Physical Mutagenesis of Arabica Coffee Seeds and Seedlings

Abstract

Coffee, a perennial tropical crop, can be grown from seed or from cloned plants in the form of cuttings, grafts or tissue cultured plants. Arabica coffee is most commonly grown from seeds while Canephora is mostly grown vegetatively from cuttings and other propagules. Improving Arabica coffee through conventional breeding is seriously limited by the lack of genetic variation within the cultivated and wild species. Mutation breeding provides great potential to induce the novel genetic variation needed for Arabica coffee improvement. Here we present protocols for mutation induction of coffee seed and seedlings using the FAO/IAEA in-house gamma (⁶⁰Cobalt) and X-ray (RS2400 irradiator) sources. Methods for mutation induction using gamma- and X-ray mutagenesis techniques are described. Methods for the preparation, seed quality control and post-radiation treatment of materials are also provided along with example data for radio-sensitivity testing of Coffea arabica seed under laboratory conditions.

1 Introduction

Coffee is a perennial crop belonging to the genus Coffea in the family Rubiacea. There are about 125 species within this genus, with Coffea arabica and Coffea canephora, representing approximately 70% and 30% of coffee production, respectively (Lashermes et al. 2008). Arabica coffee is a tetraploid and self-pollinating (autogamous), while Robusta coffee is a diploid and allogamous (Wintgens 2004). The efficiency of traditional Arabica coffee breeding approach is greatly reduced by the lack of sufficient genetic diversity and the long time needed for coffee to flower and bear fruits (Scalabrin et al. 2020). Mutation breeding provides great potential to induce the novel genetic variation needed for Arabica coffee improvement.

Coffee can be grown from seed or from cloned plants in the form of cuttings, grafts or tissue cultured plants. Arabica coffee is most commonly grown from seeds while Canephora is mostly grown vegetatively from cuttings and other propagules. Despite widely reported spontaneous mutants of Arabica coffee, there are very few studies on induced mutagenesis in coffee. The first attempt to induced mutation breeding of C. arabica was reported by Carvalho et al. (1954) using X-ray irradiation. The process of optimizing dose involves dose-response experiments where the pattern of reduction in germination (Lethal Dose, LD) or growth rate (Growth Reduction, GR) is determined in relation to an increase of absorbed dose. From these experiments, the LD/GR₃₀ and LD/GR₅₀ is calculated. In case of coffee after adjustment trials of seed and vegetative part using our in-house gamma and X-ray irradiators, we came to a range of (0, 50, 100, 150, 200, 400 Gy) for C. arabica seeds and (0, 5, 10, 15, 20, 30 and 40 Gy) for seedlings and cuttings of C. arabica and C. canephora. The protocol for seed treatment follows the general procedure which starts with sorting clean and viable seeds, moisture equilibration in a desiccator with 60% glycerol, irradiation treatments, planting the treated material in suitable set-up such as moist filter papers in petri-dish, soil in trays or pots and incubate at appropriate condition under warm condition 28–30 °C. Germination or growth rate after 30 days is recorded and plotted relative to the untreated seeds over the series of the doses. From the plotted graph the doses for LD₅₀, GR₅₀ and LD₃₀, GR₃₀ are estimated. The same follows for vegetative propagules (cuttings, seedling, embryo etc.) except that the applied doses here are relatively low in the order of 0–40 Gy. The estimated dose can be used for bulk treatment. Induced mutations are random events, implying that even adherence to published irradiation conditions might not result in the same mutation events. A way of overcoming this is to work with large populations. In case of seed, it is generally recommended to target the production of an M₂ population of a minimum of 5,000–10,000 individuals.

In this chapter, step-by-step procedures for seed quality control, irradiation treatment, and radiosensitivity testing of seed and seedlings is described. Example LD/GR values resulting from radiosensitivity testing of seed germination and growth rate under laboratory conditions are presented.

2 Materials

• 1. High quality, disease-free seeds, clean and uniform in size (Note 1).

• 2. Sterilized soil mixture.

• 3. Trays or pots of appropriate size.

• 4. Glasshouse facility.

• 5. Growth incubator with temperature control.

• 6. Gamma radiation source.

• 7. X-ray irradiator RS-2400.

• 8. Paper envelopes (air- and water-permeable).

• 9. Vacuum desiccator.

• 10. Petri dishes (90 mm diameter).

• 11. Whatman filter papers for 90 mm Petri dishes.

• 12. 60% Glycerol (v/v).

• 13. Sterile and non-sterile deionised water.

• 14. Parafilm.

3 Methods

3.1 Seed Treatment

3.1.1 Preparation and Quality Control of Seed

• (i) Use freshly harvested seeds as coffee seeds loose rapidly viability if not properly stored. Select seeds from genetically homogenous plants, after a few cycles of selfing from the targeted preferred variety. In case of freshly harvested cherries, de-pulp to remove the two seeds, separate seeds from mucilage, wash in running tap water and air dry at room temperature (under shade) for 3–7 days. Seeds are then ready for germination.

• (ii) Sort out clean seeds and remove small, shriveled, and damaged seeds from the starting seed lots.

• (iii) Perform viability test following appropriate germination test procedure; soak few seeds (50–60) from each genotype in warm water for 1–3 days and incubate on moist filter paper in petri dish (15–20 seeds/dish) or directly in light soil and keep under warm (27–29 °C) and moist condition until germination. Record germination based on visual scoring of full protrusion of the radicle and appearance of the coleoptile after about 2 weeks (Fig. 1). Proceed to mutation induction protocol with seeds having viability close to or above 90% germination.

  Fig. 1

  

  Viability test of the seed lot by germination test on moist filter paper in petri dish (a), viable seed lot is divided into number of samples equal to the planned irradiation treatment, seed sample for each treatment is placed in small paper bag and labelled (b), bags with similar treatment are grouped in a larger bag and placed in a desiccator with 60% glycerol for moisture equilibration (c), after moisture equilibration seeds are ready for irradiation (d)

• (iv) Divide the seed lot with high viability seeds (> 90%) based on the amount available and the planned set-up for radio-sensitivity test (incubator vs. soil) into 20–50 seeds per treatment and place in appropriate paper bag (Fig. 1).

• (v) Clearly label the bag with the genotype name, source of irradiation (Gamma vs. X-ray), treatment dose (0–400 Gy), replication number (R1–R3), date and the number of seeds (Fig. 1).

• (vi) Pack the prepared seed bags, in step 5 above, for different genotypes in joint groups based on the planned source of irradiation treatment (Gamma vs. X-ray), similarity in treatment dose and replications. In total we will have 6 groups of treatments multiplied by the number of replications (2–3) (Fig. 1).

• (vii) Place the prepared seeds in step 6 above in a desiccator with 60% glycerol for moisture equilibration (Fig. 1). Keep the seeds in the desiccator for time sufficient to equilibrate the moisture of the seeds to (12–14%). In cereals and legumes this takes around 3–7 days. Longer stay in the desiccator is not expected to affect the treatment (Note 2).

• (viii) Remove the seeds from the desiccator, vacuum pack (optional) and proceed to the irradiation facility for treatment. Up to this stage the seeds are known as M₀ seeds.

3.1.2 Gamma and X-ray Irradiation Treatment

The in-house Cobalt gamma-ray Cell and RAD-Source (RS 2400) was used for gamma-ray and X-ray irradiation treatment respectively, using previously described procedures (Spencer-Lopes et al. 2018). Briefly, the RS 2400 X-ray machine source is an upgraded irradiator for research and industry used by the FAO/IAEA Insect Pest Control Laboratory for the Sterile Insect Technique (Mastrangela et al. 2010; Mehta and Parker 2011) and adapted for mutation induction of plant propagules by the FAO/IAEA Plant Breeding and Genetics Laboratory. It is important in X-ray irradiation that samples are tightly packed to minimize air space and to maintain a near uniform field of X-rays through the entire sample. The dose rate of the RS2400 X-ray is about 12 Gy/min. After irradiation, the seeds and resulting plants are known as M₁ stage.

3.1.3 Post-treatment Handling and Radio-Sensitivity Testing

3.1.3.1 Viability and Growth Rate Testing of the M₁ Seeds in Petri Dish in an Incubator

• 1. Remove the paper bags containing the treated M₁ seeds from the packing set and rearrange them based on genotype, replication, and doses from 0 to 400 Gy.

• 2. Place the treated seeds in appropriate tube, add sufficient warm water (30–35°C), tightly close the tube, label for treatment, genotype, and replication, and leave in the incubator (27–29 °C) for 72 h for soaking of the M₁ seeds (Fig. 2).

  Fig. 2

  

  Post treatment handling and radiosensitivity testing of irradiated seed. Steps of water soaking of treated seeds at 28 °C and 12 h photoperiod in incubator for 3–7 days, transfer to either petri dishes or soil for monitoring and recording germination (viability) and growth responses (growth reduction) of the irradiated seeds

• 3. Remove the soaked M₁ seeds and place them on moist filter paper in petri dish. Label the dish as in the soaking step above.

• 4. Place the petri-dish with soaked M₁ seeds in the incubator under the same condition above with 12 h day length (light) and incubate for germination (Fig. 2).

• 5. Monitor the germinating M₁ seeds and ensure sufficient moisture by wetting the filter papers every 2 days or whenever needed.

• 6. Record germination and measure root and coleoptile length after 2 weeks (Fig. 2).

• 7. Calculate the germination percentage and reduction in growth relative to the growth of the seedling from the untreated seeds.

• 8. Plot the germination percentage and reduction in growth rate to estimate the optimum dose based on lethal dose for 30 and 50% of the seeds (LD₃₀ and LD₅₀, respectively) and growth reduction of 30 and 50% in the treated seeds compared to the control in root and coleoptile, and other vegetative parameters as appropriate (GR₃₀ and GR₅₀, respectively).

3.1.3.2 Viability and Growth Rate Testing of the M₁ Seeds in Soil in the Glasshouse

Repeat steps 1 and 2 as described above under Sect. 3.1.3. Then, follow below steps:

• 1. Prepare soil mix and distribute in medium size trays using the recommended soil type for coffee seeds germination: light soil containing sand, clay and peat moss with acidic pH (5–6).

• 2. Prepare necessary labels (plastic) for genotype, replication and for each treatment.

• 3. Plant seeds in rows each with about 20 seeds from each treatment. Assign each replication in a separate tray. Sufficiently water the seeds (Fig 2).

• 4. Monitor the germinating M₁ seeds and ensure sufficient moisture by wetting every 2 days or when needed.

• 5. Record germination and measure hypocotyl length on regular basis (Fig. 2).

• 6. Calculate germination percent and reduction in growth relative to the growth of the seedling from the untreated seeds.

• 7. Plot germination percentage and reduction in growth rate to estimate the optimum dose based on lethal dose for 30 and 50% of the seeds (LD₃₀ and LD₅₀, respectively) and growth reduction of 30 and 50% in the treated seeds compared to the control in root and coleoptile and other vegetative parameters as appropriate (GR₃₀ and GR₅₀, respectively).

3.2 Coffee Seedlings Treatment

• 1. Grow coffee seedlings up to 56 days after germination (DAG) (Fig. 3).

  Fig. 3

  

  Key steps in the process of seedlings irradiation starting with preparation of seedlings at 56 days after germination (DAG) (a), transfer to gamma irradiation facility for treatment (b) and transplanting in pots in a glasshouse under appropriate conditions. Seedling at 14 (c) and 28 days after irradiation treatment (DAT) are shown (d)

• 2. Tie seedlings with a rope or tape into a pack to fit in the irradiation container.

• 3. Place the seedling pack in the center of an appropriate container for X or gamma-ray such that the material is tightly packed with minimum air space in case of X-ray.

• 4. Take the treated seedling for planting in glasshouse or field (Note 3).

3.3 Example Radio-Sensitivity Testing of Arabica Coffee Seed Under Laboratory Conditions

Seed from three C. arabica varieties (Kent, Geisha and Mundo Novo) from the Cocoa Research Institute of Nigeria were freshly harvested and sent within less than one month to the FAO/IAEA PBG Laboratory, Austria. Upon arrival, the seeds were immediately inspected, cleaned, and used for gamma and X-ray radiosensitivity testing as described above. Germination was scored using the laboratory-based procedure about 2 weeks after treatment and data analysed to estimate LD₃₀ and LD₅₀ values as well as measurement of seedling growth rate relative to the untreated seeds (control) to estimate GR₃₀ and GR₅₀ values (Note 4). The viability of the seeds of the three coffee varieties exceeded 95% of germination on moist filter paper after soaking in warm water for 3 days.

As show in Fig. 4, the LD₃₀ ranged between 38 and 75 Gy among the three varieties for gamma treatment, compared to 16–52 for the X-ray, while the LD₅₀ ranged between 118 and 150 Gy for Gamma and between 105 and 136 using X-ray (Table 1).

Fig. 4

Radio-sensitivity testing based on germination percent of the M₁ seeds using gamma-ray (a) and X-ray (b) in-house irradiation sources in three C. arabica varieties. As shown, for gamma treatment, the LD₃₀ ranged between 38 and 75 Gy, compared to 16–52 Gy for the X-ray treatment, while the LD₅₀ ranges between 118 and 150 Gy for gamma, and between 105 and 136 Gy using X-ray

Table 1 Comparing LD₃₀ and LD₅₀ using the FAO/IAEA in-house gamma and X-ray sources and their relative biological effect (RBE) in three C. arabica coffee varieties

The relative biological effect (RBE) of gamma to X-ray ranged from 0.21 to 0.74 for LD₃₀ and from 0.7 to 0.95 for LD₅₀ indicating that Gamma and X-ray were relatively closer in their effect in LD₅₀ (Table 1).

4 Notes

• 1. When performing a radio-sensitivity test 15–20 seeds are placed in paper bag per replication (3 replications). In case of X-ray irradiation, bags of the same dose are rolled together with the seed well distributed at the bottom and then placed in the center of the container to ensure uniform radiation. The remaining space is filled with instant rice for vacuum establishment. For bulk irradiation large amounts of seeds are used and these may be placed in an appropriate container for uniform irradiation without the use of a filler.

• 2. Moisture content. The moisture content of the plant propagule to be irradiated is a critical factor. In barley, for instance, it has been shown that at seed moisture content below 14%, there is marked increase in mutation frequencies as the moisture content decreases. It is therefore necessary to equilibrate the moisture content of seed prior to irradiation.

• 3. For radio-sensitivity test 10–20 propagules per replication are sufficient (3 replications). For bulk irradiation use large population sizes. In case of seed, it is recommended to target the production of an M₂ population of a minimum of 5,000–10,000 individuals.

• 4. It is strongly recommended to conduct a radiosensitivity testing of coffee seed also under greenhouse conditions (e.g., in pots or trays with soil) and monitor survival or growth for longer periods, e.g., until the cotyledons or first true leaves have appeared, to mimic as closely as possible the greenhouse or field conditions where the mutant population(s) will eventually be sown. Given the long germination time of coffee seed, such radiosensitivity testing may take up to 1 to 3 months, depending on the seed quality and growth conditions.