Molecular Biology and Genetics

Plant Biotechnology

Electronic Journal of Biotechnology ISSN: 0717-3458 Vol. 11 No. 1, Issue of January 15, 2008
© 2007 by Pontificia Universidad Católica de Valparaíso -- Chile Received January 26, 2007 / Accepted May 4, 2007
DOI: 10.2225/vol11-issue1-fulltext-1
TECHNICAL NOTE

Multiple pulses improve electroporation efficiency in Agrobacterium tumefaciens

Tariq Mahmood#
Department of Plant Sciences
Quaid-i-Azam University
Islamabad, Pakistan
Tel: 92 51 4845 623
Fax: 92 51 9290 160
E-mail: taariq.mahmood@yahoo.com 

Tamkina Zar
Department of Plant Sciences
Quaid-i-Azam University
Islamabad, Pakistan
Tel: 92 51 4845 623
Fax: 92 51 9290 160

S.M. Saqlan Naqvi*
Department of Biochemistry
University of Arid Agriculture Rawalpindi
Murree Road, Rawalpindi, 46300, Pakistan
Tel: 92 51 4845 623
Fax: 92 51 9290 160
E-mail: saqlan@isb.paknet.com.pk

*Corresponding author

Financial support: This study was supported by a grant No. 01-03-01-19 from Agricultural Linkages Program of the Pakistan Agricultural Research Council.

Present address: #Department of Plant Sciences, Quaid-i-Azam University, Islamabad, Pakistan.

Keywords: Agrobacterium, electroporation, pulse number, transformation efficiency.

Abbreviations:

Kb: Kilo base pair
LB: Luria Bertani
PCR: polymerase chain reaction

Abstract
Full Text

Electroporation entails brief, high intensity pulse to create transient pores in the cell membrane to facilitate the entry of exogenous macromolecules, which may otherwise be excluded. Removal of the external field leads to the resealing of the membrane electropores permitting the survival of the electrically stimulated recipient cells. Using this technique foreign deoxyribonucleic acid (DNA) has been successfully introduced into many cell types both from prokaryotes and eukaryotes. Increase in pulse voltage and length beyond a critical limit has been reported to decrease transformation efficiency, hence in this study we have investigated another strategy i.e. increase in the number of pulses at constant high voltage and pulse duration. Commonly used Agrobacterium strains LBA4404 and EHA101 and binary vector pCAMBIA1301 were used. Transformants were selected on a combination of hygromycin and kanamycin, and confirmed by polymerase chain reaction (PCR) and restriction analysis. Increase in the number of pulses was found to show a significant and linear increase in transformation efficiency.

Supported by UNESCO / MIRCEN network