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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

Organogenesis in Peanut

Research Experience for Undergraduates

Food Biotechnology Laboratory

Alabama A&M University

Antonio Brazelton

7/3/08

introduction
Introduction

What is tissue culture?

Why is it important?

How can tissue culture be applied to peanut improvement?

what is tissue culture
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
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
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
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)

peanut and tissue culture
Peanut and Tissue Culture

Importance of Peanut

Current status of peanut organogenesis

plan of action
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
Objective

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

methods
Methods

Sterilization

Germination

Regeneration

Organogenesis

(. Source: Li. et al, 2003)

Fig 1. Flow Diagram for peanut regeneration

hormone concentrations prepare 3 medium solutions
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
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
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
slide14

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

conclusions made from regeneration
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.
organogenesis22
Organogenesis

1

2

3

BAP

2

1

3

TDZ

1

2

3

HA

conclusions of 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
Future Research
  • To find the effect of other factors such as,

- pH

- temperature

- nutrients

- vitamins and

- enzymatic poisons

on peanut germination and regeneration.

acknowledgements
Acknowledgements
  • A&M University,
  • Dr. Konan and his students.
  • North Alabama Center for Educational Excellence
  • Dr. Wang
  • NRES STAFF
  • REU Colleagues