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.
2.
3.
4.
Phytohormone Perception
Dedifferentiation of differentiated cells to
acquire competence.
Reentry of cells into the cell cycle
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*



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
1
2
3
4
5
6
Fig. 5 Steps in peanut regeneration
7
1.
2.
3.
4.
5.
Embryo in culture
Germinating embryo
Elongating shoot
well elongated shoot
Single well elongated
shoot
6. Hypocotyl explant
7. Contaminated plate
!!Contamination!!
Germination using 3 different hormones at 3 different concentrations
1
2
3
1
2
3
1
2
3
BAP
TDZ
HA
Peanut germination compared
10
9
No. of successful germination
8
7
6
5
4
3
2
1
0
T10
T15
T20
B10
B15
B20
Hormones and their conc.
HA50
HA100
HA200
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
HA
2
3
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




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A&M University,
Dr. Konan and his students.
North Alabama Center for Educational
Excellence
Dr. Wang
NRES STAFF
REU Colleagues