3192 ReprinLed from CROP SCIEi\CE Vol. 12, July-Aug. 1972, p. 440-442 Maize with Multilayer Aleurone of High Protein Contene M. J. Wolf, H. c. Cutler, M. S. Zuber, and Uheng Khoo2 ABSTRACT rangement of kernels on the cob but is also notable Coroico corn (Zea mays L.), a South American race for the range of aleurone and cob colors. of floury maize contained two to six layers of aleurone The object of the experiments reported here was cells instead of the customary single aleurone cell layer to determine the amount of aleurone tissue and the found in ordinary yellow dent corn. In Coroico with an average of 3.7 aleurone cell layers, the aleurone made up protein and amino acid content of the aleurone of 4.3% of the endosperm as against 2.1% in yellow dent Coroico in comparison with yellow dent hybrid corn corn. Total protein in Coroico aleurone is 35 to 38% as a basis for evaluating the potential of Coroico in compared with 22% in yellow dent com. Lysine levels in improving the nutritional quality of common yellow aleurone proteins of all corns examined, both Coroico and dent hybrids. yellow dent were comparable ranging from 4.0 to 4.4 g/lOO g of protein. Because of the additive effect of the multiple aleurone and the high protein content of the MATERIALS AND METHODS aleurone, lysine in Coroico endosperm was higher than in that of yellow dent corn. Multiple aleurone is transmitted Maize Varieties. A double cross yellow dent hybrid (B37 as a partial dominant character over the normal single Tcms X H84 and Oh 43 RF X AGI9) and W64A, a yellow dent aleurone condition. Improvement of protein nutritional inbred, both with a typical single-layer aleurone (Fig. la), were quality using Coroico germ plasm appears worthwhile. used as reference corns. Peru 442, a Peruvian floury maize, and variants of Coroico Additional index words: Seed proteins, Maize indige­ were obtained from the maize germ plasm center at the Colom­ nous races. bian Institute of Agriculture, Medellin, Colombia. Additional accessions of Coroico or of maize related to Coroico were ob­ tained from the ational Seed Storage Laboratory, Fort Collins, IGH-QUALITY proteins and vitamins are con­ Col.; the International Maize and Wheat Improvement Center, Mexico City, Mexico; and the Institute of Genetics, University H centrated in the outer layer of endosperm cells of Piracicaba, Sao Paulo, Brazil. of maize (15) and most common cereal grains (10, 14). Aleurone Counts. The number of rows of aleurone cells were This layer, the aleurone, usually consists of a single determined by microscopic examination of 5 to 20 kernel sam­ layer of cells. In large kernels of common maize, aleu­ ples of each multialeurone maize accession. Since yellow dent corn has only a single layer of aleurone cells, counts were un­ rone makes up only 2% or less of the total weight (9). necessary in this corn. Freehand sections were taken from one Some cereal grains have multilayer aleurone tissues. ide of each kernel barely penetrating the aleurone layer. The Barley (13), oats, and certain varieties of rice may sections were stained with an aqueous iodine solution, mounted have two to four layers of aluerone cells (II). Occa­ in 1: 1 glycerol: water, and the number of aleurone cell layers was counted at a magnification of 100. Average number of cell sionally doubling of individual aleurone cells may layers per kernel were reported. Kernels sampled in this way occur in common yellow dent maize, but multiple remained viable. layers have not previously been reported. Microscopy. Fluorescence microscopy was the most informa­ tive and convenient method of examining the aleurone layer. We found multiple aleurone layers in some kernels Thin sections of maize kernels 3 to 4 ,urn thick were prepared of a Peruvian floury maize, 'Peru 442.' This finding by dry-sectioning as described by Wolf and Khoo (17), stained appeared to be important genetically because of the for 3 min with 0.1 % aqueous Acridine Orange, and mounted possibility that increase in the proportion of aleurone on slides in silicone fluid (refractive indices 1.40 to 1.50). Aleu­ rone nuclei fluoresced bright red while the cell walls were bright tissue at the expense of starchy endosperm would in­ orange. crease the proportion of high-quality proteins and vita­ Thick sections were required to demonstrate multinucleate mins in maize and thereby improve its nutritional Coroico aleurone. Sections 10 to 12 ,urn thick were prepared by quality. Peru 442 was obviously a mixture of strains freeze-sectioning glutaraldehyde-fixed kernels. The sections were stained for protein-bound sulfhydryl groups by the method of since only a third of the kernels had more than a sin­ Barrnett and Seligman (2). Aleurone nuclei were heavily stained gle layer of aleurone cells. Because Peru 442 seemed a deep red color against a background of lighter red matrix to have been derived from the Coroico race, our search protein. Good contrast between nuclei and matrix protein was for multilayer aleurone was concentrated on speci­ obtained by photographing with a green filter ( o. 58 Wratten). Isolation of Aleurone. From 5 to 20 kernels were soaked in mens of this race and its variants. distilled water for 30 min and the pericarp was peeled off, Coroico, a primitive and highly variable race, was leaving the aleurone intact over the endosperm. The dehulled first described by Cutler (7, 12). It is found on the kernels were immersed in boiling water for 3 min to inactivate aleurone enzymes, after which the grains were steeped in 90% eastern Andean slopes of Bolivia, Peru, and Ecuador dimethyl sulfoxide for 30 min, followed by brief rinsing in dis­ and eastward into Brazil. It has been called Piricinco tilled water. The aleurone layer was stripped off with a small in descriptions of Peruvian maize (8), Pojoso in Ecua­ scalpel, and adhering bits of pericarp on the outer surface or dor (16), and Interlocked in Brazil (5). The race is starchy endosperm on the inner surface were quickly scraped off under the dissecting microscope. The clean aleurone layer was characterized partly by its unique interlocking ar- immediately frozen in a glass tube immersed in dry ice-acetone mixture. The aleurone was freeze-dried and stored over Drierite [or chemical analysis. 1 Contribution from the Northern Marketing and utrition Preparation of Endosperm. The pericarp was peeled off as Research Division, Agricultural Research Service, U.S. Depart­ described above in samples of 5 to 20 kernels. The germ em­ ment of Agriculture, Peoria, Ill. 61604. Received Oct. 18, 1971. bedded in the endosperm was dissected out with a scalpel. The 2 Principal Chemist, orthern Regional Research Laboratory, remaining tissue is whole endosperm including the inner starchy RS, USDA, Peoria, Ill.; Curator of Useful Plants, Missouri cells and the aleurone layer. The endosperm was air-dried at Botanical Garden, St. Louis, Mo. 63110; Research Agronomist, room temperature and ground to 40-mesh in a micro Wiley University of Missouri, USDA, Columbia, Mo. 65201; and Bota­ mill. The term endosperm as used in this paper refers to whole nist, orthern Regional Research Laboratory, ARS, USDA, endosperm; that is, combined aleurone layer and starchy endo­ Peoria, Ill. sperm. 440 Purchased by U. S. Dept. g[ ~~turofor Official Usc WOLF ET AL.: HIGH PROTEI]\; CONTE ;T OF MAIZE 441 Chemical Analyses. Crude protein (% Kjeldahl NX 6.25) was determined in duplicate by a micro-Kjeldahl method (I). The standard deviation was ±0.12%. Single amino acid analyses were made on 30-mg samples of isolated aleurone tissue. Endosperm was analyzed in duplicate for amino acids using 150-mg samples. Aleurone or endosperm was hydrolyzed by refluxing in constant boiling HCI for 24 hours. Hydrolysates were evaporated to dryness. The residue was dissolved in pH 2.2 citrate buffer for analysis on a Beckman Spinco" Model 120 amino acid analyzer using the 3-hour pro­ cedure of Benson and Patterson (4). Peaks were integrated elec­ tronically with an Infotronics-Integrator, and the amino acid data were computed automatically (6). The relative standard Fig. 1. Single and multiple aleurone layers in maize. a. Typi­ deviation of a single lysine analysis was ±7.8%. All chemical cal single·eelled aleurone layer in ordinary yellow dent corn. analyses were reported on a dry basis. b. Coroico with multiple aleurone cell layers. RESULTS AND DISCUSSION Average number ot aleurone layers in 15 variants of Coroico ranged trom 2.0 to 3.7 per kernel with some grains having as many as six layers. Outermost cells in the aleurone ot Coroico are quite large, and the inner ones become progressively smaller (Fig. 1b). Increase in total aleurone and protein is not proportional to the number of cell layers. In one kernel lot of Peru 442, a single-celled aleurone aver­ aged 51.6 ,urn in thickness, slightly thicker than the aleurone of yellow dent corn. A three-cell-thick aleu­ rone layer measured 101 ,urn. Although yellow dent corn aleurone cells are always Fig. 2. Cross·section through aleurone layer of Coroico showing rnononucleate, some accessions of Coroico had from multinucleate cells. one to four nuclei per: aleurone cell. The multiple nuclei are best demonstrated in relatively thick sec­ Table 1. Protein and lysine in endosperm of hand·dissected tions (Fig. 2). The multinucleate condition in multi­ multialeurone corn and in ordinary yellow dent corn. aleurone cells o[ Coroico suggests that further in­ % of endosperm .\ieu- Alcu- Endo- crease in aleurone tissue might have occurred had cell cell Alcu- Aleu- Endo- Aleu- sperm layer;;, protein, rone rone sperm rone lysine, division followed every nuclear division.