Bioscience Methods 2026, Vol.17, No.1, 9-22 http://bioscipublisher.com/index.php/bm 18 The present circumstances demonstrate progress in the field despite these existing issues. The transformation systems based on organogenesis and somatic embryogenesis have demonstrated better success rates according to He et al. (2023). The use of Cas9 protein or RNA for DNA-free genome editing results in faster processes and prevents stable transformation according to Guo et al. (2023) and Young et al. (2019). This method has worked in other monocots and may make pineapple editing easier to accept by the public. Recent research indicates that pineapple transformants remain stable and scientists have successfully made short-term modifications to a GFP marker gene (Cheng et al., 2025) which indicates that the field is advancing. 5.2 Potential solutions and innovations Several ideas may help solve pineapple’s editing problems. One is to improve transformation methods. Researchers are testing physical delivery like particle bombardment to deliver CRISPR into tissues less responsive to Agrobacterium. Using biolistic delivery of Cas9–sgRNA complexes (RNPs) could produce edited plants without transgenes if regeneration works (Sturme et al., 2022; Nadakuduti and Enciso-Rodríguez, 2021). Tissue culture can also be improved. Morphogenic regulator genes such as Baby Boom and WUSCHEL have boosted transformation in cereals and might work for pineapple. They can be added temporarily to promote embryogenic callus formation, then removed later. CRISPR tools themselves can be improved. High-fidelity Cas9 types, such as SpCas9-HF1 or eSpCas9, cut off-target DNA far less often (Wu et al., 2022). PAM-relaxed Cas9 types like xCas9, SpG Cas9, or Cas12a can reach more target sites than the normal NGG PAM (Langner et al., 2018). This is useful when the target gene has no NGG nearby. Guide design tools such as CRISPOR, together with genome scans, can also lower off-target risk (Guo et al., 2023). Transient expression or RNP delivery is another option. Cas9 RNPs could be delivered into pineapple protoplasts or through vascular injection (Modrzejewski et al., 2020). This would create edited plants without foreign DNA. Even if efficiency is low, one good edited plant can be multiplied by vegetative propagation. New CRISPR types may also help. Cas12b and Cas13 are examples. Cas13 can cut RNA and could be used to fight pineapple RNA viruses during plant propagation. In general, using better tissue culture, improved CRISPR enzymes, and new delivery methods together may remove the current limits. Sharing methods through networks like the International pineapple Working Group can make progress faster. Other crops once thought hard to edit, such as soybean and sorghum, are now edited regularly. Pineapple will likely follow the same path. 5.3 Regulatory and ethical considerations for genome-edited pineapple CRISPR-edited pineapples encounter increasing obstacles in commercial markets because of evolving legal frameworks and shifting public opinions. Regulations differ by country. CRISPR plants that contain no foreign DNA elements are not considered GMOs in certain jurisdictions. The United States treats these plants in the same manner as conventional breeding varieties (Sprink et al., 2022). The first commercial CRISPR crop available to consumers was a white mushroom variety. The researchers did not classify this product as a GMO because it contained only a minimal deletion of DNA without any foreign DNA insertion (Smith-Willis and San Martin, 2015; Ahmad et al., 2023). The European Union uses the classification system of Tomlinson (2018) to categorize most gene-edited crops as GMOs. The approval process for an edited pineapple would require detailed evaluation according to Buchholzer and Frommer (2023). Exporters of pineapple products need to understand that various markets have their own set of regulations. The GMO classification system of Japan and Australia and Brazil does not include DNA-free small edits according to Sprink et al. (2022). The world has seen an increasing number of nations implement precise gene editing technology.
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