Induction of double flowers in Pharbitis nil using a class-C MADS-box transcription factor with Chimeric REpressor gene-Silencing Technology

Abstract

The Chimeric REpressor gene-Silencing Technology (CRES-T) system is a novel reverse genetic method using a chimeric transcriptional repressor fusing an EAR transcriptional repression domain called SRDX. We sought to change the flower shape of Pharbitis nil, a model ornamental flower, using an Arabidopsis transcription factor fused with SRDX. For the first trial modulating flower shape we transformed with the class-C MADS-box transcription factor AGAMOUS (AG) fused with SRDX (AGSRDX). Defects in class-C genes cause double flowers in Arabidopsis and Pharbitis. However, when AGSRDX was expressed under the CaMV 35S promoter (p35S), the transgenic Pharbitis bore a malformed flower with a protruding pistil. We then used DUPLICATED (DP), one of the class-C genes in Pharbitis. The p35S :: DPSRDXintroduced callus were difficult to regenerate during transgenic steps, but occasionally made a perfect double flower bud showing severe growth defects. The flower buds never developed to flower opening stage. These results indicate that CRES-T is functional in Pharbitis but even using a conserved transcription factor, some species-specific variation might exist. To avoid these unwanted effects, we recruited inducible promoters to control expression of the chimeric transcription repressors in combination with the DNA-binding domain of GAL4 in yeast fused with SRDX and the GAL4 upstream activator sequence (UAS). Normal regeneration was observed by inducible repression of DPSRDX during in vitro redifferentiation, and the double-flowered Pharbitis was generated. We successfully induced a non-transformant (NT)-like flower in DPSRDX-expressing double-flowered transformants. Our approach will enable us to breed transgenic horticultural plants with inducible fertility.

Morphological changes in Ipomoea nil using chimeric repressors of Arabidopsis TCP3 and TCP5

“…We observed phenotypic variation among transgenic lines, but severe phenotypes were not always correlated with higher expression levels of the transgenes (data not shown). It has been reported that when using the CRES-T system, differences in expression level of SRDX fused cDNA do not reflect the severity of the phenotype Sage-Ono et al 2011), and we noticed a similar effect in TCP3SRDX and TCP5SRDX transformants.…”

Molecular Mechanism Regulating Floral Architecture in Monocotyledonous Ornamental Plants

“…These monocot species can be used for studies to modify floral organ development by overexpression or reduction of B-and C-class floral organ identity genes. The chimeric repressor of SRDX has been used successfully to reduce C-class gene expression and produce double-flowered mutants in morning glory and cyclamen (Sage-Ono et al, 2011;Tanaka et al, 2013). Recently, virus-induced gene silencing (VIGS) has been employed to reduce B-or C-class gene expression in ornamental plants such as Aquilegia, Phalaenopsis, and Petunia Hsieh et al, 2013;Kramer et al, 2007;Noor et al, 2014).…”

Morphological Variation and <i>AGAMOUS</i>-like Gene Expression in Double Flowers of <i>Cyclamen persicum</i> Mill.

“…In a previous study, Tanaka et al (2013) produced petaloid-stamen-type double-flowered cyclamen by AGAMOUS chimeric repressor expression (CRES-T). Similarly, petaloidstamen-type double flowers in other ornamental flowers were also produced by AG-like gene silencing (Narumi et al, 2008;Sage-Ono et al, 2011). Therefore, it is suspected that the petaloidy of stamens even in other naturally occurring double-flowered cyclamens might be caused by the reduced expression of AG-like genes in whorl 3.…”