Molecular Plant Breeding 2025, Vol.16, No.3, 191-201 http://genbreedpublisher.com/index.php/mpb 196 5.2 Targeting genes for ethylene production Ethylene is the dominant signal that triggers the ripening and softening of durian, and reducing ethylene production is considered to be one of the most effective ways to delay softening. By knocking out key enzyme genes in the ethylene synthesis pathway (such as ACO1 or ACS2) through CRISPR, the accumulation of ethylene during ripening can be greatly reduced, thereby slowing down the entire ripening process (Hu et al., 2021). It should be noted that completely blocking ethylene may cause durian to fail to ripen normally (becoming a “never ripe” state), so the strategy should pursue “slowing down” rather than “completely blocking”. For example, the key amino acids of the ACS2 enzyme are replaced with a partially inactivated form using base editing technology, so that the ethylene production rate is reduced but not zero. This fine regulation is expected to delay softening while still allowing the fruit to eventually mature and show the desired flavor. This is similar to the situation in which weak mutant alleles in tomatoes delay ripening but do not affect flavor (Hewitt and Dhingra, 2020). 5.3 Regulating transcription factors Fruit ripening is a complex gene network, in which core transcription factors such as MADS-box and NAC play the role of “master switch” (Hewitt and Dhingra, 2020). Durian ripening is likely to have master effect transcription factors similar to tomato RIN and NOR. Once knocked out, they will greatly delay or even stop the ripening process. For example, if durian has NAC transcription factors that function similar to tomato NOR genes, in theory, CRISPR knockout of this gene will greatly delay the softening of the fruit, accompanied by the postponement of apparent ripening characteristics. In early breeding, the natural tomato mutants rin and nor had extremely long shelf life due to slow ripening, but also had problems of incomplete ripening and poor flavor (Lobato-Gómez et al., 2021). Therefore, for such key transcription factors, it is not advisable to knock out all of them, but it is possible to consider creating partial loss of function or low-function alleles. The advantage of gene editing is that point mutations can be introduced precisely. In durian, attempts can be made to edit the promoters or enhancers of similar transcription factor genes to reduce their expression levels. For example, the CRISPR-Cas12a system was used to remove the key cis-elements on the promoter of the durian MADS-box gene, so that it was only weakly expressed after harvest, and the activation of downstream softening genes would be delayed or weakened (Lobato-Gómez et al., 2021). For another example, the putative durian NAC master regulator can be genetically edited to make its protein lack a transcriptional activation domain, changing from an activated type to a partially inactivated type, which can delay maturity without causing complete unripening. This refined editing scheme can be technically achieved through site-directed base editing or sequence-specific deletion. 5.4 Other emerging gene editing strategies In addition to traditional gene knockout, upgraded versions of gene editing technology (such as CRISPR/Cas base editing, in situ editing, and genome reprogramming) can also be applied to durian softening regulation (Prado et al., 2024). For example, base editors can directly mutate one base to another without cutting the DNA double strand, thereby accurately generating point mutations. This is very useful for creating the above-mentioned “weak function” alleles, such as changing the key codon of the ethylene receptor gene ETR to a mutant type (similar to the mutation with ethylene resistance phenotype in Arabidopsis), so that durian is less sensitive to ethylene but not completely unresponsive. Another example is genome reprogramming tools, which can direct the epigenetic state or transcription level of genes. Multi-gene stacking editing is also one of the strategies. As mentioned above, multiple softening enzyme genes and regulatory factors can be targeted at the same time to cultivate “slow-ripening” comprehensive trait lines (Lobato-Gómez et al., 2021). Some cutting-edge methods such as hormone combination culture medium and co-expression of morphogenesis regulatory genes can be used to improve the regeneration rate of durian and provide the necessary conditions for gene editing.
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