Thirty years of transformation

A case study exploring the impact of plant transformation technology on plant science research and the global agricultural 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 into a plant , using a of soil characteristics, which breeders are incorporating into Impact Summary called tumefaciens. new varieties with increased yield, disease resistance AFRC-funded research at the 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. 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 . mediated plant transformation’, has revolutionised plant and crop science research. It has enabled researchers This case study explores the continuing impact to explore 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 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 project going in Mary-Dell’s lab. Then I came back to 1974: 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 , known as the , 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 . 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 ), uses transformation that had been transformed. Importantly, the researchers to knock out the 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 – 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, , 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. , Mary-Dell Chilton announced their discovery 1994: Calgene’s 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 , 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 ; 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. He created the BIN19 vector, it, but he hadn’t made a vector, to the best of my for example, and showed that you could get gene The trials find more weeds, and more of 6 knowledge,” explains Bevan . transfer. A lot of people had been trying to do that, but the insects that depend on them, around Mike’s vectors made it easy. Many people throughout conventional beet and spring oilseed rape Bevan’s vector, known as BIN19, was adopted by the world started to use his vectors.” crops. There were also more weed – an researchers around the world as Bevan made it freely important food source for farmland birds. available to academic users. “It had many desirable “Discovery was one thing, but the adoption [of the features that I think made it perform pretty well, and it’s BIN19 vectors] was really the game-changing thing,” In contrast, there were more insects and weeds still widely used now. It’s been the most widely used says Flavell. “All around the world, there are thousands around GM . GM winter oilseed rape had transformation vector, at least for basic research,” says of people using this. There was enormous craving no overall effect on weed numbers. Bee and Bevan. in the 1980s to learn how to do this following those butterfly numbers are lower around this crop, although other insects are not affected. initial publications, and that was the audience we were serving by building these useful vectors and mailing out 2012: Scientists from Rothamsted Research “The key thing about envelopes with these vectors in.” begin a trial of a ‘second generation’ GM crop: modified to express a gene which Mike’s work is that it made The GUS reporter produces an aphid alarm pheromone, protecting transformation easy to do.” the plant from aphids. Professor Don Greirson Following the development of the BIN19 vector, Bevan turned his attention to genetic switches, which 2012: The global market value of transgenic could be used to regulate gene activity. Working with crops is estimated at US$14.84Bn postdoctoral researcher Richard Jefferson, who had Bevan’s 1984 paper has since been cited over 1700 recently moved to PBI from the US, Bevan developed 2013: Chilton, Fraley and Van Montagu are times7, and a 2006 report found that it was the third the β-glucuronidase, or GUS, reporter gene system9. awarded the 2013 World Food Prize for most highly-cited paper in the development of plant their work on agrobacterium-mediated plant The GUS gene encodes an enzyme from Escherichia transformation technology8. transformation. coli which, following a simple treatment produces a blue colour, allowing researchers to visually identify any cells According to Professor Don Grierson, Emeritus expressing the GUS gene. GUS allowed researchers to assess at practical application in the first UK field trial of the cellular level. By coupling the GUS reporter gene to transgenic crop plants, which was run by PBI the genes to be introduced into plant cells, researchers researchers in Cambridge in 1987 using potatoes that could quickly see which cells had been successfully expressed the GUS reporter gene from a tuber-specific transformed and in which the new genes were being promoter. expressed. As a measure of how useful this has proved to be for academic researchers and its routine use in Subsequent advances research, the most highly-cited paper in the field of plant transformation is the 1987 publication describing the These days, Agrobacterium-mediated plant GUS reporter system8, which has been cited over 5,500 transformation is used by academia and industry to times10. insert genetic material into a wide variety of plant species, including many of the most important crop The GUS reporter system also found immediate species. The technology was originally developed in tobacco plants, which could be transformed efficiently as they could withstand Agrobacterium infection. Tobacco could also be regenerated from a callus of transformed cells into a whole plant.

Following the development of the fundamental tools required to transform tobacco, Bevan began looking at transformation in crop plants. He developed a potato transformation system and, working with Grierson and researchers from ICI Seeds, a system to transform tomato plants.

However, at the time researchers did not think it was possible to transform grass plants (monocots), which Rows of conventional potato plants. Credit: Pauline Eccles include some of the most important global crops such as rice or wheat, using Agrobacterium. It took around ten years before researchers understood how to use The Ti plasmid of Agrobacterium. Credit: Wikimedia Agrobacterium in these agriculturally important crops. Transforming research

Today, plant transformation is a routine tool that agriculture and food production, genes were important underpins many of the techniques used in plant and we knew roughly how many genes there were, way “I was very excited about how . This potential for the technology before DNA sequencing had led to complete , to be used in research was clear to its creators and and so it was a good way to find out what genes did.” T-DNA worked, and how you early adopters in the early 1980s. “I was very excited could use it to understand the about how T-DNA worked, and how you could use it Anti-sense gene silencing function of genes... It ushered to understand the function of genes using molecular biology,” says Bevan. “It ushered in the era of plant With funding from AFRC and, later, BBSRC, Grierson in the era of plant molecular molecular biology.” used Agrobacterium-mediated transformation and biology.” Professor Mike Bevan Bevan’s BIN19 vectors to explore the function of several “We were among the first to follow-up Mike’s work,” genes involved in tomato fruit ripening. Together with known gene, and allows researchers to identify the says Grierson. “Everybody was interested in transferring Imperial Chemical Industries (now Syngenta), the function of that gene. new or different genes to plants, because when you researchers identified many genes being expressed manipulate genes you can find out what they do. At during tomato ripening, but they did not know at first “The key thing we were able to achieve, which was that time we knew for almost every process involved in what the genes did. important and novel, was that we were able to use transgenic technology to knockout genes using anti- Plant transformation proved to be vital to their sense and sense gene silencing. In parallel with similar investigations. Alongside other techniques, Grierson work being done in petunia, this was the first time in the used Agrobacterium to introduce to the tomato plant world this had ever been done,” explains Grierson. cells ‘anti-sense’ versions of the genes they were interested in, with the intention of silencing the gene of In 1988, Grierson used anti-sense gene silencing to interest. An anti-sense gene is a copy of the gene of knockout a gene in tomato involved with fruit ripening, interest, but with a back-to-front genetic sequence. RNA which led to the creation of the genetically modified produced from this gene binds to RNA produced from tomatoes used in the creation of the first GM food the original gene, and this RNA hybrid is degraded by product on sale in the UK – a tomato puree (see the plant cell, effectively preventing the original gene ‘Transforming crop production’, below)11. from functioning. This approach is one form of ‘reverse ’. Grierson and colleagues also used anti-sense gene Unlike forward genetics, where researchers seek to silencing to identify, for the first time in any organism, understand the genetic changes responsible for an Calli of tomato plant, tabacum, cells. Credit: Wikime- the gene for an enzyme called ACC oxidase, which was dia/Igge observed mutation, reverse genetics begins with a required for the last step in the biosynthesis of ethylene. According to Grierson, “Everyone was interested in What is Agrobacterium? ethylene and ethylene biosynthesis as it’s an important involved with ripening in many, but not all, fruits, the senescence of leaves, the senescence of flowers and all of these are important biological and agricultural processes. It’s also involved in bacterial and fungal infection, resistance responses, and other kinds of stress responses. It’s a really important hormone.”

Insertional mutagenesis

Agrobacterium-mediated plant transformation also has several other uses in modern molecular biology research. For instance, when a plant is transformed T-DNA is inserted essentially at random into the . Credit:AJC1/Wikimedia Commons Agrobacterium, a soil-dwelling bacterium, first came genome. If it is inserted anywhere within an existing to attention as the causal agent of crown gall disease gene, it can knockout that gene and create what is in plants. After infecting a plant through a wound in Now, there are more than 450,000 mapped insertions known as a loss of function mutant. Researchers can a stem or root, Agrobacterium bacteria transfer their are available for Arabidopsis alone14. The technique exploit that randomness by transforming plant cells Ti (or tumour-inducing) plasmid into the plant cell. has helped researchers understand the function of with a small piece of T-DNA to create large libraries of The T-DNA from the plasmid is inserted into the plant many Arabidopsis genes, such as those controlling plants which each carry a single . The DNA, resulting in the development of tumour-like flowering time and senescence, organ development researchers can then screen this library looking for galls and causing the plant to produce nutrients for and photosynthesis15. Many of the libraries developed mutations of interest to their work and, as they know the the bacteria. in Arabidopsis, which can each consist of tens of sequence of the T-DNA, can identify and clone the gene thousands of plants, have been made available to into which it has been inserted. Crown gall disease affects many plants of the rose researchers through the Nottingham Arabidopsis Stock family, including roses, apples, pears, cherries and Centre in the UK, established by the AFRC in 1991 Such libraries have been made in the UK and almonds, although it does not have a serious impact and now supported by BBSRC and others16, and the elsewhere and screened to discover many significant on plant productivity20. Arabidopsis Biological Resource Centre at Ohio State genes. For instance, the first such studies in the model 17 12,13 University in the USA . Image: Agrobacterium cells infecting a larger plant plant Arabidopsis took place in the late 1980s . cell. Source: The White House (public domain) Overexpression mode of action of the herbicide. The technique can also ensure that newly developed herbicides do not stray Researchers can also insert additional copies of from the intended mode of action as different chemical existing genes to ‘overexpress’ them. With multiple analogues are developed from the initial lead molecule. copies of the same gene, the plant produces much “It’s a very powerful technique, although it’s quite more of the protein encoded by the gene and this can a routine technology now.” says Syngenta scientist be observed in the physical characteristics of the adult Richard Dale. “It’s really a very clean diagnostic tool, plant, for instance larger size, or an increased tolerance and saves us a lot of resource. You can quite quickly of unfavourable environmental conditions. This can help build up a big panel for these different lines. You can researchers to explore gene function particularly where cover a large majority of the known commercial modes knocking out the gene of interest is lethal to the plant. of action, and some of the more unusual ones as well. The biggest advantage is, once you’ve generated the line, all you need to do is keep the seeds, and you can “It’s a very powerful technique, keep this going for many years,” he adds. although it’s quite a routine technology now” Richard Dale, Syngenta

The technique has several other uses. For instance, Syngenta routinely use overexpression to determine the mode of action of new herbicides. To do so, they use several lines of transgenic tobacco, transformed using derivatives of Bevan’s BIN19 vectors to overexpress one of the genes that could be targeted by the herbicide.

When the plants are sprayed with the herbicide, only the one that is overexpressing the gene targeted by the herbicide survives, showing the researchers the Transforming crop development

Agrobacterium-mediated plant transformation enabled tomato relied on knocking out the gene for the enzyme of fruit, they would remain firm for longer. The idea the creation of genetically modified (GM) crops – crops polygalacturonase, which degrades the pectin in the was that you didn’t have to pick them too early; you carrying new genes that provide plants with traits they tomato cell walls. Working with Zeneca, Grierson’s could let them develop a better flavour, but this was not otherwise could not possess. This might include the group were the first to publish the sequence of the gene commercially successful.” ability to tolerate certain herbicides, or to produce more and, in 1988, demonstrate the anti-sense knockout. nutritious food. They took this one step further and in 1990 showed that As Calgene and Zeneca each held patents for knocking transforming a plant with the sense gene (i.e. another out the polygalacturone gene, they agreed that the Flavr In 2012, the global market value of transgenic crops copy of the same gene) could also knock it out. Savr tomato would be produced using the anti-sense was estimated at US$14.84Bn. This developed over gene knockout and the sense gene knockout used in the years following the introduction in the US in 1994 “The sense gene knockout was really surprising. If the tomatoes for the puree. of the first commercial GM crop, the Flavr Savr tomato, you add a sense gene you just expect to get more of created using Agrobacterium by American agri-biotech the product. What we showed was that sometimes if The puree was later withdrawn from sale during the company Calgene, Inc18. you add a sense gene there’s a slight alteration in the public debate on GM crops and food in Europe in the structure of the gene that’s inserted and it knocks out late 1990s In the UK, the first GM product sold in supermarkets the endogenous gene,” Grierson explains. He adds, was a tomato puree produced by Zeneca and “Calgene used the same gene and the same approach Agricultural biotechnology developed by Grierson and colleagues at Nottingham for their Flavr Savr tomato because they felt if you could in the early 1990s. Both the Flavr Savr and Grierson’s stop the degradation of pectin chains in the cell walls More recently, companies such as Syngenta have used Agrobacterium-mediated transformation to produce a number of genetically modified crops that have been adopted by growers around the world. “Transformation’s used both for basic research and product development – transgenic crops such as transgenic corn, transgenic , and there are other people around the world working on transgenic rice, sugarcane, wheat, and almost all other crops have an Agrobacterium transformation system,” says Dr Qiudeng Que, the head of Syngenta’s plant transformation organisation in North Carolina, USA.

Weed control in .Credit: US Agricultural Research Sevenspot ladybird - an aphid predator attracted by the Service aphid pheromone from Rothamsted’s GM wheat. Credit: Wikimedia/Vera Buhl Although there are other techniques that could be used adopted in India and China too. Transgenic corn is to genetically modify plants, Agrobacterium remains mainly grown in South and North America, and soybean one of the most commonly used and most reliable. is the same. About 90% of corn and soy in these “There are other ways to make transgenic plants, countries is transgenic. For corn, without the Bt insect like biolistic bombardment, but Agrobacterium has resistance trait you’d lose a lot of yield,” Que explains. some advantages,” says Que. “The transgenic plants you get out using Agrobacterium are much cleaner. He adds that “if you look at China, they import a lot of Biolistics usually results in the gene being fragmented, grain from the US, Brazil or Argentina. A lot of the yield and the copy number is a little bit higher, so it’s not increase contributed to those countries’ ability to export. as clean as Agrobacterium-mediated transformation. In China, in food they are mostly self-sufficient, but for Agrobacterium-mediated plant transformation has animal feed they import a lot of the grain. Without the become the preferred tool for plant biotechnologists for yield increase, those people probably wouldn’t have product development.” enough affordable meat to eat, and the situation would Caterpillar of the European Corn Borer. Credit: US Agricultural be quite different.” “Without the Agrobacterium research, agricultural Research Service biotechnology would not be where it is today,” Que to introduce the desired traits through conventional The Rothamsted wheat trial adds. breeding as the plants do not possess the capabilities required. For example, transgenic ‘Bt corn’ expresses In 2012, Rothamsted Research, an institute that Now, Syngenta is interested in introducing traits such a gene from a natural soil bacterium that produces a receives strategic funding from BBSRC, began a field as insect resistance or herbicide tolerance, boosting toxin that kills pests like the European corn borer. These trial of transgenic wheat19. The wheat is a second yield, or helping plants adapt to abiotic stresses such pests are particularly difficult to control as they burrow generation transgenic plant, which focuses on using as drought. In these cases, it is often not possible into the plant’s stem, which means they are shielded natural plant defence mechanisms to tackle pests. It from pesticides sprayed onto fields. carries a gene for an aphid alarm pheromone known as (E)-β-farnesene that is produced naturally by some “Without the Agrobacterium Such GM crops can lead to significant yield increases plants to repel aphids that would otherwise feed on the and contribute to food security, although concerns plant and attract aphid predators such as ladybirds. research, agricultural have been raised about the environmental and health Aphids can cause significant damage to wheat crops biotechnology would not be impacts of such crops. Strict regulations, introduced as and, at the moment, are usually controlled using a a result of these concerns, mean that few GM crops are broad spectrum insecticide, which can also harm where it is today,” beneficial insects. The wheat used in the trial was Dr Qiudeng Que, Syngenta approved to be grown in Europe. “Crops like Bt are widely grown, not just in the US, but it’s widely modified using Agrobacterium-mediated transformation to introduce the (E)-β-farnesene gene. Notes and References

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