine. Osborne and Mandel (1914) showed that rats of all ages went into rapid decline and eventually died if placed on a diet in which zein was the sole source of dietary protein. The prolamin fraction is named differently as is zein in maize, gliadin in wheat, kafarin in sorghum, hordein in barley, and avenin in oats. As indicated earlier, both oat and rice have good protein quality owing to low levels of prolamin. Despite high lysine in these two cereals compared to others, lysine is still the first limiting amino acid. Proteins from both these cereals have higher biological value relative to other cereal proteins. It is further interesting to point out that high protein content in oat does not adversely affect the biological value of protein.

TABLE 6
Prolamin content of major cereals

BREEDING EFFORTS FOR IMPROVING PROTEIN QUALITY IN CEREALS

People in the developing countries, particularly in Asia, consume cereal grains as staple food and derive their calories and protein requirements from such cereals. Nutritional improvement in such cereals through plant breeding efforts have been under active consideration for the past several decades but realistic breeding efforts could not be taken up in the absence of specific genes for such traits. Altering the amino acid profile of cereal proteins and making them more balanced will impact hundreds of millions of people without altering their food habits and preferences.

Maize

To start with, germplasm accessions were screened for genetic variability for lysine content. Variation was observed in maize but differences were rather small. It would have needed many years to elevate levels sufficiently to make the protein profile reasonably balanced in manifesting superior biological value. The protein quality therefore remained a concern but no immediate solutions were in sight and no good breeding options could be deployed at that time to affect improvements. A beginning in genetic manipulation of protein quality began with the discovery of high lysine mutant opaque-2 (o2) (Mertz et al., 1964) and a year later another mutant floury-2 (Nelson et al., 1965) was discovered by Purdue University researchers. These exciting discoveries generated a lot of enthusiasm and hopes, and paved the way for improving protein quality in maize. Of interest is the fact that these mutant alleles changed protein quality of endosperm and not that of germ. These mutants were able to alter the amino acid profile of maize endosperm protein resulting in a two fold increase in the levels of lysine and tryptophan compared to normal genotypes. The phenotype of the mutants was easily recognizable from their soft chalky appearance. Alterations were noticed in other amino acids as well. An increase was observed for amino acids such as histidine, arginine, aspartic acid and glycine and a decrease in glutamic acid, alanine and leucine. Leucine:isoleucine ratio was improved and became better balanced, which in turn is considered beneficial as it helps to liberate more tryptophan for more niacin biosynthesis, thus helping to combat pellagra. These mutants bring about improvements in lysine and tryptophan by suppressing lysine-deficient zein fraction without altering the contribution of other fractions. A reduction in zein fraction causes proportional elevation of other fractions rich in lysine, thus resulting in elevation of these two amino acids in protein, but not on an absolute basis of per unit of endosperm in the grain.

The search was continued for new and better mutants, but the ones found (o7, o6, fl3) were in no way better than opaque-2. Breeding efforts were thus initially concentrated on opaque-2 and floury-2. Since floury-2 did not hold its promise it was dropped in the early 1970s. High quality protein materials developed using o2 did not show competitive performance compared to their normal counterparts. They suffered from a number of problems including lower grain yield, unacceptable soft chalky endosperm, slower drying, more vulnerable to ear rot pathogens and to stored grain pests. These agronomic deficiencies were serious enough to cause a decline in interest and even a complete abandoning of efforts in many programmes. Only a few institutions such as CIMMYT, Purdue University, Crows Hybrid Seed Company in Milford, Illinois, and University of Natal in South Africa sustained their efforts, choosing different options to develop normal looking agronomically acceptable varieties and hybrids. The success of approaches deployed at CIMMYT and the germplasm developed will be described in detail in a later section.

Barley

Discoveries of nutritionally superior mutant alleles o2 and fl2 in maize stimulated interest in other cereal crops. Screening efforts to identify similar types of mutant alleles as in maize were initiated in Sweden and Denmark. A high-lysine gene (Hily) was identified from the Hiproly source (Munck et al., 1971) and another gene Riso 1508 was identified in Denmark (Doll and Koie, 1975; Ingverson et. al., 1973). The latter mutant showed simple recessive inheritance and had 40 percent increase in lysine content. Both mutants suffered from agronomic defects. There was a reduction in seed size and also a reduction in yield. In feeding trials, Ris 1508 or hily Hiproly barley produced optimal growth of pigs without addition of protein or amino acid supplements. It may be added that normal barleys are intermediate between maize and sorghum on the one hand and rice and oats on the other. Again because of agronomic problems, widespread efforts in improving protein quality did not result in a positive outcome.

Sorghum

Thousands of accessions were screened for high lysine mutants in sorghum. Two mutants, 15-11167 and 15-11758 were identified from the Ethiopian world sorghum collections (Singh and Axtell, 1973). Later an induced mutant P721 was reported (Mohan and Axtell, 1975). The mutant allele P721 appeared to be partially dominant and had a 60 percent increase in lysine over the normal. The lysine in normal was 2.11 percent as against 2.88 percent in high lysine. P721 had soft phenotype and had reduced yield. It behaved differently in different genetic backgrounds and only in a few did yield appear to be satisfactory. Converted materials using this gene had poor acceptance because of soft kernels. Modified vitreous types have also been encountered (Ejeta, 1979) but work was not pursued rigorously. Ethiopian high lysine sorghums are proposed for for use as weaning food pending conformation of the fact that digestibility is acceptable.

Rice

Milled rice is low in protein concentration (7 percent). It contributes 40-80 percent of the calories and at least 40 percent of the protein in Asian diets. Rice has good quality protein despite its poor concentration. A lot of work has been done over the past five decades at IRRI to improve protein content and quality in rice. The Researchers concluded after many years of work that there is some hope and prospect of further improving the lysine concentration in rice protein (Coffman and Juliano, 1979). Improvement for protein concentration appeared to be a good possibility, but results so far have been disappointing as witnessed by the lack of high protein rice cultivars.

Rice protein consists mostly of glutelin (80 percent), prolamin (less than 5 percent), albumin (5 percent) and globulin (10 percent). It is of interest to point out that albumin and globulin are concentrated in the aleurone layers. The lysine content of different fractions is glutelin (3.47 percent lysine), albumin (4.92 percent lysine), globulin (2.56 percent lysine) and prolamine (0.51 percent lysine). Bran and embryo proteins are mainly albumin proteins and are rich in lysine.

Rice has more lysine and better biological value compared to other cereals (Coffman and Juliano 1979; Khush and Juliano 1984; Tanaka 1983; Frey 1977).

Oat

Oat ranks fifth in the total production following wheat, rice, corn and barley. It is mainly used for animal feed. Oat protein has good protein concentration and has excellent balance of amino acids (Robbins et al., 1971). Its protein quality and biological value is maintained even at higher protein concentrations. Genetic enhancement and manipulation for higher protein content is possible and commercial cultivars having 20 percent protein have been developed (Briggle, 1971). High yield has no adverse effect on protein content. A few high protein cultivars - Dal, Goodland, Marathan and Wright developed in Wisconsin have two-three percent increase in groats protein.

Wheat

This is chiefly used as food and its use as feed is less important. Surpluses are sometimes fed to livestock. Despite extensive research efforts, the high lysine mutants have not been encountered. There are better prospects of increasing protein content and lines exceeding 12 percent have been isolated. From by-products of wheat milling, as much as 28 percent of the grain, mainly in the form of bran and shorts, finds its way into mixed livestock feeds.

Triticale and Rye

These are mostly used as feed for livestock. Triticale has improved protein content and quality and so continues to generate optimism as a potential feed source.

QUALITY PROTEIN MAIZE SUCCESS STORY

As pointed out earlier, CIMMYT scientists used opaque-2 gene because no other genes offered any greater advantage. In the beginning emphasis was on developing soft endosperm cultivars. As the agronomic problems mentioned earlier became obvious, several different options were tried which could result in acceptable quality protein maize germplasm. These approaches are described in several CIMMYT publications and journal articles (Byarnason and Vasal, 1992; Vasal et al., 1984; Vasal et al., 1980; Vasal et al., 1979; Vasal, 1994; Vasal, 2000). Only one approach appeared promising which could resolve all problems confronting soft opaques and result in high-quality protein materials with acceptable yield performance, kernel phenotype and low vulnerability to ear rots and stored grain pests. The approach involved use of two genetic systems involving the opaque-2 gene and the genetic modifiers of opaque-2 locus. Using this approach, the initial emphasis was on developing hard endosperm opaque-2 donor stocks. Subsequently these donor’s stocks were used to convert normal maize materials to hard endosperm opaque-2. In addition several broad based gene pools were formed. By late 1978, a huge volume of quality protein maize germplasm was developed with normal looking kernel phenotype.

Merging and reorganization was attempted at this point to form a fixed number of pools and populations for systematic handling and improvement (Vasal, 1994, 2000). In all, 10 populations and 13 Quality Protein Maize (QPM) pools resulted from this effort. In the mid 1980s QPM hybrid effort was initiated. Problems were overcome and progress was attained in most traits deficient in original soft opaque-2 materials. International testing of QPM varieties and hybrids has been extensive and the results have been extremely encouraging. Several countries have identified varieties or hybrids which are competitive and are either equal or better than the best normal checks included in the trials (Table 7). Also during the mid 1990s, 55 QPM inbreds were announced and made available to public and private sectors. In the past four years at least 22 countries have released QPM materials, including China, India and Vietnam (Table 8). Successful field days were conducted in most of the countries releasing the hybrids. In many instances, high ranking politicians attended the ceremonies. There is enthusiasm and hope of covering more area under QPM in the coming years.

TABLE 7
Superior white QPM hybrids tested across fifteen locations at El Salvador, Guatemala and Mexico, 1998

Local checks: HB-83, CB-HS-5G, H-59, XM7712, GUAYOPE

TABLE 8
Recent releases involving CIMMYT germplasm

FOOD AND FEED USE OF CEREALS

Cereals are consumed principally as food for humans and feed for livestock. Total production of cereal grains in 2000 was 1870 million tonnes compared with 1581 million tonnes in 1978. It is estimated that 34 percent of the world’s grain crop is used to feed livestock raised for meat. For humans, cereal grains provide a major portion of calories and protein needed in the diet. Today the world obtains about 50 percent of its dietary protein from cereals, about 20 percent from legumes and 30 percent from animal products (Oram and Brock, 1972). In developing countries, people obtain about 26 percent of their protein from animal products and the remaining two-thirds from cereals. In contrast, people from the developed world meet 56 percent of their protein requirement from animal products.

Feed use of cereals has been steadily increasing. On a worldwide basis, roughly one-third of grain crops are used for feeding livestock. The feed use of cereals in Asia totaled 158.1 million tonnes. China was the largest user (103. million tonnes) followed in order of their use by, Japan (15.9 million tonnes), India (8.0 million tonnes), South Korea (7.6 million tonnes) and Taiwan (5.0 million tonnes). Maize use as feed is quite large in Asia and perhaps exceeds 50 percent of total production.

The consumption of meat and milk has grown many fold in the developing countries, at least in the past 3 decades. The total meat consumption in the world has risen from 139 million tonnes in 1983 to 184 million tonnes in 1993. This is projected to increase to 303 million tonnes by 2020. The meat consumption in developing countries increased from 50 in 1983 to 88 million tonnes in 1993, and the projected consumption for 2020 is 188 million tonnes. Between the mid 1970s and the mid 1990s, the consumption of meat in the developing countries grew almost three times as much as it did in the developed world (Pinstrup-Andersen et al., 1999). Consumption grew at an even faster rate in the second half of this period, with Asia in the lead (Delgado et al., 1999). Future projections suggest that meat and milk in the developing countries will grow by between 2.8 and 3.3 percent per year between the early 1990s and 2020. The corresponding developed world growth rates were 0.6 and 0.2 percent per year.

High lysine cereals in human nutrition

Most cereals have lysine as the first limiting amino acid. Naturally occurring high lysine cereals are rice and oats. The lysine values range from 3.5 to 4.0 percent in protein. Despite high lysine values, the first limiting amino acid in both cereals is lysine. As discussed earlier, conscious effects to further increase the levels of lysine have not yielded positive results. In respect of protein, rice is quite low (seven percent) but oat protein content is reasonably high. Here again breeding efforts aimed at increasing protein content in rice have not been very successful, but the prospects of developing high protein oats without sacrificing lysine are quite good. Because of high lysine values both rice and oat have demonstrated higher biological value relative to other cereals (Coffman and Juliano, 1979; Khush and Juliano, 1984; Tanaka, 1983; Frey, 1977).

Rice will continue to be a staple diet of at least half of the world’s population. Compared to all other cereals, oat grain combines the advantage of both protein content and quality and its use as a human food will increase, even though its major use is presently as feed grain. Rice will continue to be an important cereal for food and has the advantage of being high in protein quality despite being low in its concentration.

In the remaining crops, maize, sorghum, barley and millet, the protein quality is not good while the protein quantity is in the range of 9-10 percent in the whole grain. Except maize, the nutritional improvements for improved amino acid composition through breeding efforts have not been successful, so the benefits of nutritionally enhanced characteristics in sorghum, barley and millet cannot be harnessed by people and tribes consuming such cereals. The use of high lysine sorghum could be advocated as weaning food, as is the case in Ethiopia. The high lysine types are easily recognizable because they are somewhat dented. Farmers could produce high lysine sorghum grain as a protein source for weaning children and for pregnant and nursing mothers. Sorghum flour is quite indigestible by the infants, so more studies are needed before it can be recommended as a weaning food.

QUALITY PROTEIN MAIZE FOR HUMAN NUTRITION

In maize, the development of QPM has turned out to be a success story. It has similar agronomic performance, appearance and taste as the normal maize. It has a reduced prolamin fraction (25-30 percent) but elevated levels of other fractions such as glutelins, albumins and globulins. There is a two-fold increase in the levels of lysine and tryptophan with high digestibility and biological value. QPM has a balanced leucine:isoleucine ratio and thus an enhanced production of niacin to help overcome pellagra. QPM is like eggs and milk, both low in niacin, but they offer protection from pellagra because their proteins contain high levels of tryptophan. Compared to skim milk, the nutritional value of QPM is about 90 percent. It meets the requirements of pre-school children for their protein needs. In countries or communities where low protein and tuber crops make up an infant’s diet, QPM offers better prospects. There is a tendency for increased nitrogen retention when a switch over from normal to QPM is made. It should in turn translate into body weight, stature and protection from protein deficiency illnesses. Clinical studies conducted in hospitals have demonstrated that QPM can give preventative help and cure of severe protein deficiency disease (Kwashiorkor) in young children by simply using it as the only source of protein (Pradilla et al., 1973). QPM could be a great weaning food when used alone in maize diets. Substitution of normal maize with QPM will produce more benefits. QPM could be really helpful in catch-up growth, particularly in the malnourished and those who are sick, especially after diarrhoea.

QPM could have a role in improving birth rates. In addressing problems of infant mortality due to low birth weight, QPM fed to pregnant women could raise the chances of child survival. Poorer sections of society lacking resources to buy milk could rely on low cost QPM to provide very similar benefits (Singh and Jain, 1977). QPM could also be a better alternative for those groups who are unable to eat bulk food, even if it is available, as is the case in infants and children. A diet solely based on QPM is regarded adequate in meeting the energy and protein needs of infants and children (Graham et al., 1980, 1990). It is believed that QPM should be a good measure for infants and young children (ranging from three months to three years in age) to reduce mortality and improve growth rates. Studies on adults using QPM are limited, but there are indications that QPM is more efficient than normal corn in supplying the protein requirements of adults (Clark, 1966; Clark et al., 1977). QPM can also provide a high amount of usable protein as energy, 8.3-9.6 percent when a value of 8 percent is considered adequate for a one-year old child. Carotenoids, the coloured plant pigments which are precursors and give rise to vitamin A in the body, are better utilized in QPM compared to normal maize. From limited studies on humans and animals, it is well demonstrated that it has high biological value (BV), high digestibility and better food efficiency (g food intake/g weight gain). In defining an exact and further role of QPM in human nutrition, additional studies are needed to make nutritional and economic assessments.

Value of high-lysine cereals (QPM) in animal nutrition

A variety of animals have been used in demonstrating the superior performance of QPM compared to normal maize used alone or in combination with different food rations. It is fair to say that QPM has great potential in monogastric animals such as rats, chickens and swine. In experiments carried out in about the last three decades, there is clear evidence that QPM is a better feed than normal maize because its proteins are well balanced. Other advantages and roles of QPM could be seen in substituting it for high protein costly supplements like soybean or fish meal.

Feeding trials showed that rats fed on opaque-2 compared with normal maize exhibited a three to six fold increase in body weight. Bressani obtained similar results with rats in Guatemala. They also exhibited a greater food intake (162 for QPM compared with 130.5 for normal maize) and a better feed conversion efficiency (7.0 in normal compared with 9.4 in QPM).

In feeding chickens, QPM could play a much greater role because of increasing demand for poultry in several countries of Asia. In poultry feeding some special considerations must be kept in mind. Growing chicks need high protein and high methionine content diets. With only methionine supplementation, the opaque-2 fed chickens grew faster than those fed on normal maize, and produced better live weight gain and feed conversion, even at below optimal protein levels. Feed efficiency results obtained from Guatemalan trials were quite striking. The feed efficiency ratio for QPM and normal maize was 3.5:1 and 8.2:1 respectively. From limited studies that are available in Guatemala, one may conclude that QPM has great promise for feeding poultry if supplemented adequately with methionine.

Field demonstrations of QPM on swine have produced striking and convincing results. Thus pigs can be used as model animals in demonstrating the value of this special maize. For swine, QPM can be fed as the only source of protein during finishing, gestation and pre-gestation periods without reducing growth (Maner, 1975). In Colombian trials, pigs fed QPM grew 3.5 times faster than on normal maize when maize was the sole protein source. Since protein in QPM is not concentrated, it is advisable to add or mix with some supplement. Animals gain weight faster than humans especially during the early growing period. Piglets and rats, for example, put on 10 percent of their body weight per day. In contrast, an infant puts on only one percent/day of its body weight. It is therefore recommended that for growing pigs of all ages or lactating sows, opaque-2 corn must be supplemented with extra protein to produce optimum and maximum performance. The dramatic effects of QPM have been demonstrated in other countries such as Guatemala, China, Vietnam and Kenya. In Guizou province of China, feeding QPM within pig raising systems transformed the livelihoods of the poorest people in the poorest province. From the foregoing it may be concluded that rearing and production of pigs and chickens can be carried out more efficiently on QPM, and indirectly this will improve human diets by providing more meat and eggs.

The expanded demand for meat and other animal products has witnessed unprecedented growth. In the next two decades the growth is likely to continue at the rate of 3.3 percent per year. The demand for feed will thus rise rapidly and will have to be met by cereals which have potential for increased productivity and improved nutritional value through better feed efficiency. Maize will certainly play a dominant role, and QPM will have the added advantage of being superior in protein quality and higher in feed efficiency.

REFERENCES

Bjarnason, M. & Vasal, S.K., 1992. Breeding of quality protein maize (QPM) In J. Jankick, ed. Plant Breeding Review., Janick, J., ed., p. 181, 1992.

Briggle, L. W. 1971. Improving nutritional quality of oats through breeding. Agronomy Abstracts., p. 53. 1971.

Clark, H. E., Glover, D. V., Betz, J. L., & Bailey, L. B., 1977. Nitrogen retention of young men who consumed isonitrogenous diets containing normal, opaque-2, or sugary-2 opaque-2 corn. Journal of Nutrition. 107: 404, 1977.

Clark, H. E., 1966. Opaque-2 corn as a source of protein for adult human subjects, In E.T. Mertz & O.E. Nelson, eds. Proceedings. of High Lysine Corn Conference, p. 40., West Lafayette, IN, Mertz, E. T. and Nelson, O. E., Eds.,Washington, DC, Corn Refiners Association Inc., Washington, D. C., 40, 1966.

Coffman, W. R. & B. O. Jualiano, B. O. 1979. Seed protein improvement in rice: Status Report, pp. 261-75. In Cereals and Grain Legumes. Proceedings. of Symposium. On Seed Protein Improvements. In Cereals and Grain Legumes,. Neuherberg, Federal. Republic. of Germany, 4-8 Sept. 1978.

Delgado, C., Rosegrant, M., Steinfeld, H., Ehui, S., & C. Courbois, C. 1999. The Next Food Revolution. Chapter 14. In Livestock to 2020, Chapter 14.IFPRI. 1999.

Doll, H., & B. Koie, B. 1975. Evaluation of high lysine barley mutants. In Breeding for seed protein improvement using nuclear techniques, p. 55-59. Vienna, IAEA., Vienna, pp.55-59, 1975.

Ejeta, G. 1979. Selection for genetic modifiers that improve the opaque kernel phenotype of P-721 high lysine sorghum (Sorghum bicolor [L.] Moench). Lafayette, Ind. USA. Ph. D. thesis, Purdue University., Lafayette, Ind., 1979 (Ph.D. thesis).

Frey, K.J. 1977. Proteins of oats. Z. Pflanzenzucht,. 78: 185-215, 1977.

Graham, G. G., Glover, D. V., Romaña, G. L., Morales, E., & Maclean, W. C., 1980. Nutritional value of normal, opaque-2 and sugary-2, opaque-2 maize hybrids for infants and children. I. Digestibility and utilization., Journal. of Nutrition., 110:, 1061, 1980.

Graham, G. G., Lembcke, J., and Morales, E., 1990. Quality protein maize as the sole source of dietary protein and fat for rapidly growing young children., Pediatrics, 85:, 85, 1990.

Ingverson, J., B. Koie, B. & H. Doll, H. 1973 Induced seed protein mutant of barley. Experientia 29:1151-52, 1973.

Khush, G.S., and B.O. Juliano, B.O. 1984. Status of rice varietals improvement for protein content at IRRI. P. 199-202, 1984. In Nuclear techniques for cereal grain protein improvement. Proceedings. of Research. Coordination. Meeting., Vienna. IAEA, Vienna, 6-10 Dec. 1982.

Maner, J.H. 1975. Quality protein maize in swine nutrition. In High-quality protein maize. p. 58-82. Stroudsberg, PA, USA. Hutchinson Ross Publishing Co., Stroudsburg, PA. p. 58-82, 1975.

Mertz, E. T., Bates, L.S., and Nelson, O. E., 1964. Mutant gene that changes protein composition and increases lysine content of maize endosperm., Science, 145:, 279, 1964.

Mohan, D. P., and J. D. Axtell, J.D. 1975. Diethyl sulfate induced high lysine mutant in sorghum. Paper presented at Ninth Biennial Grain Sorghum Research. And Utilization. Conference., Lubbock, TXex., USA, 4-6 Mar. 1975.

Munck, L., K. E. Karlsson, K.E. and A. Hagberg, A. 1971. Selection and characterization of high protein lysine variety from the world barley collection. In R. Nilan, R. (ed.) Barley genetics II., p. 544-558. Washington, DC, Pullman, Wash., pp. 544-58, 1971.

Nelson, O.E., Mertz, E. T., and Bates, L.S., 1965. Second mutant gene affecting the amino acid pattern of maize endosperm proteins,. Science, 150:, 1469, 1965.

Oram, R. N., and R. D. Brock, R.D. 1972. Prospects for improving plant protein yield and quality by breeding. Journal. of the Australian. Institute. of Agricultural. Science. 38: 163-68. 1972.

Osborne, T.B. and Mendel, L.B., 1914. Amino acids in nutrition and growth., Journal of Biological. Chemistry.,17:, 325, 1914.

Pinstrup-Andersen, P., R. Pandya-Lorch, R. and M. W. Rosegrant, M.W. 1999. World food prospects: Critical issues for the early twenty-first century. Washington, DC, IFPRI 2020 Vision Food Policy Report, Washington, D. C., 1999.

Pradilla. A.G., C.A. Frances, C.A. and F.A. Linares, F.A. 1973. Studies on protein quality of flint phenotypes of modified maize. Arch. Latinoam. Nutrition. 23: 217-223. 1973.

Robbins, G. S., Y. Pomeranz,Y. and L. W. Briggle, L.W. 1971. Amino acid composition of oat oats. Agricultural. Food Chemistry,. 19: 536-39., 1971

Singh, J., and H. K. Jain, H.K. 1977. Studies on assessing the nutritive value of opaque-2 maize. New Delhi, Indian Agricultural. Research. Institute., New Delhi, 1977.

Singh, R., and J. D. Axtell, J.D. 1973. High lysine mutant gene (hl) that improves protein quality and biological value of grain sorghum. Crop Science., 13: 535-539. 1973.

Tanaka, S. 1983. Seed proteins of rice and possibilities of its improvement through mutant genes. In W. Gottchalk and H.P. Muller (eds.) Advances in agricultural biotechnology: Seed proteins: biochemistry, genetics, nutritive value. p. 225-244. The Hague, Nijhoff, Junk The Hague. P. 225-244, 1983.

Vasal, S. K. 2000. Quality Protein Maize Story. Food and Nutritional Bulletin, Vol. 21(4), No. 4: 445-450, 2000.

Vasal, S.K. 1994. High quality protein corn. In: A.R. Hallauer (ed.), Speciality corns. p. 80-121. CRC Press, Boca Raton, Fl., USA. CRC PressP. 80-121, 1994.

Vasal, S.K., Villegas, E., and Bauer, R., 1979. Present status of breeding quality protein maize., In Seed Protein Improvement in Cereals and Grain Legumes, p. 127. Vienna, IAEA, Vienna, 127, 1979.

Vasal, S.K., Villegas, E., Bjarnason, M., Gelaw, B., and Goertz, P., 1980. Genetic modifiers and breeding strategies in developing hard endosperm opaque-2 materials., In W.G. Pollmer and R.H. Phipps, eds. Improvement of Quality Traits of Maize for Grain and Silage Use, p. 37. The Hague, Pollmer, W.G. and Phipps, R.H., Eds., Nighoff, The Hague, 37, 1980.

Vasal, S.K., Villegas, E., Tang, C.Y., Werder, J., and Read, M., 1984. Combined use of two genetic systems in the development and improvement of quality protein maize., Kulturpflanze, 32:, 171, 1984.