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Main Specialty Corns, Second Edition
Specialty Corns, Second EditionArnel R. Hallauer
Completely revised and updated, the Second Edition of Specialty Corns includes everything in the first edition and more. Considered the standard in this field, significant changes have been made to keep all the information current and bring the references up-to-date. Two new chapters have been added to keep up with the latest trends: Blue Corn and Baby Corn. Access the latest methods in developing specialty corns with this standard-setting reference. Edited by an expert in the field who has spent his professional life working with corn, Specialty Corns, Second Edition discusses the genetic variation inherent in corn, genetic materials available, breeding methods, and special problems associated with the development of specialty corns. Hallauer has assembled a team of international experts who have contributed to this work.
Second Edition Specialty Corns Edited by Arnel R. Hallauer, Ph.D. CRC Press Boca Raton London New York Washington, D.C. © 2001 by CRC Press LLC © 2001 by CRC Press LLC Library of Congress Cataloging-in-Publication Data Specialty corns / edited by Arnel R. Hallauer.—2nd ed. p. cm. Includes bibliographical references. ISBN 0-8493-2377-0 1. Corn—Varieties. 2. Corn—Breeding. 3. Corn—Germplasm resources. 4. Corn—Utilization. I. Hallauer, Arnel. R., 1932SB191.M2 S6252 2000 633.1′523—dc21 00-039767 CIP This book contains information obtained from authentic and highly regarded sources. Reprinted material is quoted with permission, and sources are indicated. A wide variety of references are listed. Reasonable efforts have been made to publish reliable data and information, but the author and the publisher cannot assume responsibility for the validity of all materials or for the consequences of their use. Neither this book nor any part may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, microfilming, and recording, or by any information storage or retrieval system, without prior permission in writing from the publisher. All rights reserved. Authorization to photocopy items for internal or personal use, or the personal or internal use of specific clients, may be granted by CRC Press LLC, provided that $.50 per page photocopied is paid directly to Copyright Clearance Center, 222 Rosewood Drive, Danvers, MA 01923 USA. The fee code for users of the Transactional Reporting Service is ISBN 0-8493-2377-0/01/$0.00+$.50. The fee is subject to change without notice. For organizations that have been granted a photocopy license by the CCC, a separate system of payment has been arranged. The consent of CRC Press LLC does not extend to copying for general distribution, for promotion, for creating new works, or for resale. Specific permission must be obtained in writing from CRC Press LLC for such copying. Direct all inquiries to CRC Press LLC, 2000 N.W. Corporate Blvd., Boca Raton, Florida 33431. Trademark Notice: Product or corporate names may be trademarks or registered trademarks, and are used only for identification and explanation, without intent to infringe. © 2001 by CRC Press LLC No claim to original U.S. Government works International Standard Book Number 0-8493-2377-0 Library of Congress Card Number 00-039767 Printed in the United States of America 1 2 3 4 5 6 7 8 9 0 Printed on acid-free paper Preface Corn is one of the major cereal grains grown in the world, exceeded only by rice and wheat in terms of quantity produced. In the U.S., corn is produced on 70 to 80 million acres annually and plays an important role in the economy of the country. Hybrid corn was introduced in the U.S. in the 1930s, and average production per unit area increased from 35 bushels per acre for the period of 1945 to 1950 to more than 120 bushels per acre for the decade of the 1990s. In 1998, corn growers in the U.S. produced 9.77 billion bushels with an average yield of 134 bushels per acre. Nearly 60% of the increase in yield was because of genetic improvement of hybrids. Over 95% of the corn produced in the U.S. is marketed as commodity corn, which is primarily yellow dent corn. Commodity corn is fed to livestock (55%), is exported (29%) and largely used for feed, is used in wet milling (12%), and is used in dry milling (4%). Although a typical supermarket displays over 1200 items that include one or more ingredients from corn, less than 5% of the total U.S. corn production is marketed as specialty corns. Several distinct types of specialty corns are available (popcorn, sweet corn, high-protein quality corn, high waxy corn, high oil corn, etc.), but individually each has less than 1% of the total corn market. Alternative uses of the corn that can be produced in large quantities in the U.S. would have broad implications in the economy of the nation and for the individual grower. Greater emphasis is being given to the development of new processes and new products to enhance the value of corn. One new use for corn in recent years has been the extraction of ethanol for use as corn-based fuels for cars and trucks. About 600 million bushels of corn in the U.S. are converted to ethanol each year, adding at least 30 cents greater return per bushel of corn and greater returns for the producer. Development of superior specialty corns that have consistent performance in quality and in quantity has not received the same emphasis in genetics and breeding that has been given to the yellow dent corns. Each specialty corn has traits that require special emphasis, but the basic germplasm and breeding methods used to improve yellow dent corn often are used in development of improved specialty corns. However, because of the standards required for specialty corns, methods of development and improvement are usually more complex than those for yellow dent commodity corn. The same standards of performance are desired, but the genetics of the specialty traits often are in conflict with the standards used for yellow dent corns. Specialty corn programs have unique characteristics that require careful handling and monitoring during their development for specific needs. The objective of this volume is to provide a summary of the germplasm, methods of development, and specific problems involved for some specialty corns. Chapters on development of blue corn and baby corn are new to the Second Edition. Although blue corn and baby corn have specific niche markets, they are becoming more recognized in the human diets, especially for specific ethnic groups. Two chapters provide an introduction of the kernel mutants and of the different types of starch modifications available in corn to illustrate the variation in properties of the corn kernel. Eight chapters provide detailed descriptions of the germplasm and methods used to develop value-added corns that are related to food and feed uses. One chapter summarizes the processes used for food products derived from corn. The last three chapters describe methods used to develop corn for manufacture of pipes, corn for silage, and corns for temperate areas. Some traditional markets of yellow dent corn other than for feed or for export — ethanol, corn sweeteners, and corn starches — were not addressed in this volume. Most chapters have been revised to include the latest information and more recent references; exceptions were the chapters on high amylose and waxy corn and breeding white © 2001 by CRC Press LLC endosperm corn. Only minor revisions were included in the chapter on high quality protein corn. It is our desire that the information provided will serve as a guide and reference to those engaged in development of specialty corns and to those that are considering the possibilities of initiating specialty corn programs. U.S. corn producers have become extremely proficient at producing more grain per unit area, but profit margins continue to decrease. Adding value either via alternative products from the large volumes of grain produced or development of specialty corns is of interest to producers and processors. We hope the revised edition can enhance the future uses of corn. I wish to thank the contributing authors for their tireless efforts, their cooperation in preparing and revising manuscripts, and their patience during the completion of the volume. Arnel R. Hallauer © 2001 by CRC Press LLC Editor Arnel R. Hallauer is a C. F. Curtiss Distinguished Professor in Agriculture with the Department of Agronomy, College of Agriculture at Iowa State University. He is a native of Kansas who graduated from Kansas State University in 1954 with a B.S. degree in plant science. After serving with the U.S. Army (1954–1956), he enrolled at Iowa State University where he fulfilled the requirements for M.S. (1958) and Ph.D. (1960) degrees in plant breeding. His professional career started in 1958 as an agronomist with the U.S. Department of Agriculture breeding for resistance to the European corn borer. During 1961–1962, Hallauer was stationed in the Genetics Department at North Carolina State University to study the inheritance of quantitative traits. In 1962, he returned to Iowa, where his research interests focused on the inheritance of quantitative traits, selection methods for the enhancement of germplasm resources, effectiveness of methods for evaluating inbred lines for their potential in hybrids, and evaluation, adaptation, and enhancement of exotic germplasm sources for use in temperate area breeding programs. Hallauer retired from the U.S. Department of Agriculture in 1989 and accepted a position in corn breeding at Iowa State University, where he continues to pursue research on breeding methods and germplasm enhancement. He teaches graduate-level courses in plant breeding. He has been the major advisor for more than 80 students who have fulfilled the requirements for M.S. and Ph.D. degrees with majors in plant breeding, and he has served on the program of study committees for more than 145 graduate students. Hallauer has been recognized for his basic research in the application of quantitative genetic theory to applied plant breeding for line and hybrid development and germplasm enhancement. A compilation of the quantitative genetic studies conducted in corn and their applications to corn breeding were summarized and published in Quantitative Genetics in Maize Breeding. The book has become widely used by breeders of corn and other crop species in the U.S. and internationally. He has been recognized for his contributions with election to the National Academy of Sciences in 1989 and inclusion in the U.S. Department of Agriculture’s Agricultural Research Science Hall of Fame in 1992. He received the Iowa Governor’s Science Medal in 1990 and the National Council Commercial Plant Breeders/Genetics and Breeding Award in 1984. © 2001 by CRC Press LLC Contributors Chokechai Aekatasanawan National Corn and Sorghum Research Center Kasetsart University Pakchong, Nakhon Ratchasima Thailand F. Javier Betrán Associate Professor Department of Soil and Crop Sciences Texas A & M University College Station, Texas Anton J. Bockholt Department of Soil and Crop Sciences Texas A&M University College Station, Texas Charles D. Boyer Department of Horticulture Oregon State University Corvallis, Orgeon James G. Coors Professor Department of Agronomy University of Wisconsin-Madison Madison, Wisconsin Larry L. Darrah Research Leader USDA-ARS Plant Genetics University of Missouri Columbia, Missouri Virgil Fergason Retired, Former Director of Research Custom Farm Seed Division of National Starch and Chemical Co. Decatur, Illinois Marta Hilda Gomez Nabisco East Hanover, New Jersey © 2001 by CRC Press LLC L. Curtis Hannah Department of Vegetable Crops University of Florida Gainesville, Florida Robert J. Lambert Professor of Plant Breeding and Genetics Department of Crop Sciences University of Illinois Urbana, Illinois Joseph G. Lauer Associate Professor Department of Agronomy University of Wisconsin Madison, Wisconsin Charles G. Poneleit Professor Department of Agronomy University of Kentucky Lexington, Kentucky Lloyd W. Rooney Professor Cereal Quality Laboratory Department of Soil and Crop Sciences Texas A & M University College Station, Texas Sergio O. Serna-Saldivar Instituto Tecnologico y de Estudios Superiores de Monterrey Departamento de Tecnologia de Alimentos Monterrey, N.L., Mexico William F. Tracy Professor Department of Agronomy University of Wisconsin Madison, Wisconsin Forrest Troyer Vice President R & D, Retired DeKalb Genetics Adjunct Professor of Plant Breeding Crop Sciences, University of Illinois DeKalb, Illinois Pamela J. White Professor Food Science and Human Nutrition Department and Center for Crops Utilization Research Iowa State University Ames, Iowa Surinder Kumar Vasal CIMMYT Department of Agriculture Kasetsart University Bangkok, BKK 10900 Thailand Kenneth E. Ziegler Instructor Department of Agronomy Iowa State University Ames, Iowa © 2001 by CRC Press LLC Contents Chapter 1 Kernel Mutants of Corn Charles D. Boyer and L. Curtis Hannah Chapter 2 Properties of Corn Starch Pamela J. White Chapter 3 High Amylose and Waxy Corns Virgil Fergason Chapter 4 High Quality Protein Corn Surinder Kumar Vasal Chapter 5 High-Oil Corn Hybrids Robert J. Lambert Chapter 6 Sweet Corn William F. Tracy Chapter 7 Popcorn Kenneth E. Ziegler Chapter 8 Breeding White Endosperm Corn Charles G. Poneleit Chapter 9 Baby Corn Chokechai Aekatasanawan Chapter 10 Blue Corn F. Javier Betrán, Anton J. Bockholt, and Lloyd W. Rooney Chapter 11 Food Uses of Regular and Specialty Corns and Their Dry-Milled Fractions Sergio O. Serna-Saldivar, Marta Hilda Gomez, and Lloyd W. Rooney © 2001 by CRC Press LLC Chapter 12 Pipe Corn, Basis of the “Barnyard Briar” Larry L. Darrah Chapter 13 Silage Corn James G. Coors and Joseph G. Lauer Chapter 14 Temperate Corn — Background, Behavior, and Breeding Forrest Troyer © 2001 by CRC Press LLC 1 Kernel Mutants of Corn Charles D. Boyer and L. Curtis Hannah CONTENTS I. II. III. Introduction Historical Perspective The Genetic System A. Genes Affecting Starch and Protein Composition of the Kernel 1. Genic Variation 2. Allelic Variation B. Genes Affecting Kernel Color C. Genes Affecting Kernel Development IV. Biochemical Analysis A. Kernel Carbohydrates B. Kernel Proteins C. Kernel Lipids D. Interaction in the Accumulation of Kernel Components V. Gene Cloning and Biotechnology VI. Future Perspective References I. INTRODUCTION Corn is the major cereal crop of the U.S. and the second most important cereal crop worldwide. The diversity of potential uses for corn was realized by the Native Americans prior to the first voyage of Columbus. Today, corn production in the U.S. provides feed, food, and a resource for many unique industrial and commercial products. The potential to enhance use of corn for specialty purposes based on existing uses, and develop new products to meet the needs of future generations, provides the plant breeder/plant scientist with unique challenges. The grain of corn is composed of several chemicals of commercial value. The mature kernel is composed of 70 to 75% starch, 8 to 10% protein, and 4 to 5% oil.1 The two major structures of the kernel, the endosperm and the germ (embryo), constitute approximately 80 and 10% of the mature kernel dry weight, respectively. The endosperm is largely starch (approaching 90%) and the germ contains high levels of oil (30%) and protein (18%). The oil and protein often are of commercial value as by-products from the production of starch. Immature kernels contain relatively high levels of sugars and lesser amounts of starch, protein, and oil, which accumulate during development.2 The sugar content is the most clearly recognizable component of sweet corn quality as sweet corn is eaten at an immature stage of development.3 By utilizing genetic variation, the composition of the kernel can be changed for both the quantity and quality (structure and chemical diversity) of starch, protein, and oil throughout kernel development. The ability of future generations of scientists to utilize existing genetic variation and to create new avenues to designing novel variation in corn composition will provide the basis for the development of the next generation of specialty corns. © 2001 by CRC Press LLC In this chapter we will review the well-developed body of information on the genetic variation known to affect the composition, development, and structure of corn kernels. As part of this discussion we will provide some practical suggestions on the construction of various genotypes. With the increased interest in the potential of novel corns for special purposes,4–11 we believe these suggestions will be useful to biochemists, food scientists, plant scientists, and others beginning to use various genes and gene combinations in their work. In addition, we will provide a review of the relationship of the genetic variation to the modification of metabolism in the kernel. We feel the future expansion of this understanding of metabolism will provide the basis for the development of corns designed for specific uses. Finally, we will provide a brief description of the potential that the new technologies of genetic engineering provide for the creation of new variation in the future. The importance of corn as a commodity has led to a large number of reviews and books devoted to the nature of corn. Some of the previous reviews the reader may wish to consult include reviews of corn genetics,12 carbohydrate and protein metabolism,13–21 genetic control of kernel metabolism,2,22–29 regulation of kernel development,30 and various specialty types of corn.2, 31–33 High-quality photographs of the kernel phenotypes for many of the mutants can be found in the two books The Mutants of Maize and Mutants of Maize.34,35 II. HISTORICAL PERSPECTIVE As with other major cereal crops, the origin of corn as a domesticated crop was prior to the written record. It is clear that corn had evolved all the morphologically essential features that distinguish it from its nearest relative, teosinte, by the time of Columbus.36–39 By that time, the corn crop had spread throughout the New World from South America to as far north as the Canadian border of North America. In many of the religions of Native Americans, the corn god or goddess was the supreme deity and corn itself was a gift of the gods.40 The Native Americans also had developed large numbers of varieties and specialty corns by this time.38,39 In fact, all of the major types of corn — including dent, flint, flour, pop, and sweet corn — had been developed39,40 and were used in early attempts by botanists to classify races and varieties of corn.36,41 Similarly, the practice of isolation of varieties was sufficiently well developed to allow the maintenance of the genetic purity of the specialty types that are controlled by recessive genetic characters. The Native Americans had developed a number of specialty uses for the various types of corn as well. Different corns were used for the production of flour, beverages, soups, dried candies, and other purposes. For example, sweet corns were eaten green as a highly prized fresh product; immature kernels were parboiled and/or dried to produce a candy, kancha, and boiled and dried for eating during the winter months; mature kernels were ground to produce the confection pinole and as a fermentable source for the production of an alcoholic drink, chicha.38,42,43 In fact, green corns were based on sugary and flour corns.40 III. THE GENETIC SYSTEM Since the rediscovery of Mendelian principles of inheritance shortly after the turn of the century, corn has been the subject of intense genetic analysis. Collins and Kempton44 described the waxy1 gene and the recessive behavior of this gene. After this report many additional genes modifying kernel appearance and other characters were identified by genetic analysis.45–48 The fruits of these efforts are the large numbers of genes identified and mapped to the ten chromosomes of corn. During this period, genes were identified based on the distinct morphological character (the phenotype) conditioned by the mutant genotype. The nomenclature for the genes was based on a description of this mutant character. In addition, a gene symbol of one or two letters was assigned to the gene. The rules of nomenclature for maize genes was standardized in 1974, and revised in 1995 and 1997 (see Neuffer et al.35).49,50 © 2001 by CRC Press LLC A general practice is to only list the mutant genes when describing a genotype. It is assumed that unless a gene is listed, the genotype at that locus is homozygous for the wild type allele. For example, when a waxy1 (wx1) cultivar is referred to, it is assumed that the other genes influencing kernel carbohydrates are wild type. This will be the format used throughout this chapter. A second result of the intense genetic analysis of maize has been the identification of multiple alleles for various genes. These alleles are given a laboratory number or designation that is separated from the gene symbol or name by a dash. More recently, genes have been identified based on their biochemical phenotype, i.e., the protein product which the gene encodes. When genes are identified and described on this biochemical basis, there is not necessarily a morphological characteristic of the kernel associated with allelic variation of the gene. As a result, these genes are named based on the protein product of the gene (i.e., Zein protein, Zp1; and Sus1, constituitively expressed sucrose synthase). Based on the characterization of the effect of various genes on the properties of the kernel, the mutants can be divided into several groups. Gene systems which influence kernel development, kernel color/pigmentation (anthocyanins and carotenoids), kernel carbohydrates, proteins, and lipids will be discussed in this chapter. Because kernel carbohydrates and proteins are of major concern for their potential for the production of specialty corn, the major part of the chapter will be devoted to these components of the kernel and mutants affecting their quantity and quality. A. GENES AFFECTING STARCH AND PROTEIN COMPOSITION OF THE KERNEL 1. Genic Variation Many of the genes identified early in the genetic analysis of corn were genes affecting the appearance of the kernel. These mutations were easily identified, and the genetic segregation could be observed on a single F2 ear. The description of these genes beginning, with waxy1,44 was based primarily on the altered appearance of the mutant kernel when compared with the nonmutant or normal kernel phenotype (Tables 1.1 and 1.2). Essentially all of these mutants were recessive and required the homozygous genotype to be expressed. Occasionally, dosage effects were observed where the pheTABLE 1.1 Corn Genes Affecting Carbohydrate Composition of the Kernel Genea amylose extender1 brittle1 brittle-2 dull1 Gene Symbol ae1 bt1 bt2 du1 Chromosome 5 5 4 10 mn1 sh1 sh2 sh4 h1 su1 su2 wx1 2 9 3 5 — 4 6 9 miniature seed1 shrunken1 shrunken-2 shrunken-4 soft starch1 sugary1 sugary-2 waxy1 a b c Kernel Phenotypeb tarnished, translucent, or opaque; sometimes semi-full shrunken, opaque to tarnished shrunken, opaque to tarnished opaque to tarnished; S.C.c semi-collapsed translucent with some opaque sectors small, somewhat defective kernel, viable collapsed, opaque shrunken, opaque to translucent shrunken, opaque opaque wrinkled, glassy; S.C. not as extreme slightly tarnished to tarnished opaque All gene loci are named and symbolized using the revised rules for genetic nomenclature.35 Adapted from Garwood and Creech.56 S.C. Sweet corn background differs from dent background. Source: From Hallauer, A.R., Specialty Corns, CRC Press, Boca Raton, FL, 1994. With permission. © 2001 by CRC Press LLC TABLE 1.2 Corn Genes Affecting Protein Composition of the Kernel Genea floury1 floury2 floury3 opaque1 opaque2 opaque5 opaque6 opaque7 defective endosperm—B30 mucronate1 a Gene Symbol fl1 fl2 fl3 o1 o2 o5 o6 o7 De-B30 Mc1 Chromosome 2 4 4 7 7 10 7 ? Mature Kernel Phenotype opaquea opaquea opaque opaque opaque opaque opaque; lethal seedling opaque opaque opaque These loci show dosage effects to varying degrees. Source: From Hallauer, A.R., Specialty Corns, CRC Press, Boca Raton, FL, 1994. With permission. notypes of the heterozygous kernels were intermediate to the phenotypes of kernels homozygous for either allele. Kernel phenotypes/gene names were described based on the overall appearance (i.e., waxy1, dull1, shrunken1), the light transmission properties of the kernels (i.e., opaque1), or the physical properties of the kernel (i.e., brittle1 and floury1). In some instances, gene names were based on some desirable characteristic of the composition of the kernel, e.g., sugary1 and amylose extender1. With further analysis, these genes have been shown to have major effects on the accumulation of the carbohydrate or protein fractions of the kernel. Those mutants affecting the carbohydrates of the kernel fall into two groups. The first group (Class One) includes the shrunken, brittle, and miniature seed mutants. The shrunken and brittle mutants have extreme phenotypes and greatly reduce the total dry weight of the kernel.51,52 Miniature seed kernels have approximately one fifth of the dry weight of normal kernels.53 The second group of mutants (Class Two) have a more varied array of phenotypes (tarnished, opaque, and translucent) and less effect on the total dry weight.54–56 The genes influencing kernel protein fractions in general have opaque mature kernel phenotypes (Table 1.2). With the identification of increasing numbers of new mutants, mutants were identified that had phenotypes similar to existing mutants but were, in fact, mutations of different genes. Standard allele tests (tests of genetic complementation often termed cis–trans tests) were required to ascertain whether a new mutation was due to either allelic variation or a new mutation.57 In these tests, the new mutant is crossed to a known genetic stock of the previously identified mutant. If the F1 kernels had the mutant phenotype, allelism was confirmed. If the F1 kernels had a normal phenotype (complementation), allelism would be questioned and advanced generations (F2 and backcross, BC) would be analyzed. In those instances where the new mutant was found to be nonallelic to the original mutant, the new gene would be assigned the same gene name but be distinguished from the older gene by a number (i.e., sh1, sh2, and sh4). It is now recommended that the originally identified mutant carry the number one, e.g., sh1, shrunken1.35,50 As this is a new convention, many researchers have not included the number “1” when describing the first mutant in a series. Therefore, unless designated otherwise, the reader should assume that the absence of a number with a gene name implies that the gene is the originally identified mutant (e.g., sugary = sugary1, sh = sh1, brittle = brittle1). In some instances, gene names and numbers were reported before allelism was tested and the gene was later shown to be allelic to another gene. In these instances, the originally reported name would be retained and the more recently reported gene name (and number) would be removed from use. For example, o6 and proline-1 were recently shown to be allelic.58 In this case, the original name, o6, was retained. As a result, some gene names/numbers are not complete series (e.g., no shrunken-3 or opaque-3 gene exists). A recent report using Robertson’s mutator © 2001 by CRC Press LLC stocks to generate mutations demonstrates that even after 85 years of new study, new mutations are likely to continue to be identified and characterized.59 The phenotypes conditioned by different mutants are not always the same in different genetic backgrounds. This can often be observed when the mutants are backcrossed into different inbred backgrounds. For example, the phenotypes of su1 kernels are highly variable.56 The phenotype of su1 kernels in sweet corn inbreds is often less extreme than in dent corn inbreds. In some instances, su kernels in dent lines can be almost as extremely wrinkled and collapsed as Class One mutants, sh2 and bt2.60 In many instances, these extreme kernels are difficult to germinate under field conditions.61–63 In some backgrounds, other mutants (i.e., ae1, su2 , fl1, fl2, and du2) give kernel phenotypes that are difficult to distinguish from the normal kernels on a segregating ear.64 In these instances, the genotype of a line is best confirmed by allele testing with a line where the mutant produces a strong phenotype. The variation with genetic background can be due to quantitative genes differing between lines that are accumulated during the breeding process or possibly to a fewer number of modifier genes, each of which might have only small effects on the expression of a major gene. This variation can be of significant importance in the utilization of the genes in specialty corn production. For example, the apparent amylose content of ae1 lines was increased significantly by 20% (50% apparent amylose to 70%) by selection.65,66 The differences in amylose content have been proposed to be due to one or few modifier genes selected by the breeder.67 In sweet corn breeding, a specific modifier of the su1 gene has been identified, named, and originally mapped to chromosome 4.68–70 This modifier, sugary enhancer1 (se1), is difficult to positively identify in all su1 backgrounds, but usually se1 has been associated with a lighter kernel color and a slower kernel drying rate on ears segregating for se1.69,71 In addition, su1 has been shown to affect sucrose and maltose concentrations in developing kernels. Recent Restriction Fragment Length Polymorphisms (RFLP) analysis has shown that a Quantitative Trait Loci (QTL) controlling these traits maps to the distal portion of the long arm of chromosome 2.71 Numerous sweet corn cultivars have been developed using this modifier (Chapter 6). Modifier genes also have been selected in mutants affecting kernel proteins. The kernels of o2 are of poor and unacceptable quality due to their soft, low-density kernels.72–74 By selection, modifiers of o2 which give hard kernel types have been developed.75 Two modifier loci have been identified by genetic analysis.76,77 These modifiers seem to specifically increase the synthesis of gamma-zein proteins in the kernel.78–82 While these modifiers also increase gamma-zein synthesis in normal endosperm (O2), this increase does not modify the kernel phenotype.83 One of these modifiers may be the (o15) locus.84 In addition to examining kernel phenotypes of single genes, many of the genes have been combined in sets of two or more genes. These multiple gene combinations show a number of genetic interactions (Table 1.3). These include epistasis, in which the kernel phenotype of the multiple gene combination is indistinguishable from the phenotype of one of the single genes. For example, bt2 is epistatic to su1 and kernels of the double mutant bt2 su1 are shrunken. In the double mutant su1 wx1, su1 is epistatic to wx1 and the kernels are wrinkled and glassy. Other gene combinations have been described as showing complementary interactions, in which the phenotype in the multiple genotype is distinct from the phenotype of either single gene in the combination.56 The double mutant ae1 su1 is a good example of a complementary interaction. The ae1 su1 kernels are translucent and glassy but not as wrinkled as su1 kernels or as full as ae1 kernels. As additional mutant genes are added to the genotype, the phenotype generally becomes more severe. As a result, many of the phenotypes of the multiple gene combinations are difficult to clearly differentiate. Earlier, Boyer and Shannon2 divided the mutants into two classes. Class One mutants have the most extreme phenotypes and include the brittle and shrunken genes. These genes are epistatic to other genes or Class Two mutants in multiple genotypes. Within Class Two mutants, various forms of complementary or epistatic gene action are observed.56 However, when multiple Class Two mutants are combined, the phenotype becomes more extreme as additional recessive alleles are added to the genotype. In many instances, the phenotypes of kernels with multiple Class Two mutants are as extreme as Class One phenotypes. It should be noted that epistasis only is being © 2001 by CRC Press LLC TABLE 1.3 Examples of Gene Interaction in Double and Triple Mutant Gene Combinations for Genes Affecting Kernel Carbohydrates and Proteins Genotype ae1 bt1 bt2 su1 sh1 su1 o2 sh ae1 sh2 wx1 su1 wx1 ae1 su1 ae1 o2 fl1 sh2 du1 fl1 ae1 su1 su2 ae1 du1 wx1 a b c Interaction epistasis (bt1)b epistasis (bt2) complementary epistasis (sh1) epistasis (sh2) epistasis (su1) complementary epistasis (o2) epistasis (sh2) epistasis (f11) complementary complementary Mature Kernel Phenotypea shrunken, opaque to tarnished shrunken, translucent to tarnished extremely wrinkled, glassy with opaque sectors collapsed, opaque shrunken, opaque wrinkled, glassy to opaque not as full as ae, translucent (tarnished in S.C.c) may have opaque caps opaque, tarnished sectors in W23 × L317 background shrunken, opaque opaque partially wrinkled, translucent to tarnished shrunken, opaque to tarnished; S.C. semi-collapsed opaque Based on Garwood and Creech.56 Gene expressed is given in parentheses. S.C. Sweet corn. Source: From Hallauer, A.R., Specialty Corns, CRC Press, Boca Raton, FL, 1994. With permission. considered for the phenotype of the mature kernel. Epistasis at the level of kernel composition is described in Sections IV A and D. As the kernel phenotypes become more extreme, it becomes more difficult to reliably identify the kernel genotypes based on phenotype. This difficulty is further compounded by the frequent occurrence of dosage effects in multiple gene combinations.85,86 In this regard, the triploid nature of the endosperm is important. The triploid condition derives from the fertilization of two 1n polar nuclei in the egg sac by one 1n sperm nucleus from the pollen. As a result, the endosperm can have four genotypes when two alleles are segregating at a locus, e.g., Wx1 Wx1 Wx1, Wx1 Wx1 wx1, Wx1 wx1 wx1, and wx1 wx1 wx1. When only the wx1 gene is segregating, the first three genotypes produce kernels with the normal phenotype and the expected 3:1 genetic ratio (3 normal kernels to 1 wx kernel) is observed. However, if the kernel is homozygous for a second recessive allele (i.e., ae1), each of the four wx1 genotypes sometimes produce “slightly different” phenotypes, and the different phenotypes become difficult to distinguish.85 Similar results are observed when ae1 is segregating in a homozygous wx1 background. When both wx1 and ae1 are segregating on the same ear, many phenotypes are observed (16 genotypes are expected). These dosage effects, as well as the lack of distinct phenotypes in many multiple gene combinations, necessitate that the genotypes of these gene combinations be confirmed by tests of allelism. We recommend that the tests be performed for all genes thought to be present in the genotype. By making appropriate choices in selecting for multiple genotypes, the researcher can often improve the chances of success for obtaining the desired genotype. This is frequently done by the selection of kernels with the phenotype of the least extreme single mutant from the segregating ear. For example, in the case of the joint segregation of ae1 and wx1, only 1 of 16 of the kernels would be expected to be the double mutant ae1 wx1. Because errors in selection of ae1 wx1 kernels would be expected due to the dosage effects discussed above, many of the selections will continue to segregate. By selecting wx1 kernels from the segregating ear, an average of two out of three of these kernels would be expected to be heterozygous for ae1 while homozygous for wx1. In the next generation, an ear of wx1 kernels segregating for ae1 would be produced. This ear would have one fourth of the kernels ae1 wx1. In addition, the wx background is clearly tested on the ear and the uncertainties that may arise from ear-to-ear or line-to-line variation can be reduced. Again, proof of the final genotype should be confirmed by appropriate allele tests. © 2001 by CRC Press LLC Multiple combinations of genes influencing the protein fractions have also been developed. As with the single mutations, the kernel phenotypes are opaque. Various types of interactions are seen when the protein mutants are combined with the mutants affecting kernel carbohydrates (Table 1.3). The Class One mutants that affect kernel carbohydrates are epistatic to the protein mutants for kernel fullness. However, in some instances (i.e., sh1 o2) the double mutant is clearly distinguishable from either single mutant (Table 1.3). Phenotypes of double mutants of Class Two carbohydrate mutants and protein mutants are generally opaque or opaque with tarnished sectors (Table 1.3). 2. Allelic Variation The genetic analysis of newly occurring mutations resulted in the identification of multiple alleles at many of the gene loci. Often, the phenotypes of these new alleles were indistinguishable from the phenotypes of existing alleles. In some cases, these alleles have been shown to be similar with regard to their effects on the chemical composition of the kernel as well.87,88 Some allelic series, however, include novel alleles, which are now being exploited for gene cloning and may have potential for specialty corn. The most powerful alleles for gene cloning are those which result from the insertion of a transposable element in the coding regions of a gene or near a gene.89–94 These alleles are often unstable and produce a variegated phenotype. In addition to being important to unraveling the events involved in transposition of the element, these alleles have provided the means to isolate genes without complete knowledge of the biochemical/metabolic function of the gene (see Section IV). Stocks of Robertson’s mutator also have proven a rich source of new variation. In other instances, alleles of a particular gene have been shown to have distinct kernel phenotypes and composition. Interestingly, these alleles have been relatively unused in specialty corn breeding. The waxy1 gene locus was thoroughly investigated by Oliver Nelson and his associates. He identified and collected many alleles at this locus. In some cases, these alleles were functionally intermediate and had chemical phenotypes between waxy1 and normal kernels. For example, kernels homozygous for wx1-a allele were shown to have 4 to 5% amylose as compared to 0 and 25% amylose in wx1-c (the standard allele) and normal kernels, respectively.95 To our knowledge, no attempt has been made to use these alleles in specialty corn production. Hannah and Bassett96 reported that an allele of brittle1, bt1-A, had unusually good germination for these Class One type mutants. At the same time, the sugar levels of this allele were as high as other supersweet corn types. These properties were suggested to be of potential use for the development of supersweet corn with improved field germination. Again, no commercial use of this allele has occurred at this time. The laboratory of Hannah identified a number of alleles that resulted from the mutation of shrunken2 containing the transposable element dissociation (Ds).97 When Ds is removed from the gene, novel alleles arise when three to six new base pairs remain in the gene. One of these alleles, sh2–Rev 6, produced seed with 11 to 18% greater weight. The observed phenotype was explained by modified allosteric properties from the enzyme ADP-glucose pyrophosphorylase, which is partially encoded by sh2 (see Section IV). Similar results have been observed in potato transformed with allosteric-modified ADP-glucose pyrophosphorylase from E. coli.98 The potential for sh2-Rev 6 to increase yield is being investigated at this time. As additional molecular analysis of different genes occur, it is likely that variations in “wild-type” and natural alleles will become increasingly obvious. A number of examples at the O2 locus have recently been reported.99,100 However, the practical value of these alleles for specialty corns remains to be demonstrated. The most dramatic variation of alleles has been observed at the sugary1 locus.60,101–104 Alleles of su1 can range from glassy wrinkled to almost normal in phenotype (Table 1.4). To date, the alleles at the sugary1 locus fall into three distinct classes. Twelve of these alleles collected at Pennsylvania State University by Roy Creech have phenotypes indistinguishable from the su1-Ref allele and have easily identifiable glassy wrinkled kernels. Three alleles, su1-am, su1-66, and su1P, have full kernels that are indistinguishable from the normal or wild type kernels. These alleles © 2001 by CRC Press LLC TABLE 1.4 Allelic Variation at the sugary1 Locus of Corn Compositiona (mg/g dry weight) Allele Su1-Ref Su1-st su1-Bn2 su1-am a Name reference starchy brawn2 amylaceous Mature Kernel Phenotype wrinkled, glassy wrinkled, glassy crown full wrinkled, glassy less extreme then su-Ref full — near normal Sucrose 245 124 177 78 Starch 77 191 241 356 Phytoglycogen 130 122 55 4 20 days after pollination; adapted from Garwood and Vanderslice.101 Source: From Hallauer, A.R., Specialty Corns, CRC Press, Boca Raton, FL, 1994. With permission. are best observed in double mutant combinations with su2 and du1. In these combinations, the kernel phenotypes included glassy wrinkled (su1-66 su2) and extremely shrunken with opaque sectors similar to Class One mutants (du1 su1-P) and collapsed glassy (su1-P su2 and su1-am su2). Two alleles, su1-st and su1-Bn2, are intermediate to the full and standard sugary phenotypes. The phenotypes of these kernels are more variable and generally include a partially wrinkled crown of the kernel. The chemical composition of these kernels varies with the physical phenotype as well (Table 1.4). One study evaluated the potential of the su1-Bn2 allele for improved silage corn.105 It was reasoned that the intermediate phenotype of this mutant would provide enhanced nutritional value to the silage (primarily available carbohydrate). A more recent study found no improvement of su1-Bn2 over dent corn as a part of the alfalfa silage mix for dairy cows. In fact, this study reported adverse effects on ruminal forage digestion.106 B. GENES AFFECTING KERNEL COLOR The appearance of mature kernels can range from white to almost black. Two pigments are primarily responsible for determining the color of the corn kernel. The carotenoids are primarily associated with the yellow color of the kernel. Two classes of carotenoids are the carotenes and xanthophylls. Carotenoids are important as sources of vitamin A in the diet and xanthophylls as feed sources for the yellow color of poultry skins and egg yolks.107 The major carotene is B-carotene.108 The most abundant xanthophylls of the kernel are lutein and zeaxanthin.108,109 The second class of kernel pigments is the anthocyanins, which are red to blue in color. Anthocyanins are flavonoid compounds of which cyanidin, pelargonidin, and peonidin are found in the kernel. Additional variation of the anthocyanins is derived from different types, combinations, and degree of hydroxylation, glucosylation, methylation, and acylation.110 Reviews and extensive treatments of the genetic control and biochemistry of these pigments in corn have been recently published.12,110,111 Therefore, only the major genetic variation will be considered here. Many genetic loci determine the color of corn kernels. Based on the results of numerous researchers, the genes modifying anthocyanin content of the kernels have been ordered in the pathway of synthesis.110–112 These gene orders are based on both biochemical and genetic studies. Based on the placement of a mutant, color and pigment content can be explained. Several loci (C1, colored aluerone1; c2, colorless 2; a1, anthocyaninless1; a2; and R1, colored1) condition the absence of color if a homozygous recessive genotype is present for any of these loci. Some of these loci appear to be regulatory, based on numerous criteria, whereas other loci have been identified as structural genes for specific enzymes in the biosynthetic pathway.111 Many alleles have been identified for these loci, and these alleles not only modify anthocyanin accumulation in the kernel but other plant tissues as well.12,30 The effects of the loci bz1, bronze1, and Bz2 are seen when the dominant alleles of the loci above are present. The homozygous recessive condition at bz1 or bz2 results in brown kernels, while kernels of the double mutant are colorless.12 Quantitative variation in color has also been described at the genetic level. The recessive allele of In1, intensifier1, © 2001 by CRC Press LLC increases pigmentation when homozygous.30 The dominant pr1 (red aluerone1) conditions a high ratio of cyanidin glucoside to pelargonidin glucoside.30,113 In addition to accumulating in the kernel, carotenoid pigments are also important components of photosynthetic tissues. Coe and Neuffer12 classified 117 loci affecting photosynthetic pigments including the carotenoids. In this classification, mutants are classified on endosperm color (yellow vs. pale or white), embryo dormancy (dormant vs. viviparous), seedling color (green, yellow–green, yellow, white, etc.), and plant color (green, yellow–green, etc.). Four loci (Y1, yellow1; y8; c1, white cap1; and Bn1, brown aluerone1) have mutant phenotypes that are white or pale kernels, dormant seeds, and green seedlings and plants. When the recessive alleles are homozygous for y1, y8, or bn1, the kernels are white or pale yellow.114 In contrast, Wc1 is a dominant mutation and gives kernels with white crowns and pale yellow endosperm when the Y1 allele is also present. Other mutants affecting kernel carotenoids also have white or pale yellow endosperms. In addition, these mutants affect seed dormancy, and seedling and plant color.12 Many of these mutants are viviparous (precocious germination), including y7, y9, vp2 (viviparous-2), vp5, and w3 (white-3).12,30 One viviparous mutant, vp, has a similar pleiotropic effect on anthocyanin accumulation in the kernel.30 The pleiotropic effects can be explained in part by a common early pathway for the carotenoids and abscisic acid.115,116 Finally, mutants including y10, cl1 (chlorophyll), and the lemon white mutants (lw1, lw2, lw3, and lw4) have albino or pale yellow seedlings that die after germination.12 C. GENES AFFECTING KERNEL DEVELOPMENT In addition to the mutants described that have abnormal dormancy, mutants frequently have been identified which affect early kernel development.47,53,117–121 These mutants can affect embryo and endosperm development (defective kernel, dek, mutants), endosperm development, (defective endosperm, de, mutants), and embryo development (embryo specific, emb, mutants). Many of the dek mutants alter auxin and cytokinin levels, thus changing endosperm development.122 Some mutants have been given more descriptive names based on phenotypes.123 Because most of these genes are lethal and the mutant must be maintained as a heterozygote, many of the mutants identified in earlier studies have not been maintained. Recently, Nueffer and Sheridan124–129 have assembled a large number of samples of embryo development in different mutants after mutagenesis. These mutants affect embryo development at many different times and in different ways. Some of these mutants can be “germinated” by placement of immature embryos in culture. Those mutants showing some development under these conditions were termed “nutritional mutants,” while mutants failing to show some development under these conditions were termed “developmental mutants.”101 From this work, 51 embryo-specific mutations have been identified.130 The mutants affecting endosperm development have also been shown to differentially affect development in other tissues. The miniature seed1 (mn1) mutant is characterized by premature breakdown of the placental–chalazal cells at the base of the kernel around 14 days after pollination.53 This breaks the continuous bridge between the maternal and endosperm cells. This developmental breakdown, in turn, may be the result of osmotic damage from an invertase deficiency in maternal and endosperm cells.131 Other mutants delay the initiation of dry matter accumulation or reduce dry matter accumulation at various stages of kernel development.132,133 These variable mutant effects help to describe the genetic control of endosperm development. IV. BIOCHEMICAL ANALYSIS A. KERNEL CARBOHYDRATES The mature corn kernel is composed of over 70% starch. By far most (80% to 90%) of this starch is found in the endosperm, which comprises about 80% of the total kernel dry weight. Starch is a homopolymer of glucose. The molecular structure involves only two linkages of the glucose © 2001 by CRC Press LLC molecules: α-1,4 and α-1,6. A majority of the linkages are α-1,4; these linkages make up a linear arrangement of the molecules. The small amount of α-1,6 linkages provide a branching pattern to the molecule. Two distinct types of polymers are found in most starches. Amylose is essentially a linear molecule of α-1,4 linked glucose. Both linear and slightly branched amylose molecules are found in starch.134–136 In the branched amylose molecules, the long stretches of linearly linked glucose are interrupted with a branch (α-1,6 linkage) about every 200 glucose units. In general, amylose molecules are highly dispersed in molecular weight. They range in size from 100 to 1000 glucose subunits, with the larger molecules usually containing a few α-1,6 branch points.134 Amylopectin molecules are much larger than amylose and contain up to 200,000 glucose subunits. About 4% to 5% of the total glucose units of amylopectin are linked α-1,6. The linear regions of the molecule are short chains of various sizes with two populations of chain sizes of 10 to 20 glucose units and 30 to 50 glucose units.137 This arrangement of the chains gives amylopectin a cluster-like structure.138 The reader is referred to Chapter 2 for further details of the structure and functional features of starch. Although starch, on initial examination, is a fairly simple polymer to understand, the synthesis of starch is still a subject of intense study. Several factors contribute to the complexity of the process of starch synthesis. These include the synthesis of starch as part of a megamolecular assembly, the starch granule. The starch granule is cold-water insoluble, while the majority of the enzymes involved in starch synthesis are soluble. The granule is composed of large numbers of amylose and amylopectin molecules (as many as 109 and 107, respectively). Furthermore, these molecules are regularly arranged, giving the granule stability and crystalline properties. Starch is not synthesized in the cytosol of the cell, but within the subcellular organelles, the amyloplasts. These doublemembrane-bound organelles compartmentalize the process of synthesis, allowing only certain precursors for starch entry into the amyloplasts. As a result, the complete process of starch synthesis and accumulation in the corn kernel involves not only the direct synthesis of starch from glucose, but also the integrated metabolism of the sugar intermediates. The complex integrated pathway of starch synthesis accounts for the large number of genetic loci affecting kernel carbohydrate composition. As these loci are expressed in a coordinated way during development, it is not surprising that complete complex interactions between mutants may occur.139–141 Furthermore, it is the complete understanding of the nature of starch synthesis and all the associated genetic loci mutants that will allow the future manipulation of the process for the development of specialty corns based on starch types. Many reviews have proposed pathways and described the enzymes of starch biosynthesis.14,16,18,23,27–29 We previously proposed looking at the pathway as containing two types of reactions (Figure 1.1).2 In this approach, early reactions in the pathway are involved in the interconversion of sugar intermediates and the synthesis of the immediate substrate for starch synthesis, ADP-Glc. These reactions are often controlled by the Class One mutants discussed above which have extreme kernel phenotypes. Many of the reactions as drawn in the pathway are, in fact, catalyzed by different enzymes found in the cytosol or amyloplast.142 The second part of the pathway includes the reactions directly involved in the synthesis of the starch molecules from ADP-Glc. Two basic reactions are involved. The starch synthase enzymes transfer a glucose moiety from ADP-Glc to the nonreducing end of an α-1,4 chain of the starch molecule, thus increasing the length of a linear portion of the molecule by one glucose unit.143,144 The starch branching enzymes form the α-1,6 branch points of starch by breaking an internal α-1,4 linkage from a linear chain. The released chain (six or seven glucose units long) is then transferred to another chain by the formation of the α-1,6 linkage.145,146 These enzymes have been shown to be the gene product of the Class Two mutants, such as ae1 and wx1. The less extreme phenotypes of these mutants can be explained by the observation that the starch synthases and branching enzymes exist as multiple forms. Therefore, the elimination of an enzyme activity by a given mutant only partially reduces the total activity. The epistatic masking of the phenotypes of Class Two mutants by Class One mutants is explained by the early position in the pathway of the enzymes encoded by Class One mutants. The © 2001 by CRC Press LLC CLASS TWO MUTANTS CLASS ONE MUTANTS Sucrose sh1 mn1 UDP 1 Amylose 6 UDP Fructose Glucose iP ATP ATP 3 2 ADP ADP G-6-P iP (10) su1 ADP UDPG1c 9 wx1 2 3 iPP F-6-P Phytoglycogen 7 UTP 4 10 ADPG1c 5 sh2 bt2 G-1-P ATP 10 12, 13 ae1, su1 8 iP 12 du1 su1 12 (10) ADP Amylopectin 11 sh4 FIGURE 1.1 Hypothetical scheme for the formation of starch and phytoglycogen from sucrose in corn kernels. Location of various mutants affecting carbohydrate accumulation in the pathway is shown. These mutants may be structural genes for the enzymes indicated or have secondary effects on the enzyme activities. Enzyme reactions are indicated by number as follows: (1) invertase (EC 220.127.116.11), (2) hexokinase (EC 18.104.22.168.), (3) hexose-6-phosphatase (EC 22.214.171.124), (4) glucose phosphate isomerase (EC 126.96.36.199), (5) phosphoglucomutase (EC 188.8.131.52), (6) sucrose synthase (EC 184.108.40.206), (7) UDP-glucose pyrophosphorylase (EC 220.127.116.11), (8) ADPglucose pyrophosphorylase (EC 18.104.22.168), (9) starch granule bound starch synthase (EC 22.214.171.124), (10) soluble starch synthase (EC 126.96.36.199), (11) starch phosphorylase (EC 188.8.131.52), (12) starch branching enzyme (Qenzyme) (EC 184.108.40.206), (13) starch debranching enzyme (isoamylase-like) (EC 220.127.116.11). Modified from Boyer and Shannon.2 specific lesion of sh1 kernels is sucrose synthase (reaction 6). The enzymatic lesion of sh2 and bt2 is ADP-Glc pyrophosphorylase (reaction 8). As these reactions are in the earlier part of the pathway, the reduction of starch synthesis is seen regardless of the genotype at the Class Two loci. The sh4 gene was initially shown to have lowered activities of starch phosphorylases, which at one time was believed to be a starch polymerizing as well as a degradative enzyme.147 However, the lesion of sh4 has been located in the synthesis of pyridoxal phosphate.148 Pyridoxal phosphate is a cofactor for many enzyme reactions, including phosphorylase; therefore, sh4 kernels show multiple altered enzyme activities. Three additional points should be made and discussed from examination of the pathway presented in Figure 1.1. First, despite the extensive genetic analysis of corn during the past 90 years, mutants for many of the enzymes in the pathway have not been found by the isolation of mutants based on kernel phenotypes. Two possible explanations for this exist. First, the actual pathway may not require the enzymes as developed in this pathway. Some support for this view has been presented based on the studies of the enzyme phosphoglucomutase (reaction 5, Figure 1.1). This enzyme exists in two forms in the endosperm.142 One of the forms is found in the amyloplasts, while the other form is in the cytoplasm of the endosperm cell. Many standard inbred lines have been found to lack measurable activity of the cytosolic form of this enzyme.149 As the kernels of © 2001 by CRC Press LLC these inbreds are normal in appearance, the logical conclusion of these observations is that the cytosolic form of this enzyme is not necessary for the normal development of the kernel. A second possible explanation for the failure to identify mutants for many enzymes may be that the deficiency of these enzymes produces such a severe disruption of kernel development and starch synthesis that they prove lethal. Possibly some of these mutants may be identified in the dek or de mutants discussed in Section III C. Miniature seed is an example of a mutant with both developmental (placental–chalazal tissue breakdown) and biochemical (invertase, reaction 1, Figure 1.1) lesions.131 A second point is that Figure 1.1 also shows that more than one gene may be found to be associated with a particular reaction. Although many different suggestions have been made to account for these observations, most of the mutants can be explained by more than one structural gene for the enzymatic reaction. In the case of the enzyme ADPG pyrophosporylase, bt2 and sh2 kernels both contain about 10% of the activity found in normal kernels.150–154 Both of these genes encode a subunit for the functional enzyme which is thought to exist as a tetramer of the two large subunits (bt2) and two small subunits (sh2).155–157 The loss of a functional allele for either subunit reduces the observed activity. In the case of sucrose synthase, the sh1 mutant also reduces the enzyme activity to about 10% of the activity in normal kernels.158 In this case, a second enzyme is the product of a second gene, Sus1. The second gene was identified based on biochemical and genetic analysis. No mutant phenotype is known for this locus.159,160 The starch synthases and branching enzymes also are found to exist as multiple enzymes and, in some cases, the same form of these enzymes is found as both on soluble fractions and bound to the granule.161 In addition, each of the multiple forms of starch synthesis and starch branching enzymes have slightly different enzyme properties and thus are likely to play different roles in starch biosynthesis.162,163 The starch synthases are both soluble enzymes and starch-granule-bound enzymes.143,144,164–166 The wx1 mutant reduces the activity of the major starch-granule-bound enzyme.143 A minor starch-granule-bound starch synthase and the soluble starch synthases are not affected.144,167,168 Similarly, the starch branching enzymes have been shown to exist as two or three fractions.165,166,169 It is clear that two enzymes exist: branching enzyme I and branching enzyme II. Branching enzyme II also has been further divided into two fractions, branching enzyme IIa and IIb.165,166 These fractions have very similar properties including similar chromatographic affinities, amino acid composition, molecular weight, and peptide maps.165,166,170,171 The ae1 gene, however, only influences the activity of one of these fractions (branching enzyme IIb). The other branching enzyme activities are not influenced in ae1 kernels. 165,166 The effects of dosage of the recessive ae1 allele on branching enzyme IIb activity are consistent with the function of ae1 as a structural gene of this enzyme.172 Therefore, branching enzymes IIa and IIb are encoded by two very similar genes, only one of which is ae1.173 A third point from Figure 1.1 is that not all mutants have been clearly identified with a particular lesion at the biochemical level in spite of intensive efforts in many laboratories. In an earlier edition of this book, the biochemical explanations for bt2, du2, su1, and su2 were still being intensely researched and their roles in starch synthesis were being debated. The sugary1 locus of corn is probably the most extensively studied of any mutants due to its traditional importance in sweet corn cultivars. In the past 5 years, significant progress has been made toward understanding how su1, du1, and bt1 affect starch synthesis. Thus the biochemical lesion for only su2 remains to be explained. Starch from su2 kernels has increased amylose content and unusual thermal properties.174 As seen in the following discussion of su1, du1, and bt1, understanding the role of the su2 levels may provide some surprises and changes in the current views of starch biosynthesis. The major modification of the carbohydrate composition of su1 kernels is the large quantities of phytoglycogen that are accumulated.175–180 This phytoglycogen is similar to animal glycogens and is water-soluble. Over the years, several reports on a “phytoglycogen branching enzyme” in su1 kernels have appeared.181–186 These reports have appeared as recently as 1987.185 However, in our studies we have found this “phytoglycogen branching enzyme” in all genotypes whether phytoglycogen accumulates or not.165,166 More recently, Pan and Nelson187 reported that su1 kernels © 2001 by CRC Press LLC contain less starch debranching enzyme than corresponding normal kernels. This observation supported the earlier suggestion of S. Erlander188 that amylopectin was formed by the action of debranching enzyme on phytoglycogen. More careful characterization of the debranching enzymes in developing endosperm demonstrated that both pullulanase and isoamylase activities are present.189 Cloning and careful characterization of the enzyme product of su1 has clearly demonstrated that the su1 locus encodes debranching enzymes with isoamylase-type activity.190–193 The pullulanase activity is encoded by a separate gene, zpu1. While the direct activity of a debranching enzyme on phytoglycogen is not sufficient to produce amylopectin as originally suggested by Erlander,188 the action of the su1 debranching enzyme in combination with starch synthases and branching enzymes make up an enzyme complex responsible for amylopectin synthesis (see review195). The du1 mutant has been shown to decrease the activities of both soluble starch synthase and branching enzyme IIa activity.165 In these early studies, it was impossible to establish either of these enzymes as the gene product of this locus. The great interest in starches from various combinations of du1 and other kernel mutants5, 8, 9 justify future biochemical studies of this locus. The amylopectin fraction of starches from du1 endosperm have been shown to have shorter chain lengths than amylopectins from normal or wx1 endosperm in numerous studies.196,197 These observations are consistent with reduced starch synthesis activity. More recently, the cloning of the du1 gene has already demonstrated that the gene encodes the major activity in one of the two soluble starch synthase fractions in developing corn endosperm.197–199 Characterization of the bt1 locus has presented challenges and unexpected findings. Starch accumulation in kernels of this Class One mutant only reaches 25% of the starch of normal kernels.200 An early report showed that bt1 kernels were reduced in the activity of an enzyme that is capable of the formation of a short oligosaccharide from glucose-1-P.201 This enzyme has been suggested as a key reaction in the formation of the initial oligosaccharides for the initiation (priming) of starch synthesis and a mutant in this enzyme would be expected to be a Class One mutant.202 However, other priming reactions of starch synthesis have been proposed. For example, the primer for glycogen synthesis, glycogenin, has been identified.203 Glycogenin is a self-catalytic protein that provides a protein primer for glycogen synthesis. A similar protein has been reported from phytoglycogen from sweet corn.204 The role of this protein (or related proteins) in starch biosynthesis remains to be elucidated. These reactions do not, however, appear to be the function of the bt1 gene product. Cloning of the gene showed partial homology with genes encoding proteins which transport adenylate across plastid membranes.94 Other studies had demonstrated an adenylate translocator in maize amyloplast membranes.205 Subsequently, it was shown that membranes from amyloplasts from bt1 endosperm lacked three major related proteins206,207 and that the bt1 protein was target to the inner membrane of chloroplast.208 As the major substrate for starch synthesis, ADP-glucose was thought to be synthesized in the amyloplast, an obvious explanation for the extreme phenotype of bt1 difficult to account for initially. However, at the same time, other researchers found that ADP-glucose pyrophosphorylase subunits encoded by sh2 and bt2 were not modified after translation as would be expected of proteins transported into the amyloplast.209 Thus the activity of this enzyme was located both in the cytosol and amyloplast.210 Examination of sugar intermediates in bt1 kernels revealed that these kernels contained 10 times the amount of ADPglucose as wild-type kernels.211 Thus the bt1 kernels have reduced starch synthesis due to the failure of ADP-glucose synthesized in the cytosol to be transported into the amyloplast. These unexpected findings demonstrate the continued need for research on starch synthesis and critical examination of proposed pathways. The pathway presented in Figure 1.1 is also useful in providing possible explanations of the changes in the quantity and type of carbohydrate accumulating in kernels of different genotypes. It is these changes which provide the basis for the use of various mutants for the production of specialty corn starches. An extensive review of the effects of various mutants and mutant combinations on kernel dry weight, starch granule size, and glucan composition (amylose, amylopectin, © 2001 by CRC Press LLC and phytoglycogen) is available.23 Therefore, we will only consider some of the key interactions and related points. The most consistent effect of various genotypes on kernel dry weight and starch granule size is a reduction of these in the mutant kernels compared with normal kernels. Notable exceptions are wx1 kernels and starch granules, which have similar dry weights and size as normal kernels and starch granules. In this regard, it is important to consider that the amylose and amylopectin fractions of the starch are synthesized in separate processes. As wx1 blocks the less efficient pathway, amylose synthesis, there seems to be sufficient potential of the amylopectin synthetic pathway to compensate for the lost synthesis due to wx1. Therefore, amylopectin synthesis in wx1 kernels is actually increased. The separation of amylose and amylopectin synthesis also explains the epistatic action of wx1 in combinations with other mutants at the level of starch composition. All double and triple mutant combinations including wx1 contain no amylose.54,212,213 Early reports on the double mutant ae1 wx1 suggested that this starch did, indeed, contain amylose.54,212,214,215 However, these results were based on the measurement of amylose content using the blue value or a procedure based on iodinebinding of the starch. Subsequent analysis revealed that ae1 wx1 starch was, in fact, composed solely of amylopectin, but this amylopectin had longer chain lengths than normal or wx1 amylopectin.216–219 This structure of the amylopectin results in a higher iodine binding, thus an overestimate of amylose based on this property of the starch. The structure of amylopectin in starch from ae1 kernels is similar in structure to the structure of amylopectin from ae1 wx1 kernels. The ae1 kernels also contain the more obvious increased level of amylose. In ae1 kernels where the efficiency of the amylopectin pathway is reduced, increased synthesis of amylose, therefore, occurs in a separate pathway. However, unlike the case in wx1 kernels, this increased synthesis of amylose in ae1 kernels is not sufficient to compensate for the lost amylopectin synthesis, and starch levels and kernel dry weight are decreased. The relationship of amylose and amylopectin synthesis and the synthesis of phytoglycogen need to be considered as well. Neither the simple concept of amylopectin being the precursor to phytoglycogen nor phytoglycogen being the precursor to amylopectin seem to be valid. Studies of the development of su1 kernels have shown that some phytoglycogen seems to be derived from the mobilization of starch granules (amylose and amylopectin).220 However, additional synthesis and polymerization of phytoglycogen also is likely to occur. Detailed studies of the glucans from su1 kernels have revealed a range of truly soluble molecules and particulates which could be removed by increasing speeds of centrifugation. These particulate fractions contained less phytoglycogen and more amylose and amylopectin as the size of the particulates increased.175,178 Hence, these particulates likely represent various fractions of starch granules being mobilized to phytoglycogen. In the double mutant su1 wx1, phytoglycogen synthesis is slightly enhanced compared with su1 kernels.221 In contrast, ae1 is epistatic to su1 for phytoglycogen, and synthesis in the kernels of the double mutant ae1 su1 is greatly reduced.222 These interactions can be explained by the relative changes in the multiple activities of branching and debranching enzymes. In addition, the starch granules in the different genotypes are very different.220,223 In particular, high amylose granules have been shown to be relatively resistant to amylase action in comparison with normal and waxy1 starch granules.224 This relative inert nature of the starch granules from ae1 genotypes may be interfering with the mechanism leading to mobilization of the starch granule during phytoglycogen formation. Although extensive studies on the properties of starches (and sugars) from kernels of various genotypes have been reported,54,55,212,215,223–229 the reader should be aware that variation between genetic backgrounds has also been reported.218,230–233 Most reports agree on the major properties of starches from a given genotype. However, comparison of various reports often show a range in the values of the different properties of the starch studied. In some instances, this range of variation may be attributed to different methodologies employed by the researchers. In recent studies on the various features of the starch, starch granule size and morphology, amylose content, amylopectin chain-length, and amylopectin molecular weight have been shown to vary as a result of the genotype © 2001 by CRC Press LLC and the interaction of genotype and inbred line.219,230–233 Furthermore, these properties could be correlated with the physical-functional behavior of the starches, including the temperature and energy required to swell the starch in aqueous systems.230–233 Like the variation in mature kernel phenotypes for different genotypes in different backgrounds (Section III A), this variation in starch properties can be explained by either quantitative variation or modifier genes. The role of selection and modifier genes in amylose content of ae1 kernels has already been discussed.65–67 Another example of a modifier is the se1 gene, which is a modifier of su1. The effects of se1 are seen in the sugar content of immature se1 su1 kernels, which are to two to three times the sugar levels of immature su kernels.234–236 Certainly, the variation due to quantitative loci and modifier genes will be difficult to fix in specialty corns. However, with appropriate selection this variation should allow the plant breeder to fine-tune the properties of future cultivars. B. KERNEL PROTEINS The proteins of the kernel are traditionally classified based on their solubility in different solvents as initially described by Osborne237 and later modified by Landry and Moureaux.238 Different researchers have applied varying conditions for the solubilization of proteins. As a result, differences in composition and complexity of the various fractions are in the literature.15,25,26,239–244 The albumins are soluble in water and comprise about 7% of the whole kernel nitrogen. Globulins are soluble in salt solutions and comprise about 5% of the kernel nitrogen. The prolamins are soluble in alcohol with or without added reducing agents and comprise about 52% of the kernel nitrogen. Finally, the glutelins are soluble in dilute alkali and comprise about 25% of kernel nitrogen. The major protein class, the prolamins, are collectively referred to as the zeins. These proteins contain large amounts of the amino acids glutamine, proline, leucine, and alanine, and are of relatively poor nutritional quality.244–248 The zein proteins are typically separated after selective solubilization by polyacrylamide gel electrophoresis. This analysis reveals molecular weight heterogeneity with major components of MWs of 10, 14, 16, 19, 22, and 27 kD. Further heterogeneity is revealed by isoelectric focusing and gel electrophoresis.249–253 One classification of zeins is primarily by molecular weight.253 In this system, zeins are classified as A-, B-, C-, D- and E-zeins (21–26 kD, 18–24 kD, 13–18 kD, 9–11 kD, and 28 kD, respectively). More recently, the zeins have been classified based on the relatedness of their primary gene and amino acid sequences.78,239,254–256 These studies classify the zeins as α-(22 and 19 kD), β-(14 kD), γ-(27 and 16 kD), and δ-(10 kD). The α-zeins are by far the most abundant fraction and comprise up to 80% of the total zeins. Zeins are synthesized by membrane-bound polyribosomes and transported to the lumen of the endoplasmic reticulum.257,258 In the lumen, the zeins are assembled into insoluble protein bodies. Variation in the relative composition of the protein bodies in the aluerone and endosperm has been demonstrated.259 The small protein bodies in the subaluerone contain β- and γ-zein and the larger protein bodies of the endosperm contain α- , β-, and γ-zeins. The genes encoding the storage proteins are present as multiple copies; early estimates for the number of genes encoding the zein proteins ranged up to 100 genes.260–268 It is not known whether all the genes are active in the synthesis of the zein protein, and different genes may be expressed at different levels.269 The genes are distributed on several chromosomes in the corn genome. To date, 20 genes have been mapped to both arms of chromosome 4 and the short arm and long arm of chromosomes 7 and 10, respectively.270–272 With further analysis, the relationship of gene families has become clearer. The α-zeins are encoded by a large gene family. In contrast, the β-, δ-, and γzeins are encoded by one or two genes. The mutants affecting proteins of the kernel have been shown to affect zein levels to varying degrees.88,241,273–280 The reduction in zeins in the mutant kernels is 20 to 50% of the level in normal kernels. Synthesis of all zeins are reduced in fl2, Mc, and o6 kernels. In contrast, o2 kernels and De*-B30 kernels have more pronounced reductions of the 22 kD α-zeins, and o7 kernels are greatly reduced in the 19 to 20 kD α-zeins. Although the various zein components are reduced in the © 2001 by CRC Press LLC mutant kernels, it is still possible to identify all the different subunits (families) of zein. As a multigene family, mutation of a single α-zein gene would be expected to have little overall affect on the phenotype. Therefore, unlike the mutants affecting carbohydrate metabolism which usually encode enzymes of the biosynthetic pathway, the mutants influencing kernel proteins that have been identified based on kernel phenotypes are not structural genes for the storage proteins. These genes are regulatory, and the mutants affect the expression of complete sets of related genes encoding a particular class(es) of zein proteins. Studies of the mutants have established that the amounts of zein mRNAs are decreased in mutant kernels.275,281 Likewise, transcription of zein proteins is reduced in the mutants.70 Various types of interaction of the mutants have been observed, and mutant effects are correlated to a “regulatory protein” called b-32.280 More recently, the mechanism of the o2 mutant has been described at a molecular level. The Opaque2 protein is localized in the cell nucleus.282 The o2 locus encodes a DNA-binding protein with a leucine-binding motif.283 This protein can bind the 5′ flanking sequences of the genes encoding the 22 kD α-zeins and acts as a transcriptional activator.284 As this binding is thought to be part of the normal transcription process involving the o2 encoded protein and other transcription factors, a mutant would be expected to reduce but not completely eliminate transcription of multiple α-zein genes. The fl2 mutation is associated with reduced synthesis of several classes of zeins.275 This observation has resulted in speculation that the fl2 mutation, like o2 was involved in transcriptional regulation. However, fl2 has several additional effects, including reduced membrane-bound polysomes,275 the appearance of a novel 24 kD zein protein,208 irregular-shaped protein bodies,285 and elevated levels of a 70 kD cytoplasmic reticulum protein with homology to the chaperonin BiP.286 Recently, the production of the novel 24 kD zein has been shown to be the result of a defective signal peptide in a 22 kD zein transcript.287,288 This novel 24 kD protein would explain the altered protein bodies, while the alteration in the normal secretory process could result in the other associated changes in membrane polysomes and housekeeping proteins.288 Biochemical explanations for other protein mutants remain to be elucidated. C. KERNEL LIPIDS The amount and composition of lipids in corn kernels is also under genetic control. Corn kernels contain approximately 4.4% oil. In contrast, the Illinois High Oil line contained 20.4% oil after 85 generations of mass selection.108 The increase in oil from 4.7% in the original population has been continuous over the course of selection and clearly demonstrates the potential genetic variability in corn for oil quantity. More recent studies have utilized NMR for rapid screening of kernels for oil content. Using this method, oil content was increased from 4.0% to 9.1% in seven generations.289 Triacylglycerides are the major component of commercial corn oil, although a wide variety of other lipids are found in the kernel.108 The triacylglycerides contain a mixture of saturated and unsaturated fatty acids. Corn oil is generally accepted as high quality as determined by a high linolenic acid (50%) and low linolenic acid (1%) content. Other fatty acids including oleic (40%), palmitic (12%), and stearic (2%) acid also are found in corn oil. A number of different approaches have been used to demonstrate genetic variation for the fatty acid composition of the kernel. A survey of a wide range of germplasm sources showed that different lines ranged in composition from 14 to 64% for oleic acid and 19 to 71% for linoleic acid,290 while exotic breeding materials were shown to have wide variation in fatty acid composition.291 Analysis of U.S. commercial corn oil showed that the linoleic acid content increased from 55.8% to 62.8% in a 12-year period.292 Most of this variation was attributed to changes in parental inbreds during the same time, although no direct selection for oil composition is practiced in most breeding programs. A single-gene linoleic acid1 with a recessive allele, ln1, which conditions high linoleic acid levels, was identified in genetic studies involving Illinois High Oil corn.293 Single-gene inheritance has also been identified in other reports.294–296 Other studies using monosomic lines © 2001 by CRC Press LLC have identified genes controlling oleic and linoleic acid composition on chromosomes 1, 2, 4, and 5.297–299 High stearic acid and high oleic acid contents were reported to be under the control of one major gene.300,301 In essentially all studies, researchers suggested that major gene effects were being modulated by modifier genes for oil composition. Although it seems that sources of major genes for composition of corn oil can be utilized, other studies indicate that the inheritance of oleic, linoleic, palmitic, and stearic acid content when considered together is complex and under multigenic control.302–305 Additional studies are needed to more precisely identify the genes and enzymes involved in determining the composition of corn oil. D. INTERACTION IN THE ACCUMULATION OF KERNEL COMPONENTS The accumulation of starch, protein, and lipids during kernel development is not totally independent. The correlated effects of various mutants and mutant combinations have been the subject of numerous studies.306–309 Notably, the mutants affecting kernel carbohydrates also reduce the amount of zein accumulated in the kernel.25,306 Furthermore, the reduction of zein content was further enhanced in double mutants of o2 and the starch mutants, with kernels of the double mutants having less zein than kernels of either single mutant. However, kernels of the double mutants bt2 fl2 and sh2 fl2 have the same amount of zein as kernels of the single mutants bt2 and sh2. Conversely, o2 kernels have reduced levels of starch as well as a major reduction in zein content.310 The mutants affecting kernel proteins and carbohydrates alter the osmotic potential of the immature kernels in a similar fashion. The accumulation of free amino acids in the protein mutants and sugars in carbohydrate mutants increases the osmotic potential of the endosperm. Under these conditions, water flow into the kernel is favored, but movement of osmotically active sugars and amino acids would be expected to be reduced. Thus, correlated reduction in the synthesis of starch or protein would occur. Conversely, the oil percentage of the kernels of protein and carbohydrate mutants is higher. For example, bt2, fl2, and su2 kernels have increased oil. However, most of this difference can be explained by a greater reduction in endosperm size than embryo size in these mutants.311–313 V. GENE CLONING AND BIOTECHNOLOGY Many of the mutants discussed in this chapter have been cloned utilizing various techniques (Table 1.5). In addition, the genes encoding many of the enzymes of the reactions of carbohydrate metabolism and the structural genes for storage proteins have been cloned. Both cDNA and genomic clones have been isolated and characterized for most genes. Analysis of the transcribed regions of the genes and the upstream regions of these genes has identified promoters and enhancer sequences. Various combinations of promoters and enhancers with the gene of interest will allow the expression of these chimaric genes in a tissue-specific or temporal-specific manner. It is easy to envision the benefits of modulating gene expression in two ways: either elevating gene expression or eliminating gene expression. However, in other instances, the end product may require reduced gene expression and, depending upon the objective, varying levels of expression may be needed for the production of the desired phenotype at the whole kernel or biochemical level. For example, an ideal sweet corn cultivar would be one that maintained a high sugar level during the desired eating stage, but that accumulated sufficient starch late in kernel development to provide higher seed quality. This could be achieved by the temporal control of the developmental expression as well as modifying the levels of expression of various genes. The potential of manipulation of starch, protein, and other biochemical properties of corn and other crops is clear.321,322 The potential of the use of genetic engineering for the production of novel variation has recently been demonstrated in the carbohydrates of potato tubers. The development of potato lines with amylose-free starch (waxy) and low starch (reduced ADPG pyrophosphorylase) tubers has recently occurred.323,324 These studies utilized antisense RNA constructs to reduce gene expression. The amylose-free potatoes have also been transformed with an active © 2001 by CRC Press LLC TABLE 1.5 Cloned Genes Involved in Carbohydrate Metabolism of Storage Protein Accumulation in Corn Kernels Gene Carbohydrate Metabolism sh1 Sus Sh2 bt1 bt2 du1 wx1 su1 ZPU1 BEI ae Biochemical Function References Sucrose synthase (endosperm specific) Constitutive sucrose synthase ADP-Glc pyrophosphorylase (subunit) Adenylate translocator ADP-Glc pyrophosphorylase (KDA subunit) Starch synthase II Starch granule bound starch synthase Isoamylase Pullulanase Branching enzyme I Branching enzyme II 89,91 159,160 157 94 155,156 198 93,314 193 190,192 316 317 Protein Metabolism o2 Glb 1 Glb 2 glutelin-2 Zp1, Zp2, etc. Regulatory protein Leucine-zipper Major globulin protein (63 KD) Major globulin protein (45 KD) Glutelin-2 gene (28 KD) Zein protein 92,315 318 319 320 261,262,264,265,269 starch granule-bound starch synthase gene.325 The tubers of these plants contain varying levels of amylose. In order to successfully use similar methods in corn, reliable methods for genetic transformation need to be developed. Recent reports by several groups have reported methods to transform corn using different techniques.326–329 These developments provide completely new methods for the production of genetic variation for specialty corns. VI. FUTURE PERSPECTIVE Corn is clearly a diverse crop with many specialty uses and types. These types have evolved from a rich past of selection based on recognition of unique properties associated with various genetic variants. The continued analysis of genetic variation has provided additional resources for the refinement and development of specialty corn. The ability of the biotechnologist to manipulate genetic variation at the level of specific genes offers the potential to tailor genetic variation for the production of precisely designed specialty corn in the future. This process, by necessity, will go beyond the biotechnologist. 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