BpMADS4 -: a MADS box gene of birch induces flowers on transgenic apple plants in vitroq

In comparison to herbaceous plants, the breeding of fruit trees, such as apple (Males x domestica Borkh.), is more time-consuming due to its long generation time. Shortened juvenility and precocious flowering are, therefore, important breeding goals. Flower initiation has been intensively studied in Arabidopsis and orthologues/ homologues of LEAFY (LFY), APETELA1 (AP1), and TERMINAL FLOWER (TFL1) have been cloned from apple. One approach to shortening the juvenile phase of perennial crops is to reduce juvenility/vegetative maintenance factors, such as TFL1, by gene silencing. An alternative approach is to induce flowering by the over-expression of genes involved in the formation of floral meristems. In this context, the bpMADS4 gene of silver birch, which is similar to FRUITFULL of Arabidopsis, was over-expressed in apple. This gene is normally expressed in the apical meristem before the appearance of floral meristems (Elo et al., 2001). In transgenic apple, the expression of bpMADS4 was controlled by the constitutive CAMV35S promoter. Twenty transgenic lines were obtained after Agrobacterium-mediated gene transfer into apple. The integration and expression of the nptII and bpMADS4 genes were characterized by PCR analysis and RT-PCR. To quantify the level of transgene transcription a Real Time PCR procedure was carried out. Solitary flowers were detected on in vitro shoots of line T1165 thirteen weeks after transformation. Four other lines also produced flowers during in vitro propagation. The initial flowers were solitary, but shoots later developed clusters with about five flowers. Most of these flowers appeared morphologically normal but some were abnormal. In spring 2005, shoots of these lines were rooted and transferred to the greenhouse for further evaluation. To our knowledge, this is the first report of in vitro flower induction in transgenic apple.