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Organogenesis in Peanut






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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?.
Organogenesis in Peanut

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Slide 1

Organogenesis in Peanut

Research Experience for Undergraduates

Food Biotechnology Laboratory

Alabama A&M University

Antonio Brazelton

7/3/08

Slide 2

Introduction

What is tissue culture?

Why is it important?

How can tissue culture be applied to peanut improvement?

Slide 3

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

Slide 4

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

Slide 5

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)

Slide 6

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)

Slide 7

Peanut and Tissue Culture

Importance of Peanut

Current status of peanut organogenesis

Slide 8

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.

Slide 9

Objective

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

Slide 10

Methods

Sterilization

Germination

Regeneration

Organogenesis

(. Source: Li. et al, 2003)

Fig 1. Flow Diagram for peanut regeneration

Slide 11

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

Slide 12

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)

Slide 13

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

Slide 14

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

Slide 15

!!Contamination!!

Slide 16

Germination using 3 different hormones at 3 different concentrations

2

3

1

BAP

2

3

1

TDZ

2

1

3

HA

Slide 18

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.

Slide 19

Regenerated Explant

REGENERATION

Slide 20

Cut hypocotyl and reculture in same hormone concentration.

Organogenesis Protocol

Slide 21

Organogenesis

Slide 22

Organogenesis

1

2

3

BAP

2

1

3

TDZ

1

2

3

HA

Slide 23

Organogenesis

Slide 24

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.

Slide 25

Future Research

  • To find the effect of other factors such as,

    - pH

    - temperature

    - nutrients

    - vitamins and

    - enzymatic poisons

    on peanut germination and regeneration.

Slide 26

Acknowledgements

  • A&M University,

  • Dr. Konan and his students.

  • North Alabama Center for Educational Excellence

  • Dr. Wang

  • NRES STAFF

  • REU Colleagues


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