Characteristics of Various Types of Plant Breeding

Abstract

Plants have always been integral to human society and their (genetic) improvement has been carried out ever since humans became farmers. Breeders are seeking to alter plants in a permanent and heritable manner in order to enhance agricultural production relying on the scientific and technical advancements in molecular biology and biotechnology. Plant breeding simultaneously creates and exploits biological diversity (genetic variation), which are the main activities for plant breeders. Both plant domestication and traditional (conventional or classical) breeding depended on the natural processes and genetic potential of the species. However, innovations, such as mutation breeding, various biotechnological tools (e.g. in vitro techniques), and speed breeding, have been developed to enhance genetic gain and accelerate the breeding process. Furthermore, to improve selection, molecular markers were introduced. Strategies, such as molecular-assisted selection and genomic selection, are part of molecular (modern or nonconventional) breeding, which also includes two approaches based on genetic engineering: transgenesis and genome editing. The main characteristics of all these breeding tools — the essential assets for overcoming the agricultural challenges of modern civilization — and their relation to one another are presented in this chapter.

1. Introduction

Plants have always been, and still are, integral to human society because they provide food, feed, fiber, fuel, raw materials, medicines and other various bioactive compounds, esthetic pleasure, and solutions to various environmental issues. However, nearly all the plants that are useful today do not occur naturally but exist only because of human intervention that began thousands of years ago [1].

Early humans gathered what that they could find in the wild, but as their lifestyle changed from nomadic to sedentary, thousands of years ago, desirable plant species started to be selected and cultivated. This was the beginning of plant domestication [2], which represents the earliest form of plant breeding [3]. About 150 years ago, science revolutionized selection and breeding processes, facilitating and making them more efficient [4, 5]. To this day, plant traits and characteristics continue to be changed in order to better serve the needs of modern society [6]. The role of science and technology in plant breeding has increased continuously [4] and modern breeders rely on more and more sophisticated and efficient methods to create variability, discriminate among variants, and develop varieties (cultivars) for widespread cultivation [7].

The last five decades have been the most productive period in world agricultural history, saving billions of people from hunger and starvation [8]. The enhanced production has been based on overexploitation of natural resources and changing the natural environment, essentially entailing the modification of growing conditions [5, 8]. This was possible with the implementation of advanced agricultural technology, especially the application of production inputs such as fertilizers, irrigation, and pesticides [4, 5, 8]. However, this progress cannot cater to all the needs of mankind, and it will be even less sufficient in the coming years because of the increasing demands of human society [9]. So, for the future, the challenge is to ensure a sustainable rise in global agricultural production for a growing human population using finite natural resources and a shrinking agricultural land base due to industrialization, urbanization, and limiting factors, such as climatic or environmental changes [8, 9, 10, 11]. In this context, the genetic improvement of crops is more important than ever.

In contrast to farmers, whose strategy is to enhance certain traits only temporarily — without tampering with the genetics of the organism — breeders seek to alter plants in a permanent and heritable manner so that genetic modifications are transmissible from one generation to the next [12]. The integration of newly developing technologies, such as molecular markers, OMICS, transgenesis, genome editing, and RNA interference into plant breeding, will provide the basic principles for developing modern breeding methodologies [4, 5, 13]. In this way, genetics coupled with other scientific knowledge (statistics, biometrics, biochemistry, bioinformatics, biotechnology, etc.) have the potential to overcome the aforementioned threats and challenges [8].

As an educator, I am concerned first and foremost with the students and the uninitiated, for whom understanding of all aspects of plant breeding is difficult to grasp, particularly because they are at the beginning of a challenging journey. As a result, it should be noted that this chapter is not intended for expert readers or an advanced audience as it will only offer a brief overview of the fascinating world of plant breeding, in order to provide up-to-date theoretical background and definitions. The main characteristics of the various types of plant breeding strategies are presented, starting with the fundamental principles of classical breeding and all the way to the advanced technologies of modern breeding, emphasizing approaches such as artificial induction of mutations, rapid generation advancement, the use of molecular markers, transgenesis, and genomic editing.

2. Plant breeding

It should be noted that the terms plant breeding and plant improvement are used synonymously [5]. One of the best-suited definitions for plant breeding is the one given by [12]: “the art and science of improving the heredity of plants for the benefit of humankind.” Other definitions emphasize the changing, altering, or manipulation of genetic patterns, genetic make-up, genetic information, genome, or genetics of plants in order to produce desired traits or characteristics, to increase their value, or to make them a better fit for human purposes. Basically, the plant breeding process encompasses techniques for producing, selecting, and fixing superior plant phenotypes in order to develop new, improved cultivars that better meet the requirements of farmers and consumers [14, 15].

Many authors state that the scientific basis for plant breeding was established by the ground-breaking work of Gregor Johann Mendel in the middle of the nineteenth century. However, the principles put forth by Charles Darwin and Alfred Russell Wallace during the same period are equally important since breeding is nothing less than a particular form of evolution. At the beginning of the twenty-first century, the science of crop improvement is being transformed once again by molecular breeding, which integrates the latest breakthroughs in biological research — namely molecular biology and biotechnology — with traditional breeding practices [15, 16, 17, 18, 19]. As science and technology advance, modern breeders are able to make their activities more and more predictable and precise, with plant breeding becoming indispensable to modern human society [20]. Thus, the development of a new crop variety is an example of agricultural biotechnology that includes both traditional breeding techniques and modern methods [1, 21], such as molecular markers and genetic engineering.

As mentioned before, plant breeding is often likened to evolution [5]. Yet, a crucial distinction between the two is that evolution is a natural and extremely slow process, whereas plant breeding is a relatively quick artificial one [2]. Moreover, natural evolution increases the fitness of the populations or species, whereas plant breeders aim to direct the population toward specific and predetermined goals — often related to yield, nutritional value or other commercial traits — that are generally not concerned with fitness because modern farmers can grow plants under artificial conditions [5]. It should be noted, however, that in recent years, there has been a shift toward improving adaptability to abiotic or biotic stress factors in order to make better use of land resources while also taking climate change into consideration [22].

The development of new cultivars entails two basic activities: assembling genetic variability and discriminating among variants — selection — in order to identify and advance desirable genotypes (individuals) that meet the breeding objectives [7]. These two stages are followed by the evaluation and release of the cultivar [5].

Depending on the approaches and techniques employed by breeders, which keep evolving along with science and technology, there are two basic categories of plant improvement: traditional (conventional) and molecular (nonconventional) [4, 20]. These two categories and their characteristics will be presented in the remainder of this chapter.

3. Traditional plant breeding based on selective breeding

Traditional plant breeding, also called classical or conventional breeding, is the development of crop varieties by using natural processes and conservative, older, simpler, and relatively low-tech tools to modify an organism’s genetic information within the natural boundaries of its species [1, 5]. The absence of modern developments or lack in sophistication is not implied here as traditional breeding entails both basic and advanced methods [23]. In fact, practices used in traditional breeding may include features of biotechnology such as tissue and cell culture, protoplast fusion techniques for somatic hybridization, techniques for embryo rescue to overcome incompatibility barriers, advanced pollination procedures and in vitro fertilization, techniques for polyploidization a haploidization, and mutation breeding [4, 14, 23]. However, the fundamental method remains modification through selective breeding [1, 14], also called artificial or phenotypic selection, which is based on phenotype evaluation for identifying individuals with desirable traits [3, 5, 24]. Consequently, the methods and techniques used by conventional breeders rely heavily on the species’ mode of reproduction — self-pollination, cross-pollination, or clonal (vegetative) propagation — and contrast with the newer and more innovative breeding tools of molecular breeding [5]. This is why, the initial statement is defining the traditional breeding approach by actually comparing it to the most cutting-edge breeding technologies. In much the same manner, EU legislation on genetically modified (GM) organisms (GMOs) — that is. Directive 2001/18/EC — clearly delineated traditional breeding from what was, at the time, the most innovative methodology for crop improvement — transgenesis or recombinant DNA technology [23]. This idea was rightly extended to include other genetic engineering tools – that is. genome editing — which are considered to have revolutionized crop improvement and biological research and which will be presented in Subchapter 4.2.2. In conclusion, all genetic engineering methods that enable the creation and introduction of novel variation into genomes through genetic engineering should be separated from traditional breeding [25]. This is especially true from a technical point of view, but the legal implications are more nuanced; these will be discussed further in Subchapter 4.2.2.

Artificial selection can be performed on naturally occurring individuals, but, more often, on offspring resulted from controlled crosses, also called matings or hybridizations. Therefore, in conventional breeding, desirable traits are assembled into a new cultivar from different but very closely related plants — usually belonging to the same species [4]. As such, the individuals resulting from conventional breeding only display characteristics already present in the genetic potential of their species because new genes (and characteristics) are not introduced [5]. Also, when none of the individuals on which selection is performed possesses a certain gene variant controlling a particular trait, it is not possible to select that specific trait [1].

It should also be noted that in a controlled mating all of the genetic material between the two individuals being bred, which could mean tens of thousands of genes (maize, for example, has approximately 32,000 genes), is shuffled. The results of such a mix can be very unpredictable because of the large number of combinations — that is. genotypes or individuals — it can generate and finding the best one could be very difficult [1].

Artificial selection is the oldest technique for crop improvement and still remains widely used [20, 26]. This has actually been the main process through which, over the years, humans have gradually and systematically favored traits that have increased the utility of plants [1, 5].

3.1 Mutation breeding

Plant breeding relies on genetic variation — “heritable variation is the lifeblood of plant breeding” [5] — for selecting desired genotypes or traits [27, 28, 29].

In nature, mutations — the heritable changes to an individual’s genetic makeup [14, 30] — represent one of the essential mechanisms for genetic variation and evolution — individuals with a novel trait may be preferentially selected because of their superior fitness determined by the novel (mutant) adaptive features [28, 29, 31]. Mutations are in fact the new gene variants (alleles) controlling new traits that are passed on from parents to offspring [28, 29, 30].

Humans have used natural genetic variation since they started cultivating plants by actually (and also unknowingly) selecting for the alleles that were beneficial and suitable to their needs [9]. The natural rate of mutation is nevertheless very low and insufficient for generating all the variation that breeders would like to have for their breeding programs [26], which have to run at a much faster pace than natural evolution. To overcome this limitation, plant breeders can artificially induce additional mutations by using physical (i.e. different types of high-energy radiation) or chemical (e.g. ethyl methanesulfonate and dimethyl sulfate) mutation-causing agents – mutagens [28, 29, 30, 32, 33]. This way of generating new variation to be exploited in the breeding process is called mutation breeding. Consequently, the genetic variation used in plant breeding could be: (1) found in the natural, existing gene pool; (2) obtained through crossings (hybridizations) that shuffle existing variation into new combinations without creating novel gene variants; and (3) the result of artificial mutagenesis which actually means generating new alleles.

Usually, mutation breeding is considered part of conventional or traditional breeding [23, 28, 29, 30, 32, 33], but different opinions can be found as well:

• “Genetic transformation and mutation breeding, as nonconventional breeding tools for plant improvement, are outlined and selection in vitro against a fungal toxin isolated from Mycosphaerella fijiensis is presented in more detail.” [34];

• “… nonconventional breeding of banana, more specifically genetic transformation, protoplast culture, somatic hybridization and EMS-induced mutation …” [35].

EU legislation on GMOs — that is. Article 3(1), in conjunction with Annex I B of Directive 2001/18/EC — is very clear on this topic: mutagenesis — introducing variations in the plant genome using radiation or chemicals — is regarded as traditional breeding and is explicitly exempt from the scope of the Directive 2001/18/EC, on the basis that it has a long history of safe use [23, 32, 33, 36].

With more than 3000 mutant crop varieties in more than 200 plant species having been officially released worldwide [28, 29, 31, 37], mutation breeding continues to be an important tool for today’s plant improvement efforts together with the more advanced and precise nonconventional techniques [28, 29, 31].

3.2 Speed breeding

Several years are required for developing cultivars using conventional procedures for generation advancement (to the next breeding cycle). In order to achieve a rapid generation advancement (RGA) — that is. the shortening of breeding cycles — with more than three to four generations per year, a relatively unsophisticated and highly adaptable platform for plant cultivation was perfected [38, 39, 40, 41, 42]. Research on this topic had been reported as far back as 1880 [39, 41], based on the idea of growing plants under artificial light that was experimented with by botanists [40, 41]. The RGA approach was first proposed just before World War 2, then modified in the 1960s, and its most recent form — speed breeding — was introduced only a few years ago [39, 42].

Just like mutation breeding and molecular breeding (Subchapter 4), speed breeding is aimed at accelerating genetic gain [38, 39, 40]. For this, plants are cultivated under fully enclosed environmental conditions, in growth chambers or greenhouses, where crop-specific optimal light (quality, intensity, and duration), temperature, and humidity can be artificially controlled [30, 38, 39, 40, 42].

The basic and simple procedures of speed breeding can be easily adopted. However, this approach is much more effective in enhancing genetic gain when integrated with other modern strategies [39]. To produce greatly improved outcomes, speed breeding can be combined with:

• marker-assisted selection [38]; marker-assisted selection is presented in Subchapter 4.1.1;

• high-throughput genotyping — that is. genomics-assisted breeding — combination that represents the most effective strategy for quick variety development [40]; genomics-assisted breeding is presented in Subchapter 4.1.2;

• genome editing techniques [11, 40], an approach called express edit [40]; genome editing is presented in Subchapter 4.2.2;

• automated or high-throughput phenotyping [40, 42].

This type of accelerated plant breeding reduces the time, space, and manpower needed to advance plant generations more rapidly and develop varieties at a quicker pace, so speed breeding was adopted worldwide, with already well-established protocols in many important staple crops [38, 39, 40, 41, 42].

4. Molecular plant breeding

The agricultural challenges of modern society, determined by population growth, climate change, limited resources, and the constraints of conventional breeding, require increasingly innovative, modern methodologies to be applied for the genetic improvement of crops [17, 43, 44]. In the past few decades, due to the progress in science and technology — that is. the considerable increase of knowledge on genes and their function at the molecular level — advanced, new tools for plant breeding have been added to those that have been in use for a long time — that is. traditional breeding [8, 42]. The various umbrella terms describing these new, advanced breeding methods are nonconventional (or unconventional), modern, and molecular. Molecular breeding is arguably the most appropriate and commonly used of the aforementioned terms. The following are some straightforward definitions for molecular breeding:

• “Application of molecular biology in plant breeding is molecular breeding.” [3].

• “Similarly, the recent integration of advances in biotechnology, genomic research, and molecular marker applications with conventional plant breeding practices has created the foundation for molecular plant breeding, an interdisciplinary science that is revolutionizing twenty-first century crop improvement.” [15].

• “Recent progress in biotechnology and genomics has expanded the breeders’ horizon providing a molecular platform on the traditional plant breeding, which is now known as plant molecular breeding.” [16].

• “Molecular breeding applies molecular biology tools to accelerate the breeding process.” [17].

• “Molecular breeding is a modern technique that refers to the combined application of plant biotechnology and breeding for crop improvement.” [18].

• “Modern plant breeding techniques came into being when molecular techniques were integrated along with conventional breeding techniques in order to achieve higher genetic gains.” [19].

Taking into consideration the primary components of molecular breeding outlined in the previous definitions — that is. molecular biology, biotechnology, molecular markers, and genomics — it can be concluded that its essential tools are molecular biology techniques. For better understanding what the concept of molecular breeding entails it is necessary to consider other, more detailed opinions on this topic, which are quite heterogeneous. This is due in part to the fact that terms such as genetic engineering, biotechnology, and molecular marker, used to describe molecular breeding are, in their turn, not always agreed upon by all authors.

Most often, molecular breeding encompasses the use of molecular markers and genetic engineering, as shown by the following definitions:

• “The areas of molecular breeding include QTL [quantitative trait locus] mapping or gene discovery, marker-assisted selection and genomic selection, genetic engineering, and genetic transformation.” [3]; here, it should be noted that genetic transformation is not included in genetic engineering.

• “To this end, recent advances in transcriptome profiling, functional genomics, proteomics, and metabolomics approaches, coupled with molecular marker-assisted breeding and transgenic technology have made significant contributions in enhancing the efficiency of cotton breeding; these methods are collectively referred as molecular breeding.” [45]; here, the authors do omitted genome editing; this also applies to the next definition.

• “Molecular breeding in cotton includes traditional cotton breeding supplemented with marker-assisted breeding using advances in molecular-marker technology and QTL mapping (which includes marker-assisted backcrossing and marker-assisted recurrent selection), genomics (known as genomics-assisted breeding), and transgenics technology.” [45];

• “Therefore, combination of conventional and modern breeding approaches, such as backcrossing, foreground and background selection, phenotyping, gene pyramiding, marker-assisted selections, identification of quantitative trait loci, and many more can be used to have new and improved varieties. Further, biotechnological approaches such as identification of genes by using markers, genetic transformation, regulating signal transductions, and different omics approaches (genomics, transcriptomics, proteomics, and metabolomics) are choice of scientists to develop next generation crops to tackle the challenge of having sustainable agriculture, adverse effect of climate change, and to feed the looming population.” [46].

• “Molecular breeding may be defined in a broad sense as the use of genetic manipulation performed at DNA molecular levels to improve characters of interest in plants and animals, including genetic engineering or gene manipulation, molecular marker-assisted selection, genomic selection, etc.” [47].

There are instances when the authors reference only one of the elements mentioned above, either molecular markers or genetic engineering:

• “Molecular breeding is used to describe several modern breeding strategies, including marker-assisted selection, marker-assisted backcrossing, marker-assisted recurrent selection and genome-wide selection or genomic selection.” [3].

• “DNA markers are also called molecular markers in many cases play a major role in molecular breeding.” [47, 48].

• “Marker-assisted or marker-based backcrossing is regarded as the simplest form of marker-assisted selection, and it is the most widely and successfully used method in practical molecular breeding.” [47, 48].

• “The use of DNA markers in plant breeding is called marker-assisted selection and is a component of the new discipline of “molecular breeding.” [49].

• “The classical approach for molecular breeding is heavily dependent on marker-assisted selection and the trait linked DNA markers as an alternative to support phenotypic screening.” [50].

• “Molecular breeding uses molecular biology tools in breeding crop plant. It includes approaches such as marker-assisted selection and qualitative trait loci mapping.” [51].

• “Single and multigene transgenesis is the current strategy since the past one decade, which if aptly exercised alongside other molecular breeding strategies, can yield satisfactorily performing drought tolerant crop plants.” [52].

Other authors present molecular breeding as pertaining either to molecular markers or genetic engineering:

• “Molecular breeding is the DNA marker-assisted breeding that calls for sophisticated instrumentation and facilities.” [3].

• “Molecular breeding, or MAS, refers to the technique of using DNA markers that are tightly linked to phenotypic traits to assist in a selection scheme for a particular breeding objective.” [53]

• “Molecular breeding approaches target on specific regions on the DNA and therefore are called as marker-assisted breeding. This is often taken from QTL mapping of the quantitative trait. MAB involves numerous modern plant breeding strategies, comprising marker-assisted selection, marker-assisted backcrossing, marker-assisted recurrent selection, and genome-wide selection or genomic selection. Marker-assisted selection (MAS) is a breeding approach that involves integration of detection and selection of DNA marker with a conventional breeding program.” [54].

• “Called molecular plant breeding, plant breeders may now access genes from the animal kingdom for plant improvement, but not without controversy.” [5].

Sometimes molecular breeding — potentially referring to molecular markers — and genetic engineering are seen as two different components of modern breeding methods:

• “The final part has an excellent discussion of advanced techniques of plant breeding, such as tissue culture, genetic engineering, molecular breeding, and application of genomics.” [3].

• “Molecular breeding and [genetic engineering] also have benefits over conventional breeding because they make it tranquil to grow crops with many nutritional traits of interest.” [18].

• “Now, new innovative additional plant breeding tools, including molecular breeding and plant biotechnology, are available to plant breeders, which have a great potential to be used along with the conventional breeding methods for sustainable agriculture.” [55]; here, plant biotechnology most probably refers to genetic engineering.

• “For introduction of desirable traits molecular breeding and transformation technique have also been used widely.” [56].

Certainly, there are many other particular definitions for molecular breeding, its components, and the relations between them.

Conventional techniques are time-consuming, so one of the most important advantages of molecular breeding is the possibility of reducing the duration of the crop development process by years [18]. However, due to its complexity, the successful application of molecular breeding requires sophisticated, high-tech infrastructure and deep knowledge and specialization [44].

4.1 Molecular markers in plant breeding

While significant genetic improvements have been achieved using classical breeding methods — based on phenotypic selection [3, 24] — the employment of molecular biology and genomics tools — that is, molecular or DNA markers — has the potential to enhance genetic gains even more by making selection easier, faster, and more accurate, which will reduce the generation interval and the costs and will improve the overall speed and efficiency of the breeding process [57, 58, 59]. An important advantage of using molecular markers is that genotypic evaluation can be done off-season and the influence of the environment is negligible [60]. Therefore, in recent decades, the focus shifted from phenotype-based to genotype-based selection [3], making molecular markers one of the main components of modern breeding. The two primary types of molecular marker-based strategies used in plant breeding are presented in the following subchapters.

4.1.1 Marker-assisted selection and breeding

Marker-assisted or marker-aided selection (MAS) is the selection of individuals with desirable traits based on the direct analysis of their genetic makeup (genotype) and it can be employed alone or in combination with classical methods [57]. Marker-assisted breeding (MAB) includes several molecular analysis-based applications intended to enhance mating designs, genetic testing and screening, deployment strategies and overall quality control in plant improvement programs [57].

Only a few general aspects are addressed here, while the various types of molecular markers with their features, advantages and drawbacks will not be discussed. These general aspects, that specialists have different views on, are related to how the concept of molecular markers should be perceived. One such issue is the influence of the molecular marker on the target characteristic. In the following examples MAS is presented as a form of indirect selection — the assumption that the marker closely associates with one or more genes of interest, due to genetic linkage, but does not influence the target characteristic:

• “Marker-assisted selection or marker-aided selection (MAS) is a process whereby a marker (morphological, biochemical or one based on DNA/RNA variation) is used for indirect selection of a genetic determinant or determinants of a trait of interest (i.e. productivity, disease resistance, abiotic stress tolerance and/or quality).” [3].

• “One of the most important methods of molecular breeding is MAS, the use of DNA markers that are tightly linked to target loci as a substitute to assist phenotypic screening.” [17].

• “MAS is an indirect selection process, where individuals for a particular trait of interest are selected based on the known markers linked to it.” [24].

• “MAS is the process of using morphological, biochemical, or DNA markers as indirect selection criteria for selecting agriculturally important traits in crop breeding.” [61].

• “Marker-assisted selection is a strategy to accelerate genetic gain in conventional breeding programes by selecting plants with a desirable combination of genes using tightly linked markers.” [62].

However, indirect selection is only one type of MAS, direct selection being also possible:

• “All forms of indirect selection involve selection for one trait to make improvement in a different trait, called the target trait. A second and developing, form of MAS is to select directly on the individual alleles at one or more loci affecting polygenic traits. This form of marker-assisted selection requires knowledge, at the molecular level, of some or all of the genes controlling the target trait and can be viewed as direct selection, rather than indirect selection, since selection is for specific, favorable alleles at those loci.” [57].

• “Marker-assisted selection is a newly emerging approach due to which various problems of conventional breeding avoid and enhance the selection criteria of phenotypes with the selection of genes, either indirectly or directly.” [63].

• “Molecular genetic analyses of quantitative traits lead to the identification of two broadly different types of genetic loci that can be used to enhance genetic improvement programes: causal mutations and presumed non-functional genetic markers that are linked to QTL (indirect markers). […] Whereas causative polymorphisms give direct information about genotype for the QTL, the use of indirect markers for QTL mapping and for selection is based on the existence of linkage disequilibrium (LD) between the marker and the QTL” [64].

• “Secondly, some SNPs [single nucleotide polymorphism] are located in coding regions and directly affect protein function. These SNPs may be directly responsible for some of the variations among individuals in important traits.” [65].

It is also important to understand that molecular markers represent just one of the several types of markers that can be used in plant breeding. The classification of markers can vary, but the concept is rather consistent:

• “Genetic markers used in genetics and plant breeding can be classified into two categories: classical markers and DNA markers. Classical markers include morphological markers, cytological markers and biochemical markers.” [47].

• “Such variations occurring at different levels, that is. at the morphological, chromosomal, biochemical, or DNA level can serve as the genetic markers.” [66].

• “Genetic markers are classified: based on visually evaluated traits (morphological and productive traits), based on gene product (biochemical markers), and founded on DNA analysis (molecular markers).” [67].

Usually, genetic markers encompass all of the other types of markers and can be defined as:

• “… the biological features that are determined by allelic forms of genes or genetic loci and can be transmitted from one generation to another, and thus they can be used as experimental probes or tags to keep track of an individual, a tissue, a cell, a nucleus, a chromosome or a gene.” [47];

• “… any stable and inherited variation that can be measured or detected by a suitable method, and can be used subsequently to detect the presence of a specific genotype or phenotype …” [66]; this definition emphasizes only indirect selection;

• “… a broad term for any visible or assayable phenotype or the genetic basis for assessing of the observed phenotypic variability.” [67].

The definition of molecular markers also varies between authors, especially when considering different fields of study and no attempt will be made here to reconcile them. Below are some appropriate definitions for molecular markers applicable to plant breeding:

• “The markers revealing variations at the DNA level are referred to as the molecular markers.” [66].

• “Molecular marker is a term used to refer to a specific DNA variation between individuals that has been found to be associated with certain characteristics.” [67].

• “A molecular or DNA marker is the difference in DNA nucleotide sequence — between individual organisms or species — that is in proximity or tightly linked to a target gene that expresses a trait.” [68]; it should be noted that the authors emphasize only indirect selection.

MAS is probably both the least sophisticated and the most widely used approach in practical molecular breeding because of its simplicity, especially when compared to the other molecular breeding strategies.

4.1.2 Genomic selection and genomics-assisted breeding

MAS has demonstrated its practicality and feasibility in the enhancement of qualitative traits — features associated with one or very few major genes — but its usefulness in improving quantitative traits — polygenic traits controlled by hundreds or thousands of minor genes, with small effects — is limited [24, 69]. To address this issue, a powerful new approach called genomic selection (GS) or genome-wide selection (GWS) was developed [24]. This new approach became feasible only with the development of new generations of DNA sequencing technologies that revolutionized biological research and genomics by drastically reducing the costs and duration of sequencing [59]. High-throughput genotyping enables the routine implementation of GS to fully benefit crop improvement [59].

Without prior knowledge of relevant QTLs or other molecular markers [69], GS uses dense single SNPs distributed across the whole genome to estimate the genetic merit of individuals of a breeding population and to facilitate the selection of candidates for the next breeding cycle [24, 69, 70]. GS shows great potential for resolving the issue of selection of traits associated with multiple genes [71] because it can theoretically account for the effect, no matter how small, of every piece of genetic information — gene or otherwise — from a genome [72], for which conventional selection and MAS are difficult and time-consuming to apply [71]. Therefore, GS ensures high accuracy and allows for a substantial reduction in the duration of the breeding cycle, while also decreasing the costs associated with extensive phenotyping, and thus accelerating genetic gains and improving the overall efficiency of the breeding process [60, 73].

The possibility of analyzing individuals through whole genome sequencing has turned GS and other genomics-assisted breeding (GAB) tools into powerful assets [3, 60, 72], which enable the integration of genomics with conventional phenotyping in order to facilitate the prediction of phenotype from genotype [3, 60]. In this context, the breeder can make a comprehensive characterization of the genetic variation in order to find the best alleles — that is. to accumulate beneficial alleles and purge deleterious ones [11] — and combinations of alleles (haplotypes) for future crop cultivars [3, 11, 60].

Implementation of GS requires that individuals in a fully phenotyped population v generally called training population — are genotyped using genome-wide markers instead of selected molecular markers. Available phenotypic and genotypic information is employed to build a statistical predictive model that estimates the breeding values of the alternative alleles of all the markers. The additive sum of all marker effects is used to calculate the genetic merit of each individual and the genomic estimated breeding value (GEBV) of the training population. GEBV can then be utilized to estimate the phenotypic value of individuals in a breeding program employing solely their genotypic data. In this regard, the selection of individuals in subsequent generations is based purely on GEBVs. This general outline of the genomic selection process is based on information published by [11, 24, 59, 60, 70, 72, 73].

However, there are certain factors that may influence the accuracy of the genomic prediction: the size and genetic diversity of the training and breeding populations, as well as the genetic relationship between the two, the heritability of the target trait, the influence of the environment on the initial population, the density of markers, the choice of statistical models used to estimate breeding values, and the accuracy of the phenotyping [24, 69, 72].

In conclusion, there is great potential in GS and GAB, and overcoming the limitations these technologies are currently facing will make their routine implementation possible in plant breeding [24, 72, 74], which might lead to replacing phenotypic selection and MAS, at least in the analysis of complex traits [59].

4.2 Genetic engineering in plant breeding

Due to its most defining attribute — the possibility of direct and highly accurate manipulation of genetic information — genetic engineering (GE) has transformed both the way in which biological studies are performed, by becoming an essential tool for understanding the genetic basis of biological processes and the possibilities of applying acquired knowledge [9, 75, 76]. The manipulation of genetic material is intended to induce gene function-level changes — gene inactivation, overexpression of an already existing (native) gene, or synthesis of a new compound and integration of a new function after the insertion of a new gene — that will, in turn, alter the phenotype. Thus, GE has found numerous applications in all the main sectors of human activity — industry, medicine, and agriculture — including plant breeding.

GE represents the foremost biotechnological approach that is used in modern breeding for the genetic improvement of crops, with two basic components: transgenesis or transgenic technology and genome or gene editing [77]. Both of them allow twenty-first-century breeders to alter the genome of an organism and to create new heritable variability through the direct manipulation of the genes controlling the traits of interest [16]. There is, however, an essential difference between the two: the origin of the manipulated genetic material.

In the case of transgenesis, genetic material from two (or more) species is used — that is. combined. Basically, the transfer of genetic material — that is. a gene, but several genes could be transferred as well — from one (or more) species into the genome of another is carried out. This type of transfer is called horizontal or lateral transfer of genetic material and cannot take place between the species involved in transgenesis as they are not capable of sexual reproduction with each other — that is. vertical transfer. So, in essence, transgenic technology introduces novel genes into an organism in order to enhance it with new characteristics. On the other hand, most of the gene editing tools act on the genetic material of a single organism (and species) and their genetic and phenotypic effects are the same as in the case of naturally, spontaneous occurring mutations. It should be noted that the transfer of novel genes — just like in the case of transgenesis — is also possible.

Authors do not always make the same distinction between GE, transgenesis, and genome editing. Often, GE and transgenesis were used interchangeably, especially before genome editing became widespread. Today, such an approach can be somewhat confusing — “advancements in genetic engineering and genome editing techniques draw more attention from conventional plant breeding methods” [78] — and there are many other examples that may hinder even more the understanding of the differences between the three concepts mentioned earlier:

• “The process of genetic engineering or gene editing in plants starts with isolation of the desired gene from a living source, which is then incorporated within a suitable vector to make a recombinant DNA molecule, and finally this recombinant DNA molecule is inserted into the host’s (plant) genome — thus integrating a new function within the GM plant. One of the main components of gene editing tools used for production of GM crops that have the most significant impact upon the overall process of gene editing is selection of a suitable enzyme.” [79].

• “Gene editing can be defined as a process involving advanced techniques in molecular biology for site-specific, efficient, and precise modifications within a genome. The resultant plants can be precisely termed as genetically modified (GM) plants that occur through the transfer of a transgene (gene) of known function.” [79].

In the following subchapters, general aspects regarding transgenesis and genome editing will be presented briefly.

4.2.1 Transgenic breeding

During the past four decades, the asexual transfer of genes employing specific GE methods has added a new dimension to the genetic modification and improvement of plants [57]. It should be noted that genetic modification is often used to refer specifically to genetic engineering and transgenesis [14, 80]. However, the term has traditionally been used to describe any heritable (genetic) improvements in plants (or animals) irrespective of the methodology employed by the breeders [14, 80]. Modifications — insertions, deletions, or substitutions — can range from small-scale alterations, such as a single nucleotide affecting a single gene to major changes in the genetic makeup, affecting numerous genes.

The products of GE — called transgenic plants or crops and commonly referred to as GM or biotech — became the most rapidly spreading agricultural technology in history [81, 82]. They contain at least one (foreign or exogenous) gene that has been artificially inserted into their genome to determine a desired characteristic [49, 77]. The inserted gene is known as a transgene [83] and originates from different sexually incompatible species or can even be completely artificially synthesized.

Consequently, transgenic technology can overcome the reproductive barriers of transferring genetic material so that breeding resources are extended to unrelated species, creating additional genetic variation [49, 77, 84] — characteristics not available in nature in the plants to be modified are introduced from a variety of other organisms [3, 57]. In this way, transgenesis is fundamentally different from traditional, mutation-based, or molecular marker-based breeding [57]. Such an approach also produces plants with desired characteristics faster than classical breeding [3], enabling the insertion of the foreign DNA directly into elite cultivars (genotypes) [49, 83]. The two most popular techniques for plant transformation are Agrobacterium-mediated gene transfer (plant transformation) and particle bombardment [75, 84].

Two particular types of transgenesis have been developed for transferring DNA that belongs to the same species or to other closely related and sexually compatible species: cisgenesis employs natural genetic sequences (i.e. genes with their regulatory elements) for genetic modification, while intragenesis refers to the use of new combinations of coding sequences and regulatory elements [77, 85]. There are different goals for these types of transfers — for example. Overexpression of genes that are already present within the crop itself, avoiding linkage drag that occurs when gene transfer is obtained by crossing — but a very important aspect is that the gene pool exploited by these approaches is identical to the gene pool available for traditional breeding. In this way, objections to transgenesis may be overcome more easily [77].

Gene silencing is another important tool used in plant transgenesis [86], encompassing a series of mechanisms capable of suppressing or inhibiting gene expression [83, 86].

4.2.2 Genome editing

Several new (plant) breeding techniques (NPBTs or NBTs) — also termed novel genomic techniques, new genetic modification techniques, etc. — have been developed over the past few decades [32, 33, 36, 87]. NBTs is an umbrella term, encompassing different biotechnological approaches employed in research and breeding that are capable modifying an organism’s genetic makeup and that have emerged or have been created since 2001 [32, 33, 36]. Of these, genome editing or gene editing (GEd), is a particularly useful strategy for the genetic improvement of crops, allowing much faster and more precise results than other breeding techniques [32, 33, 77]. Therefore, GEd has evolved rapidly in recent years, and it has received increasingly more attention [77, 88, 89], both from researchers and the general public, not only in plant breeding but also in many other research areas as well.

There are many different molecular (genome) editors available, such as site-directed nucleases (SDNs), which include meganucleases (MNs), zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and CRISPR/Cas or CRISPR-Cas systems — CRISPR is an acronym for clustered regularly interspaced short palindromic repeat, while Cas stands for CRISPR-associated protein, prime editing (PE), base editing (BE), oligonucleotide-directed mutagenesis (ODM), transposases, recombinases, chemical (chemistry-based) DNA cutters, and peptide nucleic acids (PNAs) [90, 91, 92]. Although some editing systems have been introduced since the 1990s, with the first one even earlier, it was the CRISPR/Cas-based platform that really “revolutionized this revolutionary field.” Introduced only in 2013, the CRISPR/Cas technology became, by far, the most popular tool for editing the genetic blueprint of an organism due to its simplicity and versatility — CRISPR/Cas can be easily adapted and programmed for many different uses — and continues to drive major breakthroughs in life sciences [90, 93]. The four main classes of CRISPR/Cas-derived genome editors are nucleases (i.e. SDNs), base editors (i.e. BE), prime editors (i.e. PE), and transposases/recombinases [90].

In essence, GEd systems generate targeted DNA mutations (at predefined locations in the genome) [49, 94], ranging from one or a few nucleotides, just like naturally occurring mutations, to inserting or removing one or more entirely functional genes [9, 36, 37].

Applications of SDNs-based GEd — mainly CRISPR/Cas — are generally grouped into different types — SDN-1, SDN-2, and SDN-3 — depending on the presence of exogenous DNA, the cellular response mechanism, and the resulting change in the genetic makeup of the targeted organism [89, 93, 95, 96]. SDN-1 and SDN-2 are somewhat similar, producing plants that contain no exogenous DNA in their genome. The desired traits result from changes, such as nucleotide substitutions and small deletions and insertions, made exclusively on endogenous DNA and are indistinguishable from natural genetic variation or what can be obtained by mutation breeding [9, 95]. The European Network of GMO Laboratories concluded that without prior knowledge, it is technically impossible to detect the small DNA changes introduced by Ged, and thus to distinguish GEd plants from plants selected for certain naturally occurring mutations or plants that are obtained through mutation breeding [9, 33]. SDN-3, on the other hand, results in the insertion of exogenous DNA into the target genome at a predefined locus, and such organisms are considered to be GMOs [9, 95]. PE and BE constitute separate categories than any SDN, but the genetic changes they are producing are similar to SDN-1 and SDN-2 [9].

Breeding has always relied heavily on genetic diversity and modern breeders have continuously looked for ways to expand it [9]. So, over time, breeding efforts were augmented by introducing various means — that is. artificial mutagenesis and trangenesis — for creating new genetic variants in addition to the spontaneous mutations found in nature [9]. One of the drawbacks of artificially induced mutations is that it generates many (probably thousands) unknown and uncontrolled mutations in a genome, even deleterious ones, so isolating a desired new trait could still be time-consuming and, in some cases, virtually impossible [9, 77]. In this context, a distinction must be made between undirected methods — that is. artificial mutation methods causing random genetic changes — and the more precise site-directed methods [97] — that is. GEd, also called targeted genome engineering [94]. So, unlike artificial mutations, the changes produced by GEd are not random, being targeted at a specific predetermined locus, which gives this technology a high level of precision while generating new variability [9]. Therefore, GEd increases the efficiency of introducing single and multiple traits and removing undesirable ones without affecting genetically linked genes [37].

In many countries, the development, commercialization, and use of GM crops are severely limited due to the many regulatory, social, and ethical issues and concerns related to environmental safety and consumer health [9, 77]. As pointed out before, most GEd technologies work without introducing exogenous DNA fragments in the targeted genome and their effects on the DNA and phenotype are the same as those of conventional mutations. It can thus be concluded that “the process is genetic engineering, but the product is not” [97], which should contribute to their acceptance. Certain authors go as far as calling GEd technology and organisms transgene-free [76, 98]. In fact, one of the most intriguing aspects related to GEd is its legal status, especially in relation to transgenesis — whether or not it falls in the same category as GMOs. Two major rulings applicable GEd were issued in 2018:

1. The US did not regulate “plants that could otherwise have been developed through traditional breeding techniques” because they are considered “indistinguishable from those developed through traditional breeding methods;” so, in the US, gene editing is not part of the same regulatory oversite as GMOs; a key point in this regard is the fact that it is nearly impossible to detect whether an organism’s DNA has been edited [9, 36, 93, 95, 99]. As a result, several countries worldwide have fully or partially exempted from national biosafety regulations specific types of GEd organisms [95].

2. The ruling of the European Court of Justice (Case C-528/16) was interpreted by the European authorities to mean that organisms developed through so-called NBT, including GEd crops are not excluded from the scope of the legislation on GMOs — Directive 2001/18/EC; this opinion is based on the fact that GEd techniques alter the genome in such a way that would not occur naturally or by mating, and they do not have a long safety record [9, 36, 37, 93, 95, 99].

The European regulatory approach regarding GEd crops is considered to be completely out of line with the regulations existing in other countries [9] and may constitute an important barrier for GEd-based research and the varieties obtained by GEd [9, 36]. There is a growing consensus that risk assessment should differentiate between GEd crops that have DNA changes, which can also occur spontaneously in nature or as a result of conventional breeding, and Ged crops having genome changes, which cannot occur in nature or as a result of conventional breeding methods — that is. the insertion of a foreign gene to a predefined location in the genome (SDN-3).

Despite the tremendous potential and the promising results obtained so far, there are still enough technical hurdles that need to be overcome so that the utility of GEd can be exploited as best as possible and with as few negative consequences as possible [97]. There are continuous efforts for streamlining the delivery of editing system components into cells and for preventing off-target effects — improving the specificity or efficiency of producing only the desired edits [37, 95]. Nevertheless, it should be taken into consideration that, in plants, the off-target modifications are a relative discussion in the context of mutation breeding methods, which create many more random changes to the genome [9].

GEd arguably represents the most dynamic and rapidly evolving sector of GE and biotechnology, with new applications being published almost daily [9]. Researchers have adopted GEd technology at an unprecedented speed because of its high precision, time and cost efficiency, simplicity, and versatility that accelerated the plant breeding process — genetic variations determining a certain favorable trait are introduced in one or two generations of plants [37, 49, 77]. There is a broad consensus that GEd crops will make a critical contribution to agriculture — and to other areas as well — in the coming years [9]. But the way GEd crops are regulated internationally — whether they fall under the scope of GMO legislation or not — will have a significant impact on the development of GEd technologies and their potential to benefit mankind [77].

5. Concluding remarks

As long as the human population continues to grow, there will also be a high demand for agricultural products. Increased production can be achieved by expanding the cultivated land area, using appropriate agronomic practices (e.g. fertilizers, irrigation, and crop rotation) and cultivating superior plant varieties. However, farmlands are sometimes converted to other uses because an increasing population comes with higher needs for residential, commercial, industrial, and recreational land uses. There are also more and more limitations on available resources and protecting the environment should be taken very seriously in the coming years. In this context, the challenge is to improve agricultural yields while decreasing the use of resources. The solution to this challenge rests with the genotypes being cultivated. Developing varieties superior to the already existing ones is achieved by plant breeding, which, in its modern form, is a very systematic and highly technological approach.

Humans have carried out plant breeding since they first started farming, but its scientific basis was firmly established only at the beginning of the twentieth century when Mendel’s work on heredity and variability was rediscovered. Since then, science and technology have made plant breeding more and more efficient, and spectacular advances have been made by breeders after the implementation of new molecular and biotechnological knowledge over the past few decades.

Plant breeding simultaneously enhances and exploits biological diversity — that is. genetic variation. Therefore, many achievements in plant breeding were based on phenotypic selection, which is still widely used. However, traditional breeding is becoming more sophisticated with the addition of modern strategies. The initial integration of molecular markers in plant breeding — marker-assisted selection — allowed important progress to be made, and now, due to the availability and employment of new sequencing technologies, genomic-based approaches are likely to dramatically change the selection process.

Traditional breeding primarily exploits natural genetic variation, and mutation breeding and genome editing were designed to extend this type of variation, but not beyond species limitations. Further expansion of variability beyond the natural boundaries of sexual reproduction was enabled by other advances in molecular biology and biotechnology — transgenesis allows the transfer of genes between sexually incompatible species. In this way, there is essentially one universal gene pool from which breeders may obtain variability for crop improvement.

The implementation of the new approaches presented in this chapter has already made invaluable contributions to overcoming the aforementioned agricultural threats and challenges. Other possibilities of augmenting breeding methodologies that have not been discussed here are also available: metabolomics-assisted breeding, high-throughput or automated phenotyping, RNA and epigenetic editing (i.e. modification), etc.

This chapter provided an introduction to plant breeding by presenting the main types of genetic improvement that were devised to meet the needs of an ever-growing human civilization. There is a large variety of tools available to breeders, both simple and complex, with incredible advantages but also drawbacks. The information is, thus, a vital resource for students interested in this field of agricultural sciences.