Genome editing is a powerful technique for genome modification in molecular research and crop breeding, and has the great advantage of imparting novel desired traits to genetic resources. However, the genome editing of fruit tree plantlets remains to be established. In this study, we describe induction of a targeted gene mutation in the endogenous apple phytoene desaturase (PDS) gene using the CRISPR/Cas9 system. Four guide RNAs (gRNAs) were designed and stably transformed with Cas9 separately in apple. Clear and partial albino phenotypes were observed in 31.8% of regenerated plantlets for one gRNA, and bi-allelic mutations in apple PDS were confirmed by DNA sequencing. In addition, an 18-bp gRNA also induced a targeted mutation. These CRIPSR/Cas9 induced-mutations in the apple genome suggest activation of the NHEJ pathway, but with some involvement also of the HR pathway. Our results demonstrate that genome editing can be practically applied to modify the apple genome.

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Tree crops have a long juvenile period which is a serious constraint for genetic improvement and experimental research. For example, apple remains in a juvenile phase for more than five years after seed germination. Here, we report about induction of rapid flowering in apple seedlings using the Apple latent spherical virus (ALSV) vector expressing a FLOWERING LOCUS T (FT) gene from Arabidopsis thaliana. Apple seedlings could be flowered at 1.5-2 months after inoculation to cotyledons of seeds just after germination with ALSV expressing the FT gene. A half of precocious flowers was normal in appearance with sepals, petals, stamens, and pistils. Pollen from a precocious flower successfully pollinated flowers of 'Fuji' apple from which fruits developed normally and next-generation seeds were produced. Our system using the ALSV vector promoted flowering time of apple seedlings within two months after germination and shortened the generation time from seed germination to next-generation seed maturation to within 7 months when pollen from precocious flowers was used for pollination.

Apple (Malus  domestica Borkh.) is a perennial woody plant that undergoes a period of dormancy (in cv. Jonathan between late September and mid-December) to survive freezing temperatures of winter. DORMANCY-ASSOCIATED MADS-BOX (DAM) genes play important roles in the regulation of growth cessation and terminal bud formation in peach. To understand the role of DAM orthologs in apple, we isolated and characterized four DAM-like genes (designated as MdDAMa, MdDAMb, MdDAMc, and MdDAMd) and monitored their expression in apical buds throughout the season by real-time quantitative polymerase chain reaction analyses. The transcription of MdDAMa peaked in October and that of MdDAMc was elevated from August to October, whereas MdDAMb and MdDAMd were practically undetectable. The tandemly arranged genes MdDAMa/MdDAMb and MdDAMc/MdDAMd were localized to chromosomes 16 and 8, respectively. Based on these observations, we infer that MdDAMa and MdDAMc acted in a dominant fashion on each locus and were correlated with the period of endodormancy.

Two apple [Malus sylvestris (L.) Mill. var. domestica (Borkh.) Mansf.] homologous fragments of FLO/LFY and SQUA/AP1 (AFL and MdAP1, respectively) were analyzed to determine the relationship between floral bud formation and floral gene expression in `Jonathan' apple. The AFL gene was expressed in reproductive and vegetative organs. By contrast, the MdAP1 gene, identified as MdMADS5, which is classified into the AP1 group, was expressed specifically in sepals concurrent with sepal formation. Based on these results, AFL may be involved in floral induction to a greater degree than MdAP1 since AFL transcription increased ≈2 months earlier than MdAP1. Characterization of AFL and MdAP1 should advance the understanding of the processes of floral initiation and flower development in woody plants, especially in fruit trees like apple.