Rapid evaluation of fruit softening-related genes in precocious flowering kiwifruit

Kiwifruit softening occurs largely in the absence of increased ethylene production, but can be triggered by exogenous ethylene or cold treatment. Some variation in softening behavior is seen in the main commercial cultivars. Using gene editing techniques could be an efficient approach for re-creating new alleles in fruit softening-related genes. However, a long juvenile phase in kiwifruit is a big issue for evaluation of the gene/allele functions via transformation. Here, we examined a rapid gene/allele evaluation system with a sibling derived from precocious flowering 'Hort16A' (female) and 'Bruce' (male), which has truncated CENTRORADIALIS-like genes, which normally act as floral repressors. For the evaluation targets, we selected 58 polygalacturonase (PG) and 32 pectin lyase (PL) gene-member families in the kiwifruit genome, which are likely to be involved in various fruit softening processes. Using a combination of phylogenetic analysis and transcriptome data we found single PG- and PL-like genes which are highly responsive to ethylene or cold treatments in a lineage-specific manner. The constructs overexpressing or editing these genes were introduced into female precocious flowering lines, via Agrobacterium-mediated transformation. We successfully obtained regenerated transformants to evaluate the gene/allele functions. We discuss the cell wall-related characteristics of these transformed lines, and also the transformation and gene evaluation efficiencies of this system.