Grapevines have been selected for their favorable properties since ancient times. Only the best vines, with large bunches, great vigor, or improved disease resistance were used to grow grapes. This human selection of “wild” grapes and natural crosses has resulted in the classic wine grapes as we know them today. In recent decades, however, various genetic techniques have been developed with which this selection can be accelerated. The desired properties can even be built in by genetic modification and the CRISPR technique. But how exactly do these genetic techniques work? Are there risks associated with their use? And, what can they mean for viticulture?
There are various methods for giving a grapevine, or a plant in general, more favorable properties. The most commonly used and widely accepted form of human control is conventional plant breeding by crossing two plants. Sexual propagation – pollinating the flower with the pollen from another plant – combines the DNA, and therefore the properties of the plants. To prevent that already present favorable properties in the wine grape are lost due to the cross, they are crossed back with the original plant. After a lengthy process of selection (for the right properties), backcrossing, selection, backcrossing, etc., eventually a plant is produced that is very similar to the original wine grape, but with the additional desired property (Figure 1A).
In some cases it is known which piece of DNA is responsible for a desired property of the plant. With genetic modification, this piece of DNA, and therefore the desired property, is simply “cut and pasted” from one plant to the DNA of another plant. The long process of breeding and selection is bypassed. The new plant that is created in this way is a genetically modified organism, a GMO. A GMO can be made from two organisms that can also reproduce together in nature, for example two different wine grapes. But it is also possible to make a GMO from two organisms that cannot reproduce in nature together, for example a jellyfish and a mouse, or a bacterium and a wine grape. In all cases, “strange” DNA will be built into the DNA of another organism. The biggest barrier in this process is bringing the new DNA together with the DNA in the cell.
The introduction of the desired DNA into a new cell – the so-called transformation – can take place with a variety of techniques, from an electric field that makes the cell wall permeable, a minuscule injection of the DNA into the cell, to DNA that is bound to gold particles and shot into the cells with a ‘gene gun’. For genetic modification of plants often the Agrobacterium bacterium is used1, 2. This bacterium has the unique characteristic that it transfers part of its DNA and builds it into the DNA of the plant cell. Normally the piece of DNA that is built in causes crown gall disease, resulting in tumors on the roots of the plant. By replacing this piece of DNA with the desired DNA, the bacterium does not build in the disease, but the desired property. However, the bacterium is not very precise here, and can build in its DNA at a virtually random location in the DNA of the plant3. The plant that is grown from the Agrobacterium treated plant cell contains the new gene in the DNA of all its cells. A GMO is born (FIGURE 1B).
Since 2012, genetics (and the rest of the world) has been captivated by the CRISPR / Cas9 technology. This technique is based on the anti-virus mechanism of bacteria and can be used to directly adjust the DNA of an organism. The holy grail for genetics! No more cutting and pasting from another animal or plant. Or do they?
CRISPR / Cas9, a bacterial defence mechanism
First, an explanation of the mechanisms of the CRISPR / Cas9 technique. CRISPR is a biological library of all kinds of pieces of DNA. The bacterium has collected all these pieces of DNA when it was attacked by a virus. In the event of a new virus attack, the bacterium uses the Cas9 protein to scan its CRISPR library to see if it has a DNA section with which it can recognize the virus. Cas9 copies the correct DNA from the library and can subsequently recognize and cut the invading virus. The DNA fragment from the CRISPR library thus serves as a “guide” for the Cas9 protein (FIGURE 2A).
Researchers have come up with a clever idea based on this defence mechanism. They use this entire mechanism by which the bacterium recognizes the virus DNA, but adjust the “guide sequences”. This allows them to control exactly which part of the plant DNA is cut by Cas9REF4. The (plant) cell will then automatically attempt to repair the break in the DNA. This DNA repair causes a small “scar” on the DNA, a tiny mutation. This mutation is exactly enough to change the function of a gene or even stop its functioning altogether. When done in the right place of the DNA, this results in a plant with a different, more favorable characteristic.
Bacterial DNA, so a GMO
But of course now you wonder, what about that cutting and pasting? Isn’t the DNA from the bacterium still involved? Exactly. In fact, just as with genetic modification, the Agrobacterium is also used here to introduce the DNA that produces Cas9 and the modified guide sequence into the plant cell and subsequently build it into the DNA of the plant5 (FIGURE 2B). This causes the plant cell to produce Cas9 and the guide sequence. Cas9 then recognizes the correct DNA section of the plant cell with the help of the guide sequence and cuts it through, resulting in an adaptation of the DNA. Nevertheless, the bacterial DNA encoding Cas9 is still in the DNA of the plant, which is therefore by definition a GMO.
Not a GMO after all?
But “GMO” written on the label is not a commercially attractive concept. Fortunately, therefore, a number of methods have been devised to prevent “strange” DNA from the Agrobacterium in the plant cell. One of these methods is based on the old backcrossing that is also the case with conventional plant breeding. The built-in bacterial DNA is removed by crossing back with the original plant. Of the new generation(s) that arise, only the plants with the modified DNA, but without the bacterial Cas9 DNA, are selected6 (see also FIGURE 1C). This process can take a long time, especially when it concerns perennial plants where sexual propagation can only take place after a few years. Recently, however, a way has also been found to introduce the Cas9 DNA and the guide sequence with Agrobacterium into the plant cell without being incorporated into the DNA of the plant. As such, the plant DNA is adjusted by Cas9, but backcrossing is no longer necessary. Nevertheless, this technique has not yet been tested with grapes7, 8.
Another method is the direct insertion of ready-made Cas9 protein and guide sequence into the plant cell (FIGURE 2C)5, 9. In this way no foreign DNA is built into the DNA of the plant at all. The difficulty with this technique, however, is that it requires plant cells without cell walls, so-called protoplasts. Because these cells do not have a cell wall, it is possible to get Cas9 and the guide sequence into the cell (and Agrobacterium is not needed). However, it is often difficult to obtain the protoplasts, and to grow a complete plant out of them. If this nevertheless succeeds, the inserted Cas9 protein and the guide sequence will be broken down during cell division. Only the modified DNA is copied and passed on to the new cells.
Due to these methods, the DNA of the plant is adapted by Cas9, but the new grown plant no longer contains any trace of the bacterial CRISPR / Cas9 DNA.
How are these techniques used in viticulture?
For the time being this question can be answered fairly briefly. All traditional and new vines used for the production of wine originate from conventional breeding techniques. Grape varieties such as Johanniter, Müller-thurgau, Pinotage, Solaris, and many others were man-made through a lengthy selection and crossing process. Nevertheless, for scientific purposes, vines such as the Cabernet Sauvignon, Shiraz, Dornfelder, Riesling and Chardonnay have been genetically modified using Agrobacterium10-12. As for now, these modified grapes are not in use (and not permitted) for the production of wine.
Genetically modified yeasts
Genetic modification has also been applied to yeasts, for example to have them also take care of the malolactic fermentation. To do this, the ML01 yeast is genetically modified with a gene from the Oenococcus oeni bacterium so it can also convert malic acid to lactic acid. This modified yeast is banned in Europe, but is allowed in Canada and the United States for the production of wine13, 14. There is quite a bit of controversy about ML01, certainly because German research has shown that genetically modified yeasts (just like other yeasts) become part of the yeast culture in the wine cellar and in the vineyard15. Yeasts are therefore not simply an auxiliary substance in the production of wine, but do have an ecological effect. In addition, they are not qualified as an excipient in Europe, because they can also be present (albeit to a very limited extent) in the final wine, even when it is filtered16.
Application of CRISPR / Cas9 in viticulture
The CRISPR / Cas9 method is already being tested for applications in the wine sector. For example, it has been shown that the CRISPR / Cas9 method works in the Chardonnay grape17, and Thompson Seedless table grapes have been made more resistant to Botrytis cinerea18. Further, also commercial wine yeasts have also been adjusted using the CRISPR / Cas9 technology. Two Saccharomyces cerevisiae yeasts have been modified so that they produce less urea19. The reduced amount of urea prevents the carcinogenic substance ethyl carbamate from being formed during fermentation, for which urea is a building material.
In addition to direct adjustments to the
vines or yeasts, the use of the CRISPR / Cas9 method to combat pests and
diseases is also being investigated. For example, the effectiveness of
releasing sterile Drosophila males in the vineyard is examined. These fruit
flies have been made infertile using the CRISPR / Cas9 method, which reduces
the reproduction speed of the entire population and prevents a plague20.
An additional advantage is that this genetically modified fly cannot spread
further due to its infertility. Two
flies birds with one stone.
In addition to the above applications, there are many other possibilities where the CRISPR / Cas9 technology can contribute to a better, tastier or healthier wine through the improvement of vines, yeasts or by combating diseases and pests in the vineyard. All these adjustments are very promising in a laboratory setting, but must eventually also be approved for (large-scale) use in viticulture and wine making practise.
The ethical discussion
In general there is a lot of resistance to GMOs, because they are “artificial”, “unnatural” and their long-term ecological effects are unclear. But what if a human-modified plant can be exactly the same as a naturally occurring variety? Small mutations of the DNA as caused by Cas9 also naturally occur in the DNA of the plant, for example under the influence of UV radiation or other environmental influences. The plants with these mutations then have slightly different properties.
In the case of wine grapes, people would like to continue breeding with the plants that have these improved properties. Based on this principle, many hundreds of clones were made in Burgundy from the Pinot noir grape. Each clone has a miniscule variation in its DNA that provides a slightly different property, a slightly better resistance to mildew, more open bunches, more vigor, and so on. Because these variations can therefore also occur in nature, it is impossible without prior knowledge to determine whether a certain variation has arisen naturally, or is artificially created by using the CRISPR / Cas9 method. The question is therefore whether this modified plant should be called a GMO. When two wine grapes are crossed, humans also control the recombination of the DNA, only less precisely.
America vs. Europe
The US Department of Agriculture took a position last year in this discussion by stating that they only assess the end product. If changes to the plant DNA can also be made naturally or through conventional plant breeding techniques, and if the final plant does not have foreign (non-self) DNA, then it is not a GMO. This is regardless of whether genetic modification techniques have been used to create the plant. A mushroom modified with CRISPR has already been designated as a non-GMO in the United States. As a result of the adjustment, the mushroom turns brown much less quickly, so that its shelf life is extended21.
Last year, the European Court of Justice decided the exact opposite. It ruled that the CRISPR / Cas9 technique falls under the existing GMO regulation (of older genetic modification techniques)22. In Europe, every plant that has been processed with genetic modification techniques is therefore classified as a GMO, even if there is no trace left of these methods in the final DNA of the plant.
Mutagenic technology & enforcement
Proponents of the CRISPR / Cas9 technique disagree with the decision of the European Court of Justice and believe that it is a mutagenic technique. CRISPR / Cas9 can make mutations in the DNA of an organism without the introduction of foreign DNA. CRISPR / Cas9, like other mutagenic techniques – such as radiation exposure -, should therefore not be covered by the GMO regulation. However, opponents of the CRISPR technique believe that this is necessary, because it involves targeted human-driven mutations23. But does this matter if exactly the same natural variation can occur in the plant spontaneously?
Anyway, there is currently no worldwide consensus on how to handle the CRISPR / Cas9 technology. This may cause problems in the future with the import and export of products that have been created using this method. After all, it is impossible for governments, winegrowers and consumers to distinguish a naturally crossed vine from a CRISPR / Cas9 vine. Enforcing a CRISPR / Cas9 ban will therefore become very difficult.
1. Zambryski P, Joos H, Genetello C, Leemans J, Montagu MV, Schell J. Ti plasmid vector for the introduction of DNA into plant cells without alteration of their normal regeneration capacity. The EMBO journal. 1983;2(12):2143-50.
2. Nester E. Agrobacterium: The Natural Genetic Engineer (100 Years Later) The American Phytopathological Society APSnet Features. 2011.
3. Gelvin SB. Agrobacterium-mediated plant transformation: the biology behind the “gene-jockeying” tool. Microbiology and molecular biology reviews : MMBR. 2003;67(1):16-37, table of contents.
4. Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science (New York, NY). 2012;337(6096):816-21.
5. Osakabe Y, Liang Z, Ren C, Nishitani C, Osakabe K, Wada M, et al. CRISPR-Cas9-mediated genome editing in apple and grapevine. Nature protocols. 2018;13(12):2844-63.
6. Char SN, Neelakandan AK, Nahampun H, Frame B, Main M, Spalding MH, et al. An Agrobacterium-delivered CRISPR/Cas9 system for high-frequency targeted mutagenesis in maize. Plant biotechnology journal. 2017;15(2):257-68.
7. Chen L, Li W, Katin-Grazzini L, Ding J, Gu X, Li Y, et al. A method for the production and expedient screening of CRISPR/Cas9-mediated non-transgenic mutant plants. Horticulture research. 2018;5:13.
8. Veillet F, Perrot L, Chauvin L, Kermarrec MP, Guyon-Debast A, Chauvin JE, et al. Transgene-Free Genome Editing in Tomato and Potato Plants Using Agrobacterium-Mediated Delivery of a CRISPR/Cas9 Cytidine Base Editor. International journal of molecular sciences. 2019;20(2).
9. Malnoy M, Viola R, Jung MH, Koo OJ, Kim S, Kim JS, et al. DNA-Free Genetically Edited Grapevine and Apple Protoplast Using CRISPR/Cas9 Ribonucleoproteins. Frontiers in plant science. 2016;7:1904.
10. Iocco P, Franks T, Thomas MR. Genetic transformation of major wine grape cultivars of Vitis vinifera L. Transgenic research. 2001;10(2):105-12.
11. Torregrosa L, Locco P, Thomas MR. Influence of Agrobacterium strain, culture medium, and cultivar on the transformation efficiency of Vitis vinifera L. American Journal of Enology and Viticulture. 2002;53:183-90.
12. Bornhoff B-A, Harst M, Zyprian E, Töpfer R, Lannini C. Transformation studies on Vitis vinifera L., via Agrobacterium tumefaciens. Acta Horticulturae. 2000;528:359-60.
13. Food and Drug Administration (FDA). Gras Notice 120. 2003.
14. Government of Canada. Risk assessment summary for EAU-224: Saccharomyces cerevisiae strain ML01. Van Vuuren and Associates; 2006. p. 1-6.
15. Grossmann M, Kießling F, Singer J, Shoeman H, Schröder M-B, Von Wallbrunn C. Genetically modified wine yeasts and risk assessment studies covering different steps within the wine making process. Annals of Microbiology. 2011;61(1):103-15.
16. Nisiotou AA, Gibson GR. Isolation of culturable yeasts from market wines and evaluation of the 5.8S-ITS rDNA sequence analysis for identification purposes. Letters in applied microbiology. 2005;41(6):454-63.
17. Ren C, Liu X, Zhang Z, Wang Y, Duan W, Li S, et al. CRISPR/Cas9-mediated efficient targeted mutagenesis in Chardonnay (Vitis vinifera L.). Scientific reports. 2016;6:32289.
18. Wang X, Tu M, Wang D, Liu J, Li Y, Li Z, et al. CRISPR/Cas9-mediated efficient targeted mutagenesis in grape in the first generation. Plant biotechnology journal. 2018;16(4):844-55.
19. Vigentini I, Gebbia M, Belotti A, Foschino R, Roth FP. CRISPR/Cas9 System as a Valuable Genome Editing Tool for Wine Yeasts with Application to Decrease Urea Production. Frontiers in microbiology. 2017;8:2194.
20. Kandul NP, Liu J, Sanchez CH, Wu SL, Marshall JM, Akbari OS. Transforming insect population control with precision guided sterile males with demonstration in flies. Nature communications. 2019;10(1):84.
21. A CRISPR definition of genetic modification. Nature plants. 2018;4(5):233.
22. Court of Justice of the European Union. Judgment in Case C-528/16 2018 [Available from: https://curia.europa.eu/jcms/upload/docs/application/pdf/2018-07/cp180111en.pdf.]
23. Callaway E. CRISPR plants now subject to tough GM laws in European Union. Nature. 2018;560(7716):16.