Molecular Pharming by Chloroplast Transformation

[Guest guest]

20 Jul 2005 15:51:51 -0000

Molecular Pharming by Chloroplast Transformation

press-release

 

 

The Institute of Science in Society Science Society

Sustainability http://www.i-sis.org.uk

 

General Enquiries sam Website/Mailing List

press-release ISIS Director m.w.ho

========================================================

 

 

ISIS 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 http://www.i-sis.org.uk/full/MPBCTFull.php.

Details here http://www.i-sis.org.uk/membership.php

 

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.

 

 

========================================================

This article can be found on the I-SIS website at

http://www.i-sis.org.uk/MPBCT.php

 

If you like this original article from the Institute of

Science in Society, and would like to continue receiving

articles of this calibre, please consider making a donation

or purchase on our website

 

http://www.i-sis.org.uk/donations.

 

ISIS is an independent, not-for-profit organisation

dedicated to providing critical public information on

cutting edge science, and to promoting social accountability

and ecological sustainability in science.

 

If you would prefer to receive future mailings as HTML

please let us know. If you would like to be removed from our

mailing list at

 

http://www.i-sis.org.uk/mailinglist/.php

========================================================

CONTACT DETAILS

 

The Institute of Science in Society, PO Box 32097, London

NW1 OXR

 

telephone: [44 1994 231623] [44 20 8452 2729] [44 20

7272 5636]

 

General Enquiries sam Website/Mailing List

press-release ISIS Director m.w.ho

 

MATERIAL ON THIS SITE MAY BE REPRODUCED FOR ANY PROFIT FREE

PURPOSES WITHOUT PERMISSION, ON CONDITION THAT IT IS

ACCREDITED ACCORDINGLY AND CONTAINS A LINK TO http://www.i-

sis.org.uk/.

ANY COMMERCIAL USE MUST BE AGREED WITH ISIS