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 184.108.40.206), 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.