In order to identify genetic components in flowering pathways of highbush blueberry (Vaccinium corymbosum L.), a transcriptome reference composed of 254,396 transcripts and 179,853 gene contigs was developed by assembly of 72.7 million reads using Trinity. Using this transcriptome reference and a query of flowering pathway genes of herbaceous plants, we identified potential flowering pathway genes/transcripts of blueberry. Transcriptome analysis of flowering pathway genes was then conducted on leaf tissue samples of transgenic blueberry cv. Aurora (‘VcFT-Aurora’), which overexpresses a blueberry FLOWERING LOCUS T-like gene (VcFT). Sixty-one blueberry transcripts of 40 genes showed high similarities to 33 known flowering-related genes of herbaceous plants, of which 17 down-regulated and 16 up-regulated genes were identified in ‘VcFT-Aurora’. All down-regulated genes encoded transcription factors/enzymes upstream in the signaling pathway containing VcFT. A blueberry CONSTANS-LIKE 5-like (VcCOL5) gene was down-regulated and associated with five other differentially expressed (DE) genes in the photoperiod-mediated flowering pathway. Three down-regulated genes, i.e., a MADS-AFFECTING FLOWERING 2-like gene (VcMAF2), a MADS-AFFECTING FLOWERING 5-like gene (VcMAF5), and a VERNALIZATION1-like gene (VcVRN1), may function as integrators in place of FLOWERING LOCUS C (FLC) in the vernalization pathway. Because no CONSTAN1-like or FLOWERING LOCUS C-like genes were found in blueberry, VcCOL5 and VcMAF2/VcMAF5 or VRN1 might be the major integrator(s) in the photoperiod- and vernalization-mediated flowering pathway, respectively. The major down-stream genes of VcFT, i.e., SUPPRESSOR of Overexpression of Constans 1-like (VcSOC1), LEAFY-like (VcLFY), APETALA1-like (VcAP1), CAULIFLOWER 1-like (VcCAL1), and FRUITFULL-like (VcFUL) genes were present and showed high similarity to their orthologues in herbaceous plants. Moreover, overexpression of VcFT promoted expression of all of these VcFT downstream genes. These results suggest that VcFT’s down-stream genes appear conserved in blueberry.

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Blueberry is a woody perennial bush with a longer juvenile period than annual crops, requiring vernalization to flower normally. Few studies have been reported on the molecular mechanism of flowering in blueberry or other woody plants. Because FLOWERING LOCUS T (FT) from Arabidopsis thaliana plays a multifaceted role in generating mobile molecular signals to regulate plant flowering time, isolation and functional analysis of the blueberry (Vaccinium corymbosum L.) FT-like gene (VcFT) will facilitate the elucidation of molecular mechanisms of flowering in woody plants. Based on EST sequences, a 525-bpVcFT was identified and cloned from the cDNA of a tetraploid, northern highbush blueberry cultivar, Bluecrop. Ectopic expression of 35S:VcFT in tobacco induced flowering an average of 28 days earlier than wild-type plants. Expression of the 35S:VcFT in the blueberry cultivar Aurora resulted in an extremely early flowering phenotype, which flowered not only during in vitro culture, a growth stage when nontransgenic shoots had not yet flowered, but also in 6-10-week old, soil-grown transgenic plants, in contrast to the fact that at least 1 year and 800 chilling hours are required for the appearance of the first flower of both nontransgenic 'Aurora' and transgenic controls with the gusA. These results demonstrate that the VcFT is a functional floral activator and overexpression of the VcFT is able to reverse the photoperiodic and chilling requirements and drive early and continuous flowering.

To optimize Agrobacterium tumefaciens-mediated transformation, factors influencing gene delivery, selection of transformed cells, and plant regeneration were investigated using two major switchgrass cultivars, including a lowland tetraploid cultivar Alamo and an upland octoploid cultivar Cave-in-Rock (CIR). Transient expression studies monitored by histochemical b-glucuronidase assay in seedling segments indicated that A. tumefaciens strain EHA105 was more effective in gene delivery than LBA4404 or GV3101. Of three major selectable genes, the bialaphos resistance (bar) gene and the hygromycin phosphotransferase (hpt) allowed effective selection of transformed cells using 2 mg l -1 glufosinate ammonium herbicide and 50 mg l -1 hygromycin, respectively; whereas the neomycin phosphotransferase II gene did not yield effective selection using 100 mg l -1 kanamycin. Herbicideor hygromycin-resistant calluses were induced from seedling segments after 2-3 months of selection. Transformants of 'Alamo' with the bar or hpt were obtained 3-4 weeks after the resistant calluses were transferred onto regeneration medium; in contrast, no regenerant was produced from the calluses of 'CIR'. Most of transformants showed normal growth in the greenhouse. Low percentages of mature seeds ranging from 1.7 to 8.7% of husks were obtained from open pollinated plants. Southern blot analysis confirmed stable integration of the bar in selected T0 transformants. Reverse transcription PCR and herbicide/hygromycin tolerance tests indicated expression of transgenes. The optimized transformation protocol using basal parts of seedling as explants shortened the process by 4-5 weeks, and it has potential use for transformation of other switchgrass cultivars.