Chloroplast Vector Systems for Biotechnology Applications

Update on Plastid Transformation Vectors Chloroplast Vector Systems for Biotechnology Applications1 Dheeraj Verma and Henry Daniell* Department of Molecular Biology and Microbiology, College of Medicine, University of Central Florida, Orlando, Florida 32816–2364 Chloroplasts are ideal hosts for expression of transgenes. Transgene integration into the chloroplast genome occurs via homologous recombination of flanking sequences used in chloroplast vectors. Identification of spacer regions to integrate transgenes and endogenous regulatory sequences that support optimal expression is the first step in construction of chloroplast vectors. Thirty-five sequenced crop chloroplast genomes provide this essential information. Various steps involved in the design and construction of chloroplast vectors, DNA delivery, and multiple rounds of selection are described. Several crop species have stably integrated transgenes conferring agronomic traits, including herbicide, insect, and disease resistance, drought and salt tolerance, and phytoremediation. Several crop chloroplast genomes have been transformed via organogenesis (cauliflower [Brassica oleracea], cabbage [Brassica capitata], lettuce [Lactuca sativa], oilseed rape [Brassica napus], petunia [Petunia hybrida], poplar [Populus spp.], potato [Solanum tuberosum], tobacco [Nicotiana tabacum], and tomato [Solanum lycopersicum]) or embryogenesis (carrot [Daucus carota], cotton [Gossypium hirsutum], rice [Oryza sativa], and soybean [Glycine max]), and maternal inheritance of transgenes has been observed. Chloroplastderived biopharmaceutical proteins, including insulin, interferons (IFNs), and somatotropin (ST), have been evaluated by in vitro studies. Human INFa2b transplastomic plants have been evaluated in field studies. Chloroplast-derived vaccine antigens against bacterial (cholera, tetanus, anthrax, plague, and Lyme disease), viral (canine parvovirus [CPV] and rotavirus), and protozoan (amoeba) pathogens have been evaluated by immune responses, neutralizing antibodies, and pathogen or toxin challenge in animals. Chloroplasts have been used as bioreactors for production of biopolymers, amino acids, and industrial enzymes. Oral delivery of plant cells expressing proinsulin (Pins) in chloroplasts offered protection against development of 1 This work was supported by the U.S. Department of Agriculture (grant no. 3611–21000–017–00D) and by the National Institutes of Health (grant no. 5R01 GM 63879–06). * Corresponding author; e-mail [email protected]. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Henry Daniell ([email protected]). www.plantphysiol.org/cgi/doi/10.1104/pp.107.106690 insulitis in diabetic mice; such delivery eliminates expensive fermentation, purification, low temperature storage, and transportation. Chloroplast vector systems used in these biotechnology applications are described. ADVANTAGES OF PLASTID TRANSFORMATION Chloroplasts are members of a class of organelles known as plastids and are found in plant cells and eukaryotic algae. As the site of photosynthesis, chloroplasts are the primary source of the world’s food productivity and they sustain life on this planet. Other important activities that occur in plastids include evolution of oxygen, sequestration of carbon, production of starch, synthesis of amino acids, fatty acids, and pigments, and key aspects of sulfur and nitrogen metabolism. Chloroplasts are generally considered as derivative of a cyanobacterial ancestor that was captured early during the evolution of a eukaryotic cell. However, the chloroplast genome is considerably reduced in size as compared to that of free-living cyanobacteria, but the genomic sequences that are still present show clear similarities (Martin et al., 2002). Land plant chloroplast genomes typically contain 110 to 120 unique genes, whereas cyanobacteria contain more than 1,500 genes. Many of the missing genes are present in the nuclear genome of the host (Martin et al., 2002). In most angiosperm plant species, plastid genes are maternally inherited (Hagemann, 2004), and therefore, transgenes in these plastids are not disseminated by pollen. This makes plastid transformation a valuable tool for the creation and cultivation of genetically modified plants that are biologically contained, thus posing lower environmental risks (Daniell, 2002, 2007). This biological containment strategy is therefore suitable for establishing the coexistence of conventional and genetically modified crops. Cytoplasmic male sterility (CMS) presents a further genetic engineering approach for transgene containment (Ruiz and Daniell, 2005). Furthermore, plant-derived therapeutic proteins are free of human pathogens and mammalian viral vectors. Therefore, plastids provide a viable alternative to conventional production systems such as microbial fermentation or mammalian cell culture. Another advantage of plastid transformation is the ability to accumulate large amounts of foreign protein (up to 46% of total leaf protein) when the transgene is stably integrated (De Cosa et al., 2001). This is due to the polyploidy of the plastid genetic system with up to Plant Physiology, December 2007, Vol. 145, pp. 1129–1143, www.plantphysiol.org Ó 2007 American Society of Plant Biologists 1129 Verma and Daniell 10,000 copies of the chloroplast genome in each plant cell, resulting in the ability to sustain a very high number of functional gene copies. Furthermore, sitespecific integration into the chloroplast genome by homologous recombination of flanking chloroplast DNA sequences present in the chloroplast vector eliminates the concerns of position effect, frequently observed in nuclear transgenic lines (Daniell et al., 2002). Other advantages seen in chloroplast transgenic plants include the lack of transgene silencing despite the accumulation of transcripts at a level 169-fold higher than in nuclear transgenic plants (Lee et al., 2003) and accumulation of foreign proteins at levels up to 46% of total leaf protein (De Cosa et al., 2001). Chloroplast genetic engineering also offers the unique advantage of transgene stacking, i.e. simultaneous expression of multiple transgenes, creating an opportunity to produce multivalent vaccines in a single transformation step. Several heterologous operons have been expressed in transgenic chloroplasts, and polycistrons are translated without processing into monocistrons (Quesada-Vargas et al., 2005). Moreover, foreign proteins synthesized in chloroplasts are properly folded with appropriate posttranscriptional modifications, including disulfide bonds (Staub et al., 2000; Arlen et al., 2007; Ruhlman et al., 2007) and lipid modifications (Glenz et al., 2006). This article is focused on the various components of vectors used for stable protein production in transgenic chloroplasts. GENOME ORGANIZATION AND CONCEPTS OF CHLOROPLAST TRANSFORMATION The chloroplast genome typically consists of basic units of double-stranded DNA of 120 to 220 kb arranged in monomeric or multimeric circles as well as in linear molecules (Palmer, 1985; Lilly et al., 2001). The chloroplast genome generally has a highly conserved organization (Raubeson and Jansen, 2005), with most land plant genomes having two identical copies of a 20- to 30-kb inverted repeat region (IRA and IRB) separating a large single copy (LSC) region and a small single copy (SSC) region. Plastid transformation is typically based on DNA delivery by the biolistic process (Daniell et al., 1990; Sanford et al., 1993) or occasionally by polyethylene glycol (PEG) treatment of protoplasts (Golds et al., 1993; O’Neill et al., 1993). This is followed by transgene integration into the chloroplast genome via homologous recombination facilitated by a RecA-type (Cerutti et al., 1992) system between the plastid-targeting sequences of the transformation vector and the targeted region of the plastid genome. Chloroplast transformation vectors are thus designed with homologous flanking sequences on either side of the transgene cassette to facilitate double recombination. Targeting sequences have no special properties other than that they are homologous to the chosen target site and are generally about 1 kb in size. Both flanking sequences are essential for homologous recombina1130 tion. Transformation is accomplished by integration of the transgene into a few genome copies, followed by 25 to 30 cell divisions under selection pressure to eliminate untransformed plastids, thereby achieving a homogeneous population of plastid genomes. If the transgene is targeted into the IR region, integration in one IR is followed by the phenomenon of copy correction that duplicates the introduced transgene into the other IR as well. Transgenes have been stably integrated at several sites within the plastid genome. Transgenes were first integrated into transcriptionally silent spacer regions (Svab and Maliga, 1993). However, transcriptionally active spacer regions offer unique advantages, including insertion of transgenes without 5# or 3# untranslated regions (UTRs) or promoters. To date, the most commonly used site of integration is the transcriptionally active intergenic region between the trnI-trnA genes, within the rrn operon, located in the IR regions of the chloroplast genome. The foreign gene expression levels obtained from genes integrated at this site are among the highest ever reported (De Cosa et al., 2001). It appears that this preferred site is unique and allows highly efficient transgene integration and expression. Chloroplast vectors may also carry an origin of replication that facilitates replication of the plasmid inside the chloroplast, thereby increasing the template copy number for homologous recombination and consequently enhancing the probability of transgene integration. oriA is present within the trnI flanking region (Kunnimalaiyaan and Nielsen, 1997; Lugo et al., 2004), and this might facilitate replication of foreign vectors within chloroplasts (Daniell et al., 1990), enhance the probability of transgene integration, and achieve homoplasmy even in the first round of selection (Guda et al., 2000). This is further confirmed by the first successful Rubisco engineering obtained by integrating the rbcS gene at this site (Dhingra et al., 2004). All other earlier attempts on Rubisco engineering at other integration sites within the chloroplast genome were only partially successful. Integration of transgenes between exons of trnA and trnI also facilitates correct processing of foreign transcripts because of processing of introns present within both flanking regions. UNIVERSAL VECTOR VERSUS SPECIES-SPECIFIC CHLOROPLAST VECTORS The proposal of a ‘‘universal vector’’ containing the trnA and trnI genes from the IR region of the tobacco chloroplast genome as flanking sequences for homologous recombination to transform several other plant species (of unknown genome sequence) was suggested several years ago (Daniell et al., 1998). This concept was based on the high conservation of this intergenic spacer region among the higher plant chloroplast genomes. Vectors designed for transformation of the tobacco plastid genome have been successfully used for potato and tomato plastid transformation, Plant Physiol. Vol. 145, 2007 Plastid Transformation Vectors because the homologous flanking sequences present in these vectors showed adequate homology to the corresponding sequences of potato and tomato plastid DNA but the efficiency of transformation is significantly lower than tobacco (Sidorov et al., 1999; Ruf et al., 2001). For example, only one potato and tomato chloroplast transgenic line was obtained per 35 and 87 bombarded plates, respectively, when compared to about 15 tobacco chloroplast transgenic lines often generated from one bombarded plate (Fernandez-San Millan et al., 2003). A similar lower efficiency was observed when petunia flanking sequences (approximately 98% homologous) were used to transform the tobacco chloroplast genome (DeGray et al., 2001), revealing that a lack of complete homology may reduce the transformation efficiency to a great extent. However, comparison of intergenic spacer regions among members of Solanaceae revealed that only four regions are identical (Daniell et al., 2006). Similarly, comparison of intergenic spacer regions of nine grass chloroplast genomes revealed that not even a single spacer region is identical among all sequenced chloroplast genomes (Saski et al., 2007). Therefore, the concept of a universal vector is applicable when a higher level of homology exists among plant species but will be less efficient than species-specific chloroplast vectors. The accession numbers for several crop chloroplast genome sequences have been provided at the Web site (http://www.bch.umontreal.ca/ogmp/projects/other/ cp_list.html, http://www.ncbi.nlm.nih.gov/genomes/ static/euk_o.html, or http://chloroplast.cbio.psu.edu/ cgi-bin/organism.cgi for access to genomic sequences). Additionally, optimization of transformation protocols specific for each crop should enhance the efficiency of transformation. SELECTABLE MARKERS FOR PLASTID TRANSFORMATION At the beginning, selection of plastid transformants was carried out by spectinomycin resistance encoded in the mutant 16S ribosomal RNA (rRNA) gene (Harris et al., 1989; Svab et al., 1990). Stable integration and expression of the aadA gene was first reported in the chloroplast genome of Chlamydomonas (GoldschmidtClermont, 1991). The aadA gene encodes the enzyme aminoglycoside 3# adenylyltransferase that inactivates spectinomycin and streptomycin by adenylation and prevents binding to chloroplast ribosomes. The aadA gene was later used as a selectable marker in tobacco, and the frequency of transformation events increased to 100-fold more than the mutant 16S rRNA genes (Svab and Maliga, 1993). Due to the recessive nature of the mutant 16S rRNA marker gene, the phenotypic resistance was not expressed until sorting out of the transgenomes was essentially completed. Lack of phenotypic resistance permitted the loss of the resistant rRNA gene in 99 out of 100 potential transformation events. Although it was first explained that spectinoPlant Physiol. Vol. 145, 2007 mycin offers nonlethal selection (Svab and Maliga, 1993) by not inhibiting cell division and growth at high concentrations (approximately 500 mg mL21), it was observed to be lethal in all other plant species (Table I). The neo gene is another alternative marker for plastid transformation that confers kanamycin resistance (Carrer et al., 1993). A different kanamycin resistance gene (aphA6) with relatively high transformation efficiency was reported later (Huang et al., 2002). Another selection strategy utilizing a ‘‘double barrel’’ vector was used for cotton transformation where explant for transformation was nongreen cells (Kumar et al., 2004b). The cotton plastid transformation vector contained two different genes (aphA6 and nptII) coding for two different enzymes. The aphA6 gene was regulated by the 16S rRNA promoter and gene 10 UTR capable of expression in the dark and in nongreen tissues. The nptII gene was regulated by the psbA promoter and UTR capable of expression in the light. Both genes with different regulatory sequences facilitated detoxification of the same selection agent (kanamycin) during day and night as well as in developing plastids and mature chloroplasts. The double barrel transformation vector was reported to be at least 8-fold more efficient than single gene (aphA6)-based chloroplast vectors. To avoid potential disadvantages of antibiotic resistance genes, several studies have explored strategies for engineering chloroplasts that are free of antibioticresistance markers. The spinach (Spinacia oleracea) betaine aldehyde dehydrogenase (badh) gene has been developed as a plant-derived selectable marker gene to transform chloroplast genomes (Daniell et al., 2001b). The selection process involved conversion of the toxic compound betaine aldehyde to beneficial Gly betaine by the chloroplast-localized gene-encoding enzyme BADH. Because the BADH enzyme is present only in chloroplasts of a few plant species adapted to dry and saline environments (Rathinasabapathi et al., 1997; Nuccio et al., 1998), it is considered as a suitable selectable marker in many crop plants. The transformation study showed rapid regeneration of transgenic shoots within 2 weeks in tobacco, and betaine aldehyde selection was 25-fold more efficient than spectinomycin. In addition, the Badh enzyme conferred salt tolerance in carrot (Kumar et al., 2004a). The bacterial bar gene, encoding phosphinothricin acetyltransferase (PAT) and conferring herbicide resistance, has also been tested as a plastid-selectable marker. PAT served as an excellent marker in nuclear transformants and conferred resistance to the herbicide phosphinothricin. Expression of the bar gene in plastid conferred phosphinothricin resistance only when introduced by selection for a linked aadA gene. However, the bar gene was not found to be suitable for the direct selection of transplastomic lines, even when expressed at a higher level (approximately 7% of total soluble cellular protein). Thus, it shows that direct selection by herbicide resistance is constrained by way of subcellular localization of the gene encoding the detoxifying enzyme PAT (Lutz et al., 2001). The lethality of herbi1131 Verma and Daniell Table I. Chloroplast transformation method and selection conditions reported for different crop species Crop Explant and Method of Transformation Plant regeneration by embryogenesis Carrot Fine cell suspension cultures derived from stem Cotton Rice Soybean Grayish-green friable callus produced from hypocotyl explants of 5-d-old cotton seedlings; biolistic using 0.6-mm gold particles Calli derived from mature seeds; biolistic using 0.6-mm gold particles Embryogenic calli; biolistic using 0.6-mm gold particles Selection Agent and Conditions for First, Second, and Third Rounds of Selection Literature Cited Spectinomycin; first selection of cell lines for 2 to 3 months on 150 mg mL21 spectinomycin; second selection on 350 mg mL21 spectinomycin for a month; multiplication using 500 mg mL21 spectinomycin; transgenic shoots were produced from somatic embryos on 500 mg mL21 spectinomycin. Kanamycin; first selection with 50 mg mL21 kanamycin; second selection with 100 mg mL21 kanamycin for 4 to 5 months. Transformed calli were converted into somatic embryos and plantlets. Kumar et al. (2004a) Streptomycin; first selection after 1 to 2 d of bombardment on medium supplemented with 200 mg mL21 streptomycin; the streptomycin-resistant shoots were rooted on Murashige and Skoog medium with 500 mg mL21 streptomycin. Spectinomycin; first selection after 2 d of bombardment on medium containing 200 or 300 mg mL21 spectinomycin and subcultured every 15 d; first green spectinomycin-resistant calli appeared after 8 weeks of selection and amplified in a medium with 150 mg mL21 spectinomycin and subsequently converted to embryos; after 2 months embryos germinated into young plants on Murashige and Skoog medium with 150 mg mL21 spectinomycin. Lee et al. (2006b) Plant regeneration by organogenesis from protoplasts Cauliflower Protoplasts isolated from Spectinomycin; embryogenic calli selected with 60 mg mL21 spectinomycin. The calli subsequently formed shoots. Leaf explants from fully expanded leaves; these regenerated shoots placed on shoot regeneration medium containing PEG4000 mediated 300 mg mL21 spectinomycin regenerated further resistant shoots. Spectinomycin; first selection was initiated after 6 d of transformation in dark Lettuce Protoplasts isolated from for 1 week; second selection in light until the calli were approximately 3-week-old shoot 0.5 mm in diameter followed by growth until calli were few millimeters culture leaves; in diameter; shoot formation; all selection steps contained 500 mg mL21 PEG6000 mediated spectinomycin. Kumar et al. (2004b) Dufourmantel et al. (2004) Nugent et al. (2006) Lelivelt et al. (2005) Plant regeneration by organogenesis from leaf Liu et al. Cabbage Leaves; biolistic using Spectinomycin; first selection of calli after 1 week of bombardment (2007) 1.0-mm gold particles on medium containing 50 mg mL21 spectinomycin and subcultured every 2 weeks; second selection until shoots formed on medium with 100 mg mL21 spectinomycin and streptomycin; regenerated shoots were rooted on medium with 200 mg mL21 spectinomycin. Spectinomycin; first selection of resistant green calli on medium Kanamoto Lettuce Young leaves from 3- to supplemented with 50 mg mL21 spectinomycin for 2 months et al. 4-week-old plants; (2006) followed by shoot regeneration on same medium in few weeks. biolistic using 0.6-mm gold particles Ruhlman Lettuce Young leaves; biolistic Spectinomycin; first selection of resistant shoots on medium supplemented using 0.6-mm gold with 50 mg mL21 spectinomycin; second selection of resistant shoots et al. (2007) particles from pieces of leaves of resistant shoots from first round of selection on medium supplemented with 50 mg mL21 spectinomycin; regenerated shoots were rooted on medium with 50 mg mL21 spectinomycin. Hou et al. Spectinomycin; first selection after 3 d of bombardment with 10 mg mL21 Oilseed Green cotyledon petioles (2003) spectinomycin; the regenerated green shoots subcultured onto the same rape of 1–2 mm in length; biolistic using tungsten selection medium once every 3 weeks twice and then transferred to rooting medium and finally to soil. particles Zubko et al. Petunia Leaves; biolistic using Spectinomycin and streptomycin; first selection on medium supplemented (2004) 1.0-mm gold particles with 200 mg mL21 spectinomycin and 200 mg mL21 streptomycin for every 3 to 4 weeks. Resistant shoots first appeared after 8 weeks. Poplar Leaves; biolistic using Spectinomycin; first selection on medium containing 30 mg/L spectinomycin Okumura et al. 0.6-mm gold particles and subcultured every 2 weeks; spectinomycin-resistant calli transferred to (2006) shoot induction medium with 30 mg mL21 spectinomycin every 4 weeks until shoot formation. Regenerated shoots were transferred to root induction medium. (Table continues on following page.) 1132 Plant Physiol. Vol. 145, 2007 Plastid Transformation Vectors Table I. (Continued from previous page.) Crop Explant and Method of Transformation Potato Leaves; biolistic using 0.6-mm gold particles Potato Leaves; biolistic using 0.6-mm gold particles Tomato Young leaves; biolistic using 0.6-mm gold particles Selection Agent and Conditions for First, Second, and Third Rounds of Selection Literature Cited Spectinomycin; after 2 to 3 d of bombardment, the pieces of leaves were placed onto regeneration medium containing spectinomycin (40, 300, and 400 mg mL21). The first spectinomycin-resistant events were identified after 4 to 6 weeks of selection. Spectinomycin; first selection on medium containing 300 mg mL21 spectinomycin for 4 weeks; second selection of the leaf explants on shoot induction medium containing 300 mg mL21 spectinomycin for every 3 weeks; the spectinomycin-resistant shoots formed in 8 to 10 weeks; rooting in Murashige and Skoog medium with 400 mg/L spectinomycin. Spectinomycin; bombarded leaves were incubated on medium with 300 or 500 mg mL21 spectinomycin for 3 to 4 months to obtain resistant yellow or pale green calli and subcultured to achieve homoplasmy. Plants were regenerated from homoplasmic callus tissue. Sidorov et al. (1999) Nguyen et al. (2005) Ruf et al. (2001) cides to plastids was determined by examining plastid ultrastructure using transmission electron microscopy (Ye et al., 2003). In glyphosate-treated cells of cultured tobacco leaf discs, the reticulate network of thylakoid membranes has been lost, indicating disintegration of the photosynthetic membranes. The plastid contents spilled out into the cell cytoplasm due to the ruptured outer plastid membrane at several locations. On the other hand, spectinomycin antibiotic had no detrimental effect on plastid ultrastructure. Therefore, herbicide resistance genes could not be used to directly select plastid transformants, and herbicide resistance was achieved only when herbicide resistance genes were introduced by selection for a linked aadA gene. A negative selection scheme has also been employed for plastid transformation based on expression of the bacterial gene codA (Serino and Maliga, 1997). Cytosine deaminase (codA) catalyzes the deamination of cytosine to uracil. 5-Fluorocytosine is toxic to cells that express cytosine deaminase because this enzyme converts 5-fluorocytosine to toxic 5-fluorouracil. This negative selection scheme was utilized to identify seedlings on 5-fluorocytosine medium from which codA was removed by the P1 bacteriophage site-specific recombinase CRE-lox (Corneille et al., 2001). GFP has also been fused with AadA and used as a bifunctional visual and selectable marker (Khan and Maliga, 1999). Further, GFP has been used to test the concept of receptor-mediated oral delivery of foreign proteins. Cholera toxin B-subunit (CTB)-GFP fusion protein with a furin cleavage site in between CTB and GFP has been used to elucidate the path of CTB and GFP in the circulatory system (Limaye et al., 2006). Mice were fed with CTB-GFP-expressing plant leaf material. GFP was detected in the intestinal mucosa and submucosa, the hepatocytes of the liver, as well as various cells of spleen utilizing fluorescence microscopy and anti-GFP antibodies. In mice fed with untransformed leaf material or IFN-GFP fusion protein-expressing plant leaf material, no GFP fluorescence was observed. This confirmed the receptor-mediated oral delivery of a foreign protein (GFP) across the intestinal lumen into the systemic circulation. Moreover, GFP was not detected in any substantial amount in the liver or spleen of mice fed with IFN-GFP-expressing plants, suggesting that a transmucosal carrier such as CTB is required for delivery of an adequate amount of a foreign protein across the intestinal lumen into the systemic circulation. Thus, GFP has been used as a reporter gene in chloroplast expression and in animal studies. REPORTER GENES USED IN PLASTIDS EXCISION OF SELECTABLE MARKER GENES GUS, chloramphenicol acetyl transferase, and GFP have been used as plastid reporters (Daniell and McFadden, 1987; Daniell et al., 1990; Ye et al., 1990; Khan and Maliga, 1999). The enzymatic activity of GUS can be visualized by histochemical staining (Ye et al., 1990; Daniell et al., 1991), whereas GFP is a visual marker that allows direct imaging of the fluorescent gene product in living cells. The GFP chromophore forms autocatalytically in the presence of oxygen and fluoresces green when absorbing blue or UV light (Hanson and Kohler, 2001). GFP has been used to detect transient gene expression (Hibberd et al., 1998) and stable transformation events (Reed et al., 2001; Lelivelt et al., 2005; Limaye et al., 2006) in chloroplasts. Most of the studies involving plastid transformation have utilized antibiotic resistance gene for the recovery of transformed plastomes, but introducing such crops into the food chain may be a cause of concern. Strategies have been developed to eliminate antibiotic resistance genes after transformation, including homology-based excision via directly repeated sequences, excision by phage site-specific recombinases, transient co-integration of the marker gene, and cotransformation-segregation. Early experiments with Chlamydomonas reinhardtii showed that homologous recombination between two direct repeats enabled marker removal under nonselective growth conditions (Fischer et al., 1996). Subsequently, marker genes have been deleted from Plant Physiol. Vol. 145, 2007 1133 Verma and Daniell transplastomic tobacco via engineered direct repeats that flank them (Iamtham and Day, 2000). A variant of homology-based marker excision technology enabled direct identification of marker-free tobacco plants by herbicide resistance (Dufourmantel et al., 2007). The vector used for plastid transformation carried the aadA gene disrupting the herbicide resistance gene. The primary transplastomic clones were selected by spectinomycin resistance. Marker-free herbicide-resistant derivatives were identified after excision of the aadA marker gene by homologous recombination within the overlapping region (403 nucleotides) of the N-terminal and C-terminal halves of the herbicide resistance gene. Excision of the aadA gene led to reconstitution of an entire herbicide resistance gene and expression of the Pseudomonas fluorescens 4-hydroxyphenylpyruvate dioxygenase enzyme that conferred resistance to sulcotrione and isoxaflutole herbicides (Dufourmantel et al., 2007). A second variant of this approach facilitated visual tracking of homology-based marker excision by creation of a pigment-deficient zone due to the loss of a plastid photosynthetic gene rbcL (Kode et al., 2006). So far, two recombinases (Cre and FC31 phage integrase [Int]) have been tested for plastid marker gene excision. Using the P1 bacteriophage Cre/lox site-specific recombination system, a marker gene flanked by lox sites was removed after expression of the CRE protein was induced via the nuclear genome. The second site-specific recombinase, Int, appeared to be a better choice for the aadA marker gene removal when flanked with directly oriented nonidentical phage attP (215 bp) and bacterial attB (54 bp) attachment sites, which are recognized by Int recombinase. Efficient excision of the marker gene was shown after transformation of the nucleus with an int gene encoding plastid-targeted Int (Kittiwongwattana et al., 2007). Alternatively, a transient co-integrative vector may even be used to avoid the integration of selectable marker genes (Klaus et al., 2004). The cotransformation-segregation approach involves transformation with two plasmids that target insertions at two different ptDNA locations: one plasmid carries a selective marker and the other a nonselected gene. Selection for the marker yields transplastomic clones that also bear an insertion of the nonselected gene. The prospect of the approach was first shown in C. reinhardtii (Kindle et al., 1991). Interestingly, when the approach was tested in tobacco, a cotransformation efficiency of 20% was obtained even though tobacco has a greater number of chloroplasts (Carrer and Maliga, 1995). An application of cotransformation was His-tagging of an unlinked ndh gene following spectinomycin selection (Rumeau et al., 2005). STABILITY OF EXPRESSED PROTEINS IN CHLOROPLASTS Newly synthesized proteins are highly susceptible to proteases and require protection from chloroplast proteases. One such approach used the CRY chape1134 rone (encoded by the orf2 gene) to fold the insecticidal protein, Cry2Aa2, into cuboidal crystals. The crystal structure protected the foreign proteins from degradation, thereby increasing protein accumulation over 128-fold (from 0.36% to 46.1% of total soluble protein [tsp]; De Cosa et al., 2001). Similarly, when the human serum albumin (hsa) coding sequence was regulated by the chloroplast psbA 5# and 3# UTRs in the light, protein expression increased 500-fold, resulting in the formation of protective inclusion bodies. A 3- to 10-fold reduction in HSA protein expression was seen when leaves were harvested in the dark (FernandezSan Millan et al., 2003). This illustrated the power of regulatory sequences during illumination and protection from proteases when their access is limited. Several studies on transgenic chloroplasts did not correlate increased transcript abundance with translation efficiency. For example, chloroplast-derived RbcS transcripts were measured to be 165-fold and 143-fold more than the nuclear RbcS antisense control plants when the transgene was regulated by the psbA 5# UTR or the promoterless gene 10 UTR, respectively. Although the psbA 5# UTR transgenic lines resulted in the first successful functional Rubisco in transgenic plants, the gene 10 UTR transgenic lines performed poorly (Dhingra et al., 2004). The lack of correlation between increased transcript levels and translation efficiency suggests that transcript abundance is of less importance than protein stability in transgenic chloroplasts. Several studies have addressed the role of 5# UTRs. However, in a few cases, the amino acid sequences downstream of the translation initiation codon may play an important role in stabilizing newly synthesized proteins or enhancing translation (Kuroda and Maliga, 2001). Human insulin was unstable in transgenic chloroplasts; fusion with CTB resulted in high-level expression (up to 16% tsp) and facilitated oral delivery studies to achieve protection against the development of insulitis in nonobese diabetic mice (Ruhlman et al., 2007). Such N-terminal degradation is not unique to chloroplasts. All commercially produced insulin in bacteria or yeast is produced as a fusion protein; when expressed without fusion, insulin is rapidly degraded. Also, high-level expression of foreign proteins may have deleterious phenotypic effects and/or impose a significant burden on the plant (Magee et al., 2004), and recovery of transplastomic plants seems to be not feasible. In that case, the use of psbA UTR is lethal and conciliation of the expression levels or inducible expression of foreign protein is highly desirable. Even though the inducible systems are well known for nuclear transgenes, most existing systems for plastids rely on nuclear transgenes, usually a T7 RNA polymerase targeted to the chloroplast where it drives expression of a transgene placed under the control of a T7 promoter (McBride et al., 1995; Magee et al., 2004; Lossl et al., 2005). A Lac repressor-based isopropylthiob-galactoside-inducible expression system for plastids has been reported, although transgene repression in Plant Physiol. Vol. 145, 2007 Plastid Transformation Vectors the uninduced state was incomplete (Muhlbauer and Koop, 2005). Thus, there is a need to devise tightly controllable plastid-inducible expression systems that do not require nuclear transgenes. PLASTID TRANSFORMATION OF DIFFERENT CROP SPECIES Tobacco has been the most widely exploited plastid transformation system because of its ease in genetic manipulations. A single tobacco plant is capable of generating a million seeds and 1 acre of tobacco can produce more than 40 metric tons of leaves per year (Cramer et al., 1999; Arlen et al., 2007). Harvesting leaves before flowering can offer nearly complete transgene containment in addition to protection offered by maternal inheritance. Recent studies have reported that escape of transgenes in tobacco is 0.0087% to 0.00024% (Daniell, 2007; Ruf et al., 2007; Svab and Maliga, 2007), making this an ideal system for use of chloroplasts as bioreactors. In addition, CMS has been engineered via the tobacco chloroplast genome as a failsafe method (Ruiz and Daniell, 2005). As a bioreactor, tobacco has been estimated to be more than 50 times less expensive than the frequently used Escherichia coli fermentation systems (Kusnadi et al., 1997). Additionally, tobacco eliminates contamination of food because it is a non-food and non-feed crop. Plastid transformation in higher plants was first successfully carried out in tobacco and is now a routine procedure because many foreign genes have been expressed to engineer agronomic traits, biopharmaceuticals, vaccines, or biomaterials (Table II). However, presence of nicotine or other alkaloids has been a disadvantage for pharmaceutical production, but the chloroplast genome of low-nicotine varieties like LAMD has been used to engineer therapeutic proteins (Arlen et al., 2007). For oral delivery studies, there is a need to move beyond tobacco. Extension of the plastid transformation technology to other species is important to exploit this platform. The study of DNA delivery strategies, target tissues, selection conditions, and regeneration systems is crucial for extending the range of species in which plastid transformation could be achieved. Plastid transformation is most commonly achieved by biolistic delivery of DNA into leaf explants but has also been achieved via direct DNA uptake by protoplasts (Lelivelt et al., 2005; Nugent et al., 2006). In species other than tobacco, like petunia and oilseed rape, adventitious shoot regeneration from bombarded leaf or petiole explants generated plastid transformants. Homoplasmic plants of soybean, carrot, and cotton were regenerated via somatic embryogenesis after bombardment of embryogenic calli, combined with the use of species-specific plastid vectors. Table I summarizes the chloroplast transformation method and selection conditions for different crop species. Attempts have been made in other plants (Table I) where protein production was carried out in non-green tissues such Plant Physiol. Vol. 145, 2007 as micro-tuber (potato), fruit (tomato), and root (carrot). However, the amount of protein was lower than the level observed in leaf chloroplasts (Kumar et al., 2004a). Some progress has also been made in improving the chloroplast transformation system for tomato plants. Utilizing that plastid expression of a bacterial lycopene b-cyclase gene resulted in herbicide resistance and triggered conversion of lycopene, the main storage carotenoid of tomatoes, to b-carotene, resulting in a 4-fold enhancement of pro-vitamin A content of fruits (Wurbs et al., 2007). Stable chloroplast transformation system has also been reported for cabbage (Liu et al., 2007). Recently, edible leafy crops, including lettuce, have attracted attention toward plastid genetic engineering. Edible plant species not only minimize downstream protein processing costs but also offer an ideal system for oral delivery. The leaves of lettuce are consumed raw by humans and the time from sowing seed to edible biomass is only weeks compared to months for crops such as tomato, potato, and carrot. Furthermore, lettuce is well suited for indoor cultivation by hydroculture systems (Kanamoto et al., 2006). Accumulation of a valuable therapeutic protein, the CTB-Pins fusion, in lettuce chloroplasts was recently reported (Ruhlman et al., 2007). This is the first report of expression of a therapeutic protein in an edible crop. Further studies are required to understand the concept of oral delivery. Economically important crops such as carrot, cotton, and soybean regenerate in vitro through somatic embryogenesis (Daniell et al., 2005b). In such crops, transformation of the plastid genome was achieved through somatic embryogenesis by bombarding embryogenic non-green cells or tissues. The first stable plastid transformation of embryogenic cell cultures and somatic embryogenesis was established in carrot (Kumar et al., 2004a). Homoplasmic transgenic plants were regenerated from cell cultures bombarded with the aadA and badh genes. However, in the case of cotton, plastid transformation using the aadA gene was unsuccessful, and no transgenic cultures or plants were recovered using spectinomycin as the selection agent. Transgenic cotton cell lines were generated using a double barrel vector containing two selectable marker genes (aphA6 and nptII) to detoxify kanamycin (Kumar et al., 2004b). Transgenic lines were fertile and showed maternal inheritance of transgene. Soybean plastid transformation was achieved using embryogenic tissue as the starting material (Dufourmantel et al., 2004) and the aadA gene as the selectable marker. Phenotypically normal transgenic soybean plants were regenerated via somatic embryogenesis from spectinomycin-resistant calli and were fully fertile. Stable plastid transformation in rice was achieved using mature seed-derived calli for bombardment (Lee et al., 2006b). The transplastomic rice plants expressed GFP in their plastids and generated viable seeds, which were confirmed to transmit the transgenes to the T1 progeny plants. However, trans1135 Verma and Daniell Table II. Engineering of agronomic traits, biopharmaceuticals, vaccine antigens, and biomaterials via the plastid genome Traits/Gene Products Agronomic trait Insect resistance Herbicide resistance Disease resistance Drought tolerance Phytoremediation Salt tolerance CMS Biopharmaceutical proteins hST Insulin-like growth factor IFNa2b HSA IFN-g Monoclonal antibody Human Pins Vaccine antigens Cholera toxin Tetanus toxin Gene Promoter/5#/3# UTRs Literature Cited cry1A(c) cry2Aa2 cry2Aa2 operon cry1Aa10 cry1Ab cry9Aa2 aroA (petunia) bar MSI-99 TPS1 (yeast) merA/merB badh phaA Prrn/rbcL/rps16 Prrn/ggagg (native)/psbA Prrn/native 5# UTR/psbA Prrn/native 5# UTR/psbA Prrn/T7 gene10/rbcL Prrn/native 5# UTR/rbcL Prrn/ggagg/psbA Prrn/rbcL/psbA Prrn/ggagg/psbA Prrn/ggagg/psbA Prrn/ggagg/psbA Prrn/ggagg/rps16 Prrn/psbA/psbA McBride et al. (1995) Kota et al. (1999) De Cosa et al. (2001) Hou et al. (2003) Dufourmantel et al. (2005) Chakrabarti et al. (2006) Daniell et al. (1998) Iamtham and Day (2000) DeGray et al. (2001) Lee et al. (2003) Ruiz et al. (2003); Hussein et al. (2007) Kumar et al. (2004a) Ruiz and Daniell (2005) hST Prrn/T7 gene10/Trps16 PpsbA/Trps16 Prrn/PpsbA/TpsbA Prrn/PpsbA/TpsbA Prrn/PpsbA/TpsbA PpsbA/TpsbA Prrn/ggagg/TpsbA PpsbA/TpsbA Prrn/T7 gene10/Trps16 Staub et al. (2000) IGF-1n IGF-1s IFNa2b hsa Gus-IFN-g Guy’s 13 CTB-Pins CPV Anthrax PA CTB-2L21 GFP-2L21 pag Prrn/ggagg/TpsbA Prrn/T7gene 10/TrbcL atpB/TrbcL Prrn/PpsbA/TpsbA Prrn/PpsbA/TpsbA Amebiasis Plague lecA CaF1-LcrV Prrn/PpsbA/TpsbA Prrn/PpsbA/TpsbA VP6 NS3 OspA OspA-T Prrn/PpsbA/TpsbA Prrn/PpsbA/TpsbA PpsbA/TpsbA EG121 ubiC phb operon xynA ASA2 Prrn/T7 gene 10/TpsbA Prrn/PpsbA/TpsbA PpsbA/TpsbA PpsbA/TpsbA Prrn/rbcL/rpL32 rbcL/accD-ORF184 Prrn/PpsbA/TpsbA Rotavirus Hepatitis C Lyme disease Biomaterials Elastin-derived polymer pHBA Polyhydroxybutyrate Xylanase Tryptophan Monellin ctxB tetC bacterial and synthetic monellin plastomic rice plants were not homoplasmic, even after two generations of continuous selection. Plastid transformation of carrot, cotton, rice, and soybean opens the door for modification of the plastid genome of several crops that require embryogenesis. METHODS FOR CONSTRUCTION OF PLASTID TRANSFORMATION VECTORS AND GENERATION OF TRANSPLASTOMIC PLANTS Plastid gene expression is regulated both at the transcriptional and posttranscriptional levels. Protein levels in chloroplasts depend on mRNA abundance, 1136 Daniell et al. (2005a) Arlen et al. (2007) Fernandez-San Millan et al. (2003) Leelavathi and Reddy (2003) Daniell et al. (2004) Ruhlman et al. (2007) Daniell et al. (2001a) Tregoning et al. (2003) Molina et al. (2004, 2005) Watson et al. (2004); Koya et al. (2005) Chebolu and Daniell (2007) Y. Ding, P. Arlen, J. Adamovicz, M. Singleton, and H. Daniell (unpublished data) Birch-Machin et al. (2004) Daniell et al. (2005a) Glenz et al. (2006) Guda et al. (2000) Viitanen et al. (2004) Lössl et al. (2003) Leelavathi et al. (2003) Zhang et al. (2001) Roh et al. (2006) which is determined by promoter strength and mRNA stability. However, high mRNA levels do not result in high-level protein accumulation as posttranscriptional processes ultimately determine obtainable protein levels. Therefore, we have designed expression cassettes for transgene assembly to achieve optimal levels of protein accumulation in leaves (Fig. 1). The basic plastid transformation vector is comprised of flanking sequences and chloroplast-specific expression cassettes (Fig. 1). Species-specific chloroplast flanking sequence (e.g. trnI/trnA) is obtained by PCR using the primers designed from the available chloroplast genomes. The chloroplast expression cassette is comPlant Physiol. Vol. 145, 2007 Plastid Transformation Vectors Figure 1. Schematic representation of the chloroplast-specific expression cassette. Map of the chloroplast expression vector shows the integration sites, promoters, selectable marker genes, regulatory elements, and genes of interest. For a list of regulatory elements and genes of interest used for chloroplast transformation, refer to Table II. posed of a promoter, selectable marker, and 5#/3# regulatory sequences to enhance the efficiency of transcription and translation of the gene. The chloroplastspecific promoters and regulatory elements are amplified from the total cellular DNA using primers designed on the basis of the sequence information available for the chloroplast genome. Suitable restriction sites are introduced to facilitate gene assembly. Because of the high similarity in the transcription and translation systems between E. coli and chloroplasts, the chloroplast expression vectors are tested in E. coli first before proceeding with plant transformation. The growth of E. coli harboring the plastid transformation vector with the aadA gene in the presence of spectinomycin confirms expression of the aadA gene. Western blot with extracts from E. coli confirms expression of the gene of interest. Once expression of transgenes is confirmed in E. coli, the transformation vector is delivered into leaves (tobacco/lettuce) via particle bombardment. The leaves used for bombardment should be young, green, and healthy. The bombarded leaves are placed on selection medium with an appropriate concentration of antibiotics (RMOP in tobacco). Normally, in 3 to 10 weeks, putative transgenic shoots appear (Fig. 2, A and D). PCR analysis is used to screen the transgenic shoots and distinguish true chloroplast transgenic events from mutants or nuclear transgenic plants. Site-specific chloroplast integration of the transgene cassette is determined by using a set of primers, one of which anneals to the native chloroplast genome and Plant Physiol. Vol. 145, 2007 the other anneals within the transgene cassette. Mutants and nuclear transgenic plants are not expected to produce a PCR product with these primers (Fig. 3A). The leaf pieces from PCR-positive shoots are further selected for a second round to achieve homoplasmy (Fig. 2, B and E). The regenerated shoots are rooted with the same level of selection (Fig. 2, C and F) and checked for homoplasmy by Southern-blot analysis (Fig. 3B). The Southern blot is probed with radiolabeled flanking sequences used for homologous recombination. Transplastomic genome contains a larger size hybridizing fragment than the untransformed genome because of the presence of transgenes. If the transgenic plants are heteroplasmic, a native fragment is visible along with the larger transgenic fragment. Absence of the native fragment confirms the establishment of homoplasmy. Transgene expression is confirmed by western-blot analysis, and the effectiveness or properties or functionality of the introduced transgene is assessed. Seeds from the transgenic plants and untransformed plants are grown on spectinomycin-containing medium to check for maternal inheritance. Transgenic seeds germinate and grow into uniformly green plants. The absence of Mendelian segregation of transgenes confirms that they are maternally inherited to progeny. AGRONOMIC TRAITS ENGINEERED VIA THE CHLOROPLAST GENOME Several useful transgenes have conferred valuable agronomic traits, including insect and pathogen re1137 Verma and Daniell Figure 2. Selection of transplastomic plants. Shown are representative photographs of transplastomic tobacco and lettuce shoots undergoing first (A and D), second (B and E), and third (C and F, rooting) rounds of selection, respectively. sistance, drought tolerance, phytoremediation, salt tolerance, and CMS through chloroplast genetic engineering (Table II). Genetically engineered tobacco plants expressing an insecticidal protein Cry2Aa2 have shown resistance against target insects and insects that developed resistance against insecticidal protein (Kota et al., 1999). Expression of the Cry2Aa2 resulted in the utmost expression levels on record (approximately 46.1% of total leaf protein) and resulted in the detection of cuboidal crystals using transmission electron microscopy (De Cosa et al., 2001). In addition, soybean plastid transformants expressing Cry1Ab also conferred insecticidal activity against velvetbean caterpillar (Dufourmantel et al., 2005). The antimicrobial peptide MSI-99, an analog of magainin 2, was expressed via the chloroplast genome to obtain high levels of expression in transgenic tobacco plants. In planta assays with the bacterial pathogen Pseudomonas syringae pv tabaci and the fungal pathogen Colletotrichum destructivum showed necrotic lesions in untransformed control leaves, whereas transformed leaves showed no lesions (DeGray et al., 2001). Environmental stress factors such as drought, salinity, and freezing are perilous to plants generally because of their sessile means of existence. Attempts to confer resistance to drought by expressing trehalose phosphate synthase 1 (tps1) gene via nuclear transformation have proven futile because of undesirable pleiotropic effects even at very low levels of trehalose accumulation. However, hyperexpression of tps1 in the chloroplasts has no phenotypic variation from the untransformed control plants, and transgenic seeds sprouted, grew, and remained green and healthy in drought tolerance bioassays with 3% to 6% PEG and dehydration/rehydration assays (Lee et al., 2003). High-level expression of BADH in cultured cells, roots, and leaves of carrot via plastid genetic engineering exhibited high levels of salt tolerance. Transgenic carrot plants expressing BADH grew in the presence of high concentrations of NaCl (up to 400 mM), the uppermost level of salt tolerance reported so far among genetically modified crop plants (Kumar et al., 2004a). Chloroplast genetic engineering has also been used for the first time to our knowledge to enhance the capacity of plants for phytoremediation. This was accomplished by incorporating a native operon containing the merA and merB genes, which code for mercuric ion reductase (merA) and organomercurial lyase (merB), respectively, into the chloroplast genome in a single transformation event. Stable integration of the merAB operon into the chloroplast genome resulted in high levels of tolerance to the organomercurial compound phenylmercuric Figure 3. Evaluation of transgene integration into the chloroplast genome. DNA isolated from putative transplastomic shoots are analyzed by PCR and Southern-blot analysis. A, 3P/3M and 5P/2M primer pairs (Kumar and Daniell, 2004) are used for PCR analysis; PCR products of 3P/3M primers. Lane 1, Untransformed plant; lanes 2 to 4, transformed lines (1.6 kb); lane 1kb1, DNA marker; lanes 5 to 7, PCR product with 5P/2M primers (3.2 kb) in transformed lines. B, The chloroplast genome is probed with a radiolabeled flank fragment. Lane 1, Untransformed plant; lanes 2 and 4, homoplasmic transplastomic plant; lane 3, heteroplasmic transplastomic plant. 1138 Plant Physiol. Vol. 145, 2007 Plastid Transformation Vectors acetate when grown in soil containing up to 400 mM phenylmercuric acetate (Ruiz et al., 2003). Chloroplast transgenic lines absorbed mercury exceeding the levels in soil and translocated 100-fold more to shoots than untransformed plants (Hussein et al., 2007). Tobacco is ideal for phytoremediation of contaminated soil because it is a non-food non-feed crop. Naturally occurring CMS has been documented for over 100 years for oilseed rape, maize (Zea mays), and rice. However, such systems are not available for the majority of crops used in agriculture. In presently available CMS lines, various loci in the nuclear genome direct a range of restoration factors that are not fully understood. Moreover, risk of sterility trait dilution through segregation and the production of transgenic seeds that spread transgenic traits to nontransgenic plants cannot be ruled out because of the possibility of cross-pollination of the male-sterile line with a restorer line or wild relative. To address some of these concerns, CMS has been engineered via introduction of phaA gene coding for b-ketothiolase into chloroplast genome. The transgenic lines were normal except for the male sterility phenotype lacking pollen (Ruiz and Daniell, 2005). Further restoration of male fertility was reported by changing conditions of illumination. Continuous illumination increases acetyl-CoA carboxylase activity, thereby increasing the levels of plastidic fatty acid biosynthesis, which is especially needed for the formation of the exine pollen wall. PLASTIDS AS BIOPHARMACEUTICAL BIOREACTORS Several chloroplast-derived biopharmaceutical proteins have been reported (Daniell, 2006; Table II). Stable expression of a pharmaceutical protein in chloroplasts was first reported for GVGVP, a protein-based polymer with medical uses such as wound coverings, artificial pericardia, and programmed drug delivery (Guda et al., 2000). Human ST (hST), a secretory protein, was expressed inside chloroplasts in a soluble, biologically active and disulfide-bonded form (Staub et al., 2000). The key use of hST is in the cure of hypopituitary dwarfism in children; additional indications are treatment of Turner syndrome, chronic renal failure, and human immunodeficiency virus wasting syndrome. Another important therapeutic protein that comprises approximately 60% of the protein in blood serum is HSA, prescribed in multigram quantities to restore blood volume in trauma and other clinical conditions. Early attempts at expressing HSA have achieved inadequately low levels of HSA (0.2% of tsp) in nuclear transgenic plants (Farran et al., 2002). On the other hand, in chloroplast transgenic plants, expression levels of up to 11.2% were observed (FernandezSan Millan et al., 2003). The type I IFNs are part of the body’s first line of defense against viral attack and also invasion by bacterial pathogens, parasites, tumor cells, and allogeneic cells from grafts. IFNa2b ranks third in world Plant Physiol. Vol. 145, 2007 market use for a biopharmaceutical, behind only insulin and erythropoietin. The average annual cost of IFNa2b for the treatment of hepatitis C infection is $26,000, and is therefore unavailable to the majority of patients in developing countries. Therefore, IFNa2b was expressed in tobacco chloroplasts with levels of up to 20% of tsp or 3 mg/g of leaf (fresh weight) and facilitated the first field production of a plant-derived human blood protein (Arlen et al., 2007). Transgenic IFNa2b had comparable in vitro biological activity to commercially produced PEG-Intron when tested for its ability to protect BHK cells against cytopathic viral replication in the vesicular stomatitis virus cytopathic effect assay and to inhibit early stage human immunodeficiency virus infection in HeLa cells. Another therapeutic protein expressed in chloroplasts is human IFN-g (Leelavathi and Reddy, 2003). In a bioassay, the chloroplast-produced human IFN-g offered complete protection to human lung carcinomas against infection by the EMC virus. PLASTIDS AS VACCINE BIOREACTORS As opposed to injected subunit vaccines, oral delivery and low-cost purification make plastid-derived subunit production quite plausible (Kamarajugadda and Daniell, 2006). Subunit vaccines expressed in plants are capable of inducing a mucosal response in animal models when given orally or parenterally; these animals also withstand a pathogen challenge. The ability for plant-derived vaccines to survive in the stomach is a major concern. However, bioencapsulation can protect the vaccine in the stomach and gradually releases the antigen in the gut (Mor et al., 1998). Vaccine antigens against cholera (Daniell et al., 2001a), tetanus (Tregoning et al., 2003), anthrax (Watson et al., 2004; Koya et al., 2005), plague (Daniell et al., 2005a), amebiasis (Chebolu and Daniell, 2007), and CPV (Molina et al., 2004) have been expressed in transgenic chloroplasts (Table II). For cholera, the CTB has been shown to be an extremely powerful vaccine candidate and is encoded by Vibrio cholerae. The chloroplastexpressed CTB assembled into pentameric protein and assumed correct quaternary structure for full activity. Subsequent binding assays confirmed the ability of chloroplast-derived CTB to bind to the intestinal membrane GM1 ganglioside receptors. CTB also acts as a powerful transmucosal carrier and is very effective in delivering several vaccine antigens. In one such investigation, oral administration of chloroplast-derived CTB-Pins fusion protein protected nonobese diabetic mice against development of insulitis (Ruhlman et al., 2007). Recently, there has been an increased threat of bioterrorism in the post 9/11 world. Anthrax is always fatal if not treated immediately. Weapon-grade spores can be produced and stored for decades and can be spread by missiles, bombs, or even through the mail. Because of this, it is an ideal biological warfare agent. 1139 Verma and Daniell The currently available human vaccine for anthrax, derived from the culture supernatant of Bacillus anthracis, contains the protective antigen (PA) and traces of the lethal and edema factors. These factors may contribute to undesirable side effects linked with this vaccine. Therefore, an effective expression system that can provide a clean, safe, and efficacious vaccine is required. In an attempt to produce anthrax vaccine in large quantities and free of extraneous bacterial contaminants, PA was expressed in transgenic tobacco chloroplasts by inserting the pagA gene into the chloroplast genome (Watson et al., 2004; Koya et al., 2005). Mature leaves grown under continuous illumination contained PA up to 14.2% of tsp. Cytotoxicity measurements in macrophage lysis assays showed that chloroplast-derived PA was equivalent in potency to PA produced in B. anthracis. Subcutaneous immunization of mice with partially purified chloroplastderived or B. anthracis-derived PA with adjuvant yielded IgG titers up to 1:320,000 and both groups of mice survived (100%) challenge with lethal doses of toxin. These results demonstrated the immunogenic and immunoprotective properties of plant-derived anthrax vaccine antigen. PLASTIDS AS BIOMATERIAL BIOREACTORS Besides vaccine antigens, biomaterial and amino acids have also been expressed in chloroplasts (Table II). Normally, p-hydroxybenzoic acid (pHBA) is produced in small quantities in all plants. In E. coli, the ubiC gene encoding chorismate pyruvate lyase catalyzes the direct conversion of chorismate to pyruvate and pHBA. However, in chloroplasts, chorismate is converted to pHBA by 10 consecutive enzymatic reactions due to lack of chorismate pyruvate lyase. Stable integration of the ubiC gene into the tobacco chloroplast resulted in hyperexpression of the enzyme and accumulation of this polymer up to 25% of dry weight (Viitanen et al., 2004). In another study, the gene for thermostable xylanase was expressed in the chloroplasts of tobacco plants (Leelavathi et al., 2003). Xylanase accumulated in the cells to approximately 6% of tsp. Zymography assay demonstrated that the estimated activity was 140,755 units kg21 fresh leaf tissue. need to sequence chloroplast genomes to facilitate transformation of crop species. Regardless of the small size of the genome and availability of tools to sequence an entire genome within a single day, it is hard to understand why only a few crop chloroplast genomes have been sequenced so far. Between 1986 and 2004, only six crop chloroplast genomes were sequenced. In the past 3 years, 25 new crop chloroplast genomes have been sequenced, including major crops like soybean and cotton (Saski et al., 2005; Lee et al., 2006a). Recent studies reveal that intergenic spacer regions and regulatory sequences contribute about 40% to 45% of the chloroplast genome and that spacer regions are not highly conserved. Comparison of nine grass chloroplast genomes revealed that not even one spacer region had 100% homology. Therefore, species-specific chloroplast vectors should be made for efficient transformation of grasses (Saski et al., 2007). Plastid transformation is a tissue culture-dependent process. Therefore, it is not adequate just to have the genome information; a better understanding of DNA delivery, selection, regeneration, and progression toward homoplasmy is essential to achieve plastid transformation in different taxonomic groups. Although chloroplast genome sequences of several monocots, including wheat and maize, have been available for several years, none of their genomes has been fully transformed so far. Major obstacles include the difficulty of expressing transgenes in non-green plastids, in which gene expression and gene regulation systems are quite distinct from those of mature green chloroplasts. Moreover, it is not possible to generate homoplasmic plants via subsequent rounds of regeneration using leaves as explants. Furthermore, proplastids are used as the transformation target rather than chloroplasts that are about 5-fold smaller in size than the fully developed chloroplasts in the green leaf tissues. Therefore, plastids with irreversible physical damage due to biolistic bombardment might be greater. It may also be necessary to develop new selection markers for a monocot-specific selection scheme. However, transformation of cotton or carrot using non-green embryogenic cells containing proplastids and regeneration via somatic embryogenesis offers new hopes for success. ACKNOWLEDGMENT We thank Dr. Nameirakpam Dolendro Singh and Tracey Ruhlman for assistance with figures. CHALLENGES AHEAD Although the concept is more than 10 years old, plastid transformation has been accomplished in relatively few species. There are numerous factors that have hampered the expansion of chloroplast transformation technology to different plant species. One factor is the unavailability of the genome sequence. 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