Going bananas: how transgene‐free editing is contributing to a fruitful future

Abstract

The ability to induce precise genetic modifications through genome editing has greatly enhanced crop improvement efforts. However, the presence of transgenes in the edited crops necessitates regulatory approval for market introduction (Gao, 2021). Typically, transgene-free genome editing is achieved by inserting a nuclease-encoding T-DNA cassette into the plant genome, which is subsequently removed through Mendelian segregation. Yet, in many perennial, sterile, or clonally propagated crop species, outcrossing is not possible or undesirable, highlighting the need for other strategies that enable transgene-free genome editing in these crops. In an article recently published in New Phytologist, Van den Broeck et al. (2025; doi: 10.1111/nph.70044) present a straightforward method for transgene-free gene editing in sterile banana. As the world's most important fruit crop, banana is highly susceptible to diseases and pests, making genome editing a valuable tool for developing more resilient cultivars. The authors employed Agrobacterium-mediated delivery of a cytosine base editor targeting both ACETOLACTATE SYNTHASE (ALS) genes in banana cell cultures. Deamination of a specific cytosine in either of these genes leads to a gain-of-function mutation, conferring resistance to the herbicide chlorsulfuron. After herbicide selection, the authors detected efficient C-to-T conversions in the ALS genes of the regenerated banana shoots. Screening over 400 chlorsulfuron-resistant lines using PCR and whole-genome sequencing, they found that up to 3.2% of the lines edited in at least one ALS gene were free of the T-DNA cassette. The efficiency of transgene-free base editing in both ALS genes – used as a proxy to estimate the efficiency of multiplex editing – was 1.0%. The authors estimated that a full-time person could perform c. 18 banana transformations per year, generating up to 16 transgene-free, chlorsulfuron-resistant banana plants that are also edited in a target gene of choice.

Adopting a science-based regulatory approach will ensure that innovative breeding techniques can be fully leveraged to address urgent agricultural and environmental challenges.

A key advantage of the transient expression strategy is its broad applicability, provided the target species is susceptible to Agrobacterium tumefaciens infection (Fig. 1). Beyond its utility for sterile crops like banana, this approach is also useful for perennial and vegetatively propagated species. Many elite tree varieties, for instance, are clonally propagated. Given most tree species are outbreeding and highly heterozygous, eliminating the T-DNA through crossing would disrupt their elite genetic constitution.

Fig. 1

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Transfer of a Cas9-encoding T-DNA from Agrobacterium tumefaciens into a host cell resulting in gene editing. Upon expression of Agrobacterium's virulence (Vir) genes (located on a helper plasmid), the T-DNA is transferred into the plant nucleus as a single-stranded DNA molecule. This transfer is accompanied by effector proteins, such as VirD2 and VirE2, through a type IV secretion system (T4SS). Within the host cell, a T-complex forms to protect the T-DNA from degradation. In the plant nucleus, the T-DNA is converted into a double-stranded, transcriptionally active form that can be either transiently expressed or integrated into the host genome, allowing stable expression. Both transient and stable expression can lead to the synthesis of the Cas9 enzyme and the guide RNA (gRNA), which together edit the target gene (yellow star). This figure was created with BioRender (BioRender.com/b99g511) and was previously published (Hoengenaert et al., 2025).

While the study by Van den Broeck et al. demonstrates a promising method for transgene-free gene editing in banana, there are several areas for potential improvement. First, enhancing the efficiency of multiplex gene editing is crucial, especially for complex traits that require the simultaneous editing of multiple genes to enhance the trait. High-throughput platforms, such as Iterative Testing of Editing Reagents (ITER), have been developed to enable the simultaneous testing of multiple reagents from the gene-editing toolbox in a multi-well setup, allowing the systematic improvement of editing configurations (Gaillochet et al., 2023). In addition, the use of alternative promoters to drive guide RNA (gRNA) expression has been shown to significantly increase the gene-editing efficiency (Li et al., 2021). For example, in poplar, using the AtU3 promoter to drive gRNA expression resulted in consistently higher base-editing efficiencies compared to the AtU6 promoter (Li et al., 2021).

Second, further increasing the number of transgene-free genome-editing events remains a priority to optimize T-DNA-free gene-editing pipelines. In their study, Van den Broeck et al. report that 3.2% of their ALS-edited shoots were transgene-free. Recently, we developed a similar co-editing system using ALS genes in poplar. In our study, 49% of the ALS-edited shoots were transgene-free (Hoengenaert et al., 2025). It is likely that the frequency of obtaining transgene-free ALS-edited lines, resulting from the transient expression of the cytosine base editor, correlates with the efficiency of the base editor. Less efficient base editors, such as APOBEC1 used by Van de Broeck et al., will naturally induce fewer C-to-T conversions during their transient expression window compared to more efficient ones. As a result, a lower percentage of transgene-free shoots will be recovered upon chlorsulfuron selection (Fig. 2). To help eliminate stable transformants, a marker gene can be incorporated in the T-DNA cassette. A study by Huang et al. (2023) demonstrated this principle by including a GFP reporter gene on the T-DNA cassette, enabling easy identification and elimination of stably transformed fluorescent shoots. Alternatively, adding the RUBY reporter gene, which encodes the synthesis of betalain, a bright red natural product, would allow visual differentiation between stable transformants and transgene-free lines by eye (He et al., 2020). Furthermore, negative selection markers that convert a nontoxic compound into a toxic analog, such as CodA, could be implemented to select for shoots devoid of the T-DNA, as demonstrated in poplar (Hoengenaert et al., 2025).

Fig. 2

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Regardless of the species, the efficiency of transformation and subsequent editing varies, affecting the number of regenerated T-DNA-free shoots that can be obtained. When using Agrobacterium for transformation, cells can be stably transformed (yellow, with a red-and-blue DNA helix inside), transiently transformed (yellow, with two separate DNA helices) or nontransformed (blue, with a single blue DNA helix). Stable transformation has a higher likelihood of generating the desired mutations and thus promoting shoot development on selection media. By contrast, transiently transformed cells have a lower probability of producing the desired mutations due to the limited presence of the gene-editing reagents in the cells. The efficiency of obtaining mutated shoots from these transiently transformed cells largely depends on the effectiveness of the gene-editing reagents used, such as nucleases, nuclear localization sequences, or guide RNAs. To efficiently generate T-DNA-free mutants, either highly effective gene-editing reagents must be used or an efficient negative selection strategy should be applied to eliminate stable (T-DNA-containing) mutants. This figure was created with BioRender (BioRender.com/jbqt5ut).

Third, improvements are needed to overcome the bottleneck of plant transformation and regeneration itself. Although Agrobacterium-mediated transformation, biolistics transformation, and protoplast transfection work for a handful of species, transforming and regenerating elite varieties often remains difficult. One approach could explore the use of alternative Agrobacterium strains and the use of ternary vector systems, which enhance bacterial virulence and thus promote the delivery of transgenes (Anand et al., 2018). In addition, harnessing morphogenic regulators that promote regeneration might facilitate the genetic improvement of elite varieties (Lowe et al., 2016; Shivani et al., 2017). Recent genome-wide association studies in poplar have identified numerous genes associated with transformation and/or regeneration efficiency. Interestingly, only a few of these genes overlap with those currently known to enhance transformation in crops (Nagle et al., 2024a,b).

The development of transgene-free gene-editing technologies can significantly accelerate the breeding of novel crop varieties and facilitate regulatory approval. It is essential that regulatory frameworks are grounded in scientific evidence, focusing on the safety and benefits of the final product rather than the methods used to create it. The recent European Council's negotiating mandate on regulating plants obtained through new genomic techniques (NGTs) marks a significant step toward accepting transgene-free gene-edited crops in Europe (Council of the European Union, 2025). However, the current proposal excludes edits for herbicide tolerance, hence also when used solely for selection purposes, as in the methods described for banana by Van den Broeck et al., and for other important crops like potato, poplar and citrus (Veillet et al., 2019; Huang et al., 2023; Hoengenaert et al., 2025). As a result, transgene-free crops developed through these elegant methods would still be subject to the legislation on genetically modified organisms, significantly limiting their market introduction. Adopting a science-based regulatory approach will ensure that innovative breeding techniques can be fully leveraged to address urgent agricultural and environmental challenges (Boerjan & Strauss, 2024).