Chemical Mutagenesis of Mature Seed of Coffea arabica L. var. Venecia Using EMS

Abstract

Chemical mutagens are a major tool to generate novel genetic variation in crops, functional genomics and breeding. They are advantageous because they do not require any specialized equipment and can induce a high mutation frequency. Compared to physical methods, chemical mutagens cause point mutations rather than deletions or translocations. Point mutations can have varying effects on gene expression ranging from knockouts to changes in amino acids that may have subtle effects on protein function. Many important gene functions have been uncovered by evaluating the in vivo effect of mutated genes in a broad range of model and crop plants. Chemical mutagens have been successfully applied to induce tolerance to fungal diseases in cereals such as barley and wheat. Among the chemical mutagens used for plant mutagenesis, ethyl methanesulfonate (EMS) is the most widely applied. This protocol chapter describes the utilization of EMS for establishing kill curves and generating a mutagenized population of Coffea arabica var. Venecia via treatment of mature seed. The different steps of the mutagenesis process are described, including quality control and preparation of the seed batches, procedures for determining Lethal Dose (LD) and Growth Reduction (GR) values, and for post-treatment handling of the chemically mutagenized seed, specific for Arabica coffee.

1 Introduction

Mutation breeding has proven to be an efficient tool to develop improved crop varieties whereby various novel traits such as e.g., yield, growth, and disease resistance can be induced (Spencer-Lopes et al. 2018). Among various mutation induction techniques, mutagenesis using chemical agents has become an efficient and robust tool to induce point mutations (Ingelbrecht et al. 2018; Jankowicz-Cieslak and Till 2016). Mutations in coding regions of genes can be silent, missense, or nonsense, while mutations can equally occur in non-coding, regulatory sequences affecting gene expression, e.g., at intron splice sites. Among the different chemical mutagens, ethyl methanesulfonate (EMS) has been frequently used to induce random mutations because it is highly effective and relatively easy to handle. To date, mutation induction using EMS has been described for a wide range of plant species covering both seed and vegetatively propagated plants (Jankowicz-Cieslak et al. 2011; Jiang et al. 2022). Chemical mutagens have been successfully applied to induce tolerance to fungal diseases in cereals such as powdery mildew-resistance in barley (Molina-Cano et al. 2003) and resistance to leaf rust Puccinia sp. in wheat (Mago et al. 2017).

The workflow for chemical mutagenesis involves choosing the genotype and the appropriate tissue type to be mutated, the mutagen, and optimizing the treatment conditions and dosage. Once the optimal dose(s) have been determined, bulk mutagenesis can proceed. EMS mutagenesis of seed is widely used in diploid, self-pollinating cereals and legumes, amongst other plant species. Briefly, seed are pre-treated by soaking in water, EMS is added, and mutations are induced during a specific treatment period. Following this, seed are thoroughly washed and sown. This results in the M₁ generation which can harbor a high density of induced point mutations.

Growth and survival measurements remain the simplest route to dosage optimization and have the added appeal that they can be applied in almost any facility as little infrastructure or expertise are required. A reduction of growth rate in the early seedling stage, typically two weeks for small-seeded cereals, serves as an easy indicator (Jankowicz-Cieslak and Till 2016; Mba et al. 2010). A range between 30 and 50% growth reduction is typically chosen as optimal dose for bulk irradiation in cereals. As with any mutagenesis treatment, a compromise needs to be made between achieving a sufficiently high mutations frequency to have a reasonable chance to recover the desired mutations and suitable level of survival and fecundity (Jankowicz-Cieslak and Till 2015). It is advisable to use up to three doses of the mutagen, corresponding to ± 20% of the optimal dose found through the toxicity test.

For chemical mutagens such as EMS, both the concentration and duration of the treatment are evaluated during dose optimization. Different treatments are tested, and germination and survivability as well as growth reduction typically measured. Where possible, embryo lethality in the M₂ seed can be used as an indicator for the efficiency of mutagenesis (Till et al. 2003). There are additional issues with chemical mutagenesis that one needs to consider. Cytotoxicity may limit the efficacy of specific mutagens for certain plant species or genotypes, necessitating trials with different mutagens (Till et al. 2007). Following the mutagenesis procedure, M₂ mutant populations can be evaluated for phenotypic or genotypic variation distinct from the non-mutagenized parental line.

The advantages and limitations of different propagules for mutagenesis treatment of Arabica coffee are briefly described in Chap. Mutation Breeding in Arabica Coffee. Since Arabica coffee is self-fertilizing and mostly propagated through seeding, seed can serve as starting material for mutagenesis treatments. As with other seed crops, M₁ chimeric plants are expected which can be resolved through successive cycles of selfing (Mukhtar Ali Ghanim et al. 2018). In this chapter, the susceptibility of Arabica coffee seed var. Venecia to the chemical mutagen EMS was evaluated and optimal doses for EMS bulk irradiation were determined. Methods of evaluation of mutagenic effect at the seedling stage are presented. Further, example data on morphological and chlorophyl variegations observed at the M₁ stage are shown.

2 Materials

2.1 Chemical Toxicity Test

• 1. Chemical mutagen laboratory equipped with fume hood and flow bench (see Note 1).

• 2. Labelled waste receptacle for dry hazardous material and collection vessels for EMS waste solution.

• 3. High quality, disease-free coffee seeds (see Note 2).

• 4. Stainless steel tea-steeper.

• 5. Ethyl-methanesulphonate (EMS) AR grade, M.W. 124.2 (see Note 3).

• 6. 10% (w/v) sodium thiosulfate (Na2S2O3.5H2O) (see Note 4).

• 7. Dimethyl sulfoxide (DMSO).

• 8. Sterile water.

• 9. Deionized water.

• 10. 50 ml falcon tubes.

• 11. Syringe.

• 12. Needle.

• 13. Disposable pipettes.

• 14. Pipette bulb.

• 15. Graduated cylinders.

• 16. Bottles (100, 500 ml).

• 17. Beakers (500 and 1000 ml).

• 18. Sieves (metal, 70 mm diam., 10–100 µm pore size).

• 19. Orbital shaker.

• 20. Disposable pipettes (5, 25 ml).

• 21. Soil: light soil containing peat moss and sand (0.5–2 mm) in a ratio of 3:1 (peat: sand) with acidic pH (5–6).

• 22. Pots and multiwell trays.

• 23. Glasshouse with light and temperature control (see Note 5).

2.2 Calculation of Lethal Dose (LD) and Growth Reduction (GR)

• 1. Pen.

• 2. Notebook.

• 3. Ruler.

• 4. Standard spreadsheet software e.g., Microsoft Excel.

2.3 Bulk Mutagenesis

• 1. All materials as listed in 2.1.

• 2. High quality, disease-free coffee seeds (e.g., 1000).

2.4 Phenotyping

• 1. Pen.

• 2. Notebook.

• 3. Ruler.

• 4. Standard spreadsheet software e.g., Microsoft Excel.

• 5. Coffee descriptors.

• 6. Camera.

• 7. Photobooth (optional).

3 Methods

3.1 Seed Quality Control and Pre-treatment

• 1. Perform a germination test of seed lots as part of quality control. The germination frequency should be minimum 90%.

• 2. Select seeds from genetically homogenous plants from the targeted preferred variety.

• 3. Sort out clean, similar in size, normal shaped and disease-free seeds. Discard small, shriveled, discolored and damaged seeds from the experimental lot (Fig. 1a and see Note 6).

  Fig. 1

  

  Overview of EMS mutagenesis of mature coffee seed. The procedure involves three steps: (i) quality control and pre-treatment of the seeds, (ii) seed mutagenesis; (iii) post-treatment handling. a Ahead of chemical mutagenesis high quality coffee seeds are selected that are uniform, homozygous and isogenic, while discoloured, small or damaged seeds are discarded. b After removal of the parchment and the silverskin, the seeds are pre-soaked in sterile, distilled water for 48 h at room temperature. c After incubation, the water is decanted from the seed batches. d Seeds are placed into labelled beakers for treatment. A dilute mixture of EMS plus DMSO is prepared and stored under the fume hood. e The seeds are incubated for a specific time under orbital rotation. f The mutagen is removed, and seeds are thoroughly washed, minimum three times. g Coffee seeds are then transferred to the glasshouse and immediately planted in a light soil

• 4. Manually remove the outer parchment and the silverskin underneath from every seed.

• 5. Divide the seeds into batches, each about 50 seeds. Leave out one batch as a control (untreated). The remaining batches correspond to the possible combinations of concentrations of EMS (Fig. 1b and see Note 7).

• 6. Place seed batches in appropriately labelled 50 ml Falcon tubes filled with sterile distilled water.

• 7. Leave standing for 48 h at room temperature (see Note 8).

• 8. Carefully decant water and transfer coffee seed into either a labelled petri plate or a tea-steeper (Fig. 1c and see Note 9).

• 9. Transfer pre-soaked seed batches to the chemical mutagenesis facility.

• 10. Immediately proceed with the chemical mutagenesis experiment.

3.2 Coffee Seed Mutagenesis

• 1. Review safety procedures of the chemical mutagenesis laboratory and Consult the Materials Safety Data Sheets for all chemicals used.

• 2. Prepare the laboratory (see Note 10).

• 3. Choose appropriate concentrations of EMS solution and incubation times (Table 1 and see Note 11).

  Table 1 Example calculations for solutions containing different EMS concentrations and 2% DMSO at a final volume of 1 L used for mutagenic treatment of var. Venecia

• 4. Prior to working with EMS, test the bottles used for mixing EMS/DMSO (see Note 12).

• 5. Prepare a bottle containing 100 mM sodium thiosulfate and place in the fume hood along with a role of paper towels.

• 6. In the fume hood, prepare fresh EMS dilutions by adding the required volume of EMS solution to the water/DMSO mixture (see Table 1). Use a sterile syringe and a 0.2 µm filter for this step. Place syringe and filter into a beaker containing 100 mM sodium thiosulfate to inactivate EMS before placing in hazardous waste (see Note 13).

• 7. Seal the bottle of each prepared EMS and DMSO solution with a screw cap.

• 8. Wipe the outside of stock bottles with a paper towel soaked in sodium thiosulfate.

• 9. Return EMS and DMSO stock bottles to their storage location.

• 10. Ensure the sash is lowered on the fume hood and shake the EMS/DMSO solution vigorously for 15 s (see Note 14).

• 11. In the fume hood, place each pre-soaked coffee seed batch into empty treatment beaker labelled with the appropriate treatment code (EMS concentration and replication number).

• 12. Carefully pour the determined volume of EMS solution to every beaker (Fig. 1d and see Note 15).

• 13. Prepare control batch in the same way by adding water to the beaker.

• 14. Wipe the outside of beakers containing EMS mixture with a paper towel soaked in sodium thiosulfate.

• 15. Place beakers (including control) on a rotary shaker set at 80 rpm, record the time and temperature and start incubation (Fig. 1e and see Note 16).

• 16. Pour 100 mM sodium thiosulfate into bottles used to prepare EMS dilutions.

• 17. Wipe the outside of the bottle with a paper towel soaked in sodium thiosulfate.

• 18. Dispose of liquid and solid waste in appropriate toxic waste containers.

• 19. Wipe the fume hood with a paper towel soaked in sodium thiosulfate.

• 20. After the incubation time, transfer beakers into the fume hood.

• 21. Quickly but carefully decant each of the treatment batches into a waste beaker and rinse with sterile water (Fig. 1f and see Note 17).

• 22. Pour the EMS waste-solution into a hazardous waste container and repeat the wash steps.

• 23. Repeat steps 21 and 22 for a total of three water washes (see Note 18).

• 24. Collect all the liquid waste in a dedicated bucket labelled as hazardous waste.

• 25. Detoxify the waste and all unused EMS solution by adding sodium thiosulfate in a 3:1 ratio by volume.

• 26. Dispose of toxic waste according to your local Laboratory Safety Rules and Regulations (see Note 19).

• 27. Decontaminate all surfaces and equipment by wiping down with 100 mM sodium thiosulfate followed by a water rinse (see Note 20).

• 28. Transfer mutagenized seed into the greenhouse and immediately proceed with sowing (Fig. 1g and see Note 21).

3.3 Post-treatment Handling of Seeds

• 1. Prepare soil mix.

• 2. Distribute the soil uniformly in multiwell trays (Fig. 2a).

  Fig. 2

  

  Coffee plants grown in the FAO/IAEA Plant Breeding and Genetics Laboratory, Seibersdorf, Austria. a Mutagenized coffee seeds are sown in multiwell trays filled with light soil immediately after EMS treatment. b Seven months post-mutagenesis, plants are transplanted to bigger pots

  Fig. 3

  

  a Coffee seed are germinated in the glasshouse and the germination date as well as germination rate is scored. b At the seedling stage (e.g., 104 days post-mutagenesis) survival count is taken along with hypocotyl/seedling height and number/dimension of leaves. c At the plant stage (e.g., 208 days post-mutagenesis) the survival rate, plant height, number of leaves and leaf morphology and other variegations are recorded

• 3. Irrigate the soil and let excess water drain.

• 4. Label the trays, include the genotype, replication, EMS concentration and treatment date.

• 5. To limit any detrimental effect of EMS on seed viability, sow the seeds immediately after treatment (Fig. 2a).

• 6. Water regularly under appropriate light and temperature conditions to maximize growth and survival.

• 7. Transplant to larger size pots approximately 7 months after treatment for further growth (Fig. 2b).

3.4 Calculation of Lethal Dose (LD): Data Collection and Analyses

• 1. Maintain mutagenized coffee seeds in the greenhouse conditions.

• 2. Identify stages during which you will take measurements, starting from the germination stage, through seedling up to the plant stage (Figs. 3 and 4 c and see Note 22).

  Fig. 4

  

  Coffee seed germination scored for estimation of 50% lethality (LD₅₀): a Mutagenized materials are first grown, and the germination date is registered which serves as the basis for the calculation of the Days to Germination values (DTG). b The germination of EMS treated seed occurred between 34 and 63 days after sowing. Here the average of DTG values for each treatment is displayed which ranges between 35 days for the control and 0.5% EMS and 48 days for 4% and 6% EMS. c The number of germinated seed was recorded at the stage of the seedling emerging from the soil. d Nearly 80% of planted control seeds emerged. Germination was visibly enhanced for 4 EMS treatments (0.2% up to 2%) and then dropped to 69% at 4% EMS and to 17% at 6% EMS

• 3. After ca 30 days of incubation start monitoring the germination of coffee seeds daily (the moment when the coffee plant emerges from the soil). Record the germination date of every seed. Mutagenic treatment might delay the seed germination process. In the current experiment, germination occurred between 34 and 63 days after sowing (Fig. 3a).

• 4. Count surviving coffee plants at the seedling and plant stage (Fig. 3b, c). Surviving plants are those which show continued growth. Plants which germinate but where growth is inhibited, and no development of true leaves is observed, are being counted as non-surviving plants.

• 5. Record other observations (e.g., Fig. 3).

• 6. Enter data for each recorded trait into a spreadsheet (e.g., Microsoft Excel).

• 7. Calculate the averages for each parameter and each treatment separately.

• 8. Calculate percentage of the parameter of the plant in relation to the control (Fig. 4 and see Note 23).

• 9. Plot percentage of control against mutagenic treatment for each parameter (Fig. 4 and see Note 24).

• 10. Estimate the mutagen concentration required to obtain 50% and 70% germination or survival relative to the untreated control to determine the LD₅₀ (the dose expected to cause 50% death of an exposed population) and LD₃₀ (the dose expected to cause 30% death of an exposed population) values (see Figs. 4 and 5).

  Fig. 5

  

  Example of chemical toxicity test performed on the coffee seed. Coffee seed are exposed to 6 concentrations of EMS (0.2; 0.5; 1, 2, 4 and 6% EMS) plus the control and incubated for 2 h. Here, the percentage germination in relation to the control (100%) was plotted for each EMS concentration (blue line). The germination was recorded between 34 and 63 days after treatment. Note that germination is enhanced for 0.2, 0.5, 1 and 2% compared to the control, whereby a clear drop is visible for 4% and 6% EMS. Same for the survival scored 104- (orange line) and 208-days (grey line) post-mutagenesis, it drops as EMS concentration increases

• 11. Choose a concentration to perform larger scale mutagenesis, called bulk treatment.

3.5 Bulk Mutagenesis

• 1. Choose the appropriate EMS concentration(s) and incubation time based on the results obtained from the chemical toxicity test.

• 2. See Fig. 1 for an overview of the bulk mutagenesis procedure.

• 3. Prepare coffee seeds (e.g., 1000) per each treatment chosen (see Note 25).

• 4. Follow procedures as outlined in Sects. 3.1, 3.2 and 3.3.

Fig. 6

Examples of coffee leaf phenotypes scored 8 months post-mutagenesis for a non-mutagenized control (labelled as ‘control’) and mutant plants (no labels)

3.6 Phenotyping

• 1. Phenotyping protocols are provided in chapters “Use of Open-Source Tools for Imaging and Recording Phenotypic Traits of a Coffee (Coffea arabica L.) Mutant Population–A PCR-Based Assay for Early Diagnosis of the Coffee Leaf Rust Pathogen Hemileia vastatrix” of this book.

• 2. Example of morphological and chlorophyl variegations of 8-month-old M₁ coffee plants are illustrated in Fig. 6.

4 Notes

• 1. EMS mutagenesis must be conducted in a dedicated laboratory equipped with a ducted fume hood, toxic waste disposal and decontamination procedures. It is advisable to work with another person when handling EMS. Personal protective equipment such as laboratory coats, gloves, goggles and disposable shoe covers should be worn. It is advised to wear double gloves so that contaminated gloves can be removed while avoiding contact of contaminated materials with skin.

• 2. This protocol describes EMS mutagenesis of Coffea arabica var. Venecia, a late maturing variety with excellent cup quality. The var. Venecia originated in San Carlos, Costa Rica from a natural mutation of a Bourbon population, selected by the Instituto del Café de Costa Rica, and released in 2010 as a new variety for its increased productivity, larger fruit size and increased resistance to fruit drop in the rain. Venecia is susceptible to Coffee Leaf Rust, Coffee Berry Disease and nematodes (World Coffee Research 2023).

• 3. EMS is carcinogenic and thus extreme care needs to be taken. Carefully read the instructions on the Material Safety Data Sheet and follow the recommendation of the manufacturer.

• 4. EMS can be inactivated by treatment with sodium thiosulfate. The half-life of EMS in a 10% sodium thiosulfate solution is 1.4 h at 20 °C and 1 h at 25 °C. Keep beakers of sodium thiosulfate (100 mM) on hand during laboratory procedures to inactivate any spills, clean tips and other consumables prior to disposing in hazardous waste.

• 5. Coffee requires specific environmental conditions for optimal germination and growth (Wintgens 2012).

• 6. Use freshly harvested seed as Arabica coffee seed viability decreases rapidly after 6 months when stored at ambient temperature (Wintgens 2012). This is a critical point in case of coffee. Always perform a germination test to check seed viability.

• 7. This protocol describes treatment of Venecia seeds with 6 EMS concentrations (0.2; 0.5; 1; 2; 4; 6%); 50 seeds per treatment at one duration of 2 h. Additional concentrations can be tested, as well as various temperatures and treatment durations.

• 8. Soaking the seeds prior to the mutagenesis treatment enhances the total uptake, the rate of uptake and the distribution of EMS within the target tissue, maximizing infusion of the mutagen into the embryo tissue within the shortest possible time. The duration of pre-soaking depends primarily on the seed anatomy; hard and thick seedcoats require longer pre-soaking times than soft and thin ones. A pre-soaking test can be performed to estimate optimal pre-soaking conditions. In the case of Venecia seed, a pre-soaking duration of 48 h at room temperature was applied.

• 9. Tea-steepers were utilized for water decanting and at the same time for maintaining treatment batches. Each tea-steeper was labelled with an attached plastic indicating the EMS concentration.

• 10. It is strongly recommended to perform EMS chemical mutagenesis in a dedicated laboratory using dedicated equipment. Consult the regulations on use and disposal of hazardous chemicals. In addition, it is advised to plan and practice critical steps in advance of the actual experiment. Ensure that sufficient waste buckets and beakers are available. If using common space, inform co-workers of the experiment in advance, to avoid accidental exposures.

• 11. Concentrations of EMS for testing a specific plant species or variety can be estimated based on previously published studies, if available. Some publications use molarity rather than percentage (volume/volume) of EMS. Optimal concentrations of ~ 20–40 mM have been reported for many studies using seed mutagenesis (Jankowicz-Cieslak et al. 2011; Kurowska et al. 2011). Always include a control (no EMS) and concentrations below and above the concentration used in published studies. The duration of the exposure and temperature of EMS treatment can also be tested, but it is recommended to start first with concentrations as the optimal dosage can typically be determined by altering the mutagen concentration only.

• 12. EMS is not miscible in water. DMSO is added to 2% to improve miscibility. The EMS/DMSO solution is prepared in a bottle, sealed with a screw cap, and then shaken vigorously before adding to coffee seed. It is important that the bottle does not leak. Test the bottle with water first and mimic the shaking procedure in the fume hood.

• 13. Prepare the concentration series of EMS commencing with the lowest concentration. Practicing this step allows for the proper placement of the stock bottle, dilution bottle, pipette, and waste container, so that the EMS stock does not spill when working in the confined space of a fume hood.

• 14. Lowering the sash is necessary for proper ventilation and provides some protection against leakage from the bottle.

• 15. Avoid adding excess volume of EMS/DMSO to the beaker of seed as this may result in liquid spilling during orbital rotation.

• 16. One day before the experiment, set up a mock experiment with water to determine the best ratio of seed to liquid for the EMS incubation. Add the number of seed to be used in the main experiment to the beaker. Add water and place on the orbital shaker. The samples should be completely immersed in the mutagen solution to ensure that all seeds are equally and fully exposed to the active ingredients of the mutagen. Adjust the speed of the shaker so that all seed move freely in the water, avoid splashing, split seeds into multiple beakers if necessary. Take note of both the volume and the number of seeds and apply this during the main experiment. Generally, EMS is unstable in water with a half-life of 26 h at 30 °C at neutral to acidic pH. Hence, the temperature of the environment can influence the efficiency of the EMS mutagenic treatment due to hydrolysis of the EMS. At low temperatures, the hydrolysis rate is decreased, implying that the EMS mutagen remains stable for longer.

• 17. Be very careful when pouring off liquid. Avoid splashes. A mesh screen or a sieve should be used to capture the seed and to avoid that seed are poured out of the beaker.

• 18. The by-products of the incubation process and residual active ingredients should be promptly washed off the incubated seed after treatment. This prevents continued absorption of the mutagen beyond the intended duration, so-called dry-back, which leads to lethality.

• 19. EMS is mutagenic, teratogenic, and carcinogenic and hence must be disposed of according to the health and safety regulations of your laboratory or institution (check disposal procedures with personnel responsible for toxic materials or local health and safety authority).

• 20. All body parts or laboratory coats contaminated with EMS should be washed thoroughly with water and detergent and further neutralized with 100 mM sodium thiosulfate.

• 21. Care should be taken to avoid that any materials removed from the chemical mutagenesis laboratory are free from contamination with EMS.

• 22. Following parameters are usually scored in the process of kill curve establishment: days to germination (DTG), germination %, seedling height, seedling/plant survival %, chlorophyll mutation frequency, seed set. In the case of coffee seed, germination (as in seedling emergence from the soil, Fig. 4c) occurs approximately 30 days after planting. In the current experiment the first seeds germinated 34 days, and the last one 63 days after sowing. Average values were calculated for each treatment which resulted in DTG values of 34–63.

• 23. This calculation is made by dividing the number of germinated seeds of the mutated material (numerator) by the number of germinated seeds of the control, untreated seed measured at the same time (denominator) multiplied by 100. For example, if 12 coffee seeds germinated for a 2 h, 0.2% EMS treatment and 12 seeds germinated for the control, then the percentage is 12/12 × 100 = 100%.

• 24. In the case of Venecia, the percentage of germination and survival were suitable parameters to estimate the damage due to the mutagenic treatment and determine the LD₅₀ and LD₃₀ values.

• 25. The total number of seed treated depends on the objectives of the experiment. An excess of seed should be treated as a percentage of seed will not germinate due to incubation with EMS. The optimal population size depends on the desired density of induced mutations, the available spatial and human resources, and the objectives of the study or breeding program.