RESEARCH PROPOSAL

Vijendren KrishnanGS21956Prof. Dr. Maziah MahmoodDr. Syahida Ahmad

SPs 5903SPs 5903

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DEVELOPING PROTOCOLS FOR EFFICIENT GENETIC

TRANSFORMATION IN eurycoma longifolia.

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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.

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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.

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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

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TRANSGENIC PLANTSCORN STRAWBERRY

SOYBEANTOMATO

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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

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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

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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

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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

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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.

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Methodology

Screening of plants

Callus induction + culture

optimization

Gene transfer optimization

Verification of gene expression

Regeneration optimization

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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)

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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.

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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.

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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.

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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.

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(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.

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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

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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

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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)

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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

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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.

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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 √ √

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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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AppendixDay 0

O.sativaC.asiatica

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G.procumbens L. pumila E. longifolia

DAY 14

C. asiatica

L. pumila

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E. longifoliaG. procumbens

O. sativa

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Thanks for your kind attention