Bioscience Methods 2026, Vol.17, No.1, 9-22 http://bioscipublisher.com/index.php/bm 17 two MLO genes together with one immune suppressor gene in grapevine during a single study. The research produced plants that demonstrated effective resistance against mildew (Moffa et al., 2025). The combination of tRNA-based multiplex guide systems with Cas12a technology enables scientists to edit multiple genes simultaneously which enhances the efficiency of this approach (Schepler-Luu et al., 2023; Li et al., 2025). Additionally, new variants like Cas12f or Cas12j (ultra-small CRISPR nucleases) may allow delivery of multiple guides even in the size-constrained virus vectors if needed for pineapple (though currently transformation is via tissue culture). One can also use a sequential transformation approach: edit gene set A, then re-transform the edited plant to edit gene set B. While time-consuming, it’s faster than classical breeding by orders of magnitude. It is important to check that adding many edits does not create bad interactions or reduce plant health. If each edit alone has little effect on growth, their combination is more likely to be safe. Careful testing is still needed to choose the best lines. Scientists can use CRISPR multiplexing technology to create pineapples that protect against all major disease-causing pathogens through successful implementation of this technology. The new method would eliminate the need for decades of conventional breeding because it enables scientists to create resistance gene "pyramids" directly in the genome through one generation of targeted editing. 5 Challenges and Future Prospects 5.1 Technical hurdles in applying CRISPR to pineapple CRISPR/Cas9 technology faces multiple technical challenges when used for pineapple cultivation. The main problem exists in the steps of plant regeneration and transformation. Pineapple is difficult to modify genetically. The process of delivering DNA into cells followed by normal plant growth of edited plants proves to be challenging. Some progress has been made with Agrobacterium-based transformation of pineapple callus (He et al., 2023), but the efficiency is low and changes with genotype. The cultivation of MD2 and other popular cultivars demands sophisticated tissue culture methods because they prove challenging to grow. Researchers have worked on methods to produce embryogenic callus and adjust selective agent levels to get shoots. The number of edited plantlets remains limited despite these developments. The extended time it takes for pineapples to grow creates an additional difficulty because field-based plant resistance testing needs multiple years of assessment. The clonal nature of pineapple propagation through tissue culture makes it difficult to distinguish between genetic modifications and somaclonal variations so researchers must include wild-type controls (Wang et al., 2021). Off-target edits are another concern. The pineapple genome contains multiple repeated sequences which are combined with duplicate gene copies. A guide RNA has the potential to make errors during its cutting process which results in incorrect placement of the cut. Most plant studies show off-target rates are low (Bessoltane et al., 2022), but we need to design guides with care and confirm possible off-target sites by sequencing. The extensive heterozygous genome of pineapple creates additional challenges for this process. For a full knockout, both alleles need to be edited. Plants show mosaic patterns because their cells contain different genetic modifications between affected and unaffected cells. The process requires additional propagation operations or editing iterations according to Guo et al. (2023) and Wang et al. (2021). Some commercial cultivars are also difficult to grow in tissue culture. The MD2 line proves to be one of the most difficult to grow but laboratory tests show other lines perform better (Cheng et al., 2025). The first step involves working with an easier cultivar before performing backcrossing or using morphogenic regulator genes and optimized media for culture improvement (He et al., 2023). The tissue culture process results in numerous genetic mutations which develop within cultured cells. The grapevine genome contains multiple thousands of single nucleotide variants and indels which developed from tissue culture procedures instead of CRISPR (Wang et al., 2021). The results show that particular regenerant features develop from culture conditions instead of gene editing so proper control systems need to be implemented.
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