Introduction: Broadening the Reach of Plant Gene Editing

Gene editing technologies, particularly CRISPR-Cas systems, have transformed plant biology by enabling precise, targeted modifications to genomes. In model species such as Arabidopsis thaliana and major crops like rice, maize, and wheat, researchers routinely knock out genes, introduce precise point mutations, or insert transgenes with high efficiency. However, the vast majority of plant species—thousands of wild relatives, underutilized crops, medicinal plants, and endangered species—remain outside the reach of these tools. Developing gene editing methods for these understudied plants is not merely a technical exercise; it is a strategic imperative for biodiversity conservation, sustainable agriculture, and the discovery of novel biochemical pathways. Without tailored approaches, many species with unique traits for climate resilience, disease resistance, or pharmaceutical production will remain genetically inaccessible.

The challenge is multidimensional. Understudied species often lack high-quality reference genomes, efficient transformation protocols, or robust regeneration systems. Their reproductive biology may differ radically from that of well-studied models, and regulatory or funding constraints can further slow progress. Yet recent advances in sequencing technology, synthetic biology, and delivery systems are opening new avenues. This article explores the obstacles, strategies, and emerging successes in developing gene editing tools for understudied plant species, and outlines the path forward.

The Value of Understudied Plant Species

Understudied plants—sometimes called "orphan crops" or "neglected species"—include many that are vital for local food security, traditional medicine, and ecosystem stability. Examples include teff, finger millet, bambara groundnut, and breadfruit, as well as thousands of non-crop wild species. These plants often possess genetic adaptations to harsh environments: drought tolerance in Cleome species, pest resistance in wild relatives of tomato, or specialized metabolic pathways that produce anti-cancer compounds in plants like Podophyllum and Camptotheca.

Gene editing could accelerate the domestication of new crops, enhance nutritional profiles, or restore endangered populations by enabling genetic rescue. For instance, editing genes for shattering in wild cereals could facilitate harvesting, while editing biosynthetic pathways could increase yields of valuable secondary metabolites. The potential impact on global food security and biodiversity is enormous, but realizing it requires solving the fundamental technical bottlenecks that keep these species out of the gene-editing toolbox.

Core Challenges in Developing Gene Editing Tools for Understudied Species

Limited Genomic Resources

High-quality genome assemblies are the foundation of any gene-editing project. Without a reference genome, designing guide RNAs for CRISPR, identifying off-target sites, and interpreting editing outcomes become nearly impossible. Many understudied species lack even a draft genome, and if a sequence exists, it may be fragmented or poorly annotated. The cost and complexity of de novo genome assembly have fallen dramatically with long-read sequencing technologies (e.g., PacBio HiFi and Oxford Nanopore), but for many small research groups or projects focused on non-model species, generating a chromosome-level assembly remains a significant investment.

Transformation Bottlenecks

Delivering gene-editing components (Cas9 nuclease, guide RNA, repair templates) into plant cells is the single most limiting step. In model plants like Arabidopsis, Agrobacterium tumefaciens-mediated transformation of floral tissues is routine. In crops like maize, biolistic particle delivery into embryogenic callus is effective. But for most understudied species, no established transformation protocol exists. Tissue-specific recalcitrance, lack of efficient regeneration, and the absence of reliable selectable markers are common barriers. The plant cell wall and innate immune defenses further complicate delivery.

Regeneration Difficulties

Even if transformation is successful, regenerating whole plants from edited cells—via somatic embryogenesis or organogenesis—is often the hardest step. Each species may require unique media formulations, hormone combinations, and light regimes. The process can take months or years and frequently fails. For many wild or non-crop species, tissue culture protocols are completely unexplored.

Unique Reproductive Biology

Gene editing often alters traits that affect reproduction—flowering time, fertility, or seed development. In perennial or obligate outcrossing species, producing homozygous edited lines through traditional breeding is extremely slow or impossible. Editing in clonally propagated crops (e.g., banana, cassava) avoids some issues but introduces others, such as mosaic editing patterns due to chimeric tissues.

Delivery System Limitations

CRISPR components can be delivered as DNA (plasmids), RNA, or ribonucleoprotein (RNP) complexes. DNA-based delivery carries the risk of random integration of plasmid sequences, raising regulatory concerns. RNA-based delivery is transient but often less efficient. RNPs offer protein-only delivery with minimal off-target effects, but they require high-quality protein preparation and appropriate delivery methods. For many understudied species, the optimal delivery mode is unknown.

Strategies for Overcoming These Barriers

Investing in Genome and Transcriptome Resources

Generating a reference genome is a prerequisite. Initiatives such as the Earth BioGenome Project and the 10,000 Plant Genomes Project are accelerating sequencing of understudied species. For smaller labs, strategies like using closely related genomes as references, combined with reduced-representation sequencing or RNA-seq for transcriptome assembly, can serve as interim solutions. Long-read sequencing and optical mapping now allow relatively straightforward assembly of genomes up to several gigabases. Sharing these resources through public databases like NCBI, Ensembl Plants, and Phytozome is critical.

Optimizing Transformation and Regeneration

Systematic testing of tissue types (leaf discs, cotyledons, hypocotyls, immature embryos, somatic embryos) and Agrobacterium strains (e.g., AGL1, GV3101) can identify permissive conditions. Factors such as preculture, acetosyringone concentration, co-cultivation duration, and antibiotic selection all require empirical optimization. Morphogenic regulators such as WUSCHEL and BABY BOOM have been used to enhance regeneration in recalcitrant species, including maize and wheat. Introducing these regulators transiently or as part of the editing system can dramatically improve success rates.

Adapting CRISPR-Cas Systems

The Cas9 nuclease from Streptococcus pyogenes is not universally efficient at all temperatures or in all genome contexts. Alternatives such as Cas12a (Cpf1) offer different PAM requirements and produce sticky ends, which can aid repair. For plants with high GC content, guide RNA design must account for secondary structures. Codon-optimizing the Cas9 for the target plant species and using plant-specific promoters (e.g., CaMV 35S, Ubiquitin, or Actin) enhances expression. For RNP delivery, the protein must be stable and active in the plant cell environment.

Leveraging Transient Expression Systems

Before investing in stable transformation, researchers can use protoplasts, leaf infiltration, or callus-based transient assays to test editing efficiency. Protoplasts from understudied species can be isolated and transformed with plasmids encoding Cas9 and guide RNAs, then analyzed by PCR or deep sequencing after 48–72 hours. This approach allows rapid validation of guide RNA activity and optimization of components without the need for regeneration. A transient test can guide which delivery method to pursue for stable transformation.

Exploring Novel Delivery Methods

Nanoparticle-mediated delivery (e.g., carbon nanotubes, gold nanoparticles, or lipid-based carriers) bypasses the need for Agrobacterium and can deliver RNPs or RNAs directly to plant cells. Nanocarriers coated with polyethylene glycol or cell-penetrating peptides show promise for delivery without mechanical damage. Viral vectors, such as those based on tobacco rattle virus (TRV), can spread gene editing components systemically in some dicot species, though they are limited by host range and cargo size. These alternative delivery methods are particularly valuable for species that are recalcitrant to Agrobacterium transformation.

Case Studies: Success in Understudied Species

Gene Editing in Rare Medicinal Plants

Researchers targeting the medicinal plant Salvia miltiorrhiza (Danshen) faced a lack of efficient transformation. By optimizing Agrobacterium infection of hairy roots and using a twin-arginine translocation signal peptide to localize Cas9, they achieved targeted mutations in the SmCPS1 gene involved in tanshinone biosynthesis. The edited roots showed altered metabolite profiles, demonstrating that gene editing can be applied to modulate secondary metabolism in a species with no prior genome editing protocols. This work, published in Plant Physiology and Biochemistry, highlights the feasibility of editing non-model medicinal plants.

Adapting Gene Editing for Orphan Crops

Teff (Eragrostis tef), a staple in Ethiopia with excellent gluten-free properties, has a tetraploid genome and no robust transformation system. Scientists developed a protocol using immature embryos transiently expressing the morphogenic regulator BABY BOOM combined with Agrobacterium-mediated delivery of CRISPR-Cas9. They successfully edited the GA20-oxidase gene to produce semi-dwarf teff lines, a key trait for lodging resistance. This breakthrough, reported in Plant Methods, demonstrates that morphogenic regulators can unlock transformation in recalcitrant orphan crops.

Gene Editing in an Endangered Cycad

In a proof-of-concept for biodiversity conservation, researchers applied CRISPR-Cas9 to edit the cycad Cycas revoluta, a threatened gymnosperm. Using a combination of protoplast isolation, RNP delivery, and subsequent callus regeneration, they achieved targeted knockout of a flavonoid biosynthesis gene. The edited cells were identified by high-resolution melt analysis. Although whole plant regeneration remains challenging, this work shows that gene editing can be used for functional genomics even in phylogenetically isolated species. Details are available in a preprint on bioRxiv.

Future Directions and Emerging Technologies

Base Editing and Prime Editing

These precise editing techniques enable single-nucleotide conversions without double-strand breaks. Base editors (cytidine or adenine deaminases fused to Cas9 nickase) can introduce specific C-to-T or A-to-G changes. Prime editors (Cas9 nickase fused to reverse transcriptase with a prime editing guide RNA) allow small insertions, deletions, and substitutions. Both methods are being adapted for plants, and their application in understudied species could correct deleterious mutations or create beneficial alleles with minimal off-target effects. A recent review in The Plant Journal outlines progress.

Synthetic Biology and Gene Drives

Gene drives—elements that bias inheritance to spread a desired trait through a population—could be used to suppress invasive species or rescue endangered populations. However, ethical and ecological concerns are significant. For understudied plants, synthetic biology approaches such as recombineering of large DNA fragments or rewriting entire metabolic pathways may become feasible as DNA synthesis costs drop. Combining gene editing with synthetic circuits could enable conditional or tissue-specific edits.

Community Resources and Open Science

The speed of progress depends on sharing data, tissues, and protocols. Initiatives like the Plant Genome Editing Database (PGED), the OpenPlant project, and the New Breeding Technologies for Orphan Crops network are fostering collaboration. Open-access repositories for transformation protocols (such as Protocols.io) and sharing of transgenic seeds through genetic stock centers (like the Arabidopsis Biological Resource Center but for non-model plants) will be vital. Funding agencies should prioritize capacity building in under-resourced regions where understudied species are most diverse.

Ethical, Regulatory, and Practical Considerations

Gene editing in understudied species raises ethical questions. For endangered species, editing could reduce genetic diversity or have unintended ecological consequences. For orphan crops used in subsistence agriculture, intellectual property restrictions may limit access to improved varieties. Regulatory frameworks for gene-edited plants differ globally: some countries exempt site-directed mutagenesis from GMO regulation, while others require stringent risk assessments. Researchers must engage with local communities, Indigenous groups, and farmers to ensure that the benefits of gene editing are equitably distributed. A thoughtful, transparent, and inclusive approach is essential to avoid repeating historical injustices associated with agricultural biotechnology.

Conclusion

Developing gene editing tools for understudied plant species is a complex but tractable challenge. By investing in genomic resources, optimizing transformation and regeneration protocols with morphogenic regulators, adapting Cas variants, and using transient tests and novel delivery systems, researchers are progressively expanding the genetic frontier. Each success—whether in a rare medicinal herb, a neglected cereal, or an ancient gymnosperm—provides a reproducible template for other species. As technologies mature and collaboration intensifies, the full spectrum of plant diversity will become accessible to genetic improvement. This will not only advance fundamental understanding of plant biology but also deliver practical solutions for food security, climate adaptation, and conservation. The tools are being developed; the future lies in applying them wisely.