Research proposal

Research proposal

Vijendren KrishnanGS21956Prof. Dr. Maziah Mahmood

SPs 5903SPs 5903

DEVELOPING PROTOCOLS FOR EFFICIENT GENETIC TRANSFORMATION

OF MEDICINAL PLANT

Introduction• Malaysia as one of 12 mega bio-diversity

country.

• Rich with various medicinal herbs. (Letchumanan, 2002)

• Lack of identification for vital bioactive compounds.

.

• Boost economy by producing herbs with more important values.

• Utilize available technology to improve local herbs.

• Research aims to improve local herbs via genetic transformation.

• Gene transfer transfer beneficial characters from one organism to another organism (Dominique et al., 2002)

• Applies to all living organism.

• Improve biotic and abiotic stress tolerance and nutritional value.

• Biolistic modern and efficient technique.

• Coated gene with microcarrier will be bombarded at high speed.

• Highly efficient compared to other techniques.

• Agrobacterium-mediated transformation, electroporation, microinjection, silicon carbide fiber-mediated method

Vaccine• Virus spread in environment are very lethal

and harmful for human and living organisms.

• 200 pathogens are been reported as potential bio-weapon. Eg: Bacillus anthracis, and Yersinia pestis.

• Effective way to prevent virus spread is by producing efficient vaccines.

B. anthracis

Y. pestis

• Transgenic plant producing protective vaccines are economical, convenient vehicles to produce vaccines.

• Production of safe transgenic plant- made vaccines against emerging infectious diseases is extremely important.

(Rigano et al., 2009)

• Gene of interest will be obtained as readily constructed gene and used in gene transformation.

Problem Statement• Less improvement for local herbs

• Lack of boost from agro-biotech for economy growth.

• Lack of local herbs with value added for commercialization.

• Lack of vaccination causes high mortality rate in Malaysia.

Objectives

• To optimize tissue culture media for selected plant.

• To optimize particle bombardment for gene transfer.

• To regenerate transformed callus.

Methodology• Screening of plants

• Optimization of culture media

• Optimization of gene transfer

• Verification of gene transfer

• Optimization of regeneration media

Screening of Plants

• Labisia pumila Kacip fatimah Var alata

• Eurycoma longifolia Tongkat ali

• Ficus deltoidea Mas cotek Female

• Centella asiatica Pegaga

• Gynura procumbens Sambung nyawa

• Oryza sativa Rice MR219 (M4)

• Plant of high antioxidative properties is chosen.

• Same size explant will be cut and cultured on Murashige and Skoog media containing 10µM 2,4- dichlorophenoxyacetic acid.

• Cultured plant will be observed every alternate days.

• Plant selection will be done according to fast responding and ease of multiplying.

• Rice plant will be used as control for screening.

Optimization of culture media• Effect of different concentration of 2,4-D on

callus initiation

Plant will be cultured on MS media with different concentration of 2,4-dichlorophenoxyacetic; 0, 5,10,15, 20, 25µM.

• Callus growth measurement

Fresh weight and dry weight of callus will be measured each week continuously for 6 weeks.

• Effect of various type of auxin

Suitable auxin for callus growth will be assessed among 2,4-D, picloram, dicamba, and napthaleneacetic acid 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, 0.5, 1.0, 1.5, 2.0mg/L. Fresh and dry weight will be analyzed as for the parameters.

• Effect of various type 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.

Optimization for gene transfer

(Heiser et al. 1992)

• Optimized parameters will be used to transfer the 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

• Optimization of gene transfer will be carried out using GFP and GUS reporter marker.

He Pressure

Target distance

GFP

GUS

Verification of gene expression• GFP and beta-glucuronidase GUS pGEM.Ubi1-sgfps65T (GFP) and pANU21(GUS)

will be co-bombarded into callus culture.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)

• 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)

• Southern Blot

Southern blot analysis will also be used to verify the integration and expression of introduced gene.

Specific restriction enzyme is used to digest the DNA to estimate the number of insertion sites and hybridization with transgene specific probes.

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.

(Dennis et al., 2006)

• Effect of different concentration of kinetin

Callus cultured on different concentration of kinetin; 0 - 10µM.

Callus respond to hormone treatment in the media will be observed. Number of shoots and number of days taken for shoot initiation will be evaluated as parameter.

• Effect of different type of cytokinin

Best concentration for callus regeneration will be used to evaluate the performance on BAP, TDZ and zeatin.

Expected Result• By end of this research, transgenic plant

which can express the gene transferred will be produced and beneficial for application for human and animals.

Gantt chartActivity Period

Literature review July – August 2008

Screening of plants Sept – Dis 2008

Callus growth measurement Jan – Mac 2009

Effect of different concentration of kinetin (Regeneration)

Effect of various type of auxin April – May 2009

Effect of different type of cytokinin (regeneration)

Effect from combination of auxin and kinetin

June – July 2009

Effect from combination of auxin and cytokinin August – Sept 2009

Optimization of gene transfer Sept – Oct 2009

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

• Dominique, J., 2002. Plant biotechnology in agriculture. Biochimie. 84: 1105-1110.

• Heiser, W., 1992. Optimization of biolistic transformation using the helium-driven PDS-1000/He system. Bulletin 1688, Bio-Rad Laboratories, Hercules CA.

• Letchumanan, R., 2002. National Policy on Biological Diversity (NPBD).• Rigano, M.M., Carmela, M., Anna, G., Alessandro, V., and Teodoro c., 2009.

Plants as biofactors 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.

AppendixDay 0

O.sativa

C.asiatica

F.deltoidea

Day 0

G.procumbens

L. pumila

E. longifolia

Day 14

E. longifolia G. procumbens

O. sativa

C. asiatica F. deltoidea

L. pumila

Thanks for your kind attention