Chemical Mutagenesis of Coffea arabica L. var. Venecia Cell Suspensions Using EMS

Abstract

Arabica coffee is widely grown in Latin America where it is under threat of leaf rust, a fungal disease caused by Hemileia vastatrix. As a perennial crop, conventional breeding of Arabica coffee is challenged by its long juvenile period and narrow genetic base. Plant mutants are important resources for crop breeding and functional genomics studies. The ethylating agent ethyl methanesulfonate (EMS) is widely used for inducing random point mutations. In a wide range of species, treatment with EMS causes GC-to-AT transitions with great efficiency. These properties, combined with ease of use, make EMS a mutagen of choice for induced mutagenesis. In vitro cell and tissue culture integrated with mutation induction provide an attractive approach for broadening the genetic base and breeding purposes, especially for perennial crops such as Arabica coffee. Embryogenic cell cultures are suitable targets for mutation induction and can accelerate the development of chimera-free mutant plantlets. Here we describe a robust protocol for EMS mutagenesis of embryogenic cell suspensions of Coffea arabica var. Venecia. Dose-response curves were established within 3–4 weeks and showed LD₃₀ and LD₅₀ values in the range of 0.5% and 0.6% EMS respectively. Methods and media used for development of the treated cell suspensions and conversion to in vitro plantlets are also described.

Joanna Jankowicz-Cieslak, Florian Goessnitzer—Contributed equally.

1 Introduction

Coffee is one of the most valuable cash crops and provides employment for millions of people worldwide, especially in Latin America and parts of Africa and Asia (FAOSTAT 2021). Coffee belongs to the family Rubiaceae and the two main species of cultivated coffee are Coffea arabica L. and Coffea canephora. Coffee leaf rust (CLR) caused by the airborne fungus Hemileia vastatrix and coffee berry disease are among the most important diseases affecting coffee production. C arabica is the most severely affected by leaf rust. Leaf rust epidemic has hit countries in Mesoamerica, including Colombia, Peru, Ecuador, and Guatemala amongst others, in the past decade (Avelino et al. 2015).

Resistant varieties are perhaps the most appropriate means to manage CLR. Improvement of Arabica coffee using conventional cross breeding is challenged by its long juvenile phase and narrow genetic base (Wintgens 2012; Scalabrin et al. 2020). Induced mutagenesis is widely used an efficient method to induce genetic variability useful for genetic studies and breeding. Since the 1970s in vitro tissue culture technologies have been developed for coffee, including methods to regenerate plants from single cells through somatic embryogenesis (see Etienne et al. 2018 for a review). Both direct and indirect methods for somatic embryogenesis in Arabica coffee have been described (Quiroz-Figueroa et al. 2006; Murvanidze et al. 2021 and references therein). Single cells or cell clusters are attractive targets for mutagenesis given the high likelihood for directly yielding chimera-free, homohistont plants. In addition, in vitro systems could offer significant efficiency gains in terms of space and labour compared to greenhouse- or field-based experiments to establish large mutant populations for perennial crops and trees such as Arabica coffee. Here, a fast and reproducible protocol for EMS mutagenesis of embryogenic cell suspensions of Arabica coffee var. Venecia is presented. Protocols for converting the EMS treated somatic embryos into in vitro plantlets are also provided.

2 Materials

2.1 Culture Medium

• 1. Analytical balance.

• 2. Weighing trays.

• 3. Spatula.

• 4. Magnetic stir bar.

• 5. Hot plate.

• 6. pH meter.

• 7. Medium dispenser.

• 8. Forceps.

• 9. Surgical Blades.

• 10. Aluminium foil.

• 11. Sterile culture tubes (50 ml).

• 12. In vitro culture test tubes (30 ml).

• 13. 10 cm Petri-dish with vents.

• 14. Culture vessels for liquid media.

• 15. Laminar flow bench.

• 16. In vitro growth room.

• 17. Tissue culture grade water.

• 18. Gelling agent (e.g., Gelrite).

• 19. Coffee culture media components (Table 1).

Table 1 Media composition (mg/l) for somatic embryogenesis and plantlet regeneration of Coffea arabica var. Venecia

2.2 Chemical Toxicity Test

• 1. Coffee cell suspensions (see Note 1).

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

• 3. Labelled waste receptacle for dry hazardous material and collection vessels for EMS waste solution (see Note 3).

• 4. Ethyl-methanesulphonate (EMS) AR grade, M.W. 124.2 (see Note 4).

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

• 6. Sterile deionized water.

• 7. 50 ml falcon tubes.

• 8. Syringe.

• 9. Needle.

• 10. Sterile membrane filter for filtering EMS solution: 25 mm diam., 0.2 µm pore size.

• 11. Pipette bulb.

• 12. Graduated cylinders.

• 13. Bottles (100 ml, 500 ml).

• 14. Beakers (500 ml and 1,000 ml).

• 15. Orbital shaker.

• 16. Disposable pipettes (5 ml, 25 ml).

2.3 Calculation of Lethal Dose (LD)

• 1. Pen.

• 2. Notebook.

• 3. Ruler

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

• 5. Camera.

• 6. Photobooth (optional).

3 Methods

3.1 Preparation of Liquid Culture Medium

• 1. Prepare MS, growth regulator and chemical stock solutions according to common procedures (concentrations shown in Table 1).

• 2. Filter sterilize all stock solutions.

• 3. Dispense into 50 ml batches and freeze for further use. Store the working solution at 4 °C.

• 4. Autoclave culture vessels for CMA1 medium before dispensing liquid medium.

• 5. Take desired amounts of solutions and chemicals (Table 1) and mix well.

• 6. Place the media on the mixer and let it mix properly.

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

• 8. While stirring, adjust medium to pH 5.8 using NaOH and HCl.

• 9. Autoclave for 20 min at 120 °C.

• 10. Allow the medium to cool.

• 11. Dispense liquid CMA1 medium into previously autoclaved culture vessels.

• 12. Dispense M5 medium in culture test tubes after autoclaving.

• 13. Store the medium in a cold room.

3.2 Mutagenesis of Coffee Cell Suspensions: Chemical Toxicity Test

• 1. Prepare sufficient suspension cultures to perform the mutagenic treatment. Procedures for obtaining cell suspensions can be found in Chap. “Somatic Embryogenesis and Temporary Immersion for Mass Propagation of Chimera-Free Mutant Arabica Coffee Plantlets”.

• 2. Filter 5–6 weeks old cell suspension cultures through 0.5 × 0.5 mm mesh.

• 3. Transfer filtered suspension to fresh CMA1 medium with an end volume of 70 ml.

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

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

• 6. Choose appropriate concentrations of EMS solution and incubation times for the mutagenesis of coffee cell cultures (see Note 6).

• 7. Prior to working with EMS, test the bottles and falcon tubes used for mixing EMS and for the mutagenic treatment of cell suspension cultures (see Note 7).

• 8. Prepare 50 ml falcon tubes containing cell suspensions in final volume of 15 ml. If necessary, dilute the cultures. Here, 3 ml of filtered cell cultures were added to 12 ml of CMA1 medium. Prepare at least 3 replicates per each treatment combination.

• 9. Transfer culture tubes to the chemical mutagenesis laboratory. Care should be taken not to expose the cultures to unfavorable conditions while transferring to the facility where the mutagenesis will be performed.

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

• 11. In the fume hood, prepare fresh 10% EMS dilution by adding the required volume of EMS into the bottle containing autoclaved water (Fig. 1a). 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 inactive EMS before placing in hazardous waste (see Note 8).

• 12. Seal the bottle of prepared 10% EMS dilution with a screw cap.

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

• 14. Ensure the sash is lowered on the fume hood and shake the solution vigorously for 15 s (see Note 9).

• 15. In the fume hood, prepare 50 ml Falcon tubes containing appropriate volume of CMA1 medium for each of the EMS concentration. The volume of the culture is calculated for all replicates.

• 16. Pipette the appropriate volume of 10% EMS (in water) into the tubes labelled with the respective concentration of EMS/treatment combination (Fig. 1b).

• 17. Mix the dilutions of EMS/culture medium properly.

• 18. Carefully distribute the determined volumes (here 5 ml) of EMS solution to every falcon tube containing cell suspensions (Fig. 1c). Ensure that all the volumes are being calculated properly, so that the final concentration of EMS corresponds to the one you wish to apply. In the chemical toxicity experiment described here following EMS concentrations were used: 0.2%, 0.5%, 0.8%, 1.5% and 2% EMS. In this experiment, the final reaction volume after adding the EMS dilution was 20 ml.

• 19. Prepare control batch in the same way by adding culture medium to the falcon tubes containing cell suspensions.

• 20. Wipe the outside of falcon tubes containing EMS with a paper towel soaked in sodium thiosulfate.

• 21. Place falcon tubes (including control) on a rotary shaker set at 60 rpm, record the time and temperature, and start incubation (Fig. 1d and see Note 11). One hour incubation was chosen for the experiment described in this protocol.

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

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

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

• 25. Fifteen minutes before the end of the incubation time, transfer falcon tubes into the fume hood and let the cells sediment. A clear pellet of mutagenized coffee cells should be visible at the bottom of the falcon tube (Fig. 1e).

• 26. Carefully decant each of the treatment batches into a waste beaker (Fig. 1f and see Note 12).

• 27. Wash each coffee cell pellet with 20 ml of CMA1 medium. Pour the EMS-medium solution off to the waste beaker (Fig. 1g, h).

• 28. Repeat the wash steps for a total of three washes (see Note 13).

• 29. After the final wash, add appropriate volume of culture medium to the treated cells. Here, 3 ml of CMA1 were added to each falcon tube containing treated, as well as non-treated cells.

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

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

• 32. Dispose of toxic waste according to local regulations. Decontaminate all surfaces and equipment by wiping down with 100 mM sodium thiosulfate followed by a water rinse (see Note 14).

• 33. Move mutagenized cell cultures into the flow cabinet and immediately proceed with transferring 100 µl of treated suspension cultures to test tubes containing freshly prepared M5 medium (Fig. 1i and see Note 15). Here, 90 culture tubes containing 100 µl cells each were prepared per treatment combination.

• 34. Place tubes in the culture room with 16 h light and a temperature of 28 ± 1 °C. Observe daily for any changes in the color of cells or growth.

Fig. 1

Procedure for EMS chemical mutagenesis of in vitro coffee cell suspensions. a A 10% EMS stock mixture is prepared under the fume hood. b Final dilutions are prepared by adding appropriate volumes of the 10% EMS stock solution into the cell culture maintenance media. c The EMS dilutions are aliquoted into 50 ml falcon tubes containing 20 ml cell suspensions, replicates of 3 per treatment are prepared. d Cells are incubated for a specific time (here 1 h) under orbital rotation. e Shortly before the end of the incubation, falcon tubes containing mutagenized cells are removed from the shaker and put aside to allow cells to settle. f The supernatant is carefully decanted, not to lose the pellet. g, h Washing of mutagenized material with 40 ml maintenance liquid media, repeated at least 3 times. i After the washing step, a set volume of culture media is added to the mutagenized material. Tubes are transferred to the in vitro laboratory and 100 µl aliquots of the mutagenized cells are transferred to the regeneration media

3.3 Calculation of Lethal Dose (LD)

• 1. Monitor the cell growth daily.

• 2. Visible differences between mutagenic treatments can be observed approximately 2 weeks after mutagenesis.

• 3. Let the cells/cell clusters grow until 3–4 weeks post treatment when the scoring can be taken (Fig. 2).

• 4. Count tubes where cell growth is clearly visible.

• 5. Calculate survivability and graph data (Figs. 3 and 4).

• 6. Repeat the chemical toxicity test if the results and data obtained are not precise enough.

• 7. Choose one or more concentrations for bulk mutagenesis following this protocol. While making the selection, consider the values calculated for LD₃₀ and LD₅₀ (Fig. 4).

Fig. 2

Example data showing the response of Arabica coffee embryogenic cell suspension treated with different EMS concentrations, observed 3 weeks post EMS treatment. Growth inhibition of 100% was observed for cultures treated with 1.5% and 2% EMS

Fig. 3

Example of the survival count of cultured coffee cells taken 3 weeks after treatment with EMS. For the control material, 90/90 cultured tubes maintained growth. In the case of treated cultures, 0.2% EMS had similar growth rate to the control, a slight drop is being observed for 0.5% EMS treated cultures (66/90 tubes survived). A clear drop occurred for the material subjected to 0.8% EMS for which only 4 out of 90 cultured cell suspension tubes maintained the growth

Fig. 4

Survival calculated as percentage of the control, whereby the control is 100%. The kill curve indicates the LD₃₀ (the dose causing the death of 30% of the population) and LD₅₀ (the dose causing the death of 50% of the population) values in the range of 0.5% and 0.6% EMS respectively

3.4 Development of Somatic Embryos and Conversion into Plantlets

• 1. After 4–6 weeks embryo development can be observed.

• 2. After 2–3-months transfer developed torpedo shaped embryos to new tubes containing M5 medium.

• 3. Embryo maturation begins after 1–2-months when first foliage leaf and root formation can be observed (Fig. 5).

• 4. Transfer all torpedo shape embryos into individual culture tubes containing M5 media.

• 5. Count the number of developed embryos for every treatment.

• 6. Place tubes in the culture room with 16 h light and a temperature of 28 ± 1 °C.

• 7. Observe daily for any changes in the color or growth.

• 8. Take a count of developed plantlets.

• 9. Calculate the survival rate and compare with data obtained (Sect. 3.3; Figs. 3 and 4, Table 2).

Fig. 5

Conversion of Coffee arabica somatic embryos to plantlets, control cultures are shown. a The embryogenic cells are cultured on a solid medium for the induction of somatic embryos, b torpedo shape embryos are selected and transferred to individual culture tubes for c, d plantlet development

Table 2 The averages of torpedo shape embryos and regenerated plantlets per 100 µl cultured volume

4 Notes

• 1. This protocol describes EMS chemical mutagenesis of Coffea arabica var. Venecia, a late maturing variety with excellent cup quality. The Venecia coffee variety has its origin in San Carlos, Alajuela where it was discovered on a coffee plantation of 100% Caturra. It was selected due to its increased productivity, larger fruit size and increased resistance to fruit drop in the rain. The procedures described here utilize coffee cell suspensions generated in the Plant Breeding and Genetics Laboratory, Seibersdorf, Austria. For details on establishing the cell suspension culture, see Chap. “Somatic Embryogenesis and Temporary Immersion for Mass Propagation of Chimera-Free Mutant Arabica Coffee Plantlets”. Briefly, leaf discs served as the starting material to produce embryogenic callus. The embryogenic callus was then transferred to a liquid medium to establish a homogenous embryogenic cell suspension culture. The cell suspension culture served to maintain and multiply embryogenic cell/cell clusters and was used for EMS treatments. The EMS-treated cultures were regenerated on semi-solid media described here.

• 2. Ideally, chemical mutagenesis experiments are conducted in a dedicated laboratory, using dedicated equipment, and equipped with a ducted fume hood, toxic waste disposal and decontamination procedures. It is advisable to work with another person during the steps handling EMS to assist with the provision of equipment (e.g., pipettes) and sodium thiosulfate to deactivate EMS. Personal protective equipment such as laboratory coats, gloves, goggles, and disposable shoe covers should be worn when working with hazardous chemicals. It is advised to wear double gloves so that contaminated gloves can be removed while avoiding contact of contaminated materials with skin. Consult the biosafety regulations on use and disposal of hazardous chemicals. In addition, in advance of performing the experiment it is advised to carefully plan out what will be done and to practice the critical steps. Ensure that enough empty, liquid, and dry waste buckets are placed in the laboratory. If using common space, inform co-workers of the experiment in advance to avoid accidental exposures.

• 3. Follow the waste disposal procedures established in your institute.

• 4. EMS is carcinogenic and thus extreme care should be taken. Pay careful attention to the information on the EMS Materials Safety Data Sheet (MSDS) and follow any recommendations in case of accidental exposure.

• 5. 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 standby during the laboratory procedures to inactivate any spills and to clean tips and other consumables prior to disposing in hazardous waste.

• 6. Some publications use percentage (v/v) of EMS rather than molarity. Optimal concentrations of ~ 20–40 mM have been reported for many studies using cell cultures mutagenesis (Jankowicz-Cieslak et al. 2011; Jankowicz-Cieslak and Till 2016). It is advisable to include a no EMS control and to test concentrations below and above the concentration used in published studies of the same species. The timing and temperature of EMS treatment can also be tested, but it is recommended to start first with concentrations as optimal dosage can typically be determined by altering mutagen concentration alone. The current protocol uses five EMS concentrations (0.2%, 0.5%, 0.8%, 1.5%, 2%) plus control. Each concentration was performed in 3 replicates. The mutagenic treatment lasted 1 h.

• 7. EMS is not miscible in water. DMSO is usually added to improve miscibility (Ingelbrecht et al. 2018). In this experiment, we omitted the use of DMSO due to the very fragile nature of the material to be treated. The prepared mixture should be mixed thoroughly. The mixture is prepared in a bottle, sealed with a screw cap, and then shaken vigorously before adding to the coffee cell cultures. It is important that the bottle does not leak. Test the bottle with water first and mimic the shaking procedure in the fume hood.

• 8. 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 stock bottle containing the concentrated EMS doesn’t spill when working in the confined space of a fume hood.

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

• 10. Verify the exact volume to be added to ensure cells will be fully exposed to the chemical mutagen.

• 11. It is advisable to estimate the best ratio of cell cultures to the EMS mixture prior to the actual experiment. Add the amount of cell cultures used in the main experiment to the beaker, add water and place on the orbital shaker. The cell cultures should be completely immersed in the mutagen solution and fully exposed to the active ingredients of the mutagen. Adjust the speed of the orbital shaker so that all cell cultures can freely move, split cell cultures into multiple falcon tubes and reduce volumes if necessary. EMS is unstable in water solutions due to hydrolysis with a half-life of 26 h at 30 °C. At low temperatures, hydrolysis rate is decreased, implying that the mutagen remains stable for longer.

• 12. Arabica coffee var. Venecia cell suspensions were incubated for 1 h. Be very careful when pouring off the liquid, avoid splashing, use a mesh screen or a sieve to ensure that all cell cultures are captured.

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

• 14. EMS is a toxic chemical and must be disposed of according to current safety regulations in the laboratory (check disposal procedures with personnel responsible for toxic materials or local health authority). 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.

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