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ISIS Report 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.

  1. 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.
  2. 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].
  3. 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.
  4. There are at least 87 species of naturally transformable bacteria in the soil [10].
  5. 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.

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