In vitro callus induction in important cacao cultivars Logo ars st thomas usda 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 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

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

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.

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. Preliminary Indicar how sections were cut MS media contain all the essential macro and micro nutrients

% 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

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

% 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

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

% 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

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 - cytokinin activity: shoot formation -Low concentrations of thidiazuron (0.0022 to 0.088 mg/liter) are effective for micropropagation organogenesis micropropagation Used previously for the induction of somatic embryos in cacao Very strong

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 (4-5mm) 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

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

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 veins Mature leaves petioles: 1cm long Culture Conditions: 2,4-D (2 concentrations: 1mg/L & 2mg/L) with dark incubation at 25.0o C

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.

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 The MDC-STU Summer Research Institute that provided the funding for this internship through a MISP grant