1. Organogenesis in Peanut Research Experience for Undergraduates Food Biotechnology Laboratory Alabama A&M University Antonio Brazelton 7/3/08

2. Introduction What is tissue culture? What is tissue culture? Why is it important? Why is it important? How can tissue culture be applied to peanut improvement? How can tissue culture be applied to peanut improvement?

3. What is tissue culture? Tissue culture - is the culture and maintenance of plant cells and organs. Tissue culture - is the culture and maintenance of plant cells and organs. Important parameters in tissue culture Important parameters in tissue culture - Type of explants: leaf, stem, hypocotyl, root, petiole, etc. - Medium: - Medium:  Macro nutrient and Micronutrients  Vitamins  pH - Hormones: - Hormones:  Cytokinins - TDZ, BAP  Auxins - NAA - Photoperiod - Aseptic technique

4. Why is tissue culture important? Plant tissue culture has value in studies such as: cell biology, genetics, biochemistry, and many other research areas. Plant tissue culture has value in studies such as: cell biology, genetics, biochemistry, and many other research areas. Crop Improvement Crop Improvement Genetic Transformation Genetic Transformation Plants can be produced quickly Plants can be produced quickly Plantlets can be used for germplasm conservation Plantlets can be used for germplasm conservation

5. Pathways Organogenesis Organogenesis  Relies on the production of organs either directly from an explant or callus structure Somatic Embryogenesis Somatic Embryogenesis  Embryo-like structures which can develop into whole plants in a way that is similar to zygotic embryos are formed from somatic cells Existing Meristems Existing Meristems  Uses meristematic cells to regenerate whole plant. (Source:Victor. et al., 2004)

6. Steps in Organogenesis 1. Phytohormone Perception 2. Dedifferentiation of differentiated cells to acquire competence. 3. Reentry of cells into the cell cycle 4. Organization of cell division to form specific organs primordia in meristem (Source:Victor. et al, 2004)

7. Peanut and Tissue Culture Importance of Peanut Importance of Peanut Current status of peanut organogenesis Current status of peanut organogenesis

8. Plan of Action To use two species of peanut for comparison. To germinate, regenerate and finally use organogenesis to produce whole plant from the hypocotyledon. Use different parameters to find optimum conditions of regeneration and organogenesis.

9. Objective To compare peanut regeneration through organogenesis using different hormones and hormones at different concentrations. To compare peanut regeneration through organogenesis using different hormones and hormones at different concentrations.

10. Methods Fig 1. Flow Diagram for peanut regeneration Sterilization Germination Regeneration Organogenesis (. Source: Li. et al, 2003)

11. Hormone Concentrations *Prepare 3 medium solutions* TDZ (Thidiazuron) : 10uM, 15uM, 20uM TDZ (Thidiazuron) : 10uM, 15uM, 20uM BAP (Benzylamineopunine): 10uM, 15uM, 20uM BAP (Benzylamineopunine): 10uM, 15uM, 20uM HA (Humic Acid) :: 12.5 mg/L, 25 mg/L, 50mg/L HA (Humic Acid) :: 12.5 mg/L, 25 mg/L, 50mg/L

12. Sterilization *make sure seeds contain no fungi or bacteria* Protocol for Sterilization Soak seeds in 20% Clorox (2x) 30min Rinse with sterile water (2-3x) Soak seeds in sterile water (1 hour) Soak again and Leave Overnight Rinse with sterile water (2-3x) (Source:Victor.et al, 2004)

13. Germination Protocol for Germination Sterilize hands with 70% Iso-proponol. Remove seed, split each down the center to reveal the embryo. Use knife to cut embryo away from endosperm. Collect embryos and proceed to culture. Use 10 embryos per plate

14. 1 7 6 5 4 3 2 Fig. 5 Steps in peanut regeneration 1.Embryo in culture 2.Germinating embryo 3.Elongating shoot 4. well elongated shoot 5.Single well elongated shoot 6.Hypocotyl explant 7.Contaminated plate

15. !!Contamination!!

16. BAP TDZ HA 1 1 1 2 2 2 3 3 3 Germination using 3 different hormones at 3 different concentrations

18. Conclusions made from Regeneration All 3 hormones bring about germination. On an average HA gave the best results for germination. Change in concentration of the hormones did not necessarily change the germination success. For both root and shoot germination HA gave the best results of the 3 hormones used.

19. Regenerated Explant

20. Organogenesis Protocol Cut hypocotyl and reculture in same hormone concentration.

21. Organogenesis

22. 1 1 1 2 2 2 3 3 3 TDZ HA BAP

23. Organogenesis

24. Conclusions of Organogenesis Peanut regeneration through organogenesis has been done. Growth regulators such as TDZ, BAP, and HA stimulate plant regeneration. Both TDZ and BAP produce more viable shoots during organogenesis. Lower concentrations gave better results. Ongoing work includes replicating the procedure using other species of peanut plant.

25. Future Research To find the effect of other factors such as, - pH - temperature - nutrients - vitamins and - enzymatic poisons on peanut germination and regeneration.

26. Acknowledgements A&M University, Dr. Konan and his students. North Alabama Center for Educational Excellence Dr. Wang NRES STAFF REU Colleagues