Generation of fluorescent flowers exhibiting strong fluorescence by combination of fluorescent protein from marine plankton and recent genetic tools in <i>Torenia fournieri</i> Lind.

Abstract

Florescent proteins have been popularly used for studying genes and proteins of interest in various experiments at a cellular level, such as the analysis of intracellular localization and protein-protein interaction. However, the strength of fluorescence was insufficient for macro level observations of tissues or of the whole plant, and the fluorescent flowers that have been generated so far needed high-sensitive imaging equipment for the observation. Here we generated fluorescent Torenia flowers by the combined use of a high-performance fluorescent protein and the latest protein expression technologies, leading to the production of fluorescent proteins that can be easily and clearly observed. A coding sequence of a yellowish green fluorescent protein from the marine plankton Chiridius poppei (CpYGFP) was fused to the optimized sequences of the heat shock protein terminator and the 5′-untranslated region of the alcohol dehydrogenase gene of Arabidopsis to gain massive accumulation of the fluorescent protein. Strong fluorescence of CpYGFP was apparent in every part of the transgenic plant under the simple combination of a blue LED for excitation and an orange colored transparent acrylic filter for emission, while faint autofluorescence remained in the wild-type plants. By evaluating the combination of excitation wavelengths (excitation and emission filters) we were able to eliminate this undesired fluorescence. The fluorescent flowers could be used for ornamental purposes as well as for the analysis of fluorescent transgenic plants spatiotemporally in a nondestructive manner.

Dissecting promoter of <i>InMYB1</i> gene showing petal-specific expression

“…Genetic transformation has been used to produce novel crops with superior traits and to analyze gene function (Fukui et al 2003;Katsumoto et al 2007;Sasaki et al 2014Sasaki et al , 2016Tanaka et al 2013). For the genetic transformation, not only transgenes but also promoters, which control transgene expression in desired tissues and timings, are important (Oshima and Mitsuda 2016;Sasaki et al 2016).…”

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

“…GFP-transgenic african daisy (Osteospermum ecklonis) were reported to be appearing distinctly green fluorescence upon ultraviolet light illumination (Mercuri et al, 2002). Recently, strong yellow-green fluorescence was produced by wishbone flower [Torenia fournieri 'Crown White' (Sasaki et al, 2014)], transformed with CpYGFP originated from a marine plankton (Chiridius poppei). It is now clear that the right choice of vectors, constitutive or spatiotemporal promoters, and translational enhancers for efficient transcript and stable protein expression are vital for strong fluorescence emission in plants.…”

“…Petunia 'Mitchell Diploid' (MD), a model plant renowned for its high transformation capacity (Vandenbussche et al, 2016), was chosen for the present study because it is mostly white with reduced amounts of pigment in the corolla. This reduced the chance of complications that certain petal pigments might interfere with fluorescence by shading excitation (Sasaki et al, 2014). The purpose of this study was to select fluorescent proteins with maximum emission for the development of fluorescent flowers.…”

Production of chrysanthemum periclinal chimeras through shoot regeneration from leaf explants

“…The fluorescence activity of the CpYGFP protein is stable over a wide pH range in higher plants (Masuda et al 2006). 'Taihei' was transformed with a highly improved CpYGFP expression vector containing three tandem CpYGFPs controlled by cauliflower mosaic virus 35S promoter with optimized translational enhancer and terminator (3×CpYGFP; Sasaki et al 2014). The 3×CpYGFP expression vector can stably accumulate high amounts of the fluorescent protein CpYGFP in plant tissues (Sasaki et al 2014) which permit efficient visualization for observing chimerism.…”

“…'Taihei' was transformed with a highly improved CpYGFP expression vector containing three tandem CpYGFPs controlled by cauliflower mosaic virus 35S promoter with optimized translational enhancer and terminator (3×CpYGFP; Sasaki et al 2014). The 3×CpYGFP expression vector can stably accumulate high amounts of the fluorescent protein CpYGFP in plant tissues (Sasaki et al 2014) which permit efficient visualization for observing chimerism. We used explants from in vitro leaves of the wild type and the CpYGFP-carrying transformant to construct periclinal chimeric plants consisting of material from both plants.…”