Scientia Horticulturae 229 (2018) 148–156

Contents lists available at ScienceDirect

Scientia Horticulturae
journal homepage: www.elsevier.com/locate/scihorti

Research Paper

Evaluation of the potential of regeneration of different Colombian and MARK

commercial genotypes of cocoa (Theobroma cacao L.) via somatic
embryogenesis
⁎
Ana María Henao Ramíreza, , Tatiana de la Hoz Vasqueza, Tatiana Marcela Ospina Osoriob,
Lucía Atehortúa Garcésb, Aura Inés Urrea Trujilloa
a
Universidad de Antioquia, Laboratorio de Fisiología Vegetal, Calle 70 No. 52-21, Medellín, A. A 1226, Colombia
b
Universidad de Antioquia, Grupo de Biotecnología, Calle 70 No 52-21, Medellín, A.A 1226, Colombia

A R T I C L E I N F O A B S T R A C T

Keywords: This paper aims to establish efficient protocols for the in vitro propagation of different cocoa genotypes (T.cacao),
Theobroma cacao L. the potential of regeneration via somatic embryogenesis in the Colombian genotypes SYS12, SYS13, SYS16,
Somatic embryogenesis SYS24, SYS4 and commercial/universal genotypes CCN51, TSH565, EET8, ICS1, ICS39, ICS60, ICS95 was
Genotypes evaluated. The effects of a different culture media, kind of explant and culture time on the induction of em-
Plant regeneration
bryogenic structures (PE), somatic globular embryos (GE) and cotyledonary somatic embryos (CE) were studied
Recalcitrance
in addition to the effect of the culture time on the average number of secondary somatic embryos (SSE) and the
culture medium over the SSE conversion. Finally, different substrates on the hardening of the stage of seedlings
was evaluated. In this study a regeneration protocol via a high frequency SSE for para CCN51, TSH565, SYS4 y
SYS13 is described in addition to the development induction up to a cotyledonary stage of the genotype SYS24
and the induction of proembryogenic masses and globular embryos for SYS12 y SYS16. For the commercial
genotypes ICS60 reached the development up to cotyledonary stage but it was not possible to reach a successful
conversion and for ICS1, EET8, ICS95, ICS39 we reached the induction of globular embryos that quickly ex-
perienced a dedifferentiation process. It is expected that the high frequency found in the embryos formation
allows a contribution to the reduction of production costs and thus promote the massive utilization of superior
cocoa genotypes propagated via somatic embryogenesis.

Abridgment
Primary somatic embryogenesis (SE), Secondary somatic embry-
ogenesis (SSE), Primary callus induction medium (PCG), Induction
medium (INDI), Secondary callus induction medium (SCG), Expression
medium (INDexp), Multiplication medium (CM2), Maturation medium
(MM6), Maturation medium (ED), Murashige and Skoog (MS)
Murashige and Skoog (1962), Driver and Kuniyuki, (1984) and Tulecke
and McGranahan, (1985) (DKW), (Lloyd and McCown, 1981)
(McCown’s), Vitaminas Gamborg's (B5) Gamborg et al. (1966), Tidia-
zuron (TDZ), 2,4-Dichlorophenoxyacetic acid (2,4-D), 2,4,5-Tri-
chlorophenoxyacetic acid (2, 4, 5-T), 1-Naphthaleneacetic acid (NAA),
Gibberellic acid (GA3), Santander Yariguíes Selection (SYS), Colombian
National Chocolate Company (CNCH), somatic globular embryos (GE),
cotyledonary somatic embryos (CE), proembryogenic structures (PE).
1. Introduction
Theobroma cacao L. (2n = 20) is a neotropical plant native to
Central and South America regions and was domesticated in Central
America. Among the 26 species of the genus, T. cacao is the only plant
cultivated on a large scale to produce chocolate (Richardson et al.,
2015). Despite the significant increase in global demand for cocoa, its
production has not followed the same trend and therefore there is a
global deficit in the supply that was 150,000 tons in 2015–2016 (ICCO,
2017). Global climate change, pest infestation and diseases occur plus
the reduction of productivity of the plants, due to aging of the crops,
have caused instability in the global production. The constant produc-
tion of T. cocoa is important in production countries to maintain sta-
bility of export and to ensure the continuity of the supply of industrial
raw materials for the growing industry of chocolate. Cocoa plays an
important economic role in Colombia, however, currently the average
productivity reaches 350 kg/ha/year, which is much lower than the

⁎
Corresponding author.
E-mail address: amaria.henao@udea.edu.co (A.M. Henao Ramírez).

http://dx.doi.org/10.1016/j.scienta.2017.10.040
Received 6 July 2017; Received in revised form 18 October 2017; Accepted 27 October 2017
Available online 06 November 2017
0304-4238/ © 2017 Elsevier B.V. All rights reserved.
A.M. Henao Ramírez et al.
average yield of plants propagated by seeds which reach up to
1.800 kg/ha/year.
The use of low quality materials, the lack of modernization in crops,
pests and the increase in the occurance of diseases due to the advanced
age of the plants are the major contributors to low productivity in 2017
(FEDECACAO). In order to increase the levels of production at a na-
tional level, it is suggested that the renewal of 80,000 ha and the
generation of 50,000 ha of new crops until 2021 be implemented; in
this context, the implementation of efficient methods in the massive
multiplication of plant material is required.
In general, the use of sexual seed and grafting are the most com-
monly used methods of propagation. Given that T. cocoa reproduces
naturally by cross pollination, sowing materials from seeds generally
exhibit a highly heterogeneous genetic background and the agronomic
yield of crops is therefore highly variable. The propagated materials by
cloning through graft materials do not have high rates of multiplication
and the bushy growth pattern is more pronounced, which is considered
an undesirable feature. In this sense, the modern techniques of propa-
gation and massive multiplication such as somatic embryogenesis can
improve the production of plant material from T. cacao.
Somatic embryogenesis is a method of in vitro regeneration for
plants, which after standardized is highly efficient, achieving a high
rate of multiplication. The in vitro regeneration of seedlings through
somatic embryos has been developed successfully in different plant
species, e.g. Arabidopsis (Wójcikowska and Gaj, 2016), Zea mays
(Garrocho-Villegas et al., 2016), Bixa orellana (Monteiro et al., 2016),
Capsicum annuum (Neftali, 2016), Coffea arabica (Loyola-Vargas et al.,
2016), Agave sp. (Rodríguez, 2016), Cocos nucifera (Sáenz-Carbonell
et al., 2016), Pinus sp. (Lelu-Walter et al., 2016), Zingiber officinale
(Shajahan et al., 2016), Musa paradisiaca (Escobedo-Gracia et al.,
2016), and Jatropha curcas (Kumar et al., 2016) and others.
Regeneration of T. cocoa plants through somatic embryogenesis has
been achieved in DKW basal medium supplemented with different
growth regulators (PGR), thidiazuron (TDZ) (Li et al., 1998), 2,4-di-
chlorophenoxyacetic acid (2,4-D) (Maximova et al., 2002) and kinetin
(KIN) (Fontanel et al., 2002). However, the success percentages in the
somatic embryo formation are still low (25%) and is limited to a few
clones, Sca 6 being the most widely reported (Garcia et al., 2016). Al-
though the TDZ has been reported as effective for inducing somatic
embryogenesis in T. cocoa, the cost of this growth regulator is high,
making itunsuitable for most commercial laboratories in developing
countries.
On the other hand, the need to implement breeding programs for
this crop is known, and that one of the key requirements in different
genetic transformation methods used so far, is having a system for plant
regeneration from the transformed tissues. Somatic embryogenesis is
the most reliable since the process of formation of embryos generally
involves a set of cells, which increases the probability that regenerated
plants in the selection medium are true transformants.
As described above, it is clear that the availability of an efficient in
vitro regeneration system is crucial for the strengthening of the first link
of the cocoa production chain in the short and medium term. To con-
tribute to this achievement, the aim of this research was to evaluate the
potential of in vitro regeneration via somatic embryogenesis of 12 cocoa
regional and universal clones with the protocols described by Li et al.
(1998) and Fontanel et al. (2002) with modifications.
2. Materials and methods
2.1. Plant material and explant preparation
For this research, the experiment design included 12 genotypes in
total, 7 universal/commercial genotypes CCN51, TSH565, TSE8, ICS1,
ICS39, ICS60, ICS95 and 5 Colombian genotypes from the Colombian
National Chocolate Company (CNCH) called Santander Yariguíes
Selection (SYS), which where denominated SYS12, SYS13, SYS16,
149
Scientia Horticulturae 229 (2018) 148–156
SYS24 and SYS4. The plant material was collected at the farms La
Nacional (Tamesis, Antioquia-Colombia) and the Farm Yariguíes
(Barrancabermeja, Santander-Colombia) of the CNCH. The explants
consisted of staminodes (sterile stamens) and petals of the immature
flower buds of each one of the genotypes indicated. The flower buds
(4–5 mm) were collected between 7 and 9 in the morning, once cut they
were submerged in a previously sterilized solution and served in conical
tubes of 50 mL cold, half concentrated solution of basal salts DKW. This
collected material was kept cold until arrival at the Laboratory of
Physiology and Plant tissue culture of the University of Antioquia,
Colombia. The flower buds were superficially sterilized by immersion in
a 0.5% (v/v) sodium hypochlorite solution for 5 min, under constant
agitation; then they were washed with sterile distilled water (3 times),
and changed to new sterile conical tubes, subsequently to be submerged
in a 250 ppm streptomycin solution for 20 min under constant agita-
tion. Finally, the staminodes and the petals were extracted from the
basal portion of the flower button and were planted in petri dishes
(60 × 15 mm) containing the corresponding culture medium.
2.2. Media and culture conditions
The disinfected explants were sown in the following sequence of
culture media: PCG-SCG-ED and INDI-INDexp-MM6 (Li et al., 1998;
Fontanel et al., 2002) with amendments and supplements of
20–40 gL−1 glucose, 2–3.5 gL−1 Gellex, and specific growth regulators,
which are detailed below. The pH of the media was adjusted to 5.8 with
1 M NaOH or 1 M HCl before being sterilized at 121 °C for 20 min.
2.3. Induction of proembryogenic structures (PE) and formation of primary
somatic embryos (SE)
For the induction of embryogenic callus, the disinfected petals and
staminodes were sown in the PCG medium (macronutrients, micro-
nutrients and vitamins DKW, (250 mgL−1) L-glutamine, (2 mgL−1) 2,4-
D, (0.005 mgL−1) TDZ and in the INDI medium (DKW macronutrients,
micronutrients and vitamins), (1 mgL−1) 2,4-D, (0.25 mgL−1) Kinetin,
mixture of amino acids per Urrea et al. (2011). The cultures were
maintained in these media for 30, 40, 50 and 60 days, and then were
transferred to the expression media. Twenty-five explants were put in
every Petri dish, and each treatment consisted of 10 Petri dishes. The
experiment was repeated 5 times.
For the expression stage of the SE, tissues with callus were trans-
ferred to the SCG media (McCown's salts), supplemented with 1 mgL-1
B5, (2 mgL−1) 2,4-D, (0.3 mgL−1) KIN and coconut water (5%) and
INDexp media (DKW macronutrients, micronutrients and vitamins).
The effect of the culture medium, type of explant and culture time on
the formation of the embryos were calculated with the average number
of proembryogenic structures, somatic globular embryos (GE) and co-
tyledonary somatic embryos (CE) formed in each explant at 15, 25 and
35 days after being transferred to the expression media. Twenty-five
explants were put in every Petri dish, and each treatment consisted of
10 Petri dishes. The experiment was repeated 5 times. With both cul-
tures, induction as expression wasmaintained at 28 °C ± 2 °C under
dark conditions during the proposed cultivation times.
2.4. Proliferation and conversion of the somatic embryos
For the induction of secondary somatic embryos (SSE), the primary
embryos in cotyledonary state (1–4 mm) from the varieties TSH565,
CCN51, SYS4 and SYS13 were segmented and cultured in the callus
multiplication medium (CM2) (MS macronutrients, micronutrients and
DKW vitamins, 1 mg L−1 2, 4, 5-T, 0.25 mgL−1 adenine and the amino
acids mixture according to Urrea et al.) (2011). To evaluate culture time
on the formation of SSE, the average number of embryos at different
stages was calculated; for this analysis, we chose the CE at 10, 15, 20, 25,
30, 35 and 40 days after being transferred to the CM2 medium. For this
A.M. Henao Ramírez et al.
experiment, 50 explants were put in every Petri dish, and each treatment
consisted of 10 Petri dishes. The experiment was repeated 5 times.
For the conversion stage, the CE longer than 3 mm were isolated
and transferred to glass containers (109.3 mm × 43.7 mm) containing
the MM6 media (MS half concentrated macronutrients, micronutrients
and DKW vitamins, 0.01 mgL−1 NAA, 0.02 mgL−1 GA3 and 1 gL−1
activated charcoal) for embryos derived from the INI-INDexp, and to ED
media (macronutrients, micronutrients and vitamins DKW) for embryos
derived from the PCG-SCG media. Cultures were maintained at
28 °C ± 2 °C under a photoperiod of 12/12 hours (light/darkness cy-
cles) with a 32 mmol/m2s illumination provided by fluorescent lamps.
To evaluate the effects of the culture medium on the embryos conver-
sion, after 60 days of culture, the average percentage of SSE for SYS4,
SYS13, TSH565 and CCN51 which succesfully reached the conversion
to root seedlings (number of germinated SE/total number of SE by
container) was calculated. Each experiment consisted of 20 containers
and was repeated 3 times.
2.5. Regenerated in vitro seedlings acclimatization
To evaluate the response to the hardening off stage, seedlings of at
least 5 cm high, 3 leaves, and with the presence of roots were removed
from the culture vessels and washed with tap water to remove culture
medium excess. They were then transferred to plastic containers
(8 × 10 cm) and subsequently to bags (7 × 14 cm) with a substrate
mix cointaining only soil from the crop (1), river sand/soil (1:1) and
(1:2). Plants were transferred to greenhouse conditions and watered
with tap water twice a week for 84 days. To evaluate the effect of
substrates on seedling development, the stem length was measured
(cm), as well as the number of new leaves, and the survival percentage
(number of alive plants/total number of plants). A seedling was planted
in each container. The experiment consisted of 10 seedlings per treat-
ment and was repeated twice.
2.6. Data analysis
For the analysis of the effect of the culture medium, explant type
and culture time on the formation of proembryogenic structures
number (PE), somatic globular embryos number (GE) and cotyledonary
Fig. 1. Callogenesis, callus type and proembryogenic structures (PE) induced in the petal explant, TSH565 genotype, INDI medium. (A) After 18 days (B) After 25 days (C) After 30 days
(D) PE in ICS39 with the petal explant after 50 days in PCG induction and 25 days in SCG expression (E) PE in SYS4 with the staminode explant after 30 days in induction and 25 days in
INDexp expression (F) PE in SYS24 after 30 days on the induction medium PCG and 15 days in the expression medium SCG (G) Filamentous white callus (FC) and Round and translucent
white vitreous cells (TC), the second type consisted of waxy yellow cells (WC), the third, granulated brown waxy callus (WBC). (For interpretation of the references to colour in this figure
legend, the reader is referred to the web version of this article.)
150
Scientia Horticulturae 229 (2018) 148–156
somatic embryos number (CE), a Poisson model was applied with a
Gamma a priori distribution since the data came from countings, and
the distribution is combined. Analysis of credibility intervals was per-
formed at 95% with a significance level of P < 0,05.
For the analysis of the effect of culture time on the average number
of secondary somatic embryos of the SYS4, SYS13, CCN51 and TSH565
genotypes, a Poisson model with a prior Gamma distribution was ap-
plied, since the data came from counts and the Gamma distribution is
combined. A 95% confidence interval analysis was performed with a
significance level P < 0.05.
For the analysis of the effect of culture media on the frequency in
the conversion of embryos to SYS4 and SYS13 seedlings, a Kruskal-
Wallis Test (P < 0,05) was applied, a non-parametric test, because the
assumptions of normality and homoscedasticity were not met to apply a
variance analysis of the factorial design. For the analysis of the results
of the TSH565 and CCN51, a factorial design was applied with an
analysis of variance and the means were compared by Tukey's Honestly-
significant-difference test (P < 0.05).
Finally, for the analysis of the effect of different substrates on sur-
vival, stem elongation and number of leaves of the in vitro regenerated
seedlings, a ANOVA with factorial design was applied and means were
compared using the Tukey HSD Test (P < 0,05). All analyses were
performed using R software (R See. 3.3.3).
3. Results
3.1. Effects of the culture medium, explant type and culture time on the
induction of PE
After 5–7 days in the induction media, the explants started the
formation of callus in both culture media evaluated, a transition to
embryogenic callus and PE were observed in all the genotypes in the
different time periods. In general, and in accordance with Gallego et al.
(2016), it was possible to recognize 4 callus types, the first type com-
prised of vitreous white, round and translucent cells (TC); the second
type consisted of waxy yellow cells (WC), the third, was a granulated
brown waxy callus (WBC) and finally a white filamentous calluses (FC)
(Fig. 1). From the TC, WC and WBC calluse, it was posible to differ-
entiate the embryogenic callus, proembryogenic structures and later
A.M. Henao Ramírez et al.
somatic embryos (Fig. 1). Significant differences were found in the re-
sponse of PE induction in the 12 genotypes evaluated according to the
culture medium, explant type and culture time (Supplemental 1).
In general, the 12 evaluated genotypes showed a different capacity
of response to the embryogenic process induction, as was expected for
the evaluated culture variables. With the INDI-INDexp culture medium
and the staminode explant PE with SYS4 and CCN51 an average of 8.26
PE for SYS4 was obtained after 30 days in the induction medium and 25
in the expression medium, and an average of 9.18 PE for CCN51 after
30 days in induction and 35 in expression. In the same medium (INDI-
INDexp) and with the petal explant, we obtained for SYS16 an average
of 6.98 PE after 30 days in the induction medium and 15 days in ex-
pression and for TSH565 7.63 PE after 30 days in induction and 25 in
expression.
Using the PCG-SCG culture medium, with the staminode explant
and 30 days in induction, we obtained for SYS12 an average of 6.50 PE
after 35 days in expression; for SYS13 an average of 23.24 PE after
15 days in expression and for ICS60 an average of 3.18 PE after 40 days
in induction and 35 in expression. In this same culture medium (PCG-
SCG) using the petal explant, 5.50 PE and 6.88 PE were obtained for
SYS24 and EET8 respectively, after 30 days in induction and 15 days in
expression; for ICS39 we obtained 6.99 PE after 60 days in induction
and 25 days in expression, for ICS1 an average of 4.33 PE after 60 days
in induction and 15 days in expression and lastly for ICS95 an average
of 6.87 PE after 60 days in induction and 25 days in expression. These
results indicate that the explant selection, culture medium and an ac-
curate culture time are essential to get somatic embryos in different
T.cocoa genotypes.
3.2. Effect of culture media, explant type and culture time on the formation
of GE and CE
We found that media culture, explant type and culture time had a
significant effect on the average number of somatic embryos in globular
state formed in the 12 evaluated genotypes (Fig. 2E–H). As expected,
the effect of these variables was consistent with the formation of PE
response. With INDI-INDexp culture medium and the staminode explant
were obtained an average of 2.39 GE for SYS4 after 25 days in ex-
pression and 7.49 GE for CCN51 after 15 days in expression. In the same
medium (INDI-INDexp) and with the petal explant with SYS16 was
obtained an average of 2.00 GE after 25 days in expression and 7.99 GE
for TSH565 after 15 days in expression. Using the PCG-SCG culture
medium, with the staminode explant we obtained for SYS13 an average
of 7.95 GE after 15 days in expression; for SYS12 an average of 2.00 GE
after 25 days in expression and for ICS60 an average of 2.18 GE after 35
in expression. In this same culture medium (PCG-SCG) using the petal
explant, 2.39 GE, 3.83 GE and 3.33 were obtained for SYS24, EET8 and
ICS1 respectively, after 15 days in expression; for ICS39 we obtained
6.99 PE after 60 days in induction and 25 days in expression, an
average of 5.87 GE and 5.49 GE for ICS95 and ICS39 after 15 days in
expression (Supplemental 2).
The main changes respect PE formation were only shown because of
the time that they remained in the medium of expression for the for-
mation of the GE in the genotypes SYS16, SYS12, TSH565 and CCN51.
The process of formation of embryos was asynchronous, and different
stages of development were detected in the same explant. The lower
times for the formation of the GE, 15 days, were obtained in ICS1, EET8,
SYS24, SYS13, CCN51 and TSH565, followed by 25 days for the geno-
types ICS95, SYS4, SYS12, SYS16 and finally 35 days for ICS39 and
ICS60.
The formation of cotyledonary somatic embryos was consistent with
the response of globular embryos and proembryogenic structure for-
mation. Significant differences in the CE average number were found in
five of the evaluated genotypes, from the variations in the growth
media, explant type and culture times used both for the induction and
expression stages (Fig. 2I–L). For SYS4 and TSH565 we observed the
151
Scientia Horticulturae 229 (2018) 148–156
biggest production of CE (6.20) and (7.99) respectively, after 15 days in
the expression medium, the same time that was required for the highest
formation of globular embryos. Based on these findings, there was si-
multaneous formation of both GE and CE. On the other hand, a per-
manence of 35 days for SYS13 was required in the expression medium
for obtaining an average number of 4.99 CE, taking 20 days longer in
the transition from globular to cotyledonary. The slower response was
obtained with CCN51 which required up to 25 days for the CE forma-
tion (7.99), i.e. 10 days longer for the transition from the globular to the
cotyledonary state. The lower average CE number was obtained with
ICS60 (2.74) and SYS24 (1.74) after 35 days in the expression stage
(Supplemental 3). Due to limitations in the CE number for ICS60 and
SYS24, the evaluation of the embryo conversion stage was not con-
tinued.
The genotypes EET8, ICS1, ICS95, SYS12, SYS16, ICS39 in spite of
reaching an embryogenic development to the globular state, did not
reach the transition to a cotyledonary state, showing in addition ded-
ifferentiation from embryogenic tissues over time. In these genotypes it
was observed that the embryos shared some features such as larger size
(800–1000 μm) compared to embryos that reached the complete tran-
sition (250–500 μm), a similar epidermis to the surface of TC and WC
callus, and a clear, vitreous and bright covering color, in contrast with
the embryos that reached the regeneration, whose color was matte
white (Fig. 2A–D).
3.3. Secondary somatic embryos (SSE) proliferation and conversion
The effect of time on the average number of secondary somatic
embryos formed was calculated for TSH565, CCN51, SYS4 and SYS13
(Fig. 2 N-P). For TSH565 after 35 days in the CM2 medium, the highest
SSE production (22.63) was achieved, however, it did not show sig-
nificant differences after 25 days of culture when a production of 21.25
SSE was reached. For CCN51, an average number of 25.16 SSE per
explant was obtained after 35 days of culture, however, it did not show
differences in the number of these SSE formed after 30 and 40 culture
days. For SYS4, an average number of 15.60 SSE per explant was ob-
tained after 20 days of culture, however, it did not show differences in
the number of these SSE formed after 15 and 25 culture days. Finally,
SYS13 with an average number of 28.25 SSE per explant was obtained
after 15 days of culture (Table 1, Supplemental 4).
The conversion frequency of the SSE was higher in the MM6
medium, which has DKW salts and glucose, and is enriched with amino
acids, adenine and activated charcoal, compared to the ED medium that
contains only half of the DKW salts concentration and the carbon source
(Fig. 3A–B). The highest percentage of SSE conversion for SYS4 and
SYS13 genotypes was 66% and 76.1% respectively, and for the TSH565
and CCN51 genotypes it was 91.41% and 92.61% respectively (Table 2,
Supplemental 5). Even though the ICS39 genotype formed embryos in
the cotyledonary state, it did not reach the conversion to seedling under
the evaluated conditions.
3.4. Regenerated seedlings acclimatization
Regenerated plants via somatic embryogenesis from the SYS4,
SYS13, TSH565 and CCN51 genotypes were successfully adapted to ex
vitro conditions. The seedlings highest survival percentage was obtained
in the 1:2 substrate with 53.4%, however, it did not show significant
differences with respect to the 1:1 substrate, with which we obtained a
42.2% rate of survival. In substrate 1, we only reached a regeneration
percentage of 13.2% (Table 3, Supplemental 6).
The highest growth in terms of stem elongation, was reached in
grown plants in the 1:2 substrate, with an average lenght of 7.16 cm
followed by 6.58 cm for grown seedlings in the 1:2 substrate and 1:1
substrate respectively, without significant differences between them
and with 1 substrate it was 2.28 cm with significant differences with 1:2
and with the 1:1 substrate. Finally, the highest number of leaves (6.4)
A.M. Henao Ramírez et al. Scientia Horticulturae 229 (2018) 148–156

Fig. 2. Not regenerating globular somatic embryos (A) EET8 (B) ICS1 (C) ICS95 (D) SYS12. Regenerating globular somatic embryos (E) TSH565 (F) SYS4 (G) SYS13 (H) CCN51.
Cotyledonary somatic embryos (I) SYS13 (J) CCN51 (K) TSH565 (L) ICS60. SSE proliferation (M) SYS13. SSE elongation and development (N) TSH565 (O) CCN51 (P) SYS4.

Table 1 substrate is the most suitable for T. cacao acclimatization (Fig. 3C–E).
Effect of the culture time on the average number of secondary somatic embryos formed
from primary embryos in a cotyledonary state on the CM2 medium.
4. Discussion
Culture Time in Average number of cotyledonary secondary embryos formed by
expression (days) explant.
Cocoa has been considered a recalcitrant species in studies related
TSH565 CCN51 SYS4 SYS13 to somatic embryogenesis (Gallego et al., 2016). However, currently
complete or partial protocols for regeneration via somatic embryogen-
10 4,82e 12,47c 3.14c 10.40d
esis for numerous genotypes have been developed which include Sca 6,
15 10,95c 12,33c 12.50ab 28.25a
20 7,68d 3,58d 15.60a 20.15b Pa 300, UIT 1, ICS 13, GC 7, DR 2, ICCRI 2, Cimanggu 1, Cimanggu 2,
25 21,25a 14,66c 14.09a 14.80c inifap 1, MCCS 14-056, POUND 7, TSH 1188, TSH 565, VB 1151, CCN
30 18,35b 22,82ab 10.80b 15.40c 51, UF 613 and PS 1319, TSH 1188, ICS95, CCN51, IMC-67, BIO B, etc.
35 22,63a 25,16a 2.95c 11.50d
(Maximova et al., 2002; Monsalve et al., 2005; Silva et al., 2008; Urrea
40 4,74e 21,56b 2.95c 12.50 cd
et al., 2011; García et al., 2016; Ajijah et al., 2014; Kan et al., 2017).
The values followed by the same letter within a column indicate that they are not sig- Despite the importance of T. cacao for Colombia, currently there are
nificantly different (p < 0.05). no massive propagation systems for Colombian regional materials that
stand out due to their flavor, aroma and productivity characteristics. So
per seedling was observed in the 1:2 substrate, compared to the 1:1 far Urrea et al. (2011) proceeded with the reported protocols evaluation
substrate, where an average of 4.6 leaves per plant were formed, for SE induction in two genotypes: ICS95 and BIOB, reaching re-
without significant differences between them and an average of 1.2 generation on the BIOB genotype; and Monsalve et al. (2005) studied
leaves with 1 substrate with significant differences between the 1:2 and regeneration in THS565, ICS95, CCN51 and IMC67 genotypes, however,
1:1 substrate. Considering the survival and development of the plant only succeeded on callogenesis induction.
(height and leaves development) it can be concluded that the 1:2 Responses during the different stages of the embryogenic process
can be very diverse and therefore, can be positive during the initial

152
A.M. Henao Ramírez et al. Scientia Horticulturae 229 (2018) 148–156

Fig. 3. (A) Seedlings obtained during conversion of

SSE from SYS13 (B) sequence of SYS4 seedlings de-
velopment up to 60 days in medium MM6. Plantlets
with roots at least 60 days in MM6 ready for accli-
matization (C) THS565 (D) CCN51. (E) Plantlets ac-
climatization for 84 days in 1:2 substrate, right
TSH565 left CCN51.

Table 2
Effect of culture medium on the Conversion Frequency (%) of SSE embryos on the ED and
MM6 media.

Genotype Conversion Frequency (%) of SSE embryos

ED MM6

SSE TSH565 10.28b 91.41a

SSE CCN51 8.53b 92.61a
SSE SYS4 4b 66a
SSE SYS13 6.7b 76.1a

The values followed by the same letter within a row indicate that they are not sig-
nificantly different (p < 0.05).

Table 3
Effect of the substrate mix on survival of seedling (%), stem length (cm) and number of
leaves.

Ratio substrate mix river Survival (%) Stem elongation Number of

sand/soil (cm) leaves

1 13.2 b 2.28 b 1.2 b

1:1 53.4 a 6.58 a 4.6 a
1:2 42.2 ab 6.58 a 6.4 a
Different letters within a column represent significant differences tested by Tukey's HSD
(honest significant difference) at P < 0.05 level.
stages such as the proembryogenyc structures formation, GE formation,
and can be negative during the final stages of embryo development and
conversion. This study confirms the high variability in embryogenic
response in the different genotypes evaluated in relation to explant
type, time and culture medium used for induction, expression and SE
and SSE maturation (Fig. 4).
For 8 of the 12 evaluated genotypes in the PCG-SCG media con-
taining 2,4-D and TDZ, both PE and SE were observed, however, only in
3 genotypes (SYS24, SYS13, ICS60) the transition to the subsequent
development states such as cotyledonary was obtained, and only the
SYS13 and SYS4 genotypes reached seedling development. It is possible
that the endogenous phytohormones concentration affected the induced
Fig. 4. Working summarizing the potential of regeneration via somatic embryogenesis
along time in the Colombian genotypes SYS12, SYS13, SYS16, SYS24, SYS4 and com-
mercial/universal genotypes CCN51, TSH565, EET8, ICS1, ICS39, ICS60, ICS95. The or-
ange and pink highlighted region indicates induction of embryogenic structures (PE) with
PCG and INDI medium. The purple and red highlighted region indicates expression of
somatic globular embryos (GE) and cotyledonary somatic embryos (CE) with SCG and
INDexp medium. The yellow region indicates induction of secondary somatic embryos
(SSE) with CM2 medium. The green region indicates SSE conversion with MM6 medium
and the blue region indicates ex vitro plantlets development with 1:2 sustrates. (For in-
terpretation of the references to colour in this figure legend, the reader is referred to the
web version of this article.)
morphogenesis by TDZ as described by Guo et al. (2013). Auxins and
cytokinins are key factors to determine the embryogenic response due
to their involvement in the cell cycle regulation and cell division. TDZ
normally increases the supply of purines for cell development and the
metabolites accumulation, as well as the conversion from adenine to

153
A.M. Henao Ramírez et al.
adenosine (Victor et al., 1999). These compounds are required during
the cell division process and protein synthesis, both are fast processes
that take place during somatic embryo development. TDZ in higher
concentrations than 0.0045 mgL−1, can stimulate the formation of
callus, adventitious shoots and somatic embryos (Huetteman and
Preece, 1993). This also affects the embryogenic callus formation and
somatic embryogenesis in T. cacao where 0.005 mgL−1 TDZ has been
found to be the most effective concentration for some genotypes (Garcia
et al., 2016).
On the other hand, for the CCN51 SYS4, TSH565, SYS16 genotypes
there was PE and GE formation and the CCN51, SYS4, TSH565 geno-
types reached a complete regeneration and seedlings development in
the INDI-INDexp medium supplemented with 2,4-D and KIN. The pre-
sence of KIN in the culture medium favored the embryogenic tissues
induction and their corresponding regeneration in these genotypes, a
coordinated action with auxins, since the hormone balance and other
culture conditions like amino acids and basal culture medium can lead
to different organs regeneration (Su and Zhang, 2014). The 2,4-D
concentration in the INDexp medium is not present and in the SCG
medium is 2 mgL−1, and in accordance with Su et al. (2011) relatively
high auxin concentrations suppress the organized growth and may void
the polarity of the embryogenic mass when this spreads inside it, which
induces the inhibition of the embryogenic process started by en-
dogenous auxins (Vondráková et al., 2011). Some of the genotypes
which formed globular embryos in the SCG expression medium (with
the highest auxin concentration), did not reach the following stages of
embryo development, allowing to suggest that they were sensitive to
the 2,4-D concentration. In this sense, Santana-Buzzy et al. (2009)
suggests an inhibitory role of auxins in the apical meristems develop-
ment of Capsicum baccatum somatic embryos. This phenomenon may be
caused by genetic, epigenetic or physiological factors. In T.cacao as in
C.baccatum, the auxins elimination from the medium is the key for the
embryo’s normal development (Venkataiah et al., 2016). In conclusion,
in several T. cacao genotypes the elimination of the 2,4-D level triggers
the initiation of the embryogenic process, opposite to what happens in
most of the Poaceae where the total elimination of this auxin during the
process to facilitate the cell division is not possible (Alcantara et al.,
2014).
In the SCG-PCG medium the TDZ presence could inactivate or limit
the responses; for Cruz et al. (2003), this regulator had a high resistance
to degradation by the action of the cytokinin oxidase enzyme of the
cytokinins, which suggests that the cytokinin action of TDZ decreases
gradually, taking long periods of time. Although the medium INDexp
has no growth regulators, a positive response was obtained which may
be the result of the different amino acids such as arginine, glycine,
leucine, lysine, tryptophan. Aminoacids represent an effective and im-
mediate nitrogen source for the cells, and are considered essential for
embryogenesis (Su and Zhang, 2014).
The results obtained in the TSH565 and CCN51 varieties match with
the report made by Garcia et al. (2016), where the explant type for
TSH565 was petal and for CCN51 was staminodes, however, induction,
expression and maturation times were lower in this study. These au-
thors obtained 3 SE per explant for TSH565 and 2 SE per explant for
CCN51, while in our work we achieved 7.99 SE per explant for both
varieties TSH565 and CCN51, using the basal salts DKW and the
medium reported by Fontanel et al. (2002).
We also obtained a higher embryo production for TSH565 compared
to what is reported by Silva et al. (2008), Monsalve et al. (2005) and
Maximova et al. (2002) and for CCN51 compared to the report made by
Monsalve et al. (2005). For the above, for these two genotypes the
medium with basal salts DKW supplemented with KIN in combination
with 2,4-D, is suitable for the process of primary somatic embryogen-
esis. Li et al. (1998), affirm that the basal salts DKW are more appro-
priate for the cocoa somatic embryogenesis than the basal salts MS,
because DKW contains a higher level of calcium, sulfur and magnesium,
and these elements can be crucial during the early induction stages of
154
Scientia Horticulturae 229 (2018) 148–156
the proembryogenic structures and embryos expression (Ajijah et al.,
2014).
For genotypes SYS4, SYS13, CCN51 and TSH565 the secondary
embryogenesis was higher compared to the primary embryogenesis, in
terms of frequency and number of generated embryos by explant (data
not shown). Production times of secondary embryos was also lower
than those reported so far in other genotypes. Garcia et al. (2016) listed
a total of 180 days since the introduction of the plant material up to the
seedlings obtention for the Sca6 genotype; Azpeitia-Morales et al.
(2015) obtained an average of 6 SSE per explant with the inifap1 gen-
otype after 210 days under a treatment devoid of phytohormones. In
the present study, the times for SSE formation were 145 days for
TSH565 and 160 days for CCN51, showing more uniformity and better
development compared to primary somatic embryos.
In this study, the highest frequency of secondary somatic embry-
ogenesis was obtained with primary somatic embryos in a cotyledonary
state, where the globular, torpedo, and heart stages were less sensitive
(data not shown). One of the limitations for the somatic embryogenesis
processes standardization (primary and secondary) is the genotype
dependence. This different response to the secondary embryogenesis
induction is highly dependent on the physiological characteristics of the
explants in a specific development stage; this was also reported for Paris
polyphylla and Sapium sebiferum (Rai et al., 2007; Raomai et al., 2014).
Efficient conversion of cotyledonary somatic embryos into seedlings
is another important step for the whole plant regeneration. In this
study, primary and secondary embryos conversion percentages over
66% were achieved, superior results to those reported by Ajijah et al.
(2014), where the Sca6, Pa300, ITU 1, ICS 13, GC 7, DR 2, ICCRI 2
genotypes were used. The seedlings complete development in the MM6
medium, is opposite to the results obtained by Garcia et al. (2016) who
describes this development in ED medium.
Regenerating embryos were characterized by a matte white col-
oration, which coincides with Teixeira et al. (2002) who asserts that
these embryos have higher conversion rates into seedlings than into
translucent embryos. One of the main differences between these media
is that the MM6 macronutrients are MS while for ED the nutrients are
DKW, in addition, the MM6 medium is supplemented with activated
carbon, amino acids, NAA and GA3. One possible explanation for these
results is a higher macroelements concentrations, especially nitrogen
(KNO3 and NH4NO3) in the MS medium compared to the DKW (Aghaye
and Yadollahi, 2012).
In the MM6 medium, which contains NAA combined with GA3, an
efficient germination was achieved, opossite to the findings in the
medium without regulators (ED). This synergistic effect of auxin and
gibberellin on the promotion of embryo conversion into seedlings has
also been reported in other species (Zlenko et al., 2002; Junaid et al.,
2007; Paramageetham et al., 2004). Ali et al. (2010) managed the so-
matic embryos development of Juglans regia in a medium that contained
MS salts and that was supplemented with NAA and GA3, after two
months of culture. GA3 has also been used in the elongation of the re-
generated buds (Lakshmi et al., 2013).
For the 12 genotypes evaluated, only EET8, ICS1, ICS95, ICS39,
SYS12, SYS16 reached the formation of embryos in a globular state and
the subsequent dedifferentiation in callus. The callus from non-re-
generating genotypes were in general vitreous white and granular,
unlike the Brown and granular callus that were related to the ones from
embryo formation response, which is in line with what was described
by Gamez (2012), in T.cacao.
According to Gallego et al. (2016) in T.cacao, non-regenerating
embryos are lacking from reserve substance but have a high polyphenol
content, which are randomly located in all of the embryo tissue. On the
other side, regenerating embryos were characterized by having a high
protein and carbohydrate content, procambium vascular bundles and
apical meristems (SAM), and a differentiated root system (RAM), imi-
tating the zygotic embryo ontogenesis in which polyphenols are only
restricted to the protodermal cells.
A.M. Henao Ramírez et al.
The explants oxidation during the beginning of the culture is a
frequent cause of early in vitro recalcitrance due to a high phenolic
content. This problem is more common in woody plant tissues such as
cocoa, because they have high levels of phenols associated with sec-
ondary growth and lignification, and in other cases are related to the
secondary metabolites production. The in vitro oxidation of these phe-
nolic compounds can be increased by the effect of the cutting done on
the explants, the disinfection process, and specific components of the
culture media, among others.
It is known that during the somatic embryogenesis process, the cells
respond to different stress factors changing their development program
to a specific physiological condition, through a reprogramming of gene
expression towards the acquisition of an embryonic competence
(Nowak and Gaj, 2016). For T.cacao, it has been identified that things
like stress factors, injuries, salt content and growth regulators like TDZ,
KIN, and NAA can trigger a conversion from floral tissue somatic cells
into embryonic competent callus (Traore et al., 2003).
In addition, other factors associated with recalcitrance in the in vitro
embryogenic development process have been described, and they in-
volve the physiology of the donor plant, life cycle stage, the explant and
the rejuvenation ability. Issali et al. (2009) showed for T.cacao, the
relationship between phenological parameters of the donor plant,
suggesting that the flowering and fruiting periods are suitable to induce
a higher embryogenesis and more embryogenic tissue. This complexity
is certainly responsible for the variation effect of the phenological states
in the embryogenesis, observed from one year to another and among
different times in the same year (Alemanno et al., 1997; De Mayolo
et al., 2003; López-Baez et al., 1993; Tan and Furtek, 2003; Chatelet
et al., 1992).
In general, it was found that the genotypic background affected all
the steps in the embryogenic process. This result coincides with
Wójcikowska and Gaj (2016), who stated that the genotype is one of the
factors that determine the proliferation rate and the nature of the
proliferation cycles as well as the changes observed during the ma-
turation stage. In addition, during the maturation stage, the genotype
determines not only the total number of developed embryos by culture,
but also the embryos proportion at specific stages of development.
Therefore, a wide variation in the yield of somatic embryogenesis be-
tween different genotypes was observed.
5. Conclusions
This study describes a protocol for regeneration via secondary so-
matic embryogenesis of higher frequency for CCN51 and TSH565 gen-
otypes compared to what is reported at the time. It reports for the first
time, the regeneration through primary and secondary embryogenesis
of the Colombian regional genotypes SYS4 and SYS13. In addition, the
induction of primary somatic embryogenesis for SYS24, SYS13 and
SYS16 Colombian genotypes was achieved. For the commercial geno-
types ICS60, ICS1, EET8, ICS95, ICS39 genotypes advanced in the pri-
mary somatic embryogenesis, but not a successful conversion; and in
some genotypes quickly experienced a dedifferentiation process. This
Protocol will be helpful for the plant’s mass propagation and the genetic
improvement of the most relevant regional genotypes. In addition, this
study sets a precedent for the research of embryonic development for
these special genotypes.
Conflict of interest
The authors claim to have no conflicts of interest.
Acknowledgements
Special acknowledgement to the Committee for Research
Development of the University of Antioquia (CODI) and Compañía
Nacional de Chocolates (CNCH). This work was supported by the
155
Scientia Horticulturae 229 (2018) 148–156
Colombian Administrative Department of Science Technology and
Innovation (COLCIENCIAS), number 111570048738 and contract 359-
2015.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in the
online version, at http://dx.doi.org/10.1016/j.scienta.2017.10.040.
References
Aghaye, N.M., Yadollahi, R., 2012. Micropropagation of GF 677 rootstock. J. Agric. Sci. 4
(2012), 1916–9752. http://dx.doi.org/10.5539/jas.v4n5p131.
Ajijah, N., Rubiyo, Sudarsono, 2014. Callogenesis and somatic embryogenesis of cacao
using thidiazuron through one step of callus induction. Jurnal Penelitian Pertanian
Tanaman Industri 20, 179–186. http://dx.doi.org/10.17503/agrivita.v38i1.619.
Alcantara, G., Dibax, R., Oliveira, R., Bespalhok, J., Daros, E., 2014. Plant regeneration
and histological study of the somatic embryogenesis of sugarcane (Saccharum spp.)
cultivars RB855156 and RB72454. Acta Sci. Agron. 36 (1), 63–72. http://dx.doi.org/
10.4025/actasciagron.v36i1.16342.
Alemanno, L., Berthouly, M., Michaux-Ferriere, N., 1997. Comparison between T. cacao
L. zygotic embryogenesis and somatic embryogenesis from floral explants. In vitro
Cell. Dev. Biol. 33, 163. http://dx.doi.org/10.1007/s11627-997-0016-8.
Ali, S.B.G., Kourosh, V., Hassan, B.S., Siamak, K., Charles, L., 2010. Enhancement of
maturation and germination of somatic embryos in Persian walnut (Juglans regia l.)
using osmolites, hormones and cold treatments. Afr. J. Food Sci. 4, 735–743.
Azpeitia-Morales, A., Alejandro-Lázaro, A., Sáenz-Carbonell, L., Mirafuentes Hernández,
F., 2015. Embriogánesis somética secundaria en el genotipo de cacao (T. cacao L.)
inifap 1 y su descripción histológica. Nova Sci. 7, 398–417. http://dx.doi.org/10.
21640/ns.v7i14.35.
Chatelet, P., Michaux-Ferrière, N., Dublin, P., 1992. Potentialités embryogènes du nucelle
et du tégument interne de grains immatures de cacaoyer (T. cacao L.). Histologie
végétale/Plant histol. C. R. Acad. Sci. Paris 315, 55–62.
Cruz, I., Angarita, A., Mosquera, T., 2003. Induction of Somatic Embryogenesis in
Alstroemeria Spp. Agronomía Colombiana 21. pp. 121–128. http://www.revistas.
unal.edu.co/index.php/agrocol/article/view/19816.
De Mayolo, G. Antunez, Maximova, S.N., Pishak, S., Guiltinan, M.J., 2003. Moxalactam as
a counter-selection antibiotic for Agrobacterium mediated transformation and its
positive effects on T. cacao somatic embryogenesis. Plant Sci. 164, 607–615. http://
dx.doi.org/10.1016/S0168-9452(03)00012-8.
Driver, D., Kuniyuki, D., 1984. In vitro propagation of Paradox walnut rootstock.
HortScience 19, 507–509.
Escobedo-Gracia, R., Enríquez-Valencia, A., Youssef, M., López-Gómez, P., Cruz-
Cárdenas, C., Ku-Cauich, J., 2016. Somatic embryogenesis in banana, Musa ssp. In:
Loyola-Vargas, V., Ochoa-Alejo, N. (Eds.), Somatic Embryogenesis: Fundamental
Aspects and Applications. Springer International Publishing, Switzerland, pp.
381–400. http://dx.doi.org/10.1007/978-3-319-33705-0_21.
Fontanel, A., Gire-Bobin, S., Labbé, G., Favereau, P., Álvarez, M., Rutte, S., 2002. In vitro
multiplication and plant regeneration of Theobroma cacao L. via stable embryogenic
calli. 10 IAPTC Congress. Plant Biotechnol.
Gallego, A.M., Henao, A.M., Urrea, A.I., Atehortúa, L., 2016. Polyphenols distributions
and reserve substances analysis in cacao somatic embryogenesis. Acta Biol. Colomb.
21, 335–345. http://dx.doi.org/10.15446/abc.v21n2.50196.
Gamborg, O.L., Millar, R.A., Ojima, K., 1966. Nutrient experiment of suspension cultures
of soybean root cells. Exp. Cell Res. 50, 151–158.
Gamez, J., 2012. Somatic embryogenesis and long term conservation of cocoa
(Theobroma cacao L.) germplasm. Master’s thesis. University of Helsinki, Faculty of
Agriculture and Forestry, Department of Agricultural Sciences. http://hdl.handle.
net/10138/37431.
Garcia, C., Corrêa, F., Findley, S., Almeida, A., Costa, M., Motamayor, J.C., Schnell, R.,
Marelli, J., 2016. Optimization of somatic embryogenesis procedure for commercial
clones of Theobroma cacao L. Afr. J. Biotechnol. 15 (36), 1936–1951. http://dx.doi.
org/10.5897/AJB2016.15513.
Garrocho-Villegas, V., Almeraya, E., Sanchez, E., 2016. Maize somatic embryogenesis:
agronomic features for improving crop productivity. In: Loyola-Vargas, V., Ochoa-
Alejo, N. (Eds.), Somatic Embryogenesis: Fundamental Aspects and Applications.
Springer International Publishing, Switzerland, pp. 201–211. http://dx.doi.org/10.
1007/978-3-319-33705-0_12.
Guo, B., Abbasi, B.H., Zeb, A., Xu, L., Wei, Y., 2013. Thidiazuron: a multi-dimensional
plant growth regulator. Afr. J. Biot. 10, 8984–9000. http://www.ajol.info/index.
php/ajb/article/view/95617.
Huetteman, C.A., Preece, J.E., 1993. Thidiazuron: a potent cytokinin for woody plant
tissue culture. Plant Cell Tissue Organ Cult. 33, 105–119. http://dx.doi.org/10.1007/
BF01983223.
ICCO, 2017. ICCO Cuts Estimate for Global Cocoa Deficit in 2015–16 Season. On line:
https://www.wsj.com/articles/icco-cuts-estimate-for-global-cocoa-deficit-in-2015-
16-season-1480532529 (Accessed 17 February 2017).
Issali, A.E., Traoré, A., Ngoran, J.A.K., Koffi, K.E., Sangaré, A., 2009. Relationship be-
tween some phenological parameters and somatic embryogenesis in T. cacao L. J.
Crop Sci. Biotechnol. 11, 23–30. http://www.ajol.info/index.php/ajb/article/
viewFile/92517/81958.
Junaid, A., Mujib, A., Bhat, M.A., Sharma, M.P., Šamaj, J., 2007. Somatic embryogenesis
A.M. Henao Ramírez et al.
and plant regeneration in Catharanthus roseus. Biol. Plant 51, 641–646. http://dx.doi.
org/10.1007/s10535-007-0136-3.
Kan, K., Tokpapon, M., Daouda, K., Brahima, S., Tchoa, K., Kouablan, K., Mongomaké, K.,
2017. Effect of antioxidants on the callus induction and the development of somatic
embryogenesis of cocoa [Theobroma cacao (L.)]. Aust. J. Crop Sci. 11 (1), 25–31.
http://dx.doi.org/10.21475/ajcs.2017.11.01. pne174.
Kumar, R., Ruiz-May, E., García-Pérez, L., Quiroz-Figueroa, F., 2016. Somatic embry-
ogenesis in Jatropha curcas. In: Loyola-Vargas, V., Ochoa-Alejo, N. (Eds.), Somatic
Embryogenesis: Fundamental Aspects and Applications. Springer International
Publishing, Switzerland, pp. 401–412. http://dx.doi.org/10.1007/978-3-319-33705-
0_22.
López-Baez, O., Bollon, H., Eskes, A., Pétiard, V., 1993. Embryogenóse somatique de
cacaoyer (T. cacao L.) é partir des pióces florales. C. R. Acad. Sci. Paris, Sciences de la
vie/ Life Sci. 579–584. http://agritrop.cirad.fr/463984/.
Lakshmi, S.R., Benjamin, J.H.F., Kumar, T.S., Murthy, G.V.S., Rao, M.V., 2013.
Organogenesis from in vitro-derived leaf and internode explants of Hoya wightii ssp.
palniensis—a vulnerable species of Western Ghats. Braz. Arch. Biol. Technol. 56,
421–430. http://dx.doi.org/10.1590/S1516-89132013000300010.
Lelu-Walter, M., Klimaszewska, K., Miguel, C., Aronen, T., Hargreaves, C., Teyssier, C.,
Trontin, J., 2016. Somatic embryogenesis for more effective breeding and deploy-
ment of improved varieties in Pinus spp.: bottlenecks and recent advances. In: Loyola-
Vargas, V., Ochoa-Alejo, N. (Eds.), Somatic Embryogenesis: Fundamental Aspects and
Applications. Springer International Publishing, Switzerland, pp. 319–365. http://dx.
doi.org/10.1007/978-3-319-33705-0_19.
Li, Z., Traore, A., Maximova, S., Guiltinan, M.J., 1998. Somatic embryogenesis and plant
regeneration from floral explants of cacao (T. cacao L.) using thidiazuron. In vitro
Cell. Dev. Biol.-Plant 34 (4), 293–299. http://dx.doi.org/10.1007/BF0 2822737.
Lloyd, G., McCown, B., 1981. Commercially feasible micropropagation of Mountain
Laurel, Kalmia latifolia, by use of shoot tip culture. Comb. Proc. –Int. Plant Propag.
Soc. 30, 421–427.
Loyola-Vargas, V., Avilez-Montalvo, J., Avilés-Montalvo, R., Márquez-López, R., Galaz-
Ávalos, R., Mellado-Mojica, E., 2016. Somatic embryogenesis in Coffea spp. In:
Loyola-Vargas, V., Ochoa-Alejo, N. (Eds.), Somatic Embryogenesis: Fundamental
Aspects and Applications. Springer International Publishing, Switzerland, pp.
241–266. http://dx.doi.org/10.1007/978-3-319-33705-0_15.
Maximova, S.N., Alemanno, L., Young, A., Ferriere, N., Traore, A., Guiltinam, M., 2002.
Efficiency, genotypic variability, and cellular origin of primary and secondary so-
matic embryogenesis of Theobroma cacao L. In Vitro Cell. Dev. Biol. -Plant 38, 252.
http://dx.doi.org/10.1079/IVP2001257.
Monsalve, L., García, C., Sigarroa, A., 2005. Obtención de embriones somáticos primarios
de Theobroma cacao en clones de interés regional para el departamento Norte de
Santander. Colombia. Revista Respuestas 1, 21–28. http://revistas.ufps.edu.co/ojs/
index.php/respuestas/article/view/675.
Monteiro, E., Dias, A., Vidal, D., Nayara, L., Santos, V., Ferrerira, A., Souza, T., Rochs, D.,
Fontes, V., Borges, V., Cardoso, M., Campos, W., 2016. Maize somatic embryogenesis:
agronomic features for improving crop productivity. In: Loyola-Vargas, V., Ochoa-
Alejo, N. (Eds.), Somatic Embryogenesis: Fundamental Aspects and Applications.
Springer International Publishing, Switzerland, pp. 213–231. http://dx.doi.org/10.
1007/978-3-319-33705-0_13.
Murashige, T., Skoog, F., 1962. A revised medium for rapid growth and bios assay with
tobacco tissue culture. Physiol. Plant. 15, 473–497.
Neftali, O., 2016. Somatic embryogenesis in Capsicum spp. In: Loyola-Vargas, V., Ochoa-
Alejo, N. (Eds.), Somatic Embryogenesis: Fundamental Aspects and Applications.
Springer International Publishing, Switzerland, pp. 233–240. http://dx.doi.org/10.
1007/978-3-319-33705-0_14.
Nowak, K., Gaj, M., 2016. Transcription factors in the regulation of somatic embry-
ogenesis. In: Loyola-Vargas, V., Ochoa-Alejo, N. (Eds.), Somatic Embryogenesis:
Fundamental Aspects and Applications. Springer International Publishing,
Switzerland, pp. 53–79. http://dx.doi.org/10.1007/978-3-319-33705-0_5.
Paramageetham, C.H., Babu, G.P., Rao, J.V.S., 2004. Somatic embryogenesis in Centella
asiatica L. an important medicinal and neutraceutical plant of India. Plant Cell Tissue
Organ Cult. 79, 19–24.
Rai, M.K., Akhtar, N., Jaiswal, V.S., 2007. Somatic embryogenesis and plant regeneration
in Psidium guajava L. cv. Banarasi local. Sci. Hortic. 113, 129–133. http://dx.doi.
org/10.1016/j.scienta.2007.02.010.
Raomai, S., Kumaria, S., Tandon, P., 2014. Plant regeneration through direct somatic
embryogenesis from immature zygotic embryos of the medicinal plant, Paris
156
Scientia Horticulturae 229 (2018) 148–156
polyphylla Sm. Plant Cell Tissue Organ Cult. 118, 445–455. http://dx.doi.org/10.
1007/s11240-014-0496-2.
Richardson, J.E., Whitlock, B.A., Meerow, A.W., Madriñán, S., 2015. The age of choco-
late: a diversification history of Theobroma and Malvaceae. Front. Ecol. Evol. 3, 120.
http://dx.doi.org/10.3389/fevo.2015.00120.
Rodríguez, B., 2016. Somatic embryogenesis in Agave spp. In: Loyola-Vargas, V., Ochoa-
Alejo, N. (Eds.), Somatic Embryogenesis: Fundamental Aspects and Applications.
Springer International Publishing, Switzerland, pp. 267–282. http://dx.doi.org/10.
1007/978-3-319-33705-0_16.
Sáenz-Carbonell, L., Montero-Cortés, N., Pérez-Nuñez, T., Azpeitia-Morales, A., Andrade-
Torres, A., Córdova-Lara, I., Chan-Rodríguez, J., Sandoval-Cancino, G., Rivera-Solis,
G., Oropeza-Salín, C., 2016. Somatic embryogenesis in Agave spp. In: Loyola-Vargas,
V., Ochoa-Alejo, N. (Eds.), Somatic Embryogenesis: Fundamental Aspects and
Applications. Springer International Publishing, Switzerland, pp. 297–318. http://dx.
doi.org/10.1007/978-3-319-33705-0_18.
Santana-Buzzy, N., Lopez-Puc, G., Canto-Flick, A., Barredo-Pool, F., Balam-Uc, E., Aviles-
Vinas, S., Solıs-Marroquın, D., Lecona-Guzman, C., Bello-Bello, J., Gomez-Uc, E.,
Mijangos-Cortes, J.O., 2009. Ontogenesis of the somatic embryogenesis of habanero
pepper Capsicum Chinense Jacq. HortScience 44, 113–118. full. http://hortsci.
ashspublications.org/content/44/1/113.
Shajahan, A., Soundar, C., Thilip, C., Varutharaju, K., Faizal, K., Musfir, V., Aslam, A.,
2016. Direct and indirect somatic embryogenesis in mango ginger (Curcuma amada
roxb.). In: Loyola-Vargas, V., Ochoa-Alejo, N. (Eds.), Somatic Embryogenesis:
Fundamental Aspects and Applications. Springer International Publishing,
Switzerland, pp. 367–379. http://dx.doi.org/10.1007/978-3-319-33705-0_20.
Silva, T., Cidade, L., Alvim, Fátima C., Cascardo, J., Costa, M., 2008. Somatic embry-
ogenesis and plant regeneration in elite clones of Theobroma cacao. Pesquisa
Agropecuária Brasileira 43 (10), 1433–1436. http://dx.doi.org/10.1590/S0100-
204X2008001000024.
Su, Y.H., Zhang, X.S., 2014. The hormonal control of regeneration in plants. Curr. Top.
Dev. Biol. 108, 35–69. http://dx.doi.org/10.1016/B978-0-12-391498-9. 00010-3.
Su, Y., Liu, Y., Zhang, X., 2011. Auxin–cytokinin interaction regulates meristem devel-
opment. Mol. Plant 4, 616–625. http://dx.doi.org/10.1093/mp/ssr007.
Tan, C.L., Furtek, D.B., 2003. Development of an in vitro regeneration system for T. cacao
from mature tissues. Plant Sci. 164, 407–412. http://dx.doi.org/10.1016/S0168-
9452(02)00428-4.
Teixeira, J.B., Marbach, P.A.S., Santos De, M.O., 2002. Otimização da metodologia da
embriogênese somática visando à propagação clonal de genótipos elite de cacau (T.
cacao L.). Brasília: Embrapa Recursos Genéticos e Biotecnologia 34. http://www.
infoteca.cnptia.embrapa.br/bitstream/doc/184362/1/doc079. pdf.
Traore, A., Maximova, S.N., Guiltinan, M.J., 2003. Micropropagation of T. cacao L. using
somatic embryo-derived plants. In vitro Cell. Dev. Biol. Plant 39, 332–337. http://dx.
doi.org/10.1079/IVP2002409.
Tulecke, W., McGranahan, G., 1985. Somatic embryogenesis and plant regeneration from
cotyledons of walnut (Juglans regia L.). Plant Sci. 40, 57–63. http://dx.doi.org/10.
1016/0168-9452(85)90163-3.
Urrea, A., Gallego, A., Atehortúa, L., 2011. Regeneración vía embriogénesis somática de
una variedad colombiana élite de Theobroma cacao L. Rev. Col Biot. 13, 39–50.
http://www.revistas.unal.edu.co/index.php/biotecnologia/article/view/27916/
38317.
Venkataiah, P., Bhanuprakash, P., Kalyan, S., Subhash, K., 2016. Somatic embryogenesis
and plant regeneration of Capsicum baccatum L. J. Genet. Eng. Biotechnol. 14 (1),
55–60. http://dx.doi.org/10.1016/j.jgeb.2016.02.001.
Victor, J., Murch, S., KrishnaRaj, S., Saxena, P., 1999. Somatic embryogenesis and or-
ganogenesis in peanut: the role of thidiazuron and N6-benzylaminopurine in the
induction of plant morphogenesis. Plant Growth Regul. 28, 9–15. http://dx.doi.org/
10.1023/A:1006274615736.
Vondráková, Z., Eliášová, K., Fischerová, L., 2011. The role of auxins in somatic em-
bryogenesis of Abies alba. Cent. Eur. J. Biol. 6, 587. http://dx.doi.org/10.2478/
s11535-011-0035-7.
Wójcikowska, B., Gaj, M., 2016. Somatic embryogenesis in Arabidopsis. In: Loyola-
Vargas, V., Ochoa-Alejo, N. (Eds.), Somatic Embryogenesis: Fundamental Aspects and
Applications. Springer International Publishing, Switzerland, pp. 185–199. http://dx.
doi.org/10.1007/978-3-319-33705-0_11.
Zlenko, V.A., Kotikov, I.V., Troshin, L.P., 2002. Efficient GA3-assisted plant regeneration
from cell suspensions of three grape genotypes via somatic embryogenesis. Plant Cell
Tissue Organ Cult. 70, 295–299. http://dx.doi.org/10.1023/A.