Teruhiko Terakawa (Team Manager), Plant-Bio Team, Bioscience Research Division, Central Research Laboratories (Director: Tsuguo Uchiyama), Hokko Chemical Industry Co., Ltd. (President: Takao Maruyama) and Masaru Takagi (Principal Research Scientist) and his coworkers, Research Institute of Genome-based Biofactory (Director: Yoichi Kamagata) of the National Institute of Advanced Industrial Science and Technology (AIST; President: Tamotsu Nomakuchi) have succeeded in developing polypetalous cyclamens using a novel gene silencing technology (CRES-T method).

The shape of flowers is an important aspect in stimulating appreciation of the beauty of horticultural plants. A flower consists of floral organs such as petals, stamens, pistils, and sepals; and the formation of these organs is controlled by gene regulators, called transcription factors. Cyclamens basically have five petals. In the traditional breeding methods, the only way to create cyclamen varieties that are polypetalous, such as roses and carnations, was the use of induction of mutation, which makes it difficult to realize the production of polypetalous cyclamens.

We have isolated and analyzed genes that regulate the production of stamens and pistils of a cyclamen. Then by using our original gene transfer technology and CRES-T method, we altered stamens and pistils into petals. This led to the development of a polypetalous cyclamen with hitherto unknown flower-shape characteristics in which petals are formed in succession. The dispersion of pollen, which remains a problem for genetically modified plants, is suppressed because stamens are altered to petals.

This study was conducted as a commissioned research project of Promotion of Basic Research Activities for Innovative Biosciences (core institution: National Institute of Floricultural Science), administered by the Bio-oriented Technology Research Advancement Institution, National Agriculture and Food Research Organization.

The result of this research will be presented at The 51st Annual Meeting of The Japanese Society of Plant Physiologists held on March 18-21, 2010, at Kumamoto University.

Photo (Left) Polypetalous cyclamen produced by our new technology		Photo (Right) Ordinary cyclamen

Cyclamen is a major ornamental plant and a representative pot flower of winter, and its cultivation area and shipping volume are the most among all pot flowers in Japan. The shape of flowers is one of the important factors in appreciating the beauty of horticultural plants. In general, a cyclamen flower has five petals and is single-flowered. In recent years, however, double-flowered cyclamen species with ten petals induced by natural mutation have been produced and commercialized. However, efficient production of double-flowered cyclamens by traditional breeding methods such as cross hybridization or selection of mutants is difficult. The production of polypetalous cyclamens with many petals like roses or carnations was also difficult. Therefore, new technologies to improve the shape of flowers efficiently were required to create new varieties such as polypetalous cyclamens. Before commercializing genetically modified plants, development of technologies to prevent pollen dispersion by sterilizing them or removing stamens will be required to limit biological diversity impact to the lowest level.

In the past, Research Institute of Genome-based Biofactory of AIST developed a technology called CRES-T method that can suppress the expression of genes regulated by certain transcription factors of plants. By applying CRES-T method, it is possible to produce a repressor that suppresses the expression of target genes of a transcription factor. Introduction of this repressor into the plant enables the production of a plant in which the function of the transcription factor is inhibited.

We planned to apply this technology to improve the properties of cyclamen flowers; however, at that time, the transcription factors of cyclamen were hardly analyzed due to shortage of genetic information about cyclamen. Therefore, we started the research to modify the shape of cyclamen flowers by first isolating the transcription factors associated with the formation of floral organs of cyclamens and then applying gene-recombination technology and CRES-T method.

Floral organs forming flowers, such as petals, stamens, pistils, and sepals are known to be regulated by genetic material called transcription factors. Generally, the development of floral organs is explained by the ABC model; i.e., the shape of a flower is thought to be regulated by transcription factors categorized in three classes: class A, class B, and class C. Among them, a transcription factors called AGAMOUS, which belong to class C, are involved in the formation of stamens and pistils, and improvement of flower shapes was thought to be possible by controlling the expression of these transcription factors.

First, we tried to obtain and analyze transcription factors regulating the formation of floral organs of cyclamens, and obtained two novel AGAMOUS genes associated with the formation of stamens and pistils. Chimera repressors for AGAMOUS were produced to induce phenotype similar to deficient mutant of the AGAMOUS genes and then introduced into cyclamen cells. The result of flowering showed that the formation of stamens and pistils was inhibited and that petals were formed instead of these organs. Thus, we succeeded in producing cyclamens with more than 50 petals, as well as double-flowered cyclamens. In the polypetalous cyclamens, petals come out successively like roses or carnations (left-side photo). These varieties do not produce seeds; however, breeding of seedlings is possible by using the tissue culture technology owned by Hokko Chemical Industry Co., Ltd.

Because petals are formed instead of stamens in these new cyclamens, dispersion of pollen will be suppressed and biological diversity impact should be small.

Our technology has opened new avenues for cyclamen varieties with novel characteristics in which petals come out successively like roses. Currently, the color of the flower is pink alone. However, we are aiming to produce polypetalous cyclamens of various colors using our new technology. We are planning to commercialize the multicolored polypetalous cyclamens after biological diversity risk assessment.