Springer 2006

Transgenic Research (2006) 15:205217

DOI 10.1007/s11248-005-3997-2

Ecient and stable transformation of Lactuca sativa L. cv. Cisco (lettuce)

plastids
Hirosuke Kanamoto1,*, Atsushi Yamashita2, Hiroshi Asao3, Satoru Okumura1, Hisabumi
Takase2, Masahira Hattori2, Akiho Yokota4 & Ken-Ichi Tomizawa1
1

Resarch Institute of Innovative Technology for the Earth, 9-2 Kizugawadai, Kizu-cho Soraku-gun, Kyoto, 6190292, Japan
2
Kitasato Institute for Life Sciences, Kitasato University, 1-15-1 Kitasato, Sagamihara-shi, Kanagawa, 228-8555,
Japan
3
Nara Prefectural Agricultural Experiment Station, 88 Shijyo-cho, Kashihara, Nara, 634-0813, Japan
4
Graduate School of Biological Science, Nara Institute of Science and Technology, Ikoma, Nara, 630-0192, Japan
Received 23 May 2005; accepted 5 October 2005

Key words: lettuce, plastid genome, plastid transformation

Abstract
Transgenic plastids oer unique advantages in plant biotechnology, including high-level foreign protein
expression. However, broad application of plastid genome engineering in biotechnology has been largely
hampered by the lack of plastid transformation systems for major crops. Here we describe the development
of a plastid transformation system for lettuce, Lactuca sativa L. cv. Cisco. The transforming DNA carries a
spectinomycin-resistance gene (aadA) under the control of lettuce chloroplast regulatory expression elements, anked by two adjacent lettuce plastid genome sequences allowing its targeted insertion between the
rbcL and accD genes. On average, we obtained 1 transplastomic lettuce plant per bombardment. We show
that lettuce leaf chloroplasts can express transgene-encoded GFP to 36% of the total soluble protein. All
transplastomic T0 plants were fertile and the T1 progeny uniformly showed stability of the transgene in the
chloroplast genome. This system will open up new possibilities for the ecient production of edible vaccines, pharmaceuticals, and antibodies in plants.

Introduction
Transformation of the plastid genome has several
advantages over conventional nuclear transformation (Staub & Maliga, 1995; Daniell et al., 1998;
Scot & Wilkinson, 1999). Most importantly, this is
thought to be up to 10,000 copies of the plastid
genome per leaf cell (Bendich, 1987). This high
ploidy level results in high levels of transgene
expression, so that the corresponding foreign
protein can account for up to about 40% of the
*Author for correspondence
E-mail: kanamoto@rite.or.jp

total soluble cellular proteins (DeCosa et al.,

2001). Because of the potentially high transgene
expression levels in chloroplasts, this system is
expected to open up new possibilities for metabolic
engineering, resistance management, and the use
of plants as factories for biopharmaceuticals
(Staub et al., 2000; Horn et al., 2003; Millan et al.,
2003; Tregoning et al., 2004). These potential
applications make plastid transformation a very
attractive technology.
Recently, higher plant plastid transformation
has been attempted in Arabidopsis (Sikdar et al.,
1998), potato (Sidorov et al., 1999), rice (Khan &
Maliga, 1999), tomato (Ruf et al., 2001), oilseed

206
rape (Hou et al., 2003), Lesquerella fendleri
(Skarjinskaia et al., 2003), and carrot (Kumar
et al., 2004). However, some problems have been
pointed out with each of these plants. In Arabidopsis, oilseed rape, and rice, transplastomic plants
were obtained but those were not stable (Sikdar
et al., 1998; Khan & Maliga, 1999; Hou et al.,
2003). In tobacco, the high content of nicotine and
other toxic alkaloids has been a critical problem for
pharmaceutical production. In the other plants,
protein production was carried out in non-green
tissues such as micro-tuber (potato), fruit (tomato),
and root (carrot). It was estimated that 100-fold
less GFP protein accumulated in potato tuber
amyloplasts compared to leaf chloroplasts. In
carrot chromoplasts, betaine aldehyde dehydrogenase activity expressed from a plastid transgene
accumulated to only 74.8% the level observed in
leaf chloroplasts (Kumar et al., 2004). These ndings suggest that chloroplast-containing tissues
would be the most eective for producing proteins
of interest from plastid transgenes, and attention
has turned to leafy crops for pharmaceutical
production. However, plastid transformation has
not yet been developed for edible leafy crops. One
such crop, lettuce (Lactuca sativa L.), has been used
to produce, via nuclear transformation, a hepatitis
B virus subunit vaccine for clinical trials (Kapusta
et al., 1999). Lettuce grows quickly and can be
harvested within a few months after planting. The
movement of plastid integrated transgenes to the
nucleus has been reported. However, the frequency
of pollen derived from transplastomic plants carried the transgene that was integrated in the plastid
genome is rather low (Huang et al., 2003;
Stegemann et al., 2003; Huang et al., 2004). Furthermore, lettuce is suitable for indoor cultivation
by hydroculture systems. Thus, the horizontal
propagation of transgenes can be prevented by
fail-safe.
Transformation of the plant plastid genome
has been achieved mainly through the following 3
steps. (1) Introduction of the transformation
vector into the plastid by the biolistic method
(Svab et al., 1990; Svab & Maliga, 1993) or
polyethylene glycol treatment (Golds et al., 1993;
Koop et al., 1996). (2) Integration of the transgene
into the plastid genome by double homologous
recombination. (3) Selection of cells containing
transformed plastids and their regeneration under
strong selection pressure for a selectable antibiotic

marker. We optimized conditions for regeneration,

selection, and homologous recombination to
obtain transplastomic plants with high eciency.
First, we screened several lettuce cultivars for the
highest regeneration eciency (Cisco), and then
determined the best regeneration/selection conditions for plastid transformation of this cultivar.
Plastid transformation vectors basically consist of
integration sites for double homologous recombination and a selection cassette. To achieve plastid
transformation, we had to determine an appropriate target site for insertion of transgenes into the
plastid genome, before constructing the transformation vector. It might be thought that the gene
organization of plastid genomes in other plants
could be used as a reference for choosing a target
site for the lettuce transformation vector. However, comparison of plastid genome sequences of
higher plants has revealed diversity in gene organization (Hiratsuka et al., 1989; Shimada & Sugiura, 1991; Doyle et al., 1992). Therefore, it was
necessary to obtain specic information about the
gene organization of the lettuce plastid genome for
the selection of an appropriate target site for
transgene insertion. In addition, to achieve high
eciency plastid transformation, the nucleotide
sequence of the integration sites in the transformation vector should be identical to that of the
target sites in the plastid genome. For these
reasons, we sequenced the entire plastid genome
of lettuce for this study, the rst complete
sequence to be reported for the Asteraceae. Using
this new sequence information, we succeeded in
plastid transformation of lettuce.
Materials and methods
Selection of lettuce cultivar
Forty foliage-leaf explants (4 mm  4 mm) derived
from each cultivar were placed on lettuce regeneration medium [MS medium supplemented with
3% (wt/vol) sucrose, 0.1 mg/l 6-benzylaminopurine (BA), 0.1 mg/l alpha-naphthaleneacetic acid
(NAA), 0.2% (wt/vol) gelangum, pH 5.8]. After
incubation for 1 month under long day conditions
(16 h light/8 h darkness, 25C), the number of
regenerated shoots was counted. The lettuce cultivar that showed the highest regeneration eciency, cv. Cisco, was selected to be the host plant
for plastid transformation.

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Determination of the plastid genome sequence
of Lactuca sativa L. cv. Cisco
Lettuce chloroplasts were isolated from 1-monthold seedlings by discontinuous Percoll density
gradient centrifugation (Miyake & Asada, 1992)
using Percoll concentrations of 40% and 80%.
The chloroplast genome DNA was puried by a
cetyltrimethylammonium bromide-based method
(Ausbel et al., 1993). The nucleotide sequence of
the chloroplast genome was determined by a
whole-genome shotgun strategy. A two kilobaseinsert genomic library was constructed, and 3000
sequences (giving 10-fold coverage) were obtained from both ends of the genomic clones.
Sequences were assembled using the PHRED/
PHRAP/CONSED package. Remaining gaps
were closed by transcriptional sequencing (Nippon Genetech, Tokyo, Japan) or by primer
walking.
Construction of plastid transformation vector
A lettuce plastid transformation vector, pRL200,
was constructed using the 1.6 kb rbcL gene and the
1.1 kb accD gene from the Lactuca sativa L. plastid
genome
(DDBJ/GenBank/EMBL
Accession
AP007232) as homologous recombination targeting sequences. The DNA fragment corresponding
to the rbcL gene was PCR-amplied from total
genomic DNA of Lactuca sativa L. by using KOD
plus DNA polymerase (TOYOBO, Tokyo, Japan)
and specic primers (5-CCGAATTCAATTCA
TGAGTTGTAGGGAG-3 and 5-CCGCGGCC
GCGATCCAACCAACACAAA AAT-3; EcoRI
and NotI recognition sites were underlined). The
resulting fragment was digested with EcoRI and
NotI. The DNA fragment corresponding to the accD
gene was also PCR-amplied by using a dierent set
of specic primers (5-CCGTCGACGATCCTTA
GGATTGGGATAT-3 and 5-GGAAGCTTCCC
ATATGAGTAGAACTTTC-3; SalI and HindIII
recognition sites were underlined) and then digested with SalI and HindIII. The resulting DNA
fragments were cloned into pLD200 (Tomizawa &
Yokota, 2004 submitted) via the EcoRI and NotI
sites and the SalI and HindIII sites, respectively.
This lettuce plastid transformation vector was
named as pRL200.
The aadA spectinomycin-resistance cassette
was constructed as follows. A fragment containing

the lettuce plastid 16S rRNA operon promoter

(LsPrrn) was PCR-amplied from total genomic
DNA of Lactuca sativa L. by using KOD plus
DNA polymerase (TOYOBO) and specic primers
(5-CCGCGGCCGCGATATTTTGATTTGCTACCC-3 and 5-CCAGCGCTATTCGCCCGGAGTTCGCTCC-3). The resulting fragment was
digested with EcoRI and NotI. A fragment
containing the lettuce plastid psbA terminator
(LsTpsbA) was also PCR-amplied with specic
primers (5-GGCTGCAGGACTTTGGTCTTATTGTAAT-3 and 5-CCGTCGACGAGCATATTATTTCTTTCTT-3) and digested with PstI
and SalI. The 113 bp LsPrrn fragment and the
339 bp LsTpsbA fragment were cloned into pLD6
(DDBJ/GenBank/EMBL Accession BD174931)
via the EcoRINotI sites and PstISalI sites,
respectively. The resulting construct, which contained the aadA cassette, was named pRL6.
The lettuce plastid transformation vector
pRL1000 (Figure 2a), which carries the rbcL and
accD genes as targeting sequences and the aadA
cassette as a selection marker, was constructed as
follows. After pRL6 was digested with NotI and
SalI, the aadA cassette (LsPrrn-aadA-LsTpsbA)
was recovered and then introduced between the
NotI and SalI sites of pRL200. A second lettuce
plastid transformation vector, pRL1001 (Figure 2b), was constructed with the same targeting
sequences as pRL1000, plus aadA and GFP
cassettes under the control of tobacco regulatory
sequences. To make this construct, pLD601
(DDBJ/GenBank/EMBL Accession AB199889)
was digested with NotI and SalI, and the aadA
and GFP cassettes (Prrn-aadA-TpsbA/PpsbA-gfpTrps16) were recovered and introduced into the
NotI and the SalI sites of pRL200.
Plastid transformation of lettuce
Lettuce (Lactuca sativa L.) was grown aseptically
on MS medium containing 3% (wt/vol) sucrose
under long-day conditions (16 h/8 h light/dark) at
25C. For biolistic bombardment, young leaves
were harvested from 3- to 4-week-old plants. Six or
seven pieces of leaf were placed adaxial side up on
lettuce regeneration medium. After 1 day of incubation, the leaves were bombarded using 0.6 lm
gold particles coated with DNA using a PDS1000/He Biolistic Particle Delivery System (BIORAD, Hercules, USA). Five bombardments were

208
carried out for each of the plasmid constructs.
Two days after bombardment, the leaves were cut
into pieces (4 mm  4 mm) and placed adaxial side
down on lettuce regeneration medium containing
50 mg/l spectinomycin dihydrochloride and
500 mg/l polyvinylpyrrolidone (PVP). Regenerated shoots were transferred into boxes containing
phytohormone-free MS medium for rooting.
In order to obtain seeds, transplastomic plants
were transferred to soil in pots and cultivated
under long-day conditions (16 h/8 h light/dark)
at 25C.

Fluorescence microscopy of GFP protein

PCR analysis

Immunoblot analysis

Total leaf DNA was isolated according to the

method of Liu et al. (1995). PCR was performed
using standard conditions (95C for 30 s, 55C for
30 s, 72C for 90 s; 35 cycles) with ExTaq DNA
polymerase (Takara, Ohstu, Japan) and primer
pairs corresponding to the sequences anking the
transgene integration site in the lettuce chloroplast
genome (5-AGGATTGAGCCGAATCCAAC-3
and 5-AGGATTTGTTCTCTCCTACG-3). The
PCR products were separated by electrophoresis in
a 0.8% agarose gel.

Total cellular protein was extracted from frozen

leaf samples in ice-cold extraction buer containing 50 mM HEPES-KOH (pH7.6), 1 mM EDTA,
1 mM PMSF, 1 mM DTT, and 2% (wt/vol) PVP.
After centrifugation (21,000  g) at 4C for
30 min, the supernatant was recovered as the
soluble fraction. The soluble fraction was separated by polyacrylamide gel electrophoresis and
blotted onto polyvinylidine uoride membrane.
The membrane was then incubated with a GFP
polyclonal antibody (Clontech, Palo Alto, USA)
and the bound antibody detected by ECL chemiluminescence (Amersham, Uppsala, Sweden).

Southern blot and RFLP analysis

Total cellular DNA was isolated with a plant
DNeasy kit (QIAGEN, Hilden, Germany). DNA
samples were digested with SphI, separated by electrophoresis in a 0.8% agarose gel, and transferred
onto nylon membranes (Amersham, Uppsala,
Sweden). The aadA and rbcL probes were obtained
by PCR using chloroplast genome DNA as template. The PCR primers for amplication of the
aadA and rbcL probes were (5-ATGGCTCGTGAAGCGGTTAT-3 and 5-TTATTTGCCAACTACCTTAG-3) and (5-CAGTTCGGTGG
AGGAACTTT-3 and 5-TCCAACCAACACAAAAATAGAAA-3), respectively. The blot was
hybridized with uorescent probe generated
by ECF Random-Prime Labelling (Amersham,
Uppsala, Sweden) using ultrasensitive hybridization buer (Ambion, Austin, USA). The uorescent signal was amplied with an ECF signal
amplication module according to the manufacturers protocol (Amersham, Uppsala, Sweden).
The uorescence signal was detected with
FLA3000GF (Fujilm, Tokyo, Japan).

Thin-layered leaf samples were peeled from

transplastomic leaves using forceps. GFP uorescence was detected using U-MNIBA lter sets (BP
470490, DM 505, BA 510IF) and a uorescence
microscope, BX50 (Olympus, Tokyo JAPAN).
Chlorophyll uorescence was detected using
U-MWIG lter sets (BP 520550, DM 570, BA
590). The GFP and chlorophyll uorescent images
were merged using Photoshop Elements (Adobe,
San Jose, USA).

Results
Optimization of regeneration and selection system
of lettuce transplastomic plants
To establish an ecient leaf-based regeneration
and selection system, we rst searched for an
appropriate lettuce cultivar and medium
composition for regeneration. Leaf explants
(4 mm  4 mm) derived from 5 lettuce cultivars
were placed on several kinds of medium and
examined for regeneration stability and eciency
(Table 1). The results showed that cv. Cisco,
Olympia and Nansoubeni had higher regeneration
eciency (more than 2.1 shoots per a leaf explant)
than other lettuce cultivars when leaf explants
were regenerated on MS medium including
0.1 mg/l NAA and 0.1 mg/l BA. In this medium
condition, cv. Cisco and Olympia showed more
stable regeneration than Nansoubeni. Regeneration stability is expected to contribute the

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Table 1. Selection of lettuce cultivar
Cultivar

Mediuma

Shoot
regeneration
stability (%)b

Shoot
regeneration
eciencyc

Cisco

I
II
III
IV
V
I
II
III
IV
V
I
II
III
IV
V
I
II
III
IV
V
I
II
III
IV
V

100
68
60
60
53
100
28
33
25
3
73
25
30
0
13
93
30
18
5
5
68
3
0
0
0

2.3
1.4
1.4
1.0
0.8
2.1
0.6
0.7
0.4
0.1
0.8
0.4
0.4
nad
0.2
2.5
0.6
0.3
0.1
0.1
1.3
0.1
na
na
na

Olympia

Red re

Nansoubeni

Okayama
saladana

I:BA 0.1 mg/l, NAA 0.1 mg/l, II:BA 0.5 mg/l, NAA 0.5 mg/l,
III: BA 0.5 mg/l, NAA 1.0 mg/l, IV:BA 1.0 mg/l, NAA 0.5 mg/l,
V:BA 1.0 mg/l, NAA 1.0 mg/l.
b
Number of explant which showed regenerated shoot/total
explants x 100 (%).
c
Number of regenerated shoot/total explants.
d
No appearance.

stabilization of plastid transformation eciency.

Therefore, we adopted the cv. Cisco, which
showed higher regeneration eciency than Olympia as a host plant and the medium condition for
plastid transformation.
For selection of transplastomic lettuce, we used
the spectinomycin-resistance gene aadA as a
selectable marker because it has been successfully
used for the plastid transformation of various
plant species (Svab & Maliga, 1993; Sikdar et al.,
1998; Ruf et al., 2001; Skarjinskaia et al., 2003).
When untransformed lettuce leaf explants were
transferred onto lettuce regeneration medium containing 50 mg/l spectinomycin, the explants were
completely chlorotic within 3 weeks and no shoots

appeared. Therefore, 50 mg/l spectinomycin was

used for selection of lettuce transplastomes.
During the selection of transformants, we
observed browning of the selection medium due
to oxidation of polyphenol compounds (Fujita
et al., 1990). Because this discoloration caused
the leaf explants to wither, we added 500 mg/l PVP
to the selection medium to prevent browning.
Furthermore, only a limited number of leaf
explants were placed on the medium (less than 25
leaf explants per plate (/ 90 mm)).
Construction of plastid transformation vector
The plastid transformation vector consists of an
integration site and a selection cassette. In order to
determine an appropriate integration site, we rst
determined the complete plastid genome sequence
of Lactuca sativa L. cv. Cisco by a whole-genome
shotgun strategy and claried its gene organization. The plastid genome consisted of a circular
double-stranded DNA of 152,765 bp. The BamHI
and PstI-digestion patterns deduced from the
genome sequence were consistent with those
obtained empirically by pulsed eld gel electrophoresis analysis (data not shown). The potential
protein- and rRNA-encoding genes were assigned
by a combination of computer prediction and
similarity searches (Figure 1). The rbcL and accD
genes, whose intergenic region was shown to be a
suitable target site for the insertion of transgenes in
tobacco (Svab & Maliga, 1993; Daniell et al., 1998;
Kota et al., 1999; Tomizawa & Yokota, 2004
submitted), were adjacent on the lettuce plastid
genome (see Figure 1). Therefore, for constructing
the plastid transformation vectors, we chose the
rbcLaccD intergenic region of the lettuce plastid
as the target site for homologous recombination.
The plastid transformation vector carries an aadA
cassette that supplies spectinomycin resistance.
Because Sriraman et al. (1998) reported that
transcription of the rrn operon depends on
species-specic factors that facilitate transcription
initiation by the general transcription machinery,
we constructed a selection cassette in which aadA is
under the control of lettuce plastid regulatory
elements, specically the 16S ribosomal RNA
operon promoter (LsPrrn) fused to the 5UTR of
the rbcL gene and the psbA gene terminator
(LsTpsbA). The resulting lettuce plastid transformation vector pRL1000 is shown in Figure 2a.

210

Figure 1. Gene organization of the Lactuca sativa L. cv. Cisco plastid genome. The circular genome of lettuce plastid was opened
at the junction between IRA and LSC and is represented by a linear map starting from the junction point. The potential protein
coding regions are shown as boxes. Genes for which a putative function could be deduced by similarity search are indicated by the
gene name. rRNA and tRNA genes are also shown on the map. Genes drawn on the upper side are transcribed from left to right,
and on the lower side, from right to left. Asterisks indicate genes containing introns.

211

Figure 2. Plastid transformation vectors for lettuce. (a) Diagram of the transformation vector for expressing the aadA
gene under the control of a promoter from lettuce (pRL1000).
(b) Diagram of the transformation vector for expressing the
aadA and GFP genes under the control of promoters from
tobacco (pRL1001).

Generation and analysis of transplastomic lettuce

plants
For plastid transformation, sterile lettuce leaves
were bombarded with plasmid pRL1000 using
a 900 psi rupture disk. Primary spectinomycinresistant green calli began to appear after
1 month of incubation of bombarded leaf explants
on regeneration medium including spectinomycin

(Figure 3a). Seventeen green calli were nally

obtained after 2 months of selection. Following
a few weeks incubation of the calli on the
regeneration medium, 5 calli grew into shoots
(Figure 3b). Presence of the transgene in all 5
shoots was conrmed by PCR using primers
specic for the rbcL and the accD genes, producing
a 1.6 kb PCR product (Figure 4d). In contrast, the
remaining calli did not generate shoots and were
bleached. Before bleaching of these calli, PCR
analysis were carried out about the integration of
transgene for three calli. The results indicated that
three calli also had transgene in plastid genome
(data not shown). The reason why three calli nally
bleached is uncertain. The ve shoots were transferred to MS medium supplemented with
0.1 mg/l NAA and 50 mg/l spectinomycin for
rooting (Figure 3c). Transgene integration and
homoplasmy were ultimately conrmed by Southern blot analysis. The plastid gene organization of
transplastomic lettuce produced using pRL1000 is
shown in Figure 4a and b. Total cellular DNA
extracted from two spectinomycin-resistant lettuce
plants was digested with SphI, electrophoresed,
and analyzed by Southern blot. When the rbcL
gene was used as a probe, a 3.5 kb fragment was
detected in the spectinomycin-resistant lines and a

Figure 3. Transplastomic plants transformed with pRL1000. (a) Spectinomycin-resistant green callus indicated by arrow on selection plate. (b) Spectinomycin-resistant green shoot on selection plate. (c) Transplastomic lettuce plant with root on MS medium
containing spectinomycin.

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Figure 4. Integration of the aadA gene into the lettuce plastid genome after transformation with pRL1000. (a) Physical maps of
the pRL1000 transformation vector with the selection marker aadA and recombination sites for targeting to the plastid genome of
wild-type lettuce. Bold lines show homologous recombination targeting sites. (b) Physical map of the plastid genome of transplastomic lettuce. The aadA cassette (Prrn-aadA-TpsbA) was integrated into the plastid genome of the transformant. The location of
the rbcL and aadA probes are indicated by dotted lines below the map. (c) Southern blot analyses of wild-type and transplastomic
plants. The rbcL probe hybridized to a 2.3 kb SphI fragment in the wild-type plant and to a larger, 3.5 kb fragment in the transplastomic plants. The aadA probe hybridized to a 3.5 kb SphI fragment only in the transplastomic plants. (d) PCR analysis with a
pair of primers anking the transgene insertion site. From the wild-type plastid genome, a 0.3 kb product is amplied, whereas
from the transplastomic genome a 1.6 kb product is amplied.

2.3 kb fragment was detected in the non-transformed line (Figure 4c). When the aadA gene was
used as a probe, the 3.5 kb fragment was detected
in the spectinomycin-resistant lines but no signal
was observed in the non-transformed line (Figure 4c). These results veried that the transgenes
were inserted into the intergenic region between the
rbcL and the accD genes. Based on the Southern
blot analysis, it appeared that both transplastomic
lines had reached almost complete homoplasmy. A
very faint band was detected by PCR analysis, and
it was probably derived from residual wild-type
plastid genome or ptDNA fragments integrated in

the nucleus or mitochondria genome (Nakazano &

Hirai, 1993; Rice Chromosome 10 Sequencing
Consortium, 2003) (Figure 4d). The resulting
transplastomic plants were then planted in soil
and grown to maturity. All transplastomic plants
were fertile and produced seeds. In order to check
the stability of the aadA transgene, the T1 progeny
was tested for its ability to grow on spectinomycin.
As expected for a plastid transgene, the T1 progeny
was uniformly spectinomycin-resistant (Figure 5a
and b). Seed transmission of the transplastome to
the T1 generation was also conrmed by PCR
amplication of the transgene integrated in the

213

Figure 5. Stable heredity of the transgene in the T1 generation of transplastomic lettuce transformed with pRL1000. T1
transplastomic (a) and wild-type (b) lettuce plants were grown on MS medium containing 50 mg/l spectinomycin. Wild-type plants
were spectinomycin sensitive and all seedlings were chlorotic at 2 weeks after germination (b). (c) PCR analysis of T0 and T1 generations of transplastomic lettuce with a pair of primers anking the transgene insertion site. A 0.3 kb product is amplied from
the wild-type plastid genome, whereas a 1.6 kb product is amplied from the transplastomic genome. (d) Transplastomic lettuce of
the T1 generation grew normally on soil.

plastid genome (Figure 5c). The transplastomic T1

plants grew normally on soil (Figure 5d). These
results indicated that the transplastomic lettuce
was stable and reproducible. We detected a 0.3 kb
fragment derived from the non-transformed plastid
genome in T0 plants (Figure 5c), indicating that
lettuce plastid transformants were not completely
homoplasmic in the rst selected generation. Based
on PCR analysis, the level of heteroplasmy in the
T1 plants was almost the same as that of the T0
plants (Figure 5c). Therefore, the level of heteroplasmy may not be related to the inheritance of the
transgene by the T1 generation.
Accumulation of foreign protein in lettuce
chloroplast
To examine the level of foreign protein
accumulation in lettuce chloroplasts, we constructed the GFP expression transformation vector pRL1001. The pRL1001 vector consisted of
the lettuce plastid rbcLaccD intergenic region as a

targeting site, an aadA cassette (tobacco rrn

promoteraadA-tobacco psbA terminator) and a
GFP cassette (tobacco psbA promotergfp-tobacco rps16 terminator) (Figure 2b). Lettuce was
transformed with pRL1001 by the same methods
described for pRL1000. One shoot was obtained
from 14 green calli and the regenerated shoot gave
rise to a transplastomic line (data not shown).
Fluorescence microscopy revealed green uorescence in chloroplasts of transplastomic lettuce
upon blue light excitation (Figure 6a). Chloroplast
chlorophyll uorescence is shown in Figure 6b,
and the green and the chlorophyll uorescence
images are shown merged in Figure 6c. Total
soluble protein extracted from a transplastomic
plant was separated by SDS-PAGE. An extra
band was detected at a position corresponding to a
26 kDa protein in the transplastomic plant by
CBB staining (Figure 6d). In contrast, AadA
protein (29 kDa) accumulation was not detected.
The accumulation of GFP protein was conrmed
by immunoblot analysis using a GFP-specic

214

Figure 6. GFP protein expression in transplastomic lettuce leaf. (a) GFP uorescence in stomatal guard cells of leaf epidermis. (b)
Chlorophyll uorescence of chloroplasts in the same area as (a). (c) Panels (a) and (b) were merged. (d) Protein samples were extracted from equal amounts of leaf tissue. The extracted soluble proteins were electrophoresed and stained with CBB. Only in the
transplastomic lettuce, a single 26 kD band was detected (indicated by arrow). (e) Immunoblot analysis of GFP accumulation in
leaves of the transformant. Hundred nanogram of soluble protein from transplastomic and wild-type lettuce leaves were separated
by SDS-PAGE and probed with GFP antibody at 1:8000 dilution in the right-hand lanes. Puried GFP standard (1, 10, 50, 100,
200 ng/lane) was included for quantication. Lettuce leaf protein samples were prepared from lettuce plants grown on soil in a
chamber. Scale bars represent 10 lm.

antibody (Figure 6e). Based on the intensity of the

signals, the accumulation of GFP protein in leaves
of transplastomic lettuce reached 36% of the total
soluble leaf protein.

Discussion
Transplastomic technologies oer a tremendous
potential for conferring useful traits on plants,
including the production of foreign proteins. In
particular, successful development of plastid transformation of edible leafy crops is expected to boost
plant production of edible vaccines, antibodies,
and therapeutic substances. This report is the rst
description of a method allowing the ecient
generation of stable and fertile transplastomic
edible leafy crops.
In this work, we focused on two aspects of
plastid transformation in our development of a

protocol for lettuce. First, we examined the

regeneration eciency of several lettuce cultivars,
and chose Lactuca sativa L. cv. Cisco as the best
experimental material. The eciency of regeneration was dependent on both the cultivar and the
composition of the medium (Table 1), highlighting
the importance of both these factors for attaining
ecient plastid transformation. Second, we determined the whole genome sequence of the lettuce
plastid, because this information was necessary for
construction of the plastid transformation vector.
In plastid transformation, the transgene is inserted
into the plastid genome by homologous recombination with a specic target site. Unfortunately,
the sequence and gene organization of the plastid
genome is not necessarily conserved in all plant
species. For example, the commonly used integration site rbcLaccD is not conserved in legume
species (Kato et al., 2000). Therefore, information
about the organization of lettuce plastid genes was

215
necessary for construction of the lettuce plastid
transformation vector. We found that many target
sites which had been used previously for plastid
transformation of other higher plants, including
the trnHpsbA, trnGtrnfM, ycf3trnS, rbcL
accD, petApsbJ, 5rps12clpP, petDrpoA,
ndhBrps7, 3rps12trnV, trnVrrn16, rrn16trnI,
trnItrnA, trnRtrnN and rpl32trnL intergenic
regions (Maliga, 2004), were conserved in the
lettuce plastid genome (Figure 1). Therefore, we
were able to use the rbcLaccD insertion site with
complete condence. In addition, the complete
genome sequence of the lettuce plastid also
allowed us to use PCR to obtain DNA fragments
corresponding to the lettuce plastid regulatory
elements. As a result, we could quickly construct a
lettuce-specic plastid transformation vector.
In the plastid transformation of tobacco
(Nicotiana tabacum), 115 transgenic lines were
obtained per bombardment (Svab & Maliga, 1993;
Langbecker et al., 2004). On the other hand,
transformation of Arabidopsis, potato (Solanum
tuberosum), L. fendleri, and tomato (Lycopersicon
esculentum) was achieved by bombardment of
green leaf tissues (Sikdar et al., 1998; Sidorov
et al., 1999; Ruf et al., 2001; Skarjinskaia et al.,
2003), but the frequency of plastid transformation
was much lower than tobacco. For example, one
transgenic line was obtained per 40 or 151 bombardments in Arabidopsis, 35 bombardments in
potato, 25 bombardments in oilseed rape, and 20
bombardments in tomato. Among higher plants,
plastid transformation is routinely available only
in tobacco because of its high transformation
eciency. Therefore, we sought to develop a
reliable and ecient transformation method for
lettuce plastids.
Plastid
transformation
vectors
utilize
homologous anking regions for recombination
and insertion of foreign genes into the plastid
genome. In the case of potato, tomato, and L.
fendleri, the vectors employed for plastid transformation were constructed using anking sequences
derived from tobacco (Shinozaki et al., 1986) or
Arabidopsis (Sato et al., 1999). In tobacco, plastid
transformation eciency decreased drastically
when petunia anking sequences were used (DeGray et al., 2001). Taken together, these ndings
suggest that lack of complete homology of targeting sequences results in reduction of transformation eciency (Kavanagh et al., 1999). In contrast,

ecient transformation of lettuce (ve and one

transgenic lettuce lines per ve bombardments)
was achieved by using lettuce DNA fragments as
anking sequences to construct the plastid transformation vectors pRL1000 and pRL1001.
In this study, we constructed two transformation vectors, pRL1000 and pRL1001, whose aadA
cassettes were controlled by lettuce and tobacco
plastid regulatory elements, respectively. The
tobacco element controlled aadA cassette (tobacco
rrn promoteraadA-tobacco psbA terminator)
acted as the selection marker in the lettuce plastid
transformation by pRL1001. An aadA cassette
controlled by tobacco plastid regulatory elements
was also used in the transformation of Arabidopsis,
tomato, and L. fendleri (Sikdar et al., 1998; Ruf
et al., 2001; Skarjinskaia et al., 2003; Dufourmantel et al., 2004). Based on our data and other
examples of plastid transformation, it seems likely
that a species-specic promoter is not always
necessary for the construction of the selection
marker cassette in species-specic plastid transformation vectors.
To our knowledge, this report is the rst
description of a complete plastid genome sequence in the Asteraceae. The chloroplast genome
sequence of lettuce will be useful for designing plastid transformation vectors for other Asteraceae
species, just as tobacco and Arabidopsis plastid
genome sequences were available for plastid transformation in tomato, potato, and L. fendleri.
Other Asteraceae species of interest for plastid transformation are, for example, the oil- and
fat-producing species sunower and saower.
One of the attractive advantages of plastid
transformation is the high level of transgene
expression and foreign protein accumulation.
Transplastomic lettuce expressing GFP revealed
that foreign protein could account for up to 36%
of the total soluble protein without changing the
amount of the major chloroplast protein Rubisco
(Figure 6d). The amount of foreign protein in the
transplastomic lettuce was estimated to be 1.9 mg
protein per gram of leaf, suggesting that this
system is a viable alternative for producing edible
vaccines, antibodies, and therapeutic substances
for human consumption. Furthermore, because
plants are able to harvest solar energy through
photosynthesis, production of foreign protein in
chloroplasts consumes less fossil energy than doing
so in bacterial and animal cells. In future, the

216
development of a production method for edible
vaccine in lettuce will require nding a selectable
marker safe for human consumption or a way to
eliminate the bacterial aadA gene used here.
Combination of plastid transformation with technologies allowing elimination of the marker gene
or antibiotic-free selection (Daniell et al., 2001;
Hajdukiewicz et al., 2001; Klaus et al., 2004)
would enhance the progress of plant molecular
breeding. In tobacco, Horn et al. (2003) and
Tregoning et al. (2004) have reported a few
examples of the production of pharmaceuticals
through plastid transformation. Application of
plastid transformation technology to lettuce is
expected to open up new possibilities for metabolic
engineering and for the use of edible leafy crops as
factories for biopharmaceuticals.
Acknowledgements
This work was funded by a grant from Keihanna/
Ministry of Education, Culture, Sports, Science
and Technology.

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