Chemical Mutagenesis of Zygotic Embryos of Coffea arabica L. var. Catuaí Using EMS and NaN₃

Abstract

The genetic improvement of C. arabica L. is challenged by its low genetic diversity and autogamous reproductive biology. Induced mutagenesis offers an alternative approach to conventional cross-breeding to increase genetic variability in wild and cultivated Arabica coffee germplasm for further use in breeding programs and genetic studies. Here protocols are described for the preparation of zygotic embryos from C. arabica seed and for toxicity testing of zygotic embryos using two chemical mutagens, sodium azide (NaN₃) and ethyl methanesulfonate (EMS). Zygotic embryos were immersed for 10 min in a solution of NaN₃ (0, 2.5, 5.0, 7.5, 10.0, 12.5, 15.0 and 20. 0 mM) and for 2 h in a solution of EMS (0, 0.5, 1, 1.5, 2, 4 and 6 % v/v). The percentage survival was evaluated and the LD values for NaN₃ and EMS were determined at 12.5 mM (51.6%) and 1 % v/v (48.3%), respectively. Our protocols indicate that coffee zygotic embryos are suitable propagules for NaN₃ and EMS mutagenesis and expand the types of propagules suitable for induced mutagenesis, breeding and genetic studies in Arabica coffee.

1 Introduction

Coffee is one of the most important products around the world. Global coffee imports in 2021–2022 amounted to 133.59 million 60-kg bags with a global market value of US $107.93 billion in 2021, taking second place in international trade after crude oil. Coffee is cultivated in more than 80 countries in tropical and subtropical regions of the globe, especially in Africa, Asia, and Latin America. Coffee production generates directly or indirectly income to more than 100 million people around the world (Mishra and Slater 2012).

Approximately 60% of the world coffee production is derived from C. arabica L. because of its superior quality, aromatic characteristics, and low caffeine content compared to Robusta coffee (Mishra and Slater 2012; Alpízar 2014; Ahmed et al. 2013).

The cultivated varieties of C. arabica L. are derived from the “Typica” or “Bourbon” coffee lineages, resulting in low genetic diversity (Mishra and Slater 2012). The reproductive biology of Arabica coffee, being approximately 90% autogamous, along with historical and geographic data indicating the occurrence of genetic bottlenecks due to domestication and global spread from its center of origin in Ethiopia and a polyploidization event are responsible for the low genetic variation in Arabica coffee (Romero et al. 2010; Mendonça 2014; Scalabrin et al. 2020; Montagnon et al. 2021). Consequently, C. arabica varieties are often highly susceptible to different diseases and pests (Romero et al. 2010).

Induced mutagenesis offers a promising alternative to improve current coffee cultivars for enhanced tolerance to pathogens, as previously shown in other crops (Gressel and Levy 2006). Induced mutagenesis using chemical or physical mutagens typically introduces random changes throughout the genome and can thus generate a variety of mutations within a single plant. As opposed to cross-breeding, induced mutagenesis can be applied to a wide variety of plant propagules.

Ethylmethanesulfonate (EMS) is today the most widely used chemical mutagen. It selectively adds alkyl groups to guanine bases causing random point mutations; most of the changes (70–99%) are base-pair transitions from G/C to A/T (Jankowicz-Cieslak et al. 2016; Sikora et al. 2011). Another chemical mutagen is sodium azide (NaN₃), whose mutagenic effect is mediated through the production of an organic metabolite of the azide compound: l-azidoalanine. It creates point mutations, mostly transitions, of the type: G/C to A/T or vice versa (Prina et al. 2010; Srivastava et al. 2011).

This chapter describes protocols for the preparation and mutagenic treatment of zygotic embryos of C. arabica L. var. Catuaí using the chemical mutagens sodium azide and ethyl methanesulfonate, including methods for bulk treatment, germination, and development of mutant plantlets.

2 Materials

2.1 Plant Material

• 1. Coffea arabica L. var. Catuaí seeds (see Note 1).

2.2 Reagents

• 1. 10% (w/v) sodium thiosulfate (e.g., Sigma Cat Nr.: 217263).

• 2. 100 mM phosphate buffer (pH 3.0) (e.g., Sigma Cat Nr.: 217263).

• 3. 1 N KOH (e.g., Phytotechnology Cat Nr.: P682).

• 4. 1 N NaOH (e.g., Phytotechnology Cat Nr.: S835).

• 5. 2,3,5-Triphenyltetrazolium Chloride (TTC) stock solution (e.g., Phytotechnology Cat Nr.: T8164).

• 6. 6-benzylaminopurine (BAP) (e.g., Sigma Cat Nr.: D3408).

• 7. Biotin (e.g. Phytotechnology Cat Nr.: B140).

• 8. Citric acid (e.g., Phytotechnology Cat Nr.: C277).

• 9. Disodium phosphate (Na₂HPO₄).

• 10. d-Sucrose (e.g. Phytotechnology Cat Nr.: S829).

• 11. Ethanol 95° (v/v).

• 12. Ethyl methanesulphonate (EMS) (e.g., Sigma Cat Nr.: M0880) (see Notes 2, 3, and 4).

• 13. Gelling agent (e.g., Phytagel: Sigma Cat Nr.: P8169).

• 14. Gibberellic acid (GA₃) (e.g., Sigma Cat Nr.: G7645).

• 15. l-ascorbic acid (e.g., Phytotechnology Cat Nr.: M524).

• 16. Monopotassium phosphate (KH₂PO₄).

• 17. Murashige and Skoog (MS) basal salt mixture (e.g., Phytotechnology Cat Nr.: M524).

• 18. Nicotinic acid (e.g., Phytotechnology Cat Nr.: N765).

• 19. Phosphate buffer.

• 20. Phosphorus acid (H₃PO₃).

• 21. Pyridoxine HCl (e.g., Phytotechnology Cat Nr.: P866).

• 22. Sodium azide (NaN₃) (e.g., Sigma Cat Nr.: S2002) (see Notes 2, 3, and 4).

• 23. Sodium hypochlorite (3.5% v/v).

• 24. Sterile distilled water.

• 25. Thiamine HCl (e.g., Phytotechnology Cat Nr.: T390).

• 26. Tween 20 (e.g., Phytotechnology Cat Nr.: P720).

2.3 Glassware and Minor Equipment

• 1. 2 ml reaction tubes.

• 2. Beakers (250, 1,000 ml).

• 3. Bottles (100, 500 ml).

• 4. Box for dry hazardous material disposal.

• 5. Disposable pipettes (1 ml, 5 ml, 10 ml, 25 ml, or as needed according to calculations).

• 6. Erlenmeyer (250 ml).

• 7. Forceps.

• 8. Glass or disposable pipettes (1, 5, 10, 25 ml).

• 9. Graduated cylinder (50, 100, 500 and 1,000 ml).

• 10. Hazardous liquid waste receptacle (collection vessels for sodium azide and ethyl methanesulfonate waste solution).

• 11. Magnetic stir bar.

• 12. Personal protective equipment (disposable laboratory coat dedicated only to mutagenesis experiments, eye protection/goggles, shoe protection, nitrile gloves).

• 13. Petri dishes (100 mm × 20 mm).

• 14. Plastic boxes (88 × 42 × 16 cm).

• 15. Scalpel.

• 16. Spatula.

• 17. Volumetric flasks (1,000 ml).

• 18. Weighing trays.

2.4 Equipment

• 1. Analytical balance.

• 2. Autoclave.

• 3. Centrifuge.

• 4. Chemical mutagen laboratory equipped with fume hood and flow bench (see Notes 2, 3, and 4).

• 5. Fume Hood.

• 6. Hot plate shaker.

• 7. Orbital shaker.

• 8. pH meter.

• 9. Stereoscope.

• 10. Water bath at 65 °C.

2.5 Software

• 1. Standard spreadsheet software (e.g., Microsoft Excel or Open Office Excel).

2.6 Bulk Mutagenesis of Zygotic Embryos

All materials as listed in Sects. 2.1, 2.2, 2.3, 2.4, and 2.5.

3 Methods

3.1 Preparation of Stock Solutions

• 1. 100 mM phosphate buffer: add 13.6 g of KH₂PO₄ to 1,000 ml volumetric flask and dissolve by adding 250 ml of tissue culture grade water. Once completely dissolved, stir the solution while adding tissue culture grade water and bring to 1,000 ml. Adjust pH to 3.0 using phosphorus acid (H₃PO₃).

• 2. 6-Benzylaminopurine (BAP; 1 mg/ml stock solution): add 50 mg of the powder to a 100 ml volumetric flask and dissolve by adding 2–5 ml of 1 N NaOH. Once completely dissolved, stir the solution while adding tissue culture grade water and bring to 50 ml. Sterilize by filtering through a 0.2 μm filter and store aliquots (1 ml) at 4 °C.

• 3. Gibberellic acid (GA₃; 1 mg/ml stock solution): add 50 mg of the powder to a 100 ml volumetric flask and dissolve by adding tissue culture grade water. Once completely dissolved, stir the solution while adding tissue culture grade water and bring to 50 ml. Sterilize by filtering through a 0.2 μm filter and store aliquots (1 ml) at 4 °C.

• 4. KH₂PO₄ (0.07 M): add 907.8 mg of the powder to 250 ml volumetric flask and dissolve by adding 50 ml of tissue culture grade water. Once completely dissolved, stir the solution while adding tissue culture grade water and bring to 100 ml.

• 5. Na₂HPO₄ (0.08 M): add 1.1876 g of the powder to 250 ml volumetric flask and dissolve by adding 50 ml of tissue culture grade water. Once completely dissolved, stir the solution while adding tissue culture grade water and bring to 100 ml.

• 6. Sodium azide (500 mM): add 1.625 g of the powder to 250 ml volumetric flask and dissolve by adding 50 ml phosphate buffer (100 mM). Once completely dissolved, stir the solution while adding phosphate buffer (100 mM) and bring to 100 ml. Sterilize by filtering through a 0.2 μm filter and store at 4 °C in the dark.

• 7. 2,3,5-Triphenyltetrazolium Chloride (TTC) stock solution: add 4 ml of KH₂PO₄ (0.07 M) and 6 ml of Na₂HPO₄ (0.08 M). Adjust pH to 7. Add the amount of TTC needed to reach 1% (m/v) (100 mg for 10 ml of solution). Sterilize by filtering through a 0.2 μm filter and store aliquots in the dark at 4 °C.

• 8. Citric acid (100 mg/ml stock solution): add 1,000 mg of the powder to a 50 ml volumetric flask and dissolve by adding tissue culture grade water. Once completely dissolved, stir the solution while adding tissue culture grade water and bring to 10 ml. Sterilize by filtering through a 0.2 μm filter and store aliquots (1 ml) in the dark at 4 °C.

• 9. l-ascorbic acid (100 mg/ml stock solution): add 1,000 mg of the powder to a 50 ml volumetric flask and dissolve by adding tissue culture grade water. Once completely dissolved, stir the solution while adding tissue culture grade water and bring to 10 ml. Sterilize by filtering through a 0.2 μm filter and store aliquots (1 ml) in the dark at 4 °C.

3.2 Preparation of Tissue Culture Medium

• 1. Prepare stock solutions of BAP (1 mg/ml), GA₃ (1 mg/ml), Biotin (1 mg/ml), Calcium pantothenate (1 mg/ml), Nicotinic acid (1 mg/ml), Pyridoxine HCl (1 mg/ml), and Thiamine HCl (1 mg/ml).

• 2. Place a beaker containing 400 ml tissue culture grade water on a hot plate shaker and mix:

  • For 1 L of germination medium (EG): full-strength MS salts, 1.0 ml BAP, 1 ml GA₃, and 300 mg/l activated charcoal, and 2.5 g Phytagel™.

  • For 1 L of development medium (DEV): full-strength MS salts 1 ml thiamine HCl, 1 ml pyridoxine HCl, 1 ml nicotinic acid, 1 ml calcium pantothenate, 0.01 ml biotin, 100 mg myo-inositol, 0.3 ml BAP, 30 g sucrose, 2.5 g Phytagel™.

• 3. While stirring, add tissue culture grade water to a final volume of 1,000 ml.

• 4. Stir until the solution is homogenous and clear.

• 5. Calibrate the pH meter as per manufacturer instructions.

• 6. While stirring, adjust medium to pH 5.6 using 1 N NaOH and 1 N HCl.

• 7. For semi-solid medium, add the gelling agent and heat while stirring until the solution is homogenous and clear.

• 8. Dispense the culture medium in the respective culture vessels before or after autoclaving (depending on the application).

• 9. Sterilize all media in an autoclave at 0.1 kg/cm² for 21 min at 121 °C.

• 10. Allow the medium to cool prior to use.

• 11. Store the medium for up to a week in a cold room.

3.3 Seed Disinfection and Excision of Zygotic Embryos

• 1. Collect mature cherries from genetically homogenous mother plants maintained in the greenhouse or in the field (see Notes 1, 5 and 6 and Fig. 1).

• 2. Remove the pulp, the mucilage, and the parchment by hand.

• 3. Prepare a uniform seed stock by selecting disease-free seeds and removing any small, shriveled or damaged seeds.

• 4. Soak the seeds for 24 h in distilled water with two drops of Tween 20 with orbital rotation.

• 5. Disinfect the seeds with 3.5% (v/v) sodium hypochlorite for 1 h and finally rinse three times with sterile distilled water.

• 6. Soak seeds in sterile distilled water for 48 h.

• 7. In a laminar flow cabinet, excise zygotic embryos with the aid of tweezers and a scalpel and maintain them in a solution of citric acid and ascorbic acid (100 mg/ml each; pH 5.6) until the excision of all embryos prior to the germination experiments.

• 8. Culture the zygotic embryos in petri dishes (100 mm × 20 mm) containing 20 ml of regeneration medium.

• 9. Incubate the petri dishes under a 16 h light photoperiod at 27 ± 2 °C for 4 weeks.

• 10. Record germination after 2–4 weeks based on visual scoring of the presence of leaves (see Fig. 2).

Fig. 1

Schematic representation of the mutagenesis of coffee (Coffea arabica L. var. Catuaí) zygotic embryos. a For chemical mutagenesis and toxicity testing, normal shaped, disease-free seeds are selected and NaN₃ and EMS dosage and incubation time are optimized using seed lots with a germination rate equal to or above 90%. These steps take approximately 8 weeks. b Bulk irradiation of zygotic embryos. For both a and b, the zygotic embryos are prepared by manually removing the pulp, the mucilage, and the parchment of the seed, disinfecting the seeds, excising the zygotic embryo, and incubating the zygotic embryos using the appropriate NaN₃ or EMS concentration followed by incubating, rinsing and planting the mutagenized zygotic embryos

Fig. 2

Germination and viability test of zygotic embryos of coffee (Coffea arabica L. var. Catuaí). a Excised zygotic embryos germinated under in vitro culture conditions. b TTC viability test

3.4 Determination of the Viability of the Zygotic Embryos

• 1. Place the zygotic embryos in a 2 ml reaction tube and add 1 ml of the TTC stock solution (see Sect. 2.5 for description of the TTC viability test).

• 2. Incubate the samples for 24 h in the dark at 37 °C without shaking.

• 3. Remove the TTC solution by decanting or centrifugation and wash the sample with distilled water.

• 4. Add 1 ml of 95° (v/v) ethanol.

• 5. The evaluation of the viability of the embryos was carried out by counting the embryos that stained red (see Fig. 2).

3.5 Sodium Azide Dose Determination

• 1. Review safety procedures of the chemical mutagenesis laboratory (see Notes 2 and 3).

• 2. Autoclave all non-disposable materials (e.g., sieves, forceps).

• 3. Prepare a fresh 0.5 M NaN₃ stock solution by adding the required amount of NaN₃ to the phosphate buffer (100 mM, pH 3.0).

• 4. Select similar and normal shaped seeds that are disease-free.

• 5. Disinfect the seed and excise the zygotic embryos beforehand according to the protocol described in Sect. 3.3.

• 6. Place 100 zygotic embryos in 250 ml labelled Erlenmeyer. The number of Erlenmeyer depends on the number of treatments. Label each tube with the appropriate treatment code (concentration and incubation time).

• 7. In a fume hood, add appropriate volumes of phosphate buffer solution (100 mM) (see Table 1).

• 8. Add NaN₃ to a final concentration of 2.5, 5.0, 7.5, 10.0, 12.5, 15.0, and 20.0 mM (see Table 1).

• 9. Shake the solution vigorously.

• 10. Incubate the mixture for 10 min in the dark at 27 ± 2 °C with gentle rotation (100 rpm).

• 11. After the incubation time, quickly but carefully decant each of the treatment batches and rinse the treated zygotic embryos with 100 ml of sterile distilled water. This step and any subsequent steps must be carried out in a fume hood.

• 12. Repeat washing step 3 times.

• 13. Collect all the liquid waste in a dedicated bucket labelled as hazardous waste. Dispose of toxic waste according to local regulations.

• 14. Twenty-four hour after the NaN₃ mutagenesis treatment, determine viability of zygotic embryos and observe them using a stereoscope as described in Sect. 3.4.

• 15. Calculate the survival rate as follows: [(the number of survival explant/the number of treated explant) * 100].

• 16. Record the data for each treatment and enter it into a spreadsheet (e.g., Microsoft Excel or Open Office Excel).

• 17. Plot percentage of control against mutagenesis treatment.

• 18. Estimate the mutagen concentration required to obtain 50% viability compared to the control (see Fig. 3).

• 19. Identify concentrations suitable for bulk mutagenesis of your material according to the LD₅₀ previously estimated.

Table 1 Concentrations chosen for the toxicity test in coffee seeds

Fig. 3

Effect of NaN₃ concentration on survival and viability of coffee (C. arabica L. var. Catuaí) zygotic embryos. a Survival percentage (solid line) versus NaN₃ concentrations. Each value represents the mean ± SD of three repetitions. b Zygotic embryo viability versus NaN₃ concentrations. Zygotic embryos that stain red are considered viable. Bar, 1.0 cm

3.6 Ethyl Methanesulphonate Dose Determination

• 1. Review safety procedures of the chemical mutagenesis laboratory (see Notes 2 and 3).

• 2. Autoclave all non-disposable materials (e.g., sieves, forceps).

• 3. Select similar and normal shaped seeds that are disease-free.

• 4. Disinfect the seed and excise the zygotic embryos according to the protocol described in Sect. 3.3.

• 5. Place 100 zygotic embryos in a 250 ml labelled Erlenmeyer. The number of Erlenmeyer depends on the number of treatments. Label each tube with the appropriate treatment code (concentration and incubation time).

• 6. In a fume hood, add appropriate volumes of sterile distilled water (see Table 2).

• 7. Add EMS to a final concentration of 0.5, 1, 2, 3, 4, 5, and 6% v/v (see Table 2). Shake the solution vigorously (see Note 7).

• 8. Incubate the mixture for 2 h in the dark at 27 ± 2 °C with gentle rotation (100 rpm).

• 9. After the incubation time, quickly but carefully decant each of the treatment batches and rinse the treated zygotic embryos with 100 ml of sterile distilled water. This step and any subsequent steps must be carried out in a fume hood.

• 10. Repeat washing step 3 times.

• 11. Collect all the liquid waste in a dedicated bucket labelled as hazardous waste. Dispose of toxic waste according to local regulations.

• 12. Twenty-four hour after the EMS mutagenesis, determine viability of zygotic embryos and observe them using a stereoscope as described in Sect. 3.4.

• 13. Calculate the survival percentage as follows:

  Survival percentage = Number of survived explant/the number of treated explants) * 100.

• 14. Record the data for each treatment and enter it into a spreadsheet (e.g., Microsoft Excel or Open Office Excel).

• 15. Plot percentage of control against mutagenesis treatment.

• 16. Estimate the mutagen concentration required to obtain 50% survival compared to the control (see Fig. 4).

• 17. Identify concentrations suitable for bulk mutagenesis of your material according to the LD₅₀ previously estimated.

Table 2 Concentrations chosen for the toxicity test in coffee zygotic embryos

Fig. 4

Effect of EMS concentration on survival/viability of coffee (C. arabica L. var. Catuaí) zygotic embryos. a Survival percentage (solid line) versus EMS concentrations. Each value represents the mean ± SD of three repetitions. b Zygotic embryo viability versus EMS concentrations. Red stained zygotic embryos were considered as viable. Bar, 1.0 cm

3.7 Bulk Mutagenesis

• 1. Autoclave all non-disposable materials (e.g., sieves, forceps).

• 2. Choose appropriate NaN₃ or EMS concentration and incubation time based on the results obtained from the chemical toxicity test (see Sects. 3.4 and 3.5).

• 3. Select similar and normal shaped seeds that are disease-free.

• 4. Disinfect the seed and excise the zygotic embryos according to the protocol described in the Sect. 3.3.

• 5. Place 100 zygotic embryos in a 250 ml labelled Erlenmeyer. The number of Erlenmeyer depends on the number of repetitions.

• 6. Label each Erlenmeyer with the appropriate treatment code (NaN₃ and EMS concentration and incubation time).

• 7. Transfer Erlenmeyer containing the zygotic embryos into the chemical mutagenesis laboratory.

• 8. In a fume hood, add appropriate concentrations of NaN₃ or EMS.

• 9. Shake the solution vigorously.

• 10. Place Erlenmeyer in the dark at 27 ± 2 °C on a rotary shaker set at 100 rpm for the chosen length of time.

• 11. After incubation, quickly but carefully decant each of the treatment batches and rinse the treated zygotic embryos with 100 ml of sterile distilled water.

• 12. Repeat washing step 3 times.

• 13. Collect all the liquid waste in a dedicated bucket labelled as hazardous waste. Dispose of toxic waste according to local regulations.

• 14. After mutagenesis treatment, add 10 ml of liquid germination medium (pH 5.6) and maintain the cultures for 24 h in the dark on a rotary shaker (100 rpm) at 26 ± 2 °C.

• 15. Perform a zygotic viability test as described in Sect. 3.4.

• 16. Carefully decant the treated zygotic embryos and plate them in Petri dishes containing 20 ml of semisolid germination medium.

• 17. Incubate the Petri dishes under a 16 h light photoperiod at 27 ± 2 °C for 8–10 weeks.

• 18. Transfer the plantlets to baby food jars containing 20 ml of development medium (DEV) with 16 h light photoperiod at 27 ± 2 °C.

3.8 Acclimatization of M₁ Plantlets

• 1. After 8 weeks, do a transverse cut in the stem of the plantlets with 4 leaves and 3–4 cm tall, below a node, and remove the leaves near the cut.

• 2. Place the freshly cut basal part of the stem in a solution of indoleacetic acid (IAA) (1 mg/mL) for 30 s.

• 3. Plant the plantlets in sterile substrate (peat moss with perlite) in plastic boxes (30 cm × 20 cm × 10 cm).

• 4. Cover boxes with plastic and place them under greenhouse conditions with 12 h light photoperiod at 27 ± 2 °C.

• 5. Two weeks later, make small holes in the plastic covering the boxes.

• 6. One week later, plant the plantlets in polyethylene bags with a 3:1 mixture of substrate (peat moss with perlite: coconut fiber). Two plants per bag were planted and identified according to the original numbering.

• 7. After 3 weeks, fertilize with granular slow-release fertilizer (e.g., Osmocote 14-14-14).

• 8. Irrigate the plants twice a week according to the climatic conditions of the greenhouse and the water requirement of the crop.

4 Notes

• 1. This protocol has been established using Coffea arabica L. var. Catuaí zygotic embryos. It can be used as a reference for other Arabica coffee varieties, nevertheless, it is recommended to optimize the mutagenic parameter (NaN₃ and EMS concentration and incubation time) for each variety used.

• 2. All the mutagenesis experiments should be conducted in a dedicated chemical mutagenesis laboratory equipped with a ducted fume hood, toxic waste disposal and decontamination procedures.

• 3. Read the Materials Safety Data Sheet (MSDS) of materials being used and follow the recommendation of the manufacturer. Pay attention to the information on sodium azide and EMS and what to do in case of exposure. It is very important to wear personal protective equipment (gloves must be compatible with chemical mutagens, for instance PVC or neoprene gloves); safety glasses with side shields or chemical goggles; lab coat, closed-toe shoes, shoe protections, and full-length pants. A double glove system is advised.

• 4. Store the original EMS and NaN₃ always in an airtight colored bottle, preferably inside a sealed chamber containing a desiccant.

• 5. Coffee seeds loose viability rapidly if not properly stored. Therefore, it is recommended to use freshly harvested seeds or otherwise to store the seeds between 10 and 12% moisture at 15 °C not longer than 3 months.

• 6. Proceed to mutation induction protocol with seeds having germination rate close to or above 90%.

• 7. Some protocols use water-dimethyl sulfoxide (2% v/v DMSO) mixture in order to make the EMS mutagen more miscible.