`1 Traditional Plant Breeding`

From archeological findings, we have learned that agriculture began in the Fertile Crescent of the Middle East somewhere around ten thousand years ago.¹ Named for its quarter moon shape, the Fertile Crescent is the region in the Middle East referred to as Mesopotamia (Greek for “between two rivers”), and it flanks the Tigris and Euphrates Rivers, covering parts of modern-day southern Iraq, Syria, Lebanon, Jordan, Israel, and northern Egypt. There is evidence that barley, wheat, and lentils were domesticated during that period. Human selection of desired crops for reproduction, as noted by George Acquaah, is “the act of discriminating among biological variation” in a plant population to select desirable cultivars.²

The earliest agricultural practices included tilling the soil, planting seeds, and harvesting crops. Human societies evolved from hunter gatherers to planters of seeds, which fostered stationary settlements around cultivated soils. Reflecting on the origins of conventional plant breeding, John Bingham wrote, “The first stage of plant breeding was selection of genetically varied crops before people knew what genetics was. There were variations in both cultivated and wild crops from which farmers could choose for desired traits. Since each farm ecological system was uniquely suitable for certain crop genetics, the farmer’s role was to discover the best variant for his unique environment.”³

Although it is generally understood from archeological discoveries that humans have fashioned tools for more than two million years, the development of human observation of and intelligence about how plant seeds can produce crops is fairly recent.⁴ The earliest method of plant breeding was selection, defined as “the act of discriminating among biological variation to select desirable variants.”⁵ The traits sought were not unlike those that are sought today, such as resistance to disease, fruit size, color, and taste. Certain variants that once were highly selected have been protected. The term landrace⁶ refers to a cultivated variety with a distinctive identity that has evolved over centuries of farmer selection without formal crop improvement.⁷

The National Research Council (NRC), which is a part of the National Academies of Sciences, Engineering, and Medicine (NASEM), has classified two techniques for improving crops—selection and breeding. It does not view natural selection, which determines the survival of species, as a method of breeding. Incidental selection was eventually augmented by artificial selection. According to the NRC, “The earliest farmers selected plants having advantageous traits, such as those that bore the largest fruit or were the easiest to harvest. Perhaps through some rudimentary awareness that traits were passed from one generation to the next, the choicest plants and seeds were used to establish the next year's crop.”⁸ Artificial selection—the method used to narrow and control the available gene pool—provided an additional tool over incidental selection. In the former case, “a genetically heterogeneous population of plants is inspected and ‘superior’ individuals—plants with most desired traits, such as improved palatability and yield—are selected for continued propagation.”⁹ Thus, by selecting and isolating choice plants for cultivation, the early farmers were in essence influencing which plants would cross-pollinate. Through selection and isolation, they were narrowing, yet controlling, the available gene pool for each crop.

The next stage in plant breeding occurred when agricultural societies learned how to engage in plant reproduction. This stage in plant breeding was believed to have been practiced by the Assyrians and Babylonians around 700 BC, when farmers learned how to pollinate plants artificially by introducing pollen (male gametes) into the stigma (female reproductive part of a flower), circumventing natural (and thus random) pollination by wind or insects. What has been termed conscious classical plant breeding or crossing has resulted in many artificially pollinated crops, such as the palm and tomato.¹⁰

In 1984, the National Research Council defined breeding as hybridization where “farmers selected two plants and then crossed them to produce offspring having the desired traits of both parents. This was a trial and error process, however, since early plant breeders did not understand the genetic transmission of traits and could not predict the likely outcome of a particular cross.”¹¹ This technique improved on the accidental crossing of sexually compatible crops because it involved human intervention in the sexual life of plants.

The crossing of different varieties or species to produce new ones, referred to as deliberate hybridization, arose as an outgrowth of the knowledge of plant reproduction. Rudolf Jakob Camerer, professor of natural philosophy at the University of Tȕbingen, Germany, reached a conclusion in 1694 that plants were made up of male and female parts and that pollen could fertilize the plant.¹²

Experimental science had begun to take hold in the 1600s. For plant breeding, a major advance was the introduction of botanical gardens in the 1800s. In these gardens, plants were carefully classified and observed by botanists, who could determine by experimental trials the precise plant characteristics they desired. No longer did they have to depend on the idiosyncrasies of individual farmer choices or natural fertilization. The botanic gardens represented the first step in scientific plant breeding and were the source of many new European crops.¹³

Working within the same species, plant breeders developed inbreeding and outbreeding techniques to create desired varieties. Inbreeding occurs when the male and female gametes of the same strain are bred back (backcrossed). This creates plants of stable homogeneous properties called pure lines (which may take more than one generation), where the offspring resemble the parental lines.

In outbreeding, male gametes (sperm) that are carried through pollen tubes fertilize female gametes (eggs inside the ovary) from different genetic backgrounds (outcrossing). For example, a domesticated plant can be outcrossed with a wild type, combining two genetically different individual plants from the same species. Outcrossing maximizes genetic variations in the variety, allowing the breeder a broader genetic stock from which to work.

A breeder may combine two different traits from two individual varieties to create a single plant, which possesses the two desired traits from each variety. Although this can happen randomly in nature, the more assured way to accomplish this is first to create two pure lines through inbreeding. Then the breeder cross-pollinates the two pure lines to obtain a hybrid with the two traits in one variety. This is called hybrid seed technology. For this to work, the pure lines must be reproductively compatible. Interspecies crossing between closely related species can take place naturally (by cross-pollination) or through human intervention (by induced pollination).

The next stage in plant breeding was advanced at the turn of the twentieth century when the field of genetics gained traction in plant biology. Two developments that advanced traditional breeding are the inducing of mutations in plants and the advancement of cell culture methods. Both introduced greater variability in the plant germ plasm for crop development. Mutagenesis in plants was induced on the gametes by radiation or chemical mutagens. The induction of mutations did not introduce foreign genes into the plant but rather modified the available germ plasm of the plant variety. Although the method could increase the variability of traits, it was a trial and error process that took years to reach a useful outcome. Plant reproductive cells were exposed to gamma rays, protons, neutrons, alpha particles. or beta particles to create mutations in the cells’ genome. Scientists could then observe whether and how the mutations changed the plant’s physical characteristics (phenotype). There was no way to predict the outcome other than by observing the results of countless mutations.

A second breeding process involved culturing plant cells. This was particularly useful when plants were not by nature reproductively compatible. Scientists learned how to overcome reproductive incompatibility by developing methods to induce reluctant stigmas to be fertilized by foreign pollen. Methods for circumventing cross-incompatibility included electrical currents, wire brushes, and heat treatment. After the embryo from the mechanical tools of hybridization was created, it required special treatment in culture. This process (called embryo rescue) involves extracting the immature hybrid embryos, which ordinarily would not survive, by growing them in a culture with nutrient solutions.¹⁴

After plant cells could be grown in culture, breeders observed spontaneous mutations in the cells, also called somaclonal variations. Scientists observed that the mutations provided potentially valuable variants for new varieties. The process is imprecise and consumes a lot of time.

Thus, traditional breeding includes many unnatural methods of plant reproduction. For example, hybridization, a process of reproductively uniting two different plant varieties, can be accomplished by crossing natural breeding barriers. One such method is chromosome engineering, where portions of plant chromosomes are combined through a process of chromosome translocation. However, this process does not easily remove detrimental genes that are carried along in the transferred chromosome.

Questions that have arisen in discussions among biologists include the following: Are there limits in crossing species boundaries beyond which conventional breeders cannot go to create new cultivars? Does biotechnology increase the range of hybridization over traditional breeding? If so, how far can it be extended? What is the significance of that extension?

Before I explore these questions, I pause to discuss how biologists classify organisms and what that classification reveals about the differences between traditional breeding and molecular breeding. The biological world is classified and ranked into nine categories. At the top of the ranking is life, under which fall all living organisms from viruses to primates. The classification system continues with domain, kingdom, phylum, class, order, family, genus, and species, where specificity increases from domain to species.

Thus, humans are a species and part of the family Hominidae, which also includes chimps, gorillas, and orangutans. Cabbage is a species that is part of the family Brassicaceae, which also includes horseradish, mustard, rutabaga, broccoli, Brussels sprouts, cabbage, and cauliflower. Mushroom is a species that is part of the family Morchellaceae, which includes 146 different species and ten genera. Finally, the species common pond amoeba is in the family Amoebidae, which includes the genera Chaos, Entamoeba, Pelomyxa, and Amoeba. The classifications genus and species are the only classes within the taxanomic classification where crosses in conventional breeding take place. Conventional breeding permits the movement of genetic material between different species or closely related genera.

Wide crosses are defined as “crosses between species of the same or different genera.”¹⁵ The greater the phylogenetic distance between the species, the more difficult it becomes to engage in a wide cross. These crosses, when they occur, are usually accomplished with artificial techniques. For example, plant breeders can get the pollen of species A to fertilize the egg of species B. Because the embryo may not be able to survive naturally, the breeders use the technique of embryo rescue, as previously noted, by removing the embryo from the hybrid seed and culturing it with plant hormones and nutrients. According to a National Research Council report, “Such embryo rescue is not considered genetic engineering, and it is not commonly used to derive new varieties directly, but is used instead as an intermediary step in transferring genes from distant, sexually incompatible relatives through intermediate, partially compatible relatives of both the donor and recipient species.”¹⁶

H. C. Sharma reports that “hybrids from crosses spanning wide taxonomic boundaries” have occurred when wheat pollinated with maize and produced embryos and when hybrids were created from wheat and sorghum or from rice and wheat. In these cases, he is referring to crosses between members of different subfamilies.¹⁷ He notes that “crosses may be as wide as one can make them.”¹⁸ Wide crosses can, under unusual circumstances, cross two families. Most frequently, wide crosses can be induced among different species and less frequently across a genus. There is no evidence that wide crosses in natural processes or by the traditional non-natural methods of plant breeding can take place across groups in order, class, phylum, kingdom, and domain.

In his book Hybrid: The History and Science of Plant Breeding, Noel Kingsbury writes that

Much of twentieth-century plant breeding has been concerned with getting progressively less and less well-related relatives to cross, moving up to crosses between apparently close genera. Methods of overcoming cross-incompatibility by forcing reluctant stigmas to accept alien pollen have become increasingly bizarre (or desperate): passing electrical currents across stigmas during pollination to get Brussels sprouts and savoy cabbage to cross, “mutilating” … cabbage stigmas with a wire brush, heat treating of lily … styles prior to pollinations.¹⁹

Artificial hybridization between plants of different species and plants and organisms within different genera has expanded in the twentieth century. The methods of traditional breeding for creating crop varieties by hybridization seem to have reached their limits. As Sharma notes: “the difficulty in obtaining wide hybrids increases with the phylogenetic difference between the parental fauna involved.”²⁰ Breeders also have used vegetative propagation of stem cuttings. This has allowed them to produce clones of plants, which do not create new traits or widen genetic variability. They also have created new varieties by propagating plants in tissue culture with the use of protoplasts—plant cells with the cell wall removed enzymatically. The protoplasts can be combined or modified to regenerate a whole new plant variety by transferring traits between species. This is called somatic hybridization or cell fusion. The protoplasts derived from different plant cells are fused together by electrical techniques. Also, by processes of electroporation or microinjection, DNA can be transferred to plant cells. Electroporation sends electrical impulses through the protoplast, which increases the protoplast’s membrane permeability and allows foreign DNA to enter. Through microinjection, foreign DNA can be injected into the protoplasts.

Another method of modifying the genetics of crops is by changing the number of chromosomes in a plant variety. This can be done by crossing a plant with two sets of chromosomes (diploid) with a plant that has four sets (tetraploid), resulting in a plant with three sets (triploid), such as a banana.²¹ On the down side, the triploid banana is sterile. Alternatively, one can use chemicals such as colchicine to double the number of chromosomes.

Mutational breeding can use chemicals and radiation such as x-rays. Plant cells or seeds are exposed to mutagens at doses that are nonlethal but high enough to induce mutations resulting in new traits. The Food and Agricultural Organization of the United Nations collaborates with the International Atomic Energy Agency to maintain a mutant variety database online. From 1930 to 2014, it is estimated that more than 3,200 mutagenic plant varieties from 214 crops in 60 countries, including 1,000 varieties of staple crops, have been made available. Although mutational breeding changes the DNA of a plant cell, it is not classified as genetic engineering by regulatory agencies in the United States or Europe.

In conclusion, traditional or conventional breeding encompasses natural hybridization and selection, artificial hybridization among sexually compatible species within the same or different genera, the exposure of gametes to mutations with chemicals or radiation, the use of protoplast fusion that combines genes from the cells of two species, and chemically induced polyploidy (more than two paired sets of chromosomes).

Traditional breeding can be distinguished from biotechnology by the fact that the former operates at the cellular scale, whereas the latter operates at the molecular scale. As such, biotechnology has been aptly called “molecular plant breeding.”²² By operating at a molecular unit of analysis, scientists have been able to overcome many of the limits of genetic crosses previously discussed: “Traditional breeding focuses on individuals and populations and relies primarily on sexual reproduction to manipulate useful variability; in contrast, biotechnology focuses on the cellular and subcellular levels, capitalizing on the techniques of molecular biology both to generate and manipulate useful variability.”²³ However, the line between traditional breeding and biotechnology has been blurred with technical advances. For instance, molecular-level techniques are applied to traditional breeding through marker-assisted selection or quantitative trait loci. Without inserting new genes, gene sequencing gives breeders an opportunity to screen out for desired crops more quickly than by trial and error, which then can be bred by traditional breeding methods. Also, gene editing by CRISPR (clustered regularly interspaced short palindromic repeats) can be used to make specific mutations in plants, reducing the uncertainty of mutations by chemicals and radiation. The next chapter explores the methods used in biotechnology to create new plant varieties by molecular breeding, comparing and contrasting those with traditional breeding techniques.

Notes

1. Catherine Preece, Alexandra Livarda, Pascal-Antoine Christin, Michael Wallace, Gemma Martin, Michael Charles, Glynis Jones, Mark Rees, and Colin P. Osborne, “How Did the Domestication of Fertile Crescent Grain Crops Increase Their Yields?,” Functional Ecology 31 (2017): 387–397.

2. George Acquaah, Principles of Plant Genetics and Breeding, 2nd ed. (New York: Wiley-Blackwell, 2012), chap. 2.

3. John Bingham, “The Achievement of Conventional Plant Breeding,” Philosophical Transactions of the Royal Society London B 292 (1981): 441.

4. Hakan Ulukan, The Evolution of Cultivated Plant Species: Classical Plant Breeding versus Genetic Engineering,” Plant System Evolution 280 (2009): 123.

5. Acquaah, Principles of Plant Genetics, chap. 2.

6. Tania Carolina Camacho Villa, Nigel Maxtel, Maria Scholten, and Brian Ford-Lloyd, “Defining and Identifying Crop Landraces,” Plant Genetic Resources: Characterization and Utilization 3, no. 3 (2005): 373–384.

7. Lawrence Busch, William B. Lacy, Jeffrey Burkhardt, and Laura B. Lacy, Plants, Power, and Profit: Social, Economic, and Ethical Consequences of the New Biotechnologies (Cambridge, MA: Blackwell, 1991), 58.

8. National Research Council, Genetic Engineering of Plants: Agricultural Research Opportunities and Policy Concerns (Washington, DC: National Academy Press, 1984), 5.

9. National Research Council, Safety of Genetically Engineered Foods: Approaches to Assessing Unintended Health Effects (Washington, DC: National Academy Press, 2004), 24.

10. Ulukan, “The Evolution of Cultivated Plant Species,” 135.

11. National Research Council, Genetic Engineering of Plants, 5.

12. Herbert F. Roberts, “The Founders of the Art of Breeding—II,” Journal of Heredity 10, no. 4 (1919): 229–239.

13. Noel Kingsbury, Hybrid: The History of Plant Breeding (Chicago: University of Chicago Press, 2009).

14. Kingsbury, Hybrid, 257.

15. H. C. Sharma, “How Wide Can a Wide Cross Be?,” Euphytica 82 (1995): 43.

16. National Research Council, Safety of Genetically Engineered Foods, 26.

17. Sharma, “How Wide Can a Wide Cross Be?,” 59.

18. Sharma, 59.

19. Kingsbury, Hybrid, 257.

20. Sharma, “How Wide Can a Wide Cross Be?,” 56.

21. Rodomiro Ortiz and Dirk Vuylsteke, “Recent Advances in Musa Genetics, Breeding and Biotechnology,” Plant Breeding Abstracts 66, no. 10 (1996): 1355–1363.

22. Stephen P. Moose and Rita H. Mumm, “Molecular Plant Breeding as the Foundation for the Twenty-first-Century Crop Improvement,” Plant Physiology 147 (2008): 969–977.

23. Michael Hansen, Lawrence Busch, Jeffrey Burkhardt, William B. Lacy, and Laura R. Lacy, “Plant Breeding and Biotechnology,” BioScience 36, no. 1 (1986): 29–39.