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 usinga 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 CREST 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 plantswith inducible fertility.