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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%3Den


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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
http://wc.pima.edu/~bfiero/tucsonecology/plants/images/peanut.jpg


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1177

66

55

44

33

22

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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