lechuga Transgenica

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Efficient 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 Tomizawa11Resarch Institute of Innovative Technology for the Earth, 9-2 Kizugawadai, Kizu-cho Soraku-gun, Kyoto, 619-

0292, Japan2Kitasato Institute for Life Sciences, Kitasato University, 1-15-1 Kitasato, Sagamihara-shi, Kanagawa, 228-8555,

Japan3Nara Prefectural Agricultural Experiment Station, 88 Shijyo-cho, Kashihara, Nara, 634-0813, Japan

4Graduate 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 offer 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 sativaL. cv. Cisco. The transforming DNA carries aspectinomycin-resistance gene (aadA) under the control of lettuce chloroplast regulatory expression ele-

ments, flanked 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 efficient production of edible vac-

cines, pharmaceuticals, and antibodies in plants.

Introduction

Transformation of the plastid genome has severaladvantages over conventional nuclear transforma-

tion (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

total soluble cellular proteins (DeCosa et al.,

2001). Because of the potentially high transgene

expression levels in chloroplasts, this system isexpected 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*Author for correspondence

E-mail: [email protected]

Transgenic Research (2006) 15:205217 Springer 2006

DOI 10.1007/s11248-005-3997-2


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rape (Hou et al., 2003), Lesquerella fendleri

(Skarjinskaia et al., 2003), and carrot (Kumar

et al., 2004). However, some problems have beenpointed out with each of these plants. In Arabid-

opsis, 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. Incarrot chromoplasts, betaine aldehyde dehydroge-

nase activity expressed from a plastid transgene

accumulated to only 74.8% the level observed in

leaf chloroplasts (Kumar et al., 2004). These find-

ings suggest that chloroplast-containing tissues

would be the most effective 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 (Kapustaet 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 car-

ried the transgene that was integrated in the plastid

genome is rather low (Huang et al., 2003;

Stegemann et al., 2003; Huang et al., 2004). Fur-

thermore, 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 efficiency.First, we screened several lettuce cultivars for the

highest regeneration efficiency (Cisco), and then

determined the best regeneration/selection condi-

tions for plastid transformation of this cultivar.

Plastid transformation vectors basically consist of

integration sites for double homologous recombi-

nation and a selection cassette. To achieve plastid

transformation, we had to determine an appropri-

ate target site for insertion of transgenes into the

plastid genome, before constructing the transfor-

mation vector. It might be thought that the gene

organization of plastid genomes in other plantscould be used as a reference for choosing a target

site for the lettuce transformation vector. How-

ever, comparison of plastid genome sequences of

higher plants has revealed diversity in gene orga-

nization (Hiratsuka et al., 1989; Shimada & Sugi-

ura, 1991; Doyle et al., 1992). Therefore, it was

necessary to obtain specific 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

efficiency plastid transformation, the nucleotide

sequence of the integration sites in the transfor-

mation vector should be identical to that of thetarget sites in the plastid genome. For these

reasons, we sequenced the entire plastid genome

of lettuce for this study, the first 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) derivedfrom each cultivar were placed on lettuce regen-

eration medium [MS medium supplemented with

3% (wt/vol) sucrose, 0.1 mg/l 6-benzylaminopu-

rine (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 cul-

tivar that showed the highest regeneration effi-

ciency, 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-month-

old seedlings by discontinuous Percoll density

gradient centrifugation (Miyake & Asada, 1992)

using Percoll concentrations of 40% and 80%.

The chloroplast genome DNA was purified 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 kilobase-

insert genomic library was constructed, and 3000

sequences (giving 10-fold coverage) were ob-

tained from both ends of the genomic clones.Sequences were assembled using the PHRED/

PHRAP/CONSED package. Remaining gaps

were closed by transcriptional sequencing (Nip-

pon Genetech, Tokyo, Japan) or by primer

walking.

Construction of plastid transformation vector

A lettuce plastid transformation vector, pRL200,

was constructed using the 1.6 kbrbcLgene and the

1.1 kbaccDgene from the Lactuca sativaL. plastid

genome (DDBJ/GenBank/EMBL Accession

AP007232) as homologous recombination target-ing sequences. The DNA fragment corresponding

to the rbcL gene was PCR-amplified from total

genomic DNA ofLactuca sativaL. by using KOD

plus DNA polymerase (TOYOBO, Tokyo, Japan)

and specific 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-amplified by using a different set

of specific primers (5-CCGTCGACGATCCTTAGGATTGGGATAT-3 and 5-GGAAGCTTCCC

ATATGAGTAGAACTTTC-3 ; SalI and HindIII

recognition sites were underlined) and then di-

gested 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-amplified from total genomic

DNA of Lactuca sativa L. by using KOD plusDNA polymerase (TOYOBO) and specific primers

(5-CCGCGGCCGCGATATTTTGATTTGCTA-

CCC-3 and 5-CCAGCGCTATTCGCCCGGA-

GTTCGCTCC-3). The resulting fragment was

digested with EcoRI and NotI. A fragment

containing the lettuce plastid psbA terminator

(LsTpsbA) was also PCR-amplified with specific

primers (5-GGCTGCAGGACTTTGGTCTTA-

TTGTAAT-3 and 5-CCGTCGACGAGCATA-

TTATTTCTTTCTT-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 con-

tained 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 (Fig-ure 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-gfp-

Trps16) 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 incu-

bation, the leaves were bombarded using 0.6lm

gold particles coated with DNA using a PDS-

1000/He Biolistic Particle Delivery System (BIO-

RAD, Hercules, USA). Five bombardments were

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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 sidedown on lettuce regeneration medium containing

50 mg/l spectinomycin dihydrochloride and

500 mg/l polyvinylpyrrolidone (PVP). Regener-

ated 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.

PCR 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 flanking 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.

Southern blot and RFLP analysis

Total cellular DNA was isolated with a plant

DNeasy kit (QIAGEN, Hilden, Germany). DNA

samples were digested withSphI, separated by elec-

trophoresis in a 0.8% agarose gel, and transferred

onto nylon membranes (Amersham, Uppsala,

Sweden). TheaadAandrbcLprobes were obtained

by PCR using chloroplast genome DNA as tem-

plate. The PCR primers for amplification of the

aadA and rbcL probes were (5-ATGGCTC-

GTGAAGCGGTTAT-3 and 5-TTATTTGCC-

AACTACCTTAG-3) and (5

-CAGTTCGGTGGAGGAACTTT-3 and 5-TCCAACCAACACA-

AAAATAGAAA-3), respectively. The blot was

hybridized with fluorescent probe generated

by ECF Random-Prime Labelling (Amersham,

Uppsala, Sweden) using ultrasensitive hybridiza-

tion buffer (Ambion, Austin, USA). The fluores-

cent signal was amplified with an ECF signal

amplification module according to the manufac-

turers protocol (Amersham, Uppsala, Sweden).

The fluorescence signal was detected with

FLA3000GF (Fujifilm, Tokyo, Japan).

Fluorescence microscopy of GFP protein

Thin-layered leaf samples were peeled fromtransplastomic leaves using forceps. GFP fluores-

cence was detected using U-MNIBA filter sets (BP

470490, DM 505, BA 510IF) and a fluorescence

microscope, BX50 (Olympus, Tokyo JAPAN).

Chlorophyll fluorescence was detected using

U-MWIG filter sets (BP 520550, DM 570, BA

590). The GFP and chlorophyll fluorescent images

were merged using Photoshop Elements (Adobe,

San Jose, USA).

Immunoblot analysis

Total cellular protein was extracted from frozen

leaf samples in ice-cold extraction buffer contain-

ing 50 mM HEPES-KOH (pH7.6), 1 mM EDTA,

1 mM PMSF, 1 mM DTT, and 2% (wt/vol) PVP.

After centrifugation (21,000g) at 4C for

30 min, the supernatant was recovered as the

soluble fraction. The soluble fraction was sepa-

rated by polyacrylamide gel electrophoresis and

blotted onto polyvinylidine fluoride membrane.

The membrane was then incubated with a GFP

polyclonal antibody (Clontech, Palo Alto, USA)

and the bound antibody detected by ECL chemi-

luminescence (Amersham, Uppsala, Sweden).

Results

Optimization of regeneration and selection system

of lettuce transplastomic plants

To establish an efficient leaf-based regeneration

and selection system, we first searched for an

appropriate lettuce cultivar and medium

composition for regeneration. Leaf explants

(4 mm

4 mm) derived from 5 lettuce cultivarswere placed on several kinds of medium and

examined for regeneration stability and efficiency

(Table 1). The results showed that cv. Cisco,

Olympia and Nansoubeni had higher regeneration

efficiency (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. Regenera-

tion stability is expected to contribute the

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stabilization of plastid transformation efficiency.

Therefore, we adopted the cv. Cisco, which

showed higher regeneration efficiency than Olym-

pia as a host plant and the medium condition forplastid 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 con-

taining 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, weobserved 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 anintegration site and a selection cassette. In order to

determine an appropriate integration site, we first

determined the complete plastid genome sequence

ofLactuca sativa L. cv. Cisco by a whole-genome

shotgun strategy and clarified its gene organiza-

tion. 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 field gel electro-

phoresis analysis (data not shown). The potential

protein- and rRNA-encoding genes were assigned

by a combination of computer prediction andsimilarity 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-specific factors that facilitate transcription

initiation by the general transcription machinery,

we constructed a selection cassette in which aadAis

under the control of lettuce plastid regulatory

elements, specifically 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 transfor-

mation vector pRL1000 is shown in Figure 2a.

Table 1. Selection of lettuce cultivar

Cultivar Medium

a

Shootregeneration

stability (%)b

Shootregeneration

efficiencyc

Cisco I 100 2.3

II 68 1.4

III 60 1.4

IV 60 1.0

V 53 0.8

Olympia I 100 2.1

II 28 0.6

III 33 0.7

IV 25 0.4

V 3 0.1

Red fire I 73 0.8

II 25 0.4

III 30 0.4

IV 0 nad

V 13 0.2

Nansoubeni I 93 2.5

II 30 0.6

III 18 0.3

IV 5 0.1

V 5 0.1

Okayama

saladana

I 68 1.3

II 3 0.1

III 0 na

IV 0 na

V 0 na

aI: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.bNumber of explant which showed regenerated shoot/total

explants x 100 (%).cNumber of regenerated shoot/total explants.dNo appearance.

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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.

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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 spectinomycin-

resistant 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 finally

obtained after 2 months of selection. Following

a few weeks incubation of the calli on theregeneration medium, 5 calli grew into shoots

(Figure 3b). Presence of the transgene in all 5

shoots was confirmed by PCR using primers

specific for therbcLand theaccDgenes, 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 finally

bleached is uncertain. The five shoots were trans-ferred 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 confirmed by South-

ern 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 2. Plastid transformation vectors for lettuce. (a) Dia-

gram 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 theaadA and GFP genes under the control of promoters from

tobacco (pRL1001).

Figure 3. Transplastomic plants transformed with pRL1000. (a) Spectinomycin-resistant green callus indicated by arrow on selec-

tion plate. (b) Spectinomycin-resistant green shoot on selection plate. (c) Transplastomic lettuce plant with root on MS medium

containing spectinomycin.

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2.3 kb fragment was detected in the non-trans-

formed line (Figure 4c). When the aadA gene wasused 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 (Fig-

ure 4c). These results verified 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 SequencingConsortium, 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 aadAtransgene, 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 confirmed by PCR

amplification of the transgene integrated in the

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 transplas-

tomic 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 trans-

plastomic 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 flanking the transgene insertion site. From the wild-type plastid genome, a 0.3 kb product is amplified, whereas

from the transplastomic genome a 1.6 kb product is amplified.

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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 first 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 hetero-plasmy 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 con-

structed the GFP expression transformation vec-

tor pRL1001. The pRL1001 vector consisted of

the lettuce plastidrbcLaccDintergenic region as a

targeting site, an aadA cassette (tobacco rrn

promoteraadA-tobacco psbA terminator) and a

GFP cassette (tobacco psbA promotergfp-tobac-

co 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 fluores-

cence in chloroplasts of transplastomic lettuce

upon blue light excitation (Figure 6a). Chloroplastchlorophyll fluorescence is shown in Figure 6b,

and the green and the chlorophyll fluorescence

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 confirmed

by immunoblot analysis using a GFP-specific

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 gen-

erations of transplastomic lettuce with a pair of primers flanking the transgene insertion site. A 0.3 kb product is amplified from

the wild-type plastid genome, whereas a 1.6 kb product is amplified from the transplastomic genome. (d) Transplastomic lettuce of

the T1 generation grew normally on soil.

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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 offer a tremendouspotential for conferring useful traits on plants,

including the production of foreign proteins. In

particular, successful development of plastid trans-

formation of edible leafy crops is expected to boost

plant production of edible vaccines, antibodies,

and therapeutic substances. This report is the first

description of a method allowing the efficient

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 efficiency of several lettuce cultivars,

and chose Lactuca sativa L. cv. Cisco as the best

experimental material. The efficiency of regenera-

tion was dependent on both the cultivar and the

composition of the medium (Table 1), highlighting

the importance of both these factors for attaining

efficient plastid transformation. Second, we deter-

mined the whole genome sequence of the lettuceplastid, 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 recombi-

nation with a specific 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 integra-

tion site rbcLaccD is not conserved in legume

species (Kato et al., 2000). Therefore, information

about the organization of lettuce plastid genes was

Figure 6. GFP protein expression in transplastomic lettuce leaf. (a) GFP fluorescence in stomatal guard cells of leaf epidermis. (b)

Chlorophyll fluorescence of chloroplasts in the same area as (a). (c) Panels (a) and (b) were merged. (d) Protein samples were ex-

tracted 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. Purified GFP standard (1, 10, 50, 100,200 ng/lane) was included for quantification. Lettuce leaf protein samples were prepared from lettuce plants grown on soil in a

chamber. Scale bars represent 10 lm.

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necessary for construction of the lettuce plastid

transformation vector. We found that many target

sites which had been used previously for plastidtransformation 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 confidence. 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 regulatoryelements. As a result, we could quickly construct a

lettuce-specific 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, onetransgenic line was obtained per 40 or 151 bom-

bardments 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

efficiency. Therefore, we sought to develop a

reliable and efficient transformation method for

lettuce plastids.

Plastid transformation vectors utilize

homologous flanking regions for recombination

and insertion of foreign genes into the plastidgenome. In the case of potato, tomato, and L.

fendleri, the vectors employed for plastid transfor-

mation were constructed using flanking sequences

derived from tobacco (Shinozaki et al., 1986) or

Arabidopsis (Sato et al., 1999). In tobacco, plastid

transformation efficiency decreased drastically

when petunia flanking sequences were used (De-

Gray et al., 2001). Taken together, these findings

suggest that lack of complete homology of target-

ing sequences results in reduction of transforma-

tion efficiency (Kavanagh et al., 1999). In contrast,

efficient transformation of lettuce (five and one

transgenic lettuce lines per five bombardments)

was achieved by using lettuce DNA fragments asflanking sequences to construct the plastid trans-

formation vectors pRL1000 and pRL1001.

In this study, we constructed two transforma-

tion vectors, pRL1000 and pRL1001, whose aadA

cassettes were controlled by lettuce and tobacco

plastid regulatory elements, respectively. The

tobacco element controlled aadAcassette (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 ofArabidopsis,tomato, and L. fendleri (Sikdar et al., 1998; Ruf

et al., 2001; Skarjinskaia et al., 2003; Dufourman-

tel et al., 2004). Based on our data and other

examples of plastid transformation, it seems likely

that a species-specific promoter is not always

necessary for the construction of the selection

marker cassette in species-specific plastid transfor-

mation vectors.

To our knowledge, this report is the first

description of a complete plastid genome sequ-

ence in the Asteraceae. The chloroplast genome

sequence of lettuce will be useful for designing plas-

tid transformation vectors for other Asteraceaespecies, just as tobacco and Arabidopsis plastid

genome sequences were available for plastid trans-

formation in tomato, potato, and L. fendleri.

Other Asteraceae species of interest for plas-

tid transformation are, for example, the oil- and

fat-producing species sunflower and safflower.

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 theamount 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

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development of a production method for edible

vaccine in lettuce will require finding a selectable

marker safe for human consumption or a way toeliminate the bacterial aadA gene used here.

Combination of plastid transformation with tech-

nologies 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 metabolicengineering 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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