Chloroplast transformation for transgene containment
Chloroplasts are a class of plastids - organelles in
plant cells – apparently derived from a cyanobacteria (blue-green bacteria)
ancestor that once lived symbiotically inside the plant cell. Chloroplasts
contain chlorophyll and are found in the shoots and leaves of green plants,
while colourless plastids are found in the roots and other coloured plastids
are found in fruit. The number of plastids in each cell is variable, and each plastid
contains multiple copies of its own genome, typically 50 to 100. Many plastid
genomes have been sequenced. They resemble bacterial genomes in many respects;
though features normally found in muticellular organisms, such as interrupted
genes and RNA editing are also present. The chloroplast genome codes for the
transcription and translation machinery of the chloroplast plus numerous
structural proteins. But the vast majority of the chloroplast proteins are
encoded in the plant nucleus and imported into the chloroplast after synthesis.
Stable transformation of the chloroplast – putting foreign genes into the
chloroplast genome - was first achieved in the single cell green alga Chlamydomonas
reinhardtii in 1988, soon to be followed by tobacco plant, and more recently,
Arabidopsis thaliano . Several biotech companies, including Monsanto,
Rhone-Poulenc, Novartis, American Cyanamid, Calgene, Pioneer Hybrid have initiated
major programmes on chloroplast transformation since the late 1990s .
transformation has been touted at least as far back as 1998 as a means of “containing”
transgenes; that is, preventing them from transferring to non-GM crops or wild
relatives through pollen, and hence preventing the creation of transgenic
herbicide tolerant weeds. The theory is that chloroplasts are inherited
exclusively through the female line.
Joe Cummins has exposed the fallacy of this claim . He pointed out that
tobacco pollen does transfer chloroplast transgenes under selection with a herbicide-like
drug tentoxin. It is well known that chloroplasts are mainly inherited through
pollen in conifers, and major crops such as alfalfa inherit chloroplasts from
both pollen and egg. There is also occasional biparental inheritance of chloroplast
genes in rice, and cultivars of peas vary in the presence of chloroplast DNA
in pollen. These cases, he emphasized, are just a few examples from a large
literature showing that chloroplasts are inherited through pollen, pollen and
egg, or selectively influenced by stress to transmit chloroplast genes through
pollen where maternal transmission is usual.
Surprisingly, C.S. Prakash, later to become a major protagonist for GM crops,
co-authored a letter with C. Neal Stewart, Jr., agreeing with Cummins, which
was published on the same page of the journal Nature Biotechnology .
They pointed out in addition that pollen spreading to GM crops from weeds could
create herbicide tolerant weeds, as in the case of GM canola, which showed increased
cross-pollination by weedy relatives compared to the reciprocal cross. They
added, “Overstating the biosafety of cp [chloroplast]-transgenic crops with
regard to gene flow could lead to policy mistakes and ecological problems. We
would hope that assumptions of biosafety regarding gene flow using any system
will be empirically tested and not treated as brute fact. Second, we hope that
monitoring for transgene-introgressed weeds will become the norm for potentially
problematic crops such as canola.” We couldn’t agree more. But that advice has
fallen on deaf ears, including those of the subsequently transformed CS Prakash.
Other benefits of chloroplast transformation
Peter J. Nixon of Imperial College, London
University, in a paper published by UK’s Department for Environment, Food
& Rural Affairs (DEFRA) in February 2001 , again recommended
fallaciously, chloroplast transformation as a means of containing transgenes;
but also mentioned other advantages.
transformation involves homologous recombination. This not only minimises the
insertion of unnecessary DNA that accompanies transformation of the nuclear
genome, but also allows precise targeting of inserted genes, thereby also
avoiding the uncontrollable, unpredictable rearrangements and deletions of
transgene DNA as well as host genome DNA at the site of insertion that
characterises nuclear transformation . In practice, the inserted transgene
has short DNA sequence tails added at each end, the tails are homologous to
sequences on the chloroplast target gene, which thus initiate homologous
recombination. Once the transgene is inserted into the chloroplast chromosome,
the target gene is disrupted. The disruption of the target gene is expected to
alter the growth and metabolism of the plant.
Leaf discs are
bombarded with plasmid constructs containing a selectable antibiotic resistance
marker physically linked to the gene of interest, flanked by DNA for inserting
into the correct site of the chloroplast genome. The antibiotic resistance
marker most frequently used is the aadA
gene encoding resistance for spectinomycin and streptomycin, driven by the
promoter of the chloroplast encoded 16S rRNA gene.
Nixon, this transformation procedure applied to tobacco, Arabidopsis or oil seed rape, generates
plants in which all the chloroplast genomes are uniformly transformed (a
condition referred to as homoplasmic), despite the fact that tobacco leaf cells
may contain 100 chloroplasts, each containing 100 copies of the chloroplast
Another advantage of chloroplast transformation is that foreign genes can
be over-expressed, due to the high gene copy number, up to 100 000 compared
with single-copy nuclear genes. And there does not seem to be gene-silencing
and other instability that plague nuclear transformation. The gene product is
retained inside the chloroplasts or can in principle be targeted to a specific
compartment in the chloroplast.
However, a somewhat less
rosy picture on chloroplast transformation was painted by Pal Maliga of Waksman
Institute, Rutgers University, New Jersey in the United States,
commenting on the successful plastid transformation in tomato , in which
notable levels of transgene protein accumulated in the tomato fruit, indicating
that the tomato fruit may be a useful system for producing edible vaccines.
Until then, plastid transformation has only been successful in tobacco plants
in that fertile plants are obtained that transmitted the transgene to the next
generation. Although transplastomic potatoes, Arabidopsis
and rice have been obtained, these plants have not yet been shown to
transmit transgenes to the next generation. One major difficulty is in getting homoplasmic
plants – plants in which all the chloroplasts are uniformly transformed, for
that takes a long process of selection. The process in Arabidopsis for example, yields 100 times
fewer lines per transformed sample than tobacco.
Another problem is to get
high level of protein expression, even though the gene copy number is high. In
chloroplasts, post-transcriptional processes determine the levels of proteins
expressed, depending on translational signals.
High protein accumulation of transgene product in the tomato fruit is good
news for those interested in the protein, but bad news for those planning to
produce the transplastomic crop successfully. Because the protein levels required
for selection is greater than 10% of total soluble protein in rice, it may constitute
a significant metabolic burden on the plants. Furthermore, the high level of
expression of antibiotic resistance marker gene would greatly exacerbate public
concern over the environment release of such transplastomic plants, although
techniques for removing the antibiotic resistance marker gene, once it has served
its useful purpose, are being developed.
Nevertheless, Maliga ended on an optimistic note: “the capacity to express
foreign proteins at a high level in a consumable fruit should open new opportunities
for engineering the next generation of medicinal products that are more palatable
to the consumer.”
Molecular pharming by chloroplast transformation entails unique risks
There are currently 37
patents for molecular pharming by chloroplast transformation listed . The
first commercial exploitation of chloroplast transformation for molecular pharming
is likely to be in Chlamydomonas reinhardtii
(“GM pharmaceuticals in common green alga”).
Chloroplast transformation to produce GM pharmaceuticals entails specific
risks that are associated with its advantages.
The high level of
transgene expression that can be achieved increases the hazards of
environmental contamination and inadvertent exposure of human subjects,
domestic livestock and wild life.
The high copy number
of transgenes increases the hazards of horizontal gene transfer to
bacteria and viruses, with the potential of creating dangerous pathogens
and spreading antibiotic resistance marker genes. It is now known that DNA
persists in all environments, and transformation by direct uptake of DNA
is a major route of horizontal gene transfer among bacteria .
The close similarities
(homologies) between plastid and bacterial genomes is expected to greatly
increase the frequency of horizontal gene transfer, up to a billion-fold
. Furthermore, the horizontal transfer of non-homologous DNA occurs at
relatively high frequencies when a homologous DNA ‘anchor sequence’ is
present, which can be as short as 99bp.
There are at least
87 species of naturally transformable bacteria in the soil .
The disruption of
the target gene in transformation results in changes in the growth and
metabolism of the plant that may pose risks to health and the environment.
There can be no environmental releases of chloroplast
transformed crops or algae producing GM
pharmaceuticals. They must be firmly confined in contained use where every
precaution is taken to prevent environmental releases not only of the living
transgenic organism or cells, but also of transgenic DNA.