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[SANET-MG] pharming by hloroplast
http://www.i-sis.org.uk/MPBCT.phpISIS Press Release 20/07/05
Molecular Pharming by Chloroplast Transformation
The advantages are also its greatest hazards; no environmental releases
should be considered. Dr. Mae-Wan Ho and Professor Joe Cummins
A fully referenced version of this article is posted on ISIS members’
website. Details here
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 [1]. Several biotech
companies, including Monsanto, Rhone-Poulenc, Novartis, American
Cyanamid, Calgene, Pioneer Hybrid have initiated major programmes on
chloroplast transformation since the late 1990s [2].
Chloroplast 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 [3]. 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 [4]. 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 [1], again recommended fallaciously,
chloroplast transformation as a means of containing transgenes; but also
mentioned other advantages.
Chloroplast 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 [5]. 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.
According to 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 genome.
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.
Benefits over-stated
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 [6], 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 [7]. 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 [8].
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 [9]. 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 [10].
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.
The Institute of Science in Society, PO Box 32097, London NW1 OXR
telephone: [44 20 8452 2729] [44 20 7272 5636]
General Enquiries sam@i-sis.org.uk - Website/Mailing List
press-release@i-sis.org.uk - ISIS Director m.w.ho@i-sis.org.uk
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