Background

Maize (Zea mays L) is the queen of cereal crops with highest grain yield potential and high fodder production capacity owing to its C4 type carbon fixation. It accounts 10% to the national food stock, contributes significantly to the agriculture GDP and paves the way for huge employment. It is grown in wide range of agro-ecological environments. India ranked fifth next to USA, China, Brazil and Argentina in terms of maize production. Consumption wise it is used as poultry feed 52%, human food 24%, animal feed 11% and more than 22% is diverted towards industrial processing. Production of maize was about 12 million tonnes in early 2000s and it was increased to around 22 million tonnes in 2014. However, India is far behind in productivity (2.5 metric tonnes/ha) which is less than half of the world average (5.5 metric tonnes/ha) due to its cultivation mainly in rain-fed conditions with inadequate irrigation, quality seed and other inputs. It has been estimated to produce 55 million tonnes maize by the year 2030 to meet ever increasing demand for human consumption. Cultivation of maize hybrids with major focus on Rabi maize production and innovative technologies in maize value chain can address this target.

Nutritional security is the backbone of human health. In spite of the importance of maize as a third important cereal crop, it is nutritionally poor due to improper seed protein composition and amino acid balance. However, quality protein maize (QPM) can be an alternative. In this pursuit, I focus on the prospects of QPM for amino acid amelioration besides enhancing the productivity of maize through development of heterotic hybrids using elite QPM introgression lines.

1 Maize-an Important Food and Feed

Maize grains is an important human food (fried and boiled) and animal feed (ground dried as corn meal) in many countries (Gupta et al., 2009). It is the principal staple food for Americans. As an alternative to wheat flour, it can be used for bread making and other baked products. It serves as poultry feed (51%), human food (23%), animal feed (12%), industrial starch products (12%), beverages and seed (1% each). Corn flour is a major food component in home cooking and industrialized food products. Maize is rich in vitanmin-B, moderate in dietary fiber and the essential minerals (magnesium and phosphorus); whereas other nutrients are in low amounts. It provides 86 calories/100 gm kernels. Green ears of maize are used on large scale for roasting, and consumed as food at dough stage. The mixed feed manufacturing industry is the largest industrial user of shelled grain. About three-fourth of the mixed feed industries output is used for manufacturing poultry and dairy feeds. Maize grains serve as raw material for manufacture of starch, syrup, dextrose, maltodextrin, lactic acid, ethyl and butyl alcohol, acetone and whiskey. Besides, variety of food products e.g., corn meal, corn flakes, cake, cookies, sweeteners and porridge are prepared from maize grains. General public including tribals also consume normal maize as food mixture and green cobs. Improper amino acid balance caused malnutrition. For this purpose the lysine is added as inorganic source for preparation of poultry and cattle feed.

2 Seed Proteins in Maize

Endosperm occupies major portion (82%) of the maize grain and it is surrounded by thin pericarp (6%); while the rest constitutes the embryo (12%) (Watson, 1987). Endosperm serves as the major source of seed storage proteins. The grains contain around 9% protein, intermediate between rice and wheat. Therefore, maize seed protein turns to be a frontier area research for many years. In maize, zein (60%) followed by glutelin (34%) are the major fractions of seed storage proteins (Leite et al., 1999), while albumin and globulin occur in traces (3% each). Average Indian adults need 52 gm protein per day against the available quantity of 26-30 gm in daily diet. There is a scope to increase the protein content as high as 18% by increasing the prolamine (zein) fraction in maize endosperm (Dudley and Lambert, 1969), but unfortunately it consequently led to lysine deficiency.

A number of multigene families are meant for zein proteins (Lending and Larkins, 1989) which form protein bodies on the rough endoplasmic reticulum (Larkins et al., 1993). Each zein polypeptide is synthesized by differential structural gene (Zp). These zp genes follow simple inheritance and serve as members of a large group of genes (upto 150). Zeins are rich in glutamine (21-26%), leucine (20%), proline (10%) and alanine (10%), but deficient in important essential amino acids e.g., lysine and tryptophan leading to protein malnutrition. In maize grains, α-zeins are the major prolamin fraction, while β-15kD, γ-16-27kD and σ-10kD zeins (Coleman and Larkins, 1999; Leite et al., 1999) occur in traces. α and σ-zeins form the protein body core which is surrounded by β and γ-zeins (Esen and Stetler, 1992; Lending et al., 1992). α-zeins are in fact, constituted of two major sub-classes e.g., 19kD and 22kD zeins. Polymorphism of these zein fractions can be detected by SDS-PAGE of total seed protein.

3 Development of the Magic Quality Protein Maize (QPM)-the Story

Nutritional deficiency of maize was documented prior to 1960s. There was a dearth need to alter amino acid composition. Altering amino acid composition become difficult by conventional breeding technique. To address this problem, quest for improved protein content was first attempted. But, this turns to be futile, as protein content maintains inverse relationship with lysine and tryptophan content in seed. In 1963, Purdue university researchers isolated opaque 2 (o₂) and floury 2 (fl₂) natural mutants (Mertz et al., 1964; Nelson et al., 1965) with altered amino acid profile and composition of corn endosperm protein which resulted in two-fold tryptophan content compared to normal maize.

Later, the pleiotropic effects of opaque-2 mutation were known that resulted poor consumer preference in terms of unpleasant taste, chalky, lighter and soft endosperm. This led to damaged kernel while harvesting, increased susceptibility to pest and diseases. The mutants showed reduced cob weight and lower yield due to reduced dry matter accumulation. These undesirable features seem to be the major hindrances in genetic improvement for higher productivity with enriched nutritional quality. Subsequently, numerous modifiers togetherly contributing kernel vitreousness came to limelight. However, the mode of expression and inheritance pattern of the o2 modifiers are still unclear. Later, in mid 1980s, Surinder K. Vasal and Evangelina Villegas of CIMMYT used modified backcrossing and recurrent selection to improve kernel hardness and grain yield by combining the opaque 2 and its complex genetic modifier systems. This resulted a special category of biofortified maize named as “Quality Protein Maize” (QPM) for which Vasal and Villegas were conferred the world food prize for the year 2000 on world food day (16 October). Thereafter, serious efforts were undertaken for development of genetic modifiers in opaque 2 backgrounds through intrapopulation selection of QPM types exhibiting a higher frequency of modified o2 kernels. Subsequently, combining ability studies for QPM status (Vasal et al., 1993a; 1993b) assisted QPM hybrid development under varying climatic and growing conditions. Now, QPM are being grown on roughly 9 million acres worldwide (Kataki and Babu, 2003).

4 Nutritive Value of Normal Maize and QPM

In most of the developing countries with high population density, 70 percent of protein is provided by cereals eaten directly. Although, maize kernels harboured more protein than rice, but quality-wise it is poor due to low status of tryptophan and lysine (which our body can not synthesize), and high leucine and glutamine content, causing imbalance of amino acids and malnutrition. Maize endosperm contains about 2 percent lysine and 0.5 percent tryptophan; but there is a need of approximately double of this amount for our normal growth and development. Surprisingly, the isolation of the opaque 2 (o₂) mutants seemed to be the befitting answer to solve such nutritional starvation. Ihe lysine and tryptophan content was almost doubled in maize endosperm (Vasal, 1994; Krivanek et al., 2007) by decreasing the synthesis of zein proteins (deficient in lysine and tryptophan) and increase in the other seed protein bound lysine and tryptophan. QPM was proved to be nutritionally rich compared to common maize and almost equivalent though not equal to the growth response observed when milk protein (casein) was fed (Amorin, 1972; Valverde et al., 1981). Digestibility of seed protein of QPM is significantly higher and is better utilized (due to its better essential amino acid balance) than normal maize. Protein digestibility of processed opaque 2 maize is estimated to be 76.5% as against its true digestibility value 82%; whereas it is just 46.5% in case of non-QPM counterpart (Kies et al., 1965; Clark et al., 1967). This proves to be a commercially viable practical proposition as the daily recommended ration may be cut short to just half by using QPM. In poultry, it boosts 50% increase in body weight compared to normal maize over a 9 week-period (Mbuya et al., 2011). Hence, the merit of QPM diversified value added products for children and adults cannot be undermined to meet food and nutritional security (Atlin et al., 2011). In addition, tryptophan is readily bioavailable by way of its conversion to Niacin in our body, which theoretically reduces the incidence of Pellagra.

5 Genetic Basis of QPM

In maize, the Opaque 2 gene codes a transcriptional activator that regulates the most abundant endosperm storage protein genes. The o₂ gene is reported to have large effect on lysine and protein content while it has minor effect on oil content (Lou et al., 2005). Sequencing of the o₂ gene in a set of cultivated and teosinte (wild progenitor of cultivated maize) accessions revealed 5.4% polymorphic sites and 72 insertions/deletions, located mostly in noncoding regions (Henry et al., 2005). Besides, the o₂ transcriptional activator revealed quite high molecular diversity compared other transcription factors in maize (Henry et al., 2005).

Several spontaneous (o₁, o₂, o₅, o_9-11, o₁₃, o₁₆, o₁₇) and induced mutations affect amino acid composition in maize seed proteins. Maize carrying opaque 2 mutation can have nearly double lysine and tryptophan content compared to normal maize. A opaque 6 mutant allele also increased lysine and tryptophan content in endosperm. Recently, a transposable element ‘rbg’ brings about differential expression of opaque 2 mutant gene in two opaque 2 NILs derived from the same inbred line (Chen et al., 2014).

Expression of the o₂ gene is largely influenced by GxE interaction. Besides, background polygenes accounts for appreciable variation in amino acid composition and opaque phenotypes (Lou et al., 2005). The discovery of numerous modifiers associated with opaque 2 mutation made it possible for kernel vitreousness and increased seed yield with tolerance to abiotic stresses. Two major QTLs associated with endosperm modification have been identified. Vasal et al. (1980) produced elite germplasm with hard kernel and much higher quantity of lysine and tryptophan by combining the opaque-2 allele with these QTLs.

6 Modulation of Amino Acid Composition and Endosperm Characteristics

A protein with balanced amino acid composition is in vogue assist body building process. Ten amino acids including lysine and tryptophan are essential amino acids (which our body cannot synthesize). These allow the body to synthesize complete proteins Therefore, amino acid balance seems to be a determining factor for quality of any food and feed. Several mutants are now available with altered amino acid profile of endosperm protein. These include opaque-2 (o₂), floury 2 (fl₂), Mucronate (Mc) and Defective endosperm B30 (DEB30). The opaque mutants are recessive (o₁, o₂, o₅, o_9-11, o₁₃, o₁₆, o₁₇) and the floury mutation is semi-dominant (fl-1, fl-2 and fl-3), but ‘Mucronate’ and ‘Defective endosperm’ are dominant mutations. The opaque mutations affect the regulatory network (Mertz et al., 1964; Nelson et al., 1965; Krivanek et al., 2007) whereas floury, Mucronate and defective endosperm affect the storage proteins(Gibbon and Larkin, 2005).

The opaque 2 (o₂) down regulates the zein protein expression leading to increase in the other seed protein bound lysine and tryptophan (Henry et al., 2005). This seems to be a compensatory mechanism that activates translation of other mRNAs instead of zein mRNAs. Besides, deletion mutagenesis conditioning zein protein expression can alter amino acid composition (Holding, 2014). Down regulation of 22 kD zein protein mediated by RNA interference is reported to cause opaque phenotype more profoundly as compared to 19 kD component possibly due to greater interaction of 22 KD components with β and γ-zeins resulting in disruption in protein body formation (Segal et al., 2003; Huang et al., 2004). Besides, Zhang et al. (2010) revealed increase in synthesis of lysine and tryptophan content in maize carrying opaque-16 mutant allele. Transfer of o₁₆ allele into opaque 2 genetic background can further increase the lysine content in maize.

The details of proteome modulation that operates to alter amino acid composition is not clear. As per microarray analysis, 60 genes out of 1,400 genes are reported to be three times up-regulated in the o₂ mutant and these are related to stress responses, molecular chaperones and protein turn over (Prioul et al., 2008). Sixty six genes are down-regulated which are involved in carbon, carbohydrate metabolism and branched chain amino acids (Hunter et al., 2002). o₂ mutants revealed significant decrease in LKR/SDH (lysine-ketoglutarate reductase/saccharopine dehydrogenase)-the first enzyme of lysine catabolism, as compared to wild type (Brochetto-Braga et al., 1992; Azevedo et al., 2003). Segmental duplication in the Teosinte genome is reported to result diverged copies of the regulatory opaque-2 gene (Xu and Messing, 2008) during evolution. Besides, a 15.26kb duplication at the 27-kDa γ-zein locus is shown to be a major modifier QTL leading to its enhanced expression and endosperm hardness (Liu et al., 2016). A major QTL has been identified between the marker 0916-2 and Ch7-120.35 within a narrow interval of 100kb using fine mapping. Further, SDS-PAGE of seed proteins revealed that o₂ introgression decreased the accumulation of most of the zein proteins except for 27-kDa γ-zein (Zhang et al., 2015; Zhou et al., 2016).

Pleiotropic effects of opaque-2 gene has been recognized. In addition to alteration in amino acid composition, it affects starch organization making the kernel more soft, opaque in appearance and unpleasant taste. A set of modifier genes (QTLs) in the opaque 2 genetic background are known to improve hard and vitreous kernels (Bjarnason et al., 1976; Ortega and Bates, 1983; Burnett and Larkins, 1999) and accumulation of smaller size protein bodies (Zhou et al., 2016). Inheritance of o₂ modifiers is complex (Vasal et al., 1980). The modified opaque 2 mutants reduced the levels of 22 KD α-zeins and bring about 2-3 times higher levels of 27 KD-γ zeins (Geetha et al., 1991) along with increased hardness of the kernel (Moro et al., 1995). Besides, Krivanek et al. (2007) reported a series of amino acid modifier genes for improvement of lysine and tryptophan. Subsequently, several favourable modifiers have been accumulated in o₂ genetic background to generate modified QPM genetic stocks for their use in QPM breeding (Sofi et al., 2009).

7 Mapping of Opaque-2 Gene and Modifier Complexes

A genotype with both opaque 2 and modifier complexes is needed for biofortification. The opaque 2 gene has been identified in the short arm of Chromosome 7 and it is mapped near to the defective endosperm gene ‘DEB 30’ (Holding and Larkins, 2008; Sofi et al., 2009). While, RFLP analysis in an F₂ population indicated association of two major QTLs with endosperm modification. One of these QTL is located near to the centromere and another at near to the telomere on the long arm of above seventh chromosome (Lopes et al., 1995). Mapping of opaque 2 and QTLs conditioning endosperm modification in the same linkage group envisaged considerable inter se association which can be harnessed for QPM breeding. In fact, there exist varying degrees of endosperm modifications among available germplasm stocks (Paez et al., 1969). The mechanism underlying change of the grain structure from chalky to vitreous in modified opaque-2 mutants (mo₂) by the endosperm modifiers is yet not clear. But, breeders can accumulate above modifier complexes (QTLs) using marker assisted selection to assist QPM breeding programme (Dudley and Lambert, 2004). Vasal et al. (1980) succeeded to combine the opaque 2 allele with QTLs for genetic modifiers for development of modified version of QPM germplasm with hard kernel and much higher quantity of lysine and tryptophan.

8 Identification of Molecular Markers

Identification of molecular markers that co-inherit with the opaque 2 phenotype is a crucial step for their use in marker assisted breeding. Until recent years, several polymorphic markers have been identified in QPM lines using RAPD (Nkongolo et al., 2011; Hemavathy, 2015), ISSR (Nkongolo et al., 2011; Lenka et al., 2015), SSR (Bantte and Prasanna, 2003) and SNP (Semagn et al., 2012) primers. Bantte and Prasanna (2003) reported a few SSR markers e.g., bnlg 105, bnlg 125, bnlg 439, phi 037 and dupssr 34 with high polymorphic information content to differentiate QPM lines. But, none of these was efficient to differentiate between QPM and Normal maize inbreds. CIMMYT designed o₂-gene specific SSR primers viz., phi 057, phi 112 and umc 1066 which are located as internal repetitive elements within opaque 2 gene on short arm of chromosome 7 (www.agron.missouri.edu). Allelic polymorphism among QPM and normal maize inbreds was surveyed by several workers using the above SSR primers (Babu et al., 2005; Jompuk et al., 2006; Magulama and Sales, 2009; Gupta et al., 2013). Bantte and Prasanna (2003) detected 30 unique SSR alleles to differentiate QPM inbreds. Besides, they identified the SSR primer phi 057 to detect QPM inbreds carrying opaque 2 mutation. Phi 057 amplified a 169 bp band in QPM donors and 159 bp amplicon in normal maize inbreds (Magulama and Sales, 2009). phi 057 and Umc 1066 are reported to be co-dominant, while phi 112 is a dominant marker (Magulama and Sales, 2009). Kata et al. (1994) used RFLP technique using Hind III digestion of genomic DNA and opaque 2 locus specific cDNA probe to detect O₂/O₂, O₂/o₂, and o₂/o₂ genotypes of individual plants in breeding populations. Besides, Zhang et al. (2010) reported use of molecular marker umc1141 to trace the inheritance of the erstwhile mentioned opaque 16 mutant allele that led to elevated synthesis of lysine and tryptophan content.

9 Breeding for Quality Protein Maize (QPM)

Maize is a cross pollinated crop. The major breeding approach for increasing productivity is production of hybrids using heterosis breeding. The success from this method depends on development and identification of suitable inbred lines using an appropriate mating design; and selection of most promising heterotic normal maize hybrid. For production of QPM hybrid, the ultimate aim is to combine the advantage of heterosis along with amelioration of amino acid composition using QPM donors. To achieve this, conversion of either of the parental non-QPM inbreds to QPM status is the first step to develop a heterotic QPM hybrid. A number of reliable QPM donors are made available at CIMMYT by selection for modified grain texture in QPM backgrounds using various selection schemes. Initially, four tropical hard endosperm populations (composite K, Ver 181-Ant gp venezula-1, Thai composite and PD 9MS6) and one highland composite (Composite 1) were developed by intra-population selection of genetic modifiers in o₂ background followed by grouping of modified o₂ sources into pools. These were later recombined to maintain modified QPM status and were used for large scale conversion of non-QPM inbreds to their QPM version. The inbred lines e.g., CML 176 and CML 186 are reported to be potential QPM donors (Manna et al., 2005; Danson et al., 2006) for introgression of o₂ allele to non-QPM maize. Vivek et al. (2008) reported tryptophan content more than 0.60% and lysine content more than 2.6% in a set of CIMMYT QPM inbreds. Besides, Ortega and Villegas (1988) reported on an average 0.8% tryptophan and 3.1% lysine content among a set of QPM inbreds as against 0.4% and 1.6% respectively in normal maize lines.

CIMMYT adopted a conservative approach to develop modified opaque 2 (mo₂) genotypes to strike a balance between proteins levels and grain quality and competitive yield levels. The modifier complex is polygenic in nature. Therefore, molecular marker based screening for QPM status coupled with phenotypic selection for improving endosperm characteristics seems to be an appropriate strategy for development of QPM introgression lines. The erstwhile mentioned gene-specific co-dominant markers phi 057 (Manna et al., 2005; Danson et al., 2006; Magulama and Sales, 2009) and umc 1066 (Gupta et al., 2009; Gupta et al., 2013; Rajesh Singh and Lekha Ram, 2014) are worthwhile to trace the opaque 2 (o₂) allele in conversion programme. Distinct polymorphism revealed by both the primers can discriminate the QPM donors from respective non-QPM recurrent parents and also between homozygous (O₂O₂) and heterozygous (O₂o₂) opaque 2 back cross progeny. This paves the way for rejection of back cross progenies (dominant homozygous) resulting short cutting the breeding cycle (eliminates the need to grow F₂) and substantial savings of labour and material resources for amino acid estimation (Tanksley et al., 1989; Frisch et al., 1999; Gupta et al., 2013). This made it possible to breed a QPM hybrid in less than half the time required in conventional breeding. Besides, foreground selection for opaque 2 combined with phenotypic selection for recipient parent at early back cross generations can bring about rapid recovery of recurrent parent genotype. The introgression lines developed using marker assisted back cross breeding may serve as important breeding material for development of QPM hybrids.

The pioneering work initiated by the Breeding Program of the National Maize and Sorghum Research Center (CNPMS-EMBRAPA) led to release of two Brazian QPM varieties e.g., BR 451 and BR 473 in 1988 and 1994 respectively for commercial cultivation. The former is used as a substitute to wheat due to its white color; while the later resembling normal maize fetches huge consumers’ acceptance. Mixture of wheat flour with that of BR 451 in a suitable proportion was reported ideal for industrial production of bread, cookies, and pasta (Peixoto et al., 1989). Besides, a double cross QPM hybrid was released in Brazil during 1997 and a QPM hybrid Zhongdan 9407 and Shakti-1 were released in China and India respectively. In India, Vivek QPM-9: a hybrid product of two QPM introgression lines was released in 2008 (Gupta et al., 2009; Gupta et al., 2013). For this, the parental normal maize inbreds, CM 212 and CM 145 were converted to QPM status through marker assisted backcross breeding using CML 180 and CML 170 respectively as QPM donors. The said QPM hybrid, recorded grain yield at par, but resulted 41% increase in tryptophan and 30% increase in lysine content over the normal hybrid. Soon after, many countries participated in QPM network. The Republic of South Africa had earlier released hybrids HL-1, HL-2, and has recently released HL8 which has hard endosperm, good yield potential, and tolerance to diseases. Similarly, maize genotypes for reduced anti-nutritional factors has been developed using marker-assisted backcross breeding (MABB) (Naidoo et al., 2012). Later, Muthusamy et al. (2014) successfully attempted Marker-Assisted Introgression of ß-carotene hydroxylase Allele to develope ß-Carotene Rich Maize Hybrids. Chander et al. (2008) identified a major loci (y1) for carotinoid content using gene targeted molecular marker (Y1ssr).

10 Limitations

QPM seems to be a panacea for salvation of malnutrition, but yet it is not free from limitations. QPM at its present form (after endosperm modification) look similarly yellow grain, and retain all morphological and agronomic features of normal maize making them indistinguishable. This poses potential threat for phenotypic selection and varietal maintenance in seed plot. Besides, cross-pollination would reduce both yield and nutritional value of the QPM variety. QPM, being nutritionally balanced, is liable to be more susceptible to store grain pests and diseases than normal maize. Since QPM varieties are few, they may not be suited to variety of ecological variations. In this regard, QPM hybrid may serve as a good candidate. Hybrids generally yield better and maintain their genetic qualities more consistently than composites. For this, separate QPM lines must be generated by marker assisted back crossing and then reassembled to form hybrid combinations, which is a cumbersome job than development of normal maize hybrid.

11 Conclusion

Maize ranked third important cereal crop in the food chain. Quality protein maize has a far-reaching impact for nutritional security with the discovery of opaque 2 mutation. Such a natural recessive mutation led to selective down-regulation of specific zein genes resulting alteration in amino acid composition and opaque phenotype of endosperm. Modified Marker assisted back cross breeding made it possible to develop QPM versions of normal maize inbreds with desirable endosperm characteristics and seed yield. Development of QPM hybrids by combining elite QPM introgression lines can meet food and nutritional security in developing countries including India.

Acknowledgement

I sincerely acknowledge and thank all researchers for their valuable contributions included in the text as references.

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