Delivery efficiency remains a critical determinant

The gap

Evidence profile

Stated in the future work and cells limitations sections of the source papers, classified as general, spanning 4 journals.

Supporting evidence — 4 representative gaps

• Advancing Functional Genomics in Marchantia polymorpha: A Review of Induced Mutagenesis Toolkits and Applications (2026) · Plant Molecular Biology Reporter · doi

  The diverse mutagenesis tools available for M. polymorpha each have distinct strengths and ideal applications, summa- rized in Table 1. The choice of method depends on the spe- cific research goal. For unbiased phenotype-driven screens, random mutagenesis approaches are indispensable. Heavy- ion beam irradiation currently offers the highest efficiency and diverse mutation spectrum. For testing hypotheses about specific genes, targeted editing tools like CRISPR/Cas9 are unrivalled for their speed and precision in generating knock- outs. While still developing, base and prime editing repre- sent the future for introducing specific nucleotide changes to study protein function and for trait engineering. Abbreviations: EMS, ethyl methanesulfonate; NaN ₃, sodium azide; MNU, N-methyl-N-nitrosourea; LET, linear energy transfer; DSBs, double-strand breaks; gRNA, guide RNA; NHEJ, non-homologous end joining; HDR, homol- ogy-directed repair; CRISPR, clustered regularly interspaced short palindromic repeats; TALENs, transcription activator- like effector nucleases; T-DNA, transferred DNA Several exciting frontiers lie ahead. First, the community must develop standardized, optimized protocols for random mutagenesis (e.g., EMS, heavy-ion) to build large, publicly available mutant libraries. Second, efforts to enhance HDR efficiency and adapt base/prime editing for Marchantia will unlock precise genome engineering. Third, addressing transgene expression variability through synthetic biology approaches will be crucial for complex genetic manipula- tions. Finally, integrating these mutagenesis tools with high- throughput phenotyping and sequencing technologies will enable systems-level approaches to understand gene function in this evolutionarily pivotal plant.

  generalstated in future workevidence 5/5
  Keywords: mutagenesis tools approaches editing diverse available random heavy efficiency specific like crispr base prime function
• Breaking self-incompatibility for diploid hybrid potato breeding: advances, mechanisms, and emerging technologies (2026) · Frontiers in Plant Science · doi

  The integration of speed breeding, genomic selection (GS), and precision genome editing provides a promising framework for accelerating diploid hybrid potato breeding, all of which depend on the prior establishment of self-compatibility. Speed breeding shortens generation cycles by optimizing growth conditions, enabling more rapid generation advancement of SC materials. Genomic selection supports early and high-throughput prediction of complex traits, allowing more efficient screening of SC lines and promising hybrid combinations. When combined with targeted editing of SI-related genes, these tools can help streamline breeding pipelines; however, efficiency varies across genetic backgrounds, and long-term field performance remains to be fully validated (Xu et al., 2022). 4.4 Alternative reproductive strategies: apomixis Apomixis is an asexual reproductive system that produces clonal seeds without fertilization, representing an alternative reproductive strategy that reduces dependence on sexual reproduction and selfing rather than directly overcoming SI. Apomixis does not Future research directions toward diploid hybrid potato breeding should be rooted in a deeper understanding of SI mechanisms and the rational application of SC-related technologies. To achieve stable and efficient diploid hybrid breeding systems, research priorities should be balanced between practically achievable shortterm goals and longer-term exploratory objectives. In the short term, core efforts should focus on strengthening the efficiency and stability of SC induction systems mediated by Sli introgression and genome editing. It is critical to recognize that Slimediated SC is not universally effective across all genetic backgrounds, and its function may be modulated by additional genetic modifiers and environmental factors. This limitation is consistent with the variable performance of Sli-based SC observed in earlier germplasm evaluation and breeding application sections. Further characterization of these regulatory interactions will help improve the robustness and predictability of SC conversion in diverse elite germplasm. Additional short-term priorities include optimizing breeding pipelines for inbred line development, mitigating inbreeding depression and deleterious genetic load, and improving the reliability of phenotypic screening for SC traits.

  generalstated in future workevidence 5/5
  Keywords: breeding term hybrid genetic speed genomic selection editing diploid generation cient apomixis short integration genome
• Emerging applications of CRISPR-Cas9 genome editing in horticultural crop improvement (2026) · Discover Plants · doi

  Although CRISPR/Cas technology has emerged as a highly precise and versatile tool for targeted genome modification, it should be viewed as a complementary approach integrated with conventional breeding, marker-assisted selection (MAS), and genomic selection rather than as a complete replacement for traditional crop improvement strategies. Conventional breeding and MAS remain highly effective for improving complex quantitative traits controlled by multiple genes and for utilizing naturally available genetic diversity. In contrast, genome editing is particularly advantageous when precise modification of specific genes is required, when desirable alleles are absent in breeding populations, or when rapid trait introgression into elite cultivars is necessary. Therefore, future horticultural improvement programs will likely benefit most from integrated breeding approaches combining conventional breeding, molecular markers, genomic selection, and precision genome editing technologies. The future success of CRISPR/ Srivastava et al. Discover Plants (2026) 3:295 Page 19 of 26 Cas-mediated genome editing in horticultural crops will largely depend on overcoming major biological and technical constraints, including low transformation efficiency, genotype-dependent regeneration, polyploidy, prolonged juvenile phases, and limited genomic resources. Among these, inefficient transformation and regeneration remain the primary bottlenecks, particularly in woody perennials and clonally propagated crops. Several strategies may substantially improve transformation efficiency in recalcitrant horticultural species. Optimization of Agrobacterium-mediated transformation through genotype-specific modification of explant type, bacterial strain, virulence induction conditions, co-cultivation duration, and selection regimes can significantly enhance transformation success. The incorporation of developmental regulator genes such as WUSCHEL (WUS), BABY BOOM (BBM), SHOOT MERISTEMLESS (STM), and GROWTH-REGULATING FACTOR–GRF INTERACTING FACTOR (GRF-GIF) chimeric modules has shown considerable promise in improving somatic embryogenesis and shoot regeneration efficiency in several difficult-to-transform species. Similarly, the use of morphogenic regulators, inducible promoter systems, and transient expression approaches may improve regeneration while minimizing stable transgene integration. Alternative delivery platforms are also expected to accelerate genome editing applications in horticultural crops. DNA-free ribonucleoprotein (RNP)-mediated editing offers the advantage of precise editing without transgene integration and may improve regulatory acceptance, particularly in vegetatively propagated crops.

  generalstated in future workevidence 5/5
  Keywords: editing genome breeding selection horticultural mediated crops transformation regeneration precise modification conventional genomic genes particularly
• CRISPR–Cas9 Applications in plant science: Advances, challenges, and future perspectives (2026) · Zenodo (CERN European Organization for Nuclear Research) · doi

  Delivery efficiency remains a critical determinant of genome editing success. - Off-target effects, genotype dependency, biosafety, and regulatory acceptance are challenges that need to be addressed. - Regeneration of whole plants from protoplasts remains challenging for many crop species.

  generalstated in cells limitationsevidence 5/5
  Keywords: delivery efficiency remains critical determinant genome editing success

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