Keywords

1 Introduction

After petroleum, coffee beans are the second most economically important product traded worldwide. Approximately 11 million ha of coffee trees are cultivated in tropical regions (Déchamp et al. 2015), providing income to 125 million people. Despite the importance of coffee, several factors, which could be amplified by changing climatic conditions, hamper its production and influence the extent of yield losses, including agronomic management, growing environment, cultivar selection affected by diseases and pathogens. Pathogens are the major limiting factors in coffee production. Approximately 26% of the global annual coffee production is lost to diseases.

Cultivated coffee originated from wild populations in Africa, Madagascar, the Comoro and the Mascarene islands, the Indian subcontinent, Southern tropical Asia, South-East Asia and Australasia (Razafinarivo et al. 2013; Davis et al. 2006, 2007).

Among different coffee species, the two economically most important are the diploid C. canephora Pierre ex Froehn (2n = 2x = 22), native to Central and Western sub-Saharan Africa, and the allotetraploid C. arabica L. (2n = 4x = 44) from the Southwestern Ethiopian highlands, Mount Marsabit of Kenya and the Boma Plateau of Sudan (Anthony et al. 2002). C. arabica resulted from ancestral hybridization—dated approximately 50,000 years ago—between two diploid ecotypes, namely C. eugenioides and C. canephora (Anthony et al. 2010; Lashermes et al. 1997; 1999; Ribas et al. 2011). C. arabica is self-pollinated, while C. canephora is cross-pollinated. A recent study by Sant’Ana et al. (2018) showed a high allelic variation in wild accessions from Ethiopia, however, the mode of pollination and the history of coffee cultivation resulted in a reduction of genetic diversity in C. arabica. According to different authors, coffee was introduced from Ethiopia to Yemen between 1,500 and 300 years ago. From this point, the first reduction of diversity happened within Arabica cultivars. Genetic data analyses showed that two genetic bases spread from Mocha (Yemen) in the early eighteenth century (Chevalier and Dagron 1928; Haarer 1956). C. arabica var. arabica (also called var. typica Cramer) originated from a single plant that was introduced to Java (Indonesia) and later cultivated in the Botanical Garden of Amsterdam. C. arabica var. Bourbon (B. Rodr.) Choussy (Carvalho et al. 1969; Krug et al. 1939) was introduced to the Bourbon Island (Réunion). These were the starting points of coffee cultivars spreading rapidly to the American continent and Indonesia by the use of seeds produced by the auto-fertilization of coffee trees, which caused a further reduction in genetic diversity.

There are several major constraints in coffee breeding. As already mentioned, the vast majority of the world coffee production is based on two species, Coffea arabica (2n = 4x = 44 chromosomes) and C. canephora (2n = 2x = 22 chromosomes). This results in low genetic diversity among coffee cultivars and represents a massive limitation in case of control and management of pest and disease under climatic changes. The absence of pest resistance in the most preferred Coffea arabica cultivars can be overcome by cross-breeding, but due to the long juvenile period of tree crops this is a time-consuming process (Silva et al. 1999; 2006; Várzea et al. 2000).

Plant biotechnological interventions in coffee improvement are used to develop uniform planting material through cell and tissue culture (Krishnan 2011) since the pioneering attempts in mutation induction by Carvalho in the 1950s (Carvalho 1988).

In recent years mutation breeding programs have been initiated within the FAO/IAEA funded Coordinated Research Project (CRP) D22005 on mutation induction for coffee improvement (Dada et al. 2014, 2018; Bolívar-González et al. 2018; Bado et al. 2018a, b). In contrast to conventional breeding, taking at least 20 years to release a new cultivar, biotechnological methods offer valuable tools for coffee improvement and for speeding up the selection process of superior plants (Bado et al. 2017; Campos et al. 2017). Micropropagation by organogenesis is used for plant multiplication mainly from shoot tips and axillary buds allowing the production of large-scale populations for mutation induction and subsequent mutant line propagation and is mainly suitable for vegetatively propagated crops with a long juvenile period. This allows to reduce the time required and to accelerate mutation breeding when using single cell explants. A considerable acceleration of mutation breeding can be achieved by using single cell explants like double haploids or somatic embryos, which mark the shortest route to produce homozygous lines from heterozygous plant material.

Somatic embryos can be produced on a large-scale in suspension cultures and in bioreactors. As a matter of fact, somatic embryogenesis is an excellent system for mutation induction, since somatic embryos originate from single cells (da Câmara Machado et al. 1995).

Among the physical mutagens, gamma rays are the most commonly used for mutation breeding (Mba et al. 2012), resulting in small to large deletions, point mutations, single and double strand brakes and even chromosome deletions. When applying physical mutagens to different types of plant material, care should be taken with soft material such as in vitro shoot cultures as well as callus and embryogenic callus cultures, which require lower doses in comparison to seeds. In fact, the water content, storage time, applied mutagen dose and temperature represent important factors influencing mutagens in all types of plant material (Mba et al. 2010).

Depending on the explant type subjected to mutation induction different approaches are required for chimera dissolution. Plants originating either from unicellular or multicellular explants require different time frames for chimera dissolution ranging from 0 for plantlets stemming from somatic embryos to several generations up to M1V4 for plantlets originating from multicellular explants (Novak and Brunner 1992). Entire mutant populations are screened by either phenotypic evaluation to select the phenotype of interest or by genotypic evaluation to detect novel alleles in genes of interest (Fig. 1).

Fig. 1
A flowchart of the mutation breeding schemes for mutagenesis in Coffea species. The steps include donor material, in vitro cell or tissue cultures, mutagenesis on uni or multicellular explants, screening for traits of interest, and release of improved cultivars.

Mutation breeding scheme for mutagenesis in Coffea sp. 1 Mature donor plants provide vegetative buds, flower buds, leaves, while seeds may be established directly in vitro. 2 In vitro cell and tissue cultures may involve somatic and gametic cells. 3 Coffee explants for mutation induction may be of uni- or multicellular origin. Note that multicellular systems require an additional step for chimera dissolution, while single cell systems do not require this step. 4 Screening of mutants can be conducted either before acclimatization through early screening of irradiated cells and plantlets or after acclimatization of plantlets to greenhouse or under open field conditions. 5 Selected improved cultivars can be released as direct mutants or be further used in breeding programs by hybridization

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Pathogens are the major limiting factor in coffee production. New approaches are available to breed varieties that are resistant to a broad-spectrum of pathogens, genetically stable and high-yielding. Recently developed tools in genomic technologies allow to better understand coffee-pathogen interaction and help to identify the genes and mechanisms involved in pathogen resistance or susceptibility. Understanding the influence of individual factor and their interaction will help to select realistically interesting accessions and to accelerate breeding strategies.

2 Materials

2.1 Establishment of a Collection of Donor Material In Vivo

A collection of starting material of the two species of coffee—both as seeds and potted plants—should be initiated in the greenhouse to allow the maintenance of mother plant under controlled conditions (Fig. 2). In order to rule out major genotypic differences, in our experiments this included fourteen different cultivars of the leaf rust susceptible C. arabica (self-pollinating) as well as two genotypes of the leaf rust resistant C. canephora (self-incompatible) with different climate requirements and tolerance/susceptibility to different pathogen races. The collection of C. arabica cultivars (https://sca.coffee/research/coffee-plants-of-the-world) comprised:

  • Bourbon: A common cultivar C. arabica that developed naturally on Île Bourbon (an island in the Indian Ocean, east of Madagascar, now known as Réunion) from coffee brought to the island from Yemen by the French. Depending on the specific sub-group, this coffee can be red (Vermelho) or yellow (Amarelo). These plants generally have broader leaves and rounder fruit and seeds than Typica varieties. Stems are stronger and stand more upright than Typica. They are susceptible to all major diseases and pests.

  • Catimor: A group of pure-line cultivars originating from crosses between Hibrido de Timor and Caturra. It has been distributed since the 1980s. It is known to be highly productive and shows resistance to coffee leaf rust and to coffee berry disease (CBD).

  • Caturra: A pure-line dwarf spontaneous mutant of red Bourbon that has short internodes. It was found in 1937 in Brazil, and is highly productive. Its leaf and fruit characteristics are similar to Bourbon varieties and can produce red or yellow cherries. Like Bourbon, it is known to be susceptible to all main diseases and pests.

  • HDT (Clones 1–4) Hibrido de Timor (Timor Hybrid): A spontaneous cross of C. canephora and C. arabica var. Typica that occurred naturally on the island of Timor in Southeast Asia. These “Arabusta”-type hybrids likely originated from a single Robusta parent plant. It became popular in Timor in the 1950s due to its natural resistance to leaf rust. These hybrids were collected in Timor in 1978 and planted on the islands of Sumatra and Flores shortly thereafter, and since then some changes and mutations have occurred. Different versions of this hybrid have been utilized in breeding programs to introduce the rust resistance into new varieties, such as Catimor and Sarchimor.

  • Java (Clones 1, 2, 8, 9, 10, 12): A Typica selection suspected to be the progeny of coffee introduced from Yemen to the island of Java. From Java, this plant was first brought to neighboring islands (Timor) and later to East Africa (Cameroon), where it was released for cultivation in 1980. It has since been introduced in Central America by the Centre de Cooperation Internationale en Recherche Agronomique pour le Développement (CIRAD). It is known to be vigorous with moderate yield and shows good resistance to coffee berry disease in Cameroon. Java has elongated fruit and seeds and bronze-colored young leaves.

  • Kent: A tall Typica selection that likely arose on or from coffee bred on the Kent Estate in India. It has been widely planted in India since the 1930s and a selection from this cultivar, known as K7, is more common in Kenya. It is known as the first coffee selected for rust resistance.

  • Pacamara: A cross between Maragogype and Pacas developed in El Salvador. Similarly to Pacas, it is known to be susceptible to all main diseases and pests. Pacamara was released in 1958 but is genetically unstable, with 10–12% of plants reverting to Pacas.

  • Sarchimor: A group of pure-line cultivars originating from a cross between Villa Sarchi and one Hibrido de Timor. Some Sarchimor lines show good resistance to coffee leaf rust; some are also resistant to coffee berry disease.

  • Typica (Clones 1–3): This is a tall cultivar of Coffea arabica, originating from the coffee brought to Java from Yemen (possibly via India). The plants, most similar to what we today call Java, were spread from the island of Java in the early 1700s. It has bronze-tipped young leaves, and the fruit and seeds are large. Typica plants are known to have relatively low productivity and are susceptible to all main pests and diseases.

  • Villa Sarchi: A dwarf mutation of Bourbon found in Costa Rica and released in 1957. It is known to be susceptible to most pests and diseases.

For the maintenance of donor plants of Coffea sp. under greenhouse conditions the following items should be available:

  1. 1.

    High quality, disease-free seeds and plants, uniform in size (see Notes 1 and 2).

  2. 2.

    Sterilized soil mixture.

  3. 3.

    Glasshouse facility.

  4. 4.

    Regular fertilization and irrigation.

Fig. 2
A photo of the Coffea species plants.

Maintenance of donor plants of Coffea sp. under greenhouse conditions

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2.2 Establishment of In Vitro Shoot Cultures as Donor Material

Axenic cultures were established and prepared as source material for the specific regeneration and mutation programs. Different in vivo donor material was used with the intention to establish micropropagation for media optimization and induction of callus, somatic embryogenesis and suspension cultures: seeds, seedlings, leaves, stems, roots, flowers. Serving both as material for mutagenesis treatment, as well as for the recovery of individual mutagenized cells, these culture systems should be maintained throughout the entire period of the experiment.

The first 4 nodes of orthotropic shoots growing in the greenhouse under controlled conditions were excised as explants. Surface sterilization was achieved with a 15% sodium hypochlorite solution containing 1% Tween 80 for 20 min followed by 4 washes with distilled water. To carry out these steps, the following facilities and items are required:

  1. 1.

    High quality greenhouse grown plants of defined genotypes (see Note 1).

  2. 2.

    Laminar air flow cabinet for in vitro work (in vitro culture facility).

  3. 3.

    70% ethanol.

  4. 4.

    15% Danchlor solution.

  5. 5.

    Sterile Aqua dest.

  6. 6.

    Magenta boxes.

  7. 7.

    Culture media (see Note 3).

Axillary shoot cultures of different cultivars were established from 2 genotypes of C. canephora, Niaoulli (14 clones) and Quillou (6 clones), as well as from 15 genotypes of C. arabica: HDT (4 clones), Caturra, Catimor, Kent, Sarchimor, Typica (3 clones), Villa Sarchi, Java (6 clones) and Bourbon (Fig. 3).

Fig. 3
A photo of the three glass containers containing Coffea species plants.

Axenic in vitro cultures of Coffea sp. serving as donor material for mutation induction

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2.3 Establishment of Tissue Culture Material for Mutation Induction

Somatic embryogenesis is an excellent system for plant propagation and mutation induction, since somatic embryos originate from single cells and therefore reduce chimerism. Somatic embryos can be produced on a large-scale in suspensions in Erlenmeyer flasks and in bioreactors. Somatic embryos of Coffea can be obtained either by direct or by indirect somatic embryogenesis, the difference being the intermediate callus induction.

For the induction of coffee callus cultures and somatic embryogenesis additional multicellular explants, like leaves, stems and roots of in vitro grown seedlings (Fig. 4) and from in vitro shoots from selected cultivars are used.

Fig. 4
Two photos exhibit samples of Coffea species undergoing direct and indirect embryogenesis in Petri dishes.

Induction of direct and indirect embryogenesis from different in vitro explants of Coffea sp.

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Fig. 5
A cyclic diagram of embryogenesis of the progression from leaf discs to callus, embryogenic callus, globular embryos, cotyledonary embryos, and finally, plantlets. The embryogenic callus is marked as single-cell explants, and the embryos are marked as multi-cellular explants.

Induction of direct or indirect embryogenesis from different in vitro explants

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The conversion of embryos to plantlets from a number of selected cultivars after transfer of emerging embryos from embryogenic calli to regeneration medium yielded a high number of mutant plantlets (Fig. 6).

Fig. 6
A photo of 12 containers arranged in three rows, each containing plantlets. The bottom row displays small plantlets, while the top row consists of large plantlets.

Regeneration of plantlets/somatic embryos from irradiated embryogenic calli

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To establish an efficient regeneration from embryogenic calli into plantlets the following items are needed:

  1. 1.

    High quality embryogenic callus cultures of defined genotypes.

  2. 2.

    Laminar air flow cabinet for in vitro work (in vitro culture facility).

  3. 3.

    Culture media for indirect and direct embryogenesis (see Note 4).

  4. 4.

    70% (v/v) ethanol.

  5. 5.

    Parafilm.

  6. 6.

    Petri dishes (9 cm diameter).

  7. 7.

    Magenta boxes.

  8. 8.

    Regeneration media for the recovery of plantlets (see Note 5).

2.4 Mutagenesis by Physical Agents

Gamma ray mutagenesis may be performed using different facilities, such as gamma cell irradiator, gamma phytotron, gamma house, gamma field. The gamma cell irradiator with Cobalt-60 (or Cesium-137) as radioactive source is the most commonly available equipment worldwide (IAEA 1975, 1977).

However, the radioactive source remains the major consideration and constraint in plant mutagenesis (Bado et al. 2015).

  1. 1.

    Gamma radiation source.

  2. 2.

    Magenta boxes or petri dishes (9 cm diameter).

  3. 3.

    Parafilm.

  4. 4.

    Culture media (see Notes 4 and 5).

3 Methods

3.1 Mutagenesis by Physical Agents

Mutations were induced by gamma-irradiation of different explants of selected genotypes of C. arabica and C. canephora at different intervals with several repetitions. A Cobalt-60 Gamma irradiator was used and the irradiation was performed at the FAO/IAEA Laboratories in Seibersdorf, Austria. The workflow shown in Fig. 7 can be carried out with different plant cell and tissue cultures either in Magenta boxes or petri dishes.

Fig. 7
A pictorial flowchart has the following steps. 1. Open irradiation chamber of gamma cell irradiator. 2. Prepare in vitro explants for irradiation. 3. Insert the samples into the irradiation chamber. 4. Set the exposure time and run the irradiator.

Working steps for irradiation using a Cobalt-60 gamma cell

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3.2 Selection and Treatment of Explants

The first type of explants to be subjected to irradiation are axenic shoot cultures in order to determine the radiosensitivity of different C. canephora and C. arabica cultivars (Fig. 3, see Notes 6 and 7).

  1. 1.

    Prepare Magenta boxes with freshly micropropagated plantlets.

  2. 2.

    Place 20–25 shoots into a petri dish and a humid Whatman filter paper.

  3. 3.

    Seal the petri dishes with Parafilm to avoid contamination outside the tissue culture laboratory.

  4. 4.

    Label each petri dish with the sample ID and required dose (0, 10, 20, 40 and 60 Gy).

  5. 5.

    Prepare the regeneration media for the subsequent subculture.

  6. 6.

    Transport the next day to gamma irradiator facility for mutagenesis (see Notes 8 and 9).

  7. 7.

    Put the chamber at the irradiation stage by downing the elevator (see Note 10).

  8. 8.

    Start the time discount of exposure time for watch monitoring after the click of the elevator. In automatic conditions the irradiator timer will monitor to the full time (see Note 11).

  9. 9.

    Raise immediately the chamber to the loading stage when exposure time is completed. In automatic conditions the chamber raises when full time is reached.

  10. 10.

    Open the lead shielding collar and then sample chamber door.

  11. 11.

    Remove the irradiated plant material (see Note 12).

  12. 12.

    If necessary, repeat the treatment at defined time intervals to reach the required dose, which is function of exposure time base on the source dose rate.

  13. 13.

    Subculture irradiated plant material to fresh culture medium (see Note 13).

After having determined the dose range for entire shoots, callus cultures, embryogenic callus cultures, somatic embryos at the globular, torpedo or cotyledonary stage can be irradiated in a similar way.

  1. 1.

    Prepare petri dishes containing semi-solid of Zamarripa M3 medium with freshly subcultured callus or embryogenic callus cultures.

  2. 2.

    Alternatively plate 100 mg of somatic embryos at the globular, torpedo or cotyledonary stage per petri dish (with clearly assigned sample ID) and per replication.

  3. 3.

    Prepare three petri dishes per dose for a range of gamma irradiation doses of 0, 10, 15, 20, 25 and 30, 40, 60 and 80 Gy.

  4. 4.

    Label each petri dish with the sample ID and required dose (Fig. 8).

    Fig. 8
    A photo displays two sets of five Petri dishes containing callus cultures. Each set is labeled 0 G, 10 G, 15 G, 20 G, and 40 G.

    Callus cultures of Coffea sp. prepared for mutation induction

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  5. 5.

    Seal the petri dishes with Parafilm to avoid contamination outside the tissue culture laboratory.

  6. 6.

    Prepare the regeneration media for the subsequent subculture.

  7. 7.

    Transport the next day to gamma irradiator facility for mutagenesis.

  8. 8.

    Expose petri dishes to irradiation by applying the required dose for mutation induction.

  9. 9.

    Subculture irradiated plant material to new petri dishes containing semi-solid of Zamarripa M4 medium.

3.3 Regeneration of Mutant Plant Lines

Plant cell and tissue cultures from these irradiation experiments were cultivated further and resulted in shoot formation and plantlet regeneration. These tissues have to undergo rigorous scrutiny for visual detection of altered phenotypes and are evaluated for a range of parameters (Table 1). Additional parameters to be evaluated for regenerated plantlets are active shoot growth, axillary bud formation, secondary root formation.

Table 1 Overview of recommended Gamma doses used for different explants of Coffea sp. and evaluation parameters applied within this study
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  1. 1.

    Transfer irradiated samples to tissue culture laboratory.

  2. 2.

    Surface sterilize every petri dish with 70% ethanol before removing the Parafilm (see Note 14).

  3. 3.

    Transfer the irradiated material into appropriate culture media.

  4. 4.

    Cultivate irradiated shoots in the incubation room with 28 °C and 12 h light.

  5. 5.

    Collect data on the further development and survival rates after transplanting on a regular basis (Table 2).

    Table 2 GR30 and GR50 determined according to effects observed from different Gamma doses used for Coffea sp. shoot cultures
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  6. 6.

    Subculture the growing in vitro shoot cultures for chimera dissolution by producing M2 or higher mutant populations.

  7. 7.

    Screen mutant populations by either phenotypic or genotypic evaluation (Figs. 9 and 10).

    Fig. 9
    A set of 11 photos exhibits irradiated shoots and their subsequent root development.

    Phenotypic analyses of in vitro development of irradiated shoots with focus on root development at 0, 10, 15, 20, 40 and 60 Gy

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    Fig. 10
    Two sets of photos of the irradiated embryogenic callus of coffee in Petri dishes. Set 1 displays a close-up view of the calluses. Set 2 reveals 6 Petri dishes with markings 0, 15, 20, 40, 60, and 80.

    Irradiated embryogenic callus of coffee

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  8. 8.

    Transfer plants to rooting and acclimatization phase and subsequently to the glasshouse for further mutant evaluation (Fig. 1).

Since roots are known to respond more sensitively to different stresses, their development was carefully evaluated. The optimal dose range for shoot cultures was identified between 20 and 42 Gy (Table 2).

From the original 75 irradiated shoots finally after a period of approx. 18 months more than 600 plants could be recovered (Table 3). Interestingly, no shoot survived the treatment with 60 Gy.

Table 3 Number of shoots of Coffea canephora cv. Quillou recovered after irradiation of shoots with 0, 15, 20 and 40 Gy
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Following the dose range determined for shoot cultures, single cell explants should be handled (Fig. 10).

  1. 1.

    Transfer irradiated samples to tissue culture laboratory.

  2. 2.

    Surface sterilize petri dishes with 70% ethanol before removing the Parafilm (see Note 14).

  3. 3.

    Transfer the irradiated material into appropriate culture media.

  4. 4.

    Take the cultures to the incubation room with 28 °C under light and dark conditions.

  5. 5.

    At regular intervals record survival rates of the mutagenized tissues, the number of observed embryos per petri dish, per dose and per genotype.

  6. 6.

    Subculture the growing embryogenic callus and transfer individual embryos to Zamarripa M5 for plant regeneration at regular intervals until development of the plantlets.

  7. 7.

    Record the number of plants regenerated per replication, per dose and per genotype.

  8. 8.

    Transfer plants to rooting and acclimatization phase and subsequently to the glasshouse for further mutant evaluation (Fig. 1).

After irradiation, initial growth was observed only in untreated calli for the first month. However, with a delay, calli from all treatments recovered and survived. All treated calli showed a change in colour as response to gamma irradiation compared to control which maintained the yellow colour. In the third month of incubation cotyledonary embryos were observed with the doses up to 20 Gy, whereas from 40 to 80 Gy no embryo development was observed. The irradiation of embryogenic callus of Coffea canephora irradiated on 24.01.2018 led to the recovery of hundreds of shoots, this time of single cell origin (Table 4). Again, it was noted that only very few shoots survived the treatment with 60 Gy.

Table 4 Number of shoots of N20 (Coffea canephora cv. Quillou) recovered after irradiation of embryogenic calli with 0, 15, 20 and 40 Gy
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Irradiation of different developmental stages of somatic embryos revealed, that globular stage and cotyledonary stage embryos besides not growing anymore after being irradiated with 40 and 60 Gy, did not develop directly into actively growing plantlets. However, the circuit through a repetitive embryogenesis allows to recover plantlets also through this process. In fact, irradiation of globular and cotyledonary embryos of Coffea arabica cv. Java after 9 months led to recallusing and from there again to embryogenic calli producing new embryos and finally after 12 months approximately 200 shoots.

Globular embryos were relatively more resistant to gamma irradiation than cotyledonary and torpedo shaped embryos (Fig. 11).

Fig. 11
2 photos. A. 5 Petri dishes contain samples of the globular embryo. B. 5 Petri dishes contain samples at the cotyledonary stage.

Irradiated somatic embryos of coffee a globular stage, b cotyledonary stage

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As anticipated, the experiments allowed to confirm the higher radio-sensitivity of multi-cellular when compared to uni-cellular explants under in vitro conditions (Table 5). It was possible to:

  • define an optimum mutation induction dosage range for several in vitro explants

  • produce high numbers of different putative mutants generated of various in vitro explants

  • determine the effectiveness of mutation induction by phenotypic analyses

  • identify the most efficient in vitro explants for mutation induction in coffee.

According to the mutagenesis objectives starting from the second generation and higher after chimera dissolution, in vitro plants can be screened for the selection of candidate based on phenotypes or genotypes. Mutations can be detected with various direct and indirect methods. Direct methods such as sequencing, exome capture sequencing, restriction site associated DNA (RAD) sequencing and genotyping by sequencing (GBS) provide the necessary information for mutation detection and confirmation (Denoeud et al. 2014, Dereeper et al. 2015). Additionally, the generation of various EST sequences in C. arabica (Anthony et al. 2001; Mishra and Slater 2012; de Moro et al. 2009; Krishnan 2014; Vieira et al. 2006; Leroy et al. 2005; Lin et al. 2005; Noir et al. 2004) will allow to identify genes and their regulatory sequences responsible for mutated traits and estimate their value for further breeding programs.

Table 5 Response of different in vitro explants to gamma irradiation
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4 Notes

  1. 1.

    It is advisable to grow the donor material in a greenhouse to reduce contamination with fungi and bacteria. Plants should be grown under ideal conditions to improve the establishment rate of tissue cultures (Debergh 1987).

  2. 2.

    Consider that the different genotypes and explant types, e.g. seeds, in vitro cuttings, or embryogenic callus have different requirements and capacities. This is especially important in the case of long lived organisms like trees, and has consequences at the level of population size, dissolution of chimerism and frequency of mutation. Therefore, it is advisable to use several genotypes as control material.

  3. 3.

    Consider that the different genotypes have different requirements. Different media should therefore be compared for efficient micropropagation: Medium 1 (Priyono et al. 2010), Medium 2 (Ebrahim et al. 2007) and Medium 3 (Abd El Gawad et al. 2012). Visual observations of different micropropagation media indicated, that Medium 1 induced small plantlets, small, light green leaves, many short roots. Medium 2 induced quite vigorous plantlets, axillary buds, but no roots. Medium 3 yielded the most vigorous plantlets, with large, dark green leaves, formation of a long root with secondary roots. Media were supplemented with 30 g/L sucrose and the pH adjusted to 5.7 prior to the addition of 7 g/L Agar (Sigma). Media were autoclaved at 120 °C and 1.1 kg/cm2 for 20 min, and then 25 ml of medium was dispensed into each Magenta Box. The cultures were maintained at 26–27 °C in the dark. In vitro shoots are subcultured every 8–12 weeks by axillary cuttings.

  4. 4.

    Media according to Zamarripa et al. (1991), Etienne (2005) and Priyono et al. (2010) are indicated as suitable for indirect somatic embryogenesis, while for direct embryogenesis protocols were described by CATIE (1988), Hatanaka et al. (1991) and Lubabali et al. (2014).

  5. 5.

    Media M1 to M5 according to Zamarripa et al. (1991) are indicated as suitable for plantlet recovery from somatic embryos.

  6. 6.

    When the applied dose for the genotype is unknown, a radiation test should be performed to determine the optimal dose. To perform the radiosensitivity test on vegetative material like cuttings, select 30 cuttings per dose with a wide range from 0 to 100 Gy (Gy) for vegetatively propagated crops. However, the range of 0, 10, 20, 30, 40, 50 and 60 Gy of gamma rays may be sufficient to establish the optimal dose due to the high moisture content in comparison to seeds. The Gy unit used to quantify the absorbed dose of radiation (1 Gy = 1 J/kg).

  7. 7.

    When applying physical mutagens to different types of plant material, care should be taken with soft materials such as in vitro shoot cultures as well as callus and embryogenic callus cultures, which require lower doses in comparison to seeds. In fact, the water content, storage time, applied mutagen dose and temperature represents an important factor influencing mutagens in all types of plant material.

  8. 8.

    Radioactivity is mutagenic and carcinogenic. It should be operated by trained and authorized person and carried out in a defined lab. In fact, the safety precautions for exposing plant material to a gamma irradiation source have to be strictly observed.

  9. 9.

    Take care to observe all safety precautions before exposing tissues to irradiation.

  10. 10.

    A dose film can be included together with the samples to capture the absorbed dose.

  11. 11.

    Exposure time is equal to the required dose divided by the dose rate of the day.

  12. 12.

    The irradiated samples are safe to be held in hands because the sample chamber isolates the plant material from the source and there is no surface contamination.

  13. 13.

    Untreated samples (control) have to be prepared and kept in the same conditions as the treated samples.

  14. 14.

    Observe general rules for plant tissue culture practice.