CRISPR/Cas9-Mediated Cytosine Base Editing Using an Improved Transformation Procedure in Melon (Cucumis melo L.)

Abstract

Melon is a recalcitrant plant for stable genetic transformation. Various protocols have been tried to improve melon transformation efficiency; however, it remains significantly low compared to other plants such as tomato. In this study, the primary focus was on the optimization of key parameters during the inoculation and co-culture steps of the genetic transformation protocol. Our results showed that immersing the explants in the inoculation medium for 20 min significantly enhanced transformation efficiency. During the co-culture step, the use of filer paper, 10 mM 2-(N-morpholino)-ethanesulfonic acid (MES), and a temperature of 24 °C significantly enhanced the melon transformation efficiency. Furthermore, the impact of different ethylene inhibitors and absorbers on the transformation efficiency of various melon varieties was explored. Our findings revealed that the use of these compounds led to a significant improvement in the transformation efficiency of the tested melon varieties. Subsequently, using our improved protocol and reporter-gene construct, diploid transgenic melons successfully generated. The efficiency of plant genetic transformation ranged from 3.73 to 4.83%. Expanding the scope of our investigation, the optimized protocol was applied to generate stable gene-edited melon lines using the Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9)-mediated cytosine base editor and obtained melon lines with editions (C-to-T and C-to-G) in the eukaryotic translation initiation factor 4E, CmeIF4E gene. In conclusion, the optimized melon transformation protocol, along with the utilization of the CRISPR/Cas9-mediated cytosine base editor, provides a reliable framework for functional gene engineering in melon. These advancements hold significant promise for furthering genetic research and facilitating crop improvement in this economically important plant species.

From Sequencing to Genome Editing in Cucurbitaceae: Application of Modern Genomic Techniques to Enhance Plant Traits

“…The resulting mutants were resistant to Ipomovirus (cucumber vein yellowing virus) and potyviruses (zucchini yellow mosaic virus and papaya ring spot mosaic virus-W). A mutation in the gene encoding the cap-binding protein eIF4E induced resistance to the Moroccan watermelon mosaic virus in melon [133,138]. CRISPR/Cas9 mutations in eIF4E were successfully applied for breeding cucumber plants resistant to zucchini yellow mosaic virus, watermelon mosaic virus and papaya ringspot virus (Table 2) [117].…”

Possibility of genome editing for melon breeding

“…An RNA sequence in the 3ʹ UTR of MNSV was essential for the resistance/recessive phenotype (Díaz et al 2004). Using the CRISPR/Cas9mediated cytosine base editor, two kinds of mutations were introduced on Cm-elF4E, Cm-elF4E (C322T/C323G) with a stop codon and Cm-elF4E (C322T/C323T, P108L) (Shirazi Parsa et al 2023).…”

“…(Bastet et al 2019, Hunziker et al 2020, Shimatani et al 2017. Regarding the targeted AID method, it can also be used in melon, where base and amino acid substitutions were successfully made in the MNSV resistance gene Cm-elF4E (Shirazi Parsa et al 2023). Furthermore, prime editing, which specifies the location of the target site and replaces the target DNA nucleotide by linking reverse transcriptase to nCas9, has been developed and can be used in rice and wheat (Lin et al 2020).…”