Stable chloroplast transformation in potato: use of green fluorescent protein as a plastid marker

Abstract

SummaryWe describe here the development of a reproducible plastid transformation system for potato and regeneration of plants with uniformly transformed plastids. Two distinct tobacco-speci®c plastid vectors, pZS197 (Prrn/aadA/TpsbA) and pMON30125 (Prrn/GFP/ Trps16::PpsbA/aadA/TpsbA), designed for integration into the large single copy and inverted repeat regions of the plastid genome, respectively, were bombarded into leaf explants of potato line FL1607. A total of three transgenic lines were selected out of 46 plates bombarded with pZS197 and three transgenic lines out of 104 plates were obtained with pMON30125. Development of a high frequency leaf-based regeneration system, a stringent selection scheme and optimization of biolistic transformation protocol were critical for recovery of plastid transformants. Plastidexpressed green¯uorescent protein was used as a visual marker for identi®cation of plastid transformants at the early stage of selection and shoot regeneration. The establishment of a plastid transformation system in potato, which has several advantages over routinely used nuclear transformation, offers new possibilities for genetic improvement of this crop.

High efficiency plastid transformation in potato and regulation of transgene expression in leaves and tubers by alternative 5′ and 3′ regulatory sequences

“…An additional limitation related to the application of plastid transformation in breeding and biotechnology is the generally inefficient gene expression in non-green plastids, as a consequence of deficiencies in the expression machinery of non-green plastids compared to leaf chloroplasts (Brosch et al 2007;Kahlau and Bock 2008;Valkov et al 2009). Transgene expression with the plastid rrn and the bacterial trc promoters in potato tuber amyloplasts ranged from 1 to 20% of that in leaf chloroplasts, respectively, although GFP concentration attainable with the prokaryotic promoter was lower than with rrn one (0.004 vs. 0.05% of total soluble proteins), due to its low efficacy in both tissues (Sidorov et al 1999;Nguyen et al 2005). In transplastomic tomato plants, the fusion protein P24-Nef from HIV accumulated up to 40% TSP in leaves, but significantly less in green and ripe fruits (2.5% and nil, respectively) (Zhou et al 2008).…”

“…In many cases, however, low transformation efficiencies have been reported for species other than tobacco (Sikdar et al 1998;Sidorov et al 1999;Ruf et al 2001;Hou et al 2003;Skarjinskaia et al 2003;Zubko et al 2004;Nguyen et al 2005;Nugent et al 2006;De Marchis et al 2009). Low recovery of transplastomic shoots has been attributed to multiple reasons, such as a relatively inefficient homologous recombination system, non-optimal homology and length of flanking regions, the promoter used for the expression of the selectable marker gene, the kind of explant, or the regeneration protocol.…”

“…Despite recent progress, potato plastid transformation is still limited by low transformation frequencies and low transgene expression in tubers of transplastomic plants (Sidorov et al 1999;Gargano et al 2003;Nguyen et al 2005;Gargano 2006). Plastid transformation is a long and complex process requiring organelle sorting out during repeated cell divisions in vitro, in order to achieve regeneration of homoplasmic transplastomic shoots (Bock 2001;Maliga 2004).…”

Plastid transformants of tomato selected using mutations affecting ribosome structure

“…Plastid transformation of tobacco (Nicotiana tabacum) is routine in a number of laboratories, but there are only a limited number of isolated reports relating to genera outside Nicotiana. These include tomato (Ruf et al 2001), rice (Khan and Maliga 1999), potato (Sidorov et al 1999), carrot (Kumar et al 2004) and several Brassicaceae, including Arabidopsis (Sikdar et al 1998), Brassica napus (Hou et al 2002) and Lesquerella fendleri (Skarjinskaia et al 2003). All of these investigators used biolistics to deliver DNA into chloroplasts.…”

Enhanced green fluorescent protein (egfp) gene expression in Tetraselmis subcordiformis chloroplast with endogenous regulators

“…Prrn (Svab and Maliga 1993;Sidorov et al 1999), PpsbA (Li et al 2011), TpsbA (Svab and Maliga 1993;Sidorov et al 1999), and TrbcL (Doetsch et al 2001;Cui et al 2014) are commonly used endogenous regulators. Prrn, as a promoter, is derived from plastid-encoded 16S RNA, and TpsbA, as a terminator, comes from the psbA gene that encodes endogenous PS II protein D1 (Ruhlman et al 2010).…”