1. In vitro callus induction in important cacao cultivars
Logo ars st thomas usda
Natalia Pelaez
Claudia Arévalo
Mentor: Dr. Pilar Maul

2. Introduction
Cacao - important to the Global economy: chocolate industry
Cacao Genome Project and the Cacao Breeding Program at the USDA
Mars, Inc; USDA-ARS. SHRS; IBM
Released to the public on Sept. 15, 2010
Plant tissue culture
Production of callus
Previous work in cacao from flower staminoides
DNA and RNA isolation for genetic studies
Cacao is mainly grown in tropical rain forest around the globe
Plant tissue culture is a technique in which plant cells, tissues or organs are grown on artificial nutrient medium under sterile conditions.
Thanks to the release of the Cacao Genome Sequence by Mars, Incorporated , the (USDA-ARS), and IBM on September 15,2010.
Researchers now have access to information that will allow them to identify genes.
It is possible that young callus grown under dark conditions will provide good material for DNA extraction because young actively-dividing cells contain underdeveloped chloroplasts and less secondary metabolites.
Callus: Mass of undifferentiated cells that when grown under dar conditions are white, which means that their cloroplasts are underdeveloped and not yet producing clorophyll

3. Purpose of This Project
To induce callus in different cacao genotypes as a non-photosynthetic source for DNA and RNA isolation
The purpose of using tissue culture in our project is to produce large amounts of young actively-dividing cacao cells (callus) that can later be used to extract DNA and RNA for specific genomic studies

4. Experiments- 2 stages Decontamination Bleach(NaClO) treatments
Concentration
Length of Treatment
II. Callus Induction
Various Explants (initial tissues)
Various Genotypes
Various Plant Growth Regulators
Since plant tissue culture requires sterile growing conditions , the first step is to decontaminate tissues from bacteria and fungi. We used bleach (NaClO) treatments .
The purpose of our first experiments (Expt 1, 2 and 3) was to find the optimal decontamination treatment in order to introduce cacao tissue from greenhouse in tissue culture conditions.

5. Stage 1 - Decontamination
1. Several tissues from young plants - cultivars Amelonado & Iquitos (IMC 20)
2. Leaves and petioles - cultivar Matino 1-6
3. Flowers - cultivar SCA-6

6. 1. Decontamination experiment in cultivars Amelonado & Iquitos (IMC 20)
Bleach treatments: 5%,10% and 15% bleach for 20 minutes.
Explants from young plants:
-Stems
-Shoots
-Petioles
-Leaves: 1cm2
Culture Conditions:
Murashige and Skoog (MS) media
Light incubation at 25°C.
Preliminary
Indicar how sections were cut
MS media contain all the essential macro and micro nutrients

7. % Contamination in Amelonado and IMC 20 tissues
Results
% Contamination in Amelonado and IMC 20 tissues
Treatment
# of Plates
Contamination Wk1
Percentage Contamination
5 % 20 min 6
100%
10 % 20 min
15 % 20 min 4
67%
Total 18 16
89%
Most of the cultures were contaminated
Therefore we decided to raise the levels of decontamination treatment.
15% bleach treatment for 15 min was not strong enough to decontaminate cacao tissues

8. 2. Decontamination experiment in cultivar Matino 1-6
Bleach treatments:
20% for 15min
20% for 20 min
20% for 15min x2
Explants:
Leaves: 1cm2 , reddish and green, w/ veins and w/o veins
Petioles: 1cm long.
Culture conditions:
MS + 2, 4-D (1mg/L or 2mg/L)
Dark incubation at 25.0o C
Importance Genomic Sequence was found with Matino 1-6
We had no seedlind material for Matino 1-6

9. % Contamination in Matino 1-6 (with plant growth regulator 2, 4-D)
Results
% Contamination in Matino 1-6 (with plant growth regulator 2, 4-D)
leaves and petioles
Treatment
# of plates
Contamination
Total
Contamination percentage
Wk 2
Wk 3
Wk 4
20 % 15 min 16 0%
20 % 20 min 12
20 % 15 min x2 13 7 1 9
69.23%
20% for 15min best decontamination treatment
Less tissue damage

10. 3. Decontamination experiment in cultivar SCA-6
Explants: Flowers
Staminoides
Petals
Bleach treatments:
10% for 5 min
5% for 10 min
Culture conditions:
MS+ 2,4-D culture media
Dark incubation at 25.0o C
Explain why we decided to use 2 4 d simultaneously

11. % Contamination in SCA-6 Flowers
Results:
% Contamination in SCA-6 Flowers
Treatment
Number Flowers
Contamination
Percentage
Wk 1
Wk 2
5% for 10 min 4 1
25%
10% for 5 min 0%
Total 8
13%
10% for 5 min best bleach treatment for flowers:
No contamination
Tissue had less damage
percentage

12. Stage 2. Callus induction in Cacao tissues using plant growth regulators

13. Plant growth regulators that induce rapid cell division
TDZ (Thidiazuron)
- Cytokinin activity
- Organogenesis
- Micropropagation
- Somatic embryos in cacao
2,4-D(dichlorophenoxyacetic acid)
- Auxins
- Rapid cell division
- cytokinin activity: shoot formation
-Low concentrations of thidiazuron ( to mg/liter) are effective for micropropagation
organogenesis
micropropagation
Used previously for the induction of somatic embryos in cacao
Very strong

14. Callus Experiments I. Inducing callus in Cacao flowers
2,4-D +TDZ
II. Inducing callus in Cacao leaves
2,4-D( Matino 1-6)
2,4-D + TDZ

15. I. Inducing callus in Cacao flowers

16. 1. Callus Induction in SCA-6 flowers using 2,4-D
Explants: Cacao unopened immature flowers (4-5mm) from greenhouse
-Staminoides
-Petals
Bleach treatment:
10% for 5min
5% for 10 min
Conditions:
2,4-D hormone (1M & 2M)
incubation in dark at 25.0o C

17. SCA-6 (2,4-D Hormone) Callus Induction
Results
SCA-6 (2,4-D Hormone) Callus Induction
Tissue
# of Explants
Response
Percentage
Wk 1
Wk 2
Staminoides 48 13
27.00%
Petals 45 10
22%
Response on callus induction (2,4-D):
Staminoides: 27%
Petals: 22%
Change green to lighter green
Take out total row
Percentages in a column

18. 2. Callus induction in Flowers using 2,4-D and TDZ
Tissue: Cacao unopened immature flowers (4-5mm) from greenhouse one
Genotypes:
- Iquitos
- Marañon
-Nacional/Curaray
Bleach treatment:
-10% for 5min
Explants:
-Staminoides
-Petals
Culture conditions: MS +
-No PGRs
-9 mM 2,4-D
-9 mM 2,4-D & 22.7 nM TDZ
-9 mM 2,4-D & 45.5 nM TDZ

19. Inducing callus in Cacao leaves

20. 1. Callus induction in Matino 1-6 leaves with 2,4-D Hormone
Genotype:
Matino 1-6 (CC267)
Explants:
Leaves: 1cm2 ,
Young leaves: reddish and green, w/t veins – w/o veins
Mature leaves
petioles: 1cm long
Culture Conditions:
2,4-D (2 concentrations: 1mg/L & 2mg/L)
with dark incubation at 25.0o C

21. Callus inducion in Matino (CC267) with 2, 4-D
Results
Callus inducion in Matino (CC267) with 2, 4-D
Tissue
2,4-D
# of plates
Callus formation
Wk 2
Wk 3
Young leaves
Green 
1 mg/L 4
2 mg/L
Red 2 1
Old leaves 6 7
Petioles
Total 41 3
Red young leaves and petioles have more tendency to produce callus.

22. 2. Callus induction in young leaves and petioles using 2,4-D and TDZ
Tissue: Cacao unopened immature flowers (4-5mm) from greenhouse
Genotypes:
- Iquitos
- Marañon
-Nacional/Curaray
- Matino 1-6
Bleach treatment:
- 20% for 15min
Explants:
-Young Reddish Leaves
- Petioles
Culture conditions: MS +
-No PGRs
-9 mM 2,4-D
-9 mM 2,4-D & 22.7 nM TDZ
-9 mM 2,4-D & 45.5 nM TDZ

23. Conclusions Decontamination Experiment 1:
15% bleach treatment for 15 min was not strong enough to decontaminate cacao tissues
Decontamination Exp. 2:
20% for 15min best decontamination treatment for leaves and petioles
Less tissue damage
Decontamination Exp.3:
10% for 5 min best bleach treatment for flowers
No contamination ,
Tissue had less damage
Callus Induction in flowers:
Response on callus induction (2,4-D):
Staminoides: 27% Petals: 22%
Callus Induction Leaves:
Red young leaves and petioles have more tendency to produce callus.

24. Future Steps
Continue Experiment on Callus Induction with 2,4-D and TDZ in Flowers and Leaves in different cultivars.
Induce callus in more important cacao cultivars
Test DNA and RNA extraction protocols in callus

25. References
Huetteman, C. and A. Preece Thidiazuron: a potent cytokinin for woody plant tissue culture. Plant Cell Tissue Organ Cult. 33:
Tuleke, W. and G. McGranahan Somatic embryogenesis and plant regeneration from cotyledons of walnut, Juglans regia L. Plant Science 40:
Zhijian, L. , Traore, A., Maximova, S. and Guiltinan, M Somatic embryogenesis and plant regeneration from floral explants of cacao (Theobroma cacao L.) using thidiazuron. In Vitro Cell Dev. Biol.-Plant 34:

26. Acknowledgments: USDA: Dr. Heath Dr. Schnell Dr. Kuhn Cecile Tondo
Barbie Freeman
Wilber Quintanilla
Dayana Rodezno
STU:
Dr. Maul
School of Science
The Science Fellows Program
The MDC-STU Summer Research for funding the internships through a MSIP grant
The MDC-STU Summer Research
Institute that provided the funding for this internship through a MISP grant