Keywords

1 General Principles of Plant Mutation Breeding

Mutations can be defined as sudden heritable changes in the DNA of living organisms, not caused by genetic recombination or segregation. Mutational events can be easily produced in the laboratory with two principal types of mutagens: physical or chemical. Among the physical methods gamma and X-rays are the most frequently used (reviewed by Spencer-Lopes et al. 2018). Alkylating agents, especially EMS, and sodium azide are the most frequently used chemical mutagens (reviewed by Ingelbrecht et al. 2018). Mutations induced through physical or chemical mutagens occur randomly throughout the genome. Induced mutagenesis has generated a vast amount of genetic variability with a significant role in plant breeding, genetics, and functional genomics. Applied mutagenesis has been particularly successful for the genetic improvement of annual seed crops such as barley, rice, wheat, and sorghum amongst many others. Records from the Joint FAO/IAEA Centre for Nuclear Applications in Food and Agriculture, Austria, maintained in the Mutant Variety Database (MVD), show that over 3,300 crop varieties with one or more traits resulting from induced mutagenesis have been released since the 1960s (IAEA 2023).

The history of plant mutation breeding has been reviewed in several publications (van Harten 1998; Forster and Shu 2012; Bado et al. 2015) and thus will only be briefly described here. In 1901, Hugo de Vries, a Dutch botanist and one of the first plant geneticists, coined the term “mutation” to describe seemingly new forms that suddenly arose in his experiments on the evening primrose. Proof of the mutation theory of de Vries was firmly established by the pioneering work of Lewis John Stadler who induced mutations in barley and maize using X-rays (Stadler 1928). Up to that time, only natural spontaneous mutations were selected to generate novel genetic diversity in plants.

Following the spread of Arabica coffee from Africa to other continents, natural spontaneous Arabica coffee mutants appeared within the widely grown plantations. Several of these mutants attracted the attention of breeders and were described as new varieties in the different regions where they were grown. The most important spontaneous mutants are those affecting plant height, fruit shape and -colour, and leaf colour. Examples include Caturra Vermelho and Caturra Amarelo, dwarf, high-yielding mutants of Bourbon Amarelo observed in the 1930s in Brazil and officially registered as varieties in 1999 (Guimarães Mendes et al. 2007); Pacas, a dwarf mutant of Bourbon found in 1949 in El Salvador; Villa Sarchi, a dwarf mutation of Bourbon found in Costa Rica and released in 1957; and, Maragogype, a large bean size mutant within the Typica variety discovered in Brazil. In addition, a few male sterile plants have been found in Brazil and in Ethiopian accessions in the CATIE collection in Costa Rica (Wintgens 2012; Arabica Coffee Varieties | Variety Catalog; (worldcoffeeresearch.org) (https://varieties.worldcoffeeresearch.org/varieties)). More recently, natural mutations conferring very low caffeine content were discovered at the Instituto Agronômico de Campinas, Brazil. Out of 3,000 coffee trees representing 300 C. arabica accessions from Ethiopia, three plants contained only 0.07% caffeine, in contrast to the normal caffeine content of 1.2% in C. arabica (Silvarolla et al. 2004). These plants have the potential of being the basis for the development of a new coffee varieties giving rise to “naturally decaffeinated” coffee.

The Joint FAO/IAEA Centre of Nuclear Applications in Food and Agriculture has been promoting and disseminating the efficient use of mutation techniques as a tool for crop improvement since the 1960s. Several authors have documented the global impact of induced mutant varieties (Ahloowalia et al. 2004; Kharkwal and Shu 2009). From the 1980s onwards plant mutagenesis has become increasingly integrated into a range of enabling biotechnology and genomics/bioinformatics tools to fast-track the breeding process, mutant selection or mutant trait discovery (Mokry et al. 2011; Schneeberger and Weigel 2011; Ghosh et al. 2018; Knudsen et al. 2022).

Merits of induced mutagenesis as a complementary tool for crop improvement are:

  • Different plant propagules can be subjected to mutagenesis treatments, including seed, entire plants and vegetative propagules, regenerable tissues or single cells

  • It can be (much) faster than conventional breeding

  • Mutation induction is simple and can rapidly produce novel variation (e.g., gamma and X-ray mutagenesis, EMS treatments)

  • Desired traits are introduced directly into well adapted, elite lines/varieties

  • Track record of success for numerous crops and traits, including morphological and physiological traits, yield, disease resistance

  • A public domain, non-GMO technology greatly facilitating adoption of end products.

Limitations of plant mutation breeding include:

  • Not all types of useful variation can be induced as mutations

  • Mutations are random and the desired mutation is rare thus large mutant populations are required to recover the desired mutation or trait

  • Mutant selection mostly relies on phenotyping starting at the M2 generation though recent developments could provide more efficient, early selection systems

  • The space and labour required to grow out large mutant populations, particularly in case of horticultural crops and trees

  • The length of time required to develop mutant varieties in crops with a long reproductive cycle as in perennial crops.

Mutation breeding has been especially successful with annual, inbreeding, diploid crops that are seed-propagated, because it is relatively quick to advance populations from the initial mutant population (M1) to advanced mutant lines. However, vegetatively propagated crops and perennial species—including Arabica coffee—have lagged, due to the limitations listed above. Recent advances in in vitro cell culture offer new opportunities and strategies for vegetative crops and trees through single-cell mutagenesis as described further in this protocol book for Arabica coffee. Likewise, new genomic, bioinformatics and genotyping tools enable screening mutant populations in early generations and can provide a means for short-cutting generations and fast-tracking mutation breeding. This is especially relevant for perennial crops and trees with long juvenile periods.

2 Breeding Limitations in Arabica Coffee

Arabica coffee production is facing multiple threats including the interrelated challenges of climate change and transboundary pests and diseases such as Coffee Leaf Rust (CRL) and Coffee Berry Disease (CBD) (Bunn et al. 2015; Läderach et al. 2017; Solymosi and Techel 2019). Pests and diseases affecting Arabica coffee cultivation with special reference to CLR are reviewed in Chapter “Coffee Leaf Rust Resistance: An Overview”. Protocols for Leaf Rust screening and molecular diagnostics are presented in Chapters “Screening for Resistance to Coffee Leaf Rust”, “Inoculation and Evaluation of Hemileia vastatrix Under Laboratory Conditions”, “Evaluation of Coffee (Coffea arabica L. var. Catuaí) Tolerance to Leaf Rust (Hemileia vastatrix) Using Inoculation of Leaf Discs Under Controlled Conditions” and “A PCR-Based Assay for Early Diagnosis of the Coffee Leaf Rust Pathogen Hemileia vastatrix”.

Nearly all coffee is grown between the Tropics of Cancer and Capricorn where conditions for coffee cultivation are ideal. This band of latitudes is known as the coffee belt. Arabica coffee is usually cultivated in relatively cool mountain climates at 400–2800 m asl. Arabica coffee is sensitive to environmental factors such as exposure to direct sunlight, temperature, and rainfall (Muschler 2001). It is within the coffee belt that the most drastic changes in climate have occurred in recent years. The implications of these changes in coffee production can range from physiological and phenological disorders of plants, to the reduced adaptability of plants to areas with limiting conditions. In addition, pests and diseases also see their physiology and phenology altered, sometimes promoted favorably which implies greater pressure on the production systems. Overall, climate change is impacting coffee production both through changes in weather patterns, viz. rising temperatures, excessive rainfall, or longer droughts, and through changed/expanded habitats of important coffee diseases such as CLR (Avelino et al. 2015; van der Vossen et al. 2015). Coffee Berry Disease, still limited to the African continent, is a latent threat for the Americas in view of the favorable agroecological conditions offered by Latin America for this fungus.

Plant breeding requires genetic variation of useful traits to improve crops. However, the genetic diversity within the primary gene pool of C. arabica is very narrow (Scalabrin et al. 2020) so the required genetic variability to address abovementioned constraints is lacking. Most of the genetic diversity is found in Ethiopia and South Sudan, the centres of origin of C. arabica (Sylvain 1958; Thomas 1942). Since the 1960s coffee yields have stagnated in all coffee producing countries except Brazil, Colombia and Vietnam (Montagnon et al. 2019). Other challenges of Arabica coffee breeding are inherent to the perennial nature of this crop. The generation time factor—3–5 years from seed to seed—remains a major issue for coffee breeding programs.

Currently, two main approaches are followed for the genetic improvement of Arabica coffee. Since the 1950s the traditional varieties formed the basis for pedigree breeding mainly with the ‘Timor Hybrid’ (an interspecific hybrid of C. arabica and C. canephora resulting from a natural cross) that has resistance to CLR (Bettencourt 1973; Silva et al. 2018). However, pedigree breeding is a long process requiring 30 years or more to release a stable, homogeneous and distinguishable variety. To date, most Arabica coffee plantations around the world are established with the varieties resulting from breeding efforts initiated some 50 years ago. However, these varieties are susceptible to disease outbreaks, especially CLR, and are poorly adapted to the changing climatic conditions observed in many coffee growing regions during the past decade. Obtaining CLR resistant varieties will allow to produce coffee with reduced pesticide use or in organic farming systems (Arrieta 2014). Since the 1990s, F1 hybrids are being developed as an alternative breeding strategy in view of their improved performance over traditional varieties in terms of yield and disease resistance, and because of the reduced timeframe of 10–20 years from breeding to commercial release (Frédérick et al. 2019). However, unlike the traditional varieties, F1 hybrids are not true breeding and thus require a different mechanism for mass production, typically via clonal propagation (World Coffee Research | F1 Hybrid Trials (https://worldcoffeeresearch.org/programs/next-generation-f1-hybrid-varieties)). More recently, male sterility has been used for F1 hybrid seed production in Arabica coffee (Frédérick et al. 2019).

3 Mutation Breeding in Arabica Coffee

3.1 Background

Arabica coffee production is threatened by disease outbreaks and climate change while conventional breeding is hampered by the very narrow genetic base within its primary gene pool, as summarized above. Induced mutagenesis may have significant value for Arabica coffee by increasing genetic variability for genetic studies and breeding purposes. The plant Mutant Variety Database (MVD) lists over 3300 released mutant varieties in a wide range of crop plants. Over 80% of these resulted from exposure to physical mutagens. Ionizing radiation such as gamma rays and X-rays have been the most widely used techniques for mutation induction. Example successes in mutation breeding of woody plants reported in the MVD are shown in Table 1. Note that Arabica coffee is not listed in the MVD. To our knowledge no Arabica mutant variety has been released following induced mutagenesis. Thus, Arabica coffee remains a major crop that has not been improved by mutation breeding, though Arabica coffee varieties resulting from natural, spontaneous mutations are being grown commercially.

Table 1 Examples of perennial crops and trees improved using radiation breeding (IAEA 2023)
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According to the MVD, induced mutagenesis has been successful for inducing resistance to fungal diseases in 334 cases (Fig. 1). These include tree crops such as pear (Sanada et al. 1993; Saito 2016) and crops with polyploid genomes such as wheat (Sigurbjörnsson and Micke 1974) and sugarcane (IAEA 2023).

Fig. 1
A pie chart illustrates the distribution of plant species that have released mutant varieties. Rice occupies the largest proportion in the chart, while Japanese pear has the smallest proportion.

Released mutant varieties with induced resistance to fungal diseases according to the FAO/IAEA Mutant Variety Database (IAEA 2023). Only plant species with at least three released varieties are listed with rice, wheat, barley and maize having the highest counts; ‘other’ includes plant species with one or two released mutant varieties

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So far, there has been limited research on induced mutagenesis of Arabica coffee. The first attempt to induce new mutations in C. arabica was reported by Carvalho et al. (1954) using X-ray irradiation of seeds with doses up to 1,500 Gray (Gy, see below Sect.3.2). Main effects established were early termination of seedling growth if treatment was higher than 125 Gy and a general slow growth of mutagenized seedlings as compared to controls. Among the surviving seedlings, variation in the form of abnormal leaves was also observed. Moh and Orbegosos (1960) used thermal neutrons, X-rays and gamma-rays for induced mutagenesis in C. arabica and frequently obtained angustifolia (ag) mutants characterized by long and narrow leaves. Appearance of this phenotype already in the M1 generation was explained by possible chromosomal aberrations. Interestingly, the mutant leaf type was similar for the entire plant and not sectorial, which excludes the presence of chimerism. Recently, similar observations were made in M1 mutant coffee plants derived from gamma-ray irradiation at the FAO/IAEA Plant Breeding and Genetics Laboratory, Seibersdorf, Austria (Fig. 2). Moh (1961) speculates that the lack of chimerism in the M1 plants indicates that the coffee plant originates from only one initial cell in the embryo shoot apex. This would, however, be one of very rare cases among the angiosperms. It is also conceivable that the uniform mutant leaf phenotype is the result of diplontic selection between cells of the meristem or that only one initial cell survived after irradiation. A final answer to the question of M1 uniformity is yet to be given. In later induced mutation breeding experiments, analysis of traits of economic importance such as yield were put forward and monitored over several mutant generations. However, apart from the occurrence of leaf mutations, no correlation between varying yield and radiation dose could be established (Carvalho et al. 1984). These early experiments in coffee mutation breeding however, do suffer from the relatively small number of plants analyzed. A protocol for phenotypic characterization of an M1 Arabica coffee greenhouse-based mutant population is presented in Chapter “Use of Open-Source Tools for Imaging and Recording Phenotypic Traits of a Coffee (Coffea arabica L.) Mutant Population”.

Fig. 2
4 photos of Arabica coffee plants grown in pots.

Arabica coffee M1 mutants obtained from gamma-ray irradiation of seed at the FAO/IAEA Plant Breeding and Genetics Laboratory, Seibersdorf, Austria, 29 months after irradiation. a Wild type; b dwarf and leaf morphology mutant; c, d leaf morphology mutants. Note that the mutant leaf morphology characteristic is not sectorial but is similar for the entire plant

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3.2 The Need for Radiosensitivity Testing

Treatment of a plant or plant part with a mutagen affects its vigour, growth rate, germination, and fertility. Mutation rates vary with mutagen dosage. The higher the dosage of a mutagen, the more frequent the mutations and hence also, the greater the chance of undesired damage and lethality. The optimal dose is the one that, on the one hand, limits adverse effects that prevent the creation of a sufficiently large and vigorous mutant population, and, on the other hand, produces sufficient mutations to have a reasonable chance of recovering the desired mutation or mutant trait in the population, while preserving the (elite) genetic background. Hence, dose optimization is typically the first step in experimental or applied mutagenesis. Here, key principles and considerations for optimizing physical mutagenesis relevant to Arabica coffee will be briefly described. General principles and protocols for dose optimization using physical (Spencer-Lopes et al. 2018) and chemical mutagens (Ingelbrecht et al. 2018; Jankowicz-Cieslak and Till 2016) and subsequent mutant population development (Ghanim et al. 2018) have been published.

Common units in physical mutagenesis include Gray (Gy), used to quantify the dose of radiation absorbed by the plant material and Gy/s or Gy/min, which is the unit for absorbed dose rate, a characteristic of the radiation source and irradiator used for mutagenesis. Radiosensitivity is a property of the target material, e.g., seed versus vegetative tissues, and of the species/variety. In addition, radiosensitivity is subject to external factors, such as, for example, the water content of the target material. Depending on the explant, the water content can be regulated. For example, the water content of seed can be adjusted to ca 12–14% through equilibration in a desiccator containing a 60% glycerol solution prior to irradiation, which is standard procedure at the FAO/IAEA PBG Laboratory, Seibersdorf, Austria.

Radiosensitivity testing refers to the determination of the optimum dose(s) of radiation of a particular plant propagule to be used as a basis for selecting the dose levels for bulk irradiation. In practice, radiosensitivity testing is performed across a series of mutagen doses in the lab or greenhouse over a short period of time. Growth responses or lethality is measured compared to a non-radiated control, to determine the GR30 (30% Growth Reduction) and GR50 (50% Growth Reduction) or LD30 (30% Lethal Dose) and LD50 (50% Lethal Dose) values respectively. These ranges have been observed to preserve the fitness of the M1 plants (first mutant generation ) while inducing sufficient stable, genetic variability for genetic studies or breeding purposes. In case of radiosensitivity testing of seed, the GR value is usually determined from the reduction of seedling height or leaf growth of the M1 plants compared to untreated M0 controls. In case of radiosensitivity testing of lethality of seed, seedling survival is measured over a range of doses compared to untreated controls. Importantly, the biological effects observed at the M1 stage are the result of transient physiological effects and from genetic effects that are passed on to the next M2 generation. Mutations are single cell events and thus mutagenic treatment of seed or other multicellular tissues may carry one or several mutations, each occupying a small part of the resulting M1 plant. Such M1 plants are therefore chimeras. Plant scientists or breeders need to be aware of the complications caused by chimerism and apply techniques to resolve them. Mutagenic treatment of single cells followed by plant regeneration does in principle result in chimera-free plants.

The doses chosen for bulk irradiation and development of the M1 population, depend on different factors, such as the available resources to grow out and screen the mutant populations. The breeding system of the species under study plays a key role (van Harten 1998). For example, for annual diploid, self-fertile crops such as barley or sorghum, background mutations can be relatively easily removed through backcrossing. This is much more challenging or impossible for (obligately) vegetatively propagated crops or trees as in the case of Arabica coffee due to their long juvenile phase. Ideally up to three different doses are applied for bulk irradiation, including doses lower than LD50 or GR50, to ensure that at least one level will yield a sufficient number of the required mutant types. The frequency of induced mutations depends on the type of mutagen, the applied dose and the target materials. The plant species or variety, ploidy level, developmental stage, physiological state, etc. may all result in differences in response to radiation. Therefore, standardization of the target material and keeping records of all relevant information about the radiation source and treatment conditions is critical.

3.3 Choice of Material for Mutation Induction

Since Arabica coffee is self-fertilizing, the cheapest and most appropriate propagation system, especially in a commercial setting, is through seeding. However, for research and experimentation purposes, other plant propagules such as seedlings, cuttings or grafts can also be applied. In vitro cells and tissues are another attractive target in case of Arabica coffee given the availability of methods for de novo regeneration through somatic embryogenesis. Different plant propagules that can be used as targets for mutation induction in Arabica coffee with their advantages and limitations are summarized in Fig. 3.

Fig. 3
A chart exhibits the challenges and opportunities associated with different target materials, including seed, seedling, plant, in vivo cuttings, pollen, in vitro somatic cell, and in vitro propagules.

Target materials for mutagenesis treatment of Arabica coffee with limitations and advantages

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In choosing the target material for dose optimization and mutagenesis treatments, it is important to consider the life cycle of a coffee tree, the seed and germination process. The coffee plant takes approximately three years from seed germination to produce the first fruit. It takes 6–9 months from flowering to mature cherries ready for harvest. The coffee cherry is the whole fruit, and has a skin, pulp, and parchment that cover the seed of the coffee. Inside the fruit are usually two seeds. Protocols for the establishment of in vitro tissue culture systems for Arabica coffee and methods for mutation induction using in vitro tissues and cells are described in Chapters “In Vitro Plantlet Establishment of Coffea arabica from Cut Seed Explants”, “Somatic Embryogenesis and Temporary Immersion for Mass Propagation of Chimera-Free Mutant Arabica Coffee Plantlets”, “Protocol on Mutation Induction in Coffee Using In Vitro Tissue Cultures”, “Mutation Induction Using Gamma-Ray Irradiation and High Frequency Embryogenic Callus from Coffee (Coffea arabica L.)”, “Chemical Mutagenesis of Embryogenic Cell Suspensions of Coffea arabica L. var. Catuaí Using EMS and NaN3”, “Chemical Mutagenesis of Coffea arabica L. var. Venecia Cell Suspensions Using EMS” and “Chemical Mutagenesis of Zygotic Embryos of Coffea arabica L. var. Catuaí Using EMS and NaN3”. Protocols for mutation induction of seed and ex vitro vegetative propagules are described in Chapters “Physical Mutagenesis of Arabica Coffee Seeds and Seedlings”, “Mutation Induction in Coffea arabica L. Using In Vivo Grafting and Cuttings”, “Chemical Mutagenesis of Mature Seed of Coffea arabica L. var. Venecia Using EMS” and “Chemical Mutagenesis of Coffee Seeds (Coffea arabica L. var. Catuaí) Using NaN3”.

3.4 Enabling Biotechnology and Genomics Tools

In vitro plant cell and tissue culture techniques offer the possibility of rapid true-to-type multiplication. Somatic embryogenesis (SE) is an in vitro vegetative propagation technique that can produce clones of plants in large quantities. Research on in vitro tissue culture of Arabica coffee began in the 1970s. Since then, protocols for in vitro regeneration of Arabica coffee through SE have been developed, both direct and indirect methods have been reported (Barry-Etienne et al. 2002; Murvanidze et al. 2021). Somatic embryos can be produced from leaves of trees as starting material. Two innovations aimed at developing commercial scale multiplication systems include the use of bioreactors and direct sowing of somatic embryos in nurseries (Barry-Etienne et al. 1999; de Rezende Maciel et al. 2016; Etienne and Berthouly 2002; Etienne et al. 2013, 2018; Menendez-Yuffa et al. 2010). These micropropagation techniques are intended to enable mass propagation of elite Arabica coffee materials, such as F1 hybrids which cannot be propagated by seeding. The availability of protocols for in vitro regeneration of Arabica coffee offers exciting opportunities to integrate advanced cell culture techniques with induced mutagenesis to produce chimera-free mutant plants, a major bottleneck in induced mutagenesis of perennial crops with a long juvenile phase such as coffee. Some horticultural techniques, such as cuttings, can also enable cloning and multiplication of coffee plants.

The analysis of segregating molecular markers has confirmed earlier genetic and cytogenetic evidence that C. arabica is a functional diploid (Lashermes et al. 2008). Alkimim et al. (2017) and Saavedra et al. (2023) reported the use of marker-assisted selection for pyramiding multiple CLR and CBD resistance alleles. Genomic tools and large-scale sequencing enable a better understanding and characterization of the diversity and function of the Coffea genetic resources. This knowledge can then be utilised by breeders to select the best parental materials for incorporation into breeding programmes. Genomic selection (GS) allows breeders to select traits that are influenced by large numbers of small-effect alleles in a wide range of genotypes. Using GS in the context of resistance breeding for perennial crops increases the efficiency of breeding programs by shortening the breeding cycles (Alves et al. 2015). The release of reference genomes of C. canephora and C. arabica broadened the possibilities and facilitated significant progress for C. arabica genomic analysis (Denoeud et al. 2014; Dereeper et al. 2015; https://worldcoffeeresearch.org/resources/coffea-arabica-genome; Scalabrin et al. 2020). Sant’Ana et al. (2018) used the C. canephora reference genome to find SNP markers in the C. arabica genome associated with lipids and di-terpenes composition in a GWAS study of 107 diverse C. arabica genotypes. Knowledge of the molecular genetic structure of genes of interest to coffee breeders can then be applied for molecular breeding of Arabica coffee, including for example, for gene-based selection in mutation breeding programs. Protocols on the development and use of a Coffee Exome Capture kit, the application of High-Resolution Melt analysis, and the use of molecular cytogenetics for the detection of induced mutations in coffee are described in Chapters “Targeted Sequencing in Coffee with the Daicel Arbor Biosciences Exome Capture Kit”, “High Resolution Melt (HRM) Genotyping for Detection of Induced Mutations in Coffee (Coffea arabica L var. Catuaí)” and “Protocols for Chromosome Preparations: Molecular Cytogenetics and Studying Genome Organization in Coffee”, respectively.