Chloramphenicol acetyltransferase as selectable marker for plastid transformation

Abstract

Chloroplast transformation remains a demanding technique and is still restricted to relatively few plant species. The limited availability of selectable marker genes and the lack of selection markers that would be universally applicable to all plant species represent some of the most serious technical problems involved in extending the species range of plastid transformation. Here we report the development of the chloramphenicol acetyltransferase gene cat as a new selectable marker for plastid transformation. We show that, by selecting for chloramphenicol resistance, tobacco chloroplast transformants are readily obtained. Transplastomic lines quickly reach the homoplasmic state (typically in one additional regeneration round), accumulate the chloramphenicol acetyltransferase enzyme to high levels and transmit their plastid transgenes maternally into the next generation. No spontaneous antibiotic resistance mutants appear upon chloramphenicol selection. Several lines of evidence support the assumption that plant mitochondria are also sensitive to chloramphenicol suggesting that the chloramphenicol acetyltransferase may be a good candidate selectable marker for plant mitochondrial transformation.

A mutant Synechococcus gene encoding glutamate 1-semialdehyde aminotransferase confers gabaculine resistance when expressed in tobacco plastids

“…Plant 1Bs was heteroplasmic due to the presence of an additional 4.4 kb fragment typical of non-transformed plastome, while the presence of the 5.2 kb fragment and the absence of the 2.1 kb fragment in this plant indicated that the probe binding site on the trnA gene was missing or that the BglII site on the trnA gene was absent. The presence of the faint 4.5 kb fragment in the transplastomic plants 1Bg, 5Bg and 5Bs was likely due to plastid DNA inserted in the nuclear genome (Ayliffe and Timmis 1992;De Marchis et al 2011) or recombination between duplicated expression elements (the endogenous and the introduced prrn promoters) (Rogalski et al 2006;Li et al 2011). These results confirmed that plant 1Bs, besides being heteroplasmic, underwent a genetic rearrangement in the hemL-trnA region.…”

“…1), harboring both aadA and hemL, conferring spectinomycin and gabaculine resistance, respectively. This allowed us to perform a direct comparison between the two selections systems, an experimental design described in Li et al (2011) to demonstrate chloramphenicol selection for plastome transformation.…”

“…A few other SMGs have been reported to allow for selection of transplastomic cells in vitro (reviewed in Rosellini 2012): the spinach betaine aldehyde dehydrogenase (BADH) gene for betaine aldehyde resistance , the E. coli nptII and aphA-6 genes for kanamycin resistance (Carrer et al 1993;Huang et al 2002), a tobacco mutant anthranilate synthase a subunit gene (ASA) for 5-methyltryptophan resistance (Barone et al 2009), and the E. coli chloramphenicol acetyltransferase (cat) gene for chloramphenicol resistance (Li et al 2011). Recently, the use of Agrobacterium tumefaciens ipt, encoding the enzyme isopentenyl transferase, as a selectable marker for plastid transformation has been demonstrated (Dunne et al 2014).…”

Synthetic Lethality in the Tobacco Plastid Ribosome and Its Rescue at Elevated Growth Temperatures

“…genes remains more technically challenging because it requires either recycling of the aadA marker, cotransformation with two plasmids, or sequential transformation using two different selection markers (supertransformation). Although aadA is the only marker gene that is routinely used in plastid transformation experiments, a few alternative selectable marker genes have become available (Carrer et al, 1993;Huang et al, 2002;Li et al, 2011), enabling us to employ a supertransformation approach to analyze the effects of the combined inactivation of two nonessential plastid ribosomal protein genes. To this end, we used the aphA-6 marker gene, which confers kanamycin resistance (Huang et al, 2002) to disrupt a second ribosomal protein gene in the rps15, rpl33, and rpl36 knockout plants that had previously been generated with the aadA marker (Rogalski et al, 2008;Fleischmann et al, 2011).…”

Plastid Biotechnology: Food, Fuel, and Medicine for the 21st Century

“…The most commonly used selective marker gene is aadA, encoding spectinomycin resistance . Kanamycin (Carrer et al, 1993), chloramphenicol (Li et al, 2011), and the amino acid analogs 4-methylindole and 7-methyl-DL-Trp (Barone et al, 2009) have also been successfully employed as selective agents.…”