In vitro callus induction in important cacao cultivars

Natalia PelaezClaudia ArévaloMentor: Dr. Pilar Maul

• 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

Introduction

Purpose of This Project

• To induce callus in different cacao genotypes as a non-photosynthetic source for DNA and RNA isolation

Experiments- 2 stages

I. Decontamination Bleach(NaClO) treatments –Concentration– Length of Treatment

II. Callus Induction • Various Explants (initial tissues)• Various Genotypes • Various Plant Growth Regulators

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

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.

Results

15% bleach treatment for 15 min was not strong enough to decontaminate cacao tissues

% Contamination in Amelonado and IMC 20 tissues

Treatment # of Plates Contamination Wk1

Percentage Contamination

5 % 20 min 6 6 100%

10 % 20 min 6 6 100%

15 % 20 min 6 4 67%

Total 18 16 89%

 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

Results

• 20% for 15min best decontamination treatment Less tissue damage

% Contamination in Matino 1-6 (with plant growth regulator 2, 4-D)

leaves and petioles

Treatment # of plates

Contamination

TotalContamination

percentageWk 2 Wk 3 Wk 420 % 15

min 16 0 0 0 0 0%20 % 20

min 12 0 0 0 0 0%20 % 15 min x2 13 7 1 1

9 69.23%

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

Results:

•10% for 5 min best bleach treatment for flowers: No contamination Tissue had less damage

% Contamination in SCA-6 Flowers

TreatmentNumber Flowers

Contamination PercentageWk 1 Wk 2

5% for 10 min

4 0 1 25%

10% for 5 min 4 0 0 0%

Total 8 0 1 13%

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

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

Callus Experiments • I. Inducing callus in Cacao flowers – 2,4-D– 2,4-D +TDZ

• II. Inducing callus in Cacao leaves– 2,4-D( Matino 1-6)– 2,4-D + TDZ

I. Inducing callus in Cacao flowers

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

Results

• Response on callus induction (2,4-D): Staminoides: 27% Petals: 22%

SCA-6 (2,4-D Hormone) Callus Induction

Tissue # of Explants

Response PercentageWk 1 Wk 2

Staminoides 48 0 13 27.00%

Petals 45 0 10 22%

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

Inducing callus in Cacao leaves

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 veinsMature leaves

petioles: 1cm long

• Culture Conditions: 2,4-D (2 concentrations: 1mg/L & 2mg/L) with dark incubation at 25.0o C

Results

• Red young leaves and petioles have more tendency to produce callus.

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 0 02 mg/L 4 0 0

Red 1 mg/L 4 2 12 mg/L 4 2 1

Old leaves 1 mg/L 6 0 02 mg/L 7 0 0

Petioles 1 mg/L 6 1 12 mg/L 6 2 0

Total   41 7 3

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

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.

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

References

Huetteman, C. and A. Preece. 1993. Thidiazuron: a potent cytokinin for woody plant tissue culture. Plant Cell Tissue Organ Cult. 33: 105-119

Tuleke, W. and G. McGranahan. 1985. Somatic embryogenesis and plant regeneration from cotyledons of walnut, Juglans regia L. Plant Science 40: 57-63.

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.

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