Organogenesis in Peanut

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Organogenesis in Peanut

• Research Experience for Undergraduates
• Food Biotechnology Laboratory
• Alabama AM University
• Antonio Brazelton
• 7/3/08

2
Introduction

• What is tissue culture?
• Why is it important?
• How can tissue culture be applied to peanut
  improvement?

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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

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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

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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.

(SourceVictor. et al., 2004)
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Steps in Organogenesis

• 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

(SourceVictor. et al, 2004)
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Peanut and Tissue Culture

• Importance of Peanut
• Current 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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Objective

• To compare peanut regeneration through
  organogenesis using different hormones and
  hormones at different concentrations.

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Methods
(. Source Li. et al, 2003)
Fig 1. Flow Diagram for peanut regeneration
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Hormone ConcentrationsPrepare 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

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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)

(SourceVictor.et al, 2004)
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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

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• Embryo in culture
• Germinating embryo
• Elongating shoot
• well elongated shoot
• Single well elongated shoot
• Hypocotyl explant
• Contaminated plate

Fig. 5 Steps in peanut regeneration
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!!Contamination!!
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Germination using 3 different hormones at 3
different concentrations
2
3
1
BAP
2
3
1
TDZ
2
1
3
HA
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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
REGENERATION
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Organogenesis Protocol

• Cut hypocotyl and reculture in same hormone
  concentration.

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Organogenesis
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Organogenesis
1
2
3
BAP
2
1
3
TDZ
1
2
3
HA
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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

• AM University,
• Dr. Konan and his students.
• North Alabama Center for Educational Excellence
• Dr. Wang
• NRES STAFF
• REU Colleagues