Chemical Mutagenesis of Coffee Seeds (Coffea arabica L. var. Catuaí) Using NaN₃

Abstract

Coffee (Coffea arabica L.) is one of the most important crops in the world and one of the main export products in several developing countries. Coffee is a perennial crop threatened by multiple, serious diseases and pests. Induced mutagenesis of seeds is widely used for increasing the genetic diversity and improvement of annual seed crops and could equally be applied to Arabica coffee breeding and genetic studies. Here we describe protocols to induce genetic variability in Arabica coffee seeds through mutagenesis using sodium azide (NaN₃). Methods for NaN₃ chemical toxicity testing and bulk irradiation are described. Briefly, the coffee seeds were immersed for 4, 8 and 12 hours in a NaN₃ solution at different concentrations (0, 25, 50, 75, and 100mM). Two controls were used: one with distilled water and the other with the phosphate buffer (KH₂PO₄). Effects of the chemical mutagen on seed germination, seedling height, and root length were evaluated. As the concentration of applied NaN₃ increased, the germination, seedling height, and root length decreased. Eight hours exposure was determined as an adequate immersion time. The LD₅₀ values for NaN₃ were between 50–75 mM. Our results indicate that NaN₃ is an effective mutagen for Arabica coffee seeds and can be applied to coffee breeding and to study gene function in coffee.

1 Introduction

Mutations can be induced in plants by exposure of seeds or meristematic cells, tissues, and organs, to both physical and chemical agents with mutagenic properties (Mba et al. 2010). Chemical mutagenesis is the exposure of plant material to chemical agents such as alkylating agents and azides under optimized doses. The mutagenic effect of sodium azide (NaN₃) is mediated through the creation of an organic intermediate (L-azidoalanine) which incorporates the azide group and interacts with DNA to mainly produce simple base substitutions (Gruszka et al. 2012). Sodium azide’s mutagenic effect greatly depends upon the acidity of the working solution, which must be prepared at low pH values (Gruszka et al. 2012).

Reducing the time required to develop improved plants through mutation breeding is a desirable characteristic, especially in long-life cycle plant species such as coffee. Coffee cultivation has great socio-economic impact; it is positioned as the world’s second most exported commodity, only surpassed by oil (FAOSTAT 2018). The better cup quality and higher market value are associated to Coffea arabica L., the only allotetraploid (2n = 4x = 44) species among Coffea. Arabica coffee is an autogamous plant mostly incompatible with the remainder of Coffea species (Anthony et al. 2002). These characteristics, along with severe bottlenecks that happened during coffee domestication led to reduced genetic variability in C. arabica populations; this reduction enhances the general susceptibility of many C. arabica genotypes to diseases (Hendre and Aggarwal 2007). Conventional breeding faces limitations due to the long-life cycle of the coffee plant, requiring nearly 30 years to develop new cultivars.

This chapter describes the application of the chemical mutagen sodium azide for mutagenesis of C. arabica seeds as well as the germination, and development of mutant plantlets.

2 Materials

2.1 Plant Material

• 1. All experiments were conducted using seeds from Coffea arabica L. var. Catuaí (see Note 1).

2.2 Reagents

• 1. 10% (w/v) sodium thiosulfate (e.g. Sigma Cat Nr.: 217,263).

• 2. 100 mM phosphate buffer (pH 3.0).

• 3. Peat substrate.

• 4. Distilled water.

• 5. KH₂PO₄ (e.g. Sigma Cat Nr.: P5655).

• 6. Phosphorus acid (H₃PO₃) (e.g. Sigma Cat Nr.: 176,680).

• 7. Sodium azide (NaN₃) (e.g. Sigma Cat Nr.: S2002) (see Note 2).

• 8. Sodium hypochlorite (3.5% v/v).

• 9. Sterile distilled water.

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

2.3 Glassware and Minor Equipment

• 1. Beakers (250 ml).

• 2. Bottles (100, 500 ml).

• 3. Box for disposal of dry hazardous material.

• 4. Disposable pipettes (1, 5, 10, or 25 ml, as required).

• 5. Erlenmeyer (250 ml).

• 6. Graduated cylinders (100 ml or as needed according to calculations).

• 7. Hazardous liquid waste receptacle (collection vessels for the sodium azide waste solution).

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

• 9. Plastic boxes (88 × 42 × 16 cm).

2.4 Equipment

• 1. Analytical balance.

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

• 3. Fume Hood.

• 4. Germination chamber (100% humidity and 30 °C temperature).

• 5. Orbital shaker.

2.5 Software

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

3 Methods

3.1 Preparation of Stock Solutions

• 1. 100 mM Phosphate buffer: add 13.61 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 1000 ml. Adjust pH to 3.0 using phosphorus acid (H₃PO₃).

• 2. Sodium azide (0.1 M): add 6.48 g of the powder to 500 ml volumetric flask and dissolve in 100 ml phosphate buffer (100 mM). Once completely dissolved, stir the solution while adding phosphate buffer (100 mM) and bring to 200 ml. Make solution fresh as required.

3.2 Seed Germination Test

• 1. See Fig. 1 for an overview of the seed germination test.

  Fig. 1

  

  Schematic representation of the process of mutagenesis of coffee seeds. a In advance of chemical mutagenesis, the NaN₃ concentration and incubation time are optimized using seeds having germination rate close to or above 90% (preferably normal shaped seeds that are disease-free). These steps take approximately 8 weeks. b bulk mutagenesis of coffee seed is a 6 steps procedure that consists of the seed sorting and selection, processing (manual removal of the pulp, the mucilage, and the parchment), disinfection of seeds, incubation of seeds with the appropriate NaN₃ concentration, post-treatment washing and planting of mutagenized seeds

• 2. Collect mature cherries from genetically homogenous mother plants maintained in the greenhouse or the field (see Note 3).

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

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

• 5. Disinfect the seeds with 3.5% (v/v) sodium hypochlorite for 1 h and rinse three times with sterile distilled water (see Note 4).

• 6. Soak 100 seeds in sterile distilled water for 48 h.

• 7. Sow the seeds in plastic boxes (88 × 42 × 16 cm) containing autoclaved peat substrate.

• 8. Place the boxes in the germination chamber (100% humidity and 30 °C temperature) for 8 weeks in darkness. Alternatively, the boxes could be placed under greenhouse conditions.

• 9. Record germination after 8 weeks based on visual scoring of full protrusion of the radicle and appearance of the coleoptile (see Note 5).

3.3 Sodium Azide Toxicity Test and Dose Optimization

• 1. Review safety procedures of the chemical mutagenesis laboratory (see Note 6 and 7).

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

• 3. Prepare a fresh 0.1 M NaN₃ stock solution by adding the required amount of NaN₃ to the phosphate buffer (100 mM, pH 3.0).

• 4. Select normal shaped seeds that are disease-free. The seeds should have good germination (i.e. > 90%).

• 5. Disinfect the seed beforehand according to the protocol described in 3.2 (see Note 4).

• 6. Place 100 seeds in a 250 ml labelled Erlenmeyer. The number of Erlenmeyers depends on the number of treatments. Label each flask 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).

  Table 1 NaN₃ concentrations and incubation time evaluated for the toxicity test of Arabica coffee seeds

• 8. Add NaN₃ to a final concentration of 25, 50, 75, and 100 mM (see Table 1). Shake the solution vigorously.

• 9. Incubate the mixture for 4, 8, or 12 h in the dark at 27 ± 2 °C with gentle rotation (100 rpm).

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

• 11. Repeat washing step 3 times.

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

• 13. After NaN₃ mutagenesis, the seeds should be planted immediately in plastic boxes (88 × 42 × 16 cm) containing autoclaved peat substrate.

• 14. Place the boxes in a germination chamber (100% humidity and 30 °C temperature) for 8 weeks and incubate in darkness. Alternatively, the boxes could be placed under greenhouse conditions.

• 15. Record the germination percentage, hypocotyl emergence percentage (seedlings with hypocotyl emerged from the substrate), aerial length (cm) and root length (cm) after 8 weeks.

• 16. Plot the germination percentage, hypocotyl emergence percentage, aerial length (cm), and root length of the control against each mutagenic treatment (see Fig. 2).

  Fig. 2

  

  a Germination percentage, b hypocotyl emergence percentage, c aerial length (cm), and d root length (cm) of the arabica coffee plantlets 8 weeks after incubation of seeds treated with different concentrations of NaN₃ and immersed for 4, 8, and 12 h. Each value represents the mean ± SD of four repetitions

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

  Fig. 3

  

  Effect of NaN₃ treatment on germination of coffee seeds 8 weeks after incubation. Different concentrations of NaN₃ (25, 50, 75, and 100 mM) and incubation times (4, 8, and 12 h) were used. Bar, 1 cm

3.4 Bulk Mutagenesis

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

• 2. Choose appropriate concentration of NaN₃ and incubation time based on the results obtained from the chemical toxicity test.

• 3. Select similar and normal shaped seeds that are disease-free. The seeds should have good germination (i.e. > 90%).

• 4. Disinfect the seed beforehand according to the protocol 3.2.

• 5. Place 100 seeds 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 concentration and incubation time).

• 7. Transfer Erlenmeyers containing seed material into the chemical mutagenesis laboratory.

• 8. In a fume hood, add appropriate concentrations of NaN₃ and shake the solution vigorously.

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

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

• 11. Repeat washing step 3 times.

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

• 13. After mutagenic treatment, seeds should be planted immediately in plastic boxes (88 × 42 × 16 cm) containing autoclaved peat substrate.

• 14. Place the boxes in the germination chamber (100% humidity and 30 °C temperature) for 8 weeks in darkness. Alternatively, the boxes could be placed under greenhouse conditions.

• 15. Record the germination percentage, hypocotyl emergence percentage (seedlings with hypocotyl emerged from the substrate), aerial length (cm) and root length (cm) into a spreadsheet (e.g., Microsoft Excel or Open Office Excel) (see Note 3).

• 16. Plot the germination percentage of the control against each mutagenesis treatment.

3.5 Planting Mutagenized M₁ Seedling

• 1. After 8 weeks, transfer coffee seedlings to pots containing autoclaved peat substrate.

• 2. Maintain the pots under greenhouse conditions with 12 h light photoperiod at 27 ± 2 °C.

• 3. Characterize the mutagenized M₁ population using following parameters: duration of seed germination, the germination percentage, hypocotyl emergence (seedlings with hypocotyl emerged from the substrate), aerial length (cm) and root length (cm).

4 Notes

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

• 2. Store NaN₃ in an airtight colored bottle, preferably inside a sealed chamber containing a desiccant.

• 3. Coffee seeds rapidly loose viability 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 month.

• 4. Disinfection is recommended only if stored seed is used, on the other hand, disinfection is not necessary for freshly harvested seeds.

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

• 6. 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.

• 7. Lab safety precautions: read the Materials Safety Data Sheet (MSDS) of materials being used and follow the recommendation of the manufacturer. Pay careful attention to the information on sodium azide and what to do in case of exposure. It is very important to wear personal protective clothing: 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.