OBPC SYMPOSIUM: MAIZE 2004 & BEYOND –RECENT ADVANCES IN CHLOROPLAST GENETIC ENGINEERING

VIJAY KOYA AND HENRY DANIELL*

Department of Molecular Biology & Microbiology, Biomolecular Science Building # 20, Room 336,

University of Central Florida, Orlando, FL 32816-2364

(Received 22 February 2005; accepted 14 March 2005; editor T. A. Thrope)

Summary

The chloroplast genetic engineering approach offers a number of unique advantages, including high-level transgene

expression, multi-gene engineering in a single transformation event, transgene containment via maternal inheritance, lack

of gene silencing, position and pleiotropic effects and undesirable foreign DNA. Thus far, more than 40 transgenes

have been stably integrated and expressed via the tobacco chloroplast genome to confer several agronomic traits and

produce vaccine antigens, industrially valuable enzymes, biomaterials, and amino acids. Functionality of chloroplast-

derived vaccine antigens and therapeutic proteins have been demonstrated by in vitro assays and animal studies. Oral

delivery of vaccine antigens has been facilitated by hyperexpression in transgenic chloroplasts (leaves) or non-green

plastids (carrots) and the availability of antibiotic-free selectable markers or the ability to excise selectable marker genes.

Additionally, the presence of chaperones and enzymes within the chloroplast help to assemble complex multi-subunit

proteins and correctly fold proteins containing disulfide bonds, thereby drastically reducing the costs of in vitro processing.

Despite such significant progress in chloroplast transformation, this technology has not been extended to major crops. This

obstacle emphasizes the need for plastid genome sequencing to increase the efficiency of transformation and conduct basic

research in plastid biogenesis and function. However, highly efficient soybean, carrot, and cotton plastid transformation

has been recently accomplished via somatic embryogenesis using species-specific chloroplast vectors. Recent

advancements facilitate our understanding of plastid biochemistry and molecular biology. This review focuses on exciting

recent developments in this field and offers directions for further research and development.

Key words: chloroplast-derived agronomic traits; chloroplast-derived therapeutic proteins; maternal inheritance; non-green

plastids; plastid transformation; transgene containment.

Introduction

The practice of agriculture by man first began around 8000 BC.

Agriculture still remains the primary source of food for the human

population. With the current world population reaching above 6

billion, there is an ever-increasing demand for food production.

Mankind has bred plants for thousands of years with the aim of

improving their quality and quantity. There have been advances in

crop production through classical breeding using the selection

method. Although classical breeding has been fairly effective over

the years and is still being implemented in certain parts of the

world, it has not been able to keep up with the growing need for

agricultural products. Classical breeding requires a lot of time and

many factors must be taken into consideration, including the

random exchange of genes. For this reason, genetic engineering is

now being used to meet the increasing demands for agricultural

products in a shorter period of time.

Nuclear transformation. The genetic engineering of plants

through nuclear transformation has been routinely achieved by

Agrobacterium-mediated transformation, microprojectile bombard-

ment, and direct protoplast transformation. Each of these methods

has unique advantages and disadvantages that have led to the

development of novel alternative systems such as infiltration,

electroporation of cells and tissues, electrophoresis of embryos,

microinjection, pollen-tube pathway, and silicon carbide- and

liposome-mediated transformation (Rakoczy-Trojanowska, 2002).

However, these alternative methods are not widely used due to

lower transformation efficiency. Although genetic engineering of

plants is being achieved through nuclear transformation, this

method has a few disadvantages. To introduce multigenes via

nuclear transformation, the individual genes have to be introduced

first in separate plants to create an independent transgenic line.

These independent transgenic lines harboring individual genes are

brought together into a single host by repetitive breeding. This way

of achieving successful multigene transgenic plants is a very slow

process. The approach of introducing multigenes together in a

single transformation event is not feasible because of the lack of

processing of polycistrons in the nuclear genome. Besides, this

approach of multigene engineering is further complicated by the

gene silencing and position effect. Gene silencing is a phenomenon

observed in transgenic plants that results in reduced or lack of*Author to whom correspondence should be addressed: Email daniell@

mail.ucf.edu

In Vitro Cell. Dev. Biol.—Plant 41:388–404, July–August 2005 DOI: 10.1079/IVP2005660q 2005 Society for In Vitro Biology1054-5476/05 $18.00+0.00

388

transgene because of the use of repetitive regulatory sequences,

integration of multiple copies of the transgene, etc. (Daniell and

Dhingra, 2002). The position effects are due to the random

integration of the transgene that would lead to variable expression

levels (Daniell et al., 2002). Apart from the technical hurdles in

creating a transgenic plant, there are other challenges. There have

been potential environmental concerns about transgene introgres-

sion via pollen to weeds or related crops. Besides, there are

concerns due to the low expression levels that could lead to the

possibility of development of insect resistance from the plant

expressing low levels of insecticidal protein (Bogorad, 2000;

Daniell, 2000). All these concerns underscore the need for

development of alternative approaches.

Chloroplast transformation. The chloroplast transformation

approach offers attractive advantages of introducing multiple

transgenes in a single transformation step because of the

chloroplast’s ability to transcribe the operon into polycistronic

mRNA and translate the polycistronic mRNA with or without

further processing. In addition, the chloroplast genome can

accommodate many transgene copies, as high as 10 000 copies

per cell, because each chloroplast has 10–100 genomes and each

cell has 10–100 chloroplasts. This high polyploidy leads to

exceptionally high transcript levels and accumulation of abundant

translated products, up to 46% of total leaf protein (DeCosa et al.,

2001). Furthermore, the position effect is not observed due to site-

specific integration of transgenes into the spacer region of the

chloroplast genome through homologous recombination.

The phenomenon of gene silencing has not been observed despite

abundant accumulation of foreign transcripts or proteins. It has been

shown that there is no gene silencing in chloroplast transgenic lines at

the transcriptional level in spite of accumulation of transcripts 150–

200-fold higher than nuclear transgenic plants (Lee et al., 2003;

Dhingra et al., 2004). Similarly, there is no transgene silencing at the

translational level in spite of accumulation of foreign protein up to

46.1% of the total plant protein in chloroplast transgenic lines

(DeCosa et al., 2001). Chloroplasts also offer compartmentalization of

toxic foreign proteins, thereby preventing any adverse effects. For

example, the osmoprotectant trehalose proved to be very toxic when it

accumulated in the cytosol but was nontoxic when it was

compartmentalized within transgenic chloroplasts (Lee et al.,

2003). Similarly, the cholera toxin vaccine antigen was highly toxic

in the cytosol but not when expressed within plastids (Daniell et al.,

2001b). When hyperexpressed in the chloroplast, xylanase, an

enzyme important in many industrial applications, did not cause cell

wall degradation as seen in nuclear transformants; therefore, plant

growth was not affected (Leelavathi et al., 2003).

In most angiosperm plant species, plastid genes are inherited

uniparentally in a strictly maternal fashion (Zhang et al., 2003;

Hagemann, 2004). Even though transgenic chloroplasts may be

present in pollen, plastid DNA is eliminated from the male germ

line at different points during sperm cell development, depending

upon the plant species (Hagemann, 2004). This minimizes the

possibility of outcrossing transgenes to related weeds or crops (Scott

and Wilkenson, 1999; Daniell, 2002) and reduces the potential

toxicity of transgenic pollen to nontarget insects (DeCosa et al.,

2001). Thus, maternal inheritance offers containment of chloroplast

transgenes due to lack of gene flow through pollen. This

environmentally friendly feature should minimize concerns of

outcrossing with wild species and related crops.

Multigene engineering via the chloroplast genome is possible in a

single transformation event. For example, the cry operon from

Bacillus thuringiensis, coding for the insecticidal protein, was

introduced and expressed up to 46% of the total leaf protein

(DeCosa et al., 2001). Two bacterial enzymes that confer resistance

to different forms of mercury, mercuric ion reductase (merA) and

organomercurial lyase (merB), were expressed as an operon in

transgenic chloroplasts and shown to confer resistance to very high

levels of mercury and organomercurial compounds (Ruiz et al.,

2003). Furthermore, three bacterial genes coding for the

polyhydroxy-butyrate (PHB) operon, when expressed via the

chloroplast genome, accumulated significant quantities of this

polymer (Nawrath et al., 1994).

Milestones in Chloroplast Engineering

The concept of chloroplast genetic engineering was first

conceived in mid-1980s with the introduction of isolated intact

chloroplasts into protoplasts and regeneration of transgenic plants

with modified organelles. Therefore, the primary focus was on the

development of chloroplast systems capable of efficient, prolonged

protein synthesis (Daniell et al., 1986) and the expression of foreign

genes in isolated chloroplasts (Daniell and McFadden, 1987).

However, invention of the gene gun that could deliver foreign DNA

directly into organelles made it possible to transform plastids

without the need to isolate them. The first successful chloroplast

genome complementation was reported for the unicellular green

alga having a single chloroplast, Chlamydomonas reinhardtii

(Boynton et al., 1988). For this, the photosynthetically incompetent

mutants that lack the atpB gene were used. These mutants lacked

chloroplast ATP synthase activity. The wild-type atpB gene was

introduced into the cells using tungsten microprojectiles coated

with the atp gene. The single large chloroplast provided an ideal

target for DNA delivery. The wild-type atpB gene introduced into

chloroplasts was able to correct the deletion mutant phenotype by

the restoration of photoautotrophic growth upon selection in the

light.

While the complementation of a deletion mutant was successful,

it was not clear whether foreign genes with appropriate regulatory

sequences could be introduced and expressed within chloroplasts.

Using the uidA gene, it was demonstrated that the foreign gene

flanked by chloroplast DNA sequences can be stably integrated into

the C. reinhardtii chloroplast genome. Although the introduced uidA

gene was transcribed, translated product could not be detected

(Blowers et al., 1989). After the unsuccessful effort to express

foreign genes through chloroplast genome of C. reinhardtii, attempts

were made to transform the higher plant chloroplast genome using

the gene gun. The first expression of a foreign gene (cat) in plastids

of cultured tobacco cells was accomplished using autonomously

replicating chloroplast vectors (Daniell et al., 1990). Chloroplast

vectors containing a chloroplast origin of replication (ori) expressed

chloramphenicol acetyl transferase for prolonged duration com-

pared to those that did not contain an ori. This work was followed by

demonstration of expression of the uidA gene in wheat leaves,

calluses, and somatic embryos (Daniell et al., 1991), which was not

possible in C. reinhardtii. C. reinhardtii chloroplast genome was

then transformed with the aadA gene conferring spectinomycin or

streptomycin resistance (Goldschmidt-Clermont, 1991). This is a

significant observation because the aadA gene facilitated stable

RECENT ADVANCES IN CHLOROPLAST GENETIC ENGINEERING 389

transformation of the chloroplast genome in higher plants. Stable

integration of the same aadA gene into the tobacco chloroplast

genome was then demonstrated (Svab and Maliga, 1993).

When the transgenes were introduced via the chloroplast

genome, it was hypothesized that foreign genes could be inserted

only into transcriptionally silent spacer regions between divergent

genes within the chloroplast genome (Zoubenko et al., 1994).

However, Daniell’s laboratory (Daniell et al., 1998) developed the

concept of inserting transgenes into transcriptionally active spacer

regions within native chloroplast operons. This approach rapidly

advanced this field and resulted in the development of chloroplast

transgenic lines that conferred herbicide resistance (Daniell et al.,

1998), insect resistance (Kota et al., 1999), disease resistance

(DeGray et al., 2001), drought tolerance (Lee et al., 2003),

phytoremediation (Ruiz et al., 2003), and salt tolerance (Kumar

et al., 2004a). This approach also facilitated the insertion of

multiple genes under the control of a single promoter, enabling the

coordinated expression of transgenes and accumulation of foreign

proteins as high as 46% of the total leaf protein (DeCosa et al.,

2001). In addition, insertion of transgenes into transcriptionally

active spacer regions resulted in expression and assembly of fully

functional vaccine antigens against cholera (Daniell et al., 2001b),

anthrax (Watson et al., 2004), canine parvo virus (Molina et al.,

2004), and plague (Daniell et al., 2005). Several human blood

proteins that are highly susceptible to proteolytic degradation were

successfully expressed via the chloroplast genome by integrating

into transcriptionally active spacer regions (Fernandez-san Milan

et al., 2003; Chebolu and Daniell, 2005). Foreign genes expressed

via the plastid genome now bestow useful agronomic traits (Table 1),

biopharmaceutical proteins (Table 2), and vaccines (Table 3).

Engineering the Plastid Genome for Agronomic Traits

Chloroplast genetic engineering has explored the agronomic

traits of crop plants (Daniell et al., 2004a, c) and successfully

developed insect-resistant plants (McBride et al., 1995), herbicide

resistance (Daniell et al., 1998), disease-resistant plants (De Gray

et al., 2001), drought tolerance (Lee et al., 2003), and salt tolerance

(Kumar et al., 2004a). The major advancement in the field of plant

genetic engineering is the transition from the insertion of a single

gene to the insertion of multiple genes in a single transformation

event (gene stacking; Van Bel et al., 2001; Daniell and Dhingra,

2002). The recent advances in genetic engineering in plants have

helped in the manipulation of multigene traits and introduction of

metabolic pathways and bacterial operons. The following

paragraphs discuss specific agronomic traits engineered via the

plastid genome.

Insect resistance. Cry2Aa2 is an insecticidal protein produced

by the bacterium B. thuringiensis. It was anticipated that the

prokaryotic nature of the chloroplast should allow the expression of

bacterial operons. The Cry2Aa2 protein is encoded within an

operon and has been expressed in the chloroplast genome both as a

single gene (Kota et al., 1999) and as an operon (DeCosa et al.,

2001). The 4.0 kb operon consists of three genes, with cry 2Aa2 as

the distal gene. The open reading frame, orf 2, immediately

upstream of the cry gene, codes for a putative chaperonin that is

necessary for folding the protein into cuboidal crystals (that are

resistant to proteolytic degradation). The expression cassette

contained the aadA gene (selectable marker gene conferring

resistance to spectinomycin) and the cry2Aa2 operon (comprising

all three genes), under the regulation of the Prrn promoter. DeCosa

et al. (2001) demonstrated the presence of cuboidal crystals upon

expression of the cry2Aa2 operon, using transmission electron

microscopy (Fig. 1). In addition to the ORF 2 protein, crystal

formation was also facilitated by hyperexpression of the insecticidal

protein through chloroplast genetic engineering. Upon expression of

the cry2Aa2 operon in chloroplast transgenic plants, the Cry2Aa2

protein had accumulated up to 46.1% of the total leaf protein and

this is the highest expressed foreign protein in transgenic plants to

date. The chloroplast transgenic plants expressing the single

cry2Aa2 gene or the complete operon showed very high insecticidal

TABLE 1

LIST OF AGRONOMICAL TRAITS ENGINEERED VIA CHLOROPLAST GENOME

Agronomic traits Gene Site of integration Promoter 50/30 regulatory elements Reference

Insect resistance Cry1A(c) trnV/rps12/7 Prrn rbcL/Trps16 McBride et al., 1995Herbicide resistance CP4 (petunia) rbcL/accD Prrn ggagg/TpsbA Daniell et al., 1998Insect resistance Cry2Aa2 rbcL/accD Prrn ggagg (native)/TpsbA Kota et al., 1999Herbicide resistance CP4 (bacterial or synthetic) trnV/rps12/7 Prrn rbcL or T7 gene 10/Trps16 Ye et al., 2001Insect resistance Cry2Aa2 operon trn I/trnA Prrn Native 50-UTRs/TpsbA DeCosa et al., 2001Disease resistance MSI-99 trn I/trnA Prrn ggagg/TpsbA DeGray et al., 2001Drought tolerance tps trn I/trnA Prrn ggagg/TpsbA Lee et al., 2003Phytoremediation merAa/merBb trn I/trnA Prrn ggagga,b/TpsbA Ruiz et al., 2003Salt tolerance badh trn I/trnA Prrn-F ggagg/rps16 Kumar et al., 2004a

a,bRefers to genes and their respective regulatory sequences.

FIG. 1. Transmission electron micrographs. A, Accumulation of foldedCry2A protein as cuboidal crystals in transgenic chloroplasts; B, detection ofCry2A protein by immunogold-labeling using Cry2A antibody.

390 KOYA AND DANIELL

TABLE2

LISTOFBIOPHARMACEUTICALPROTEINSEXPRESSEDVIA

CHLOROPLASTGENOME

Biopharmaceutical

proteins

Gene

Siteof

integration

Promoter

50 /30regulatory

elem

ents

%tspexpression

Functionalityassay

Reference

Elastin-derived

polym

erE

G12

1tr

nI/

trnA

Prrn

T7gene10/T

psbA

ND

Inversetemp.transition

assay

Gudaet

al.,2000

Human

somatotropin

hS

Ttr

nV/r

ps12/7

Prrna,P

psbA

bT7gene10aor

psbA

b/T

rps16

7.0%

a,1.0%

bPositivegrow

thresponse

ofNb2cellline

Staubet

al.,2000

Antimicrobialpeptide

MSI-

99

trnI/

trnA

Prrn

ggagg/T

psbA

21.5–43%

Minimum

inhibitoryconc.

(MIC)of

P.

aer

ug

inos

aDeG

rayet

al.,2001

Insulin-likegrow

thfactor

IGF-1

trnI/

trnA

Prrn

Pps

bA/T

psbA

33%

ND

Ruiz,2002

Interferon

alpha5

INFalpha5

trnI/

trnA

Prrn

Pps

bA/T

psbA

ND

ND

Daniell,2002

Interferon

alpha2b

INFalpha2B

trnI/

trnA

Prrn

Pps

bA/T

psbA

18.8%

Protectionof

HeLAcells

againstcytopathic

effects

ofEMCvirus

Falconer,2002

Human

serum

albumin

hsa

trnI/

trnA

Prrna,P

psbA

bggagga,

psbA

b/

Tps

bA0.02%

a,11.1%

bND

Fernandez-san

Milan

etal.,2003

Interferon

gamma

IFN

-grb

cL/a

ccD

Pps

bAP

psbA

/Tps

bA6%

Protectionof

human

lung

carcinom

acellsagainst

infectionbyEMCvirus

LeelavathiandReddy,

2003

Monoclonal

antibodies

Gu

y’s

13

trnI/

trnA

Prrn

ggagg/T

psbA

ND

ND

Daniellet

al.,2001a

a,bRefersto

genes

andtheirrespective

regulatory

sequencesand%

tsp(total

soluble

protein).

ND,not

determined.

TABLE3

LISTOFVACCINEANTIGENSEXPRESSEDVIA

CHLOROPLASTGENOME

Vaccineantigens

Gene

Siteof

integration

Promoter

50 /30regulatory

elem

ents

%tspexpression

Functionalityassay

Reference

Choleratoxin

CtxB

trnI/

trnA

Prrn

ggagg/T

psbA

4%

GM1-ganglioside

bindingassay

Daniellet

al.,2001a

Tetanustoxin

TetC(bacterial

andsynthetic)

trnV/r

ps12/7

Prrn

T7gene10a,

atpBb/ T

rbcL

25%

a,10%

bPositivesystem

icimmuneresponse

Tregoninget

al.,2003

Canineparvovirus(CPV)

CT

B-2

L21,

GF

P-2

L2

1tr

nI/

trnA

Prrn

Pps

bA/T

psbA

31.1%,22.6%

Immuneresponse

Molinaet

al.,2004

Anthraxprotectiveantigen

Pa

gtr

nI/

trnA

Prrn

Pps

bA/T

psbA

4–5%

Macrophagelysisassay

Watsonet

al.,2004

Plaguevaccine

CaF1,

LcrV

trnI/

trnA

Prrn

Pps

bA/T

psbA

14.8%

ND

Singleton,2003

a,bRefersto

genes

andtheirrespective

regulatory

sequencesand%

tsp(total

soluble

protein).

ND,not

determined.

RECENT ADVANCES IN CHLOROPLAST GENETIC ENGINEERING 391

activity when compared to the untransformed wild-type plants. The

wild-type control plant leaf material was fed to the tobacco

budworm (Heliothis virescens) and cotton bollworm (Helicoverpa zea).

The leaves were totally devoured within 24 h (Fig. 2A, D). When the

tobacco budworm was fed with chloroplast transgenic leaves

expressing the single gene, all the insects died after 5 d (Fig. 2B),

whereas the insects fed with the leaf material expressing the Bt

operon died in 3 d (Fig. 2C). Similar results were obtained when the

same assays were applied to cotton bollworm (Fig. 2D–F). These

results show that the hyperexpression of the Cry2Aa2 protein in the

chloroplast of transgenic plants conferred 100% resistance to

insects. Most importantly, chloroplast transgenic plants killed

insects that were 40 000-fold resistant to insecticidal proteins, even

when a single cry gene was expressed (Kota et al., 1999).

Herbicide resistance. Glyphosate is a broad-spectrum herbicide

that kills both the weeds and crop plants. Glyphosate inhibits 5-

enolpyruvylshikimate-3-phosphate synthase (EPSPS), a nuclear-

encoded chloroplast-localized enzyme in the shikimic acid pathway

of plants and microorganisms that is required for the biosynthesis of

aromatic amino acids. The wild-type petunia EPSPS gene was

integrated into the chloroplast genome. Transgenic plants were then

confirmed to be homoplasmic by Southern blot analysis. Eighteen-

week-old wild-type and control plants were sprayed with 0.5–5mM

glyphosate; the wild-type plants died within 7 d. On the other hand,

the chloroplast transgenic lines were able to survive concentrations

as high as 5mM, which is ,10 times the lethal concentration

(Daniell et al., 1998). This was the first report of a eukaryotic

nuclear gene expressed within the prokaryotic chloroplast

compartment and the transit peptide was properly cleaved within

chloroplasts. Agrobacterium strain CP4 EPSPS gene was expressed

in tobacco plastids and resulted in 250-fold higher levels of the

glyphosate-resistant EPSPS enzyme than the levels achieved via

nuclear transformation (Ye et al., 2001). However, higher levels of

EPSPS expression did not proportionately enhance tolerance to

glyphosate; both nuclear and chloroplast transgenic lines showed

similar levels of herbicide tolerance.

Pathogen resistance. The antimicrobial peptide MSI-99, an

analog of magainin, has been expressed in the chloroplast genome

of transgenic tobacco (DeGray et al., 2001). MSI-99 offers

protection against prokaryotic organisms due to its high specificity

for negatively charged phospholipids found mostly in bacteria.

Although the chloroplast has a prokaryotic environment, the

potential toxic activity against the chloroplast has been ruled out

because of the presence of neutral lipids in chloroplast and

thylakoid membranes, which are in contrast to the presence of

negatively charged lipids in bacteria. In vitro assay revealed that

the cell-free extracts of transgenic plants inhibited growth up to

96% of Pseudomonas syringae pv tabaci effectively as opposed to

the wild type. In another assay, the extracts of the transformed

plants reduced the colonies of the fungi Aspergillus flavus, Fusarium

moniliforme, and Verticillium dahliae greater than 95% compared

to wild-type control. In planta antimicrobial assay was performed on

plants expressing MSI-99 in the chloroplast. The leaves were

inoculated with the phytopathogen Pseudomonas syringae pv tabaci,

and then observed for necrosis around the site of inoculation. No

necrotic tissue could be observed in transgenic plants even when

8 £ 105 cells were used. Whereas, a large necrotic area could be

seen in wild-type plants inoculated with only 8 £ 103 cells. In

planta antifungal assay was also performed with Colletotrichum

destructivum, a plant fungal pathogen (Fig. 3). The untransformed

wild-type plants developed anthracnose lesions whereas the

transgenic plants expressing MSI-99 did not develop the lesions.

In sharp contrast, nuclear transgenic plants expressing MSI-99 were

not very effective in controlling the aforementioned pathogens, due

to low levels of expression (Li et al., 2001; Chakrabarti et al., 2003).

Drought tolerance. Trehalose is an osmoprotectant found to

accumulate in algae, bacteria, yeast, fungi, animals, and plants during

drought or salinity stress. It stabilizes proteins and membrane

structures under stress because of the glass transition temperature,

greater flexibility, and chemical stability. Efforts were made to

produce stress-resistant transgenic plants by expressing the genes for

trehalose biosynthesis. Trehalose phosphate synthase (encoded by

FIG. 2. Insect bioassays. A, D, Untransformed tobacco leaves; B, E, single gene-derived cry2Aa2 transformed leaves; C, F, operon-derived cry2Aa2 transformed leaves. A–C, Bioassays with Heliothis virescens (tobacco budworm); D–F, bioassays with Helicoverpa zea(cotton bollworm).

392 KOYA AND DANIELL

TPS1 gene) catalyzes the reaction to form the osmoprotectant

trehalose. Unfortunately, trehalose accumulation in the cytosol when

engineered via the nuclear genome resulted in stunted growth, lancet-

shaped leaves, sterility, and loss of apical dominance. In order to

overcome the pleiotrophic effects due to accumulation of trehalose in

the cytosol, its compartmentalization within the chloroplast was

achieved by integrating the TPS1 gene into the chloroplast genome

(Lee et al., 2003). The high expression of theTPS1 gene in chloroplast

transgenic plants showed no pleiotrophic effects and the phenotype of

transgenic plants was similar to that of the wild type. Drought

tolerance bioassays in which transgenic and wild-type seeds were

germinated in MS medium (Murashige and Skoog, 1962) containing

concentrations of 5–10% polyethylene glycol (PEG) showed that the

chloroplast transgenic plants producing high levels of trehalose

germinated, grew, maintained green color, and remained healthy.

Untransformed wild-type seeds germinated under similar conditions

showed severe dehydration, loss of chlorophyll (chlorosis), and

retarded growth that resulted in the death of the seedlings. Loss of

chlorophyll in the untransformed plants suggests that drought

destabilizes the thylakoid membrane, but accumulation of trehalose

in transgenic chloroplasts conferred thylakoid membrane stability.

When seedlings from chloroplast transgenic and untransformed

tobacco plants were dried for 7 h, they showed symptoms of

dehydration, but when the seedlings were rehydrated in MS medium

for 48 h, all chloroplast transgenic plants that accumulated trehalose

recovered and grew normally. In contrast, the untransformed controls

became bleached out and died. Additionally, when potted chloroplast

transgenics and untransformed plants were not watered for 24 d and

were then rehydrated for 24 h, the chloroplast transgenic plants

recovered while the untransformed control plants did not recover.

These results clearly show that expression of the enzyme trehalose

phosphate synthase in the chloroplast of transgenic plants confers

drought tolerance.

Salt tolerance. Salinity is one of the major factors that limits

geographical distribution of plants and adversely affects crop

productivity and quality. The metabolic pathway for glycine betaine

synthesis in higher plants involves two enzymes, i.e., choline

monooxygenase (CMO) and betaine aldehyde dehydrogenase

(BADH), that are compartmentalized within the chloroplast

(Rathinasabapathi et al., 1997; Nuccio et al., 1998). b-Alanine

betaine is produced after methylation of b-alanine that is derived

from 3-aminopropionaldehyde in a reaction catalyzed by the BADH

enzyme (Rathinasabapathi et al., 2001). Overexpression of betaine

by manipulation of badh via chloroplast genetic engineering may

prove to be an important strategy in order to confer salt tolerance to

desired crops. Therefore, homoplasmic chloroplast transgenic

plants exhibiting high levels of salt tolerance were regenerated

from bombarded cell cultures via somatic embryogenesis (Kumar

et al., 2004a). Transformation efficiency of carrot somatic embryos

was very high, with one transgenic event per approximately four

bombarded plates under optimal conditions. In vitro transgenic

carrot cells transformed with badh transgene were visually green in

color when compared to untransformed carrot cells and this offered

a visual selection for transgenic lines. Additionally, BADH enzyme

activity was enhanced 8-fold in transgenic carrot cell cultures, grew

7-fold more and accumulated 50–54-fold more betaine (93–

101mmol g21 dry weight of b-alanine betaine and glycine betaine)

than untransformed cells grown in liquid medium containing

100mM NaCl. Transgenic carrot plants expressing BADH grew in

the presence of high concentrations of NaCl (up to 400mM), the

highest level of salt tolerance reported so far among genetically

modified crop plants. There are at least 15 prior reports where

attempts have been made to manipulate the glycine betaine

biosynthesis pathway via nuclear genetic engineering in order to

enhance salt tolerance (Flowers, 2004). In order to provide a broad

range of salinity tolerance to plants, a better strategy may be to

engineer genes that confer other mechanisms involved in signal

transduction of salt stress, in addition to osmoprotection. Therefore,

engineering plants for salt tolerance either by large accumulation of

betaine via the chloroplast genome (50–54-fold higher than

untransformed control) or in combination with an antiport

mechanism (Zhang and Blumwald, 2001) should be an attractive

option for future strategies.

Phytoremediation

Plants can be genetically modified to improve phytoremediation

by the expression of several plant and bacterial genes in transgenic

plants. The excessive use of organomercurial compounds (e.g., in

fertilizers and pesticides) is known to have severe effects on plants.

The primary site of action of Hg damage appears to be the

chloroplast thylakoid membranes and photosynthesis. Organomer-

curials are the most toxic form of mercury, presenting a serious

hazard to the environment. Current methods of chemical and

physical remediation as well as bacterial bioremediation have thus

far proven to be less effective due to the high cost and

environmental concerns. An alternative method, phytoremediation,

has been proposed as a safe and cost-effective system for the

remediation of toxic chemicals in the environment. Plants have

been engineered with modified bacterial mercuric ion reductase

(merA) and organomercurial lyase (merB) genes; these enzymes are

capable of converting highly toxic methyl-Hg into the much less

toxic Hg(0), which may be volatilized (Rugh et al., 1996; Bizily

et al., 1999, 2000). The operon containing both merA and merB

C

D

FIG. 3. In planta bioassays for disease resistance. A, B, Fungal diseaseresistance. Leaves were inoculated on the adaxial surface with eight drops of10ml each of the culture containing 1 £ 106 spores per ml of the fungalpathogen Colletotrichum destructivum. A, Wild-type leaf; B, transgenic leaf;C, D, bacterial disease resistance: 8 £ 105, 8 £ 104, 8 £ 103, and 8 £ 102

cell cultures of bacterial pathogen Pseudomonas syringae pv tabaci wereadded to a 7mm scraped area in nontransgenic and transgenic tobacco lines.Photographs were taken 5 d after inoculation.

RECENT ADVANCES IN CHLOROPLAST GENETIC ENGINEERING 393

genes were integrated into the chloroplast genome (Ruiz et al.,

2003). The integrated gene cassette has three genes, the merA,

merB, and aadA gene, which confers resistance to spectinomycin

under the control of 16S ribosomal RNA promoter. The transgenic

lines were tested in a bioassay using the organomercurial, phenyl

mercuric acetate (PMA). The transgenic plants were shown to be

substantially more resistant than wild-type tobacco plants grown

under identical conditions. Transgenic seedlings were able to

survive concentrations as high as 200mM, even though they

absorbed several hundred times more PMA than the untransformed

plants that struggled to survive even at a concentration of 50mM

(Fig. 4). When nuclear transgenic seedlings containing merA and

merB genes were germinated in a medium containing PMA, they

were only capable of resisting concentrations of 5mM (Bizily et al.,

2000). In contrast, when chloroplast transgenic plants were treated

with concentrations of 100, 200, 300, and 400mM PMA, they

showed an increase in total dry weight when compared with the

untransformed plants grown at similar concentrations, which

progressively decreased with each increase in PMA concentration.

The total dry weight of the transgenic lines remained much higher

(Ruiz et al., 2003). This is the first report of chloroplast genetic

engineering for the purpose of phytoremediation.

Cytoplasmic Male Sterility

Naturally occurring cytoplasmic male sterility (CMS) has been

reported for several major crops (Kriete et al., 1996), and has been

documented for over 100 years. Such systems are not available for

most crops used in agriculture. In currently available CMS lines,

the mitochondrial genomes confer male sterility and the nuclear

genome directs the various restoration factors, which are often

under the control of multiple loci and are not fully understood. CMS

has been associated with diseases like southern corn blight (CMS-T)

and cold susceptibility (CMS Ogura). Male sterility can be conferred

via multiple mechanisms, although most affect the tapetum and

pollen development. The anti-sense technology has been used to

deplete flavonoid pigments in the anthers, thereby arresting pollen

maturation (Kriete et al., 1996), or to inhibit mitochondrial pyruvate

dehydrogenase, causing tapetum perturbation that leads to male

sterility (Yui et al., 2003). Additionally, the nuclear expression of

the barnase gene caused degradation of the tapetum and lack of

pollen formation (Kriete et al., 1996). More recently, pollen

development was arrested by the disruption of AtGPAT1, a gene

involved in the initial step of glycerolipid biosynthesis (Zheng et al.,

2003).

The possibility of the production of transgenic seeds that spread

transgenic traits to nontransgenic plants, when a restorer line or

wild relative cross-pollinates the male sterile plant, is a major

drawback of the current forms of male sterility systems. Nuclear

encoded male sterility systems are also more vulnerable because of

genetic segregation, a factor inherent to nuclear transformation that

will eventually dilute the male sterility trait. Also, many of these

systems incur drawbacks such as interference with general

development or metabolism, and are often restricted to certain

species. Thus, there is a strong need for a male sterility system

without debilitating effects or the possibility of producing viable

offspring. Recently, the overexpression of the phaA gene, encoding

b-ketothiolase in tobacco chloroplasts, addresses some of these

concerns (Ruiz and Daniell, 2005). This resulted in transgenic

plants with normal phenotype except for the lack of pollen.

Examination using scanning electron microscopy (SEM) revealed a

collapsed morphology of the pollen grains. Floral developmental

studies showed that transgenic lines underwent an accelerated

pattern of anther development, resulting in aberrant tissue patterns

during maturation. Further studies revealed an abnormal thickening

of the outer wall, enlarged endothecium, and vacuolation, thereby

decreasing the inner space of the locules, causing the pollen grains

to form an irregular shape. Surprisingly, this phenotype can be

reversed from continuous lumination by changing the ratios of the

thiolase to synthase, resulting in viable pollen and subsequent

fertility. This research provides a new tool for efficient transgene

containment of both nuclear and organelle genomes, while also

offering a convenient mechanism for F1 hybrid seed production.

Chloroplast Genetic Engineering for Production of

Biopharmaceutical Proteins

Plants are safe and economical expression systems for the

production of biopharmaceuticals, polymers, vaccines, enzymes,

plasma proteins, and antibodies. Plants offer attractive advantages

in the production of recombinant proteins. Plant systems are more

economical because they can be produced on a larger scale than the

expensive industrial methods via fermentation of bacteria, yeast, or

cultured animal or human cell lines. The recombinant protein

production is safe and free of contaminants as opposed to bacterial

and mammalian expression systems. There is also minimized risk of

contamination from potential human pathogens as plants are not

FIG. 4. Effect of PMA concentration on the growth of wild-type ortransgenic tobacco lines. Plants were treated with 200ml of Hoagland’snutrient solution supplemented with 0, 50, 100, and 200mM PMA.Photographs were taken 14 d after treatment. WT, negative control PetitHavana; T, pLDR-MerAB transgenic line.

394 KOYA AND DANIELL

hosts for human infectious agents. Additionally, plants have the

ability to carry out post-translational modifications similar to

naturally occurring systems. However, in order to meet the

economical and commercial needs, the expression levels should be

quite high. Chloroplast genetic engineering has been developed to

produce a wide variety of biopharmaceuticals (Daniell et al., 2004b;

Kumar and Daniell, 2004; Chebolu and Daniell, 2005). The

chloroplast transformation as opposed to nuclear transformation

allows high expression levels up to 46% of foreign recombinant

protein relative to total protein (DeCosa et al., 2001). The list of

biopharmaceuticals expressed via chloroplast genome is given is

Table 2. The following paragraphs discuss a few such examples.

Human somatotropin. Human somatotropin (hST) is a thera-

peutic protein useful in the treatment of hypopituitarism, Turner

syndrome, and chronic renal failure. The somatotropin requires

proper folding and disulfide bond formation. Staub et al. (2000)

expressed the chimeric hST in chloroplasts of transgenic tobacco

and proved that the chloroplasts efficiently fold the protein and form

the disulfide bonds. Using the biolistics method, the hST gene was

introduced into the tobacco chloroplast genome, targeting insertion

between the trnV gene and rps7/30-rps12 operon. The maximum

expression level observed was 7% of the total soluble protein (tsp).

Upon purification, chloroplast-derived hST had proper disulfide

bond formation and performed identically to native human hST.

This work demonstrated that plastids possess the proper machinery

to fold eukaryotic proteins and add disulfide bonds, possibly

utilizing the chloroplast enzyme protein disulfide isomerase.

Human serum albumin. Human serum albumin (HSA) accounts

for approximately 60% of proteins in the blood serum. It is the most

widely distributed intravenous protein and is prescribed to replace

blood volume in trauma and in various other clinical situations.

HSA is a monomeric 66.5-kDa protein, which contains 17 disulfide

bonds. Currently, albumin is primarily produced by the

fractionation of the blood serum. The lack of glycosylation makes

it convenient for its production in prokaryotic systems. The current

production of HSA in microbial systems is not commercially

feasible. Sijmons et al. (1990) first expressed HSA in transgenic

plants, but very low levels were detected. HSA could not be

detected in cytoplasm, which could be due to its high susceptibility

to proteolytic degradation. The proteolytic degradation of foreign

recombinant proteins in heterologous hosts is a serious concern for

industrial processing. Therefore, HSA was expressed in transgenic

chloroplasts; transgenes were integrated between the trn I/trnA

genes within the inverted repeat region of the chloroplast genome

using two different 50 regulatory sequences (Fernandez-san Milan

et al., 2003). The Shine Dalgarno (SD) ribosome binding site (ggagg)

placed upstream of aadA and the hsa gene was regulated by the Prrn

promoter and the light-regulated psbA 50-untranslated region (UTR,

a translation enhancer) inserted immediately upstream of the hsa

gene. The chloroplast transgenic plants showed a 360-fold

difference in expression levels, with the SD construct at 0.02%

total leaf protein (tlp), and the 50UTR construct at 7.2% tlp. The

maximum HSA levels were observed when the transgenic plants

were exposed to 50 h of continuous illumination; expression levels

reached up to 11.2% tlp in the mature green leaves. Such

phenomenal expression resulted in the formation of large inclusion

bodies resulting in noticeable increase in the size of the transgenic

chloroplasts and thereby offering protection against proteolytic

degradation (Fig. 5).

Human interferon alpha. Human cytokines of the immune

system termed interferon alphas (IFNas) are known to interfere with

viral replication and cell proliferation. They are also known as potent

enhancers of the immune response and have many uses in clinical

treatments. Currently, the recombinant IFNa2b is produced by an

Escherichia coli expression system, and due to the necessary in vitro

processing and purification processes, the average cost for treatment

is $26 000 per year. IFNa2b is administered by injection and severe

side-effects are quite common. It has been demonstrated that oral

administration of natural human IFNa is therapeutically useful for

the treatment of various infectious diseases. Therefore, a recombinant

IFNa2b containing a polyhistidine tag and a thrombin cleavage site

was generated and introduced into the tobacco chloroplast genome of

Petit Havana and into a low nicotine variety of tobacco, LAMD-609,

for studies on oral delivery (Falconer, 2002). It is well known that

chloroplasts possess the ability to correctly process and fold human

proteins as well as to form disulfide bonds. Western blots detected

both monomers and multimers of IFNa2b in both the tobacco

varieties, using interferon alpha monoclonal antibody. Quantification

of IFNa2b recombinant protein by ELISA showed up to 18.8% tsp in

Petit Havana and up to 12.5% in LAMD-609 in T0 transgenic plants

(Fig. 6). The next generation (T1) of the Petit Havana chloroplast

transgenic lines had accumulated even higher levels, clearly

observable in a Coomassie-stained SDS-PAGE. IFNa2b functionality

was determined by the ability of IFNa2b to protect HeLa cells against

the cytopathic effect of encephalomyocarditis (EMC) virus and RT-

PCR. Chloroplast-derived IFNa2b was shown to be just as active as

the commercially produced Intron A. The expression levels observed

and proper functionality are ideal for purification and further use in

oral IFNa2b delivery or preclinical trials (Daniell, 2004; Daniell

et al., 2004b; Chebolu and Daniell, 2005).

Human interferon gamma. INF-g is a major cytokine of the

immune system that interferes with viral replication, prevents

proliferation, and has several other roles in immunoregulatory actions

in response to pathogenic bacteria and viruses. Researchers

generated nuclear or chloroplast transgenic lines, expressing INF-

g, either independently or as a GUS fusion protein (Leelavathi and

Reddy, 2003). Eight tobacco nuclear transgenic lines expressing

INF-gwere screened and the highest level of expression obtained was

0.001% tsp. In order to compare GUS protein accumulation along

with INF-g, the two genes were expressed in separate tobacco

chloroplast transgenic lines. The estimated levels of expression of

GUS protein and INF-g were 3 and 0.1% (100-fold greater than

nuclear transgenic lines), respectively. A recombinant fusion protein

FIG. 5. HSA accumulation in transgenic chloroplasts. Electronmicrographs of immunogold-labeled tissues from untransformed (A) ortransformed (B) mature leaves using the chloroplast vector pLDA psbAHSA.

RECENT ADVANCES IN CHLOROPLAST GENETIC ENGINEERING 395

GUS/INF-g was expressed in tobacco chloroplast transgenic lines.

Based onWestern blot analysis, it was estimated that the GUS/INF-g

fusion protein had accumulated up to 6% tsp. GUS/INF-g fusion

protein was purified to homogeneity by a two-step His-tag-based

chromatography purification scheme. Utilizing this procedure,

researchers were able to obtain ,360mg of fusion protein from 1 g

of leaf tissue with a.75% recovery. Upon cleavage of the fusion with

factor Xa, GUS estimated recovery was ,210mg g21 fresh weight

tissue and INF-g had an estimated recovery of ,40mg g21 fresh

weight tissue. A bioassay was performed with the purified INF-g, and

it was shown to offer complete protection to human lung carcinomas

against infection with the EMC virus. This demonstrated that INF-g

produced in the chloroplast of tobacco plants was just as biologically

active as native human INF-g.

Human antibody. Monoclonal antibodies for passive immu-

notherapy have been the most widely studied therapeutic proteins

produced in transgenic plants. The anti-Streptococcus mutans

secretory antibody for the prevention of dental caries is the only

plant-derived antibody currently in Phase II clinical trials (Larrick

and Thomas, 2001). Chloroplasts have the ability to fold, process,

and assemble foreign proteins with disulfide bridges. Guy’s 13 was

developed to prevent dental caries, which is caused by Streptococcus

mutans (Daniell and Wycoff, 2001; Daniell et al., 2001b; Daniell,

2004). A codon-optimized and humanized gene Guy’s 13 encoding a

chimeric monoclonal antibody (IgA/G) under the control of a

specific 50-untranslated region was used to synthesize monoclonal

antibodies in transgenic chloroplasts. Western blot analysis showed

the expression of heavy and light chains as well as fully assembled

antibody, suggesting the presence of chaperones for proper protein

folding and enzymes for formation of disulfide bond in transgenic

chloroplasts (Fig. 7). However, expression levels should be

enhanced further to facilitate industrial-scale production.

Chloroplast Genetic Engineering for Vaccine Production

The use of plant systems for vaccine production offers several

attractive advantages. Unlike the bacterial and mammalian

expression systems, plants are ideal for the production of clean

and safe vaccine antigens free of contaminants. The production of

vaccines in the edible plant parts like tomato fruit and carrot root

would enable the oral delivery of vaccine antigens. This reduces the

cost of vaccine dramatically due to elimination of purification costs,

storage, and cold chain maintenance. Bioencapsulation of

pharmaceutical proteins within plant cells offers protection against

digestion in the stomach but allows successful delivery (Walmsley

and Arntzen, 2000; Yu and Langridge, 2001). Nuclear-transformed

tomato and potato plants are capable of synthesizing the subunit

vaccines for Norwalk virus, enterotoxigenic E. coli, Vibrio cholerae,

and hepatitis B surface antigen (Langridge, 2000). The hepatitis B

surface antigen and Norwalk virus capsid protein expressed via

nuclear genome in potato have been shown to elicit a serum

immunoglobulin response when fed to mice and humans (Mason

et al., 1996; Kapusta et al., 1999; Richter et al., 2000; Tacket et al.,

2000). The expression levels seen in nuclear transformants were

very low and an increased dosage is required for an effective

immune response; therefore, the most logical step is to hyperexpress

subunit vaccines in the chloroplasts of transgenic plants (Daniell

et al., 2005). The list of vaccine antigens expressed via chloroplast

genome is shown in Table 3. The following paragraphs discuss a few

examples.

Cholera vaccine. Cholera is a disease that causes acute watery

diarrhea by colonizing the small intestine and producing the

enterotoxin, cholera toxin (CT). Cholera toxin is a hexameric AB5

protein consisting of one toxic 27-kDa A subunit having ADP

ribosyl transferase activity and a nontoxic pentamer of 11.6-kDa B

subunits that binds to the A subunit and facilitates its entry into the

intestinal epithelial cells. CTB is a potent mucosal immunogen

because it enters the eukaryotic cell surfaces via GM1 ganglioside

receptors present on the intestinal epithelial surface, eliciting a

mucosal immune response. To achieve higher expression levels, the

ctxB gene was integrated into the inverted repeat regions through

homologous recombination events between the trn I and trnA genes.

The CTB expression level was 4.1% of the tsp (Daniell et al.,

2001b). Higher expression levels (up to 31.1% tsp) were obtained

FIG. 6. Coomassie-stained SDS-PAGE. Untransformed or transgeniclines expressing high levels of IFNa2b. Lanes 1, 2, total soluble protein; laneM, protein marker; lane PH, untransformed Petit Havana; lanes 3, 4, totalprotein.

FIG. 7. Western blot analysis of transgenic lines showing the assembledantibody in transgenic chloroplasts. Lane 1, extract from a transgenic line;lane 2, negative control – extract from an untransformed plant; lane 3,positive control – human IgA. The gel was run under nonreducingconditions. The blot was developed with AP-conjugated goat anti-humankappa antibody.

396 KOYA AND DANIELL

when CTB-2L21 fusion protein was expressed in transgenic

chloroplasts (Molina et al., 2004). CTB synthesized from transgenic

chloroplasts in both investigations assembled into functional

oligomers and were antigenically identical to purified native CTB.

CTB has the highest affinity to bind with gangliosides (GM1), which

are the natural toxin receptors in the intestinal epithelial cells. The

functionality of CTB was determined by the GM1-ganglioside ELISA

binding assay (Fig. 8). The bacterial CTB and chloroplast-

synthesized CTB showed a strong affinity for the GM1-ganglioside,

confirming that the antigenic sites necessary for the binding of CTB

to GM1 were preserved by proper assembly of the pentamers and

formation of disulfide bonds. With such high levels of expression of

an efficient transmucosal carrier molecule like CTB in chloroplasts,

fusion proteins can be synthesized and also plant-derived vaccines

can be commercialized.

Anthrax vaccine. Anthrax is a zoonosis, a disease shared by

animals and humans. Animals contract the disease through

ingestion of soil-borne Bacillus anthracis spores and may die

acutely. Bacillus anthracis is a Gram-positive, nonmotile, aerobic or

facultatively anaerobic, spore-forming bacterium. The exotoxins

produced from these bacteria lead to symptoms and possible death.

In addition to the immunogenic protective antigen, the current

vaccine contains trace amounts of edema factor (EF) and lethal

factor (LF) that may contribute to the high rate of local reactions at

the site of the subcutaneous injection or reported to be toxic,

causing side-effects (Baillie, 2001). Therefore, there is a clear need

and urgency for an improved vaccine for anthrax and for improved

production methods that allow it to be mass-produced at reasonable

cost. Using chloroplast transformation technology, large quantities

of anthrax protective antigen (PA) could be produced in transgenic

plants due to the presence of thousands of copies of transgenes per

cell as opposed to only a few copies via nuclear transgenic plants.

This is in sharp contrast to nuclear expression of protective antigen

gene ( pag) that would require extensive codon modifications

because of high AT content, unfavorable codons, presence of mRNA

destabilizing sequences, and cryptic polyadenylation or splice sites.

Therefore, pag was expressed in transgenic tobacco chloroplasts in

order to obtain large amounts of PA free of contaminants and to

facilitate vaccine production to stockpile for times of crisis. PA

gene ( pag) has been introduced into the chloroplasts of transgenic

tobacco plants (Watson et al., 2004). Chloroplast transgenic plants

contained up to 2.5mg PA g21 fresh weight. The functionality of PA

was confirmed by macrophage lysis assays (Fig. 9). Chloroplast-

derived PA was capable of efficiently binding to anthrax toxin

receptor (formed heptamers), underwent proper cleavage, bound to

LF, followed by internalization of LF; completion of a sequence of

these events resulted in macrophage lysis. Quantitative analysis of

these events yielded up to 25mg of functional PA per ml in

transgenic leaves. With observed expression levels, 400 million

doses of vaccine (free of EF and LF) could be produced per acre of

transgenic tobacco using an experimental cultivar in a greenhouse,

which could be further enhanced 18-fold utilizing a commercial

cultivar in the field to combat bioterrorism.

Plague vaccine. Yersinia pestis, a Gram-negative bacterium, is

the causative agent of plague and has been listed by the Center for

Disease Control (www.bt.cdc.gov/agent/plague/) as one of the six

category A biological agents. Several subunit vaccines have been

evaluated for immunogenicity against Y. pestis. CaF1 and LcrV are

the most effective subunit vaccines so far against Y. pestis. F1 is a

capsular protein located on the surface of the bacterium with anti-

phagocytic properties. The V antigen is a component of the Y. pestis

Type III secretion system and it may form part of an injectosome.

The fusion protein of F1-V expressed in E. coli has been shown to

be safe and immunogenic when the mice were challenged with Y.

pestis. The fusion protein F1-V was expressed in transgenic

chloroplasts consisting of the F1 antigen fused at its carboxy

terminus to the amino terminus of the V antigen. Western blot and

ELISA were performed and samples were collected from plants

under continuous illumination from days 0–5 from young, mature,

and old leaves. The maximum expression levels, which were as high

as 14.8% of the tsp, were observed in mature leaves (Singleton,

FIG. 8. CTB–GM1-ganglioside binding ELISA assay. Plates, coated firstwith GM1-ganglioside and bovine serum albumin (BSA), respectively, wereplated with total soluble plant protein from chloroplast transgenic lines (3and 7) and untransformed plant total soluble protein and 300 ng of purifiedbacterial CTB. The absorbance of the GM1-ganglioside–CTB antibodycomplex in each case was measured at 405 nm.

FIG. 9. Macrophage cytotoxic assays for extracts from untransformedand chloroplast transgenic plants. Supernatant samples from T1 pLD-JW1tested (proteins extracted in buffer containing no detergent and MTT addedafter 5 h). ( ) pLD-JW1 (extract stored 2 d); ( ) pLD-JW1 (extract stored7 d); ( £ ) PA 5mgml21; ( ) control wild type (extract stored 2 d); ( ) controlwild type (extract stored 7 d); ( ) Control wild type no LF (extract stored2 d); ( ) control wild type no LF (extract stored 7 d); (X) control pLD-JW1no LF (extract stored 2 d); (X) control pLD-JW1 no LF (extract stored 7 d).

RECENT ADVANCES IN CHLOROPLAST GENETIC ENGINEERING 397

2003). Further studies should be done to confirm the

immunogenicity.

Canine parvo viral vaccine. Canine parvovirus (CPV) infects

dogs and other Canidae such as wolves, South American dogs, and

Asiatic raccoon dogs, producing hemorrhagic gastroenteritis and

myocarditis. The 2L21 synthetic peptide, coupled to KLH carrier

protein, was studied extensively and has been shown to be effective in

protecting dogs and minks against parvovirus (Langeveld et al., 1994,

1995). The 2L21 peptide, which confers protection to dogs against

virulent canine parvovirus (CPV), was expressed in tobacco

chloroplasts as a fusion protein with cholera toxin B (CTB) and with

the green fluorescent protein (GFP). The maximum levels of protein

expression were achieved with CTB-2L21, 7.49mg g21 of fresh

weight (equivalent to 31.1% of tsp) and with GFP-2L21, 5.96mg g21

of fresh weight (equivalent to 22.6% of tsp) (Molina et al., 2004). The

expression levels were also dependent on the age of the plant. Mature

plants accumulated the highest amounts of protein when compared to

the young and senescent plants. This shows the importance of the

harvesting time of the plants. Also, the chimeric protein retained the

pentamerization andGM1-ganglioside binding properties of the native

CTB. When mice were immunized intraperitoneally with the leaf

extracts from CTB-2L21, mice immunized with CTB-2L21 elicited

anti-2L21 antibodies able to recognize VP2 protein from CPV

(Fig. 10). This is the first report of an animal vaccine and viral antigen

expressed in transgenic chloroplasts.

Biomaterials and Enzymes

Besides biopharmaceuticals and vaccines, chloroplast genetic

engineering has been applied to produce cost-effective industrially

valuable biomaterials and enzymes. The multigene engineering of

chloroplast genome has successfully led to the efficient production

of many valuable biomaterials.

Chorismate pyruvate lyase. p-Hydroxybenzoic acid (pHBA) is

the principal monomer found in all commercial thermotropic liquid

crystal polymers. Chorismate pyruvate lyase (CPL) catalyzes the

direct conversion of chorismate to pyruvate and pHBA. CPL is an

enzyme encoded by the ubiC gene present in E. coli. Chorismate, the

substrate for CPL, is an intermediate of the shikimate pathway, which

is located in chloroplasts. CPL is not normally present in plants;

instead, the entire reaction leading from chorismate to pHBA involves

up to 10 successive enzymatic reaction steps as opposed to the direct

conversion with CPL. In order to accumulate high levels of pHBA,

tobacco plants were transformed to express the CPL enzyme via the

chloroplast genome (Viitanen et al., 2004). The pHBA in the T0 line,

in normal light/dark cycle (16 h light/8 h dark), continued to steadily

increase up to 100 d in soil, reaching a dry weight of ,15% pHBA

glucose conjugates in mature leaves. Upon exposure to continuous

illumination for 22 d, this further increased pHBA accumulation to

about 25% dry weight. This shows that the 50-UTR in continuous light

increases translation of the ubiC gene. The yield of pHBA in the total

plant leaf material (young, mature, and old) or the total stalk was

determined. In T0 transgenic lines, the pHBA levels varied between

10.87 and 15.22% dry weight in total leaves and between 1.65 and

2.87% dry weight in total stalk. The T1 generation plants grown in a

normal light/dark cycle were then transferred to continuous

illumination. After 27 d in continuous light, pHBA accounted up to

17.44% dry weight in old leaves (100 d in soil). By day 93 in

continuous light, the pHBA content had reached about 25% dry

weight. The T1 plants were morphologically indistinguishable from

untransformed tobacco plants even after 93 d in continuous light

(167 d in soil). The highest CPL activity observed in T1 chloroplast

transgenic lines in the total leaf material was 50 783 pkat per mg of

protein and this equates to approximately 35% tsp. Such high levels of

expression are not surprising because foreign proteins up to 46.1% of

total protein have been reported in transgenic chloroplasts (DeCosa

et al., 2001). In sharp contrast, the highest level observed in nuclear

transgenic plants was only 208 pkat per mg of protein. The pHBA

accumulation in leaf sample is 50-fold higher in transgenic

chloroplasts when compared to nuclear expression, whereas the

CPL enzyme activity in leaves is about 240-fold higher than in nuclear

transgenic plants. The 5-fold difference between CPL enzyme activity

and pHBA accumulation indicates that chorismate may be the

limiting factor; thus there is an upper limit to pHBA production in

transgenic plants, but observed levels are well within commercial

feasibility. The CPL activity in the T1 generation whole stalk was

analyzed and the results showed 8378 pkat per mg of protein (,5%

tsp); the CPL activity reported by Viitanen et al. (2004) in the whole

stalk is 40-fold higher than any other data published in the literature

and it is 250-fold higher in the leaf material than previous reports.

Integration of CPL into the chloroplast genome provides a dramatic

demonstration of the high-flux potential of the shikimate pathway for

chorismate biosynthesis, and could prove to be a cost-effective route

to pHBA. Moreover, exploiting this strategy to create an artificial

metabolic sink for chorismate could provide new insight on regulation

of the plant shikimate pathway and its complex interactions with

downstream branches of secondary metabolism, which is currently

poorly understood.

Xylanase. Leelavathi et al. (2003) expressed the alkali-

thermostable xylanase gene from Bacillus sp. strain NG-6 within

FIG. 10. Titers of antibodies at day 50 induced by chloroplast-derivedCTB-2L21 recombinant protein. Balb/c mice were intraperitoneallyimmunized with leaf extract from CTB-2L21 transgenic plants. Animalswere boosted at days 21 and 35. Each mouse received 20mg of CTB-2L21recombinant protein. Individual mice sera were titrated against 2L21synthetic peptide, VP2 protein and control peptide (amino acids 122–135 ofhepatitis B virus surface antigen). Titers were expressed as the highestserum dilution to yield two times the absorbance mean of preimmune sera.T1–T6, mouse 1 to 6; 2L21, epitope from the VP2 protein of the canineparvovirus; VP2, protein of the canine parvovirus that includes the 2L21epitope.

398 KOYA AND DANIELL

the chloroplast of tobacco plants. Alkali-thermostable xylanases are

of considerable interest because of their uses in the paper, fiber,

baking, brewing, and animal feed industries (Biely, 1985). In spite of

such uses for xylanases, they are not routinely utilized because of their

high production cost. Previous nuclear transgenic plants expressing

xylanases encountered the problem of degradation of cell wall

component, thereby affecting their growth. Therefore, xylanases were

targeted to the apoplast (Herbers et al., 1995), seed oil body (Liu et al.,

1997), or by secretion of enzyme through roots into the culture

medium (Borisjuk et al., 1999). In all the above nuclear transgenic

approaches, the expression levels were too low to make it

economically feasible. Therefore researchers transformed the xynA

gene into the chloroplast genome of tobacco plants. A zymography

assay demonstrated direct evidence for the presence of xylanase in its

biologically active form in leaf tissues. It was estimated that the

expression of xylanase was,6% tsp, and the estimated activity was

140 755U per kg of fresh leaf tissue. Nuclear expression of a

thermostable xylanase from Clostridium thermocellum accumulated

only 0.1–0.3% tsp. Since chloroplast-derived xylanase was

thermostable, researchers devised a purification technique that

utilized heat in the first step in order to possibly increase yield by

reducing proteolytic degradation by plant proteases. Several

generations of transgenic lines were tested and all of them appeared

to be no different from untransformed control in height, chlorophyll

content, flowering time, and biomass. Drying out of the transgenic

leaves in the sun or at 42 8C showed that greater than 85% activity

could be recovered. The same results were seen in leaves undergoing

senescence (85% recovery). Characterization of chloroplast-derived

xylanase showed that the enzyme is as biologically active as the

bacterially produced enzyme in the pH range of 6–11, with peak

activity at pH 8.4 (Leelavathi et al., 2003). Also, the chloroplast-

derived xylanase retained substrate specificity. This study demon-

strates that chloroplasts are quite capable of expressing industrially

important cellulolytic enzymes that cannot be expressed at high levels

via the nuclear genome because of adverse affects.

Amino acid biosynthesis: ASA2 – anthranilate synthase alpha

subunit. It is well known that plastids possess the ability to

synthesize some of the essential amino acids in higher plants. The

majority of enzymes involved in amino acid biosynthesis are

encoded in the nucleus, synthesized in the plant cytosol, and then

transported to plastids. Tryptophan (Trp) biosynthesis branches off

the shikimate pathway at chorismate, which is the last common

precursor of many aromatic compounds. Anthranilate synthase (AS)

catalyzes the first committed reaction for Trp biosynthesis,

converting chorismate to anthranilate, and undergoes feedback

inhibition by the end product (Haslam, 1993). Zhang et al. (2001)

expressed the ASA2 gene within the plastid genome of transgenic

tobacco plants. Their theory was that the regulation of ASA2

transcription within the nucleus could be overcome by expression

within plastids. Researchers also wanted to determine what effects a

plastid-expressed ASA2 gene would have on the expression of

nuclear-encoded AS genes and the b subunit genes, and to

determine if Trp biosynthesis can be increased in plastids. The

chloroplast transgenic plants showed a high level of accumulation of

ASA2 mRNA, an increased expression of the AS a subunit protein,

and demonstrated a 4-fold increase in AS enzyme activity that was

less sensitive to feedback inhibition by Trp. Researchers also

observed a 10-fold increase in free Trp in chloroplast transgenic

tobacco leaves. Western blots detected a much higher level of the a

subunit compared to wild type, indicating that the abundance of a

subunit encoded by ASA2 may stabilize the b subunit, which may

explain the increase in the holoenzyme (Zhang et al., 2001).

Plastid Genetic Engineering in Different Crop Species

Despite such significant progress in this field, chloroplast

transformation has been routinely done only in tobacco. With the

exception of carrot, all of the aforementioned studies reported plastid

transformation in tobacco. Major obstacles in extending this

technology to crop plants that regenerate through somatic

embryogenesis include inadequate tissue culture and regeneration

protocols, lack of selectable markers, and inability to express

transgenes in non-green plastids (Bogorad, 2000; Daniell et al.,

2002). The first challenge is to introduce foreign DNA into non-green

tissues containing several kinds of plastids, namely proplastids,

leucoplasts, amyloplasts, etioplasts, chromoplasts, elaioplasts, and

gerontoplasts, in which gene expression and gene regulation systems

are quite different from mature green chloroplasts (Bogorad, 2000).

Identification of appropriate regulatory sequences that function in

non-green plastids is necessary to achieve foreign gene expression.

Yet another major challenge is the ability to regenerate chloroplast

transgenic plants via somatic embryogenesis and achieve homo-

plasmy, which lacks the benefit of subsequent rounds of regeneration

offered by organogenesis, because segments of somatic embryos

cannot be regenerated into plants.

The chloroplast transformation vectors utilize homologous

flanking sequences for recombination and insertion of foreign

genes. Transformation of Arabidopsis, potato, and tomato chloroplast

genomes was achieved via organogenesis but the efficiency was

much lower than tobacco (Sikdar et al., 1998; Sidorov et al., 1999;

Ruf et al., 2001). In Arabidopsis, one chloroplast transgenic line per

40 or 151 bombarded plates was obtained but they were not fertile.

Likewise, in potato, one chloroplast transgenic line per 25

bombarded plates, and in tomato one transgenic line per 10

bombarded plates, was obtained (six clones in 520 selection plates).

In contrast, up to 14 chloroplast transgenic lines were obtained per

bombarded leaf in tobacco (Daniell et al., 2001b). In the case of

Lesquerella, transgenic clones had to be grafted onto Brassica napus

rootstock to obtain seeds. Only two chloroplast transgenic clones

were obtained from 51 bombarded samples (Skarjinskaia et al.,

2003). In soybean, only one heteroplasmic event was recovered out

of 984 bombardments performed on embryogenic suspension

cultures (Zhang et al., 2001) but this could not be regenerated. In

rice, transient integration of the transgene was demonstrated by

PCR analysis while stable integration was not achieved (Khan and

Maliga, 1999); transgenic plants were highly heteroplasmic and the

transgenes were not carried over to the next generation. Similarly, in

oilseed rape, Southern analysis of the transgenic chloroplast

genome was not shown to confirm homoplasmy (Hou et al., 2003).

Tissue culture limitations and use of non-green explants have often

been cited as primary reasons that have limited chloroplast

transformation to solanaceous crops (Bogorad, 2000).

In spite of aforementioned failures or limitations, plastid

genetic engineering has been achieved recently in several major

crops, including cotton and soybean (Dufourmantel et al., 2004;

Kumar et al., 2004a). In addition, the carrot plastid genome has

been transformed and transgenic plants were able to grow on

NaCl (up to 400mM), the highest level reported in the literature,

RECENT ADVANCES IN CHLOROPLAST GENETIC ENGINEERING 399

and accumulated betaine as high as in halophytes (Kumar et al.,

2004a). Therefore, the following paragraphs focus on recent

exciting developments in this field and offer directions for further

research and development.

Crop species. In an effort to transform a food crop, the concept of

chloroplast genetic engineering was used to insert a foreign gene into

potato plastid genome (Sidorov et al., 1999). Daniell et al. (1998)

developed the concept of the universal vector in which it should be

possible to transform chloroplast genomes of one species using

flanking sequences of a related species. The vectors used for potato

plastid transformation were originally designed for transformation of

the tobacco plastid genome. The homologous flanking sequences

present in these vectors show very high homology (98%) to the

corresponding sequences of potato. Therefore the integration of such

sequences in potato via homologous recombination was anticipated.

Another consideration of the vectors is the presence of tobacco

regulatory signals that drive the chimeric genes in potato plastids.

However, the transformation efficiency was very low, one event per 25

bombarded plates. In contrast, up to 14 chloroplast transgenic lines

were obtained per bombarded leaf in tobacco (Daniell et al., 2001b).

This low transformation efficiency could be due to poor homologous

recombination obtained with tobacco flanking sequences and the

difference in efficiency of shoot regeneration from potato leaf

explants.

Carrot is a biennial plant having a vegetative phase in the first year

and the reproductive phase in the second year. Harvesting the crop

containing the transgene at the end of first year prevents gene flow

through seeds or pollen due to lack of the reproductive system. Also,

maternal inheritance of carrot chloroplast genomes adds to the

environmentally friendly approach. Somatic embryos of carrot are

single-cell derived and multiply through recurrent embryogenesis,

which provides a uniform source of cell culture and homogeneous

single source of origin. In solanaceous crops, chloroplast genomes

have been transformed using fullymature chloroplasts as recipients of

foreign DNA and regeneration was achieved via direct organogenesis.

However, in order to transform other economically important crops, it

is essential to transform non-green cells that contain proplastids,

regenerate plants via somatic embryogenesis, and achieve homo-

plasmy without subsequent rounds of regeneration. The successful

transformation of carrot chloroplast genome was achieved by

expressing BADH through somatic embryogensis (Kumar et al.,

2004a). The expression of the BADH enzyme not only conferred the

highest level of salt tolerance (up to 400mM), but also greatly

facilitated the visual selection of the transgenic green cells from the

nontransformed yellow cells. High levels of foreign gene expression in

chromoplasts (up to 75% of expression observed in leaves) should

facilitate oral delivery of therapeutic proteins (Fig. 11).

Friable grayish callus induced from hypocotyl segments of fully

embryogenic cotton (Gossypium hirsutum cv. Coker 310FR) cultures

was bombarded with chloroplast vector pDD-Gh-aphA6/nptII. The

aphA is driven by regulatory elements gene 10 and trp1630UTR that

function in non-green plastids and the nptII is driven by the light-

regulated psbA 50- and 30-UTRs. This provides the ability to

overcome selection pressure day and night, without any interrup-

tion, in all tissue types. The flanking sequences were doubled (2 kb)

on either side to increase the efficiency of homologous

recombination and one of the flanks contained the complete

chloroplast origin of replication. Cotton chloroplast transgenic

FIG. 11. Transformation of the carrot plastid genome. Effect of salt (50–500mM NaCl) on untransformed ‘U’ and transgenic ‘T’ carrotlines grown on different concentrations of NaCl. Plants were irrigated with water containing different concentrations for up to 4 wk.

400 KOYA AND DANIELL

plants regenerated via somatic embryogenesis were fertile, flowered,

and set bolls/seeds as control untransformed plants. Transgenes

stably integrated into cotton plastid genomes were maternally

inherited and were not passed through transgenic pollen when

tested by cross-pollination with untransformed control plants

(Kumar et al., 2004b). This highly efficient and reproducible

process for plastid transformation through somatic embryogenesis

using various selectable markers should pave the way to engineer

the plastid genome of several major crops in which regeneration is

mediated through somatic embryogenesis (Fig. 12).

Recently soybean (Glycine max), a leguminous crop, has been

transformed using chloroplast transformation. Chloroplasts from

green embryogenic tissue of Glycine max were successfully

transformed by particle bombardment using the aadA gene under

the control of the tobacco plastid light regulatory sequences, flanked

by two adjacent soybean plastome sequences allowing its targeted

insertion between the trnVgene and the rps12/7 operon. All generated

spectinomycin-resistant plants were transformed and no remaining

wild-type plastome copies were detected. No spontaneous mutants

were obtained. All transplastomic T0 plants were fertile and T1

FIG. 12. Transformation of the cotton plastid genome using double barrel vector. Transgenic cotton calluses were converted intosomatic embryos (A) and elongated somatic embryos (B). Transgenic (C) and nontransgenic control cotton plant (D) at the stage of floweringand setting seeds. Different floral parts of transgenic cotton (E–G) and nontransgenic control cotton (H–J). F1 seedlings of crossesbetween transgenic F £ C nontransgenic cotton.

RECENT ADVANCES IN CHLOROPLAST GENETIC ENGINEERING 401

progeny was uniformly spectinomycin-resistant, confirming the

stability of the plastid transgene (Dufourmantel et al., 2004).

Plastid genomics. The concept of a ‘universal vector’ containing

plastid DNA flanking sequences from one plant species to transform

another species (of unknown genome sequence) was proposed in the

past decade (Daniell et al., 1998). Although this concept has been

used to successfully transform both potato and tomato plastid

genomes with flanking sequences from tobacco, the efficiency of these

transformations is significantly lower when compared to that of

tobacco (Sidorov et al., 1999; Ruf et al., 2001). Similar reduction in

efficiency was seen when Petunia flanking sequences (also ,98%

homologous) were used to transform the tobacco chloroplast genome

(DeGray et al., 2001), suggesting that a lack of complete homology

may greatly reduce the transformation efficiency. Lack of complete

chloroplast genome sequence is still one of the major limitations to

extending this technology to useful crops; only six published crop

chloroplast genomes are now available, although 200 non-crop

genomes have been sequenced or are in progress. Chloroplast genome

sequences are necessary for identification of spacer regions for

integration of transgenes at optimal sites via homologous recombina-

tion, as well as endogenous regulatory sequences for optimal

expression of transgenes. In higher plants, about 40–50% of each

chloroplast genome contains non-coding spacer and regulatory

regions. Therefore, we sequenced the soybean chloroplast genomes

using readily available BAC clones. The complete chloroplast

genome size of Glycine is 152 218 bp. The genome includes a pair of

inverted repeats (IRs) of 25 574 bp (IRa and IRb) of identical

sequence separated by a SSC region of 17 895 bp, and a LSC region of

83 175 bp. The IR extends from rps19 through a portion of ycf1 and

thus includes duplications of 19 genes. The Glycine chloroplast

genome contains 111 unique genes, and 19 of these are duplicated in

the IR, giving a total of 130 genes. There are 30 distinct tRNAs and

seven of these are duplicated in the IR. The genome consists of 60%

coding regions (52% protein coding genes and 8% RNA genes) and

40% non-coding regions, including both intergenic spacers and

introns. Gene content of the three sequenced legumes (Glycine,

determined in our study, Lotus NC_002694, and Medicago

NC_003119) is identical except that Medicago does not have

duplicate copies of the 19 genes in the IR because one copy of the IR

has been lost. The rpl22 gene is missing from all three legumes and

Medicago is also missing rps16. The gene order inGlycine differs from

the usual gene order for angiosperm chloroplast genomes by the

presence of a single, large inversion of approximately 51 kb that

reverses the order of the genes between rbcL and rps16. This same

inversion is also present in Lotus and Medicago. The rates and

patterns of sequence evolution were also compared among the three

completely sequenced legume genomes. Photosynthetic genes are

highly conserved across all genomes compared. Ribosomal protein

genes show almost the same levels of sequence divergence, whereas

RNA polymerase, NADH dehydrogenase genes, matK and ccsA have

diverged two to three times faster than photosystem genes. In

Medicago, which has only one copy of the IR, genes that are in the IR

evolved nearly two times faster than the same genes that are

duplicated in the IR of Lotus and Glycine. Synonymous substitution

rate in IR genes is two to four times lower than single-copy genes.

Many of the repeats in the intergenic spacer regions and introns are

found in the same location in the other legumes and in Arabidopsis,

suggesting that they may play some functional role in the genome.

Several lines of evidence also suggest that the psbA and rbcL repeats

did not play a role in causing the deletion of IR (Saski et al., 2005).We

have now completed the chloroplast genomes of tomato, potato,

barley, and sorghum. Sequencing of 50 other crop chloroplast

genomes is now in progress.

Conclusions

Chloroplast genetic engineering of plants opens the door for the

development of transgenic crop plants with desired agronomic traits.

The concept of introducing foreign genes into the plastid genome has

led to the newera ofmolecularmedicine for the production of vaccines

and biopharmaceuticals. The higher expression levels facilitate

inexpensive and human pathogen-free production of pharmaceutical

proteins in plants. Recent developments in transforming the plastid

genomes of edible crop plants open doors for inexpensive oral delivery

of therapeutic proteins, eliminating the expensive step of protein

purification. The concept ofmaternal inheritance of transgenes should

facilitate an environmentally friendly approach to producing

genetically modified crops and minimize public misconception

about genetically modified crops. However, the current protocols for

transforming different species of crop plants are limited by low

transformation efficiency and lack of information on chloroplast

genomes. It is surprising that among 40 published chloroplast

genomes and 200 chloroplast genome sequencing efforts, only six are

crop chloroplast genomes. With generation of more information on

crop chloroplast genomes and improved transformation protocols on

major crops, plastid transformation is likely to overcome current

concerns about genetically modified crops.

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