Environmental Risk Assessment Scenarios of Specific NGT Applications in Brassicaceae Oilseed Plants

1. Introduction

Brassicaceae is a diverse plant family and includes many domesticated plants as well as widespread wild plants, such as thale cress (Arabidopsis thaliana), capsella (Capsella bursa-pastoris), wild radish (Raphanus raphanistrum) and pennycress (Thlaspi arvense). The cultivated species of this plant family are global sources of i. a. vegetables (cabbage, radish), seeds harvested for example to produce mustard and vegetable oil for food and industrial purposes. The latter is mainly obtained from the oilseed crops, i. e. oilseed rape (Brassica napus) and camelina/false flax (Camelina sativa).There is a strong possibility of cross-hybridisation between different species within the Brassicaceae plant family (FitzJohn et al., 2007).

Breeders have been working for many years to improve both the quality of food and feed as well as raw material used in industry. They have, for example, attempted to alter traits such as the oil content and the fatty acid composition of the oilseed crops. The oil obtained from Brassicaceae oilseed crops consists mainly of saturated fatty acids (palmitic, stearic and eicosanoic acids), monounsaturated fatty acids or MUFA (oleic, erucic and gondoic acids) and polyunsaturated fatty acids or PUFA (linoleic and linolenic acids) (Sharafi et al., 2015; Rodríguez-Rodríguez et al., 2021). The latter are also known as omega-6 and omega-3 fatty acids, respectively. Both PUFAs are known to have numerous health benefits (Abedi and Sahari, 2014), but are also associated with oxidative instability (Kamal-Eldin, 2006). Depending on the intended purpose, some projects are aiming to either increase (in food and feed, see, e.g. (Xie et al., 2020)) or decrease (for food or industrial purposes, see, e.g. (Knothe, 2008; Okuzaki et al., 2018; Esfahanian et al., 2021)) the PUFA content in Brassicaceae crops. In addition, attempts are being made to further reduce the erucic acid content, as there is evidence that it is toxic (Galanty et al., 2023). Besides altering oil content and fatty acid composition, breeders are also focussing on improving yield, growth and resistance to biotic and abiotic stress, including resistance to plant pathogens and herbicides (see Section 3 and references in Table 1 and Table 2 and A1–A3). Changes in these traits often imply positive fitness effects (See Section 3).

The genomes of some Brassicaceae family members are particularly complex, which means that conventional breeding methods (including non-targeted mutagenesis) are somewhat restricted leading to difficulties in breeding (Li et al., 2022). Several Brassicaceae crops are polyploid (up to allohexaploid), i.e. they have more than two sets of paired chromosomes and, thus more than two copies of each gene. In addition, some have undergone genome triplication, which has resulted in a high number of duplicated genes (Wang et al., 2011). In order to achieve a desired breeding characteristic, it is frequently necessary to knock out all homologous genes (Wells et al., 2014), and from case to case it is doubtful whether is at all possible.

New genomic techniques (NGTs), such as CRISPR/Cas9, have been successfully applied several times in Brassicaceae (Li et al., 2022; Tian et al., 2022; Ahmad et al., 2023). The technology has made it possible to alter multiple copies of a gene, or even alter several different genes simultaneously (multiplexing). Unlike conventional breeding, NGTs can be used to introduce genetic changes in genomic regions that are difficult to access and are highly protected (Kawall, 2019). This technical potential allows genotypes and traits to be modified within short periods of time.

While some of the Brassicaceae genotypes developed by NGTs were also achieved using conventional breeding (e. g. EMS (ethyl methane sulphonate) breeding), others are completely novel (see, e. g. (Bellec et al., 2022; He et al., 2023)). In future, it is expected that different NGT-derived genotypes can be combined and stacked, thus leading to even more extensive overall genomic changes in NGT plants (Raitskin and Patron, 2016; Zetsche et al., 2017; Kawall et al., 2020).

In addition to scientific publications dealing with basic and/or applied research on NGT Brassicaceae, which will be evaluated as part of this study, various projects are already at a more or less advanced stage of commercialisation and have been tested in field trials. For example, in 2023, the US company Pairwise brought CRISPR mustard plants (B. juncea) to the market, whose leaves are supposed to taste less pungent (Karlson et al., 2022), which is the only commercialised Brassicaceae NGT plant worldwide.

The following sections provide an overview of current NGT applications in Brassicaceae oilseed crops, i. e. oilseed rape (B. napus), camelina (C. sativa) and pennycress (T. arvense). For the purpose of this review, market-orientated NGT applications in Brassicaceae oil plants were identified in the scientific literature and all NGT plants without permanent insertion of transgenes were listed. Basic research projects and applications using oligonucleotide directed mutagenesis (ODM) methods were not considered.

2. Biological Basics

2.1. The Biology of Brassica Napus

Oilseed rape (B. napus) is allotetraploid (2n=38, AACC) (An et al., 2019) and the result of a natural cross between the diploid Brassica species B. oleracea (2n=18, CC) and B. rapa (2n=20, AA). The evolutionary relationship between different Brassica species sharing three core genomes (A, B and C) is described in the ‘Triangle of U’ (Nagaharu, 1935). Europe is the centre of origin and genetic diversity for oilseed rape and several other Brassica species. Due to its origin, B. napus is well adapted to European climatic conditions. However, oilseed rape is grown globally and economically one of the most important oilseed crops (Hu et al., 2021). As ‘double-low’ oilseed rape (low erucic acid and low glucosinolate content in seeds), also known as canola, it provides a healthy and nutritionally balanced cooking oil; it is also an important source of biodiesel, industrial oil and protein for animal feed (Hu et al., 2021).

Weedy characteristics

All cultivated Brassica species have certain weedy characteristics, e.g. increased dispersal abilities, long seed dormancy, the ability to invade disturbed areas and natural plant communities. Additionally, several relatives of oilseed rape, e.g. Sinapis arvensis, Raphanus raphanistrum and Hirschfeldia incana, are regarded as weeds. Weedy forms of B. napus, B. rapa and B. oleracea also exist, e. g. oilseed rape has some wild (or weedy) plant characteristics, such as seed shedding and long secondary dormancy of the seeds in the soil. Although weedy oilseed rape is predominantly found on arable land, ruderal (feral) populations can persist over longer periods of time (Pascher et al., 2010, 2017; Banks, 2014): they have been found, for example, in various European countries (see, amongst others, (SCRI, 2004; Squire et al., 2011; Frieß et al., 2020)).

Interspecific hybridization

Oilseed rape has pronounced hybridization potential and can form hybrids with various congeneric (i.e. Brassica) species, such as B. rapa (turnip rape, field mustard, birdseed rape, etc.), B. juncea (various mustard varieties) and others, but also with wild and weedy Brassicaceae from different genera (Chèvre et al., 2004; Pascher et al., 2006). So far, spontaneous hybridization was confirmed in a total of eight native or cultivated species in Europe (Theenhaus et al., 2002; Breckling et al., 2003; FitzJohn et al., 2007; Devos et al., 2009; OECD, 2012; COGEM, 2019; Marotti et al., 2020).

Pollen flow

Oilseed rape is self-compatible and predominantly self-pollinated by wind and insects. The outcrossing rate is controversially discussed – estimates in literature range from between 12 and 55 percent (Becker et al., 1992; Darmency, 1997). It can thus be inferred that up to half of the pollen load may be dispersed by wind and, above all, by insects. Cross-pollination over very long distances has been demonstrated in oilseed rape (several kilometres) (Beckie et al., 2003; Ramsay et al., 2003). It is also a sought-after source of food, especially for bees and outcrossing can be expected in the collection area of both social and solitary bees (Pasquet et al., 2008).

Secondary dormancy

A considerable number of seeds are lost when oilseed rape is harvested, and end up back on the fields as volunteer rapeseed. According to Gulden et al. (Gulden et al., 2003), this loss can be up to around 6 % of the harvest; according to the EU research project SIGMEA, this can amount to a total of 6000 seeds per square meter (Gruber et al., 2007). Around 80 % of volunteers germinate during the first twelve months after harvest, but a considerable proportion of the seeds remain viable for up to 15 years in the soil (Lutman et al., 2003, 2005; D’Hertefeldt et al., 2008; Belter, 2016).

2.2. The Biology of Camelina Sativa

Camelina (C. sativa) is an annual Brassicaceae plant that is mainly cultivated in Europe and North America. C. sativa is allohexaploid (2n = 6x = 40, AABBCC). It is an ancient crop that has been used since prehistoric times (Zanetti et al., 2021). A recent study found that the plant was probably first cultivated in the Caucasus region (Brock et al., 2022). In contrast to other plants of the Brassicaceae family, camelina has not been widely used in plant breeding in the past, so only a small number of varieties are available for agricultural purposes (Vollmann and Eynck, 2015).

The oil from camelina seeds is used as food and animal feed, but also for biofuels and industrial compounds (Shonnard et al., 2010; Iskandarov et al., 2014; Vollmann and Eynck, 2015). It contains large amounts of polyunsaturated fatty acids (PUFAs) such as essential linoleic and linolenic acids (Abramovic and Abram, 2005; Iskandarov et al., 2014).

Pollen flow

Camelina is primarily self-pollinating, but there is also evidence of cross-pollination by bees, bumblebees and other insects (see references in (CFIA, 2017)). Outcrossing in camelina decreases rapidly with distance. For example Walsh et al. (Walsh et al., 2015) demonstrate that the outcrossing rate in camelina populations was only 0.09 % at a distance of 20 meters. However, this does not rule out the possibility of rare but ecologically significant long-distance pollination events due to insects that are able to fly considerable distances (Pasquet et al., 2008).

Dormancy

Seed shattering and the small size of camelina seeds (seed rain from the combine harvester) contribute to harvest losses. Thus, due to primary or secondary dormancy the likelihood of volunteer plants and persistence may increase. There are differing statements in literature with regard to secondary dormancy, i.e. dormancy that is induced, for example, by non-germinated seeds buried in the soil. While Ehrensing & Guy (Ehrensing and Guy, 2008) found no dormancy in camelina seeds at all, Walsh et al. (Walsh et al., 2013) showed that volunteer populations disappear only after two years, even when herbicides are used, thus suggesting survival in the seed bank. Similarly another experiment conducted in south-eastern France found that 85.8 % of wild camelina seeds at a depth of 10 cm had survived after two and a half years (Saatkamp et al., 2009). Thus, while dormancy has been documented, the longevity of camelina seeds in the seed bank still needs to be investigated.

Hybridization potential

Camelina can hybridize with wild relatives such as C. microcarpa and C. alyssum (Séguin-Swartz et al., 2013; Julié-Galau et al., 2014; Zhang and Auer, 2020). According to results of a large number of studies, interspecific hybridization with other Brassicaceae such as oilseed rape, field mustard, Arabidopsis and others, seems unlikely (see overview in (CFIA, 2017)).

2.3. The Biology of Thlaspi Arvense

Pennycress (T. arvense) is another member of the Brassicaceae family currently being engineered with NGTs. It is closely related to the thale cress Arabidopsis thaliana and native to Eurasia, but can now be found worldwide as crop and weed (Ma et al., 2023). Uses include feed and the production of agrofuels (Keadle et al., 2023). Due to its high vitamin C content, it is also added to salads or cooked like spinach (Mitich, 1996). Pennycress is diploid, the plant’s genome consists of seven chromosomes (2n=14) (McGinn et al., 2019). It has a high seed oil content and a favourable fatty acid composition that makes it suitable for use as oil crop for industrial purposes (Warwick et al., 2002). In agriculture, pennycress is mainly used as winter cover crop since it can tolerate very low temperatures (McGinn et al., 2019; López et al., 2021). As a cover crop, it provides important ecosystem services because it reduces soil erosion and the leaching of nutrients.

Weediness

Pennycress has only recently been domesticated and current cultivars still harbour many weed traits (McGinn et al., 2019). In North America, it is listed in several regions as a noxious weed which considerably reduces crop yields (Mitich, 1996).

Pollen flow

Pennycress is mainly wind-, but also self- or insect pollinated. In a study conducted in Germany, flowers were visited by different bee species, but not by honey bees (Groeneveld and Klein, 2014).

Seed dormancy

Due to its nearly non-domesticated history, T. arvense still inherits weedy characteristics, for example very long secondary seed dormancy. The seed bank can last 20 years or more (CABI, 2014).

Interspecific hybrids

Interspecific hybridization is not reported in T. arvense (Warwick et al., 2002).

Pollinator interaction

Pennycress has a high feed value for pollinating insects, and is especially valuable as early-season food source (Eberle et al., 2015).

3. Overview of NGT Applications in Brassicaceae Oilseed Plants

3.1. Brassica napus (Oilseed Rape)

So far, around 45 market-orientated NGT applications in B. napus are described in scientific literature (see Table A1). The agronomic importance as an oilseed crop has resulted in its genome being edited several times with NGTs in order to adapt the fatty acid composition and total oil content to individual intended uses (14 applications, shown in orange in Table A1 and Figure 1). Attempts to reduce PUFAs and erucic acid by knocking out FAD (fatty acid dehydrogenase) and FAE (fatty acid elongase) genes are particularly frequent. Several NGT applications are related to the survival and fitness of the plants, e. g. increased drought tolerance, superior growth, longer roots and a higher number of siliques and seeds (green in Table A1 and Figure 1).

3.2. Camelina sativa (Camelina)

Camelina has already been genetically engineered several times using NGTs (10 NGT applications, see Table A2). Similar to oilseed rape, attempts are being made to alter the fatty acid content and composition (8 applications, orange in Table A2 and Figure 1) and NGT applications to reduce PUFAs and erucic acid (FAD and FAE genes) are particularly frequent as well. In addition, different knockouts (5 genes, multiple copies) were established in a multiplexing approach resulting in early-flowering plants (green in Table A2 and Figure 1).

3.3. Thlaspi arvense (Pennycress)

In pennycress, NGTs were used to knock out FAE1, thus making it possible to abolish erucic acid production (see Table A3 and Figure 1). This knockout strain was further combined with NGT-derived FAD2 and ROD1 knockouts to increase oleic acid content and to decrease PUFA content.

3.4. Field Trials and Commercialisation Pipeline

In the USA and also in Europe, various projects are underway to secure the market release of NGT-Brassicaceae. In Europe, there is currently no commercially viable NGT-Brassicaceae product. However, applications for field trials were filed in both the EU and the UK (Napier, 2021). All trials were carried out with plants modified with the CRISPR/Cas9 gene scissors (Table 1). After its withdrawal from the EU, the UK on a new legal basis and under simplified conditions under which NGT plants can be released into the fields. Current field trials in the EU and the UK include oilseed rape as well as camelina and broccoli (B. oleracea).

In the US, companies or research institutions can submit a deregulation request to the US Department of Agriculture (USDA). The Agency determines whether a transgenic or NGT plant that is to be placed on the market needs to legally regulated or can be marketed without restrictions. In the meantime, a number of NGT Brassicaceae have been deregulated in this way (see Table 2). However, deregulation does not automatically mean that a NGT plant will actually be commercialised. So far, only brown mustard (B. juncea) developed by the company Pairwise has been commercialised. In addition to oilseed rape, camelina and pennycress have been deregulated, most of which have had their oil content modified. In nearly half of the applications for deregulation, all the relevant information was declared to be confidential business information (CBI), so that no statements can be made here about the possible objectives of a project (Table 2).

4. Findings and Scenarios Relevant for Risk Assessment

4.1. Risks for Pollinators

Brassicaceae oilseed crops are highly attractive for pollinators: They offer easily accessible and highly abundant floral resources and are an important food resource (Eberle et al., 2015; Van Reeth et al., 2019). Brassicaceae pollen has lower protein:lipid values (Vaudo et al., 2020) and is particularly rich in PUFA linolenic acid (Manning, 2001).

It has been shown that fatty acids play key roles in the development, communication, reproduction and health of pollinators, including in their colonies (see, e. g. (Arien et al., 2015, 2020; Muth et al., 2018)). Studies indicate that the specific composition of fatty acids in pollen is important for host-plant choice and bee-flower interactions (Vaudo et al., 2020). Further, pollinators seem to regulate their dietary intake by relying on lipids as nutritional cue. This is likely to be caused by the impact of fatty acids have on their overall fitness (Ruedenauer et al., 2020).

Changes in the dietary composition of pollinators should be carefully examined for risks to the insects, as suboptimal nutrient balance is thought to be one of the threats to their populations (Vanbergen and Initiative, 2013).

4.1.1. Decreased Amounts of PUFA Can Negatively Affect the Health of Pollinators

Several NGT applications in Brassicaceae oilseed crops focus on fatty acid composition in efforts to obtain plants with a low PUFA content. Like other fatty acids, PUFAs have different important functions in many biological processes, both in plants and animals. Animals cannot synthesize PUFAs themselves so they need to be part of their diet (Hulbert et al., 2012; Arien et al., 2020).

The PUFA content of seeds is mainly reduced by knocking out FAD genes (Jiang et al., 2017; Morineau et al., 2017; Okuzaki et al., 2018; Huang et al., 2020; Jarvis et al., 2021; Lee et al., 2021; Liu et al., 2022; Shi et al., 2022). Another study successfully decreased the PUFA content by knocking out CIPK9 genes (Wang et al., 2023). In B. napus it was shown that FAD genes are highly expressed in pollen, thus explaining the high levels of PUFAs (Piffanelli et al., 1997). Therefore, it can be assumed that at least the knockout of FAD genes will significantly decrease the PUFA content, including pollen.

In habitats with extensive agricultural monocultures of plants with reduced PUFA content, bees may suffer from PUFA deficiency due to a severely limited variety of pollen (Arien et al., 2015, 2020). It was shown that honey bees with a linolenic acid dietary deficiency had smaller hypopharyngeal glands; they also showed poor performance in both olfactory and tactile associative learning assays (Arien et al., 2015). Results from experimental feeding studies showed that total lipid concentration and the linoleic:linolenic acid ratio are further factors affecting brood development and adult longevity in honey bees. A balanced PUFA diet is necessary for maintaining proper colony development (Arien et al., 2020).

In addition, PUFAs seem to have some antimicrobial properties capable of inhibiting growth of honey bee pathogens, e. g. Paenibacillus larvae and fungal diseases (Ababouch et al., 1992; Feldlaufer et al., 1993; Manning, 2001). PUFAs also have an important role as pheromones, e.g. in recognizing and discriminating between nestmates and non-nestmates (Breed, 1998), or in attracting workers to form the retinue group (Keeling et al., 2003).

Taken together, a sufficient supply of PUFAs appears to be very important for the health, colony development and communication of honey bees. Malnutrition resulting from dietary deficiency of certain fatty acids may therefore cause adverse effects in honey bees and other wild pollinators, and should be analysed in risk assessment before NGT crops with an altered fatty acid content are brought to market.

4.1.2. Increasing Amounts of Oil and Unsaturated Fatty Acids Can Negatively Affect the Health of Pollinators

In addition to approaches aiming to lower the PUFA content in oilseed crops, there are several studies that have used NGTs to successfully increase the total amount of oil and, in particular, unsaturated fatty acids in seeds.

For example, oleic acid contents were elevated by knocking out FAD genes (Jiang et al., 2017; Morineau et al., 2017; Okuzaki et al., 2018; Huang et al., 2020; Jarvis et al., 2021; Lee et al., 2021; Liu et al., 2022; Shi et al., 2022).

PUFA contents were elevated by knocking out different TT genes (Xie et al., 2020; Zhai et al., 2020; Li et al., 2023), SAD gene (Huang et al., 2023), KANT3 gene (He et al., 2023), GIF1 gene (He et al., 2023), AGP11 gene (He et al., 2023) or EDA32 gene (He et al., 2023). These knockouts probably also have a considerable influence on the fatty acid composition in Brassicaceae pollen, and thus on the diet of pollinators.

Studies have shown that the reproduction and survival rate of bumble bees was significantly reduced when they ingested too many fatty acids (Vaudo et al., 2016; Ruedenauer et al., 2020). This may explain why bumble bees strongly avoided consuming pollen enriched with fatty acids in feeding experiments (Ruedenauer et al., 2020). In honey bees, feeding studies found significant increases in the mortality rate above a certain amount of oleic or linoleic acid (Manning et al., 2007).

The negative effects of fatty acids on survival and reproduction could be the result of intoxication with excessive amounts of fatty acid (Ruedenauer et al., 2020). Intake of high amounts of PUFAs, for example, might lead to lipid peroxidation and cell damage (Vaudo et al., 2016). On the other hand, a lack of other nutrients as a consequence of reduced overall pollen consumption due to fat avoidance, might further increase mortality (Ruedenauer et al., 2020). Hence, in habitats with extensive agricultural monocultures of plants with highly increased oil and PUFA content in pollen, pollinators, such as honey bees and bumble bees, may either over-consume fatty acids or suffer nutrient deficiencies due to fat avoidance.

As mentioned above, changes in oil composition of plants can also be achieved with conventional breeding (including random mutagenesis (see also (Havlickova et al., 2023)). However, NGTs offer new potential for the targeted editing of Brassicaceae genomes (He et al., 2023). In regard to individual NGT traits, it may be difficult without in-depth case-by case investigation to conclude on whether the specific genotype and phenotype could also be obtained from conventional breeding.

Furthermore, the overall number of NGT applications in Brassicaceae oilseed plants and the technological potential of NGTs (Kawall 2019) shows a new dimension in the scale (in terms of space and time) of potential releases of plants (Koller et al., 2023) with novel genotypes and phenotypes as for example shown by He et al. (He et al., 2023). This will complicate any prediction about future impact of NGT-plants, especially if compared with conventionally bred plants: Both spontaneous and intended crossings can lead to new combinations of traits causing unintended interactions without precedent (see Section 4.2.).

4.1.3. Further Observations Regarding Changes in Oil Composition

Changed oil content and composition may result in further unintended and undesirable effects. Fatty acids play important roles in plants, e. g. in the biosynthesis of secondary metabolites found in cellular membranes and phytohormones.

For example, the phytohormone jasmonate and its derivates have important roles as signalling molecules in plant defence, particularly against insect herbivores (Glazebrook, 2005; Howe and Jander, 2008). Linolenic acid is a precursor molecule of jasmonic acid (Gfeller et al., 2010). Changes in oil composition can therefore affect plant communication, signalling pathways and plant resistance to biotic or abiotic stressors (Kawall, 2021). In addition, there are further NGT applications with altered plant composition which could be relevant to interactions with pollinators, e. g. traits with a significant impact on hormone signalling (Sun et al., 2018; Wang et al., 2023) or flavonoid content (Xie et al., 2020; Zhai et al., 2020). In addition, changes in oil composition may also have effects on various other species which feed on plants, such as pest insects (Hixson et al., 2016) or other wild species (Colombo et al., 2018).

4.2. Persistence and Spread

It may not be easy to control the cultivation of NGT oilseed Brassicaceae, especially under European conditions. This is due to the biology of Brassicaceae species, such as oilseed rape (B. napus) and others. Some of the problems are:

• A broad range of Brassica species can hybridise with each other;
• Many Brassicaceae have weedy characteristics;
• Seeds can exhibit prolonged dormancy;
• Pollination by insects and wind, as well as dispersal, can occur over long distances.

Oilseed rape is also a good example of the difficulties associated with the cultivation of genetically engineered Brassica plants. Sohn et al. (Sohn et al., 2021) show that the uncontrolled spread of (transgenic) genetically engineered (GE) oilseed rape is already happening in different countries (Japan, Canada, USA, Switzerland, Argentina). The list includes countries that do not even allow the cultivation or import of transgenic plants, as transgenic populations near ports and along transport routes can originate from transgenic seeds that are imported legally or from contamination (Schulze et al., 2015).

GE herbicide-tolerant oilseed rape was recently shown to have hybridised with weedy B. rapa (bird rape mustard) in Canada (Laforest et al., 2022). Oilseed rape and B. rapa are intercrossable, and hybrids of both were previously confirmed (see, e g. (Jorgensen and Andersen, 1994; Warwick et al., 2008)). It was assumed that the hybrid plants had reduced fertility and were, therefore, unable to become permanently established in the environment. Contrary to this assumption, Laforest et al. showed that the genetically engineered trait is now detectable in purebred and weedy B. rapa plants in Canada (Laforest et al., 2022).

The oilseed rape example shows that genetically engineered Brassicaceae plants can persist in the environment as well as spread into wild or weedy populations. Assumptions of non-establishment should therefore be treated with caution. Other studies appear to indicate fitness advantages of GE oilseed rape or its hybrids with related species (see examples in (Bauer-Panskus et al., 2020)).

A closer look at NGT applications in Brassicaceae species shows there are several examples of fitness related traits (green in Table A2 and Table A3). It can be hypothesised that these traits might further increase the risk of spread and persistence of genetically engineered Brassicaceae plants. Their potential to spread may be dependent on environmental conditions: This has been, for example, discussed for semi-dwarf phenotypes (Fan et al., 2021; Song et al., 2022) which might show improved survival in ruderal areas (Reuter et al., 2008).

4.3. Spontaneous Crossings and Stacking

NGTs make it possible to induce small, targeted changes in plant genomes to generate new properties. This is, in particular, the case with site-directed nuclease 1 applications (SDN 1, i. e. short deletions and/or insertions of a few base pairs), which also enable the alteration of several different DNA sequences – and thus several properties – simultaneously (multiplexing).

As shown in the overview of applications used in Brassicaceae oilseed plants, most traits are currently based on the knock out of single genes, gene copies, members of a particular gene family or genes associated with a distinct phenotype (e. g. (Kawall, 2021; Li et al., 2022)). The two most prominent trait categories, i. e. altered fatty acid metabolism and positive fitness effects (orange and green in Table A1, Table A2 and Table A3), comprise a variety of different traits, ranging from early/late flowering and the reduction of flavonoids to the increase/reduction of unsaturated fatty acids (see Table A1, Table A2 and Table A3).

These traits are influenced by many different genes that are frequently involved in multiple biological processes, which is why their alteration may lead to undesirable pleiotropic effects and unintended consequences in some circumstances. Therefore, the potential negative impacts on ecosystems, food webs and the health of wild species, livestock and humans need to be assessed on a case-by-case basis, i. e. individually for each event. In addition, possible combinations of the traits and impacts thereof need to be assessed and monitored in the affected areas and in wider environments. These potential combinations may occur through intentional crossings (stacked event) or in the case of Brassicaceae, also spontaneously (see for example (Zhang et al., 2021)). Consequently, it is likely that new combinations of geno- and phenotypes will emerge that were neither intended nor previously considered or tested for safety.

If there were to be large scale releases of NGT Brassicaceae oilseed plants, the likelihood of hybridisation within crossable species would increase. Furthermore, the chances of spontaneous and unintended combinations of different NGT traits occurring would also increase. Similar outcrossing phenomena have already been reported in the cultivation of transgenic oilseed rape plants where spontaneous combinations of herbicide resistances have been observed. These spontaneous combinations resulted in the occurrence of transgenic Brassica oilseed plants that were not approved for release (e. g. (Aono et al., 2006; Schafer et al., 2011)).

The stacking of different NGT events via conventional breeding or spontaneous crossings can all result in NGT offspring with new biological characteristics that are absent in the parental plants (see (Bauer-Panskus et al., 2020)). These biological characteristics could, for example, affect plant species composition with unintended and possibly undesirable consequences for ecosystems, food webs, the health of wild species and consumers. They may also cause NGT plants to become invasive. Even if each of the initial NGT events were individually classified as ‘safe’ or ‘unlikely to become invasive’ in risk assessment, their offspring may show next-generation effects associated with unexpected risks caused by genomic interactions. As with transgenic plants, spontaneous crossings of NGT plants may result in new combinations of traits, such as higher fitness and/or novel changes in oil composition.

In this context, unpredictable interactions at the genome level may also depend on the genetic background (including cryptic gene variants, see for example (Aguirre et al., 2023)) and the accumulation of unintended genetic changes caused by the NGT processes (for overview, see (Koller and Cieslak, 2023; Koller et al., 2023)).

5. Conclusions

A closer look at NGT applications in Brassicaceae oilseed crops described in scientific literature, shows that various, often fitness-relevant traits have been altered, e. g. growth and yield, oil and protein composition, abiotic and biotic resistance and fertility.

Currently, most of these traits are conferred by knocking out single genes or gene copies. Some of the traits can, therefore, also be achieved with conventional breeding methods. However, the increasing complexity of NGT applications, which may involve several different genes (Bellec et al., 2022), means that it is becoming increasingly difficult to achieve the same or ‘equivalent’ results using conventional methods. This is due, in particular, to the complexity of some Brassicaceae genomes, but also to the specific technical potential of NGTs (Kawall, 2019) that may rapidly evolve further (He et al., 2023).

The use of NGTs opens up new possibilities to modify the genome (Raitskin and Patron, 2016; Zetsche et al., 2017; Kawall, 2019; Kawall et al., 2020), and often results in new genotypes and phenotypes that are unlikely to result from conventional breeding even if no additional DNA sequences are inserted and no new proteins are produced by the plants (ANSES, 2023; Bohle et al., 2023; Eckerstorfer and Heissenberger, 2023). Furthermore, the spatio-temporal scale of potential releases of Brassicaceae NGT oilseed plants may also cause new combinations and interactions to occur that were neither intended nor assessed – all of which could have inherently unpredictable ecological consequences (Bauer-Panskus et al., 2020; Koller et al., 2023).

In case of a changed oil composition an increase or decrease of PUFAs demonstrates risks for health and survival of pollinators. The likelihood of outcrossing and spread, and environmental risks would increase depending on a couple of factors: a) the number of released new genotypes, b) the spatio-temporal scale of release if new genotypes, c) the fitness relevance of the new traits and d) the novelty of traits compared to the wildtype. Persistence and spread of novel genotypes can hardly be controlled and also hazard mitigation will be extremely difficult due to the hybridization potential, seed dormancy, persistence in the wild, and large pollen transfer distances.

Environmental risk assessment scenarios of specific NGT applications in Brassicaceae oilseed crops as outlined in this review, show that the technological specificity (with the potential for increasing trait complexity) as well as the plants’ biology plus the scale of releases have to be seen in combination. Therefore, the potential release of NGT plants for agricultural purposes requires risk assessment and monitoring not only of singular traits, but also of combinatorial and long-term cumulative effects as foreseen in Directive 2001/18/EC. In addition, risk management will require new concepts and measures to control and potentially restrict the scale of releases. This is especially relevant for potential releases of NGT Brassicaceae in Europe which is a centre of diversity of this plant family.