Research and analysis

Technical guidance on using genetic technologies (such as gene-editing) for making ‘qualifying higher plants’ for research trials

Updated 8 September 2023

Applies to England

The Genetically Modified Organisms (Deliberate Release) (Amendment) (England) Regulations 2022 concern field trials of genetically modified (GM) plants that could have been produced by traditional breeding techniques or could have arisen through natural processes.

The statutory instrument describes these plants as ‘qualifying higher plants’ (QHPs).

This guidance is to assist researchers and developers in this area to understand whether the plants they wish to grow outside in research trials are QHPs and can therefore be notified and grown as such.

Field trials of GM plants that are not QHPs are regulated under the The Genetically Modified Organisms (Deliberate Release) (Amendment) (England) Regulations 2019. QHPs released for purposes other than research and development (for example, marketing and commercialisation) are also regulated under Deliberate Release Regulations.

Material from QHP field trials must not be allowed to enter the human food chain or be fed to animals.

1. QHP notifications

Defra has established a system for notifying QHPs for research trials. You may ‘self-determine’ whether your plants are QHPs, having considered the guidance provided here. Such a determination should be based on an assessment of detailed experimental data generated and recorded during the development and molecular analysis of the plants.

No formal confirmation of QHP status from Defra is required. However, you must notify Defra that you intend to grow QHPs outside for research trials by completing and submitting a notification form to Defra’s GM and Genetic Resources team.

You can request a notification form by emailing the GM and Genetic Resources team at [email protected].

If you need advice about the QHP status of your plants, contact the Defra GM and Genetic Resources team to discuss specific cases. Defra’s views on the interpretation of the relevant legislation are to be seen as advice and will not hold any special force in law.

The notifications will be published on the Genetically modified organisms: applications, decisions and notifications page on GOV.UK.

2. Scope of guidance

This guidance explains which types of genetic changes can result in the generation of a QHP. It also highlights published examples to illustrate how key criteria relating to natural processes and traditional methods and selection might be applied to understand whether your plant is a QHP.

There is a significant evidence base which describes the broad range of genetic changes that result from natural processes and the dynamic nature of genetic material, including the following the publications:

• ACRE advice on Defra’s consultation on the regulation of genetic technologies
• ACRE’s report: Genetically modified organisms: the case for new regulations
• Della Coletta, R., Qiu, Y., Ou, S. et al. How the pan-genome is changing crop genomics and improvement. Genome Biol 22, 3 (2021).
• Sun, H., Jiao, W. B., Krause, K. et al. Chromosome-scale and haplotype-resolved genome assembly of a tetraploid potato cultivar. Nat Genet 54, 342 to 348 (2022)

These phenomena are exploited by plant breeders, alongside other techniques such as those listed in regulation 5(2) of the Deliberate Release Regulations, to generate and capture genetic variation for producing improved crops. Genetic technologies such as gene-editing introduce similar types of genetic changes, often as a result of the same fundamental mechanisms.

3. Section 1: site directed nuclease (SDN)-mediated changes to genetic material

3.1 SDN1-type changes to genetic material

QHPs may be developed using bacterially derived site directed nucleases (SDNs). These gene-editing systems (such as CrisprCas9) can create DNA strand breaks at locations within a genome that are known to be related to a specific trait. Breeders and scientists rely on small errors commonly made by the cell’s own DNA repair mechanism to produce changes to the DNA sequence that result in useful characteristics.

The DNA repair mechanism usually exploited during SDN1 editing is called non-homologous end joining (NHEJ). Errors during NHEJ usually result in small insertions or deletions (indels) to the DNA sequence. These mutations affect the functioning of the targeted sequence, producing the desired trait when the cell is regenerated into a whole organism.

Because they arise through the same biological mechanism, such mutations precisely mimic those that arise in natural processes and traditional techniques, such as those listed in regulation 5(2) of the Genetically Modified Organisms (Deliberate Release) Regulations 2002.

Examples of gene-edited plants that have been created using SDN1-type changes to genetic material

• Raffan, S., et al (2021). Wheat with greatly reduced accumulation of free asparagine in the grain, produced by CRISPR/Cas9 editing of asparagine synthetase gene TaASN2. Plant Biotechnology Journal, 19(8), pp.1602 to 1613
• Nonaka, S., et al (2017). Efficient increase of ɣ-aminobutyric acid (GABA) content in tomato fruits by targeted mutagenesis. Scientific Reports, (online) 7(1), p.7057
• Chen, Y., et al (2020). High‐oleic acid content, nontransgenic allotetraploid cotton (Gossypium hirsutum L.) generated by knockout of GhFAD2 genes with CRISPR/Cas9 system. Plant Biotechnology Journal, 19(3), pp.424 to 426
• Do, P. T., et al (2019). Demonstration of highly efficient dual gRNA CRISPR/Cas9 editing of the homeologous GmFAD2–1A and GmFAD2–1B genes to yield a high oleic, low linoleic and α-linolenic acid phenotype in soybean. BMC Plant Biology, 19(1)

These publications generally describe genetic improvements to crops that are in the early stages of development and some individual lines described may therefore still contain the bacterial editing genes.

These plants are not QHPs and would only be classed as such when all transgenes have been removed, for example by segregation.

Find out more about transgenes in the transgenic changes to genetic material section of this guidance.

DNA repair that follows breakage at a single location typically results in small indels as described. Lesions at two spatially separated locations within a chromosome can also be made using SDNs. In this way larger stretches of DNA can be deleted, potentially removing one or more whole genes or non-coding regions.

This approach also mimics natural processes and traditional breeding techniques, such as transposon activity or exposure to mutagens that are known to result in significant rearrangements, deletions or translocations of genetic material.

Examples of gene-edited plants that have been created using SDN1 to generate larger indels

• Chandrasekaran, J., et al (2016). Development of broad virus resistance in non-transgenic cucumber using CRISPR/Cas9 technology. Molecular Plant Pathology, 17(7), pp.1140 to 1153
• Li, S., et al (2022). Genome-edited powdery mildew resistance in wheat without growth penalties. Nature, (online) 602(7897), pp.455 to 460
• Zhao, Y., et al (2016). An alternative strategy for targeted gene replacement in plants using a dual-sgRNA/Cas9 design. Scientific Reports, 6(1)
• Zhou, H., et al (2014). Large chromosomal deletions and heritable small genetic changes induced by CRISPR/Cas9 in rice. Nucleic Acids Research, 42(17), pp.10903 to 10914
• Brooks, C., et al (2014). Efficient Gene Editing in Tomato in the First Generation Using the Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-Associated9 System. Plant Physiology, (online) 166(3), pp.1292 to 1297

Again, to be counted as a QHP, there can be no transgene elements present, including the gene-editing cassettes, selectable markers and vector genes.

3.2 SDN2- and SDN3-type changes

Another kind of DNA repair (homologous end joining) is sometimes exploited to create targeted changes to the DNA sequence within a genome. In this case, as with SDN1-type changes, the DNA sequence is broken at a predetermined location, but in addition, a DNA template is introduced to the cell, designed to help direct the DNA repair machinery to generate the precise sequence change that is required.

In many cases that sequence change may be a small insertion or deletion. However, it is possible to use this strategy either to replace a whole or partial gene or allele with an alternate version or to insert a cisgene at a predetermined location.

Each of these scenarios mimics genetic changes that are possible to achieve naturally, and via traditional breeding techniques, when the resulting genetic composition remains within that which is accessible through crossing sexually compatible species.

Find out more about cisgenesis, in the Cisgenic changes to genetic material section of this guidance.

Examples of gene-edited plants that have been created using SDN2 and SDN3

• Shi, J., et al (2017). ARGOS8 variants generated by CRISPR-Cas9 improve maize grain yield under field drought stress conditions. Plant biotechnology journal, (online) 15(2), pp.207 to 216
• Li, J., et al (2018). Efficient allelic replacement in rice by gene editing: A case study of the NRT1.1B gene. Journal of Integrative Plant Biology, (online) 60(7), pp.536 to 540

To be counted as a QHP, there can be no transgene elements present, including the gene-editing cassettes, selectable markers and vector genes.

3.3 Prime or base editing

Using an adaptation of the approach described above, it is possible to precisely direct which specific nucleotide or nucleotides are changed within a genome, without generating a double strand DNA break. To achieve this, a SDN such as CrisprCas9 is modified so that it ‘nicks’ one of the DNA strands at a predetermined location, and through the presence of a linked enzyme, replaces the incumbent nucleotide with an alternate one. As with SDN1 type alterations, these changes mimic what occurs naturally and is exploited by breeders.

3.4 Epigenetic changes

Some research, still in its early stages, has demonstrated that SDNs can be engineered to carry out targeted epigenetic changes to genetic material such as (de)methylation. These alterations do not result in a change in DNA sequence but affect gene expression and give rise to phenotypic changes which have been observed to be inherited up to three generations.

In traditional plant breeding, pure epigenetic changes are rare and often unstable. Epialleles are thought more commonly to result from the indirect effects of nucleotide sequence changes either in an ‘obligate’ or ‘facilitate’ fashion. Pure epialleles have therefore played a relatively small role in crop improvement.

Nevertheless, for the purposes of guidance to developers, it is appropriate to note that SDN-mediated epigenetic changes rely on the same biochemical mechanisms for altering DNA and chromatin (for example, (de)methylation and (de)acetylation) as those that occur naturally or as a result of chemical induction. Consequently, plants altered in this way are likely to be QHPs.

Examples of epigenetic changes in plants

• Ghoshal, B., et al (2021). CRISPR-based targeting of DNA methylation in Arabidopsis thaliana by a bacterial CG-specific DNA methyltransferase. Proceedings of the National Academy of Sciences, 118(23)
• Shin, H., et al (2022). Epigenome editing: targeted manipulation of epigenetic modifications in plants. Genes & Genomics
• House, M. and Lukens, L. (2019). The Role of Germinally Inherited Epialleles in Plant Breeding: An Update. Epigenetics in Plants of Agronomic Importance: Fundamentals and Applications, pp.115 to 128

3.5 Summary

Plants containing the type of genetic changes mediated by SDNs, as described above, are likely to be QHPs when any transgenes such as selectable markers and vector genes have been removed (for example, by segregation). This is because the genetic changes mimic those that occur naturally or through traditional techniques and selection.

You should carefully consider cases where multiple simultaneous or sequential edits that are precisely targeted have been made. Where these result in a trait that is substantially different to one that might be reasonably expected to arise through natural processes or traditional methods, such plants may not be QHPs.

Examples of this are, when:

• the conversion of an endogenous protein to one that shares a more significant homology to one that is not native
• the substantial alteration of a metabolic pathway that results in a new or non-native compound

If you are uncertain whether a specific plant generated using a genetic technology such as gene-editing would be considered a QHP, you should contact us by emailing the Defra GM and Genetic Resources team at [email protected] for advice.

4. Section 2: the insertion of genetic material following its in vitro manipulation

4.1 Transgenic changes to genetic material

There are clear examples of genetic changes that do not result in a QHP. Usually referred to as transgenic, they involve the manipulation of a DNA molecule from a non-crossable species outside of the cell (in vitro), followed by its insertion into the genome using recombinant DNA technology.

The resulting plant is not a QHP because its genetic composition does not fall within the variation that could occur naturally within that species (or a related species) or as a consequence of traditional techniques and selection.

An example of this that is relevant to many gene-editing approaches is the insertion of the bacterial CrisprCas9 gene into a plant genome. The genetic composition of the resulting plant is very unlikely to have occurred naturally or as a result of traditional techniques and selection, so the plant would not be a QHP.

It follows that where this gene is subsequently removed, for example through segregation (and no other transgenes are present, such as selectable markers or other vector derived genes), that plant would then be a QHP if it meets the other required criteria outlined in this guidance.

The example of insertion of the CrisprCas9 gene shows an important criterion that you should consider, which is whether the inserted DNA molecule originated from a species that is sexually compatible (or crossable) with the recipient species. If it did not originate from a species that is sexually compatible (or crossable), then the plant is not a QHP. There are other clear examples of transgenesis, for example, where a plant contains a gene from an animal or a fungus.

Further examples include wheat with an inserted gene from sunflower ^{[footnote 1]} and maize with an inserted gene from Arabidopsis ^{[footnote 2]}.

A further approach that results in a transgenic organism (which is therefore not a QHP), is where the inserted DNA molecule encodes a synthetically designed peptide or protein that is not known to exist in nature. Such approaches are used to develop sustainable resistance to pests and diseases such as the potato cyst nematode ^{[footnote 3]}, the root knot nematode of aubergine ^{[footnote 4]}, and Aspergillus fungus in maize ^{[footnote 5]}.

In most cases it will be clear that the plant you have produced is not a QHP on the basis of the detailed experimental information recorded during the development and molecular analysis of the plant.

If you are uncertain whether a specific inserted gene would be considered a transgene, contact Defra for advice by emailing [email protected].

4.2 Cisgenic changes to genetic material

As described, the genetic relationship between the donor of an inserted DNA fragment and its recipient species is important for determining QHP status. Whereas transgenesis refers to inserted DNA originating from a sexually incompatible species, cisgenesis refers to inserted DNA originating from the same or a related species that is sexually compatible. Cisgenes generally include the introns and flanking native promoter and terminator in the same orientation.

Cisgenic plants are QHPs because their genetic composition is consistent with the genetic variation that could occur naturally within that species or as a result of traditional techniques and selection that are listed in regulation 5(2) of the Deliberate Release Regulations.

These include the sexual fertilisation and spontaneous mutation that occurs within and between individuals in the wild with no human intervention, as well as crosses that are made possible using a range of interventions and approaches exploited by breeders for a number of years. For example, it is possible to breed with distant wild relatives using a series of ‘bridging’ crosses (when an intermediate cross with a third species, which is compatible with both species, is used to bridge the crossing barrier).

In vitro fertilisation, cell fusion and embryo rescue are all non-GM laboratory techniques used by breeders to increase intra- and inter-specific genetic variation through manipulating somatic and sex cells in culture, followed by whole plant regeneration. More distant relatives may have progressively compromised chromosome pairing but a low proportion may give rise to fertile hybrids.

Polyploidy induction is another important breeding tool which involves increasing the number of homologous chromosome pairs by stopping cell division immediately after DNA replication.

When determining whether your cisgenic plants are QHPs, you should take into consideration the wide range of sources of genetic variation that are available to breeders from within the ‘gene pool’ of a plant species and made accessible using any combination of the methods outlined.

It is common for cisgenic plants to contain more than one inserted gene from a sexually compatible relative. Thus, a QHP may contain multiple cisgenes at a single locus or single cisgenes at multiple loci. Such a QHP may have varying structural arrangements as long as the resulting genetic composition remains consistent with the genetic variation that could occur naturally within sexually compatible relatives or as a result of traditional techniques and selection.

Plants that contain cisgenes and, in addition, short DNA sequences introduced as a by-product of the transformation process that are expected to have no observable effect on the plant’s phenotype, may also be defined as QHPs. Such sequences could include the left and right T-DNA borders, plus a few base pairs of insertional ‘read through’ on either or both sides, for example. Technically these small sequence additions would be classed as exogenous.

However, there is a strong scientific rationale for suggesting that their presence may be discounted because exogenous sequences are naturally present in the genomes of modern cultivated varieties, some of which are derived from widely divergent species, including from other phylogenetic kingdoms^{[footnote 6]}.

Examples of plants containing cisgenes

• Jo, K et al (2014). Development of late blight resistant potatoes by cisgene stacking. BMC Biotechnology, 14(1), p.50
• Gao, Y., et al (2018). Cisgenic overexpression of cytosolic glutamine synthetase improves nitrogen utilisation efficiency in barley and prevents grain protein decline under elevated CO 2. Plant Biotechnology Journal, 17(7), pp.1209 to 1221
• Maltseva, E., et al (2021). A Cisgenic Approach in the Transformation of Bread Wheat cv. Saratovskaya 29 with Class I Chitinase Gene. The Open Biotechnology Journal, 15(1), pp.29 to 35
• Vanblaere, T., et al (2011). The development of a cisgenic apple plant. Journal of Biotechnology, 154(4), pp.304 to 311
• Holme, I.B., et al (2011). Cisgenic barley with improved phytase activity. Plant Biotechnology Journal, 10(2), pp.237 to 247

4.3 Intragenic changes to genetic material

Intragenic changes to genetic material also involve the in vitro manipulation of a nucleic acid molecule followed by its insertion into the genome using recombinant DNA technology. The main difference between intragenic changes and cisgenesis is that the insert usually comprises a full or partial coding region of a gene or genes (with or without introns) combined with the promoters and/or terminators from a different genes of the same species or a crossable species.

Specifically engineered combinations of individual genetic elements such as these are unlikely to result from natural processes or traditional breeding techniques. They are therefore unlikely to match the definition of a QHP.

4.4 Summary

Transgenesis, cisgenesis and intragenesis describe cases where a DNA fragment is manipulated in vitro before being inserted into the genome using recombinant DNA technology. As outlined in the guidance, cisgenic plants are QHPs because their genetic composition is consistent with the genetic variation that could occur naturally within that species or as a result of traditional techniques and selection.

Transgenic plants, however, contain genetic material from species that are sexually incompatible or non-crossable and are therefore not QHPs. Intragenic plants are also not QHPs because their genetic composition is unlikely to occur naturally or as a result of traditional techniques and selection

1. Fernanda Gabriela González, Matías Capella, Karina Fabiana Ribichich, Facundo Curín, Jorge Ignacio Giacomelli, Francisco Ayala, Gerónimo Watson, María Elena Otegui, Raquel Lía Chan. Field-grown transgenic wheat expressing the sunflower gene HaHB4 significantly out yields the wild type. Journal of Experimental Botany, Volume 70, Issue 5, 15 February 2019, pages 1669 to 1681 ↩

2. Chen, Z., Liu, Y., Yin, Y. et al. Expression of AtGA2ox1 enhances drought tolerance in maize. Plant Growth Regul 89, pages 203 to 215 (2019) ↩

3. Green J., Wang D., Lilley C. J., Urwin P. E., Atkinson H. J. (2012). Transgenic Potatoes for Potato Cyst Nematode Control Can Replace Pesticide Use without Impact on Soil Quality. PLoS ONE 7(2) ↩

4. Pradeep K. Papolu, Tushar K. Dutta, Alkesh Hada, Divya Singh, Uma Rao. The production of a synthetic chemodisruptive peptide in planta precludes Meloidogyne incognita multiplication in Solanum melongena. Physiological and Molecular Plant Pathology, Volume 112, 2020 ↩

5. Kanniah Rajasekaran, Ronald J. Sayler, Christine M. Sickler, Rajtilak Majumdar, Jesse M. Jaynes, Jeffrey W. Cary. Control of Aspergillus flavus growth and aflatoxin production in transgenic maize kernels expressing a tachyplesin-derived synthetic peptide, AGM182. Plant Science, Volume 270, 2018, pages 150 to 156 ↩

6. Jianchao Ma, Shuanghua Wang, Xiaojing Zhu, Guiling Sun, Guanxiao Chang, Linhong Li, Xiangyang Hu, Shouzhou Zhang, Yun Zhou, Chun-Peng Song, Jinling Huang. Major episodes of horizontal gene transfer drove the evolution of land plants. Molecular Plant, 2022 ↩