DEVELOPING PROTOCOLS FOR EFFICIENT GENETIC TRANSFORMATION IN EURYCOMA LONGIFOLIA
-
Upload
vijendren-krishnan -
Category
Science
-
view
213 -
download
0
description
Transcript of DEVELOPING PROTOCOLS FOR EFFICIENT GENETIC TRANSFORMATION IN EURYCOMA LONGIFOLIA
RESEARCH PROPOSAL
Vijendren KrishnanGS21956Prof. Dr. Maziah MahmoodDr. Syahida Ahmad
SPs 5903SPs 5903
1
DEVELOPING PROTOCOLS FOR EFFICIENT GENETIC
TRANSFORMATION IN eurycoma longifolia.
2
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
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
Eurycoma longifolia
Kingdom : PlantaeDivision : MagnoliophytaClass : MagnoliopsidaOrder : SapindalesFamily : SimaroubaceaeGenus : EurycomaSpecies : 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)
Current Therapeutic Protein Producing Platforms
Mammalian Cells
Yeast
Bacteria
Transgenic
animal
Transgenic
plant
6
TRANSGENIC PLANTSCORN STRAWBERRY
SOYBEANTOMATO
7
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
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
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)
Problem Statement
Production of therapeutic protein in animals, and bacteria are inconvenient and expensive.
No established protocol for genetic transformation of E. longifolia
11
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
Methodology
Screening of plants
Callus induction + culture
optimization
Gene transfer optimization
Verification of gene expression
Regeneration optimization
13
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
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
Callus selection will be done according to fast responding, ease of multiplying and morphology.
L. pumila E. longifolia16
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 measurementFresh weight and dry weight of callus will be
measured each week continuously for 6 weeks.
17
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
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
Optimization of regeneration media Pretreatment for callusCallus 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)
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 cytokininBest concentration of kin from previous
experiment will be used to evaluate the performance of callus on BAP, TDZ and zeatin.
21
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
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)
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
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
Southern BlotSouthern blot analysis will also be used to
verify the integration and expression of the introduced gene.
26Figure 1: Southern blotting process
Preliminary Result
0µM 5µM 10µM 15µM 20µM 25µM0
5
10
15
20
25
30
35
40
45
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
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
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
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.
30
AppendixDay 0
O.sativaC.asiatica
31
G.procumbens L. pumila E. longifolia
DAY 14
C. asiatica
L. pumila
32
E. longifoliaG. procumbens
O. sativa
33
Thanks for your kind attention