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Procedure of Chloroplast Genetic Engineering

A novel system that appears to circumvent the concerns about nuclear modification is genetic engineering
of chloroplast DNA. In chloroplast genetic engineering, the recombinant DNA plasmid is bound to small
gold nanoparticles that are then injected into the chloroplasts of a leaf using a “gene gun” (Gaglani,
2006). This device uses high pressure to insert the plasmid-coated particles into the cell. These plasmids
contain multiple important genes: the therapeutic gene, a gene for antibiotic resistance, a gene that
increases expression of the therapeutic gene, and two flanking sequences that ensure that the plasmid is
not randomly integrated into the chloroplast genome. The flanking sequences guide the human
recombinant DNA into a specific place on the chloroplast genome by binding to corresponding parts on
the genome. The leaf is then grown on a plate containing an antibiotic, which ensures that the only
surviving plant cells will be those that contain the gene for antibiotic resistance and—therefore—contain
the therapeutic gene as well. These cells are then exposed to regenerative factors that induce them to start
sprouting shoots and grow into full plants that express the desired protein. Figure 14 provides details of
the procedure of chloroplast genetic engineering.

Elimination of marker genes from the plastid genome

Incorporation of a selectable marker gene during transformation is essential to obtain transformed
plastids. However, once transformation is accomplished, having the marker gene becomes undesirable.
Corneille et al. (2001) reported on adapting the P1 bacteriophage CRE-lox site-specific recombination
system for the elimination of marker genes from the plastid genome. The system was tested by the
elimination of a negative selectable marker, codA, which is flanked by two directly oriented lox sites
(codA). Highly efficient elimination of codA was triggered by introduction of a nuclear-encoded plastid-
targeted CRE by Agrobacterium transformation or via pollen. Excision of codA in tissue culture cells was
frequently accompanied by a large deletion of a plastid genome segment which includes the tRNA-Val UAC
gene. However, the large deletions were absent when cre was introduced by pollination. Thus pollination
is our preferred protocol for the introduction of cre. Removal of the codA coding region occurred at a
dramatic speed, in striking contrast to the slow and gradual build-up of transgenic copies during plastid
transformation. The nuclear cre gene could subsequently be removed by segregation in the seed progeny.
The modified CRE-lox system will be a highly efficient tool to obtain marker-free transplastomic plants.

Another method to remove the selection gene from higher plant plastids is based on loop-out
recombination, a process difficult to control because selection of homoplastomic transformants is
unpredictable. Another method uses the CRE/lox system, but requires additional retransformation and
sexual crossing for introduction and subsequent removal of the CRE recombinase. Klaus et al. (2004)
describe the generation of marker-free chloroplast transformants in tobacco using the reconstitution of
wild-type pigmentation in combination with plastid transformation vectors, which prevent stable
integration of the kanamycin selection marker. One benefit of a procedure using mutants is that marker-
free plastid transformants can be produced directly in the first generation (T0) without retransformation or

Some Examples of GeneticallyEngineered Chloroplasts

Chloroplast transformation in oilseed rape

The chloroplast transformation vector pNRAB carries two expression cassettes for the spectinomycin
resistance gene aadA and the insect resistance gene cry1Aa10. The two cassettes are sited between the
rps7 and ndhB targeting fragments. Biolistic delivery of the vector DNA, followed by spectinomycin
selection, yielded chloroplast transformants at a frequency of four in 1,000 bombarded cotyledon petioles.
PCR analysis and Southern blot of PCR products confirmed the site-specific integration of aadA and
cry1Aa10 into the chloroplast genomes of transgenic oilseed rape. When transgenic oilseed rape leaves
were fed to second instar Plutella xylostera larvae, 47% mortality was observed against this insect and the
surviving larvae had significantly lower weight than the control. This is the first report of chloroplast
transformation in oilseed rape and the introduction of novel genes between the rps7 and ndhB genes in the
chloroplast genome (Hou et al., 2003).

Chloroplast transformation in tobacco

Efficient plastid transformation has been achieved in Nicotiana tabacum using cloned plastid DNA of
Solanum nigrum carrying mutations conferring spectinomycin and streptomycin resistance (Kavanagh et
al., 1999) by use of the incompletely homologous (homeologous). Physical mapping and nucleotide
sequence analysis of the targeted plastid DNA region in the transformants demonstrated efficient site-
specific integration of the 7.8-kb Solanum plastid DNA and the exclusion of the vector DNA. The
integration of the cloned Solanum plastid DNA into the Nicotiana plastid genome involved multiple
recombination events as revealed by the presence of discontinuous tracts of Solanum-specific sequences
that were interspersed between Nicotiana-specific markers.
Huang et al. (2002) reported on the development of a new dominant selection marker for plastid
transformation in higher plants using the aminoglycoside phosphotransferase gene aphA-6 from
Acinetobacter baumannii. Vectors containing chimeric aphA-6 gene constructs were introduced into the
tobacco chloroplast using particle bombardment of alginate-embedded protoplast-derived micro colonies
or polyethylene glycol (PEG)-mediated DNA uptake. Targeted insertion into the plastome was achieved
via homologous recombination, and plastid transformants were recovered on the basis of their resistance
to kanamycin. Variations in kanamycin resistance in transplastomic lines were observed depending on the
5' and 3' regulatory elements associated with the aphA-6 coding region. Transplastomic plants were fertile
and showed maternal inheritance of the transplastome in the progeny.

Advantages of Chloroplast Genetic Engineering

Technique is environmentally friendly. Unlike nuclear transformation, this method ensures that the
recombinant transgenes are contained within the chloroplast and therefore will not spread to other plants.
Chloroplasts (and the genes they contain) are not passed in the sperm (i.e., pollen) of a plant, so they
cannot be spread by pollination. Researchers demonstrated that, even though chloroplasts in leaves were
modified to express an insecticidal protein, called CRY, at very high levels (47% total soluble protein),
the pollen did not contain any traces of the protein (DeCosa et al. 2001; Daniell, 2006). This signifies that
the recombinant genes and proteins are contained within the chloroplast so this technique is
environmentally friendly.

Allows large-scale protein production. Chloroplast engineering also allows for large-scale protein produc-
tion. The levels of pharmaceutical proteins produced in nuclear-modified plants are less than 1% of the
levels needed for the purified protein to be commercially feasible (Kusnadi et al., 1997). Each plant cell
contains approximately 100 chloroplasts and each chloroplast contains about 100 copies of its genome.
So, chloroplast genetically-engineered plants have high levels of integration of transgenes—up to 10,000
copies per cell—which elevate expression levels of recombinant proteins (up to 47% of the plant’s total
soluble protein) (Bendich, 1987b).

Applications of Chloroplast Genetic Engineering

Therapeutic proteins produced. A number of therapeutic proteins have been produced using the
chloroplast genetic engineering system. These include human somatotropin (growth hormone), serum
albumin (blood protein), insulin-like growth factor (hormone), antimicrobial peptides (proteins that kill
pathogens), interferon alpha/gamma (cytokines in the immune system which are effective against
hepatitis and leukemia), monoclonal antibodies (immune system molecules that fight off invading
pathogens and toxins), and vaccines to cholera, plague, canine parvovirus (dog virus) and anthrax
(Grevich & Daniell, 2005). Each of these proteins is clinically relevant and has not been produced
efficiently in nuclear modified plants. Somatotropin, also known as human growth hormone, is used as
therapy for stunted growth and even to help maintain muscle mass in patients. Serum albumin is the most
widely used intravenous protein that is administered to replace blood volume since it accounts for 60% of
blood protein composition (Fernandez-San Millan et al. 2003). Insulin is a crucial hormone that regulates
carbohydrate metabolism and therefore energy production. It was the first marketed therapeutic protein
produced through genetic engineering (the pharmaceutical company Eli Lilly sold it as Humulin
beginning in 1982). Most of the therapeutic proteins produced by chloroplast genetic engineering are still
in the developmental stage and need to be tested in humans. Chlorogen Inc. is working to commercialize
this technology and bring the plant-produced therapeutic proteins to the market. According to
Chlorogen’s site, their first product will be human serum albumin for the non-therapeutic market
(Chlorogen Inc., URL: More work is required before chloroplast genetic
engineering can be applied commercially. This work will probably include modifying more types of crops
and plants as well as ensuring the functionality of the resultant therapeutic proteins in humans. But it may
not be too far in the future when mothers may nag their children not only to eat their broccoli, but to eat
their transgenes.

Vaccine production. Vaccines are, of course, needed to immunize people from harmful pathogens, such
as the polio virus, but many times there is a shortage of the amount of vaccine available. Plague vaccine,
which immunizes against Yersinia pestis, has been expressed in transgenic tobacco plants at
commercially feasible levels. In addition, the canine parvorvirus vaccine (CPV), which protects dogs
against CPV and stomach complications, has been expressed highly. Recently, a team of scientists
working on chloroplast genetic engineering reported achieving such high levels of expression of the
anthrax protective antigen that, according to their extrapolation, one acre of transgenic tobacco could
produce about 400 million doses of the vaccine (Watson et al., 2004). This is a crucial property of vaccine
production due to modern concerns over viral epidemics such as avian flu.

The possibility to directly manipulate chloroplast genome-encoded information has paved the way to
detailed in vivo studies of virtually all aspects of plastid gene expression (Bock, 2001). Moreover, plastid
transformation technologies have been intensely used in functional genomics by performing gene
knockouts and site-directed mutageneses of plastid genes. These studies have contributed greatly to our
understanding of the physiology and biochemistry of biogenergetic processes inside the plastid
compartment. Plastid transformation technologies have also stirred considerable excitement among plant
biotechnologists, since transgene expression from the plastid genome offers a number of most attractive
advantages, including high-level foreign protein expression and transgene containment due to lack of
pollen transmission.

Plastid transformation is becoming more popular and an alternative to nuclear gene transformation
because of various advantages like high protein levels, the feasibility of expressing multiple proteins from
polycistronic mRNAs, and gene containment through the lack of pollen transmission. Recently, much
progress in plastid engineering has been made. In addition to model plant tobacco, many transplastomic
crop plants have been generated which possess higher resistance to biotic and abiotic stresses and
molecular pharming. (For some more examples of transplastomic plants developed so far through plastid
engineering and the various applications and advantages of plastid transformation refer to Wani et al.