Establishment Of Cell Suspension Callus Culture from Leaves Andrographis paniculata sp. and Micropropagation of Borreria laevicaulis sp.

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

2. INTRODUCTION

3. 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.

4. 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.

5. 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.

6. 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.

7. 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.

8. MATERIAL AND APPARATUS

9. MATERIAL AND APPARATUS OF
MICROPROPAGATION
1)
2)
3)
4)
5)
Tween 20
Ethanol 75%
Mercury chloride 0.5%
MS media
Hormonebenzylaminopurine
(BAP )and Kinetin(Kn)
1) Forceps
2) Scalper
3)
4)
5)
6)
7)
Surgical blade
Petri disk
Spirit lamp
Beaker
Internode from
Borreria laevicaulis
sp.

10. MATERIAL AND APPARATUS FOR CALLUS
CULTURE
1) Tween 20
2) Ethanol 75%
3) Mercury
chloride 0.5%
4) MS media
5) Hormonenaphthalene
acetic acid
(NAA)
6) Forceps
7) Scalper
8) Surgical blade
9) Petri disk
10) Spirit lamp
11) Beaker
12) Leaves of
Andrographis
paniculata sp.
(Hempedu
Bumi)

11. MEDIA PREPARATION

12. • (macro,
micro,
vitamin,
hormones
)
Stock
Solutions
Volume
adjustment
• Adjust
volume
to 800
mL
• (5.4-5.8)
• Use
either
HCl or
NaOH
Check pH
Organic
elements,
gelling
agent
• Add 30 g
Sucrose
– carbon
source
• 8 g Agar
– gelling
agent
• Dispense
into
culture
vessels
• petri
dish, test
tube,
glass jar
Pouring
media
Autoclave
• Autoclav
e the
medium
• Make it
slang

13. MICROPROPAGATION

14. 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

15. 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 threefourth full with
the bleach
solution

16. RESULT

17.  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

18. a) Callus without root
Before
b) Callus with root
After

19. 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

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

21. 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

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

23. DISCUSSION

24. 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

25. 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.

26. The reasons for contamination occur
1) Sterilization process
2) Airborne particles
3) Dirty lab coat and cloth
4) Water source
5) Equipment
6) Non-sterile apparatus
7) Careless in culturing

27. 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

28. 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

29. CONCLUSION

30. 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.

31. 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.

32.  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.

33. THANK YOU