Bioscience Methods 2026, Vol.17, No.1, 9-22 http://bioscipublisher.com/index.php/bm 14 These findings show that CRISPR can copy known resistance changes in perennial fruit crops without bringing in outside genes. For pineapple, if matching MLOgenes (AcMLO) are identified, this method may work. They also suggest that CRISPR fits clonal fruit crops, using tissue culture and DNA-free methods to follow rules and current regulations. 3.2 Banana (Musaspp.) – resistance to banana streak virus via eBSV sequence editing Banana suffers from Banana streak virus (BSV). In some cultivars, the virus comes from “endogenous” viral DNA (eBSV) inside the genome. Under stress, these sequences can activate and cause disease. Commercial bananas are sterile, so breeding to remove eBSV is not possible. Tripathi et al. (2019) used CRISPR/Cas9 to disrupt the eBSV DNA in plantain (Musa AAB genome). They designed sgRNAs to target three conserved regions of the viral genome inside the plant’s chromosomes. Embryogenic cells from the cultivar ‘Gonja Manjaya’ were transformed with Cas9 and sgRNAs. The edited plants had indels in the eBSV sites and did not develop BSV symptoms, even under stress. Control plants still showed disease. The edited bananas grew normally, with no off-target problems. This was one of the first CRISPR uses in a clonally propagated polyploid crop, solving a problem breeding could not. The method shows how precise editing can remove harmful sequences in the plant’s own DNA. It also suggests similar strategies could be used in pineapple for viruses or other genetic disease risks. 3.3 Citrus (Citrus sinensis) – resistance to citrus canker via CsLOB1 promoter editing Citrus canker, caused by Xanthomonas citri, is a major disease in sweet orange. CRISPR was used to give resistance by editing the promoter of CsLOB1, a susceptibility gene. The pathogen uses TAL effectors to bind to the CsLOB1 promoter and activate it, leading to disease. Jia and Wang (2020) used CRISPR/Cas9 to change the effector-binding elements (EBEs) in the CsLOB1 promoter. They delivered Cas9 and sgRNAs into citrus cells with Agrobacterium. The edited plants had small indels at the binding site and showed no canker symptoms after infection, while controls did. Peng et al. (2017) edited CsLOB1 in Duncan grapefruit, changing both the promoter and coding regions. Some plants had both alleles edited in the first generation and showed strong resistance. Editing the promoter kept the normal function of CsLOB1 for growth but blocked pathogen attack. In some cases, the editing was transgene-free, which is important for consumer and regulatory acceptance. This case shows CRISPR can protect crops by removing a pathogen’s “entry point” in the genome. For pineapple, a similar promoter-editing strategy could help protect against bacterial diseases like heart rot. 4 Potential of CRISPR/Cas9 in Pineapple Disease Resistance Breeding 4.1 Enhancing defense gene function via promoter editing or activation One way to improve pineapple’s disease resistance is to make its own defense genes work harder. CRISPR/Cas9 can do this by changing the promoter of a gene to make it more active, or by using CRISPR systems that turn on a gene without changing its DNA. In traditional breeding, overexpression of genes like those in the phenylpropanoid pathway or PR proteins can give stronger resistance, but adding extra copies by transgenics often causes regulation problems. With CRISPR, promoters can be adjusted so a defense gene is active more often or responds faster when the plant is infected. In tomato, Rivera-Toro et al. (2025) used a dCas12a activator to boost SlPAL2, a gene in the lignin pathway. The treated plants showed thicker cell walls, smaller lesions, and fewer bacteria, while still keeping normal growth. In pineapple, good targets may include AcPAL, AcWRKY transcription factors, and AcPR1. Editing promoters to cut repressor sites or add stronger motifs could raise PR proteins or lignin enzymes in tissues. CRISPR activation (CRISPRa) is another option. This method uses a dead Cas9 linked to an activator to raise gene expression
RkJQdWJsaXNoZXIy MjQ4ODYzNA==