Somatic Embryogenesis and Temporary Immersion for Mass Propagation of Chimera-Free Mutant Arabica Coffee Plantlets

Abstract

Coffee is one of the most valuable cash crops providing employment for millions of people worldwide. Arabica coffee is widely grown in Latin America where it is under threat of leaf rust. Conventional breeding of Arabica coffee is challenged by its narrow genetic base and long reproductive cycle, and it can take up to 30 years for variety development and release. In vitro somatic embryogenesis is a propagation technique whereby a single plant somatic cell can give rise to a somatic embryo under appropriate culture conditions. For tree crops such as Arabica coffee, single-cell mutagenesis using embryogenic cell cultures provides a powerful approach to produce chimera-free mutant lines directly from cells. Here we describe protocols to induce friable embryogenic callus, establish embryogenic cell suspensions, and convert somatic embryos into plantlets using a RITA^® bioreactor for Coffea arabica  var. Venecia. In addition, methods for gamma-ray mutagenesis of regenerable cell suspensions are described.

1 Introduction

Coffee is a global commodity providing employment for millions of people worldwide (FAOSTAT 2021). The Coffea genus belongs to the family Rubiaceae and the two main cultivated species are Coffea arabica L. (2n = 4x = 44) and Coffea canephora Pierre ex A. Froehner (2n = 2x = 22). Arabica coffee is a self-pollinating species, widely cultivated in South America, Africa and Asia. During the past decade, coffee leaf rust, a fungal disease caused by Hemileia vastatrix, has devastated C. arabica plantations across Latin America (Avelino et al. 2015).

Conventional breeding of Arabica coffee is challenging due to its long reproductive cycle and narrow genetic base (Wintgens 2012; Scalabrin et al. 2020). Mutation-assisted breeding offers an attractive alternative to induce genetic diversity useful for coffee breeding and genetic studies. Since the 1990s in vitro tissue culture technologies have been developed for coffee, including methods to regenerate plants through somatic embryogenesis (Campos et al. 2017; Etienne et al. 2018). Both direct and indirect methods for somatic embryogenesis in coffee have been described (Quiroz-Figueroa et al. 2006; Murvanidze et al. 2021). However, to our knowledge so far, Arabica coffee single-cell micropropagation techniques have not been integrated with mutation induction techniques using gamma-ray irradiation.

Single cells or cell clusters derived from embryogenic callus or somatic cell suspensions are attractive targets for induced mutagenesis given that they are expected to (mostly) produce chimera-free, homo-histont plants, as opposed to mutagenesis of multicellular tissues such as seed which results in chimeras. Dissolving chimeras through successive cycles of selfing is possible in C. arabica as it is a self-compatible species, however this is a lengthy process given its long reproductive cycle. Here we present protocols to produce friable embryogenic callus, establish embryogenic cell suspensions and convert somatic embryos into plantlets using a RITA^® bioreactor in Arabica coffee var. Venecia. Induced mutagenesis of embryogenic cell suspensions using gamma-ray irradiation is also described.

2 Materials

2.1 Plant Material

• 1. Mature coffee plants of C. arabica var. Venecia (see Note 1).

2.2 Supplies, Reagents and Basic Equipment

• 1. Aluminum foil.

• 2. Beakers (100, 500, and 1000 ml).

• 3. Bottles (100, 500, and 1,000 ml).

• 4. Casein hydrolysate.

• 5. Culture tubes (30 ml).

• 6. Culture vessels for liquid media.

• 7. Distilled Water.

• 8. 70% Ethanol (v/v).

• 9. 40—mesh filter (Sigma-Aldrich^®).

• 10. Forceps.

• 11. Gelling agent (phytagel).

• 12. Glycine.

• 13. Magnet for stirring.

• 14. Microfuge tubes (e.g. Eppendorf).

• 15. Myo-inositol.

• 16. MS media (Murashige and Skoog media).

• 17. Petri dishes (60 × 15 mm; 100 × 20 mm).

• 18. Parafilm.

• 19. Pipettor.

• 20. Scissors.

• 21. Sieves.

• 22. Sterile distilled water.

• 23. Surgical blades.

• 24. Stirrer.

• 25. Thiamine.

2.3 Equipment

• 1. Analytical balance.

• 2. Autoclave.

• 3. Centrifuge.

• 4. Gammacell 220 irradiation unit (see Note 2).

• 5. In vitro growth room.

• 6. Laminar flow bench.

• 7. Orbital shaker.

• 8. pH meter.

• 9. RITA^® bioreactor.

• 10. Stereomicroscope.

2.4 Stock Solutions and Tissue Culture Media

• 1. 1-Naphthaleneacetic acid (NAA) 1 mM.

• 2. 2,4-Dichlorophenoxyacetic acid (2,4 D) 1 mM.

• 3. 6-(γ,γ-Dimethylallylamino)Purine (2iP) 1 mM.

• 4. 6-Benzylaminopurine (BAP) 1 mM.

• 5. Indole-3-butyric acid (IBA) 1 mM.

• 6. Sodium hydroxide 1 N.

• 7. Sodium hypochlorite solution 2% (v/v).

• 8. Semi-solid callus induction medium (C) (see Table 1).

  Table 1 Media composition (mg/l) for somatic embryogenesis and plantlet regeneration of Coffea arabica var. Venecia (van Boxtel and Berthouly, 1996)

• 9. Semi-solid embryogenic callus induction medium (E) (see Table 1).

• 10. Liquid proliferation medium, flasks (CP) (see Table 1).

• 11. Liquid regeneration media, RITA^® (R) (see Table 1).

• 12. Liquid development media, RITA^® (EG) (see Table 1).

3 Methods

The procedures described below utilize leaf discs as starting material to produce friable embryogenic callus on semi-solid medium. The friable embryogenic callus is then transferred to a liquid medium in Erlenmeyer flasks to establish a homogenous embryogenic cell (cluster) suspension culture. The cell suspension culture serves to multiply the embryogenic cells and cell clusters and is used for gamma-ray irradiation. Next, globular-stage somatic embryo cell cultures are transferred to a 1-L RITA^® bioreactor for further development of the somatic embryos and conversion to rooted plantlets. The different steps of the procedure are illustrated in Table 2 and Figs. 1, 2, 3.

Table 2 Steps in the in vitro regeneration and mutagenesis procedure with indicative timeline

Fig. 1

Somatic embryogenesis process in Coffea arabica from ex vitro leaf disks. a Leaf disk in callus induction medium. b Leaf disk with primary calli. c–d Close-up of friable embryogenic calli

Fig. 2

Preparation of embryogenic cells/cell clusters for irradiation treatment. a 40-Mesh filter. b Embryogenic cell suspension after sieving. c Cell suspension in microcentrifuge tubes ready for gamma-ray irradiation

Fig. 3

Coffea arabica somatic embryogenesis process. a Friable embryogenic callus indicated with the arrow. b SE in proliferation medium. c Embryogenic cells for radiation treatment. d Germination of somatic embryos in a RITA^® bioreactor. e–g Rooted plantlets

3.1 Media Preparation

• 1. Prepare the macro- and micro-nutrient stock solutions and iron stock solution according to Table 1 in advance and store at 4 °C (see Note 3).

• 2. Prepare the vitamin and growth regulator solutions according to Table 1 and keep in 15 ml plastic tubes and store at − 20 °C.

• 3. Autoclave the semi-solid and liquid media or filter sterilize (see Note 4).

• 4. For media preparations, start with less water of the required final volume in a flask or beaker and add the specified number of stocks, amino acids, sugar, and growth regulators.

• 5. Adjust pH to 5.6 and add distilled water to the required final volume.

3.2 Tissue Collection and Disinfection

• 1. Select well developed, healthy leaves from mature, greenhouse-grown plants.

• 2. In the lab or washroom, rinse the leaves with water and soap.

• 3. Perform all subsequent steps in a laminar flow cabinet; disinfect the cabinet with 70% ethanol prior to use.

• 4. In the laminar air flow cabinet place the leaves in a sterile beaker.

• 5. Disinfect the leaves with a sodium hypochlorite solution 2% (v/v) for 30 min.

• 6. Decant the solution, and rinse 3–4 times with sterile deionized water, each 5 min.

• 7. Transfer leaves to petri plates.

3.3 In Vitro Leaf Disc Preparation and Primary Callus Induction

• 1. Using a sterile cork-borer punch leaf discs measuring ca 5 mm diameter, avoiding main and secondary veins and leaf margins.

• 2. Place 6–7 leaf discs, upper surface down, in 60 × 15 mm petri plates containing callus induction medium (C); seal petri dish with parafilm or similar self-sealing plastic (Fig. 1a).

• 3. Place in culture room at 28 °C in complete darkness.

• 4. After 7–10 days callus starts to grow (Fig. 1b).

• 5. After 4–5 weeks leaf discs with primary calli can be moved to friable embryogenic callus induction medium.

3.4 Embryogenic Callus Induction

• 1. Subculture leaf disks from callus induction medium to friable embryogenic callus medium (E).

• 2. Seal petri dishes and place in dark at 28 °C.

• 3. Within 2–5 months, friable embryogenic callus (yellowish) develops on explants on E medium (Fig. 1c, d).

3.5 Cell Multiplication and Establishing the Embryogenic Cell Suspension

• 1. Isolate embryogenic calli and culture in liquid proliferation medium (CP) for multiplication purposes (Fig. 3b).

• 2. Culture about 10 mg calli per 1 ml CP medium in a 250 ml flask containing embryogenic callus medium (E).

• 3. Maintain flasks in an orbital shaker at 90–100 rpm at 28 °C in the dark.

• 4. Subculture every 3 weeks for 2–3 months.

3.6 Gamma-ray irradiation of the Embryogenic Cell Suspension

• 1. After 4 weeks of growth, pass the embryogenic suspension cultures through a 40—mesh filter (Sigma-Aldrich^®) (Fig. 2a, b).

• 2. Once the material is obtained, divide it into sterile 1.5 ml microcentrifuge tubes.

• 3. Add 0.5 ml of regeneration media (R).

• 4. Seal the microcentrifuge tubes with Parafilm, place inside a petri dish and seal the petri dish (Fig. 2c).

• 5. Transport sealed petri dishes for irradiation in the dark at room temperature and irradiate using a Gammacell 220 irradiaton unit (see Note 2).

3.7 Somatic Embryo Differentiation and Germination

• 1. Maintain flask of irradiated cells in the dark on a rotary shaker (100 rpm) at 26 ± 2 °C.

• 2. Embryos can be transferred to the RITA^® bioreactor after developing to globular stage (Fig. 3d).

3.8 Development of Somatic Embryos and Conversion to Plantlets

• 1. Place about 200 mg of the embryogenic aggregates in the RITA^® bioreactor along with 200 ml of the regeneration medium (R).

• 2. Subculture embryos once every 2 months for embryo development and germination under light conditions (12 h photoperiod, 50 μmol photons m⁻² s⁻¹) at 26 ± 2 °C.

• 3. Set the immersion frequency to 1 min every 12 h.

• 4. After 3–4-months cotyledonary shaped embryos will develop.

• 5. After 3 months, rooted plantlets will develop (Fig. 3e–g).

4 Notes

• 1. Well-developed leaves were harvested from mature, greenhouse-grown Coffea arabica var. Venecia plants, second node from the top.

• 2. The in vitro cultures were irradiated using a self-contained Gammacell 220 Cobalt-60 research irradiator at a dose rate of ~ 53.3 Gy/min. The following doses were applied: 0, 5, 10, 20, 40 and 80 Gy.

• 3. The MS micro and micro mineral solutions as well iron solutions were prepared separately as individual stock solutions and then combined to half- or full-strength MS (Murashige and Skoog 1962). The B5 vitamins were purchased as a commercial ready-for-use powder (Gamborg et al. 1968).

• 4. Check the liquid media for any contamination by keeping the freshly made liquid media at room temperature overnight and then store in the fridge.