Thirty years of plant transformation A case study exploring the impact of plant transformation technology on plant science research and the global agricultural biotechnology industry Credits, left to right, Dominik Maenni, Jason Yardley, Toony, US Agricultural Research Service Thirty years of plant transformation In 1983 researchers demonstrated that they could insert role in identifying the genetic basis of important crop new genes into a plant genome, using a species of soil characteristics, which breeders are incorporating into Impact Summary bacteria called Agrobacterium tumefaciens. new varieties with increased yield, disease resistance AFRC-funded research at the Plant Breeding or which produce healthier, more nutritious food. Institute (PBI), Cambridge, published in 1983 The breakthrough was made simultaneously by three Much of this work relies on genetic tools developed by enabled researchers to create transgenic research groups, including a team at the Plant Breeding researchers at PBI. plants using Agrobacterium-mediated plant Institute (PBI) in Cambridge UK, which was funded transformation. by BBSRC’s predecessor the Agricultural and Food Plant transformation technology also led to the creation Research Council (AFRC). The genetic tools developed of the agricultural biotechnology industry, which in at PBI became freely available to academics, ensuring 2012 had a global market value of US$14.84Bn1. The the technique was adopted by research groups around genetically modified crops produced by this industry are The technology, including novel vectors and the world. now grown in countries such as the USA, Brazil, India reporter genes developed at PBI, revolutionised and China, although different regulations and on-going research around the world, and now forms a Since then, the technology, known as ‘Agrobacterium- public debate mean few are grown in Europe. fundamental tool for plant molecular biology. mediated plant transformation’, has revolutionised plant and crop science research. It has enabled researchers This case study explores the continuing impact to explore gene function, furthering our understanding of research into Agrobacterium-mediated plant Two of the top three most-cited papers in plant of how plants develop, adapt to their environment and transformation at the PBI in the 1980s, funded by the transformation research arose from research at fight off pests and diseases. It has also played a vital AFRC. PBI. Plant transformation technology also enabled the formation of the global agricultural biotechnology industry, which now has a market value of US$14.84Bn. Tobacco plant, a tomato and a US field of corn. All three crops have played an important role in the development of plant transformation tech- nology and the agri-technology industry. Credit: Dominik Maenni/David Besa, Sonoma, USA/Graylight. A history of the discovery In 1983, three groups published research papers Agrobacterium T-DNA to drive the expression [of the demonstrating that they could stably introduce and new gene in the plant cell],” Bevan explains. “I got that Timeline of key events express bacterial genes into the genome of a tobacco project going in Mary-Dell’s lab. Then I came back to 1974: Marc Van Montagu and Jeff Schell at plant, a commonly-used model plant2,3,4. One of those the UK and I got a job at the PBI as a post-doc and I Ghent University show that Agrobacterium uses papers was authored by Mike Bevan and Dick Flavell continued the project there.” a plasmid, known as the Ti plasmid, to transfer at the UK’s PBI in Cambridge, which was supported its genetic material into a plant cell. by the AFRC, and Mary-Dell Chilton at Washington Independently, Dick Flavell’s research group at the PBI University in the USA, who pioneered research into had also started to investigate the possibility of inserting 1979: Working at the AFRC-funded PBI in Agrobacterium4. They showed not only that they could novel genes into plants to aid plant breeders, building Cambridge, Dick Flavell is the first researcher use Agrobacterium to insert bacterial genes reliably on work in the 1970s that allowed researchers to isolate anywhere in the world to successfully clone plant into tobacco plants, but that the inserted genes were specific genes. DNA. inherited and expressed by subsequent generations of the plants. “If you’re sitting in a plant breeding institute, it is clear 1983: Three groups, including Mike Bevan and that the goal of plant breeders is to introduce new Dick Flavell at PBI, UK, publish research papers Bevan’s research began when he joined Mary-Dell and more useful genes into crops. When it started to demonstrating that they can stably introduce and express bacterial genes into a plant genome. Chilton’s group at Washington University, St Louis in 1980. He was interested in the genes in T-DNA – the 1984: Mike Bevan creates the BIN19 binary length of DNA transferred from the Agrobacterium to the vector and begins distributing it to academic plant. Bevan and Chilton had the idea that they could researchers around the world. use elements of the genes in the T-DNA to express a bacterial gene in the plant cells. In particular, they chose 1987: Creation of the GUS reporter system by a bacterial gene that would confer antibiotic resistance. Richard Jefferson and Mike Bevan. The paper As a result, the researchers could use the gene as a describing their work is now the most highly-cited ‘selectable marker’, because adding an antibiotic (at plant transformation paper to date. lethal levels) would eliminate any cells that had not been transformed. 1987: First field trial of a GM crop (potatoes) in the UK, by researchers at PBI. The transgenic potatoes included the GUS reporter gene. Bevan identified a promoter and a terminator sequence in the T-DNA, which worked as genetic switches to control the expression of a gene in the plant. “The The former PBI offices in Trumpington, Cambridge, UK. innovation was to use a genetic element from the Credit: Wikimedia/James Yardley become obvious that there might be a way of moving In 1982, Bevan joined the PBI to continue his work. By genes, in the laboratory, from other organisms in to crop the end of the year he had shown that it was possible Timeline of key events plants, then it was a technology that was destined to be to express the bacterial antibiotic resistance gene in 1988: Don Grierson at the University of extremely useful,” explains Flavell. “So my group in the tobacco plant cells, and that the antibiotic gene could be Nottingham, working with agri-tech company PBI set about attempting to do that,” he adds. used as a selectable marker to identify only those cells Zeneca (now Syngenta), uses transformation that had been transformed. Importantly, the researchers to knock out the polygalacturonase gene in Their first forays into plant transformation in the late also demonstrated that the genes could be inherited by tomatoes, which degrades the pectin in the 1970s were focussed on using protoplasts – plant subsequent generations of the plants according to the tomato fruit cell wall. cells with their tough cellulose cell walls removed – in rules of Mendelian inheritance. collaboration with the AFRC-funded work of Edward 1994: EU approves the first GM crop, a tobacco Cocking at the University of Nottingham. However, with Alongside two other groups, one led by Jeff Schell plant carrying resistance to a herbicide, to be the discovery that Agrobacterium could transfer its own and Marc Van Montagu at Ghent University, Belgium, grown commercially in Europe. DNA into plants, Flavell’s group altered the focus of its and the other by Rob Fraley at agri-tech company research. Monsanto, Mary-Dell Chilton announced their discovery 1994: Calgene’s Flavr Savr tomatoes go on sale at the Miami Winter Symposium in January 1983. in the USA. The tomatoes use GM to knock out the polygalacturonase gene, enabling the fruit Bevan’s landmark paper was published in the journal to remain firm for longer so they could be picked Nature in July of that year4. later, letting them develop a better flavour. Vectors 1996: Tomato puree made from GM tomatoes with the polygalacturonase gene knocked To introduce its genetic material to plant cells, out goes on sale in Safeway and Sainsbury’s Agrobacterium relies on a loop of DNA called a plasmid. supermarkets in the UK, based on work by To replicate this feat in the laboratory, Bevan had Grierson and Zeneca. The GM tomatoes mean created a DNA ‘vector’. “Once you’ve got the selectable the puree is thicker and less of the fruit is wasted marker gene, the idea then is to make a vector. A vector during production. is a piece of DNA you can use to transfer DNA from one organism to another; in this case from Agrobacterium to 1999: The UK begins the farm scale evaluations to investigate the effects of the management a plant cell,” Bevan explains. practices of herbicide-tolerant GM crops on wildlife when compared to weed management In 1984, he published a paper describing the binary practices for conventional crops. Protoplasts from a petunia leaf. Credit: Wikimedia/Mnolf vector he had created5. Binary vectors consist of two plasmids; one containing the T-DNA with the genes Professor at the University of Nottingham and one of the to be introduced to the plant, and the other containing early adopters of the plant transformation techniques Timeline of key events the genes required to transfer the T-DNA into the plant developed at PBI, including the BIN 19 vectors. “The 2005: The final results of the farm scale genome. “The original idea came from Rob Schilperoort key thing about Mike [Bevan]’s work is that it made evaluations are published. in Leiden. He had the idea, and had even patented transformation easy to do.