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 - PowerPoint PPT Presentation
Inducing callus from various Cacao Genotypes for Mitochondrial DNA Extraction
In vitro callus induction in important cacao cultivars
Natalia PelaezClaudia ArvaloMentor: Dr. Pilar Maul
Logo ars st thomas usda1 Cacao - important to the Global economy: chocolate industry
Cacao Genome Project and the Cacao Breeding Program at the USDAMars, 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 Cacao is mainly grown in tropical rain forest around the globePlant 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
2Purpose of This ProjectTo 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 studies3Experiments- 2 stages
Decontamination Bleach(NaClO) treatments Concentration Length of Treatment
II. Callus Induction Various Explants (initial tissues) Various Genotypes Various Plant Growth RegulatorsSince 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.
4Stage 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 25C.
PreliminaryIndicar how sections were cutMS media contain all the essential macro and micro nutrients6Results15% bleach treatment for 15 min was not strong enough to decontaminate cacao tissues% Contamination in Amelonado and IMC 20 tissuesTreatment # of Plates Contamination Wk1 Percentage Contamination5 % 20 min 66100%10 % 20 min 66100%15 % 20 min 6467%Total 181689%Most of the cultures were contaminated
Therefore we decided to raise the levels of decontamination treatment.
72. 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 CImportance Genomic Sequence was found with Matino 1-6We had no seedlind material for Matino 1-68Results 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 1600000%20 % 20 min 1200000%20 % 15 min x2 13711969.23%93. Decontamination experiment in cultivar SCA-6Explants: 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 10Results: 10% for 5 min best bleach treatment for flowers: No contamination Tissue had less damage
% Contamination in SCA-6 Flowers TreatmentNumber Flowers Contamination PercentageWk 1Wk 25% for 10 min40125%10% for 5 min4000%Total80113%percentage11
Stage 2. Callus induction in Cacao tissues using plant growth regulators
12Plant growth regulators that induce rapid cell divisionTDZ (Thidiazuron) - Cytokinin activity - Organogenesis - Micropropagation - Somatic embryos in cacao2,4-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 micropropagationorganogenesis micropropagation Used previously for the induction of somatic embryos in cacaoVery strong
13Callus Experiments I. Inducing callus in Cacao flowers 2,4-D2,4-D +TDZII. Inducing callus in Cacao leaves2,4-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 InductionTissue# of Explants Response PercentageWk 1Wk 2Staminoides 4801327.00%Petals4501022%
Change green to lighter greenTake out total rowPercentages in a column17
2. Callus induction in Flowers using 2,4-D and TDZ
Tissue: Cacao unopened immature flowers (4-5mm) from greenhouse one Genotypes:- Iquitos- Maraon-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 platesCallus formation Wk 2 Wk 3Young leavesGreen1 mg/L4002 mg/L400Red 1 mg/L4212 mg/L421Old leaves1 mg/L6002 mg/L700Petioles 1 mg/L6112 mg/L620Total 4173
2. Callus induction in young leaves and petioles using 2,4-D and TDZTissue: Cacao unopened immature flowers (4-5mm) from greenhouse Genotypes:- Iquitos- Maraon-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 TDZConclusionsDecontamination Experiment 1: 15% bleach treatment for 15 min was not strong enough to decontaminate cacao tissuesDecontamination Exp. 2: 20% for 15min best decontamination treatment for leaves and petioles Less tissue damageDecontamination Exp.3: 10% for 5 min best bleach treatment for flowers No contamination , Tissue had less damageCallus 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 StepsContinue Experiment on Callus Induction with 2,4-D and TDZ in Flowers and Leaves in different cultivars. Induce callus in more important cacao cultivarsTest DNA and RNA extraction protocols in callusReferencesHuetteman, 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.
Dayana RodeznoSTU: 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