Organogenesis in

Peanut
Research Experience for Undergraduates
Food Biotechnology Laboratory
Alabama A&M University
Antonio Brazelton
7/3/08
Introduction
 What is tissue culture?

 Why is it important?

 How can tissue culture be applied to peanut

improvement?
 What is tissue culture?
 Tissue culture - is the culture and maintenance of plant cells
and organs.

 Important parameters in tissue culture

- Type of explants: leaf, stem, hypocotyl, root, petiole, etc.
- Medium:
 Macro nutrient and Micronutrients
 Vitamins
 pH

- Hormones:
 Cytokinins - TDZ, BAP
 Auxins - NAA
- Photoperiod
- Aseptic technique
Why is tissue culture important?

 Plant tissue culture has value in studies such as: cell biology,
genetics, biochemistry, and many other research areas.

 Crop Improvement

 Genetic Transformation

 Plants can be produced quickly

 Plantlets can be used for germplasm conservation

 Pathways
 Organogenesis
 Relies on the production of organs either directly from an explant or callus
structure

 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
 Uses meristematic cells to regenerate whole plant.

(Source:Victor. et al., 2004)

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)

Peanut and Tissue Culture

 Importance of Peanut

 Current status of peanut organogenesis

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.
Objective

To compare peanut regeneration through

organogenesis using different hormones
and hormones at different concentrations.
Methods Sterilization

Germination

Regeneration

Organogenesis

(. Source: Li. et al, 2003)

Fig 1. Flow Diagram for peanut regeneration

Hormone Concentrations
*Prepare 3 medium solutions*
*Prepare 3 medium solutions*

 TDZ (Thidiazuron) : 10uM, 15uM, 20uM

 BAP (Benzylamineopunine): 10uM, 15uM, 20uM
 HA (Humic Acid) :: 12.5 mg/L, 25 mg/L, 50mg/L
 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)

 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
 7
1 4

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

Fig. 5 Steps in peanut regeneration

!!Contamination!!
Germination using 3 different hormones at 3 different concentrations
1 2 3

BAP

1 2 3

TDZ

1 2 3

HA
 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.
Regenerated Explant
Organogenesis Protocol

 Cut hypocotyl and

reculture in same
hormone
concentration.
Organogenesis
Organogenesis
1 2 3
BAP

1 2 3
TDZ

1 2 3
HA
Organogenesis
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.
Future Research
 To find the effect of other factors such as,
- pH
- temperature
- nutrients
- vitamins and
- enzymatic poisons
on peanut germination and regeneration.
Acknowledgements
 A&M University,
 Dr. Konan and his students.
 North Alabama Center for Educational
Excellence
 Dr. Wang
 NRES STAFF
 REU Colleagues