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Wednesday, March 14, 2007

Roundup Ready® Transgenic Plants

By the late 1990's it was apparent that recombinant DNA technology [see Nobel Laureate: Paul Berg] had advanced to the point where it was feasible to consider the production of genetically modified crops. One of the first targets was the creation of plants that were resistant to the herbicide glyphosate or Roundup® [How Roundup® Works].

Surprisingly, in spite of extensive spraying with Roundup® no resistant plant species had been detected. Since the target of glyphosate, EPSP synthase (EC, is also present in bacteria, a search for resistant bacteria was undertaken. The idea is that if a glyphosate-resistant enzyme from bacteria could be transferred to plants it might make the plants resistant to the herbicide. Such Roundup Ready® transgenic plants be an enormous advantage for farmers since a crop of, say Roundup Ready® soybean, could be sprayed with Roundup® to kill all weeds without affecting the crop.

Coincidently , it would be of enormous advantage to Monsanto, the manufacturer of Roundup®, especially if they could control the distribution of the genetically modified plants.

The C4 strain of Agrobacterium sp. proved to be just the thing. This is a species of bacteria that was found growing in the waste-fed column at a factory that made glyphosate. The EPSP synthase enzyme from this bacterium (C4 EPSP synthase) was almost completely insensitive to glyphosate.

The C4 EPSP bacterial gene was cloned and inserted into a bacterial plant vector in order to prepare for cloning into plants. The details of one of the Monsanto C4 EPSP cloning vectors are shown in the first patent filed on September 13, 1994 [US Patent 05633435].

This is a modifed bacterial plasmid vector designed to be propagated in E. coli (for cloning and construction) and Agrobacterium tumefaciens (for transforming plants). Ori-322 is an origin of replication from plasmid pBr322. It is used in E. coli to replicate the plasmid. Ori-V is an origin from plasmid RK2, a plasmid that can propagate in a wide variety of gram negative bacteria, including Agrobacterium tumefaciens. Rop is a small gene that encodes a protein requried to maintain plasmid copy number in bacteria.

There are two selectable markers. SPC/STR encodes a protein conferring spectinomycin/streptomycin resistance. The gene is derived from transposon Tn7. AAC(3)-III encodes bacterial gentamycin-3-N-acetyl transferase type III allowing selection for gentamycin resistance in plants. The bacterial AAC(3)-III gene has to be modified in order to allow effient expression in plant cells. A plant promoter (P-35S) is inserted at the 5' end. This promoter is the 35S promoter from cauliflower mosaic virus (CaMV). The 3' end of the gene is modified by inserting the polyadenylation site (NOS 3') from the nopaline synthase gene of the tumor-inducing (Ti) plasmid from Agrobacterium tumefaciens.

Similarly, the bacterial C4 EPSP gene was modified to have a strong plant promoter (P-e35S, related to P-35S) and a polyadenylation site (NOS 3'). One additional modification is necessary because the plant EPSP synthase is in chloroplasts where synthesis of chorimsate takes place. The bacterial gene has to have an N-terminal leader sequence that targets the protein to the chloroplast. This is supplied by CTP2, the chloroplast transit peptide from the Arabidopsis (wall cress) EPSP synthase gene.

The shuttle plasmid is built in E. coli then purified plasmid DNA is used to transform Agrobacterium tumefaciens. This bacterium infects plants and injects DNA from a Ti-like plasmid into plant cells where it enters the nucleus and becomes incorporated into the plant chromsomes. Under normal circumstances Agrobacterium tumefaciens causes gall tumors in plants but in this case the recombinat DNA is transferred and no tumors are formed. The transformation is mediated by cutting the plasmid at the RIGHT BORDER to produce a linear DNA molecule. Defective Ti plasmids in the bacterial cell are required to promote the transfer of the recombinant DNA.

The interesting feature of this transformation is that it is mediated by the bacteria. All you need to do is expose the plant cells to the bacteria under the right conditions and your gene of interest will end up in a plant chromsome.

The complete process begins with the isolation of small bits of plant tissue. They are grown on nutrient plates before being exposed to the bacteria carying the recombinant DNA plasmid.

Transformed cells will start to grow and they can eventually be isolated and transferred to a liquid that promotes shoot growth. After a few weeks you end up with an entire plant carrying the recombinant DNA. This plant is then propagated to produce thousands of genetically modified plants and seeds.

Roundup Ready® soybean was the first crop plant produced by Monsanto. Today, 90% of the soybean crop in the USA consists of Roundup Ready® plants. You can't buy soybean products that don't come from genetically modified plants.

Two thirds of the cotton and a quarter of the corn crop are Roundup Ready® plants. There is some resistance to growing Roundup Ready® wheat.


Torbjörn Larsson said...

"a protein conferring spectinomycin/streptomycin resistance"

So a couple of antibiotic resistance genes gets spread outside the fabrication process because gene analysis (or perhaps bioluminescence markers) and mechanical sorting is expensive. Or why not select by using Roundup? The results from the corporate world are mysterious.

Larry Moran said...

Why do you say they're "mysterious"? Can you think of a better way it could have been done in 1992-1993?

I have no idea how they do it today. Do you?

Torbjörn Larsson said...

It was tongue in cheek.

No, I don't know how they do it, which is why I am curious about the alternatives.

Tony Jackson said...

It’s claimed that antibiotic resistance marker genes might end up in bacteria and thus contribute to the problem of antibiotic resistance. Frankly, this hypothetical scenario seems unlikely as a major problem, especially when compared to the current massive over use of antibiotics in medicine and agriculture - which really is promoting the evolution of antibiotic resistance in a big way.

Nonetheless, plant biotechnologists are well aware of the public concern and have been quietly developing alternatives.

According to my plant scientist friends, there are several different approaches being looked at. One is to simply insert a plant intron into the antibiotic resistance gene. That way, even if it did ‘escape’ and found itself in a bacterial chromosome (and also next to a convenient bacterial promoter), the gene still wouldn’t work.

Another possibility is to move from a selection to a screening-based system. eg the GUS (beta glucuronidase) gene encodes an enzyme whose activity can be detected with a suitable dye.

A third approach is to remove the selection gene altogether before commercial release. To do this, there have been attempts to develop something similar to the Cre/Lox system. ie the antibiotic selectable marker gene is flanked by special DNA sequences recognised by an enzyme that cuts the marker out. The gene for this excision enzyme is inserted into the plant genome together with the selectable marker, but it’s expression is controlled by a pollen/seed-specific promoter. That way, the antibiotic selection marker is removed from the pollen and seed.

Of course, each of these methods will have to undergo their own safety trials etc, so I don’t know how close they are to commercial reality. In any case, even if these alternatives were widely adopted, I have a feeling that anti-GM activists will simply come up with some other reason why they’re still a bad idea...

Anonymous said...

Thank you for your articles about Roundup and the related articles it helped us a lot to write our personal and professional opinion for our ethics course
Thanks a lot!

Anonymous said...

I just stumbled on this post, looking for details on how GM plants are actually made. Wondering if you compiled this or where I could get reference citations?

Anonymous said...

Do you know if this means that the GM plant is making both the EPSPS (native) and the c4-EPSPS (resistant) gene product, so that it is both making glyphosate-susceptible and glyphosate-resistant proteins?

Keith Reding said...

Yes. The plant makes both EPSPS proteins. The native protein is inhibited by glyphosate and the resistant protein makes the aromatic amino acids.