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Organogenesis inOrganogenesis in
PeanutPeanutResearch Experience for UndergraduatesResearch Experience for Undergraduates
Food 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 peanutHow can tissue culture be applied to peanutimprovement?improvement?
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What is tissue culture?What is tissue culture? Tissue cultureTissue culture - is the culture and maintenance of plant cells- is the culture and maintenance of plant cells
and organs.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, BAPCytokinins - 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
OrganogenesisOrganogenesis
Relies on the production of organs either directly from an explant or callusRelies on the production of organs either directly from an explant or callus
structurestructure
Somatic EmbryogenesisSomatic Embryogenesis Embryo-like structures which can develop into whole plants in a way that isEmbryo-like structures which can develop into whole plants in a way that is
similar to zygotic embryos are formed from somatic cellssimilar to zygotic embryos are formed 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 useorganogenesis to produce whole plant from
the hypocotyledon.
Use different parameters to find optimumconditions of regeneration and
organogenesis.
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ObjectiveObjective
To compare peanut regeneration throughTo compare peanut regeneration throughorganogenesis usingorganogenesis using different hormonesdifferent hormones
and hormonesand 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 SterilizationSoak seeds in 20% Clorox (2x) 30minRinse with sterile water (2-3x)Soak seeds in sterile water (1 hour)
Soak again and Leave OvernightRinse with sterile water (2-3x)
(Source:Victor.et al, 2004)(Source:Victor.et al, 2004)
http://images.google.com/imgres?imgurl=http://www.thenutfactory.com/photos/nuts-peanuts-blanched-ns.JPG&imgrefurl=http://www.thenutfactory.com/section.asp%3Fcategory%3Dnuts-peanuts&h=300&w=400&sz=12&hl=en&start=13&um=1&tbnid=gr1e3OVz7wfXFM:&tbnh=93&tbnw=124&prev=/images%3Fq%3Dpeanuts%26um%3D1%26hl%3Den8/7/2019 Antonio Ppt [Search Engine com
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GerminationGermination
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
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1177
66
55
44
33
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Fig. 5 Steps in peanut regenerationFig. 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 explant7. Contaminated plate
1. Embryo in culture
2. Germinating embryo
3. Elongating shoot
4. well elongated shoot
5. Single well elongated
shoot
6. Hypocotyl explant7. Contaminated plate
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BAP
TDZ
HA
1
1
1
2
2
2
3
3
3
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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Organogenesis Protocol
Cut hypocotyl and
reculture in same
hormone
concentration.
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Organogenesis
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Organogenesis
11
11
11
22
22
22
33
33
33
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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