Mutation Induction in Coffea arabica L. Using in Vivo Grafting and Cuttings

Abstract

Coffee leaf rust (CLR) caused by the obligate parasite, the biotrophic Hemileia vastatrix Berk. & Broome (Basidiomycetes: Pucciniales), is the most devastating disease of Coffea arabica L. Breeding resistant varieties is one of the most economic and environment friendly means to control the disease. However, this is challenged by the loss of resistance after a short period in commercial production. Catimor CIFC7963, an elite, leaf rust resistant Coffea arabica L. variety, has been cultivated in China for decades, which has resulted in the breakdown of its disease resistance. Due to the lengthy breeding process of coffee, the development of new resistant varieties is arduous. Physical and chemical mutagenesis offers an alternative means to more rapidly create novel and beneficial genetic variations. Bud grafting is a propagation technique frequently used for woody plants whereby a bud of one plant is attached to the rootstock of another plant. Likewise, cutting is a frequently used propagation technique. In coffee, physical irradiation of the bud followed by grafting or cutting can accelerate the mutation breeding process, as cutting or grafting increases the growth rate without affecting the major traits of the background varieties. Here, we present protocols to induce mutations on buds of the C. arabica variety Catimor CIFC7963 by gamma-ray irradiation and their subsequent propagation through cutting or bud grafting.

1 Introduction

Coffee is one of the most important beverages in the world. The genus Coffea has over 70 different species. Of these, only two species, namely Coffea arabica (Arabica) and C. canephora (Robusta) dominate global coffee production. C. arabica is a dominant species with a demand for genetic improvement, especially for disease resistance such as coffee leaf rust (CLR) (Tran et al. 2018). CLR caused by the fungus Hemileia vastatrix is one of the most important diseases of C. arabica. A few rust-resistant varieties have been developed, e.g. Oeiras MG 6851 (Pereira et al. 2000), however, the resistance was soon broken down by the newly emerged race XXXIII of H. vastatrix (Capucho et al. 2012). Thus, widening the genetic base would be key for developing sustainable disease resistance in coffee, given that Coffee-H. vastatrix rust interactions follow the gene-for-gene relationship (Flor 1942). Transcriptome and proteome methods have been used to identify leaf rust resistance genes (Guerra-Guimarães et al. 2015; Florez et al. 2017). While various H. vastatrix effector candidate genes (HvECs) have been reported, the knowledge of leaf rust resistance genes in coffee (S_H1-S_H9) is still limited (Maia et al. 2017). There is still insufficient knowledge to develop functional markers for marker-assisted selection for CLR resistance. Mutation breeding seems to be a promising approach for coffee improvement to help address the leaf rust resistance breakdown due to rapidly evolving H. vastatrix races.

Compared to conventional breeding techniques such as traditional crossing, induced mutagenesis can more efficiently generate novel variations and introduce new traits (Harten 1998). Gamma irradiation has proven to be effective in improving important agronomic traits such as yield, quality and disease resistance. The FAO/IAEA Joint Centre has significantly promoted the application of mutation breeding in agriculture, which generated more than 3400 varieties in 210 plant species and commercially planted in more than 70 countries (https://mvd.iaea.org/).

It takes over three years for C. arabica from planting until fruit production. This makes its propagation and breeding an extremely lengthy process. For tree crops, grafting offers an efficient way to propagate and maintain elite germplasm (Parlak 2017). Grafting technique has already been successfully applied in coffee for preventing damage by root-lesion nematodes (Villain et al. 2000). In addition, cutting is also widely used to propagate tree plants for maintaining outstanding traits. Cutting is also applied for clonal propagation of coffee plants, which is more stable than seed propagation for maintaining yield traits (Priyono et al. 2010). The survival rate of cutting is influenced by environmental factors such as exogenous phytohormones, humidity, temperature, etc. It results in a lower survival rate compared to grafting. On the other hand, cutting is a simpler method than grafting as it does not require a rootstock. Here, we present a protocol for gamma irradiation of coffee in combination with grafting and cutting techniques.

2 Materials

2.1 Grafting

• 1. Catimor CIFC7963 coffee plants.

• 2. Two-year-old Catimor CIFC7963 plant (rootstock).

• 3. Plastic strips.

• 4. Plastic label.

• 5. Grafting knife.

• 6. Tree pruner scissors.

• 7. Floral foam for flower arrangement.

• 8. Plastic storage box.

2.2 Cutting

• 9. Catimor CIFC7963 coffee plants.

• 10. Floral foam for flower arrangement.

• 11. Plastic bin.

• 12. River sand.

• 13. Flowerpot.

• 14. Difenoconazole (CAS: 119446-68-3).

• 15. 3000 mg/L of naphthaleneacetic acid (NAA) solution.

• 16. White plastic bag (keep moisture and transparency).

• 17. Newspaper.

• 18. Watering can.

• 19. Plastic greenhouses.

3 Methods

3.1 Mutagenesis and Propagation of Coffee Through Bud Grafting

3.1.1 Preparation of Straight Branches

• 1. Remove the lateral branches and leaves using tree pruner scissors, retain only the top leaves (see Note 1 and Fig. 1a).

  Fig. 1

  

  Grafting procedure. a Straight shoot; b the floral foam for arrangement of the coffee shoots; c irradiation; d preparation of the rootstock; e–g grafting; h successful graft

• 2. Cut the bottom of the straight branches into a tongue-shape and put them into the floral foam for flower arrangement (see Note 2 and Fig. 1b).

• 3. Transfer floral foam with the coffee shoots into plastic containers for gamma-ray irradiation using Cobalt-60 source (see Note 3).

3.1.2 Mutagenic Treatment

• 1. Take the straight branches out of the plastic container and divide into 6 sets, each containing 70 shoots, for irradiation using different dosages.

• 2. Perform mutagenic treatment using 0, 5, 10, 15, 20, and 25 Gy of gamma rays at the rate of 1 Gy/min (Fig. 1c).

• 3. Transfer the treated shoots into a greenhouse for grafting.

3.1.3 Preparation of the Rootstock

• 1. Remove the trunk of a 2-year-old Catimor CIFC7963 with only 10–15 cm stump retained (Fig. 1d).

• 2. Use a grafting knife to cut an incision on one side of the rootstock up to the xylem with a length of about 3 cm (Fig. 1e).

3.1.4 Preparation of the Scion

• 1. Take the irradiated shoots, retain the green unpinched cork with 1–2 buds on the top by cutting off the lower part.

• 2. Cut the scion with a grafting knife by two chops. The length of the two cutting faces should be about 3 cm, each side should be uneven with the other side in length as well as thickness. The width of the cutting side should be slightly narrower or equal to the diameter of the rootstock (Fig. 1f).

3.1.5 Grafting Method

• 1. Once the scion and rootstock have been cut, insert the graft in the rootstock, ensuring that the graft union is well aligned.

• 2. Immediately and tightly wrap the joint of the scion and stock with plastic strips (see Note 4 and Fig. 1g).

3.1.6 Post-grafting Management

• 1. Grow the grafted plants in a plastic greenhouse with temperature 20–25 °C and natural light.

• 2. Keep the rootstock clean by removing any emerging buds and shoots (see Note 5).

3.1.7 Survival Rate Statistics

• 1. One to two months after grafting, the successful graft is identified by the presence of emerged leaves on the scion and healing of the contact point between the scion and rootstock (see Note 6 and Fig. 1h).

3.1.8 Unbundling

• 1. Remove the wrapping tape when the scion tip is over 10 cm long and the interface is completely healed.

3.2 Mutagenesis and Propagation of Coffee Through Cuttings

3.2.1 Preparation of Straight Branches

• 1. Remove the lateral branches and leaves using tree pruners, retain only the top leaves on the shoots (see Note 1 and Fig. 2a).

  Fig. 2

  

  Cutting procedures. a Straight shoot; b arrange coffee shoots in the floral foam; c irradiation; d 0.1% difenoconazole sterilizing solution; e NAA solution treatment; f stick cutting into the most rooting substrate; g–h set up wire support and cover with plastic bag to maintain moisture and humidity; i shading; j established cutting

• 2. Cut the bottom of the straight branches into a tongue-shape and arrange the cuttings in the floral foam (see Note 2 and Fig. 2b).

• 3. Transfer the floral foam with the coffee cuttings into plastic containers for gamma-ray irradiation using a Cobalt-60 source (see Note 3).

3.2.2 Mutagenesis

• 1. Take the straight branches out of the plastic container and divide into six sets, each containing 70 shoots, for irradiation using different dosages.

• 2. Perform the mutagenic treatment using 0, 5, 10, 15, 20, and 25 Gy of gamma rays at the rate of 1 Gy/min (Fig. 2c).

• 3. Transfer the treated shoots to a greenhouse for cutting (see Note 4).

3.2.3 Preparation of the Cutting Substrate

• 1. Fill pots with river sand until it is 5–10 cm away from the top of the pot.

• 2. Level the sand bed and keep moist.

• 3. Spray 0.1% difenoconazole solution to sterilize the sand bed.

• 4. Perforate the sand bed at 10 cm ± 2 cm distance, and a depth of about 20 cm using bamboo skewers slightly thicker than the cutting strip.

3.2.4 Sterilization

• 1. Remove the irradiated shoots from the floral foam and soak them in 0.1% difenoconazole solution for 5–10 min (Fig. 2d).

• 2. Remove the corky part on the bottom of the straight branch and retain the upper, green part with 2–3 buds, i.e. the scion.

3.2.5 Cultivation

• 1. Cut the end of the cuttings at an angle of about 45° and dip in a solution of 3,000 mg/L NAA for 3–6 s (Fig. 2e).

• 2. Insert the cuttings vertically into prepared holes in the sand bed at a depth of 15–20 cm (Fig. 2f).

• 3. Wet the sand bed. Build a mini greenhouse environment with a height of 50–60 cm by using two wire brackets and plastic bags to maintain humidity and moisture (Fig. 2g, h).

• 4. Keep the cuttings in the dark for 2 days (Fig. 2i).

• 5. Maintain the temperature at 20–25 °C and observe the growth of the cuttings regularly.

• 6. Water every 3–5 days, and tightly wrap the plastic bags to maintain humidity and moisture.

• 7. The culture of the cuttings takes around 2–4 months.

• 9. Spray 0.1% difenoconazole for sterilization every 20–25 days.

• 10. Immediately remove any necrotic or dead cuttings.

3.2.6 Survival Rate Statistics

• 1. After 6 months, successful cuttings are identified by the newly emerging leaves and the presence of fibrous roots (Fig. 2j).

4 Notes

• 1. The shoots of Catimor CIFC7963 were collected from the Coffee Germplasm Resource Garden in Ruili, Ministry of Agriculture and Rural Areas. Coffee shoots containing 5–6 buds were used in this protocol.

• 2. Collect only straight branches, as side branches are not able to develop into upright trees.

• 3. In cases of long-distance transportation, try to avoid cold stress on the cuttings.

• 4. To ensure high survival rate, optimal timing for coffee grafting or cutting should be set during spring season, between March and April, when the coffee plants are not flowering, or in the fall, September to October, prior to the fruit harvest.

• 5. To ensure that the new leaves originate from the scions, newly emerging buds should be regularly removed from the rootstock once the grafting process is completed.

• 6. Dry scions or scions that do not grow or drop are typical features of failed grafting.