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BABY BOOM (BBM) is a member of the APETALA2/ETHYLENE RESPONSE FACTOR (AP2/ERF) family and its expression has been shown to improve herbaceous plant transformation and regeneration. However, this improvement has not been shown clearly for tree species. This study demonstrated that the efficiency of transgenic apple (Malus domestica Borkh.) plant production was dramatically increased by ectopic expression of the MdBBM1gene. “Royal Gala” apple plants were first transformed with a CaMV35S-MdBBM1construct (MBM) under kanamycin selection. These MBMtransgenic plants exhibited enhanced shoot regeneration from leaf explants on tissue culture media, with most plants displaying a close-to-normal phenotype compared with CaMV35S-GUStransgenic plants when grown under greenhouse conditions, the exception being that some plants had slightly curly leaves. Thin leaf sections revealed the MBMplants produced more cells than the GUSplants, indicating that ectopic-expression of MdBBM1enhanced cell division. Transcriptome analysis showed that mRNA levels for cell division activators and repressors linked to hormone (auxin, cytokinin and brassinosteroid) signalling pathways were enhanced and reduced, respectively, in the MBMplants compared with the GUSplants. Plants of eight independent MBMlines were compared with the GUSplants by re-transforming them with an herbicide-resistant gene construct. The number of transgenic plants produced per 100 leaf explants was 0–3% for the GUSplants, 3–8% for five MBMlines, and 20–30% for three MBMlines. Our results provided a solution for overcoming the barriers to transgenic plant production in apple, and possibly in other trees.

MicroRNA172 (miR172) plays a role in regulating a diverse range of plant developmental processes, including flowering, fruit development and nodulation. However, its role in regulating flavonoid biosynthesis is unclear. In this study, we show that transgenic apple plants over-expressing miR172show a reduction in red coloration and anthocyanin accumulation in various tissue types. This reduction was consistent with decreased expression of APETALA2homolog MdAP2_1a(a miR172target gene), MdMYB10, and targets of MdMYB10, as demonstrated by both RNA-seq and qRT-PCR analyses. The positive role of MdAP2_1ain regulating anthocyanin biosynthesis was supported by the enhanced petal anthocyanin accumulation in transgenic tobacco plants overexpressing MdAP2_1a, and by the reduction in anthocyanin accumulation in apple and cherry fruits transfected with an MdAP2_1avirus-induced-gene-silencing construct. We demonstrated that MdAP2_1a could bind directly to the promoter and protein sequences of MdMYB10in yeast and tobacco, and enhance MdMYB10promotor activity. In Arabidopsis, over-expression of miR172reduced flavonoid (including anthocyanins and flavonols) concentration and RNA transcript abundance of flavonoid genes in plantlets cultured on medium containing 7% sucrose. The anthocyanin content and RNA abundance of anthocyanin genes could be partially restored by using a synonymous mutant of MdAP2_1a, which had lost the miR172target sequences at mRNA level, but not restored by using a WT MdAP2_1a. These results indicate that miR172inhibits flavonoid biosynthesis through suppressing the expression of an AP2 transcription factor that positively regulates MdMYB10.

ABSTRACTmicroRNA172 (miR172) expression has been shown to have a positive effect on Arabidopsis fruit (siliques) growth. In contrast, over-expression of miR172 has a negative influence on fruit growth in apple, resulting in a dramatic reduction in fruit size. This negative influence is supported by the results of analyzing a transposable element (TE) insertional allele of a MIR172gene that has reduced expression of the miRNA and is associated with an increase in fruit size. Arabidopsis siliques are a dry fruit derived from ovary tissues, whereas apple is a fleshy pome fruit derived mostly from hypanthium tissues. A model has been developed to explain the contrasting impact of miR172 expression in these two plant species based on the differences in their fruit structure. Transgenic apple plants with extremely high levels of miR172 overexpression produced flowers consisting of carpel tissues only, which failed to produce fruit. By comparison, in tomato, a fleshy berry fruit derived from the ovary, high level over-expression of the same miR172 resulted in carpel-only flowers which developed into parthenocarpic fruit. These results further indicate that the influence of miR172 on fruit growth in different plant species depends on its fruit type.