Efficient production of novel floral traits in torenia by collective transformation with chimeric repressors of Arabidopsis transcription factors

Abstract

Flower colors and shapes are important commercial characters in floricultural plants. Many flowers with novel floral traits have been generated by crossfertilization, selective breeding, and mutation breeding (Shibata 2008). However, such traditional breeding methods require enormous time and effort. In contrast, genetic engineering in molecular breeding enables production of novel floral traits in floricultural plants that cannot be obtained by traditional breeding more efficiently and with less effort. For instance, attempts have been made for centuries to produce blue roses by traditional breeding, but this has proved impossible because roses lack a key enzyme, flavonoid 3Ј,5Ј-hydoxylase (F3Ј5ЈH), for delphinidin biosynthesis necessary for blue pigmentation. Katsumoto et al. (2007) succeeded in producing a delphinidin-accumulating rose with color that was blue-hued because of viola F3Ј5ЈH gene expression. Flower colors in carnation, petunia, torenia, and gentian have been modified by manipulating flavonoid biosynthesis genes (Nishihara and Nakatsuka 2010; Tanaka et al. 2009). Suppression by antisense gene or RNAi methods and overexpression by sense genes are typical strategies to manipulate genes of interest. However, information on DNA sequences is required for these strategies and genomic or EST analyses are still to be performed for most floricultural plants. On Abstract Molecular breeding with genetic modification enables the production of novel floral traits in floricultural plants that could not be obtained by traditional breeding. To facilitate novel flower production, we collectively introduced 2 sets of 42 and 50 chimeric repressors of Arabidopsis transcription factors into Agrobacterium and then used these to co-transform torenia (Torenia fournieri). We generated 750 transgenic torenias, and identification of the transgenes revealed that more than 80% of the transgenic torenias had a single transgene. A total of 264 plants showed phenotypic modification, and 91.2% displayed modified flower colors and/or shapes, such as altered color patterns, curled petal margins, and wavy petals. These results indicated that the collective transformation system can be applied to molecular breeding of flowers. Detailed analysis of the phenotypes revealed that PETAL LOSS could control blotch sizes and that modification of cell shape could change the texture of petals. We found that the chimeric repressors of functionally unknown transcription factors also induced novel floral traits, and therefore, the transgenic torenias provide an understanding of the functions of transcription factors that could not be revealed by previous studies in Arabidopsis.

A chimeric repressor of petunia PH4 R2R3-MYB family transcription factor generates margined flowers in torenia

“…The chimeric repressor of AtRL2, a single-type MYB gene from Arabidopsis, seems to strongly down-regulate anthocyanin accumulation in torenia petals in an earlier report. 7 A contrastingly restricted activity of PhPH4-RD in downregulation of genes involved in anthocyanin synthesis, could lead to margined flowers.…”

“…Pale margins appear in transgenic torenia flowers harbouring T-DNA insertions of the chimeric repressors of AtAGL6 and AtGNL genes, together with other phenotypic alterations. 7 Although transgene expressions and reproducibility of the phenotype were not examined, these chimeric repressors could be said to induce pale margins. Margined flowers also appear in the bulk of ionbeam induced torenia mutants.…”

“…In a way similar to AtTCP3 repressor, other repressors also have pleiotropic effects on flower and leaf phenotypes, including the generation of bizarre flowers. 7 In this study with torenia, we sought new constructs that might affect vacuolar pH and so change flower color -especially toward blue. Against expectation, anthocyanin color did not change to blue with these constructs.…”

The new FioreDB database provides comprehensive information on plant transcription factors and phenotypes induced by CRES-T in ornamental and model plants

“…In the individual project, we made multiple transgenic plants by transforming each gene construct one by one. In the bulk project, mixtures of 40 to 60 gene constructs were transformed into Arabidopsis and torenia and several hundred transgenic plants were cultivated to identify, in an efficient manner, TFs that induce intriguing phenotypes (Mitsuda et al 2008;Shikata et al 2011). In Arabidopsis, mixtures of T1 transgenic seeds for the more than 1,500 TFs that were prepared one by one in the individual project, were grown on a large scale (more than 10,000 plants) (Mitsuda et al 2008).…”

Promotion of Efficient Molecular Breeding Using Chimeric Repressors in Ornamental Flowers

“…Therefore, CRES-T may be effective for a large variety of monocot and dicot ornamental crops. In addition, a collective transformation (CT) system (Shikata et al 2011) would accelerate development to keep pace with changing consumer preferences, while still controlling the costs of commercialization such as in performing massive screening using the CT system.…”