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 chloroplast
genome occurs via homologous recombination of
flanking sequences used in chloroplast vectors. Iden-
tification of spacer regions to integrate transgenes and
endogenous regulatory sequences that support opti-
mal expression is the first step in construction of
chloroplast vectors. Thirty-five sequenced crop chlo-
roplast 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 agro-
nomic traits, including herbicide, insect, and disease
resistance, drought and salt tolerance, and phytore-
mediation. 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 [Sola-
num tuberosum], tobacco [Nicotiana tabacum], and to-
mato [Solanum lycopersicum]) or embryogenesis (carrot
[Daucus carota], cotton [Gossypium hirsutum], rice [Oryza
sativa], 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 been
evaluated 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), 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 bio-
polymers, 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 daniell@mail.ucf.edu.
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 (daniell@mail.ucf.edu).
www.plantphysiol.org/cgi/doi/10.1104/pp.107.106690
Plant Physiology, December 2007, Vol. 145, pp. 1129–1143, www.plantphysiol.org Ó 2007 American Society of Plant Biologists
insulitis in diabetic mice; such delivery eliminates
expensive fermentation, purification, low temperature
storage, and transportation. Chloroplast vector sys-
tems 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, chlo-
roplasts 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, produc-
tion 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 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 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 suit-
able for establishing the coexistence of conventional
and 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 mammalian
viral vectors. Therefore, plastids provide a viable al-
ternative 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
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, site-
specific integration into the chloroplast genome by
homologous recombination of flanking chloroplast
DNA 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 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. simulta-
neous expression of multiple transgenes, creating an
opportunity to produce multivalent vaccines in a
single transformation step. Several heterologous op-
erons have been expressed in transgenic chloroplasts,
and polycistrons are translated without processing
into monocistrons (Quesada-Vargas et al., 2005). More-
over, 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 fo-
cused 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 con-
served 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 pro-
cess (Daniell et al., 1990; Sanford et al., 1993) or occa-
sionally by polyethylene glycol (PEG) treatment of
protoplasts (Golds et al., 1993; O’Neill et al., 1993). This
is followed by transgene integration into the chloro-
plast 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 recombina-
1130
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 cor-
rection 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, includ-
ing insertion of transgenes without 5# or 3# untrans-
lated regions (UTRs) or promoters. To date, the most
commonly used site of integration is the transcription-
ally 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 repli-
cation that facilitates replication of the plasmid inside
the chloroplast, thereby increasing the template copy
number 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 vectors
within chloroplasts (Daniell et al., 1990), enhance the
probability of transgene integration, and achieve ho-
moplasmy 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 inte-
gration sites within the chloroplast genome were only
partially successful. Integration of transgenes be-
tween 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 ho-
mologous 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 chlo-
roplast 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

because the homologous flanking sequences present in
these vectors showed adequate homology to the cor-
responding sequences of potato and tomato plastid
DNA but the efficiency of transformation is signifi-
cantly 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 (approxi-
mately 98% homologous) were used to transform the
tobacco chloroplast genome (DeGray et al., 2001), re-
vealing that a lack of complete homology may reduce
the transformation efficiency to a great extent. How-
ever, comparison of intergenic spacer regions among
members of Solanaceae revealed that only four regions
are identical (Daniell et al., 2006). Similarly, compari-
son of intergenic spacer regions of nine grass chloro-
plast 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 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 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 (Goldschmidt-
Clermont, 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 phe-
notypic resistance permitted the loss of the resistant
rRNA gene in 99 out of 100 potential transformation
events. Although it was first explained that spectino-
Plant Physiol. Vol. 145, 2007
Plastid Transformation Vectors
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 plas-
tid transformation that confers kanamycin resistance
(Carrer et al., 1993). A different kanamycin resistance
gene (aphA6) with relatively high transformation effi-
ciency 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 con-
tained 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 detox-
ification of the same selection agent (kanamycin) dur-
ing 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 resis-
tance genes, several studies have explored strategies
for engineering chloroplasts that are free of antibiotic-
resistance markers. The spinach (Spinacia oleracea) be-
taine 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 transfor-
mation study showed rapid regeneration of transgenic
shoots within 2 weeks in tobacco, and betaine alde-
hyde selection was 25-fold more efficient than specti-
nomycin. 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 re-
sistance, 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 in-
troduced 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 subcellu-
lar localization of the gene encoding the detoxifying
enzyme PAT (Lutz et al., 2001). The lethality of herbi-
1131
Verma and Daniell

Table I. Chloroplast transformation method and selection conditions reported for different crop species
Explant and Method Selection Agent and Conditions for First, Literature
Crop
of Transformation Second, and Third Rounds of Selection Cited

Plant regeneration by embryogenesis

Carrot Fine cell suspension Spectinomycin; first selection of cell lines for 2 to 3 months on 150 mg mL21 Kumar et al.
cultures derived spectinomycin; second selection on 350 mg mL21 spectinomycin for a (2004a)
from stem month; multiplication using 500 mg mL21 spectinomycin; transgenic shoots
were produced from somatic embryos on 500 mg mL21 spectinomycin.
Cotton Grayish-green friable Kanamycin; first selection with 50 mg mL21 kanamycin; second selection Kumar et al.
callus produced from with 100 mg mL21 kanamycin for 4 to 5 months. Transformed calli (2004b)
hypocotyl explants of were converted into somatic embryos and plantlets.
5-d-old cotton seedlings;
biolistic using 0.6-mm
gold particles
Rice Calli derived from mature Streptomycin; first selection after 1 to 2 d of bombardment on Lee et al.
seeds; biolistic using medium supplemented with 200 mg mL21 streptomycin; the (2006b)
0.6-mm gold particles streptomycin-resistant shoots were rooted on Murashige and Skoog
medium with 500 mg mL21 streptomycin.
Soybean Embryogenic calli; biolistic Spectinomycin; first selection after 2 d of bombardment on medium Dufourmantel
using 0.6-mm gold containing 200 or 300 mg mL21 spectinomycin and subcultured every et al.
particles 15 d; first green spectinomycin-resistant calli appeared after 8 weeks of (2004)
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.
Plant regeneration by organogenesis from protoplasts
Cauliflower Protoplasts isolated from Spectinomycin; embryogenic calli selected with 60 mg mL21 Nugent et al.
fully expanded leaves; spectinomycin. The calli subsequently formed shoots. Leaf explants from (2006)
PEG4000 mediated these regenerated shoots placed on shoot regeneration medium containing
300 mg mL21 spectinomycin regenerated further resistant shoots.
Lettuce Protoplasts isolated from Spectinomycin; first selection was initiated after 6 d of transformation in dark Lelivelt et al.
3-week-old shoot for 1 week; second selection in light until the calli were approximately (2005)
culture leaves; 0.5 mm in diameter followed by growth until calli were few millimeters
PEG6000 mediated in diameter; shoot formation; all selection steps contained 500 mg mL21
spectinomycin.
Plant regeneration by organogenesis from leaf
Cabbage Leaves; biolistic using Spectinomycin; first selection of calli after 1 week of bombardment Liu et al.
1.0-mm gold particles on medium containing 50 mg mL21 spectinomycin and subcultured (2007)
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.
Lettuce Young leaves from 3- to Spectinomycin; first selection of resistant green calli on medium Kanamoto
4-week-old plants; supplemented with 50 mg mL21 spectinomycin for 2 months et al.
biolistic using 0.6-mm followed by shoot regeneration on same medium in few weeks. (2006)
gold particles
Lettuce Young leaves; biolistic Spectinomycin; first selection of resistant shoots on medium supplemented Ruhlman
using 0.6-mm gold with 50 mg mL21 spectinomycin; second selection of resistant shoots et al.
particles from pieces of leaves of resistant shoots from first round of selection on (2007)
medium supplemented with 50 mg mL21 spectinomycin; regenerated
shoots were rooted on medium with 50 mg mL21 spectinomycin.
Oilseed Green cotyledon petioles Spectinomycin; first selection after 3 d of bombardment with 10 mg mL21 Hou et al.
rape of 1–2 mm in length; spectinomycin; the regenerated green shoots subcultured onto the same (2003)
biolistic using tungsten selection medium once every 3 weeks twice and then transferred to
particles rooting medium and finally to soil.
Petunia Leaves; biolistic using Spectinomycin and streptomycin; first selection on medium supplemented Zubko et al.
1.0-mm gold particles with 200 mg mL21 spectinomycin and 200 mg mL21 streptomycin for (2004)
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
0.6-mm gold particles and subcultured every 2 weeks; spectinomycin-resistant calli transferred to et al.
shoot induction medium with 30 mg mL21 spectinomycin every 4 weeks (2006)
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.)

Explant and Method Selection Agent and Conditions for First, Literature
Crop
of Transformation Second, and Third Rounds of Selection Cited

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
Spectinomycin; after 2 to 3 d of bombardment, the pieces of leaves were Sidorov
placed onto regeneration medium containing spectinomycin (40, 300, and et al.
400 mg mL21). The first spectinomycin-resistant events were identified after (1999)
4 to 6 weeks of selection.
Spectinomycin; first selection on medium containing 300 mg mL21 Nguyen
spectinomycin for 4 weeks; second selection of the leaf explants on shoot et al.
induction medium containing 300 mg mL21 spectinomycin for every (2005)
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 Ruf et al.
500 mg mL21 spectinomycin for 3 to 4 months to obtain resistant yellow (2001)
or pale green calli and subcultured to achieve homoplasmy. Plants were
regenerated from homoplasmic callus tissue.

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 detrimen-
tal 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 cyto-
sine to uracil. 5-Fluorocytosine is toxic to cells that
express cytosine deaminase because this enzyme con-
verts 5-fluorocytosine to toxic 5-fluorouracil. This neg-
ative selection scheme was utilized to identify seedlings
on 5-fluorocytosine medium from which codA was re-
moved by the P1 bacteriophage site-specific recombi-
nase 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 sub-
mucosa, 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 de-
tected in any substantial amount in the liver or spleen
of mice fed with IFN-GFP-expressing plants, suggest-
ing 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 circula-
tion. 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.
Plant Physiol. Vol. 145, 2007
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 ex-
cision 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
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 spec-
tinomycin 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 di-
oxygenase enzyme that conferred resistance to sulco-
trione 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) attach-
ment sites, which are recognized by Int recombinase.
Efficient excision of the marker gene was shown after
transformation of the nucleus with an int gene encod-
ing 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 inser-
tions 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 chape-
1134
rone (encoded by the orf2 gene) to fold the insecticidal
protein, Cry2Aa2, into cuboidal crystals. The crystal
structure protected the foreign proteins from degra-
dation, 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 (Fernandez-
San Millan et al., 2003). This illustrated the power of
regulatory sequences during illumination and protec-
tion from proteases when their access is limited.
Several studies on transgenic chloroplasts did not
correlate increased transcript abundance with transla-
tion 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. Al-
though 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 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 newly
synthesized proteins or enhancing translation (Kuroda
and 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 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 poly-
merase 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 isopropylthio-
b-galactoside-inducible expression system for plastids
has been reported, although transgene repression in
Plant Physiol. Vol. 145, 2007

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 of-
fered 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 ge-
nome 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 suc-
cessfully carried out in tobacco and is now a routine
procedure 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 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 cru-
cial for extending the range of species in which plastid
transformation could be achieved. Plastid transforma-
tion 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 to-
bacco, like petunia and oilseed rape, adventitious
shoot regeneration from bombarded leaf or petiole
explants generated plastid transformants. Homoplas-
mic plants of soybean, carrot, and cotton were regen-
erated 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
Plastid Transformation Vectors
as micro-tuber (potato), fruit (tomato), and root (car-
rot). 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 improv-
ing the chloroplast transformation system for tomato
plants. Utilizing that plastid expression of a bacterial
lycopene b-cyclase gene resulted in herbicide resis-
tance and triggered conversion of lycopene, the main
storage carotenoid of tomatoes, to b-carotene, result-
ing in a 4-fold enhancement of pro-vitamin A content
of 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, have
attracted attention toward plastid genetic engineering.
Edible plant species not only minimize downstream
protein 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 seed
to edible biomass is only weeks compared to months
for crops such as tomato, potato, and carrot. Further-
more, lettuce is well suited for indoor cultivation by
hydroculture systems (Kanamoto et al., 2006). Accu-
mulation 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 em-
bryogenic 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 cot-
ton, 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 select-
able marker genes (aphA6 and nptII) to detoxify kana-
mycin (Kumar et al., 2004b). Transgenic lines were
fertile and showed maternal inheritance of trans-
gene. 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 embryo-
genesis from spectinomycin-resistant calli and were
fully fertile. Stable plastid transformation in rice was
achieved using mature seed-derived calli for bom-
bardment (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, trans-
1135
Verma and Daniell

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 trait
Insect 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 proteins
hST hST Prrn/T7 gene10/Trps16 Staub et al. (2000)
PpsbA/Trps16
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 Ruhlman et al. (2007)
gene10/Trps16
Vaccine antigens
Cholera toxin ctxB Prrn/ggagg/TpsbA Daniell et al. (2001a)
Tetanus toxin tetC bacterial and synthetic Prrn/T7gene 10/TrbcL Tregoning et al. (2003)
atpB/TrbcL
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)
Biomaterials
Elastin-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 Lössl et al. (2003)
Xylanase xynA PpsbA/TpsbA Leelavathi et al. (2003)
Tryptophan ASA2 Prrn/rbcL/rpL32 Zhang et al. (2001)
rbcL/accD-ORF184
Monellin monellin Prrn/PpsbA/TpsbA Roh et al. (2006)

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
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 cas-
settes 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 cas-
settes (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 com-
Plant Physiol. Vol. 145, 2007

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 tran-
scription and translation of the gene. The chloroplast-
specific 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 restric-
tion sites are introduced to facilitate gene assembly.
Because of the high similarity in the transcription
and translation systems between E. coli and chloro-
plasts, the chloroplast expression vectors are tested in
E. 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 of
spectinomycin 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. 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
Plastid Transformation Vectors
the other anneals within the transgene cassette. Mu-
tants 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 radiola-
beled flanking sequences used for homologous recom-
bination. 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 proper-
ties or functionality of the introduced transgene is
assessed. Seeds from the transgenic plants and untrans-
formed 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 re-
1137
Verma and Daniell

Figure 2. Selection of transplastomic plants.

Shown are representative photographs of
transplastomic tobacco and lettuce shoots un-
dergoing 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 engi-
neering (Table II). Genetically engineered tobacco plants
expressing an insecticidal protein Cry2Aa2 have shown
resistance against target insects and insects that de-
veloped 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 detec-
tion 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 ex-
pression in transgenic tobacco plants. In planta assays
with the bacterial pathogen Pseudomonas syringae pv
tabaci and the fungal pathogen Colletotrichum destructi-
vum showed necrotic lesions in untransformed control
leaves, 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 to
confer resistance to drought by expressing trehalose
phosphate synthase 1 (tps1) gene via nuclear transfor-
mation 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 ex-
hibited 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 geneti-
cally modified crop plants (Kumar et al., 2004a). Chlo-
roplast 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


acetate when grown in soil containing up to 400 mM
phenylmercuric acetate (Ruiz et al., 2003). Chloroplast
transgenic lines absorbed mercury exceeding the lev-
els in soil and translocated 100-fold more to shoots
than untransformed plants (Hussein et al., 2007). To-
bacco 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 con-
cerns, 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. Con-
tinuous 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 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-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 indi-
cations 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, 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 of
defense against viral attack and also invasion by
bacterial pathogens, parasites, tumor cells, and allo-
geneic cells from grafts. IFNa2b ranks third in world
Plant Physiol. Vol. 145, 2007
Plastid Transformation Vectors
market use for a biopharmaceutical, behind only in-
sulin 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 immu-
nodeficiency virus infection in HeLa cells. Another
therapeutic 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 against
infection by the EMC virus.
PLASTIDS AS VACCINE BIOREACTORS
As opposed to injected subunit vaccines, oral deliv-
ery 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, 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 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 chloroplast-
expressed 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 mem-
brane GM1 ganglioside receptors. CTB also acts as a
powerful transmucosal carrier and is very effective in
delivering several vaccine antigens. In one such in-
vestigation, 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 an-
thracis, 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 con-
taminants, PA was expressed in transgenic tobacco
chloroplasts by inserting the pagA gene into the chlo-
roplast genome (Watson et al., 2004; Koya et al., 2005).
Mature leaves grown under continuous illumination
contained PA up to 14.2% of tsp. Cytotoxicity mea-
surements in macrophage lysis assays showed that
chloroplast-derived PA was equivalent in potency to
PA produced in B. anthracis. Subcutaneous immuni-
zation of mice with partially purified chloroplast-
derived 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 pro-
duced in small quantities in all plants. In E. coli, the
ubiC gene encoding chorismate pyruvate lyase cata-
lyzes the direct conversion of chorismate to pyruvate
and pHBA. However, in chloroplasts, chorismate is
converted to pHBA by 10 consecutive enzymatic re-
actions 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.
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 that
have hampered the expansion of chloroplast transfor-
mation technology to different plant species. One
factor 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
1140
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 soy-
bean 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 chlo-
roplast genomes revealed that not even one spacer
region had 100% homology. Therefore, species-specific
chloroplast vectors should be made for efficient trans-
formation 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 to-
ward homoplasmy is essential to achieve plastid trans-
formation 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 diffi-
culty of expressing transgenes in non-green plastids,
in which gene expression and gene regulation systems
are quite distinct from those of mature green chloro-
plasts. Moreover, it is not possible to generate homo-
plasmic plants via subsequent rounds of regeneration
using leaves as explants. Furthermore, proplastids are
used as the transformation target rather than chloro-
plasts 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, trans-
formation of cotton or carrot using non-green embryo-
genic 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.
Received August 3, 2007; accepted September 30, 2007; published December 6,
2007.
LITERATURE CITED
Arlen PA, Falconer R, Cherukumilli S, Cole A, Cole AM, Oishi KK, Daniell H
(2007) Field production and functional evaluation of chloroplast-
derived interferon-a2b. Plant Biotechnol J 5: 511–525
Birch-Machin I, Newell CA, Hibberd JM, Gray JC (2004) Accumulation of
rotavirus VP6 protein in chloroplasts of transplastomic tobacco is
limited by protein stability. Plant Biotechnol J 2: 261–270
Plant Physiol. Vol. 145, 2007

Carrer H, Hockenberry TN, Svab Z, Maliga P (1993) Kanamycin resistance
as a selectable marker for plastid transformation in tobacco. Mol Gen
Genet 241: 49–56
Carrer H, Maliga P (1995) Targeted insertion of foreign genes into the
tobacco plastid genome without physical linkage to the selectable
marker gene. Nat Biotechnol 13: 791–794
Cerutti H, Osman M, Grandoni P, Jagendorf AT (1992) A homolog of
Escherichia coli RecA protein in plastids of higher plants. Proc Natl Acad
Sci USA 89: 8068–8072
Chakrabarti SK, Lutz KA, Lertwiriyawong B, Svab Z, Maliga P (2006)
Expression of the cry9Aa2 B.t. gene in tobacco chloroplasts confers
resistance to potato tuber moth. Transgenic Res 15: 481–488
Chebolu S, Daniell H (2007) Stable expression of Gal/GalNAc lectin of
Entamoeba histolytica in transgenic chloroplasts and immunogenicity in
mice towards vaccine development for amoebiasis. Plant Biotechnol J 5:
230–239
Corneille S, Lutz K, Svab Z, Maliga P (2001) Efficient elimination of
selectable marker genes from the plastid genome by the CRE-lox site-
specific recombination system. Plant J 27: 171–178
Cramer CL, Boothe JG, Oishi KK (1999) Transgenic plants for therapeutic
proteins: linking upstream and downstream strategies. Curr Top Micro-
biol Immunol 240: 95–118
Daniell H (2002) Molecular strategies for gene containment in transgenic
crops. Nat Biotechnol 20: 581–586
Daniell H (2006) Production of biopharmaceuticals and vaccines in plants
via the chloroplast genome. Biotechnol J 1: 1071–1079
Daniell H (2007) Transgene containment by maternal inheritance: effective
or elusive? Proc Natl Acad Sci USA 104: 6879–6880
Daniell H, Carmona-Sanchez O, Burns B (2004) Chloroplast derived
antibodies, biopharmaceuticals and edible vaccines. In R Fischer, S
Schillberg, eds, Molecular Farming. Verlag Publishers, Weinheim, Germany,
pp 113–133
Daniell H, Chebolu S, Kumar S, Singleton M, Falconer R (2005a)
Chloroplast-derived vaccine antigens and other therapeutic proteins.
Vaccine 23: 1779–1783
Daniell H, Datta R, Varma S, Gray S, Lee SB (1998) Containment of
herbicide resistance through genetic engineering of the chloroplast
genome. Nat Biotechnol 16: 345–348
Daniell H, Khan MS, Allison L (2002) Milestones in chloroplast genetic
engineering: an environmentally friendly era in biotechnology. Trends
Plant Sci 7: 84–91
Daniell H, Krishnan M, McFadden BA (1991) Transient expression of
b-glucuronidase in different cellular compartments following biolistic
delivery of foreign DNA into wheat leaves and calli. Plant Cell Rep 9:
615–619
Daniell H, Kumar S, Dufourmantel N (2005b) Breakthrough in chloroplast
genetic engineering of agronomically important crops. Trends Biotech-
nol 23: 238–245
Daniell H, Lee SB, Grevich J, Saski C, Quesada-Vargas T, Guda C,
Tomkins J, Jansen RK (2006) Complete chloroplast genome sequences
of Solanum bulbocastanum, Solanum lycopersicum and comparative anal-
yses with other Solanaceae genomes. Theor Appl Genet 112: 1503–1518
Daniell H, Lee SB, Panchal T, Wiebe PO (2001a) Expression of the native
cholera toxin B subunit gene and assembly as functional oligomers in
transgenic tobacco chloroplasts. J Mol Biol 311: 1001–1009
Daniell H, McFadden BA (1987) Uptake and expression of bacterial and
cyanobacterial genes by isolated cucumber etioplasts. Proc Natl Acad
Sci USA 84: 6349–6353
Daniell H, Muthukumar B, Lee SB (2001b) Marker free transgenic plants:
engineering the chloroplast genome without the use of antibiotic selec-
tion. Curr Genet 39: 109–116
Daniell H, Vivekananda J, Nielsen BL, Ye GN, Tewari KK (1990) Transient
foreign gene expression in chloroplasts of cultured tobacco cells after
biolistic delivery of chloroplast vectors. Proc Natl Acad Sci USA 87: 88–92
De Cosa B, Moar W, Lee SB, Miller M, Daniell H (2001) Overexpression of
the Bt cry2Aa2 operon in chloroplasts leads to formation of insecticidal
crystals. Nat Biotechnol 19: 71–74
DeGray G, Rajasekaran K, Smith F, Sanford J, Daniell H (2001) Expres-
sion of an antimicrobial peptide via the chloroplast genome to control
phytopathogenic bacteria and fungi. Plant Physiol 127: 852–862
Dhingra A, Portis AR Jr, Daniell H (2004) Enhanced translation of a
chloroplast-expressed RbcS gene restores small subunit levels and
Plant Physiol. Vol. 145, 2007
Plastid Transformation Vectors
photosynthesis in nuclear RbcS antisense plants. Proc Natl Acad Sci
USA 101: 6315–6320
Dufourmantel N, Dubald M, Matringe M, Canard H, Garcon F, Job C, Kay
E, Wisniewski JP, Ferullo JM, Pelissier B, et al (2007) Generation and
characterization of soybean and marker-free tobacco plastid transform-
ants over-expressing a bacterial 4-hydroxyphenylpyruvate dioxygenase
which provides strong herbicide tolerance. Plant Biotechnol J 5: 118–133
Dufourmantel N, Pelissier B, Garcxon F, Peltier G, Ferullo JM, Tissot G
(2004) Generation of fertile transplastomic soybean. Plant Mol Biol 55:
479–489
Dufourmantel N, Tissot G, Goutorbe F, Garcxon F, Muhr C, Jansens S,
Pelissier B, Peltier G, Dubald M (2005) Generation and analysis of
soybean plastid transformants expressing Bacillus thuringiensis Cry1Ab
protoxin. Plant Mol Biol 58: 659–668
Farran I, Sanchez-Serrano JJ, Medina JF, Prieto J, Mingo-Castel AM (2002)
Targeted expression of human serum albumin to potato tubers. Trans-
genic Res 11: 337–346
Fernandez-San Millan A, Mingo-Castel A, Miller M, Daniell H (2003) A
chloroplast transgenic approach to hyper-express and purify human
serum albumin, a protein highly susceptible to proteolytic degradation.
Plant Biotechnol J 1: 71–79
Fischer N, Stampacchia O, Redding K, Rochaix J (1996) Selectable marker
recycling in the chloroplast. Mol Gen Genet 251: 373–380
Glenz K, Bouchon B, Stehle T, Wallich R, Simon MM, Warzecha H (2006)
Production of a recombinant bacterial lipoprotein in higher plant
chloroplasts. Nat Biotechnol 24: 76–77
Golds T, Maliga P, Koop HU (1993) Stable plastid transformation in PEG-
treated protoplasts of Nicotiana tabacum. Nat Biotechnol 11: 95–97
Goldschmidt-Clermont M (1991) Transgenic expression of aminoglycoside
adenine transferase in the chloroplast: a selectable marker of site-directed
transformation of chlamydomonas. Nucleic Acids Res 19: 4083–4089
Guda C, Lee SB, Daniell H (2000) Stable expression of a biodegradable
protein-based polymer in tobacco chloroplasts. Plant Cell Rep 19: 257–262
Hagemann R (2004) The sexual inheritance of plant organelles. In H
Daniell, CD Chase, eds, Molecular Biology and Biotechnology of Plant
Organelles. Springer, Dordrecht, The Netherlands, pp 93–113
Hanson MR, Kohler RH (2001) GFP imaging: methodology and applica-
tion to investigate cellular compartmentation in plants. J Exp Bot 52:
529–539
Harris EH, Burkhart BD, Gillham NW, Boynton JE (1989) Antibiotic
resistance mutations in the chloroplast 16S and 23S rRNA genes of
Chlamydomonas reinhardtii: correlation of genetic and physical maps of
the chloroplast genome. Genetics 123: 281–292
Hibberd JM, Linley PJ, Khan MS, Gray JC (1998) Transient expression of
green fluorescent protein in various plastid types following micro-
projectile bombardment. Plant J 16: 627–632
Hou BK, Zhou YH, Wan LH, Zhang ZL, Shen GF, Chen ZH, Hu ZM
(2003) Chloroplast transformation in oilseed rape. Transgenic Res 12:
111–114
Huang FC, Klaus S, Herz S, Zou Z, Koop HU, Golds T (2002) Efficient
plastid transformation in tobacco using the aphA-6 gene and kanamycin
selection. Mol Genet Genomics 268: 19–27
Hussein SH, Ruiz ON, Terry N, Daniell H (2007) Phytoremediation of
mercury and organomercurials in chloroplast transgenic plants: en-
hanced root uptake, translocation to shoots and volatilization. Environ
Sci Technol (in press)
Iamtham S, Day A (2000) Removal of antibiotic resistance genes from
transgenic tobacco plastids. Nat Biotechnol 18: 1172–1176
Kamarajugadda S, Daniell H (2006) Chloroplast-derived anthrax and
other vaccine antigens: their immunogenic and immunoprotective
properties. Expert Rev Vaccines 5: 839–849
Kanamoto H, Yamashita A, Asao H, Okumura S, Takase H, Hattori M,
Yokota A, Tomizawa K (2006) Efficient and stable transformation of
Lactuca sativa L. cv. Cisco (lettuce) plastids. Transgenic Res 15: 205–217
Khan MS, Maliga P (1999) Fluorescent antibiotic resistance marker for
tracking plastid transformation in higher plants. Nat Biotechnol 17:
910–915
Kindle KL, Richards KL, Stern DB (1991) Engineering the chloroplast
genome: techniques and capabilities for chloroplast transformation in
Chlamydomonas reinhardtii. Proc Natl Acad Sci USA 88: 1721–1725
Kittiwongwattana C, Lutz K, Clark M, Maliga P (2007) Plastid marker
gene excision by the phiC31 phage site-specific recombinase. Plant Mol
Biol 64: 137–143
1141
Verma and Daniell
Klaus SMJ, Huang FC, Golds TJ, Koop HU (2004) Generation of marker-
free plastid transformants using a transiently cointegrated selection
gene. Nat Biotechnol 22: 225–229
Kode V, Mudd EA, Iamtham S, Day A (2006) Isolation of precise plastid
deletion mutants by homology-based excision: a resource for site-
directed mutagenesis, multi-gene changes and high-throughput plastid
transformation. Plant J 46: 901–909
Kota M, Daniell H, Varma S, Garczynski SF, Gould F, Moar WJ (1999)
Overexpression of the Bacillus thuringiensis (Bt) Cry2Aa2 protein in
chloroplasts confers resistance to plants against susceptible and
Bt-resistant insects. Proc Natl Acad Sci USA 96: 1840–1845
Koya V, Moayeri M, Leppla SH, Daniell H (2005) Plant-based vaccine:
mice immunized with chloroplast-derived anthrax protective antigen
survive anthrax lethal toxin challenge. Infect Immun 73: 8266–8274
Kumar S, Daniell H (2004) Engineering the chloroplast genome for
hyperexpression of human therapeutic proteins and vaccine antigens.
Methods Mol Biol 267: 365–383
Kumar S, Dhingra A, Daniell H (2004a) Plastid-expressed betaine alde-
hyde dehydrogenase gene in carrot cultured cells, roots, and leaves
confers enhanced salt tolerance. Plant Physiol 136: 2843–2854
Kumar S, Dhingra A, Daniell H (2004b) Stable transformation of the cotton
plastid genome and maternal inheritance of transgenes. Plant Mol Biol
56: 203–216
Kunnimalaiyaan M, Nielsen BL (1997) Fine mapping of replication origins
(ori A and ori B) in Nicotiana tabacum chloroplast DNA. Nucleic Acids
Res 25: 3681–3686
Kuroda H, Maliga P (2001) Sequences downstream of the translation
initiation codon are important determinants of translation efficiency in
chloroplasts. Plant Physiol 125: 430–436
Kusnadi AR, Nikolov ZL, Howard JA (1997) Production of recombinant
proteins in transgenic plants: practical considerations. Biotechnol Bio-
eng 56: 473–484
Lee SB, Kaittanis C, Jansen RK, Hostetler JB, Tallon LJ, Town CD, Daniell
H (2006a) The complete chloroplast genome sequence of Gossypium
hirsutum: organization and phylogenetic relationships to other angio-
sperms. BMC Genomics 7: 61
Lee SB, Kwon HB, Kwon SJ, Park SC, Jeong MJ, Han SE, Byun MO,
Daniell H (2003) Accumulation of trehalose within transgenic chloro-
plasts confers drought tolerance. Mol Breed 11: 1–13
Lee SM, Kang K, Chung H, Yoo SH, Xu XM, Lee SB, Cheong JJ, Daniell H,
Kim M (2006b) Plastid transformation in the monocotyledonous cereal
crop, rice (Oryza sativa) and transmission of transgenes to their progeny.
Mol Cells 21: 401–410
Leelavathi S, Gupta N, Maiti S, Ghosh A, Reddy VS (2003) Overproduc-
tion of an alkali- and thermo-stable xylanase in tobacco chloroplasts and
efficient recovery of the enzyme. Mol Breed 11: 59–67
Leelavathi S, Reddy VS (2003) Chloroplast expression of His-tagged GUS-
fusions: a general strategy to overproduce and purify foreign proteins
using transplastomic plants as bioreactors. Mol Breed 11: 49–58
Lelivelt C, McCabe M, Newell C, deSnoo C, Dun K, Birch-Machin I, Gray
J, Mills K, Nugent J (2005) Stable plastid transformation in lettuce
(Lactuca sativa L.). Plant Mol Biol 58: 763–774
Lilly JW, Havey MJ, Jackson SA, Jiang J (2001) Cytogenomic analyses
reveal the structural plasticity of the chloroplast genome in higher
plants. Plant Cell 13: 245–254
Limaye A, Koya V, Samsam M, Daniell H (2006) Receptor-mediated oral
delivery of a bioencapsulated green fluorescent protein expressed in
transgenic chloroplasts into the mouse circulatory system. FASEB J 20:
959–961
Liu CW, Lin CC, Chen J, Tseng MJ (2007) Stable chloroplast transformation
in cabbage (Brassica oleracea var. capitata L.) by particle bombardment.
Plant Cell Rep 26: 1733–1744
Lossl A, Bohmert K, Harloff H, Eibl C, Muhlbauer S, Koop HU (2005)
Inducible trans-activation of plastid transgenes: expression of the R.
eutropha phb operon in transplastomic tobacco. Plant Cell Physiol 46:
1462–1471
Lössl A, Eibl C, Harloff HJ, Jung C, Koop HU (2003) Polyester synthesis in
transplastomic tobacco (Nicotiana tabacum L.): significant contents of
polyhydroxybutyrate are associated with growth reduction. Plant Cell
Rep 21: 891–899
Lugo SK, Kunnimalaiyaan M, Singh NK, Nielsen BL (2004) Required
sequence elements for chloroplast DNA replication activity in vitro and
in electroporated chloroplasts. Plant Sci 166: 151–161
1142
Lutz KA, Knapp JE, Maliga P (2001) Expression of bar in the plastid
genome confers herbicide resistance. Plant Physiol 125: 1585–1590
Magee A, Coyne S, Murphy D, Horvath E, Medgyesy P, Kavanagh T
(2004) T7 RNA polymerase-directed expression of an antibody fragment
transgene in plastids causes a semi-lethal pale-green seedling pheno-
type. Transgenic Res 13: 325–337
Martin WRT, Richly E, Hansen A, Cornelson S, Lins T, Leister D, Stoebe
B, Hasegawa M, Penny D (2002) Evolutionary analysis of Arabidopsis,
cyanobacterial, and chloroplast genomes reveals plastid phylogeny and
thousands of cyanobacterial genes in the nucleus. Proc Natl Acad Sci
USA 99: 12246–12251
McBride KE, Svab Z, Schaaf DJ, Hogan PS, Stalker DM, Maliga P (1995)
Amplification of a chimeric Bacillus gene in chloroplasts leads to an
extraordinary level of an insecticidal protein in tobacco. Biotechnology
(N Y) 13: 362–365
Molina A, Hervas-Stubbs S, Daniell H, Mingo-Castel AM, Veramendi J
(2004) High-yield expression of a viral peptide animal vaccine in
transgenic tobacco chloroplasts. Plant Biotechnol J 2: 141–153
Molina A, Veramendi J, Hervás-Stubbs S (2005) Induction of neutralizing
antibodies by a tobacco chloroplast-derived vaccine based on a B cell
epitope from canine parvovirus. Virology 342: 266–275
Mor TS, Gomez-Lim MA, Palmer KE (1998) Perspective: edible vaccines-a
concept coming of age. Trends Microbiol 6: 449–453
Muhlbauer SK, Koop HU (2005) External control of transgene expression
in tobacco plastids using the bacterial lac repressor. Plant J 43: 941–946
Nguyen TT, Nugent G, Cardi T, Dix PJ (2005) Generation of homoplasmic
plastid transformants of a commercial cultivar of potato (Solanum
tuberosum L.). Plant Sci 168: 1495–1500
Nuccio ML, Russell BL, Nolte KD, Rathinasabapathi B, Gage DA,
Hanson AD (1998) The endogenous choline supply limits glycine
betaine synthesis in transgenic tobacco expressing choline monooxyge-
nase. Plant J 16: 487–496
Nugent GD, Coyne S, Nguyen TT, Kavanagh TT, Dix PJ (2006) Nuclear
and plastid transformation of Brassica oleracea var. botrytis (cauliflower)
using PEG-mediated uptake of DNA into protoplasts. Plant Sci 170:
135–142
Okumura S, Sawada M, Park YW, Hayashi T, Shimamura M, Takase H,
Tomizawa K (2006) Transformation of poplar (Populus alba) plastids and
expression of foreign proteins in tree chloroplasts. Transgenic Res 15:
637–646
O’Neill C, Horvath GV, Horvath E, Dix PJ, Medgyesy P (1993) Chloroplast
transformation in plants: polyethylene glycol (PEG) treatment of
protoplasts is an alternative to biolistic delivery systems. Plant J 3:
729–738
Palmer JD (1985) Comparative organization of chloroplast genomes. Annu
Rev Genet 19: 325–354
Quesada-Vargas T, Ruiz ON, Daniell H (2005) Characterization of heter-
ologous multigene operons in transgenic chloroplasts: transcription,
processing, and translation. Plant Physiol 138: 1746–1762
Rathinasabapathi B, Burnet M, Russell BL, Gage DA, Liao PC, Nye GJ,
Scott P, Golbeck JH, Hanson AD (1997) Choline monooxygenase, an
unusual iron-sulfur enzyme catalyzing the first step of glycine betaine
synthesis in plants: prosthetic group characterization and cDNA clon-
ing. Proc Natl Acad Sci USA 94: 3454–3458
Raubeson LA, Jansen RK (2005) Chloroplast genomes of plants. In
RJ Henry, ed, Diversity and Evolution of Plants-Genotypic and Phenotypic
Variation in Higher Plants. CABI Publishers, Cambridge, MA, pp
45–68
Reed ML, Wilson SK, Sutton CA, Hanson MR (2001) High-level expres-
sion of a synthetic red-shifted GFP coding region incorporated into
transgenic chloroplasts. Plant J 27: 257–265
Roh KH, Shin KS, Lee YH, Seo SC, Park HG, Daniell H, Lee SB (2006)
Accumulation of sweet protein monellin is regulated by the psbA 5#UTR
in tobacco chloroplasts. J Plant Biol 49: 34–43
Ruf S, Hermann M, Berger IJ, Carrer H, Bock R (2001) Stable genetic
transformation of tomato plastids and expression of a foreign protein in
fruit. Nat Biotechnol 19: 870–875
Ruf S, Karcher D, Bock R (2007) Determining the transgene containment
level provided by chloroplast transformation. Proc Natl Acad Sci USA
104: 6998–7002
Ruhlman T, Ahangari R, Devine A, Samsam M, Daniell H (2007) Ex-
pression of cholera toxin B-proinsulin fusion protein in lettuce and
Plant Physiol. Vol. 145, 2007

tobacco chloroplasts—oral administration protects against development of
insulitis in non-obese diabetic mice. Plant Biotechnol J 5: 495–510
Ruiz ON, Daniell H (2005) Engineering cytoplasmic male sterility via the
chloroplast genome by expression of b-ketothiolase. Plant Physiol 138:
1232–1246
Ruiz ON, Hussein HS, Terry N, Daniell H (2003) Phytoremediation of
organomercurial compounds via chloroplast genetic engineering. Plant
Physiol 132: 1344–1352
Rumeau D, Becuwe-Linka N, Beyly A, Louwagie M, Garin J, Peltier G
(2005) New subunits NDH-M, -N, and -O, encoded by nuclear genes, are
essential for plastid Ndh complex functioning in higher plants. Plant
Cell 17: 219–232
Sanford JC, Smith FD, Russell JA, Ray W (1993) Optimizing the biolistic pro-
cess for different biological applications. Methods Enzymol 217: 483–509
Saski C, Lee SB, Daniell H, Wood TC, Tomkins J, Kim HG, Jansen RK
(2005) Complete chloroplast genome sequence of Glycine max and
comparative analyses with other legume genomes. Plant Mol Biol 59:
309–322
Saski C, Lee SB, Fjellheim S, Guda C, Jansen RK, Luo H, Tomkins J,
Rognli OA, Daniell H, Clarke JL (2007) Complete chloroplast genome
sequences of Hordeum vulgare, Sorghum bicolor and Agrostis stolonifera,
and comparative analyses with other grass genomes. Theor Appl Genet
115: 571–590
Serino G, Maliga P (1997) A negative selection scheme based on the
expression of cytosine deaminase in plastids. Plant J 12: 697–701
Sidorov VA, Kasten D, Pang SZ, Hajdukiewicz PTJ, Staub JM, Nehra NS
(1999) Stable chloroplast transformation in potato: use of green fluores-
cent protein as a plastid marker. Plant J 19: 209–216
Staub JM, Garcia B, Graves J, Hajdukiewicz PTJ, Hunter P, Nehra N,
Paradkar V, Schlittler M, Carroll JA, Spatola L, et al (2000) High-yield
production of a human therapeutic protein in tobacco chloroplasts. Nat
Biotechnol 18: 333–338
Svab Z, Hajdukiewicz P, Maliga P (1990) Stable transformation of plastids
in higher plants. Proc Natl Acad Sci USA 87: 8526–8530
Plant Physiol. Vol. 145, 2007
Plastid Transformation Vectors
Svab Z, Maliga P (1993) High-frequency plastid transformation in tobacco by
selection for a chimeric aadA gene. Proc Natl Acad Sci USA 90: 913–917
Svab Z, Maliga P (2007) Exceptional transmission of plastids and mito-
chondria from the transplastomic pollen parent and its impact on
transgene containment. Proc Natl Acad Sci USA 104: 7003–7008
Tregoning JS, Nixon P, Kuroda H, Svab Z, Clare S, Bowe F, Fairweather N,
Ytterberg J, van Wijk KJ, Dougan G, et al (2003) Expression of teta-
nus toxin Fragment C in tobacco chloroplasts. Nucleic Acids Res 31:
1174–1179
Viitanen PV, Devine AL, Khan MS, Deuel DL, Van Dyk DE, Daniell H
(2004) Metabolic engineering of the chloroplast genome using the
Escherichia coli ubiC gene reveals that chorismate is a readily abundant
plant precursor for p-hydroxybenzoic acid biosynthesis. Plant Physiol
136: 4048–4060
Watson J, Koya V, Leppla SH, Daniell H (2004) Expression of Bacillus
anthracis protective antigen in transgenic chloroplasts of tobacco, a non-
food/feed crop. Vaccine 22: 4374–4384
Wurbs D, Ruf S, Bock R (2007) Contained metabolic engineering in
tomatoes by expression of carotenoid biosynthesis genes from the
plastid genome. Plant J 49: 276–288
Ye GN, Colburn SM, Xu CW, Hajdukiewicz PTJ, Staub JM (2003) Persis-
tence of unselected transgenic DNA during a plastid transformation
and segregation approach to herbicide resistance. Plant Physiol 133:
402–410
Ye GN, Daniell H, Sanford JC (1990) Optimization of delivery of foreign
DNA into higher-plant chloroplasts. Plant Mol Biol 15: 809–819
Zhang XH, Brotherton JE, Widholm JM, Portis AR (2001) Targeting a
nuclear anthranilate synthase a-subunit gene to the tobacco plastid
genome results in enhanced tryptophan biosynthesis. Return of a gene
to its pre-endosymbiotic origin. Plant Physiol 127: 131–141
Zubko M, Zubko E, Zuilen K, Meyer P, Day A (2004) Stable transformation
of petunia plastids. Transgenic Res 13: 523–530
1143