Chapter 6

Theobroma cacao: Somatic Embryogenesis

Caroline Guillou and Dorothée Verdier

Abstract
A two-step process combining direct and indirect somatic embryogenesis, on solid and liquid medium,
respectively is described for Theobroma cacao L. Staminodes and petals from unopened bud flowers are used
to induce primary direct embryos. Then, these primary embryos are cut to produce embryogenic calli which
will develop secondary embryos. This step of indirect SE allows us to produce large quantities of embryos
and to do mass propagation using liquid culture medium. Despite a very strong clone dependency and high
batch-to-batch variability, about 80% of T. cacao cultivars respond to somatic embryogenesis and can be
propagated by this method.

Key words Theobroma cacao, Somatic embryogenesis, Direct SE, Indirect SE, Solid culture medium,
Liquid culture medium

1 Introduction

Somatic embryogenesis (SE) has been widely studied on T. cacao

for 20 years in NR-Plant Science-Tours [1–5]. 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 treat-
ments, the limited embryo-to-plant conversion rate, and the envi-
ronmental factors impacting the mother tree. In the progress made
during our previous T. cacao projects, about 80% of T. cacao culti-
vars respond to somatic embryogenesis and hence it can be propa-
gated by this method. The embryo-to-plant conversion rate
reached 20 to 40% with a very strong clone dependency and high

Marco A. Ramı́rez-Mosqueda (ed.), Somatic Embryogenesis: Methods and Protocols,

Methods in Molecular Biology, vol. 2527, https://doi.org/10.1007/978-1-0716-2485-2_6,
© The Author(s) 2022

69
70 Caroline Guillou and Dorothée Verdier

batch-to-batch variability (unpublished work). Moreover, contam-

inations 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 germi-
nation; and (4) plantlet acclimatization.

2 Material

2.1 Instrumentation, Flow cabinet, 250
C Glass beads sterilizer, scalpels blades and
Glassware, and Other handle, micro and macro surgical forceps, food plastic wrap cut at
Materials 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), (Domi-
nique 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, Disinfection solution: A 12 g/l active sodium hypochlorite solution

Solutions, and Culture prepared with a commercial bleach or sodium hypochlorite.
Media

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.
 Theobroma cacao: Somatic Embryogenesis 71

Table 1
Culture media composition (PCG: Primary callus growth and SCG: Secondary callus growth, CC21,
CC2, G800, and ENR8 are internal codifications)

Induction
Multiplication Expression Maturation Germination
Culture media name PCG SCG CC21 CC2 G800 ENR8
Macronutrients (mg/l) DKW WPM MS MS MS/2 MS
CaCl2·2H2O 112.500 72.500 440.000 440.000 220.000 440.000
Ca(NO3)2·4H2O 1664.640 471.260
KH2PO4 265.000 170.000 170.000 170.000 85.000 170.000
K2SO4 1559.000 990.000
KNO3 1900.000 1900.000 950.000 1900.000
MgSO4·7H2O 361.490 180.540 370.000 370.000 185.000 370.000
NH4NO3 1416.000 400.000 1650.000 1650.000 825.000 1650.000
Micronutrients (mg/l) DKW WPM DKW DKW MS DKW
CoCl2·6H2O 0.025
CuSO4·5H2O 0.250 0.250 0.250 0.250 0.025 0.250
Fe-Na-EDTA 44.630 36.700 44.630 44.630 44.630
Na2-EDTA·2H2O 37.300
FeSO4·7H2O 27.800
H3BO3 4.800 6.200 4.800 4.800 6.200 4.800
MnSO4·1H2O 33.800 22.300 33.800 33.800 16.900 33.800
Na2MoO4·2H2O 0.390 0.250 0.390 0.390 0.250 0.390
ZnSO4·7H2O 17.000 8.600 17.000 17.000 10.600 17.000
KI 0.830
Vitamins (mg/l)
Myo-inositol 200.0 100.0 100.0 100.0 100.0 100.0
Nicotinic acid 1.0 1.0 1.0 1.0 0.5 1.0
Thiamine 2.0 10.0 2.0 2.0 0.7 2.0
Pyridoxine-HCl 1.0 0.5
Amino acids (mg/l)
Glycine 2.0 2.0 2.0 2.0 2.0 2.0
L-Lysine 0.4 0.4 0.4 0.4
L-Leucine 0.4 0.4 0.4 0.4
L-Arginine 0.4 0.4 0.4 0.4
L-Tryptophan 0.2 0.2 0.2 0.2
Glutamine 250.0
Plant growth regulators (mg/l)
2,4,5-Trichloro 1.000
phenoxyacetic acid
2,4- 2.000 2.000
Dichlorophenoxyactic
acid
Kinetin 0.250
Thidiazuron 0.005
Adenine-H2SO4 0.250 0.025
Coconut milk (mg/l) 50.0

(continued)
72 Caroline Guillou and Dorothée Verdier

Table 1
(continued)

Induction
Multiplication Expression Maturation Germination
Culture media name PCG SCG CC21 CC2 G800 ENR8
Other (g/l)
Activated charcoal 1
Glucose 20 20 30 30 40 30
Gelrite™ 3 3 3 3
pH 5.6 5.6 5.6 5.6 5.8 5.8

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

3 Method

3.1 Culture Media 1. Prepare the culture media according to quantities given in
Table 1.
2. Adjust the pH.
3. Add the gelling agent and/or active charcoal.
4. Sterilize by autoclaving at 121
C for 20 min.
 Theobroma cacao: Somatic Embryogenesis 73

5. For solid culture media: Pour hot culture media into Petri
dishes or plastic boxes under sterile conditions in a flow cabi-
net. Let it cool and remove condensation in the laminar flow,
then close the Petri dishes with plastic food wrap.
6. Store culture media at room temperature in a dark clean area.

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

Conditions 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 supple-
mented with). Temperature should be maintained between 25 and
30
C with 80 to 100% relative humidity.

3.3 Direct Somatic 1. Remove bugs, dirties, opened and damaged buds (Fig. 1a).
Embryogenesis 2. Put unopened floral buds in the half full tea balls.
3.3.1 Bud Disinfection 3. Pour the disinfecting solution in a sterile recipient.
4. Completely immerge the tea ball in the disinfecting solution for
7–11 min shaking occasionally (see Note 2).
5. Rinse three times the buds in sterile water for 1 min (Fig. 1b).
6. Place the buds in a Petri dish (Fig. 1c).

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

Fig. 1 (a) Buds collected in greenhouse before disinfection. (b) Buds in a tea ball during rinsing. (c)
Disinfected buds.
74 Caroline Guillou and Dorothée Verdier

Fig. 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)

55 mm Petri dishes containing PCG culture medium, in the

dark at 25
C (Fig. 2d, see Note 3).
3. After few days, check the contamination.
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. After two weeks on SCG medium, transfer the explants to CC2
culture medium (55 mm Petri dishes). Reactive explants dis-
play morphological changes due to cellular proliferation (callo-
genesis), whereas non-reactive floral pieces remain unchanged
(Fig. 2e).
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).
 Theobroma cacao: Somatic Embryogenesis 75

Fig. 3 At least 12 weeks after induction. (a) Reactive staminods with roots formation. (b) Reactive staminods
with embryo formation

3.4 Indirect Somatic 1. Take the primary embryos and place the pieces on CC21
Embryogenesis culture medium in 55 mm Petri dishes, in the dark at 25
C.
3.4.1 Onto Solid Culture 2. CC21 culture medium is renewed every 5 weeks for at least
Medium 5 cycles without selection. Depending on the clone reactivity,
cycles can be adapted from 5 to 8 weeks.
Embryogenic Calli 3. After 5 cycles, different types of calli have developed cut
Production 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.

Embryo Expression 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. 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 1. Select and suspend 0.05 to 0.10 g of fresh embryogenic calli
Medium into 10 ml of liquid multiplication medium (CC21) in 25-ml
Erlenmeyer flasks.
Embryogenic Calli 2. Renew the medium every 3 weeks by doubling the volume of
Production the suspensions: 20 ml in 50-ml flasks, then 50 ml in 100-ml
flasks, and 100 ml in 250-ml flasks.
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. 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).
76 Caroline Guillou and Dorothée Verdier

Fig. 4 (a) Clusters of calli onto solid culture medium. (b) Embryogenic calli. (c) Secondary somatic embryos

Fig. 5 Glass filter holder for plant material in liquid medium. Filter is a nylon
mesh with a 50 μm porosity
 Theobroma cacao: Somatic Embryogenesis 77

Embryo Expression 1. By filtration (0.50 μm) remove CC21 culture medium.

2. Collect 0.2 g of embryogenic calli and subculture in 100 ml
CC2 liquid culture medium in a 250-ml flask.
3. Renew culture media every 3 weeks until expression of second-
ary somatic embryos.

3.5 Embryo-to-Plant At this stage, the process is achieved on a solid medium.

Conversion

3.5.1 Embryo 1. Collect embryos at torpedo or cotyledonary stage from solid

Development 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. 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.

Fig. 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
78 Caroline Guillou and Dorothée Verdier

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

3.5.2 Plantlet 1. The day before acclimatization, soak the substrate plugs thor-
Acclimatization oughly and place under the tunnel at 80 to 100% humidity.
2. The day of acclimatization, place and wash the selected plant-
lets 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. Put the plantlets in the plugs, ensuring that all the plant root is
in contact with the substrate.
4. Soak the plants thoroughly and close the tunnel (Fig. 7a).
5. Monitor the growth and the plug humidity for two months
and add nutritive solution every two weeks.
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%.

3.6 Process The timeframe to obtain an acclimatized plant starting from

Efficiency unopened buds flower could be between one to two years
(Fig. 8). In 170 clones evaluated by our team, around 80%

Fig. 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
 Theobroma cacao: Somatic Embryogenesis 79

Fig. 8 Process efficiency and timing. (w weeks, SEmb somatic embryos)

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. Multipli-
cation 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.

4 Notes

1. The delay between the bud collection and the somatic embryo-
genesis induction should be no more than five days. After this
time, buds open during travel and reactivity to SE is lower.
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. 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. 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 contamina-
tion from bacteria, explants can be saved under vertical flow
cabinet. In case of fungi development, explants must be
eliminated.
80 Caroline Guillou and Dorothée Verdier

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

Acknowledgments

A particular thank you to Audrey Fillodeau, David Breton, Nathaly

Vial, Jessica Logan, Rafael Chan, and Fabrizio Arigoni for their
input. Also, a big thank you to everyone in the team for the work
done to improve the Cocoa Se process over the past fifteen years.

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