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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?.

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Organogenesis in peanut l.jpg

Organogenesis in Peanut

Research Experience for Undergraduates

Food Biotechnology Laboratory

Alabama A&M University

Antonio Brazelton

7/3/08


Introduction l.jpg
Introduction

What is tissue culture?

Why is it important?

How can tissue culture be applied to peanut improvement?


What is tissue culture l.jpg
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 l.jpg
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.

(Source:Victor. 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

(Source:Victor. 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

Sterilization

Germination

Regeneration

Organogenesis

(. Source: Li. et al, 2003)

Fig 1. Flow Diagram for peanut regeneration


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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 l.jpg
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 l.jpg
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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2

1

3

5

6

4

7

  • Embryo in culture

  • Germinating embryo

  • Elongating shoot

  • well elongated shoot

  • Single well elongated shoot

  • Hypocotylexplant

  • Contaminated plate

Fig. 5 Steps in peanut regeneration



Slide16 l.jpg

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 concentrations

  • 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 concentrations

REGENERATION


Organogenesis protocol l.jpg

Cut hypocotyl and reculture in same hormone concentration. concentrations

Organogenesis Protocol


Organogenesis l.jpg
Organogenesis concentrations


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Organogenesis concentrations

1

2

3

BAP

2

1

3

TDZ

1

2

3

HA


Organogenesis23 l.jpg
Organogenesis concentrations


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Conclusions of Organogenesis concentrations

  • 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 concentrations

  • 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 concentrations

  • A&M University,

  • Dr. Konan and his students.

  • North Alabama Center for Educational Excellence

  • Dr. Wang

  • NRES STAFF

  • REU Colleagues