This research proposal aims to develop genetic transformation protocols for Eurycoma longifolia using particle bombardment. The objectives are to transform E. longifolia with therapeutic protein genes, analyze transformants for gene insertion and expression, and facilitate production of therapeutic proteins in medicinal plants. The methodology involves optimizing callus induction, regeneration, and genetic transformation parameters. Preliminary results show E. longifolia and L. pumila have the best callus growth. Upon completing the optimization and verification steps, transgenic E. longifolia expressing the therapeutic protein gene will be produced.

1. RESEARCH PROPOSAL
Vijendren Krishnan
GS21956
Prof. Dr. Maziah Mahmood
Dr. Syahida Ahmad
SPs 5903
1

2. DEVELOPING PROTOCOLS FOR
EFFICIENT GENETIC TRANSFORMATION
IN eurycoma longifolia.
2

3. Introduction
• Genetic transformation in medicinal plant
is a new emerging field for Malaysian
scientist to venture.
• Genetic transformation has led to
production of therapeutic proteins from
plant sources.
• Biopharming  Using organism to
produce therapeutic proteins.
3

4.  Wide range of valuable proteins such as
vaccines, blood substitutes, enzymes and
hormone can be expressed in plants
 Introducing gene in plant using particle
bombardment has become very convincing
and promising.
4

5. Eurycoma longifolia
Kingdom : Plantae
Division : Magnoliophyta
Class : Magnoliopsida
Order : Sapindales
Family : Simaroubaceae
Genus : Eurycoma
Species : E. longifolia
 Known to pose anti malaria, anti ulcer, anti
tumor and anti parasitic properties.
 Also known for its aphrodisiac properties.
5
(Kuo et al., 2003)

6. Current Therapeutic Protein
Producing Platforms
Mammalian
Cells
Yeast
Bacteria
Transgenic
animal
Transgenic
plant
6

7. TRANSGENIC PLANTS
CORN STRAWBERRY
SOYBEANTOMATO
7

8. 1 cell = 50 – 10000 plastids
1 plastids = ~100 genomes
1 cell = ~5000 – 10000 protein genes
1 plant can produce >250mg of protein
Safflower
Tobacco
Sunflower seed
8

9. Advantages of using plant as
biofactory
 Reduced cost compared to current method
 Cost effective – sunlight and water is required
for growth.
 Medium time scale (months)
 Unlimited scale up potential
9
Sunflower

10.  Much faster than transgenic animals
 Post-translational modification(s)
 Safe, no risk of pathogen contamination
 Edible crops  Oral vaccine
 Robust and reliable – high level
of production
10
Corn
(Rigano et al., 2009)

11. Problem Statement
 Production of therapeutic protein in
animals, and bacteria are inconvenient
and expensive.
 No established protocol for genetic
transformation of E. longifolia
11

12. OBJECTIVES
 To develop genetic transformation
protocol for E. longifolia using particle
bombardment system.
 To analyze transformants for insertion
and expression of transgenes.
 To facilitate the introduction of gene
related to therapeutic protein production
in medicinal plant.
12

13. Methodology
Screening of
plants
Callus induction
+ culture
optimization
Gene transfer
optimization
Verification of
gene expression
Regeneration
optimization
13

14. Screening of Plants
 Labisia pumila Kacip fatimah Var alata
 Eurycoma longifolia Tongkat ali
 Gynura procumbens Sambung nyawa
 Centella asiatica Pegaga
 Oryza sativa Rice MR219 (M4)
( Control plant)
14

15.  The choice of plants for screening are
based on high antioxidative properties
which have been done in our lab.
 Same size explant will be cut and
cultured on Murashige and Skoog media
containing different concentration of 2,4-
dichlorophenoxyacetic acid (0 - 25µM).
 Cultured explant will be observed every
alternate days.
15

16.  Callus selection will be done according to
fast responding, ease of multiplying and
morphology.
L. pumila E. longifolia
16

17. Optimization of culture
media Effect of different concentration of
2,4-D on callus initiation
Explants will be cultured on MS media
with different concentration of 2,4-D;
0 - 25µM.
 Callus growth measurement
Fresh weight and dry weight of callus will
be measured each week continuously for 6
weeks.
17

18.  Effect of various type of auxin
Suitable auxin for callus growth will be
assessed among 2,4-D, pic, dic and naa by
measuring the fresh and dry weight of
callus in culture respectively.
 Effect from combination of auxin
and kinetin
Best auxin from previous experiment will
be used with combination of different
concentration of kinetin; 0- 2.0mg/L. Fresh
and dry weight will be analyzed as for the
parameters.
18

19.  Effect from combination of auxin
and cytokinin.
Most suitable auxin with different type of
cytokinin such as zeatin,
benzylaminopurine, and thidiazuron will be
analysed using fresh and dry weight of
callus.
19

20. Optimization of regeneration
media
 Pretreatment for callus
Callus will be cultured on MS media with
10µM 2,4-D and sub-cultured to 5µM 2,4-D
then 0µM 2,4-D after 3 weeks respectively.
20
(Dennis et al., 2002)

21.  Effect of different concentration of
kinetin
Callus cultured on different concentration of
kinetin; 0 – 2mg/L.
Number of shoots and number of days taken
for shoot initiation will be evaluated as
parameter.
 Effect of different type of cytokinin
Best concentration of kin from previous
experiment will be used to evaluate the
performance of callus on BAP, TDZ and zeatin.
21

22. Optimization for gene
transfer
(Heiser et al., 1992)
 Optimized parameters will be used to
transfer the therapeutic protein producing
gene into the plant callus.
Parameter Measurement
He Pressure 650,900,1100,1300psi
Target distance 6,9,12cm
Microparticle size 0.6, 1.0, 1.6µM
22
Table 1: Parameters for gene transfer optimization

23.  Optimization of gene transfer will be
carried out using GFP and GUS reporter
marker.
He Pressure
Target distance
GFP
GUS
23
Particle Bombardment system
(PDS1000/He)

24. Verification of gene
expression Green fluorescent protein (GFP) and
beta-glucuronidase (GUS)
Pcambia 1304 vector with GFP and GUS
will be used for optimization purpose.
GFP as reporter marker will be observed
under fluorescent microscope equipped
with GFP filter set.
Histochemical GUS staining will be
carried out to visualize blue spots of GUS
using stereo microscope.
(Sreeramanan et al., 2006)
24

25.  Reverse Transcriptase polymerase
chain reaction (RT-PCR)
PCR analysis is used to confirm the
integration of the introduced protein
producing gene in the transformed plant.
Molecular weight marker will be used to
ensure the integrated plasmid.
PCR will be carried out using DNA thermal
cycler 480 device.
(Sreeramanan et al., 2006)
25

26.  Southern Blot
Southern blot analysis will also be used to
verify the integration and expression of the
introduced gene.
26Figure 1: Southern blotting process

27. Preliminary Result
0
5
10
15
20
25
30
35
40
45
50
0µM 5µM 10µM 15µM 20µM 25µM
L.pumila
E. longifolia
C. asiatica
G. procumbens
O. sativa
Concentration of 2,4 D
Days
27
Figure 2: Study of callus performance.
Results are stated as mean ± sd. N=3

28. Expected Result
 By end of this research, transgenic
plant which can express the gene
transferred will be produced and
beneficial for application in humans.
28

29. Gantt chart
2008 2009 2010
Activity Q3 Q4 Q1 Q2 Q3 Q4 Q1 Q2 Q3
Literature review √ √ √ √
Screening of plants √ √
Optimization of
culture media
√ √ √
Optimization of
regeneration media
√ √
Optimization of gene
transfer
√ √
Verification of gene
expression
√ √
Thesis writing √ √
29

30. References
 Dennis, T.T., Maseena, E.A., 2006. Callus induction and plant
regeneration in Cardiospermum halicacabum Linn. An important
medicinal plant. Scientia horticulturae. 108: 332-336
 Heiser, W., 1992. Optimization of biolistic transformation using the
helium-driven PDS-1000/He system. Bulletin 1688, Bio-Rad
Laboratories, Hercules CA.
 Kuo. P.C., Damu. A.G., Lee. K.H., and Wu. T.S., 2004. Cytotoxic and
antimalarial constituents from the roots of Eurycoma longifolia.
Bioorganic and medicinal chemistry. 12: 537-544.
 Rigano, M.M., Carmela, M., Anna, G., Alessandro, V., and Teodoro c.,
2009. Plants as biofactories for the production of subunit vaccines against
bio-security related bacteria and viruses. Journal of Vaccine. 01.120
 Sreeramanan, S., Maziah, M., Rosli, N.M., Sariah, M., Xavier,R., 2006.
Particle bombardment-mediated co-transformed of chitinase and β-1,3
glucanase genes in banana. Journal of Biotechnology. 5(2): 203-216.
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31. Appendix
Day 0
O.sativa
C.asiatica
31
G.procumbens L. pumila E. longifolia

32. DAY 14
C. asiatica
L. pumila
32
E. longifoliaG. procumbens
O. sativa

33. 33
Thanks for your
kind attention