Organogenesis in Organogenesis in PeanutPeanut

Research Experience for UndergraduatesResearch Experience for UndergraduatesFood Biotechnology LaboratoryFood Biotechnology Laboratory

Alabama A&M UniversityAlabama A&M UniversityAntonio BrazeltonAntonio Brazelton

7/3/087/3/08

IntroductionIntroduction

What is tissue culture?What is tissue culture?

Why is it important?Why is it important?

How can tissue culture be applied to peanut How can tissue culture be applied to peanut improvement?improvement?

What is tissue culture?What is tissue culture? Tissue cultureTissue culture - is the culture and maintenance of plant - is the culture and maintenance of plant

cells and organs. cells and organs.

Important parameters in tissue cultureImportant parameters in tissue culture

- - Type of explants:Type of explants: leaf, stem, hypocotyl, root, petiole, etc. leaf, stem, hypocotyl, root, petiole, etc.

- - Medium: Medium: Macro nutrient and Micronutrients Vitamins pH

- - Hormones:Hormones: Cytokinins - TDZ, BAP Cytokinins - TDZ, BAP Auxins - NAAAuxins - NAA

- Photoperiod- Photoperiod

- Aseptic technique- Aseptic technique

Why is tissue culture important?Why is tissue culture important?

Plant tissue culture has value in studies such as: cell biology, Plant tissue culture has value in studies such as: cell biology, genetics, biochemistry, and many other research areas.genetics, biochemistry, and many other research areas.

Crop ImprovementCrop Improvement

Genetic TransformationGenetic Transformation

Plants can be produced quicklyPlants can be produced quickly

Plantlets can be used for germplasm conservationPlantlets can be used for germplasm conservation

PathwaysPathways

Organogenesis Organogenesis Relies on the production of organs either directly from Relies on the production of organs either directly from

an explant or callus structurean explant or callus structure

Somatic EmbryogenesisSomatic Embryogenesis Embryo-like structures which can develop into whole Embryo-like structures which can develop into whole

plants in a way that is similar to zygotic embryos are plants in a way that is similar to zygotic embryos are formed from somatic cellsformed from somatic cells

Existing MeristemsExisting Meristems Uses meristematic cells to regenerate whole plant.Uses meristematic cells to regenerate whole plant.

(Source:Victor. et al., 2004)(Source:Victor. et al., 2004)

Steps in Organogenesis

1. Phytohormone Perception

2. Dedifferentiation of differentiated cells to acquire competence.

3. Reentry of cells into the cell cycle

4. Organization of cell division to form specific organs primordia in meristem

(Source:Victor. et al, 2004)(Source:Victor. et al, 2004)

Peanut and Tissue CulturePeanut and Tissue Culture

Importance of PeanutImportance of Peanut

Current status of peanut organogenesisCurrent 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.

ObjectiveObjective

To compare peanut regeneration through To compare peanut regeneration through organogenesis using organogenesis using different hormones different hormones

and hormones and hormones at different concentrationsat different concentrations..

MethodsMethods

Fig 1. Flow Diagram for peanut regeneration

Sterilization

Germination

Regeneration

Organogenesis

(. Source: Li. et al, 2003)(. Source: Li. et al, 2003)

Hormone ConcentrationsHormone Concentrations*Prepare 3 medium solutions**Prepare 3 medium solutions*

TDZ (Thidiazuron) : 10uM, 15uM, 20uMTDZ (Thidiazuron) : 10uM, 15uM, 20uM BAP (Benzylamineopunine): 10uM, 15uM, 20uMBAP (Benzylamineopunine): 10uM, 15uM, 20uM HA (Humic Acid) :: 12.5 mg/L, 25 mg/L, 50mg/LHA (Humic Acid) :: 12.5 mg/L, 25 mg/L, 50mg/L

SterilizationSterilization*make sure seeds contain no fungi or bacteria**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)(Source:Victor.et al, 2004)

GerminationGerminationProtocol 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

11 77

66

55

44

33

22

Fig. 5 Steps in peanut regenerationFig. 5 Steps in peanut regeneration

1. Embryo in culture2. Germinating embryo3. Elongating shoot4. well elongated shoot5. Single well elongated

shoot6. Hypocotyl explant7. Contaminated plate

1. Embryo in culture2. Germinating embryo3. Elongating shoot4. well elongated shoot5. Single well elongated

shoot6. Hypocotyl explant7. Contaminated plate

!!Contamination!!

BAP

TDZ

HA

1

1

1

2

2

2

3

3

3

Germination using 3 different hormones at 3 different concentrations

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

Organogenesis11

11

11

22

22

22

33

33

33

TDZTDZ

HAHA

BAPBAP

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 A&M University, Dr. Konan and his students. North Alabama Center for Educational

Excellence Dr. Wang NRES STAFF REU Colleagues