establishment of cell suspension callus culture from leaves andrographis paniculata sp. and...

FPE 2153

TISSUE CULTURE TECHNOLOGY

Establishment Of Cell Suspension Callus Culture from Leaves Andrographis paniculata sp.

&

Micropropagation of Borreria laevicaulis sp.

1) NURUL AIN NABILAH BINTI MOHAMAD ZAWAWI (B11A329)2) AZIFAH BINTI MOHD SUHAIMI (B11A038)3) NOR SYAKIRA BINTI MAT (B11A289)4) FARAH NUR AYU BINTI AHMAD (B11A068)5) MOHD ADIB BIN MOHD NAFI (B11A201)

INTRODUCTION

Plant Tissue Culture

Tissue culture is a technique used for the growth and

maintenance of tissue and organ growth in aseptic culture (in

vitro).Plant tissue culture is a culturing

of any part of plants in a growth media.

Micropropagation

Micropropagation is one of the techniques in plant tissue

culture. It is the practice of rapidly

multiplying stock plant material to produce a large number of progeny plants, using modern plant tissue culture methods.

Borreria laevis sp.

B. laevis (Lam.) Griseb. (Syn.: S. laevis Roxb. and S. assurgens Ruiz and Pavon) is a small herb found

in the tropical regions of Asia.used to treat kidney pain.

Cell suspension of callus culture

A plant cell callus consists of somatic undifferentiated cells from an adult

subject plant . The callus can be suspended in a solid callus induction media containing all the required nutrients and elements to allow for optimal growth which acts to turn all

cells into undifferentiated cells.

The cells will continuously grow until one of the factors becomes limiting causing

cell growth to slow.

Andrographis paniculata sp. (Hempedu Bumi)

Uses : Treatment for diabetes, high blood

pressure. asthma, arteriosclerosis, chest pain due to heart disease,

malaria, fever, stimulates the activity of the stomach, tonic, restoring body functions, snake bites and venomous

stings.

MATERIAL AND APPARATUS

MATERIAL AND APPARATUS OF MICROPROPAGATION

1) Tween 202) Ethanol 75%3) Mercury chloride

0.5%4) MS media5) Hormone-

benzylaminopurine (BAP )and Kinetin(Kn)6) Forceps

7) Scalper8) Surgical blade9) Petri disk10)Spirit lamp11)Beaker12)Internode from

Borreria laevicaulis sp.

MATERIAL AND APPARATUS FOR CALLUS CULTURE

1) Tween 202) Ethanol 75%3) Mercury

chloride 0.5%4) MS media5) Hormone-

naphthalene acetic acid (NAA)

6) Forceps7) Scalper

8) Surgical blade9) Petri disk10)Spirit lamp11)Beaker12)Leaves of

Andrographis paniculata sp. (Hempedu Bumi)

MEDIA PREPARATION

• (macro, micro, vitamin, hormones)

Stock Solutions

• Adjust volume to 800 mL

Volume adjustment

• (5.4-5.8) • Use

either HCl or NaOH

Check pH

• Add 30 g Sucrose – carbon source

• 8 g Agar – gelling agent

Organic elements,

gelling agent

• Dispense into culture vessels

• petri dish, test tube, glass jar

Pouring media

• Autoclave the medium

• Make it slang

Autoclave

MICROPROPAGATION

SELECTION OF PLANT

MATERIAL

* Cell, tissue or organ of a plant that is used to start in vitro

cultures – axillary bud

* >95% of all micropropagation,Genetically stable,

Simple and straightforward

SURFACE STERILISATION

* Washing removes endemic surface contaminants* Bacteria and fungi will overgrow the explant on the medium unless they are removed

* Pre-treatments to clean up the explant

* Detergents, Sterilants and Antibiotics, HgCl2, H2O2 , Chlorax

MULTIPLICATION

* Direct adventitious

organ formation

*no intervention of callus

* the cells initiation begin to develop

The leaf is quickly

rinsed under cool tap water

Then the leaf is wash

in water with 0.1% detergent for 3-4

minute and the leaf is gently agitated

every 20-30 seconds during the

washing step.

The leaf is gently

agitated in 10% bleach

solution for 10 minutes. The beaker should be filled three-fourth full with

the bleach solution.

RESULT

Result of Callus Culture : Leaves of Hempedu Bumi

Week

Total of plate

Callus with

root

Callus without

root

Explant without

respond 1

(18/11/2013)

18

4

10

4

2

(25/11/2013)

18

6

8

4

3

(2/12/2013)

18

7

7

4

a) Callus without root b) Callus with root

Before After

Result of Micropropagation Borrheria laevicallus

1st Batch (Use BAP Hormone)

Hormone concentration

Green in colour

Brown in colour

Contamination

BAP 1.0

4

2

2

BAP 1.5

2

0

3

BAP 2.0

2

4

1

Total of tube culture

8

6

6

a) Green in colour b) brown in colour c) contamination

2nd Batch (Use KN Hormone)

Hormone

concentration

Green in colour

Brown in colour

Contamination

KN 1.0

3

4

3

KN 2.0

2

6

1

KN 3.0

6

2

1

Total of tube culture

11

12

5

a) Green in colour b) brown in colour c) contamination

DISCUSSION

Advantages of tissue culture :

Reduce time to propagate plant

Save time for crop improvement selection

Can create potential disease-free plants

Save space and reduce cost for land use

Has pharmaceutical properties

Can conserve endangered species

Can be use to manage genetic resources

Is not limited by seasonal change

The most crucial part in micro propagation is when establish an

aseptic culture.It is really important to fully sterilize the parent plant to make sure it does not bring any harmful pathogen that can contaminate the cultured tissue.Safety precautions have to be fully

utilised to avoid any further contamination.

The reasons for contamination occur

1) Sterilization process2) Airborne particles

3) Dirty lab coat and cloth4) Water source

5) Equipment6) Non-sterile apparatus7) Careless in culturing

How to reduce contamination?Sterilization of explant should be done thoroughly

at least twice which is in the washing area and second time in the laminar flow hood.

Make sure that the air flow from outside when entering is as low as possible to avoid airborne

contaminate from outside.

Wear a clean lab coat and cloth at all time inside the lab

Create a properly aseptic working area inside the laminar flow

Autoclave apparatus properly to kill all contaminants

Use water that is quality ensured

Distilled water has to be distill properly to remove all particles and autoclaved for

secondary sterilization on explant.

Use double or triple distillation, RO, ion exchange or ultrafiltration to remove

trace metal, bacteria and other organic compound.

Use a sterile chemical

CONCLUSION

Result of Callus Culture : Leaves of Hempedu Bumi

The media used in this propagation is NAA 1.0, 1.5, 2.0 and 3.0.

Based on the result, we can conclude that the media used for NAA 1.0 and 1.5 is the most ideal media for the callus culture of Hempedu

Bumi

Because the callus in media NAA 1.0 and 1.5 has produced more roots.

Result of Micropropagation Borreria laevicaulis

1st Batch (Use BAP Hormone)

Media used in this propagation containing

BAP(benzylaminopurine) 1.0, 1.5, and 2.0.

The shoots produced more in media BAP 1.0 rather than 1.5 and 2.0.

2nd Batch (Use KN Hormone)Using kinetin hormone with concentration 1.0,

2.0 and 3.0. media which containing KN 3.0 has more active

shooting explant rather than KN 1.0 and 2.0.

KN 3.0 can be considered as ideal media for micropropagation of Borreria sp.

Explant without respond(turn to brown) is higher than green in color.

Because of the aseptic technique mistakes in sterilisation process.

THANK YOU