A Guide to Choosing Vectors for Transformation of the Plastid Genome of Higher Plants 1[C][W][OA]

Plastid transformation, originally developed in tobacco ( Nicotiana tabacum ), has recently been extended to a number of crop speciesenablingin vivo probing of plastid functionand biotechnological applications. Inthis articlewe reportnewplastid vectors that enable insertion of transgenes in the inverted repeat region of the plastome between the trnV and 3 # rps12 or trnI and trnA genes. Efﬁcient recovery of transplastomic clones is ensured by selection for spectinomycin ( aadA ) or kanamycin ( neo ) resistance genes. Expression of marker genes can be veriﬁed using commercial antibodies that detect the accumulation of neomycin phosphotranseferase II, the neo gene product, or the C-terminal c-myc tag of aminoglycoside-3 $ -adenylytransferase, encoded by the aadA gene. Aminoglycoside-3 $ -adenylytransferase, the spectinomycin inactivating enzyme, is translationally fused with green ﬂuorescent protein in two vectors so that transplastomic clones can be selected by spectinomycin resistance and visually identiﬁed by ﬂuorescence in ultraviolet light. The marker genes in the new vectors are ﬂanked by target sites for Cre or Int, the P1 and phiC31 phage site-speciﬁc recombinases. When uniform transformation of all plastid genomes is obtained, the marker genes can be excised by Cre or Int expressed from a nuclear gene. Choice of expression signals for the gene of interest, complications caused by the presence of plastid DNA sequences recognized by Cre, and loss of transgenes by homologous recombination via duplicated sequences are also discussed to facilitate a rational choice from among the existing vectors and to aid with new target-speciﬁc vector designs.

Delivery of transformation vectors to chloroplasts is by the biolistic process (Boynton et al., 1988) or by polyethylene glycol treatment (Golds et al., 1993). Transformation of ptDNA is based on targeted insertion of the transforming DNA by homologous recombination (Fig. 1A), followed by enrichment of the transformed ptDNA copy by growing the cells on selective tissue culture medium. The gradual process of organelle (plastid) and genome (ptDNA) sorting ultimately yields genetically stable homoplastomic cells carrying only transformed ptDNA copies. Genetically stable plants are obtained by regenerating plants from the homoplastomic cells.
The objective of engineering the plastid genome is either to alter (or delete) the DNA sequence of native plastid genes or to incorporate new functions. Engineering of native plastid genes is accomplished by including a modified ptDNA sequence in the vector plastid-targeting region. Examples include testing a spinach (Spinacia oleracea) psbF editing segment in tobacco chloroplasts (Bock et al., 1994) and replacing the tobacco rbcL gene with cognate genes from sunflower (Helianthus annuus) or photosynthetic bacteria (Kanevski et al., 1999;Whitney and Andrews, 2001). When engineering of a native gene is the goal, the marker gene is always adjacent to the target gene to minimize the probability of recombination between the mutant sequence and the marker gene. The vectors for these manipulations are gene specific and are outside the scope of this article.
We report here on new vectors that are suitable for incorporation of novel functions in the plastid genome and are equipped with sequences for posttransformation removal of the marker genes (Fig. 1B). Excision of marker genes by phage recombinases reported here is not the only approach to obtain marker-free transplastomic plants: The alternatives are excision by homologous recombination via direct repeats (Iamtham and Day, 2000), transient cointegration facilitated by visual-assisted marker selection (Klaus et al., 2003(Klaus et al., , 2004, and cotransformation segregation (Ye et al., 2003; for review, see Lutz and Maliga, 2007a). For additional reviews on plastid transformation and its applications in basic science and biotechnology see Maliga (2004), Daniell et al. (2005), Herz et al. (2005), and Bock (2007).
This report is an update on plastid repeat vectors (pPRV) described in 1994 (Zoubenko et al., 1994). It lists five vectors we still recommend from the earlier article, four vectors described in the meantime, and nine new vectors we report here. Novel features of the new vectors are sequences for posttransformation excision of the marker genes, a new insertion site in the trnI-trnA intergenic region and vectors with alternative marker genes.

Insertion Sites
The site of insertion in the plastid genome is determined by the choice of ptDNA segment flanking the marker gene and the gene of interest. Insertion of foreign DNA in intergenic regions of the plastid genome has been accomplished at more than 14 sites (Maliga, 2004).Only three of the insertion sites have been developed into more sophisticated vectors in which a marker gene is adjacent to a polycloning site so that new chimeric genes can be directly assembled in the transformation vector in Escherichia coli. Two of the plastid vectors target the 25-kb inverted repeat (IR) region between the trnV-3#rps12 and trnI-trnA genes and one targets the intergenic region between trnfM-trnG genes located in the large single copy region of the 155-kb tobacco ptDNA (Fig. 2). Any gene inserted into one of the repeats is rapidly copied over into the second repeat copy by gene conversion, thus a gene targeted to the IR is present in two copies per genome (both transformed and nontransformed copies may be templates for gene conversion, thus gene conversion may also eliminate a transgene).
We selected the trnV-3#rps12 intergenic region in the IR for insertion of transgenes because there is no readthrough transcription from the plastid ribosomal RNA (rrn) operon (Zoubenko et al., 1994;Fig. 2, pPRV111A). Thus, promoter activity at this site could be studied without interference by read-through transcription. Originally, we constructed two vectors with different expression signals for the aadA marker gene: In vectors pPRV111A/B aadA is expressed in a psbA cassette (PpsbA and TpsbA derive from the psbA gene promoter and terminator, respectively) and in vectors pPRV112A/B aadA is expressed in a Prrn/Trps16 cassette. Having alternative aadA expression signals enabled avoiding duplication of expression signals on the gene of interest, which may lead to deletion of sequences between the direct repeats by homologous recombination (Iamtham and Day, 2000; Kode et al., 2006). The pPRV vectors targeting insertions at the trnV-3#rps12 intergenic region are the most commonly used vectors. They yield high levels of protein expression (Maliga, 2003) and are now endowed with signals for marker gene excision (see below; Fig. 3; Table I).
Several laboratories have inserted transgenes between the trnI and trnA genes in the IR region of ptDNA. These two tRNAs are located between the small (rrn16) and large (rrn23) rRNA subunit genes and the operon is transcribed from promoters upstream of rrn16 ( Fig. 2; Vera and Sugiura, 1995;Suzuki et al., 2003). The polycistronic rrn operon mRNA is efficiently processed, releasing transgenic mRNA inserted between the two tRNAs. The first vector targeting insertions in the trnI-trnA intergenic region, pSBL-CTV2, was developed in the Daniell laboratory (Daniell et al., 1998) and was used to express several proteins (Daniell et al., 2005). Transgenes in the Daniell laboratory are typically expressed by cloning genes and operons into an XbaI site downstream of aadA, which is expressed in a cassette consisting of the rrn operon promoter (Prrn) and the psbA gene 3#-untranslated region (UTR; TpsbA; Fig. 2). The aadA marker gene and the inserted transgenes are expressed from two mRNAs: The mRNA transcribed from the ectopic Prrn promoter driving the aadA marker gene and from the read-through mRNA derived from the native rrn operon promoter. Stability of the transgenic mRNA is ensured by the 5#-UTR and 3#-UTR sequences of the Prrn-TpsbA cassette; protein accumulation from the transgene depends on the 5#-UTR inserted upstream of the reading frame encoding the gene(s) of interest. For information on protein expression with these vectors see Daniell et al. (2005).
Vectors targeting insertion of two complete genes in the trnI/trnA intergenic region were also reported from the Hanson laboratory (Yu et al., 2007) and from our laboratory. Our 300-series PRV vectors give the freedom to either utilize read-through transcription (pPRV323Clox, pPRV324Clox) or to insert a complete gene upstream (pPRV323Clox, pPRV324Clox) or downstream (pPRV323Blox) of the c-myc-tagged aadA marker gene ( Fig. 4; Table I). Read-through transcription was utilized, for example, for expression of the Cry9Aa2 protein in a pPRV323Clox (formerly pPRV312L) vector derivative (Chakrabarti et al., 2006). In our vectors the marker genes are flanked by loxP sites (floxed), thus they can be excised when transformation is complete. Transgene expression at this insertion site in the repeated region of the ptDNA benefits from duplication of the gene copy number and from increased mRNA levels due to read-through transcription from the upstream rrn operon promoter. However, more important for protein accumulation is the choice of 5#-UTR that may affect protein yields in a 10,000-fold range (Maliga, 2003).
The trnfM-trnG intergenic region in the large single copy region is utilized in the pRB94 and pRB95 plastid vectors developed in the Bock laboratory (Ruf et al., 2001;Fig. 2). This vector is used to study RNA editing (Bock, 1998) and metabolic engineering in chloroplasts (Bock, 2007). Recently a portable intercistronic expression element was characterized using this vector that mediates intercistronic cleavage into stable monocistronic mRNAs. The short (50 bp) element derived from the psbT-psbH intergenic region facilitates translation of monocistronic mRNAs at predictable levels (Zhou et al., 2007).

Marker Genes
There are two classes of plastid marker genes: primary selective markers that are suitable for direct selection of transplastomic clones and secondary selective markers that confer a phenotype when present in most ptDNA copies but are not suitable for recovery of transplastomic clones when present in only a few ptDNA copies (Maliga, 2004). Currently known pri- Figure 2. Plastid transformation vectors targeting insertions at three commonly used insertion sites. A, Plastid transformation vector pPRV111A targets insertion into the trnV-3#rps12 intergenic region (Zoubenko et al., 1994). A gene of interest (goi) can be cloned into the multiple cloning site upstream of the aadA marker gene. Red, wavy lines symbolize transcripts. Note that there is no read-through transcription from the rrn operon. Restriction sites marked with asterisks are not unique. B, Plastid transformation vector pSBL-CTV2 targets insertion into the trnI-trnA intergenic region (Daniell et al., 1998). Genes for expression are cloned into the XbaI site in the 3#-UTR (TpsbA) of the (aadA) marker gene. Transgenes are transcribed from the rrn operon promoter and from the promoter upstream of aadA. Vertical arrows above transcripts mark processed ends. C, Plastid transformation vector pRB94 targets insertion into the trnfM-trnG intergenic region. Genes for expression may be cloned into the multiple cloning site. [See online article for color version of this figure.] mary markers are resistance to spectinomycin, streptomycin, and kanamycin. Resistance to spectinomycin and streptomycin in plastids is conferred by the expression of the aadA gene (Goldschmidt-Clermont, 1991;Svab and Maliga, 1993); resistance to kanamycin is due to expression of the neo [aph(3#) IIa; Carrer et al., 1993;Lutz et al., 2004] or aphA-6 gene (Huang et al., 2002).
The original pPRV plastid vectors carry aadA genes, which express aminoglycoside-3$-adenylytransferase (AAD) at a relatively low level. The aadA genes in the new pPRV series are expressed from the PrrnLatpB 1 DS and PrrnLrbcL 1 DS translation control regions, which yield proteins in the 7% to 10% total soluble protein range (Kuroda and Maliga, 2001b). Recovery of transplastomic clones with the new markers is efficient and the AAD gene product can be quantified using the commercially available antibody to the C-terminal c-myc 9E10 (EQKLISEEDL) epitope tag (Kolodziej and Young, 1991). The Prrn promoter in the new vectors is in an inverted orientation relative to the native rrn promoter, an arrangement that prevents deletion of intervening sequences (Kittiwongwattana et al., 2007). The aad-gfp fusion gene is an aadA derivative that enables selection for spectinomycin resistance and is visually traceable by GFP fluorescence (Khan and Maliga, 1999). The new plastid vector pair pPRV131A and pPRV131B carry the aadA-gfp fusion gene as a selective marker ( Fig. 3; Table I).
Although kanamycin resistance could be used to recover transplastomic clones after bombardment with the plasmid pTNH32, the neo gene was not included in advanced vectors because the transformation efficiency with this neo gene was low (Carrer et al., 1993). We report here a dramatic improvement in plastid transformation efficiency with a highly expressed (7% neomycin phosphotranseferase II) neo gene derived from plasmid pHK30 (Kuroda and Maliga, 2001b). Bombardment of 25 leaves with the vector that carries the new neo gene (pAAK201) yielded 34 kanamycin resistant clones. Out of these, DNA gel-blot analyses confirmed plastid transformation in 27 clones (Supplemental Fig. S1) whereas only three out of 99 kanamycin resistant clones were transplastomic after transformation with vector pTNH32. Interestingly, the kanamycin resistant clones appear later (after 6-12 weeks) than the spectinomycin resistant clones (3-12 weeks). These data confirm the earlier report about the efficiency of kanamycin selection with the new neo gene in plastid transformation experiments (Lutz et al., 2004). The PrrnLatpB 1 DS promoter driving neo in plasmid pAAK201 is in tandem with the native Prrn promoter. We therefore shall release vectors pPRV145C and pPRV145D, in which the orientation of neo is inverted relative to the rrn operon (Fig. 3).

Expression of the Gene of Interest
Levels of protein expression from plastid transgenes depend on mRNA abundance determined by promoter strength and mRNA turnover. The stability of mRNAs depends on protective stem-loop secondary structures in the 5#-and 3#-UTRs and their interactions with RNA binding proteins (Barkan and Goldschmidt-Clermont, 2000). More important for protein expression is the translatability of the mRNA determined by the 5# translation control region that may affect protein yield in a 10,000-fold range (Maliga, 2003).
Thus far, expression signals, such as promoters and 5#-and 3#-UTRs are derived from the source organism, which results in duplicated ptDNA sequences. One option to minimize duplications is to build polycistronic expression units in which several genes may be expressed in the same promoter-terminator cassette. One example is pPRV110lox (formerly pPRV110L; Fig.  3; Table I), a vector with a promoterless aadA gene, in which transcription of the marker gene is from the operon inserted upstream. The utility of the vector has been shown by introduction of herbicide resistance genes into the plastid genome, and subsequent removal of the marker genes by the Cre site-specific recombinase (Lutz et al., 2006a). Vectors suitable for polycistronic expression are pPRV323Clox and pPRV324Clox, in which suitably engineered operons may be expressed in the trnI-trnA intergenic region. Again, these vectors are equipped for excision of the marker genes by the Cre-loxP site-specific recombination system. The utility of the pPRV323Clox vector ( Fig. 4; Table I), formerly designated as pPRV312L vector, was shown by expression of the cry9Aa2 Bacillus thuringiensis gene as part of the rrn operon (Chakrabarti et al., 2006). A second option to minimize duplications is incorporation of expression signals from heterologous sources. An early example for successful use of a heterologous signal is the T7 phage gene 10 leader that promotes high-level translation in chloroplasts (Staub et al., 2000;Kuroda and Maliga, 2001a). A heterologous source of promoters may be prokaryotes or mitochondria, which have a transcription machinery similar to the plastid NEP. Interesting in this regard is the demonstration that the mitochondrial atpA promoter is faithfully recognized in plastids (Bohne et al., 2007).

Choices for Marker Excision
Marker genes are essential for the selective amplification of the initially transformed ptDNA copies. When the transplastomic plants carry only transformed ptDNA, the marker gene is no longer needed. Most of the new vectors listed in Table I are equipped with sequences that are necessary for posttransformation removal of the marker genes utilizing phage site-specific recombinases. The transformed ptDNA, in which recombinase target sites flank the marker gene, is stable in the absence of the recombinase (Fig. 1A). When removal of the marker gene is desired, the phage enzymes are expressed from a nuclear gene. Plastid targeting is achieved by fusing the recombinase at its N terminus with the plastid-targeting region of a nuclear-encoded, plastidtargeted gene, such as the Rubisco small subunit transit peptide. The recombinase, translated on cytoplasmic ribosomes, enters all plastids and simultaneously excises the marker genes (Fig. 1B).
Thus far two recombinases have been tested for plastid marker gene excision, the Cre and the Int. The Cre enzyme derives from the P1 bacteriophage and excises target sequences flanked by directly oriented 34-bp loxP sites (Corneille et al., 2001;Hajdukiewicz et al., 2001;Kuroda and Maliga, 2003;Lutz et al., 2006a;Tungsuchat et al., 2006). The Cre gene has been introduced into the plant nucleus by three methods: (1) stable transformation of the nucleus using an Agrobacterium binary vector (Corneille et al., 2001;Hajdukiewicz et al., 2001); (2) by pollination (Corneille et al., 2001); (3) or by transiently expressing Cre from T-DNA by agroinfiltration (Lutz et al., 2006a). There are seven vectors in Table I that carry a floxed marker gene, each of which can be removed by any of the three approaches using a Cre vector listed in Table II. The second site-specific recombinase that has been tested for marker gene excision is Int, the phiC31 phage site-specific recombinase (integrase). To facilitate excision of the aadA marker gene, it was flanked with directly oriented nonidentical phage attP (215 bp) and bacterial attB (54 bp) attachment sites. Efficient excision of the marker gene was shown after transformation of the nucleus with an Int gene encoding a plastid-   Lutz et al. (2004) targeted Int enzyme (Kittiwongwattana et al., 2007). At this time two vectors, pPRV111Aatt and pPRV111Batt, are available for release ( Fig. 3; Table I). The plastidtargeted nuclear Int is encoded in Agrobacterium Int vector pKO117 (Table II). Out of the two site-specific recombinases, Int appears to be the better choice since the ptDNA contains pseudo-lox sites recognized by the Cre but no sequences recognized by the Int (Corneille et al., 2003;Lutz et al., 2004;Kittiwongwattana et al., 2007). Thus Cre, but not Int, may induce deletions between target sites and ptDNA sequences. A second problem observed during recombinase-mediated marker excision was enhanced homologous recombination between repeated (nontarget) ptDNA sequences (Corneille et al., 2001;Tungsuchat et al., 2006). Deletions via repeated sequences could be avoided when the repeated sequences were in an inverted orientation (Kittiwongwattana et al., 2007).

Loss of Transgenes via Repeated Sequences
We recently found that both the bar and aadA genes are lost in a small fraction of the seed progeny when the bar gene is expressed from a Prrn promoter after transformation with plasmid pMBC12, a pPRV111Batt vector derivative (Fig. 5A). The deletion was noticed because loss of the bar gene restores the normal green color to leaf cells that are golden yellow when expressing the bar gene in chloroplasts ( Fig. 5C; Kittiwongwattana et al., 2007). Loss of the bar gene was detected by formation of green sectors and by faster growth in approximately 0.6% of the seed progeny (10 variegated seedlings found among 1,584 selfed seed progeny; Fig. 5D). Sequencing of the PCR-amplified recombination junction revealed that the deletion occurred by homologous recombination between the Prrn promoter driving the bar gene and the native rrn operon promoter (data not shown). Deletion of sequences between the rrn operon repeats was also confirmed by DNA gel-blot analyses in each of the 10 lines. A representative blot is shown in Figure 5B for one of the lines.
In plasmid pMBC12 the Prrn promoter and the rrn operon promoter are 2.7-kb apart sharing 84 bp sequence as a direct repeat (PP-BamHI promoter sequence deposited in GenBank under accession no. EF416278). In the pPRV112A/B vectors described in 1994 the Prrn promoter contains a 118 bp repeat 1.3 kb upstream of the native rrn operon promoter. Although never shown to be unstable experimentally, we assume that deletion of aadA and trnV also occurs in plants transformed with pPRV112 vectors via the direct Prrn repeats. That is because probability of deletion via direct repeats is dependent on the length and the distance of the repeats. To obtain reasonably frequent deletions 649 bp repeats placed 5.4 kb apart were used because a 418 bp sequence spaced 1 kb Figure 5. Deletion of bar by homologous recombination via 84 bp direct repeat yields green sector in seedling. A, ptDNA map to show deletion via direct Prrn promoter repeat. Shown are: aadA, spectinomycin resistance gene; bar, bialaphos resistance gene, delays growth and causes golden leaf color; PrrnPclpP and PpsbA are promoters; TrbcL and TpsbA are 3#-UTRs; BB' and PP' are the attB and attP sequences; rrn16, trnV, and 3#rps12 are plastid genes. B, DNA gel-blot analyses confirms deletion of bar gene. Probing with ApaI-StuI ptDNA fragment (Fig. 5A) identifies 1.5-kb EcoRI fragment resulting from deletion. Variegated Nt-pPMBC12 plant is heteroplastomic and also contains the parental 4.1-kb Nt-pMBC12 fragment. C, Golden-yellow (aurea) Nt-pMBC12 parent. D, Larger variegated seedling among smaller aurea siblings. apart barely yielded any deletions (Iamtham and Day, 2000;Kode et al., 2006). We now only recommend vectors targeting the trnV-3#rps12 intergenic region, in which the Prrn promoter is in an inverted orientation relative to the native rRNA operon.
If essential plastid genes are deleted from the plastid genome, ptDNA copies lacking essential genes disappear in the absence of direct selection . The trnV-3#rps12 insertion site is special because the trnV gene between the insertion site (nucleotide 102,312 in the tobacco plastid genome; GenBank accession no. z00044) and the rrn operon promoter is dispensable (Corneille et al., 2001;Hajdukiewicz et al., 2001;Tungsuchat et al., 2006). While it is worth taking note of the potential problem caused by tandemly repeated sequences, the adverse consequences can be readily avoided by constructing only IRs or placing the duplicated sequence at a distance.

Pros and Cons of Species-Specific Vectors
Is it necessary to use vectors with species-specific targeting sequences and expression signals? The answer is no, although there are benefits in doing so. Targeting sequences are typically 1 to 2 kb long and flank the marker gene and gene of interest (Fig. 1A). Recombination may take place via sequences adjacent to the marker gene (sites no. 1 in Fig. 1A) or via sequences distal to the marker gene (site no. 2 in Fig. 1A). Sequencing of the ptDNA of multiple genetic lines indicates that there is significant intraspecies sequence variation in the plastid genome in at least some taxonomic groups. For example, intraspecies variation of the plastid genome in rice (Tang et al., 2004) and Arabidopsis (Sall et al., 2003) is well characterized. To obtain only one type of recombinant in such species requires transformation with strain-specific rather than species-specific vectors. This is definitely not worth the effort, particularly since individual distinct ptDNA may be a useful identifier for patent protection.
When transformation is carried out with a vector having heterologous targeting sequences, there is sufficient sequence conservation in the coding region of all higher plant plastid genomes to ensure integration of the marker gene and the gene of interest in any region of the ptDNA. This does not mean that the new transplastomic plants contain any heterologous ptDNA sequence. If recombination is via sequences proximal to the marker gene (site no. 1 in Fig. 1A), no sequences are incorporated from the vector targeting region. However, if recombination is via distal sequences (site no. 2 in Fig. 1A), the vector targeting sequence may replace the native ptDNA sequence (in this case the rrn16 and trnV genes). Caution is advised when the targeting region contains edited genes in which posttranscriptional C to U conversion restores a codon for a conserved amino acid. There are about 18 edited plastid genes carrying a total of approximately 30 edited C nucleotides (Tsudzuki et al., 2001;Kahlau et al., 2006). Highly edited genes make a bad targeting sequence for a heterologous transformation vector, as the new host may or may not have the capacity for editing the sites (Bock et al., 1994;Schmitz-Linneweber et al., 2005).

CONCLUSION
Plastid transformation vectors and marker excision systems are developed coordinately. Our intent has been to provide a simple vector system that enables transformation of the plastid genome in wild-type plants. While most of the vectors in Table I are new, the marker genes and the elements of the marker excision systems have been tested before. Both the Cre-lox and Int-att site-specific recombination systems are efficient, although Int appears to be the better choice.
High-level protein expression from plastid transgenes may come at a cost to the plant. Delayed development was shown in at least one transgenic line expressing the Cry9Aa2 B.t. protein in chloroplasts (Chakrabarti et al., 2006), but high-level protein accumulation is apparently compatible with normal growth in lines expressing other proteins. Delayed development is not a disadvantage if the goal is high-level expression of recombinant proteins but it may be a drawback in agronomic applications. The metabolic burden imposed by chloroplast expression is yet to be evaluated for most agronomic traits. It will be necessary to determine the protein level desired for each application and develop new tools that ensure protein accumulation at the desired level. Another new research direction will be exploration of the limits of increasing the size of ptDNA. Since insertion into the IR automatically doubles the size of the foreign DNA a task ahead will be development of new vectors for insertion of foreign DNA in the single copy regions.

Vector Construction
The backbone of the new pPRV100 series vectors, targeting insertions in the trnV-3#rps12 region, is derived from plasmid pPRV1 (Zoubenko et al., 1994). The vectors were obtained by ligating the marker genes and multiple cloning sites into the ScaI site of vector pPRV1. The kanamycin resistance gene in vectors pPRV145C and pPRV145D derived from plasmid pHK30 and is expressed in a PrrnLatpB 1 DS promoter and TrbcL terminator cassette (Kuroda and Maliga, 2001b). The two (C, D) vectors differ with respect to the relative orientation of the multiple cloning sites. Plastid transformation vector pAAK201 is identical with vector pPRV145C or D, other than the promoter of neo was cloned in tandem with the native rrn operon promoter and the neo gene is flanked by loxP sites (Supplemental Fig. S1).
The pPRV123Clox vector was obtained by cloning the SwaI fragment present in plasmid pPRV323Clox (formerly pPRV312L; Chakrabarti et al., 2006) into the ScaI site in plasmid pPRV1. Vectors pPRV123Blox and pPRV123Clox differ with respect to the position of multiple cloning site and the marker gene (in vectors A and B the cloning sites are on the right; in vectors C and D the cloning sites are on the left of the marker gene). In the vectors the aadA coding region is translationally fused at its C terminus with the 9E10 c-myc tag (EQKLISEEDL; Kolodziej and Young, 1991). The extension lox indicates that the aadA marker gene is flanked by the 34-bp wild-type P1 phage loxP sequences (Corneille et al., 2001). The pPRV124Clox vector is identical with pPRV123Clox, other than the aadA gene is expressed from the rbcL leader and downstream sequence (LrbcL 1 DS; PrrnLrbcL 1 DS/TrbcL cassette) derived from plasmid pHK34 (Kuroda and Maliga, 2001b). Vectors pPRV131A and pPRV131B are identical with vectors pPRV111A and pPRV111B, other than the aadA gene in the pPRV111 plasmids was replaced with the aadA-gfp fusion gene from plasmid pMSK56 (Khan and Maliga, 1999).
Construction of the pPRV323Clox (formerly pPRV312L) has been described (Chakrabarti et al., 2006). Vector pPRV324Clox is identical with pPRV323Clox, other than aadA is expressed from the PrrnLrbcL 1 DS promoter derived from plasmid pHK34 (Kuroda and Maliga, 2001b). Vector pPRV323Blox is identical with vector pPRV323Clox, other than the multiple cloning site is downstream of the marker gene.
The pMBC12 plastid transformation vector carries a bar gene that causes the aurea phenotype in a pPRV111Batt vector (Fig. 5A). This construct is identical to the bar gene in plasmid pCK2, other than the bar gene in plasmid pCK2 was cloned into a pPRV111Aatt vector (Kittiwongwattana et al., 2007).

Plastid Transformation
Plastid transformation with plasmid pAAK201 was carried out as described (Lutz et al., 2006b;Lutz and Maliga, 2007b). Briefly, leaves of tobacco (Nicotiana tabacum) 'Petit Havana' grown in sterile culture were placed abaxial side up on filter paper and the transforming DNA was introduced by the biolistic process using 1.0 mm gold particles. Two days after bombardment the leaf sections were transferred to RMOP medium containing 50 mg/L kanamycin sulfate. Kanamycin inhibited growth and greening of leaf calli from the leaf sections. The kanamycin resistant clones were identified as green shoots and proliferating green calli 6 to 12 weeks after bombardment. Leaves from regenerating shoots transferred onto the same selective RMOP medium, regenerated and characterized by DNA gel-blot analyses.
Plastid transformation and identification of transplastomic clones after bombardment with the pMBC12 vector was carried out as described for transformation with plasmid pCK2 (Kittiwongwattana et al., 2007).

DNA Gel-Blot Analyses of ptDNA
DNA gel-blot analysis was carried out as described Lutz et al., 2006b;Lutz and Maliga, 2007b). Briefly, total leaf cellular DNA was digested with the appropriate restriction endonucleases. The DNA fragments were separated by electrophoresis in 0.8% agarose gels and transferred to Hybond-N membranes (GE Healthcare) using the PosiBlot Transfer apparatus (Stratagene). Hybridization with the probes was carried out in Rapid Hybridization buffer (GE Healthcare) overnight at 65°C. DNA probe was prepared by random-primed 32 P labeling. DNA isolated from Nt-pMBC12 leaves was digested with the EcoRI restriction endonuclease and probed with the ApaI-StuI plastid-targeting region probe (Fig. 5B). DNA isolated from Nt-pAAK201 leaves was digested with the BamHI restriction endonuclease and probed with the 1.5-kb ApaI-BstEII fragment derived from the targeting region (Supplemental Fig. S1).

Identification of Seedlings with bar Gene Deletion
Approximately 2,000 Nt-pMBC12 seeds were spread on the surface of Pro-Mix general purpose growing medium code 0432 (Premier Horticulture Inc.) in plastic flats (10 3 20 inches) and grown in the greenhouse with supplemental lighting (16 h daylight). During the first 5 d of cultivation the germinating seedlings were kept moist by covering the flats with a plastic dome. Seedlings with green sectors could be identified 3 weeks after planting the seed (Fig. 5D).

Supplemental Data
The following materials are available in the online version of this article.
Supplemental Figure S1. DNA gel-blot analyses confirmed plastid transformation in plants selected by kanamycin resistance in pAAK201transformed leaf cultures.