Mutation Induction Using Gamma-Ray Irradiation and High Frequency Embryogenic Callus from Coffee (Coffea arabica L.)

Abstract

Mutation induction through chemical or physical mutagenesis has been widely used for crop improvement for more than 70 years. Coffee is one of the most important crops in Latin-America, and, as any other crop, it can be affected by pests and diseases. Coffee leaf rust (CLR), caused by the biotrophic fungus Hemileia vastatrix, is the most important disease affecting Arabica coffee leading to significant losses for growers. As a perennial crop, conventional breeding of Arabica coffee is time-consuming. Plant tissue culture in combination with mutation induction techniques can provide an alternative approach to increase genetic variability of Arabica coffee for breeding applications. The present chapter describes protocols to establish embryogenic callus suspensions from Arabica coffee cv Venecia and for gamma ray irradiation of callus suspension cultures to achieve genetic improvement in the crop.

1 Introduction

Coffea arabica L. (coffee) belongs to the Rubiaceae family which comprises about 500 genera and more than 6000 species, mostly tropical trees and shrubs (Jiménez and Carril 2014). The Coffea genus includes more than 100 species from which only C. arabica and C. canephora are grown commercially (Mishra and Slater 2012). Central America is the world's fifth largest Arabica coffee producer, where Costa Rica stands out in terms of production and quality (ICAFE 2016).

More than 80% of Arabica coffee produced in Latin America comes from varieties derived from a narrow genetic base, being highly susceptible to diseases and pests, caused by microorganisms such as fungi, bacteria, viruses and nematodes (Bertrand et al. 2011). In the region, the majority of the diseases are caused by phytoparasitic fungi and around 300 diseases affecting the crop have been detected worldwide (Canet Brenes et al. 2016). Coffee leaf rust (CLR), caused by the fungus Hemileia vastatrix, is one of the main limiting factors of Arabica coffee production in all coffee growing countries (Mishra and Slater 2012).

Despite ongoing efforts for resistance breeding, chemical control is still the most widely used method to contain pests and diseases, including CLR (Neto and da Cunha 2016). Therefore, the development of alternative, environmentally friendly solutions for control of CLR is important. A long-term solution is through the development of resistant varieties, which is the focus of many breeding programmes in Arabica coffee (Mishra and Slater 2012). However, due to the perennial nature of coffee it can be difficult and time consuming to breed for disease resistance through conventional breeding methods (Barrueto Cid et al. 2004).

Plant breeders can use different tools to induce genetic variation in crops (Bermúdez-Caraballoso et al. 2016). Given the perennial nature of Arabica coffee, an effective way to induce variability, can be plant tissue culture in combination with mutation induction (Muthusamy et al. 2007). Combined, these techniques could increase genetic variability and reduce the time needed to develop new plant varieties (Bolívar-González et al. 2018).

Mutations can be induced by physical mutagens such as X-rays, gamma rays, neutrons and by chemical mutagens such as ethyl methanesulfonate (EMS). Physical mutagens appear more frequently used than chemical mutagens (Beyaz and Yildiz 2017). Since the 1960s gamma-ray mutagenesis has been the most commonly used method in plant mutation breeding (Li et al. 2019). Gamma rays are ionizing radiation (Beyaz and Yildiz 2017); they interact with atoms or molecules producing free radicals in cells that induce physiological, biochemical, cytological, genetic and morphogenetic changes in cells and tissues of plants (Chusreeaeom and Khamsuk 2019).

Somatic embryogenesis (SE) is a plant tissue culture technique where embryos are obtained from cells that are not the product of gametic fusion. Through SE thousands of seedlings identical to the mother plant can be produced (Bartos et al. 2018). Induced mutations are single cell events and thus the mutagenic treatment of seeds will result in chimeric M₁ plants, i.e., they may carry different mutations, each occupying a (small) part of the plant. Since somatic embryos regenerate from single cells, somatic embryogenesis is considered to be an effective method for eliminating chimeras (Roux et al. 2004).

The optimal irradiation dose(s) leading to genetic improvement of a specific crop or trait may vary depending on the genetic constitution of the plant species and cultivar. Until now only the work of Sari et al. (2019) has referred to the use of gamma rays for mutation induction of Robusta coffee embryogenic callus suspensions. It is necessary and essential to conduct radiosensitivity testing to determine the optimal dose(s) of gamma-ray irradiation of Arabica coffee embryogenic cell cultures before conducting bulk irradiation experiments (Bermúdez-Caraballoso et al. 2016; Spencer-Lopes et al. 2018). The present chapter describes a protocol on how to obtain the embryogenic callus suspensions of Coffea arabica and to determine the optimal irradiation dose.

2 Materials

2.1 Plant Material

• 1. In these experiments Coffea arabica cv Venecia.

2.2 Explants Collection and Disinfection

• 1. Young leaves from a donor plant.

• 2. Soap and distilled water.

• 3. Beakers (1,000 ml).

• 4. Sodium hypochlorite (NaOCl) 2% (v/v).

• 5. Antioxidant sterile solution (350 mg/L ascorbic acid, 200 mg/L citric acid y 20 g/L saccharose) + fungicide (3 g/L AMISTAR).

• 6. Sterile deionized water.

• 7. Laminar airflow cabinet.

2.3 Induction of Embryogenic Callus

• 1. Dissecting instruments (scalpels, blades, and forceps).

• 2. Glass jars (72.5 × 84.5 mm) with 15 ml of V1 media culture (see Table 1).

• 3. Glass jars (72.5 × 84.5 mm) with 20 ml of V2 media culture (see Table 1).

• 4. Laminar airflow cabinet.

• 5. Room with environmental control.

Table 1 Composition of media culture (mg/l) for indirect somatic embryogenesis in Coffee (Coffea arabica) (see Note 2)

2.4 Embryogenic Callus Multiplication

• 1. Erlenmeyer flasks (25, 50, 125, 250, 500, 1,000 and 2,000 ml).

• 2. Aluminum foil.

• 3. Parafilm.

• 4. Graduated cylinder (100 ml).

• 5. Falcon tubes (50 ml).

• 6. Orbital shake.

• 7. Analytical balance.

• 8. Laminar airflow cabinet.

• 9. Room with environmental control.

2.5 Embryogenic Callus Irradiation

• 1. Filter paper (Diameter: 9 cm).

• 2. Funnel.

• 3. Erlenmeyer (250 ml).

• 4. Microcentrifuge tubes (1.5 ml).

• 5. Micropipette (100–1,000 µl).

• 6. Micropipette tips (100–1,000 µl).

• 7. Media culture V4 (see Table 1).

• 8. Parafilm.

• 9. Petri dishes (100 × 15 mm).

• 10. Forceps.

• 11. Analytical balance.

• 12. Laminar airflow cabinet.

• 13. Irradiation source (see Note 1).

2.6 Regeneration of the Irradiated Embryogenic Callus

• 1. Micropipette tips (100–1,000 µl).

• 2. Micropipette (100–1,000 µl).

• 3. Erlenmeyer flasks (250 ml).

• 4. Media culture V4 (see Table 1).

• 5. Graduated cylinder (100 ml).

• 6. Laminar airflow cabinet.

• 7. Orbital shaker.

• 8. Room with environmental control.

2.7 Embryo Germination

• 1. Filter paper (Diameter: 9 cm).

• 2. Funnel.

• 3. Erlenmeyer (250 ml).

• 4. Forceps.

• 5. Glass jars (59.5 × 68.0 mm) with 15, 20, 25 ml of V5 media culture (see Table 1).

• 6. Plastic film.

• 7. Laminar airflow cabinet.

• 8. Room with environmental control.

2.8 Development of Somatic Embryos into Plantlets

• 1. Forceps.

• 2. Glass jars (59.5 × 68.0 mm) with 25 ml of V6 media culture (see Table 1).

• 3. Plastic film.

• 4. Laminar airflow cabinet.

• 5. Room with environmental control.

2.9 Media Culture: Preparation

• 1. pH meter.

• 2. Autoclave.

• 3. Glass jars.

• 4. Microwave.

• 5. Analytical balance.

• 6. Magnetic stirrers.

3 Methods

3.1 Explants Collection and Disinfection

• 1. Remove well developed and healthy leaves from the first or second node from the donor plant (see Note 3).

• 2. Rinse with tap water and soap.

• 3. Transfer into the laminar airflow cabinet and place leaves in a sterile beaker.

• 4. Add the fungicide-antioxidant solution and frequently swirl the solution by hand during 10 min.

• 5. Decant the solution and rinse three times with sterile deionized water.

• 6. Disinfect the explants with a sodium hypochlorite solution 2% (v/v) and swirl gently during 30 min.

• 7. Decant the solution and rinse three times with sterile deionized water and decant.

3.2 Induction of Embryogenic Callus

• 1. Cut square leaf segments of 0.5 cm² avoiding the main, secondary veins and leaf borders.

• 2. Place 7 segments in each flask, with the upper side in contact with V1 media culture.

• 3. Incubate in the dark at 28 °C for 22 days.

• 4. Remove the leaf segments from the V1 media culture and transfer on the V2 media.

• 5. Incubate in the dark at 28 °C until embryogenic callus is formed (friable callus, white color of dusty consistency) can be observed.

3.3 Embryogenic Callus Multiplication

• 1. After 4–5 months, in the laminar airflow cabinet, remove the somatic embryogenic callus and weigh using an analytical balance.

• 2. Once the weight is determined, choose an Erlenmeyer flask that can contain the necessary volume (10% of the actual capacity) maintaining a ratio of 10 mg embryogenic callus/ml of V3 culture medium. Seal with parafilm.

• 3. Place the flask containing the embryogenic callus in a liquid media on an orbital shaker and incubate in the dark at 90 rpm and 28 °C.

• 4. Subculture every 20 days.

• 5. Transfer the embryogenic callus into a 50 ml centrifuge tube, carefully decant the old media keeping a minimum amount to avoid loss of callus.

• 6. Duplicate the media culture volume and transfer to a new Erlenmeyer flask.

• 7. Seal with parafilm and incubate in the dark on an orbital shaker at 90 rpm and 28 °C.

• 8. Repeat steps 4–7 for a maximum of 5 subcultures (3 months incubation).

3.4 Irradiation of the Embryogenic Callus

• 1. Take an Erlenmeyer containing the embryogenic suspension culture from Coffea arabica, in multiplication stage, with a maximum of five subcultures (3 months) (see Fig. 1a).

• 2. Carefully open the Erlenmeyer in the laminar airflow cabinet and decant the material.

• 3. Filter the material in a filter paper-funnel-Erlenmeyer system to eliminate as much culture medium as possible (see Fig. 1a, b).

• 4. Once the material is obtained, divide it into sterile 1.5 ml microcentrifuge tubes with 20 mg each.

• 5. Add 0.5 ml of regeneration media culture (V4).

• 6. Seal the microcentrifuge tubes with Parafilm, place inside a petri dish and seal the petri dish for transportation (see Fig. 1d).

• 7. Transport the culture in dark condition and at room temperature to the irradiation facility (see Fig. 1e) and irradiate the samples using different doses (see Note 4).

Fig. 1

Coffea arabica embryogenic callus irradiation; a embryogenic suspension; b filtration system; c filtrated embryogenic callus; d embryogenic callus divided into microcentrifuge tubes to irradiate; e callus irradiation on Ob-ServoIgnis irradiator; f regeneration of the irradiated material

3.5 Regeneration of the Irradiated Embryogenic Callus

• 1. Use a sterile micropipette to remove the irradiated material from the microcentrifuge tube.

• 2. Transfer to an Erlenmeyer (250 ml) maintaining the ratio 1 mg per ml of regeneration media (V4).

• 3. Place the Erlenmeyer flask on a rotary shaker and incubate in the dark at 100 rpm and 28 °C.

• 4. Incubate until the regeneration of the embryos is observed (1–3 months) (see Fig. 1f).

3.6 Embryo Germination

• 1. Remove the regenerated embryos from the Erlenmeyer flask as described in Sect. 3.4, step 3.

• 2. Place individual embryos on glass jars containing V5 media.

• 3. Incubate the material at 28 °C with a 12 h photoperiod for 30 days.

• 4. Subculture the viable material and discard the amorphous material. Keep track of the data to determine the LD₅₀ (see Note 4).

3.7 Regeneration of Plantlets

• 1. Once the embryos have been developed correctly (foliar and root development) subculture them in semi-solid V6 media culture.

• 2. Incubate the material at 28 °C with 12 h photoperiod, subculture every 30 days until the plantlets are developed (2–3 pairs of true leaves and 1–2 root cm) (Fig. 2).

Fig. 2

Somatic embryogenesis process in Coffea arabica

4 Notes

• 1. The irradiation of the materials was conducted at the Gamma radiation laboratory at the facilities of the Instituto Tecnológico de Costa Rica (TEC), using a Ob-ServoIgnis irradiator (Cobalt 60 radioactive source and an activity of 4.4 × 10¹⁴ Bq) (Becquerel).

• 2. For the preparation of the media culture the following procedures are followed: The components for each media culture are specified in Table 1, each amount shown is in mg/L. Place all media components in a volumetric flask and stir until fully dissolved. Complete the volume with water until the mark. Decant to a beaker and adjust the pH to 5.6. If the media is semi-solid add phytagel (the amount of phytagel may vary between 3.6 and 5.0 g/L), microwave until boiled (8 min/L). Dispense in glass jars and sterilize at 121 °C 1.5 lb of pressure.

• 3. Healthy leaves were collected from a donor plant established in the field. Young leaves from the first or second internode were removed from branches of the coffee plant. The selected branches were positioned at the middle part of the plant.

• 4. The doses used in the experiment were: 5, 10, 15, 20, 25, 30, 35, 40, 60, 80 and 100 Gy. The LD₅₀ is 40 Gy.