chloroplast vector systems for biotechnology applications1

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 trans-genes. Transgene integration into the chloroplastgenome occurs via homologous recombination offlanking sequences used in chloroplast vectors. Iden-tification of spacer regions to integrate transgenes andendogenous regulatory sequences that support opti-mal expression is the first step in construction ofchloroplast vectors. Thirty-five sequenced crop chlo-roplast genomes provide this essential information.Various steps involved in the design and constructionof chloroplast vectors, DNA delivery, and multiplerounds of selection are described. Several crop specieshave stably integrated transgenes conferring agro-nomic traits, including herbicide, insect, and diseaseresistance, drought and salt tolerance, and phytore-mediation. Several crop chloroplast genomes havebeen transformed via organogenesis (cauliflower[Brassica oleracea], cabbage [Brassica capitata], lettuce[Lactuca sativa], oilseed rape [Brassica napus], petunia[Petunia hybrida], poplar [Populus spp.], potato [Sola-num tuberosum], tobacco [Nicotiana tabacum], and to-mato [Solanum lycopersicum]) or embryogenesis (carrot[Daucus carota], cotton [Gossypium hirsutum], rice [Oryzasativa], and soybean [Glycine max]), and maternal inher-itance of transgenes has been observed. Chloroplast-derived biopharmaceutical proteins, including insulin,interferons (IFNs), and somatotropin (ST), have beenevaluated by in vitro studies. Human INFa2b trans-plastomic 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), andprotozoan (amoeba) pathogens have been evaluatedby immune responses, neutralizing antibodies, andpathogen or toxin challenge in animals. Chloroplastshave been used as bioreactors for production of bio-polymers, amino acids, and industrial enzymes. Oraldelivery of plant cells expressing proinsulin (Pins) inchloroplasts offered protection against development of

insulitis in diabetic mice; such delivery eliminatesexpensive fermentation, purification, low temperaturestorage, and transportation. Chloroplast vector sys-tems used in these biotechnology applications aredescribed.

ADVANTAGES OF PLASTID TRANSFORMATION

Chloroplasts are members of a class of organellesknown as plastids and are found in plant cells andeukaryotic algae. As the site of photosynthesis, chlo-roplasts are the primary source of the world’s foodproductivity and they sustain life on this planet. Otherimportant activities that occur in plastids includeevolution of oxygen, sequestration of carbon, produc-tion of starch, synthesis of amino acids, fatty acids, andpigments, and key aspects of sulfur and nitrogenmetabolism. Chloroplasts are generally considered asderivative of a cyanobacterial ancestor that was cap-tured early during the evolution of a eukaryotic cell.However, the chloroplast genome is considerably re-duced in size as compared to that of free-living cya-nobacteria, but the genomic sequences that are stillpresent show clear similarities (Martin et al., 2002).Land plant chloroplast genomes typically contain 110 to120 unique genes, whereas cyanobacteria contain morethan 1,500 genes. Many of the missing genes are presentin the nuclear genome of the host (Martin et al., 2002).

In most angiosperm plant species, plastid genes arematernally inherited (Hagemann, 2004), and therefore,transgenes in these plastids are not disseminated bypollen. This makes plastid transformation a valuabletool for the creation and cultivation of geneticallymodified plants that are biologically contained, thusposing lower environmental risks (Daniell, 2002, 2007).This biological containment strategy is therefore suit-able for establishing the coexistence of conventionaland genetically modified crops. Cytoplasmic male ste-rility (CMS) presents a further genetic engineering ap-proach for transgene containment (Ruiz and Daniell,2005). Furthermore, plant-derived therapeutic pro-teins are free of human pathogens and mammalianviral vectors. Therefore, plastids provide a viable al-ternative to conventional production systems such asmicrobial fermentation or mammalian cell culture.

Another advantage of plastid transformation is theability to accumulate large amounts of foreign protein(up to 46% of total leaf protein) when the transgene isstably integrated (De Cosa et al., 2001). This is due tothe polyploidy of the plastid genetic system with up to

1 This work was supported by the U.S. Department of Agriculture(grant no. 3611–21000–017–00D) and by the National Institutes ofHealth (grant no. 5R01 GM 63879–06).

* Corresponding author; e-mail [email protected] author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policydescribed in the Instructions for Authors (www.plantphysiol.org) is:Henry Daniell ([email protected]).

www.plantphysiol.org/cgi/doi/10.1104/pp.107.106690

Plant Physiology, December 2007, Vol. 145, pp. 1129–1143, www.plantphysiol.org � 2007 American Society of Plant Biologists 1129

10,000 copies of the chloroplast genome in each plantcell, resulting in the ability to sustain a very highnumber of functional gene copies. Furthermore, site-specific integration into the chloroplast genome byhomologous recombination of flanking chloroplastDNA sequences present in the chloroplast vector elim-inates the concerns of position effect, frequently ob-served in nuclear transgenic lines (Daniell et al., 2002).Other advantages seen in chloroplast transgenic plantsinclude the lack of transgene silencing despite theaccumulation of transcripts at a level 169-fold higherthan in nuclear transgenic plants (Lee et al., 2003) andaccumulation of foreign proteins at levels up to 46% oftotal leaf protein (De Cosa et al., 2001).

Chloroplast genetic engineering also offers theunique advantage of transgene stacking, i.e. simulta-neous expression of multiple transgenes, creating anopportunity to produce multivalent vaccines in asingle transformation step. Several heterologous op-erons have been expressed in transgenic chloroplasts,and polycistrons are translated without processinginto monocistrons (Quesada-Vargas et al., 2005). More-over, foreign proteins synthesized in chloroplasts areproperly folded with appropriate posttranscriptionalmodifications, including disulfide bonds (Staub et al.,2000; Arlen et al., 2007; Ruhlman et al., 2007) and lipidmodifications (Glenz et al., 2006). This article is fo-cused on the various components of vectors used forstable protein production in transgenic chloroplasts.

GENOME ORGANIZATION AND CONCEPTS OFCHLOROPLAST TRANSFORMATION

The chloroplast genome typically consists of basicunits of double-stranded DNA of 120 to 220 kbarranged in monomeric or multimeric circles as wellas in linear molecules (Palmer, 1985; Lilly et al., 2001).The chloroplast genome generally has a highly con-served organization (Raubeson and Jansen, 2005), withmost land plant genomes having two identical copiesof a 20- to 30-kb inverted repeat region (IRA and IRB)separating a large single copy (LSC) region and a smallsingle copy (SSC) region. Plastid transformation istypically based on DNA delivery by the biolistic pro-cess (Daniell et al., 1990; Sanford et al., 1993) or occa-sionally by polyethylene glycol (PEG) treatment ofprotoplasts (Golds et al., 1993; O’Neill et al., 1993). Thisis followed by transgene integration into the chloro-plast genome via homologous recombination facilitatedby a RecA-type (Cerutti et al., 1992) system betweenthe plastid-targeting sequences of the transformationvector and the targeted region of the plastid genome.Chloroplast transformation vectors are thus designedwith homologous flanking sequences on either side ofthe transgene cassette to facilitate double recombination.Targeting sequences have no special properties otherthan that they are homologous to the chosen target siteand are generally about 1 kb in size. Both flankingsequences are essential for homologous recombina-

tion. Transformation is accomplished by integration ofthe transgene into a few genome copies, followed by25 to 30 cell divisions under selection pressure toeliminate untransformed plastids, thereby achieving ahomogeneous population of plastid genomes. If thetransgene is targeted into the IR region, integration inone IR is followed by the phenomenon of copy cor-rection that duplicates the introduced transgene intothe other IR as well.

Transgenes have been stably integrated at severalsites within the plastid genome. Transgenes were firstintegrated into transcriptionally silent spacer regions(Svab and Maliga, 1993). However, transcriptionallyactive spacer regions offer unique advantages, includ-ing insertion of transgenes without 5# or 3# untrans-lated regions (UTRs) or promoters. To date, the mostcommonly used site of integration is the transcription-ally active intergenic region between the trnI-trnAgenes, within the rrn operon, located in the IR regionsof the chloroplast genome. The foreign gene expressionlevels obtained from genes integrated at this site areamong the highest ever reported (De Cosa et al., 2001).It appears that this preferred site is unique and allowshighly efficient transgene integration and expression.Chloroplast vectors may also carry an origin of repli-cation that facilitates replication of the plasmid insidethe chloroplast, thereby increasing the template copynumber for homologous recombination and conse-quently enhancing the probability of transgene inte-gration. oriA is present within the trnI flanking region(Kunnimalaiyaan and Nielsen, 1997; Lugo et al., 2004),and this might facilitate replication of foreign vectorswithin chloroplasts (Daniell et al., 1990), enhance theprobability of transgene integration, and achieve ho-moplasmy even in the first round of selection (Gudaet al., 2000). This is further confirmed by the firstsuccessful Rubisco engineering obtained by integratingthe rbcS gene at this site (Dhingra et al., 2004). All otherearlier attempts on Rubisco engineering at other inte-gration sites within the chloroplast genome were onlypartially successful. Integration of transgenes be-tween exons of trnA and trnI also facilitates correctprocessing of foreign transcripts because of processingof introns present within both flanking regions.

UNIVERSAL VECTOR VERSUS SPECIES-SPECIFICCHLOROPLAST VECTORS

The proposal of a ‘‘universal vector’’ containing thetrnA and trnI genes from the IR region of the tobaccochloroplast genome as flanking sequences for ho-mologous recombination to transform several otherplant species (of unknown genome sequence) wassuggested several years ago (Daniell et al., 1998). Thisconcept was based on the high conservation of thisintergenic spacer region among the higher plant chlo-roplast genomes. Vectors designed for transformationof the tobacco plastid genome have been successfullyused for potato and tomato plastid transformation,

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1130 Plant Physiol. Vol. 145, 2007

because the homologous flanking sequences present inthese vectors showed adequate homology to the cor-responding sequences of potato and tomato plastidDNA but the efficiency of transformation is signifi-cantly lower than tobacco (Sidorov et al., 1999; Rufet al., 2001). For example, only one potato and tomatochloroplast transgenic line was obtained per 35 and 87bombarded plates, respectively, when compared toabout 15 tobacco chloroplast transgenic lines oftengenerated from one bombarded plate (Fernandez-SanMillan et al., 2003). A similar lower efficiency wasobserved when petunia flanking sequences (approxi-mately 98% homologous) were used to transform thetobacco chloroplast genome (DeGray et al., 2001), re-vealing that a lack of complete homology may reducethe transformation efficiency to a great extent. How-ever, comparison of intergenic spacer regions amongmembers of Solanaceae revealed that only four regionsare identical (Daniell et al., 2006). Similarly, compari-son of intergenic spacer regions of nine grass chloro-plast genomes revealed that not even a single spacerregion is identical among all sequenced chloroplastgenomes (Saski et al., 2007). Therefore, the concept of auniversal vector is applicable when a higher level ofhomology exists among plant species but will be lessefficient than species-specific chloroplast vectors. Theaccession numbers for several crop chloroplast ge-nome 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 protocolsspecific for each crop should enhance the efficiency oftransformation.

SELECTABLE MARKERS FORPLASTID TRANSFORMATION

At the beginning, selection of plastid transformantswas carried out by spectinomycin resistance encodedin the mutant 16S ribosomal RNA (rRNA) gene (Harriset al., 1989; Svab et al., 1990). Stable integration andexpression of the aadA gene was first reported in thechloroplast genome of Chlamydomonas (Goldschmidt-Clermont, 1991). The aadA gene encodes the enzymeaminoglycoside 3# adenylyltransferase that inactivatesspectinomycin and streptomycin by adenylation andprevents binding to chloroplast ribosomes. The aadAgene was later used as a selectable marker in tobacco,and the frequency of transformation events increasedto 100-fold more than the mutant 16S rRNA genes(Svab and Maliga, 1993). Due to the recessive natureof the mutant 16S rRNA marker gene, the phenotypicresistance was not expressed until sorting out of thetransgenomes was essentially completed. Lack of phe-notypic resistance permitted the loss of the resistantrRNA gene in 99 out of 100 potential transformationevents. Although it was first explained that spectino-

mycin offers nonlethal selection (Svab and Maliga,1993) by not inhibiting cell division and growth at highconcentrations (approximately 500 mg mL21), it wasobserved to be lethal in all other plant species (Table I).

The neo gene is another alternative marker for plas-tid transformation that confers kanamycin resistance(Carrer et al., 1993). A different kanamycin resistancegene (aphA6) with relatively high transformation effi-ciency was reported later (Huang et al., 2002). Anotherselection strategy utilizing a ‘‘double barrel’’ vectorwas used for cotton transformation where explant fortransformation was nongreen cells (Kumar et al.,2004b). The cotton plastid transformation vector con-tained two different genes (aphA6 and nptII) coding fortwo different enzymes. The aphA6 gene was regulatedby the 16S rRNA promoter and gene 10 UTR capable ofexpression in the dark and in nongreen tissues. ThenptII gene was regulated by the psbA promoter andUTR capable of expression in the light. Both geneswith different regulatory sequences facilitated detox-ification of the same selection agent (kanamycin) dur-ing day and night as well as in developing plastids andmature chloroplasts. The double barrel transformationvector was reported to be at least 8-fold more efficientthan single gene (aphA6)-based chloroplast vectors.

To avoid potential disadvantages of antibiotic resis-tance genes, several studies have explored strategiesfor engineering chloroplasts that are free of antibiotic-resistance markers. The spinach (Spinacia oleracea) be-taine aldehyde dehydrogenase (badh) gene has beendeveloped as a plant-derived selectable marker geneto transform chloroplast genomes (Daniell et al.,2001b). The selection process involved conversion ofthe toxic compound betaine aldehyde to beneficial Glybetaine by the chloroplast-localized gene-encodingenzyme BADH. Because the BADH enzyme is presentonly in chloroplasts of a few plant species adapted todry and saline environments (Rathinasabapathi et al.,1997; Nuccio et al., 1998), it is considered as a suitableselectable marker in many crop plants. The transfor-mation study showed rapid regeneration of transgenicshoots within 2 weeks in tobacco, and betaine alde-hyde selection was 25-fold more efficient than specti-nomycin. In addition, the Badh enzyme conferred salttolerance in carrot (Kumar et al., 2004a).

The bacterial bar gene, encoding phosphinothricinacetyltransferase (PAT) and conferring herbicide re-sistance, has also been tested as a plastid-selectablemarker. PAT served as an excellent marker in nucleartransformants and conferred resistance to the herbicidephosphinothricin. Expression of the bar gene in plastidconferred phosphinothricin resistance only when in-troduced by selection for a linked aadA gene. However,the bar gene was not found to be suitable for the directselection of transplastomic lines, even when expressedat a higher level (approximately 7% of total solublecellular protein). Thus, it shows that direct selection byherbicide resistance is constrained by way of subcellu-lar localization of the gene encoding the detoxifyingenzyme PAT (Lutz et al., 2001). The lethality of herbi-

Plastid Transformation Vectors

Plant Physiol. Vol. 145, 2007 1131

Table I. Chloroplast transformation method and selection conditions reported for different crop species

CropExplant and Method

of Transformation

Selection Agent and Conditions for First,

Second, and Third Rounds of Selection

Literature

Cited

Plant regeneration by embryogenesisCarrot Fine cell suspension

cultures derivedfrom stem

Spectinomycin; first selection of cell lines for 2 to 3 months on 150 mg mL21

spectinomycin; second selection on 350 mg mL21 spectinomycin for amonth; multiplication using 500 mg mL21 spectinomycin; transgenic shootswere produced from somatic embryos on 500 mg mL21 spectinomycin.

Kumar et al.(2004a)

Cotton Grayish-green friablecallus produced fromhypocotyl explants of5-d-old cotton seedlings;biolistic using 0.6-mmgold particles

Kanamycin; first selection with 50 mg mL21 kanamycin; second selectionwith 100 mg mL21 kanamycin for 4 to 5 months. Transformed calliwere converted into somatic embryos and plantlets.

Kumar et al.(2004b)

Rice Calli derived from matureseeds; biolistic using0.6-mm gold particles

Streptomycin; first selection after 1 to 2 d of bombardment onmedium supplemented with 200 mg mL21 streptomycin; thestreptomycin-resistant shoots were rooted on Murashige and Skoogmedium with 500 mg mL21 streptomycin.

Lee et al.(2006b)

Soybean Embryogenic calli; biolisticusing 0.6-mm goldparticles

Spectinomycin; first selection after 2 d of bombardment on mediumcontaining 200 or 300 mg mL21 spectinomycin and subcultured every15 d; first green spectinomycin-resistant calli appeared after 8 weeks ofselection and amplified in a medium with 150 mg mL21 spectinomycinand subsequently converted to embryos; after 2 months embryosgerminated into young plants on Murashige and Skoog medium with150 mg mL21 spectinomycin.

Dufourmantelet al.(2004)

Plant regeneration by organogenesis from protoplastsCauliflower Protoplasts isolated from

fully expanded leaves;PEG4000 mediated

Spectinomycin; embryogenic calli selected with 60 mg mL21

spectinomycin. The calli subsequently formed shoots. Leaf explants fromthese regenerated shoots placed on shoot regeneration medium containing300 mg mL21 spectinomycin regenerated further resistant shoots.

Nugent et al.(2006)

Lettuce Protoplasts isolated from3-week-old shootculture leaves;PEG6000 mediated

Spectinomycin; first selection was initiated after 6 d of transformation in darkfor 1 week; second selection in light until the calli were approximately0.5 mm in diameter followed by growth until calli were few millimetersin diameter; shoot formation; all selection steps contained 500 mg mL21

spectinomycin.

Lelivelt et al.(2005)

Plant regeneration by organogenesis from leafCabbage Leaves; biolistic using

1.0-mm gold particlesSpectinomycin; first selection of calli after 1 week of bombardment

on medium containing 50 mg mL21 spectinomycin and subculturedevery 2 weeks; second selection until shoots formed on mediumwith 100 mg mL21 spectinomycin and streptomycin; regeneratedshoots were rooted on medium with 200 mg mL21 spectinomycin.

Liu et al.(2007)

Lettuce Young leaves from 3- to4-week-old plants;biolistic using 0.6-mmgold particles

Spectinomycin; first selection of resistant green calli on mediumsupplemented with 50 mg mL21 spectinomycin for 2 monthsfollowed by shoot regeneration on same medium in few weeks.

Kanamotoet al.(2006)

Lettuce Young leaves; biolisticusing 0.6-mm goldparticles

Spectinomycin; first selection of resistant shoots on medium supplementedwith 50 mg mL21 spectinomycin; second selection of resistant shootsfrom pieces of leaves of resistant shoots from first round of selection onmedium supplemented with 50 mg mL21 spectinomycin; regeneratedshoots were rooted on medium with 50 mg mL21 spectinomycin.

Ruhlmanet al.(2007)

Oilseedrape

Green cotyledon petiolesof 1–2 mm in length;biolistic using tungstenparticles

Spectinomycin; first selection after 3 d of bombardment with 10 mg mL21

spectinomycin; the regenerated green shoots subcultured onto the sameselection medium once every 3 weeks twice and then transferred torooting medium and finally to soil.

Hou et al.(2003)

Petunia Leaves; biolistic using1.0-mm gold particles

Spectinomycin and streptomycin; first selection on medium supplementedwith 200 mg mL21 spectinomycin and 200 mg mL21 streptomycin forevery 3 to 4 weeks. Resistant shoots first appeared after 8 weeks.

Zubko et al.(2004)

Poplar Leaves; biolistic using0.6-mm gold particles

Spectinomycin; first selection on medium containing 30 mg/L spectinomycinand subcultured every 2 weeks; spectinomycin-resistant calli transferred toshoot induction medium with 30 mg mL21 spectinomycin every 4 weeksuntil shoot formation. Regenerated shoots were transferred to rootinduction medium.

Okumuraet al.(2006)

(Table continues on following page.)

Verma and Daniell


cides to plastids was determined by examining plastidultrastructure using transmission electron microscopy(Ye et al., 2003). In glyphosate-treated cells of culturedtobacco leaf discs, the reticulate network of thylakoidmembranes has been lost, indicating disintegration ofthe photosynthetic membranes. The plastid contentsspilled out into the cell cytoplasm due to the rupturedouter plastid membrane at several locations. On theother hand, spectinomycin antibiotic had no detrimen-tal effect on plastid ultrastructure. Therefore, herbicideresistance genes could not be used to directly selectplastid transformants, and herbicide resistance wasachieved only when herbicide resistance genes wereintroduced by selection for a linked aadA gene.

A negative selection scheme has also been employedfor plastid transformation based on expression of thebacterial gene codA (Serino and Maliga, 1997). Cytosinedeaminase (codA) catalyzes the deamination of cyto-sine to uracil. 5-Fluorocytosine is toxic to cells thatexpress cytosine deaminase because this enzyme con-verts 5-fluorocytosine to toxic 5-fluorouracil. This neg-ative selection scheme was utilized to identify seedlingson 5-fluorocytosine medium from which codA was re-moved by the P1 bacteriophage site-specific recombi-nase CRE-lox (Corneille et al., 2001).

REPORTER GENES USED IN PLASTIDS

GUS, chloramphenicol acetyl transferase, and GFPhave been used as plastid reporters (Daniell andMcFadden, 1987; Daniell et al., 1990; Ye et al., 1990;Khan and Maliga, 1999). The enzymatic activity ofGUS can be visualized by histochemical staining (Yeet al., 1990; Daniell et al., 1991), whereas GFP is a visualmarker that allows direct imaging of the fluorescentgene product in living cells. The GFP chromophoreforms autocatalytically in the presence of oxygen andfluoresces green when absorbing blue or UV light(Hanson and Kohler, 2001). GFP has been used todetect 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.

GFP has also been fused with AadA and used as abifunctional visual and selectable marker (Khan andMaliga, 1999). Further, GFP has been used to test theconcept of receptor-mediated oral delivery of foreignproteins. Cholera toxin B-subunit (CTB)-GFP fusionprotein with a furin cleavage site in between CTB andGFP has been used to elucidate the path of CTB andGFP in the circulatory system (Limaye et al., 2006). Micewere fed with CTB-GFP-expressing plant leaf material.GFP was detected in the intestinal mucosa and sub-mucosa, the hepatocytes of the liver, as well as variouscells of spleen utilizing fluorescence microscopy andanti-GFP antibodies. In mice fed with untransformedleaf material or IFN-GFP fusion protein-expressingplant leaf material, no GFP fluorescence was observed.This confirmed the receptor-mediated oral delivery of aforeign protein (GFP) across the intestinal lumen intothe systemic circulation. Moreover, GFP was not de-tected in any substantial amount in the liver or spleenof mice fed with IFN-GFP-expressing plants, suggest-ing that a transmucosal carrier such as CTB is requiredfor delivery of an adequate amount of a foreign proteinacross the intestinal lumen into the systemic circula-tion. Thus, GFP has been used as a reporter gene inchloroplast expression and in animal studies.

EXCISION OF SELECTABLE MARKER GENES

Most of the studies involving plastid transformationhave utilized antibiotic resistance gene for the recoveryof transformed plastomes, but introducing such cropsinto the food chain may be a cause of concern. Strategieshave been developed to eliminate antibiotic resistancegenes after transformation, including homology-based ex-cision via directly repeated sequences, excision by phagesite-specific recombinases, transient co-integration ofthe marker gene, and cotransformation-segregation.

Early experiments with Chlamydomonas reinhardtiishowed that homologous recombination betweentwo direct repeats enabled marker removal undernonselective growth conditions (Fischer et al., 1996).Subsequently, marker genes have been deleted from

Table I. (Continued from previous page.)

CropExplant and Method

of Transformation

Selection Agent and Conditions for First,

Second, and Third Rounds of Selection

Literature

Cited

Potato Leaves; biolistic using0.6-mm gold particles

Spectinomycin; after 2 to 3 d of bombardment, the pieces of leaves wereplaced onto regeneration medium containing spectinomycin (40, 300, and400 mg mL21). The first spectinomycin-resistant events were identified after4 to 6 weeks of selection.

Sidorovet al.(1999)

Potato Leaves; biolistic using0.6-mm gold particles

Spectinomycin; first selection on medium containing 300 mg mL21

spectinomycin for 4 weeks; second selection of the leaf explants on shootinduction medium containing 300 mg mL21 spectinomycin for every3 weeks; the spectinomycin-resistant shoots formed in 8 to 10 weeks;rooting in Murashige and Skoog medium with 400 mg/L spectinomycin.

Nguyenet al.(2005)

Tomato Young leaves; biolistic using0.6-mm gold particles

Spectinomycin; bombarded leaves were incubated on medium with 300 or500 mg mL21 spectinomycin for 3 to 4 months to obtain resistant yellowor pale green calli and subcultured to achieve homoplasmy. Plants wereregenerated from homoplasmic callus tissue.

Ruf et al.(2001)



transplastomic tobacco via engineered direct repeatsthat flank them (Iamtham and Day, 2000). A variant ofhomology-based marker excision technology enableddirect identification of marker-free tobacco plants byherbicide resistance (Dufourmantel et al., 2007). Thevector used for plastid transformation carried the aadAgene disrupting the herbicide resistance gene. Theprimary transplastomic clones were selected by spec-tinomycin resistance. Marker-free herbicide-resistantderivatives were identified after excision of the aadAmarker gene by homologous recombination within theoverlapping region (403 nucleotides) of the N-terminaland C-terminal halves of the herbicide resistance gene.Excision of the aadA gene led to reconstitution of anentire herbicide resistance gene and expression of thePseudomonas fluorescens 4-hydroxyphenylpyruvate di-oxygenase enzyme that conferred resistance to sulco-trione and isoxaflutole herbicides (Dufourmantel et al.,2007). A second variant of this approach facilitatedvisual tracking of homology-based marker excision bycreation of a pigment-deficient zone due to the loss of aplastid photosynthetic gene rbcL (Kode et al., 2006).

So far, two recombinases (Cre and FC31 phageintegrase [Int]) have been tested for plastid markergene excision. Using the P1 bacteriophage Cre/loxsite-specific recombination system, a marker geneflanked by lox sites was removed after expression ofthe CRE protein was induced via the nuclear genome.The second site-specific recombinase, Int, appeared tobe a better choice for the aadA marker gene removalwhen flanked with directly oriented nonidenticalphage attP (215 bp) and bacterial attB (54 bp) attach-ment sites, which are recognized by Int recombinase.Efficient excision of the marker gene was shown aftertransformation of the nucleus with an int gene encod-ing plastid-targeted Int (Kittiwongwattana et al., 2007).Alternatively, a transient co-integrative vector mayeven be used to avoid the integration of selectablemarker genes (Klaus et al., 2004).

The cotransformation-segregation approach involvestransformation with two plasmids that target inser-tions at two different ptDNA locations: one plasmidcarries a selective marker and the other a nonselectedgene. Selection for the marker yields transplastomicclones that also bear an insertion of the nonselectedgene. The prospect of the approach was first shown inC. reinhardtii (Kindle et al., 1991). Interestingly, whenthe approach was tested in tobacco, a cotransformationefficiency of 20% was obtained even though tobaccohas a greater number of chloroplasts (Carrer andMaliga, 1995). An application of cotransformationwas His-tagging of an unlinked ndh gene followingspectinomycin selection (Rumeau et al., 2005).

STABILITY OF EXPRESSED PROTEINSIN CHLOROPLASTS

Newly synthesized proteins are highly susceptibleto proteases and require protection from chloroplastproteases. One such approach used the CRY chape-

rone (encoded by the orf2 gene) to fold the insecticidalprotein, Cry2Aa2, into cuboidal crystals. The crystalstructure protected the foreign proteins from degra-dation, thereby increasing protein accumulation over128-fold (from 0.36% to 46.1% of total soluble protein[tsp]; De Cosa et al., 2001). Similarly, when the humanserum albumin (hsa) coding sequence was regulatedby the chloroplast psbA 5# and 3# UTRs in the light,protein expression increased 500-fold, resulting inthe formation of protective inclusion bodies. A 3- to10-fold reduction in HSA protein expression was seenwhen leaves were harvested in the dark (Fernandez-San Millan et al., 2003). This illustrated the power ofregulatory sequences during illumination and protec-tion from proteases when their access is limited.

Several studies on transgenic chloroplasts did notcorrelate increased transcript abundance with transla-tion efficiency. For example, chloroplast-derived RbcStranscripts were measured to be 165-fold and 143-foldmore than the nuclear RbcS antisense control plantswhen the transgene was regulated by the psbA 5# UTRor the promoterless gene 10 UTR, respectively. Al-though the psbA 5# UTR transgenic lines resulted inthe first successful functional Rubisco in transgenicplants, the gene 10 UTR transgenic lines performedpoorly (Dhingra et al., 2004). The lack of correlationbetween increased transcript levels and translationefficiency suggests that transcript abundance is of lessimportance than protein stability in transgenic chloro-plasts. Several studies have addressed the role of 5#UTRs. However, in a few cases, the amino acid se-quences downstream of the translation initiation co-don may play an important role in stabilizing newlysynthesized proteins or enhancing translation (Kurodaand Maliga, 2001).

Human insulin was unstable in transgenic chloro-plasts; fusion with CTB resulted in high-level expres-sion (up to 16% tsp) and facilitated oral deliverystudies to achieve protection against the developmentof insulitis in nonobese diabetic mice (Ruhlman et al.,2007). Such N-terminal degradation is not unique tochloroplasts. All commercially produced insulin inbacteria or yeast is produced as a fusion protein; whenexpressed without fusion, insulin is rapidly degraded.Also, high-level expression of foreign proteins mayhave deleterious phenotypic effects and/or impose asignificant burden on the plant (Magee et al., 2004),and recovery of transplastomic plants seems to be notfeasible. In that case, the use of psbA UTR is lethaland conciliation of the expression levels or inducibleexpression of foreign protein is highly desirable. Eventhough the inducible systems are well known fornuclear transgenes, most existing systems for plastidsrely on nuclear transgenes, usually a T7 RNA poly-merase targeted to the chloroplast where it drivesexpression of a transgene placed under the control of aT7 promoter (McBride et al., 1995; Magee et al., 2004;Lossl et al., 2005). A Lac repressor-based isopropylthio-b-galactoside-inducible expression system for plastidshas been reported, although transgene repression in

Verma and Daniell


the uninduced state was incomplete (Muhlbauer andKoop, 2005). Thus, there is a need to devise tightlycontrollable plastid-inducible expression systems thatdo not require nuclear transgenes.

PLASTID TRANSFORMATION OF DIFFERENTCROP SPECIES

Tobacco has been the most widely exploited plastidtransformation system because of its ease in geneticmanipulations. A single tobacco plant is capable ofgenerating a million seeds and 1 acre of tobacco canproduce more than 40 metric tons of leaves per year(Cramer et al., 1999; Arlen et al., 2007). Harvestingleaves before flowering can offer nearly completetransgene containment in addition to protection of-fered by maternal inheritance. Recent studies havereported that escape of transgenes in tobacco is0.0087% to 0.00024% (Daniell, 2007; Ruf et al., 2007;Svab and Maliga, 2007), making this an ideal systemfor use of chloroplasts as bioreactors. In addition, CMShas been engineered via the tobacco chloroplast ge-nome as a failsafe method (Ruiz and Daniell, 2005). Asa bioreactor, tobacco has been estimated to be morethan 50 times less expensive than the frequently usedEscherichia coli fermentation systems (Kusnadi et al.,1997). Additionally, tobacco eliminates contaminationof food because it is a non-food and non-feed crop.Plastid transformation in higher plants was first suc-cessfully carried out in tobacco and is now a routineprocedure because many foreign genes have been ex-pressed to engineer agronomic traits, biopharmaceut-icals, vaccines, or biomaterials (Table II). However,presence of nicotine or other alkaloids has been a dis-advantage for pharmaceutical production, but the chlo-roplast genome of low-nicotine varieties like LAMDhas been used to engineer therapeutic proteins (Arlenet al., 2007). For oral delivery studies, there is a need tomove beyond tobacco.

Extension of the plastid transformation technologyto other species is important to exploit this platform.The study of DNA delivery strategies, target tissues,selection conditions, and regeneration systems is cru-cial for extending the range of species in which plastidtransformation could be achieved. Plastid transforma-tion is most commonly achieved by biolistic deliveryof DNA into leaf explants but has also been achievedvia direct DNA uptake by protoplasts (Lelivelt et al.,2005; Nugent et al., 2006). In species other than to-bacco, like petunia and oilseed rape, adventitiousshoot regeneration from bombarded leaf or petioleexplants generated plastid transformants. Homoplas-mic plants of soybean, carrot, and cotton were regen-erated via somatic embryogenesis after bombardmentof embryogenic calli, combined with the use ofspecies-specific plastid vectors. Table I summarizesthe chloroplast transformation method and selectionconditions for different crop species. Attempts havebeen made in other plants (Table I) where proteinproduction was carried out in non-green tissues such

as micro-tuber (potato), fruit (tomato), and root (car-rot). However, the amount of protein was lower thanthe level observed in leaf chloroplasts (Kumar et al.,2004a). Some progress has also been made in improv-ing the chloroplast transformation system for tomatoplants. Utilizing that plastid expression of a bacteriallycopene b-cyclase gene resulted in herbicide resis-tance and triggered conversion of lycopene, the mainstorage carotenoid of tomatoes, to b-carotene, result-ing in a 4-fold enhancement of pro-vitamin A contentof fruits (Wurbs et al., 2007). Stable chloroplast trans-formation system has also been reported for cabbage(Liu et al., 2007).

Recently, edible leafy crops, including lettuce, haveattracted attention toward plastid genetic engineering.Edible plant species not only minimize downstreamprotein processing costs but also offer an ideal sys-tem for oral delivery. The leaves of lettuce are con-sumed raw by humans and the time from sowing seedto edible biomass is only weeks compared to monthsfor crops such as tomato, potato, and carrot. Further-more, lettuce is well suited for indoor cultivation byhydroculture systems (Kanamoto et al., 2006). Accu-mulation of a valuable therapeutic protein, the CTB-Pinsfusion, in lettuce chloroplasts was recently reported(Ruhlman et al., 2007). This is the first report ofexpression of a therapeutic protein in an edible crop.Further studies are required to understand the conceptof oral delivery.

Economically important crops such as carrot, cotton,and soybean regenerate in vitro through somaticembryogenesis (Daniell et al., 2005b). In such crops,transformation of the plastid genome was achievedthrough somatic embryogenesis by bombarding em-bryogenic non-green cells or tissues. The first stableplastid transformation of embryogenic cell culturesand somatic embryogenesis was established in carrot(Kumar et al., 2004a). Homoplasmic transgenic plantswere regenerated from cell cultures bombarded withthe aadA and badh genes. However, in the case of cot-ton, plastid transformation using the aadA gene wasunsuccessful, and no transgenic cultures or plantswere recovered using spectinomycin as the selectionagent. Transgenic cotton cell lines were generatedusing a double barrel vector containing two select-able marker genes (aphA6 and nptII) to detoxify kana-mycin (Kumar et al., 2004b). Transgenic lines werefertile and showed maternal inheritance of trans-gene. Soybean plastid transformation was achievedusing embryogenic tissue as the starting material(Dufourmantel et al., 2004) and the aadA gene as theselectable marker. Phenotypically normal transgenicsoybean plants were regenerated via somatic embryo-genesis from spectinomycin-resistant calli and werefully fertile. Stable plastid transformation in rice wasachieved using mature seed-derived calli for bom-bardment (Lee et al., 2006b). The transplastomic riceplants expressed GFP in their plastids and generatedviable seeds, which were confirmed to transmit thetransgenes to the T1 progeny plants. However, trans-



plastomic rice plants were not homoplasmic, evenafter two generations of continuous selection. Plastidtransformation of carrot, cotton, rice, and soybeanopens the door for modification of the plastid genomeof several crops that require embryogenesis.

METHODS FOR CONSTRUCTION OF PLASTIDTRANSFORMATION VECTORS AND GENERATIONOF TRANSPLASTOMIC PLANTS

Plastid gene expression is regulated both at thetranscriptional and posttranscriptional levels. Proteinlevels in chloroplasts depend on mRNA abundance,

which is determined by promoter strength and mRNAstability. However, high mRNA levels do not result inhigh-level protein accumulation as posttranscriptionalprocesses ultimately determine obtainable proteinlevels. Therefore, we have designed expression cas-settes for transgene assembly to achieve optimal levelsof protein accumulation in leaves (Fig. 1). The basicplastid transformation vector is comprised of flankingsequences and chloroplast-specific expression cas-settes (Fig. 1). Species-specific chloroplast flankingsequence (e.g. trnI/trnA) is obtained by PCR usingthe primers designed from the available chloroplastgenomes. The chloroplast expression cassette is com-

Table II. Engineering of agronomic traits, biopharmaceuticals, vaccine antigens, and biomaterials via the plastid genome

Traits/Gene Products Gene Promoter/5#/3# UTRs Literature Cited

Agronomic traitInsect resistance cry1A(c) Prrn/rbcL/rps16 McBride et al. (1995)

cry2Aa2 Prrn/ggagg (native)/psbA Kota et al. (1999)cry2Aa2 operon Prrn/native 5# UTR/psbA De Cosa et al. (2001)cry1Aa10 Prrn/native 5# UTR/psbA Hou et al. (2003)cry1Ab Prrn/T7 gene10/rbcL Dufourmantel et al. (2005)cry9Aa2 Prrn/native 5# UTR/rbcL Chakrabarti et al. (2006)

Herbicide resistance aroA (petunia) Prrn/ggagg/psbA Daniell et al. (1998)bar Prrn/rbcL/psbA Iamtham and Day (2000)

Disease resistance MSI-99 Prrn/ggagg/psbA DeGray et al. (2001)Drought tolerance TPS1 (yeast) Prrn/ggagg/psbA Lee et al. (2003)Phytoremediation merA/merB Prrn/ggagg/psbA Ruiz et al. (2003); Hussein et al. (2007)Salt tolerance badh Prrn/ggagg/rps16 Kumar et al. (2004a)CMS phaA Prrn/psbA/psbA Ruiz and Daniell (2005)

Biopharmaceutical proteinshST hST Prrn/T7 gene10/Trps16

PpsbA/Trps16Staub et al. (2000)

Insulin-like growth factor IGF-1n IGF-1s Prrn/PpsbA/TpsbA Daniell et al. (2005a)IFNa2b IFNa2b Prrn/PpsbA/TpsbA Arlen et al. (2007)HSA hsa Prrn/PpsbA/TpsbA Fernandez-San Millan et al. (2003)IFN-g Gus-IFN-g PpsbA/TpsbA Leelavathi and Reddy (2003)Monoclonal antibody Guy’s 13 Prrn/ggagg/TpsbA Daniell et al. (2004)Human Pins CTB-Pins PpsbA/TpsbA Prrn/T7

gene10/Trps16Ruhlman et al. (2007)

Vaccine antigensCholera toxin ctxB Prrn/ggagg/TpsbA Daniell et al. (2001a)Tetanus toxin tetC bacterial and synthetic Prrn/T7gene 10/TrbcL

atpB/TrbcLTregoning et al. (2003)

CPV CTB-2L21 GFP-2L21 Prrn/PpsbA/TpsbA Molina et al. (2004, 2005)Anthrax PA pag Prrn/PpsbA/TpsbA Watson et al. (2004); Koya

et al. (2005)Amebiasis lecA Prrn/PpsbA/TpsbA Chebolu and Daniell (2007)Plague CaF1-LcrV Prrn/PpsbA/TpsbA Y. Ding, P. Arlen, J. Adamovicz,

M. Singleton, and H. Daniell(unpublished data)

Rotavirus VP6 Prrn/PpsbA/TpsbA Birch-Machin et al. (2004)Hepatitis C NS3 Prrn/PpsbA/TpsbA Daniell et al. (2005a)Lyme disease OspA OspA-T PpsbA/TpsbA Glenz et al. (2006)BiomaterialsElastin-derived polymer EG121 Prrn/T7 gene 10/TpsbA Guda et al. (2000)pHBA ubiC Prrn/PpsbA/TpsbA Viitanen et al. (2004)

Polyhydroxybutyrate phb operon PpsbA/TpsbA Lossl et al. (2003)Xylanase xynA PpsbA/TpsbA Leelavathi et al. (2003)Tryptophan ASA2 Prrn/rbcL/rpL32

rbcL/accD-ORF184Zhang et al. (2001)

Monellin monellin Prrn/PpsbA/TpsbA Roh et al. (2006)

Verma and Daniell


posed of a promoter, selectable marker, and 5#/3#regulatory sequences to enhance the efficiency of tran-scription and translation of the gene. The chloroplast-specific promoters and regulatory elements areamplified from the total cellular DNA using primersdesigned on the basis of the sequence informationavailable for the chloroplast genome. Suitable restric-tion sites are introduced to facilitate gene assembly.

Because of the high similarity in the transcriptionand translation systems between E. coli and chloro-plasts, the chloroplast expression vectors are tested inE. coli first before proceeding with plant transforma-tion. The growth of E. coli harboring the plastid trans-formation vector with the aadA gene in the presence ofspectinomycin confirms expression of the aadA gene.Western blot with extracts from E. coli confirms ex-pression 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. Theleaves used for bombardment should be young, green,and healthy. The bombarded leaves are placed onselection medium with an appropriate concentrationof antibiotics (RMOP in tobacco). Normally, in 3 to 10weeks, putative transgenic shoots appear (Fig. 2, Aand D). PCR analysis is used to screen the transgenicshoots and distinguish true chloroplast transgenicevents from mutants or nuclear transgenic plants.Site-specific chloroplast integration of the transgenecassette is determined by using a set of primers, one ofwhich anneals to the native chloroplast genome and

the other anneals within the transgene cassette. Mu-tants and nuclear transgenic plants are not expected toproduce a PCR product with these primers (Fig. 3A).The leaf pieces from PCR-positive shoots are furtherselected for a second round to achieve homoplasmy(Fig. 2, B and E). The regenerated shoots are rootedwith the same level of selection (Fig. 2, C and F) andchecked for homoplasmy by Southern-blot analysis(Fig. 3B). The Southern blot is probed with radiola-beled flanking sequences used for homologous recom-bination. Transplastomic genome contains a larger sizehybridizing fragment than the untransformed genomebecause of the presence of transgenes. If the transgenicplants are heteroplasmic, a native fragment is visiblealong with the larger transgenic fragment. Absenceof the native fragment confirms the establishment ofhomoplasmy. Transgene expression is confirmed bywestern-blot analysis, and the effectiveness or proper-ties or functionality of the introduced transgene isassessed. Seeds from the transgenic plants and untrans-formed plants are grown on spectinomycin-containingmedium to check for maternal inheritance. Transgenicseeds germinate and grow into uniformly green plants.The absence of Mendelian segregation of transgenesconfirms that they are maternally inherited to progeny.

AGRONOMIC TRAITS ENGINEERED VIATHE CHLOROPLAST GENOME

Several useful transgenes have conferred valuableagronomic traits, including insect and pathogen re-

Figure 1. Schematic representation of the chloroplast-specific expression cassette. Map of the chloroplast expression vectorshows the integration sites, promoters, selectable marker genes, regulatory elements, and genes of interest. For a list of regulatoryelements and genes of interest used for chloroplast transformation, refer to Table II.



sistance, drought tolerance, phytoremediation, salttolerance, and CMS through chloroplast genetic engi-neering (Table II). Genetically engineered tobacco plantsexpressing an insecticidal protein Cry2Aa2 have shownresistance against target insects and insects that de-veloped resistance against insecticidal protein (Kotaet al., 1999). Expression of the Cry2Aa2 resulted in theutmost expression levels on record (approximately46.1% of total leaf protein) and resulted in the detec-tion of cuboidal crystals using transmission electronmicroscopy (De Cosa et al., 2001). In addition, soybeanplastid transformants expressing Cry1Ab also conferredinsecticidal activity against velvetbean caterpillar(Dufourmantel et al., 2005). The antimicrobial peptideMSI-99, an analog of magainin 2, was expressed viathe chloroplast genome to obtain high levels of ex-pression in transgenic tobacco plants. In planta assayswith the bacterial pathogen Pseudomonas syringae pvtabaci and the fungal pathogen Colletotrichum destructi-vum showed necrotic lesions in untransformed controlleaves, whereas transformed leaves showed no lesions(DeGray et al., 2001).

Environmental stress factors such as drought, salin-ity, and freezing are perilous to plants generally be-cause of their sessile means of existence. Attempts toconfer resistance to drought by expressing trehalose

phosphate synthase 1 (tps1) gene via nuclear transfor-mation have proven futile because of undesirablepleiotropic effects even at very low levels of trehaloseaccumulation. However, hyperexpression of tps1 inthe chloroplasts has no phenotypic variation from theuntransformed control plants, and transgenic seedssprouted, grew, and remained green and healthy indrought tolerance bioassays with 3% to 6% PEG anddehydration/rehydration assays (Lee et al., 2003).High-level expression of BADH in cultured cells, roots,and leaves of carrot via plastid genetic engineering ex-hibited high levels of salt tolerance. Transgenic carrotplants expressing BADH grew in the presence of highconcentrations of NaCl (up to 400 mM), the uppermostlevel of salt tolerance reported so far among geneti-cally modified crop plants (Kumar et al., 2004a). Chlo-roplast genetic engineering has also been used for thefirst time to our knowledge to enhance the capacity ofplants for phytoremediation. This was accomplishedby incorporating a native operon containing the merAand merB genes, which code for mercuric ion reductase(merA) and organomercurial lyase (merB), respectively,into the chloroplast genome in a single transformationevent. Stable integration of the merAB operon into thechloroplast genome resulted in high levels of toleranceto the organomercurial compound phenylmercuric

Figure 2. Selection of transplastomic plants.Shown are representative photographs oftransplastomic tobacco and lettuce shoots un-dergoing first (A and D), second (B and E), andthird (C and F, rooting) rounds of selection,respectively.

Figure 3. Evaluation of transgene integration into the chloroplast genome. DNA isolated from putative transplastomic shoots areanalyzed by PCR and Southern-blot analysis. A, 3P/3M and 5P/2M primer pairs (Kumar and Daniell, 2004) are used for PCRanalysis; PCR products of 3P/3M primers. Lane 1, Untransformed plant; lanes 2 to 4, transformed lines (1.6 kb); lane 1kb1, DNAmarker; lanes 5 to 7, PCR product with 5P/2M primers (3.2 kb) in transformed lines. B, The chloroplast genome is probed with aradiolabeled flank fragment. Lane 1, Untransformed plant; lanes 2 and 4, homoplasmic transplastomic plant; lane 3,heteroplasmic transplastomic plant.

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acetate when grown in soil containing up to 400 mM

phenylmercuric acetate (Ruiz et al., 2003). Chloroplasttransgenic lines absorbed mercury exceeding the lev-els in soil and translocated 100-fold more to shootsthan untransformed plants (Hussein et al., 2007). To-bacco is ideal for phytoremediation of contaminatedsoil because it is a non-food non-feed crop.

Naturally occurring CMS has been documented forover 100 years for oilseed rape, maize (Zea mays), andrice. However, such systems are not available for themajority of crops used in agriculture. In presentlyavailable CMS lines, various loci in the nuclear genomedirect a range of restoration factors that are not fullyunderstood. Moreover, risk of sterility trait dilutionthrough segregation and the production of transgenicseeds that spread transgenic traits to nontransgenicplants cannot be ruled out because of the possibility ofcross-pollination of the male-sterile line with a restorerline or wild relative. To address some of these con-cerns, CMS has been engineered via introduction ofphaA gene coding for b-ketothiolase into chloroplastgenome. The transgenic lines were normal except forthe male sterility phenotype lacking pollen (Ruiz andDaniell, 2005). Further restoration of male fertility wasreported by changing conditions of illumination. Con-tinuous illumination increases acetyl-CoA carboxylaseactivity, thereby increasing the levels of plastidic fattyacid biosynthesis, which is especially needed for theformation of the exine pollen wall.

PLASTIDS AS BIOPHARMACEUTICAL BIOREACTORS

Several chloroplast-derived biopharmaceutical pro-teins have been reported (Daniell, 2006; Table II).Stable expression of a pharmaceutical protein in chlo-roplasts was first reported for GVGVP, a protein-basedpolymer with medical uses such as wound coverings,artificial pericardia, and programmed drug delivery(Guda et al., 2000). Human ST (hST), a secretoryprotein, was expressed inside chloroplasts in a soluble,biologically active and disulfide-bonded form (Staubet al., 2000). The key use of hST is in the cure ofhypopituitary dwarfism in children; additional indi-cations are treatment of Turner syndrome, chronic renalfailure, and human immunodeficiency virus wastingsyndrome. Another important therapeutic protein thatcomprises approximately 60% of the protein in bloodserum is HSA, prescribed in multigram quantities torestore blood volume in trauma and other clinicalconditions. Early attempts at expressing HSA haveachieved inadequately low levels of HSA (0.2% of tsp)in nuclear transgenic plants (Farran et al., 2002). Onthe other hand, in chloroplast transgenic plants, expres-sion levels of up to 11.2% were observed (Fernandez-San Millan et al., 2003).

The type I IFNs are part of the body’s first line ofdefense against viral attack and also invasion bybacterial pathogens, parasites, tumor cells, and allo-geneic cells from grafts. IFNa2b ranks third in world

market use for a biopharmaceutical, behind only in-sulin and erythropoietin. The average annual cost ofIFNa2b for the treatment of hepatitis C infection is$26,000, and is therefore unavailable to the majority ofpatients in developing countries. Therefore, IFNa2bwas expressed in tobacco chloroplasts with levels ofup to 20% of tsp or 3 mg/g of leaf (fresh weight) andfacilitated the first field production of a plant-derivedhuman blood protein (Arlen et al., 2007). TransgenicIFNa2b had comparable in vitro biological activity tocommercially produced PEG-Intron when tested forits ability to protect BHK cells against cytopathic viralreplication in the vesicular stomatitis virus cytopathiceffect assay and to inhibit early stage human immu-nodeficiency virus infection in HeLa cells. Anothertherapeutic protein expressed in chloroplasts is hu-man IFN-g (Leelavathi and Reddy, 2003). In a bioassay,the chloroplast-produced human IFN-g offered com-plete protection to human lung carcinomas againstinfection by the EMC virus.

PLASTIDS AS VACCINE BIOREACTORS

As opposed to injected subunit vaccines, oral deliv-ery and low-cost purification make plastid-derivedsubunit production quite plausible (Kamarajugaddaand Daniell, 2006). Subunit vaccines expressed inplants are capable of inducing a mucosal response inanimal models when given orally or parenterally;these animals also withstand a pathogen challenge.The ability for plant-derived vaccines to survive in thestomach is a major concern. However, bioencapsula-tion can protect the vaccine in the stomach and grad-ually 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 transgenicchloroplasts (Table II). For cholera, the CTB has beenshown to be an extremely powerful vaccine candidateand is encoded by Vibrio cholerae. The chloroplast-expressed CTB assembled into pentameric protein andassumed correct quaternary structure for full activity.Subsequent binding assays confirmed the ability ofchloroplast-derived CTB to bind to the intestinal mem-brane GM1 ganglioside receptors. CTB also acts as apowerful transmucosal carrier and is very effective indelivering several vaccine antigens. In one such in-vestigation, oral administration of chloroplast-derivedCTB-Pins fusion protein protected nonobese diabeticmice against development of insulitis (Ruhlman et al.,2007).

Recently, there has been an increased threat ofbioterrorism in the post 9/11 world. Anthrax is alwaysfatal if not treated immediately. Weapon-grade sporescan be produced and stored for decades and can bespread by missiles, bombs, or even through the mail.Because of this, it is an ideal biological warfare agent.



The currently available human vaccine for anthrax,derived from the culture supernatant of Bacillus an-thracis, contains the protective antigen (PA) and tracesof the lethal and edema factors. These factors maycontribute to undesirable side effects linked with thisvaccine. Therefore, an effective expression system thatcan provide a clean, safe, and efficacious vaccine isrequired. In an attempt to produce anthrax vaccine inlarge quantities and free of extraneous bacterial con-taminants, PA was expressed in transgenic tobaccochloroplasts by inserting the pagA gene into the chlo-roplast genome (Watson et al., 2004; Koya et al., 2005).Mature leaves grown under continuous illuminationcontained PA up to 14.2% of tsp. Cytotoxicity mea-surements in macrophage lysis assays showed thatchloroplast-derived PA was equivalent in potency toPA produced in B. anthracis. Subcutaneous immuni-zation of mice with partially purified chloroplast-derived or B. anthracis-derived PA with adjuvant yieldedIgG titers up to 1:320,000 and both groups of micesurvived (100%) challenge with lethal doses of toxin.These results demonstrated the immunogenic andimmunoprotective properties of plant-derived anthraxvaccine antigen.

PLASTIDS AS BIOMATERIAL BIOREACTORS

Besides vaccine antigens, biomaterial and aminoacids have also been expressed in chloroplasts (TableII). Normally, p-hydroxybenzoic acid (pHBA) is pro-duced in small quantities in all plants. In E. coli, theubiC gene encoding chorismate pyruvate lyase cata-lyzes the direct conversion of chorismate to pyruvateand pHBA. However, in chloroplasts, chorismate isconverted to pHBA by 10 consecutive enzymatic re-actions due to lack of chorismate pyruvate lyase.Stable integration of the ubiC gene into the tobaccochloroplast resulted in hyperexpression of the enzymeand accumulation of this polymer up to 25% of dryweight (Viitanen et al., 2004). In another study, thegene for thermostable xylanase was expressed in thechloroplasts of tobacco plants (Leelavathi et al., 2003).Xylanase accumulated in the cells to approximately6% of tsp. Zymography assay demonstrated that theestimated activity was 140,755 units kg21 fresh leaftissue.

CHALLENGES AHEAD

Although the concept is more than 10 years old,plastid transformation has been accomplished in rel-atively few species. There are numerous factors thathave hampered the expansion of chloroplast transfor-mation technology to different plant species. Onefactor is the unavailability of the genome sequence.The chloroplast transformation vectors utilize homol-ogous flanking regions for recombination and inser-tion of foreign genes. Therefore, there is an urgent

need to sequence chloroplast genomes to facilitatetransformation of crop species. Regardless of the smallsize of the genome and availability of tools to sequencean entire genome within a single day, it is hard tounderstand why only a few crop chloroplast genomeshave been sequenced so far. Between 1986 and 2004,only six crop chloroplast genomes were sequenced. Inthe past 3 years, 25 new crop chloroplast genomeshave been sequenced, including major crops like soy-bean and cotton (Saski et al., 2005; Lee et al., 2006a).Recent studies reveal that intergenic spacer regionsand regulatory sequences contribute about 40% to 45%of the chloroplast genome and that spacer regions arenot highly conserved. Comparison of nine grass chlo-roplast genomes revealed that not even one spacerregion had 100% homology. Therefore, species-specificchloroplast vectors should be made for efficient trans-formation of grasses (Saski et al., 2007).

Plastid transformation is a tissue culture-dependentprocess. Therefore, it is not adequate just to have thegenome information; a better understanding of DNAdelivery, selection, regeneration, and progression to-ward homoplasmy is essential to achieve plastid trans-formation in different taxonomic groups. Althoughchloroplast genome sequences of several monocots,including wheat and maize, have been available forseveral years, none of their genomes has been fullytransformed so far. Major obstacles include the diffi-culty of expressing transgenes in non-green plastids,in which gene expression and gene regulation systemsare quite distinct from those of mature green chloro-plasts. Moreover, it is not possible to generate homo-plasmic plants via subsequent rounds of regenerationusing leaves as explants. Furthermore, proplastids areused as the transformation target rather than chloro-plasts that are about 5-fold smaller in size than thefully developed chloroplasts in the green leaf tissues.Therefore, plastids with irreversible physical damagedue to biolistic bombardment might be greater. It mayalso be necessary to develop new selection markersfor a monocot-specific selection scheme. However, trans-formation of cotton or carrot using non-green embryo-genic cells containing proplastids and regeneration viasomatic embryogenesis offers new hopes for success.

ACKNOWLEDGMENT

We thank Dr. Nameirakpam Dolendro Singh and Tracey Ruhlman for

assistance with figures.

Received August 3, 2007; accepted September 30, 2007; published December 6,

2007.

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