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JXB Advance Access published online on June 24, 2009
Journal of Experimental Botany, doi:10.1093/jxb/erp160
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Published by Oxford University Press [on behalf of the Society for Experimental Biology] 2009.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. This paper is available online free of all access charges (see http://jxb.oxfordjournals.org/open_access.html for further details)

RESEARCH PAPER

Tobacco plastid transformation using the feedback-insensitive anthranilate synthase [{alpha}]-subunit of tobacco (ASA2) as a new selectable marker

Pierluigi Barone1 *,{dagger}, Xing-Hai Zhang2 and Jack M. Widholm1

1University of Illinois, Department of Crop Sciences, Edward R. Madigan Lab, 1201 W Gregory Dr, Urbana, IL 61801, USA
2Florida Atlantic University, Department of Biological Sciences, 777 Glades Road, Boca Raton, FL 33431, USA

{dagger} To whom correspondence should be addressed: E-mail: pierluigibarone{at}gmail.com

Genetic engineering of chloroplasts normally requires the stable introduction of bacterial derived antibiotic or herbicide-resistance genes as selective markers. Ecological and health concerns have been raised due to the presence of such genes within the environment or the food supply. One way to overcome this issue is the use of plant genes able to confer a metabolic or developmental advantage to the transformed cells manipulating the plant's biosynthetic pathways. We explored the feasibility of using, for plastid transformation, the selection system based on the feedback-insensitive anthranilate synthase (AS) {alpha}-subunit gene of tobacco (ASA2) as a new selective marker and the indole analogue 4-methylindole (4MI) or the tryptophan analogue 7-methyl-DL-tryptophan (7MT) as the selection agents. An expression cassette containing Prrn-ASA2 was effectively integrated into the region between accD and ycf4 of the tobacco plastome by the biolistic process. Plastid transgenic plants were obtained on medium supplemented with 300 µM 7MT or 4MI. Transplastomic plants showed normal phenotype and fertility and the resistance to the selection agents 7MT and 4MI was transmitted maternally. The plastid transformed lines also exhibited a higher level of AS enzyme activity that was less sensitive to Trp-feedback inhibition and, consequently, increased free Trp levels in leaves about 7-fold.

Key words: Anthranilate synthase, 7-methyl-DL-tryptophan, 4-methylindole-tryptophan, non-antibiotic selection, plastid transformation, selectable marker

* Present address: Chromatin, Inc. 3440 South Dearborn St. Suite 280 Chicago, Illinois 60616, USA.

Received 23 February 2009; Revised 17 April 2009 Accepted 20 April 2009


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