organogenesis in peanut research experience for undergraduates food biotechnology laboratory alabama...
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Organogenesis in Organogenesis in PeanutPeanut
Research Experience for UndergraduatesResearch Experience for UndergraduatesFood Biotechnology LaboratoryFood Biotechnology Laboratory
Alabama A&M UniversityAlabama A&M UniversityAntonio BrazeltonAntonio Brazelton
7/3/087/3/08
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IntroductionIntroduction
What is tissue culture?What is tissue culture?
Why is it important?Why is it important?
How can tissue culture be applied to peanut How can tissue culture be applied to peanut improvement?improvement?
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What is tissue culture?What is tissue culture? Tissue cultureTissue culture - is the culture and maintenance of plant - is the culture and maintenance of plant
cells and organs. cells and organs.
Important parameters in tissue cultureImportant parameters in tissue culture
- - Type of explants:Type of explants: leaf, stem, hypocotyl, root, petiole, etc. leaf, stem, hypocotyl, root, petiole, etc.
- - Medium: Medium: Macro nutrient and Micronutrients Vitamins pH
- - Hormones:Hormones: Cytokinins - TDZ, BAP Cytokinins - TDZ, BAP Auxins - NAAAuxins - NAA
- Photoperiod- Photoperiod
- Aseptic technique- Aseptic technique
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Why is tissue culture important?Why is tissue culture important?
Plant tissue culture has value in studies such as: cell biology, Plant tissue culture has value in studies such as: cell biology, genetics, biochemistry, and many other research areas.genetics, biochemistry, and many other research areas.
Crop ImprovementCrop Improvement
Genetic TransformationGenetic Transformation
Plants can be produced quicklyPlants can be produced quickly
Plantlets can be used for germplasm conservationPlantlets can be used for germplasm conservation
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PathwaysPathways
Organogenesis Organogenesis Relies on the production of organs either directly from Relies on the production of organs either directly from
an explant or callus structurean explant or callus structure
Somatic EmbryogenesisSomatic Embryogenesis Embryo-like structures which can develop into whole Embryo-like structures which can develop into whole
plants in a way that is similar to zygotic embryos are plants in a way that is similar to zygotic embryos are formed from somatic cellsformed from somatic cells
Existing MeristemsExisting Meristems Uses meristematic cells to regenerate whole plant.Uses meristematic cells to regenerate whole plant.
(Source:Victor. et al., 2004)(Source:Victor. et al., 2004)
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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)(Source:Victor. et al, 2004)
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Peanut and Tissue CulturePeanut and Tissue Culture
Importance of PeanutImportance of Peanut
Current status of peanut organogenesisCurrent status of peanut organogenesis
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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.
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ObjectiveObjective
To compare peanut regeneration through To compare peanut regeneration through organogenesis using organogenesis using different hormones different hormones
and hormones and hormones at different concentrationsat different concentrations..
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MethodsMethods
Fig 1. Flow Diagram for peanut regeneration
Sterilization
Germination
Regeneration
Organogenesis
(. Source: Li. et al, 2003)(. Source: Li. et al, 2003)
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Hormone ConcentrationsHormone Concentrations*Prepare 3 medium solutions**Prepare 3 medium solutions*
TDZ (Thidiazuron) : 10uM, 15uM, 20uMTDZ (Thidiazuron) : 10uM, 15uM, 20uM BAP (Benzylamineopunine): 10uM, 15uM, 20uMBAP (Benzylamineopunine): 10uM, 15uM, 20uM HA (Humic Acid) :: 12.5 mg/L, 25 mg/L, 50mg/LHA (Humic Acid) :: 12.5 mg/L, 25 mg/L, 50mg/L
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SterilizationSterilization*make sure seeds contain no fungi or bacteria**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)(Source:Victor.et al, 2004)
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GerminationGerminationProtocol 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
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11 77
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Fig. 5 Steps in peanut regenerationFig. 5 Steps in peanut regeneration
1. Embryo in culture2. Germinating embryo3. Elongating shoot4. well elongated shoot5. Single well elongated
shoot6. Hypocotyl explant7. Contaminated plate
1. Embryo in culture2. Germinating embryo3. Elongating shoot4. well elongated shoot5. Single well elongated
shoot6. Hypocotyl explant7. Contaminated plate
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!!Contamination!!
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BAP
TDZ
HA
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Germination using 3 different hormones at 3 different concentrations
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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.
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Regenerated Explant
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Organogenesis Protocol
Cut hypocotyl and reculture in same hormone concentration.
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Organogenesis
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Organogenesis11
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TDZTDZ
HAHA
BAPBAP
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Organogenesis
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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.
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Future Research To find the effect of other factors such as,
- pH
- temperature
- nutrients
- vitamins and
- enzymatic poisons
on peanut germination and regeneration.
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Acknowledgements A&M University, Dr. Konan and his students. North Alabama Center for Educational
Excellence Dr. Wang NRES STAFF REU Colleagues