Somatic Embryogenesis in Theobroma cacao cultivars from the National Germplasm Repository

Claudia Arevalo (1), Jose Judy Stervil (1), David Kuhn (2), Osman Gutierrez (2) and D. Pilar Maul (1).
(1) School of Science, St. Thomas University, 16401 NW 37th Avenue, Miami Gardens, FL 33054. (2) USDA-ARS-SHRS, 13601 Old Cutler Road, Miami, FL 33158.

I.Introduction

III. Results

Cacao (Theobroma cacao) is one of the most widely consumed agricultural

crops around the world due to its importance in the production of chocolate and
confectionary products. Genetic improvement of cacao has been difficult because
of its long-lasting reproductive cycle and narrow genetic base which increases its
vulnerability to pests and pathogens. The USDA, ARS, Subtropical Horticulture
Research Station in Miami, FL is the National Germplasm Repository of cacao.
Their breeding program aims at identifying and improving genotypes of high yield
and high resistance to multiple biotic and abiotic stresses.
Somatic embryogenesis, a tissue culture technique in which an embryo is
generated from somatic cells, provides a system of clonal propagation of selected
genotypes to plant breeders. Similar to zygotic embryos derived from seeds,
somatic embryos go through four stages of embryo development: globular, heart,
torpedo and cotyledonary. Embryos are induced by incubating somatic plant tissues
in specialized culture media containing plant growth regulators. In cacao, flower
staminoides have been found to be the most responsive explant to somatic
embryogenesis conditions. Induction and embryo development media that induce
somatic embryos in various genotypes have been reported (Zhijian et al.).
Staminoides are incubated in Primary Callus Growth Media ( PCG) in the dark for
two weeks. After transfer to Secondary Callus Growth Media (SCG) and incubated
for two additional weeks, explants are transferred to Embryo Development Media
(ED) every two weeks for 24 weeks. Secondary somatic embryogenesis consists in
using somatic embryo sections as explants for the production of more somatic
embryos.
In this study, we report the use of a somatic embryogenesis protocol to
determine the response of various genotypes from the National Germplasm
Repository to both primary and secondary somatic embryogenesis.

1. Primary Somatic Embryogenesis

Number of responsive explants/flower

1.8
Average of
Embryogenic
Staminoide per
Flower

1.6
1.4
1.2
1
0.8

Average of
Embryogenic
Petals per flower

0.6
0.4
0.2
0

Figure 1. Somatic embryos were induced from flower staminoides in five out of nine
cacao cultivars tested. Petal explants produced somatic embryos in four out of the nine
cultivars. Staminoides and petal explants of 5-7 mm unopened flowers were used to
follow a well established protocol for somatic embryo induction in cacao. Responsive
explants were those that produced at least one somatic embryo in 24 weeks.

Figure 5. Primary somatic

embryos after 7 weeks in
culture in cacao cultivars UF
676 (A) and EET387 (B).

Figure 3.
Unopened cacao
flowers for
somatic
embryogenesis.

Percentage of explants responsive to secondary somatic

embryogenesis in four cacao cultivars

Number of secondary somatic embryos from cotyledonary

sections of primary somatic embryos in cacao
10

80.0

70.0

% responsive explants per plate

II. Materials and Methods

7
6
5
4
3
2
1

60.0

EET

UF 676

UF 667

AMAZ15

30.0
20.0
10.0

Secondary somatic embryogenesis . Sterile cotyledonary sections 4 mm long

from 24-week primary somatic embryos of four cacao genotypes were
incubated on petri dishes containing culture media. Genotypes tested for
secondary embryogenesis were EET387, UF676, UF667, and AMAZ 15.
Three plates were prepared for each genotype with six pieces of embryo
cotyledons per plate.

UF 676

UF 667

AMAZ15

-10.0

Cacao Genotypes

Cacao Genotypes

Figure 6. Number of secondary somatic embryos per responsive cotyledonary sections

22 weeks after induction.

To optimize culture conditions in

order to increase number of somatic
embryos induced in cacao cultivars.

2.

To test the response of additional

genotypes to somatic embryogenesis
conditions.

3.

To test the conversion of somatic

embryos to normal plantlets and to
transfer to the greenhouse.

4.

The determine the genetic stability of

somatic embryos generated.

5.

To test the potential of various

genotypes to tertiary somatic
embryogenesis.

VI. Acknowledgements

40.0

EET
0

1.

50.0

0.0

Incubation. All plates were incubated under dark conditions in an incubator set at
25oC.

V. Future Steps

2. Secondary Embryogenesis

The objectives of this study were:

a) To determine which of the selected genotypes are responsive to primary
embryogenesis
b) To determine which of the selected genotypes are responsive to secondary
embryogenesis

Culture Media. Explants were incubated on PCG(Primary Callus Growth ) media

for two weeks after which they were transferred to SCG(secondary Callus
Growth) media. Two weeks later, the explants were transferred to ED(Embryo
Development) media for two weeks. Every two weeks, the explants were
transferred to fresh ED media for an additional two weeks. After week 6,
somatic embryo formation was recorded up to 24 weeks for primary
embryogenesis and up to 18 weeks for secondary somatic embryogenesis

Figure 4. Cacao explants in culture. A.

Staminoides and petals in PCG media.
B. Staminoides with embryogenic
callus after 2 weeks in culture.

Cacao genotypes

Secondary Embryogenesis
(Explants: cotyledon sections of
primary somatic embryos)

Primary somatic embryogenesis . Unopened immature flowers 5 to 7 mm long

from various cacao accessions were collected from the USDA,ARS,SHRS
greenhouse. Genotypes tested included: UF676, UF667, EET387, PA137RUQ,
Amelonado, AMAZ15, PA120XGNVLO, ICS95. Flower decontamination
consisted of a 10% bleach treatment for 15 minutes followed by three rinses
with sterile water. Petals and staminoides were separated from the rest of the
flower and placed on petri plates containing culture media.

Figure 2. Timetable for induction of primary

and secondary embryogenesis in cacao

Number of embryos per responsive explant

Primary Embryogenesis
(Explants: floral parts)

Response of selected cacao genotypes to primary somatic

embryogenesis induction

Figure 7. Percentage of cotyledonary explants of primary embryogenesis responsive to

secondary embryogenesis 22 weeks after induction.

Figure 8. Secondary embryos

from 4mm primary cotyledon
explants. A. (UF-667 at 14 weeks
incubation) B. (EET at 10 weeks
incubation.) C. (EET at 14 weeks
incubation) D. (UF-676 at 16
weeks incubation)

IV. Conclusions:
1. Primary somatic embryogenesis is genotype-dependent in cacao. Genotypes UF676, UF667, EET387, AMAZ15 and PA120 responded to primary embryogenesis induction,

whereas PA137RUQ, Amelonado, PA120XGNVLO, and ICS95 were irresponsive.

2. Secondary somatic embryogenesis is genotype-dependent in cacao. Genotypes UF676, UF667, EET387, responded to secondary embryogenesis induction, whereas and AMAZ15
was irresponsive.
3. Genotype EET387 was the most responsive genotype to both primary and secondary embryogenesis induction out of all genotypes tested.
4. This study represents a first step toward cloning cacao genotypes with desirable genetic traits for the USDA breeding program

The authors want to thank Dr. Mark

Guiltinan and Dr. Siela Maximova. This
project would not have been possible
without the support of the STU Summer
Research Institute and the USDA-HSI
funded grant consortium FCCAgE, its
staff, the USDA agency directors and
supervisors.

VII. References
Zhijian, L. , Traore, A., Maximova, S.
and Guiltinan, M. 1998. Somatic
embryogenesis and plant
regeneration from floral explants
of cacao (Theobroma cacao L.)
using thidiazuron. In Vitro Cell
Dev. Biol.-Plant 34:293-299.
PennState College of Agricultural
Sciences. 2010. Integrated system
for vegetative propagation of
cacao. Protocol Book.
Maximova, S.N., Alemanno L., Young,
A. Ferriere, N., Traore, A. and
Guilinan, M.J. 2002. Efficiency,
genotypic variability and cellular
origin of primary and secondary
somatic embryogenesis of
Theobroma cacao L. In Vitro
Cellular and Developmental
Biology- Plant 38:252-259.