Strong Fluorescence Expression of ZsGreen1 in Petunia Flowers by Agrobacterium tumefaciens–mediated Transformation

Abstract

Fluorescent proteins (FT) have become essential, biological research tools. Many novel genes have been cloned from a variety of species and modified for effective, stable, and strong expression in transgenic organisms. Although there are many applications, FT expression has been employed most commonly at the cellular level in plants. To investigate FT expression at the whole-plant level, particularly in flowers, petunia ‘Mitchell Diploid’ [MD (Petunia ×hybrida)] was genetically transformed with seven genes encoding FTs: DsRed2, E2Crimson, TurboRFP, ZsGreen1, ZsYellow1, rpulFKz1, or aeCP597. Each gene was cloned into a pHK-DEST-OE vector harboring constitutive figwort mosaic virus 35S promoter and NOS-terminator. These plasmids were individually introduced into the genome of MD by Agrobacterium tumefaciens–mediated transformation. Shoot regeneration efficiency from the cocultured explants ranged from 8.3% to 20.3%. Various intensities of red, green, and yellow fluorescence were detected from TurboRFP, ZsGreen1, and ZsYellow1-transgenic flowers, respectively, under ultraviolet light for specific excitation and emission filters. More than 70% of plants established from the regenerated shoots were confirmed as transgenic plants. Transgenic ZsGreen1 petunia generated strong, green fluorescence in all flower organs of T0 plants including petals, stigmas, styles, anthers, and filaments. Most of the chromophores were localized to the cytoplasm but also went into the nuclei of petal cells. There was a positive linear relationship (R2 = 0.88) between the transgene expression levels and the relative fluorescent intensities of the ZsGreen1-transgenic flowers. No fluorescence was detected from the flowers of DsRed2-, E2Crimson-, rpulFKz1-, or aeCP597-transgenic petunias even though their gene transcripts were confirmed through semiquantitative reverse transcriptase-polymerase chain reaction. T1 generation ZsGreen1 plants showed green fluorescence emission from the cotyledons, hypocotyls, and radicles, which indicated stable FT expression was heritable. Four homozygous T2 inbred lines were finally selected. Throughout this study, we demonstrated that ZsGreen1 was most suitable for generating visible fluorescence in MD flowers among the seven genes tested. Thus, ZsGreen1 may have excellent potential for better utility as a sensitive selectable marker.