In vitro callus induction in important cacao cultivars

In vitro callus induction in important cacao cultivars Natalia Pelaez Claudia Arévalo Mentor: Dr. Pilar Maul

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

Purpose of This Project • To induce callus in different cacao genotypes as a nonphotosynthetic source for DNA and RNA isolation

Experiments- 2 stages I. Decontamination Bleach(Na. Cl. O) 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: 1 cm 2 • Culture Conditions: Murashige and Skoog (MS) media Light incubation at 25°C.

Results % Contamination in Amelonado and IMC 20 tissues Treatment # of Plates Contamination Wk 1 Percentage Contamination 5 % 20 min 6 6 100% 10 % 20 min 6 6 100% 15 % 20 min 6 4 67% Total 18 16 89% 15% bleach treatment for 15 min was not strong enough to decontaminate cacao tissues

2. Decontamination experiment in cultivar Matino 1 -6 • Bleach treatments: 20% for 15 min 20% for 20 min 20% for 15 min x 2 • Explants: Leaves: 1 cm 2 , reddish and green, w/ veins and w/o veins Petioles: 1 cm long. • Culture conditions: MS + 2, 4 -D (1 mg/L or 2 mg/L) Dark incubation at 25. 0 o C

Results % Contamination in Matino 1 -6 (with plant growth regulator 2, 4 -D) leaves and petioles Treatment 20 % 15 min 20 % 20 min 20 % 15 min x 2 Contamination # of plates Wk 2 Wk 3 Wk 4 16 0 0 0 12 0 0 0 13 7 1 1 Total Contamination percentage 0 0% 9 69. 23% • 20% for 15 min best decontamination treatment Less tissue damage

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. 0 o C

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

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 +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 -5 mm) from greenhouse -Staminoides -Petals • Bleach treatment: 10% for 5 min 5% for 10 min • Conditions: 2, 4 -D hormone (1 M & 2 M) incubation in dark at 25. 0 o C

Results SCA-6 (2, 4 -D Hormone) Callus Induction Tissue Response # of Explants Wk 1 Wk 2 Percentage Staminoides 48 0 13 27. 00% Petals 45 0 10 22% • Response on callus induction (2, 4 -D): Staminoides: 27% Petals: 22%

2. Callus induction in Flowers using 2, 4 D and TDZ • • Tissue: Cacao unopened immature flowers (4 -5 mm) from greenhouse one Genotypes: - Iquitos - Marañon -Nacional/Curaray • Bleach treatment: -10% for 5 min • Explants: -Staminoides -Petals • Culture conditions: MS + -No PGRs -9 m. M 2, 4 -D & 22. 7 n. M TDZ -9 m. M 2, 4 -D & 45. 5 n. M TDZ

Inducing callus in Cacao leaves

1. Callus induction in Matino 1 -6 leaves with 2, 4 -D Hormone • Genotype: Matino 1 -6 (CC 267) • Explants: Leaves: 1 cm 2 , Young leaves: reddish and green, w/t veins – w/o veins Mature leaves petioles: 1 cm long • Culture Conditions: 2, 4 -D (2 concentrations: 1 mg/L & 2 mg/L) with dark incubation at 25. 0 o C

Results Callus inducion in Matino (CC 267) with 2, 4 -D Tissue Young leaves Green Red Old leaves Petioles Total 2, 4 -D 1 mg/L 2 mg/L # of plates 4 4 6 7 6 6 41 Callus formation Wk 2 Wk 3 0 0 2 1 0 0 1 1 2 0 7 3 • Red young leaves and petioles have more tendency to produce callus.

2. Callus induction in young leaves and petioles using 2, 4 -D and TDZ • • Tissue: Cacao unopened immature flowers (4 -5 mm) from greenhouse Genotypes: - Iquitos - Marañon -Nacional/Curaray • Explants: - Matino 1 -6 -Young Reddish Leaves - Petioles • Bleach treatment: - 20% for 15 min • Culture conditions: MS + -No PGRs -9 m. M 2, 4 -D & 22. 7 n. M TDZ -9 m. M 2, 4 -D & 45. 5 n. M TDZ

Conclusions • • • Decontamination Experiment 1: – 15% bleach treatment for 15 min was not strong enough to decontaminate cacao tissues Decontamination Exp. 2: – 20% for 15 min 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. Mc. Granahan. 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: : STU: Dr. Maul Dr. Heath School of Science Dr. Schnell The Science Fellows Program Dr. Kuhn Cecile Tondo Barbie Freeman Wilber Quintanilla Dayana Rodezno The MDC-STU Summer Research for funding the internships through a MSIP grant