Cells in a developing embryo are sufficiently mature to germinate in only 15 days after pollination if separated from the endosperm. Early germination (vipipary) is inhibited by the presence of the plant hormone, abscisic acid, in the endosperm which negates the growth stimulant hormone, gibberellic acid, in the embryo. This allows normal seed desiccation as water is replaced with starch deposits and eventual seed germination inhibition because of low water content. Seed producers know that each genotype differs in the percent moisture to be used to harvest for optimum seed quality. Generally, for most corn dent hybrids, that moisture is higher (+/-36%) than normal black layer moisture of 32%. From there the moisture level must be rapidly decreased to less than 13 % to prevent excessive aging caused by damage to the cell membranes. It is the art and science of the seed producer to bring the moisture down rapidly without using excess heat which also could damage the membranes.
Cell membranes being the main structural component of embryo cells needed for energy conversion from starch in the mitochondria and translation of DNA to proteins, have a major effect on the germination quality of seeds. Genetics of mitochondrial membranes, at least partly affected by the mitochondrial DNA are probably one of the major reasons that seed quality varies between hybrid parents. It is generally known that the seed quality of a hybrid is associated with the choice of the seed parent.
Another major factor affecting on seed quality is the growing environment in the seed production field. Stress, from drought or disease during the seed maturation period shows later in seed germination quality. Such seed often becomes evident in germination tests done 4-5 months after seed harvest, sometimes in contrast to tests done only a few months earlier. It is as if these seed had some membrane pre-harvest injury that when added to normal aging during these 4-5 months surpassed the minimum for normal germination. These individual seeds either fail to germinate or are slower to emerge than other seed in the same seedlot.
Membranes dominate the structures in corn cells, being major components of the endoplasmic reticulum, mitochondria and plastids such as chloroplasts. The nucleus of the cell also includes a double layer of membrane, composed of lipids and proteins. It functions as a gateway for movement of complex molecules and minerals in and out of the nucleus. As a segment of the chromosome DNA for a gene is activated to produce a RNA code for a protein, the RNA moves to a ribosome to hook the amino acids together forming a protein. Although some of the ribosome action occurs within the nucleus, much happens after the RNA moves through the nuclear membranes into other ribosomes in the cytoplasm. Auxins and other plant hormones interact on the activation of the DNA, requiring regulation through the nuclear membranes.
Membranes for each organelle of the cell require very specific proteins, each dictated by the DNA code. Many of those proteins are coded from the nuclear chromosomes but are also affected by the single chromosomes in mitochondria and chloroplasts. Amino acids with differing nitrogen, hydrogen and oxygen ions arranged around carbon chains determine the composition of proteins and the phosphor-lipids that compose membranes are critical to all cellular function. Eventual germination of the seed is dependent on formation during seed development and maintenance of membranes during seed storage.
Another membrane component of the corn cell in the seed embryo is the chloroplast. A few billion years ago, a bacterium species had an inner membrane layer that could capture energy from light while releasing oxygen. We know these organisms as blue green algae but their single circular chromosome and membrane structure classifies them as Prokaryotes instead of a Eukaryote, like the nucleus-containing algae. Perhaps a billion years ago, these photosynthesis- producing single-celled organisms gained a symbiotic relationship in other single cell organisms and, with that event, the algae and the rest of the plant kingdom had begun. Chloroplasts have their own bacteria-like chromosome, with its own DNA, and are filled with membranes in which photosynthesis occurs.
Having their own DNA, RNA and ribosomes, chloroplasts function as an independent organism in that they can divide and produce proteins, but their symbiotic relationship with the cell still depends greatly on proteins and lipids from the host cell. The single chromosome of chloroplasts has only about 100 genes as the nuclear genes of the host supply much (95%?) of the protein for their function. Chloroplast DNA can have mutants but this is uncommon.
Chloroplasts, like mitochondria, are transmitted to the new embryo only through the female parent of seed. Protoplastids in the egg cell and, later in the developing seed, can multiply but do not produce pigments or become photosynthetically active until exposed to light in leaf tissue after germination. Plastids in cells destined to become part of non-photosynthetic tissue such as kernel endosperm, stem and root parenchyma cells generally become starch storing components of cells. Membranes structure and function of plastids are essential to the life of the corn seed.
It is difficult to imagine 32000 genes distributed among the 10 chromosomes in the nucleus of a single cell within the embryo of the corn seed. But the microscopic cell also contains many other substances that contribute to cell function once it is activated with germination. Proteins and lipids contribute to the function of the outer plasma membrane surrounding the cell, but membrane-like structures also are intertwined within the cells. Endoplasmic reticulum is used to transport cell products. Ribosomes are attached to the outside of ‘rough’ endoplasmic reticulum. These ribosomes are the organelles in which RNA codes, originating from the DNA, are used to link the amino acids to form proteins. Adjacent endoplasmic reticulum is used to transport the newly formed proteins to sites in the cell appropriate for that protein’s function.
Mitochondria, independent organelles within the cell, are the site of transferring glucose molecules in the chemical energy used by other cell functions. These organelles, carried along in the egg cell from the maternal parent plant, have their own DNA for genetics but are dependent on the rest of the cell and nuclear DNA to provide the glucose, proteins and lipids for structure and function. This symbiotic relationship is in all animal, plant and fungal species, originating a few billion years ago and certainly is significant in corn performance. Mutations in the mitochondria DNA are the source of cytoplasmic male sterility, at least partly because of a genetic defect in the outer membrane of the mitochondria results in defective pollen production. This sterility affect can be overcome by products coded in the nuclear DNA of corn, the male sterile restorer genes. However, the specific mutation to URF13 gene in mitochondria with T cytoplasm, not only cause sterility but also increased susceptibility to certain pathogen toxins such that produced by race T of Bipolaris maydis, resulting the disastrous epidemic of 1970 corn crop. This toxin destroyed mitochondrial function, reducing the plant’s ability to produce normal pathogen-inhibiting resistance chemicals.
DNA of chromosomes in the cell nucleus is, by far, the largest, affecting most cell functions, but the much smaller amounts of DNA in mitochondria and plastids are essential and interdependent with the nuclear genes.
More than 32000 genes on 10 pairs of chromosomes in each living cell of the corn seed embryo! Plus more genetics in each protoplastid, that later becomes the chloroplast, and each mitochondrion. Genes code for proteins that become enzymes participating in biochemical processes resulting in specialized cells for each corn plant structure, in hormones to direct the flow of sugars, and in the photoreceptor protein such as phytochrome that senses daylength and consequently affects flowering time in tropical corn. Some proteins are part of cell structure and some regulate the process for switching the gene to ‘on’ at the right time.
Living plant cells are surrounded by non-living cell walls. Inside these cells, proteins are major components of cell plasma membranes. The amino acid components, as coded in the DNA, have major roles in affecting which molecules enter the cells. This becomes especially important in active transport of sugars, for example, from lower concentration into higher concentration, in contradiction of usual passive transport from high concentration of molecules to lower concentration. Specific arrangements of specific amino acids allow the active transport of molecules across cell membranes.
Enzyme activity is also specifically related to allowing reactions to occur with less energy requirements and at fast speeds as they perform as catalysts. Such proteins often include more than 100 amino acids attached to each other but eventually folded in many ways allowing the pertinent charges on the enzyme molecules bind to a critical molecule assisting a chemical reaction to occur.
Cell function, and therefore, plant growth, is obviously dependent on protein structure. Those 32000 plus genes in corn, mostly dormant along with the seed when kept at low moisture, have a lot to do after seed germination begins. Mutations may have insignificant effects but some changes to just a few amino acids can cause major functional problems.
We have benefited greatly from the mutations that occurred over the thousands of years in multiple environments where people selected desirable characteristics in corn. With isolation and self-pollination, populations became distinct with certain genes ‘fixed’ with homozygous dominant or recessive versions of some genes. Mutations and cross pollination in the varieties maintained variability, presenting advantages but also disadvantages as the genetic variability presented problems in dependable replication of the favorable genetics. Selfing these plants in attempt to gain complete homozygosity became the goal, with the objective of getting repeatability in inbred and hybrid characters for each generation. Consequently, development of inbreds involved 7-10 generations of selfing, with complete homozygosity as the goal. The limit is the low rate of mutations occurred at each generation, and even with most mutations not affecting the performance of the line it did allow for gradual ‘genetic drift’. This was witnessed when a few popular public inbreds such as B73 and A632 were maintained by many separate public and private breeders and then compared for minor characters. The basic heterosis between these stiff stalk lines with Lancaster inbreds remained but minor character differences were revealed. Although some of this experience could be attributed to outside pollen contamination, it is assumed the accumulation of mutations over the years of isolation attributed to the differences among these established inbreds.
Significance of variability in parent seed presents an enigma for hybrid corn seed producers. Genetic drift in parent seed as seed is increased from a single ear to the large number needed to produce only 10000 bags of hybrid seed. By the time of hybrid seed production, it is unlikely that any seed is precisely genetically like the original hybrid between two newly developed inbreds. These changes probably have no measureable effect on heterosis or performance of the commercial hybrid. The seed industry does struggle to differ between inconsequential genetic differences from the original hybrid genotype and those that affect performance.
Dihaploid breeding produces perfectly homozygous inbreds with the first generation of doubling the haploid plant’s chromosomes. However, further increasing the seed for hybrid testing and eventual hybrid production allows for slight mutations.
Single cross hybrids produced by the crossing of two inbreds are rarely completely genetically pure due to minor mutations that have occurred during the increasing of the parent seed, but the performance difference due to the genetics from the original experimental hybrid is rarely measureable. Mutations are a blessing and an enigma to all involved in hybrid corn seed development, production and use.
Corn advanced from that first mutation in Teosinte, allowing and exposed kernel to be easily used as grain. More mutations that occurred with each annual reproduction was utilized by people over the past 9-10000 years. Mutations occur naturally in all organisms. For example, each new human baby, on average, has 100-150 new mutations different from either parent. Majority of the human mutations are unnoticed and insignificant but a few can be drastic. But human generation reproduction is once every 20 years whereas an annual plant such as corn produces new mutants with each seed generation. Although the mutation rate per gene may be low in comparison with some organisms, having 32000 genes allows for a probability of some mutations to occur with each generation. Not all of these mutations will be expressed because they will usually occur in one strand of the paired DNA strands, allowing the other dominant version of the gene to affect the trait associated with the gene.
Mutation causes are associated with errors that can happen during meiosis and recombination of gametes during reproduction. Point mutations occur when a different nucleic acid is substituted during DNA replication. This small change can code for a different amino acid when placed in the eventual protein produced from the DNA-RNA-protein process. This can lead to a difference in some biochemical process that the original protein, acting as an enzyme, would affect. It may affect drastic and very visible differences in the plant but in most cases, it is insignificant and not noticed by most observers. Point mutations are probably the most common cause of mutations but a few other more drastic causes can be related to major errors in DNA duplication as part of meiosis. It is common, however for some breakage in one of the pair allowing a segment to be exchanged with a portion of the member of that pair. This process, called a crossover, is utilized by corn breeders in backcrossing procedures in which the objective is to cross a specific gene, such as a BT gene, into a desirable inbred without disturbing most of the genetics of the original inbred. Backcrossing in a gene, long used as a breeding procedure before use of GMOs, has been relatively successful in recovering the essential genetics of the original inbred but now with the desired gene such as the Ht gene for resistance to the fungus causing northern leaf blight or wx (waxy corn gene).
Hundreds of maize mutations have been identified and stocks of these mutants have been stored at the Maize Genetics Cooperation Stock Center http://maizecoop.cropsci.uiuc.edu. These stocks are made available to researchers for study and possible use in commercial corn products.
Francis Crick and James D. Watson produced their double helix model of DNA in 1953. It took a decade after that to put together the relation between the nucleic acid codes of the DNA to RNA to protein synthesis from the amino acids coded in the DNA. These discoveries triggered the advance of biotechnology during the 70’s. Researchers concentrating on whole plant physiology, development, taxonomy and environmental interactions were suspect that researchers concentrating on the chemical nature of genes were not adequately acknowledging the complexity of plant biology. Vice-versa, gene specialists were looking at the traditional researchers as old school. Francis Crick is quoted in a book entitled ‘The Gene’ by Siddhartha Mukherjee as saying “each generation needs its own music” implying that the new music was with molecular structure and function of DNA.
My academic training included much more old school with traditional botany and pathology during the 60’s. The new genetics story was fascinating but my primary interests were, and are, with the whole plant. Advances in the knowledge of genetics remain fascinating, as not only has the corn genome size been defined (32,000+ genes) and 2.3 billion nucleic acid base pairs and now, using the CRISPR methodology, some of these genes can be modified. Inserting single genes for controlling certain insects or resistance to a few herbicides is now old technology.
Despite these gains by the ‘new musicians’ some of us old school types are still impressed by the complexity of corn biology. Proteins coded by DNA function in multiple physiological pathways to construct the vascular bundle cells, allowing for the movement of nutrients for developing tissue, the photosynthesis process that converts light energy into sugar molecules, and the movement of that sugar to kernel endosperm and the synthesis of starch from the sugars. Each of these processes are affected by the right genes turned on at the right time. It is impressive that in the 50+ years since we understood the DNA-RNA-protein synthesis process occurring in cells that now we can count the genes and even construct new ones, but there remains a lot of science left to understand how we select the best hybrid for the predicted corn environment in the summer of 2017. Should it have deep roots to better uptake of water, or spreading shallow roots to avoid root lodging in wet summers in high organic soils? Will good resistance to northern leaf blight be a factor or will there be a new corn disease making an appearance. Perhaps the music of the current generation is “it takes two to tango”.
Intentionally self-pollinating corn caused increasing revelation of mutations, some of which had negative effects on plant development. Corn has at least 32000 genes. As a diploid plant, having 2 copies of each chromosome, if the mutation only occurred on one DNA code in one of the pair, there would be an opportunity for the dominant form on the other member of the pair to cover up the deficiency. Self-pollination offered the chance (25%) of the new seed would be homozygous recessive and result in the expression of the mutation. What could be the effects of these mutations? Perhaps it is beneficial to our needs, like the sugary gene that gives us sweet corn, but homozygous recessives could also affect root growth, or vascular tissue size, photosynthetic rates or movement of sugars to the grain. Whereas keeping desirable ears for corn shows essentially maintained some heterosis within a variety, the open pollination nature of corn also allowed some selfing and therefore more homozygous genes for some negative characters in terms of grain production.
The move to inbred development was increasing the probability of homozygous negative recessives not being covered with dominant forms of the gene within the inbred. Not only could mutations be occurring during meiosis in the selfing generation but also those accumulated within the initial breeding population, whether from an F1 cross of two inbreds or from an established variety. Inbreds inevitably will accumulate an expression of mutations that have a negative effect on grain production performance. However, with the right combination, each hybrid parent will have a dominant form of enough negative recessive forms to overcome the major disadvantages of the other parent. Experience has shown that inbreds developed from Stiff Stalk Synthetic background have a high probability of showing hybrid vigor when crossed with inbreds developed from Lancaster Sure Crop background. However, corn breeders also are aware that the combinations for inbreds from these two backgrounds that show adequate performance to be commercially competitive are not frequent. This is apparently because of the frequency of negative recessives that become homozygous during the selfing process. Given the large number of genes in corn this does seem reasonable. Although a few inbreds may combine with a few inbreds of the other heterotic group to produce close to superior commercial hybrids, and thus be considered a general combiner, ultimately there will be one specific combination giving a truly superior hybrid, at least in the environments used for that hybrid.
One such hybrid 30-40 years ago was B73 X Mo17. Either of these inbreds would successfully combine with other inbreds but this specific combination was dominant in the Midwest with plant densities used during that period. However, this hybrid would also have stalk rot problems when stressed with higher plant densities or photosynthetic stress (cloudiness) more frequent in the Eastern corn belt. It is as if each combination of inbreds does not sufficiently cover all the pertinent negative genes, at least for the ultimate performance under all commercial environments but some specific crosses may be acceptable under some environments.
This blog issue emphasizes significance of gene dominance in explaining superior hybrid vigor. There are other genetic aspects as well, which will be discussed in future issues.
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About Corn Journal
The purpose of this blog is to share perspectives of the biology of corn, its seed and diseases in a mix of technical and not so technical terms with all who are interested in this major crop. With more technical references to any of the topics easily available on the web with a search of key words, the blog will rarely cite references but will attempt to be accurate. Comments are welcome but will be screened before publishing. Comments and questions directed to the author by emails are encouraged.