Key words

1 Introduction

Somatic embryogenesis (SE) has been widely studied on T. cacao for 20 years in NR-Plant Science-Tours [1,2,3,4,5]. Our method is inspired from a protocol developed by a Penn State University team [6, 7] that obtained few primary somatic embryos using staminods or petals from unopened flower buds (Direct SE). Because of the low efficiency of the direct SE, indirect SE is initiated using the primary embryos as explants to produce embryogenic calli [3, 7]. Calli cultures are then maintained for several months and differentiated into secondary somatic embryos. This technic, developed in a liquid medium, allows us to do mass propagation in the frame of the Nestlé Cocoa Plan [3, 8]. The main issues for this method are the very strong genetic effect on SE induction treatments, the limited embryo-to-plant conversion rate, and the environmental factors impacting the mother tree. In the progress made during our previous T. cacao projects, about 80% of T. cacao cultivars respond to somatic embryogenesis and hence it can be propagated by this method. The embryo-to-plant conversion rate reached 20 to 40% with a very strong clone dependency and high batch-to-batch variability (unpublished work). Moreover, contaminations occur more frequently during the rainy season and flowers reactivity depends on the seasons, the flowering cycle, and the freshness of the buds (unpublished work). On T. cacao, other explants were evaluated to induce SE as cotyledons from immature zygotic embryos with low efficiency for clonal propagation [9, 10]. An efficient protocol was developed on three elite cultivars starting from leaves of germinated zygotic embryos cultured in vitro to insure juvenile and aseptic material [11]. Litz was able to induce SE from T. cacao leaf explants but did not report plant regeneration [12] as well our work did not provide results on such explants (unpublished work).

In this chapter a protocol of direct SE followed by indirect SE is described using solid and liquid medium, respectively. Several steps are necessary in this process: (1) SE Induction (direct and indirect); (2) SE secondary calli multiplication and embryo expression; (3) Embryo conversion to plant as embryo maturation and germination; and (4) plantlet acclimatization.

2 Material

2.1 Instrumentation, Glassware, and Other Materials

Flow cabinet, 250 °C Glass beads sterilizer, scalpels blades and handle, micro and macro surgical forceps, food plastic wrap cut at 3 cm width, orbital shaker, autoclave, binocular, ultra-pure and reversed osmosis water.

Borosilicate Erlenmeyer flasks of 25 ml, 50 ml, 100 ml, 250 ml and 1000 ml, cellulose stoppers adapted to the size of the flasks (sterilized by autoclaving at 121 °C for 30 min), 55 and 90 mm Petri dishes, 50 μm filters for liquid medium culture, 500 ml culture boxes: ref.017002 (container) and ref.017002 (cover), (Dominique Dutscher S.A, Issy-les-Moulineaux, France).

LED tubes (Green Power Deep Led/White/Medium blue, 18 W, 24 μmol/s, Philips Lighting, Zurich, Switzerland).

2.2 Reagents, Solutions, and Culture Media

Disinfection solution: A 12 g/l active sodium hypochlorite solution prepared with a commercial bleach or sodium hypochlorite.

2.3 Culture Media

All the chemicals are from Duchefa®, Haarlem, The Netherlands and Sigma-Aldrich, Saint-Louis, Missouri, USA.

Macronutrients and micronutrients used for culture media are DKW [13], WPM [14], and MS [15] with some modifications according to the supplier.

PCG, SCG, CC2, CC21, G800, and ENR8 culture media described in Table 1.

Table 1 Culture media composition (PCG: Primary callus growth and SCG: Secondary callus growth, CC21, CC2, G800, and ENR8 are internal codifications)
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Ready-to-use mixes of macronutrients, micronutrients, and vitamins order to Duchefa® are used to prepare PCG, CC21, CC2, G800, and Enr8 culture media. The reference M0219 (WPM) from Duchefa® is used for SGC culture medium.

2.4 Greenhouses

Tunnels equipped with an aquanappe (350 g/m2 and a retention capacity of 4.5 l/m2, Caahmro, Saint-Cyr-sur-Loire, France) under a micro perforated black film (ref.1626998BX Puteaux SA, Villefranche sur Saone, France) and covered by a transparent plastic sheeting (100 μM ref.063851D, Caahmro, Saint-Cyr-sur-Loire, France).

Preforma plugs (M 66 VECO1, Jiffy Products International BV, Zwijndrecht, Netherlands) to subcultured plants.

Artificial light: HPS SHP-TS 400W Gro-lux claire (E40.0020807, Gro-Lux®, Feilo Sylvania, Gennevilliers, France.

Nutritive solution: 1 g/l [Nitrogen:5%/Potassium:11%/Phosphore:26%].

2.5 Plant Material

Cocoa unopened floral buds proceed from (see Note 1):

  1. 1.

    Producing countries where they are collected directly from fields. Be sure to obtain all the phytosanitary documents and insure alignment with local procedures.

  2. 2.

    Greenhouses.

3 Method

3.1 Culture Media

  1. 1.

    Prepare the culture media according to quantities given in Table 1.

  2. 2.

    Adjust the pH.

  3. 3.

    Add the gelling agent and/or active charcoal.

  4. 4.

    Sterilize by autoclaving at 121 °C for 20 min.

  5. 5.

    For solid culture media: Pour hot culture media into Petri dishes or plastic boxes under sterile conditions in a flow cabinet. Let it cool and remove condensation in the laminar flow, then close the Petri dishes with plastic food wrap.

  6. 6.

    Store culture media at room temperature in a dark clean area.

3.2 Culture Conditions

Temperature is maintained at about 25 °C in all culture rooms.

Light condition in culture room is 65 μmol/m2/s with two LED tubes with a photoperiod of 16 h light/8 h dark.

Petri dishes for solid culture medium are sealed with two layers of plastic food wrap.

Liquid culture rotations are insured at 120 rpm on an orbital shaker.

Greenhouses: At least 12 h light per day (natural light supplemented with). Temperature should be maintained between 25 and 30 °C with 80 to 100% relative humidity.

3.3 Direct Somatic Embryogenesis

3.3.1 Bud Disinfection

  1. 1.

    Remove bugs, dirties, opened and damaged buds (Fig. 1a).

  2. 2.

    Put unopened floral buds in the half full tea balls.

  3. 3.

    Pour the disinfecting solution in a sterile recipient.

  4. 4.

    Completely immerge the tea ball in the disinfecting solution for 7–11 min shaking occasionally (see Note 2).

  5. 5.

    Rinse three times the buds in sterile water for 1 min (Fig. 1b).

  6. 6.

    Place the buds in a Petri dish (Fig. 1c).

Fig. 1
figure 1

(a) Buds collected in greenhouse before disinfection. (b) Buds in a tea ball during rinsing. (c) Disinfected buds.

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3.3.2 Somatic Embryogenesis Induction

  1. 1.

    Staminodes and petal bases (cucullate bases) are used as explants for SE induction (Fig. 1a).

  2. 2.

    Open the buds cutting at the larger part of the bud base (Fig. 2b, c) using a sterile scalpel and take one by one the staminods and petals using thin forceps. Place the explants on 55 mm Petri dishes containing PCG culture medium, in the dark at 25 °C (Fig. 2d, see Note 3).

  3. 3.

    After few days, check the contamination.

  4. 4.

    After two weeks of culture on PCG culture medium, transfer the explants to SCG culture medium (55 mm Petri dishes) and place in the same culture conditions (see Note 4).

  5. 5.

    After two weeks on SCG medium, transfer the explants to CC2 culture medium (55 mm Petri dishes). Reactive explants display morphological changes due to cellular proliferation (callogenesis), whereas non-reactive floral pieces remain unchanged (Fig. 2e).

  6. 6.

    Between four to eight weeks after the last transfer on CC2 medium, the explants are observed. On reactive explants, roots (Fig. 3a) and embryos can develop (Fig. 3b). Primary embryos at torpedo stage are collected (Fig. 3b).

Fig. 2
figure 2

(a) Cocoa flower anatomy (Staminodes (S) and petal bases (P) are selected and subcultured). (b) Unopened bud before dissection. (c) Dissected buds (cross section) before opening to collect the staminods and petals. (d) Petal bases (on the left) and staminods (on the right) placed on 55 mm Petri dishes containing PCG culture medium. (e) Staminodes and petals 4 weeks after induction. NRS non-reactive staminods, RS Reactive staminods, (NRP: non-reactive petals. RP Reactive petals)

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Fig. 3
figure 3

At least 12 weeks after induction. (a) Reactive staminods with roots formation. (b) Reactive staminods with embryo formation

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3.4 Indirect Somatic Embryogenesis

3.4.1 Onto Solid Culture Medium

3.4.1.1 Embryogenic Calli Production
  1. 1.

    Take the primary embryos and place the pieces on CC21 culture medium in 55 mm Petri dishes, in the dark at 25 °C.

  2. 2.

    CC21 culture medium is renewed every 5 weeks for at least 5 cycles without selection. Depending on the clone reactivity, cycles can be adapted from 5 to 8 weeks.

  3. 3.

    After 5 cycles, different types of calli have developed cut embryos (see Note 5). Only embryogenic calli are selected using a binocular and subcultured on CC21 culture medium every 5 to 8 weeks (Fig. 4a, b). Depending on quantities obtained, 90 mm Petri dishes can be used.

Fig. 4
figure 4

(a) Clusters of calli onto solid culture medium. (b) Embryogenic calli. (c) Secondary somatic embryos

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3.4.1.2 Embryo Expression
  1. 1.

    Transfer embryogenic calli onto 90 mm Petri dishes containing CC2 culture medium subculturing 8 clusters of about 1 cm diameter (Fig. 4a, b).

  2. 2.

    Renew culture medium every 5 to 8 weeks on new petri dishes containing CC2 culture medium until embryo expression (Fig. 4c).

3.4.2 Into Liquid Culture Medium

3.4.2.1 Embryogenic Calli Production
  1. 1.

    Select and suspend 0.05 to 0.10 g of fresh embryogenic calli into 10 ml of liquid multiplication medium (CC21) in 25-ml Erlenmeyer flasks.

  2. 2.

    Renew the medium every 3 weeks by doubling the volume of the suspensions: 20 ml in 50-ml flasks, then 50 ml in 100-ml flasks, and 100 ml in 250-ml flasks.

  3. 3.

    After 12 weeks of culture in liquid medium, collect the biomass by filtration through a 50-μm nylon mesh (Fig. 5). The calluses are selected and transferred into 100 ml of CC21 culture medium with a density of 20 g/l.

  4. 4.

    Thereafter, the embryogenic cell lines are subcultured every 3 weeks into 250-ml flasks by transferring 2 g of calli into 100 ml of fresh CC21 culture medium. The suspensions are cultured on an orbital shaker at 120 rpm in the dark (see Note 6).

Fig. 5
figure 5

Glass filter holder for plant material in liquid medium. Filter is a nylon mesh with a 50 μm porosity

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3.4.2.2 Embryo Expression
  1. 1.

    By filtration (0.50 μm) remove CC21 culture medium.

  2. 2.

    Collect 0.2 g of embryogenic calli and subculture in 100 ml CC2 liquid culture medium in a 250-ml flask.

  3. 3.

    Renew culture media every 3 weeks until expression of secondary somatic embryos.

3.5 Embryo-to-Plant Conversion

At this stage, the process is achieved on a solid medium.

3.5.1 Embryo Development

  1. 1.

    Collect embryos at torpedo or cotyledonary stage from solid medium or by filtration in case of liquid culture and transplant them individually onto 90 mm Petri dishes (30 to 40 embryos per Petri dishes) (Fig. 6a) containing maturation culture medium (G800). Embryos are grown in the dark at 25 °C.

  2. 2.

    After four weeks, transfer the embryos to boxes containing rooting medium (20 to 30 embryos) (Fig. 6b, c). Place the boxes under artificial light with a photoperiod of 16 h light/8 h dark) at 25 °C.

  3. 3.

    After 6 to 8 weeks depending on the growth, select the embryos which have developed a root system, at least a 2-cm stem height and one pair of leaves with a minimum length of 1 cm (Fig. 6d).

  4. 4.

    Leave the smaller plantlets in boxes for a farther 2–3 weeks period of growth.

Fig. 6
figure 6

(a) Secondary embryos on maturation medium; (b and c). Plantlets 6 to 8 weeks after transfer in boxes containing rooting culture medium; (d) Plantlet ready to be acclimatized

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3.5.2 Plantlet Acclimatization

  1. 1.

    The day before acclimatization, soak the substrate plugs thoroughly and place under the tunnel at 80 to 100% humidity.

  2. 2.

    The day of acclimatization, place and wash the selected plantlets for a few +onds in a nutritive solution (see Note 7) in order to prepare the plant for acclimatization and to remove the gelling agent from roots.

  3. 3.

    Put the plantlets in the plugs, ensuring that all the plant root is in contact with the substrate.

  4. 4.

    Soak the plants thoroughly and close the tunnel (Fig. 7a).

  5. 5.

    Monitor the growth and the plug humidity for two months and add nutritive solution every two weeks.

  6. 6.

    After two months, open the tunnel progressively for one month (Fig. 7b), so three months after the acclimatization, plastics can be totally removed (Fig. 7c). Humidity must be maintained at a minimum of 80%.

Fig. 7
figure 7

(a) Closed tunnel the first two months after acclimatization. (b) Tunnel is opened progressively during the third month after acclimatization (hardening off). (c) Acclimatized plants, the tunnel is removed

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3.6 Process Efficiency

The timeframe to obtain an acclimatized plant starting from unopened buds flower could be between one to two years (Fig. 8). In 170 clones evaluated by our team, around 80% responded positively to SE and 95% of the clones which produced primary somatic embryos produced secondary somatic embryos. The use of solid or liquid culture medium depends on the number of plants to produce. Liquid medium is the most adapted for mass propagation. From our experiment it can be inferred that 9000 plants could be produced by one person per year using solid culture medium and 20,000 plants using liquid culture medium. Multiplication rate of embryogenic calli is about 1.5 to 2 (unpublished work) and the most efficient cell lines can produce between 1500 to 2000 embryos per gram of embryogenic calli [3]. Once embryos obtained, embryo conversion to plant is about 20% to 40% with a large batch-to-batch variability.

Fig. 8
figure 8

Process efficiency and timing. (w weeks, SEmb somatic embryos)

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4 Notes

  1. 1.

    The delay between the bud collection and the somatic embryogenesis induction should be no more than five days. After this time, buds open during travel and reactivity to SE is lower.

  2. 2.

    Buds collected in greenhouses should be disinfected for no less than 7 min. Buds from producing countries which come directly from the fields have to be disinfected for 11 min. To have a good reactivity, SE has to be induced the day of disinfection.

  3. 3.

    It is recommended to separate petals from staminods because reactivity could be different depending on the clones. For easier dissection, use of glass petri dishes is the most adapted because of the resistance to the scalpel cuts.

  4. 4.

    To limit the contaminations, forceps must be changed and placed in the glass beads sterilizer every five buds or less and petals are separated from the staminods. In case of contamination from bacteria, explants can be saved under vertical flow cabinet. In case of fungi development, explants must be eliminated.

  5. 5.

    T. cacao calli are generally friable, brown-black structures. Embryogenic calli with a big multiplication rate are made of little grains attached to firm brown-black calli. The grains are firm and occasionally slightly colored with a red pigmentation depending on the clone. Embryogenic calli can be obtained within 2 to 12 months depending on the clone reactivity. In order that each subculture can multiply embryogenic calli, embryos at the torpedo and cotyledonary stages are rejected. Also eliminated are the watery, spongy and soft tissues with light beige, white, or brown colors.

  6. 6.

    After 18 to 24 months of multiplication cycles, cell lines should be eliminated to limit the risk of somaclonal variations due to plant growth regulators in culture media.

  7. 7.

    The proportion of nutritive solution can be modified although it is important not to have too much nitrogen which is a root formation inhibitor.