1 / 26

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

ornice
Download Presentation

In vitro callus induction in important cacao cultivars

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. In vitro callus induction in important cacao cultivars 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

  3. Purpose of This Project • To induce callus in different cacao genotypes as a non-photosynthetic source for DNA and RNA isolation

  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

  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: Murashigeand Skoog(MS) media Light incubation at 25°C.

  7. Results 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

  9. Results • 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

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

  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

  14. 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

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

  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 mM2,4-D -9 mM2,4-D & 22.7 nM TDZ -9 mM2,4-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. Results • 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 mM2,4-D -9 mM2,4-D & 22.7 nM TDZ -9 mM2,4-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. 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, Juglansregia 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.

  26. Acknowledgments: • USDA: • Dr. Heath • Dr. Schnell • Dr. Kuhn • Cecile Tondo • Barbie Freeman • Wilber Quintanilla • DayanaRodezno • STU: • Dr. Maul • School of Science • The Science Fellows Program • The MDC-STU Summer Research for funding the internships through a MSIP grant

More Related