JARQ 50 (2), 79 - 86 (2016) http://www.jircas.affrc.go.jp

REVIEW

Promotion of Efficient Molecular Breeding
Using Chimeric Repressors in Ornamental Flowers
Katsutomo SASAKI1*and Norihiro OHTSUBO1,2
1

NARO Institute of Floricultural Science (NIFS),
National Agriculture and Food Research Organization (NARO)(Tsukuba, Ibaraki 305-8519, Japan)

Abstract

Useful novel floral traits have recently been generated in various ornamental crops by using Chimeric
REpressor gene Silencing Technology (CRES-T). CRES-T is a plant-specific gene silencing methodthat targets transcription factors (TFs), causing a dominant loss-of-function phenotype. In our groupproject, we applied CRES-T to various ornamental crops and mainly elucidated the following points: 1)
CRES-T was effective for the modification of floral traits, 2) it was useful in higher polyploidy crops,and 3) it enabled the creation of commercially valuable flowers. In CRES-T, the attachment of a short
repression domain derived from a transcriptional repressor to the C-terminal region of target TFs
converts transcriptional activators to dominant chimeric repressors. In the target flower species, someArabidopsis chimeric repressors were effective for the production of new floral traits without cloning
the TF genes of interest. Therefore, the use of Arabidopsis chimeric repressors could be widely effective
for many ornamental crops, even when the genome and/or expressed sequence tag (EST) informationof target crops is unavailable or lacking. In addition, the collective transformation of chimeric repressors into ornamental crops is effective in isolating useful and/or target floral traits, such as petal colors,
petal shapes, and color patterns. This technology could help to accelerate the developmental period andreduce the cost of developing new floral traits.
Discipline: Biotechnology
Additional key words: CRES-T, floral trait, promoter, transcription factor

Introduction
Genetically modified (GM) flowers such as blue carnations (Tanaka et al. 2009) and blue roses (Katsumoto et al.
2007) were commercialized in Japan from 1997 and 2007,
respectively. Thus, GM flowers have almost gained public
acceptance in Japan. However, the conventional method ofmolecular breeding for the development of these GM flowers requires a lot of time (sometimes more than a decade;Ohtsubo 2011), and the costs are very high. Therefore, it isdifficult to keep up with consumer preferences that change
every year and cope with the high costs of developing evenone new variety. At present, there is an urgent need for the
next generation of molecular breeding technology to accelerate the developmental period and reduce development costs.
Efficient massive screening methods would also be important

for reducing costs required for developing a new variety.
Transcription factors (TFs) are master genes that
control various floral traits, such as flower colors, flower
shape, petal shapes, and fragrance (Petroni & Tonelli 2011,
Preston & Hileman 2009, Colquhoun & Clark 2011).
Therefore, the modification of TF functions has great
potential to dynamically change floral traits. Chimeric
REpressor gene Silencing Technology (CRES-T; Hiratsu
et al. 2003, 2004) is a plant-specific gene silencing methodthat dominantly suppresses the functions of TFs. CRES-T
has been applied to create target traits in the model plant
Arabidopsis and in rice (Mitsuda et al. 2006, Mito et al.
2011). In addition, the flower CRES-T project in Japan(Ohtsubo 2011) reported that CRES-T can be applied tovarious ornamental crops, such as Torenia, rose, gentian,lisianthus, cyclamen, chrysanthemum, and morning glory

Present address:2
Graduate School of Life and Environmental Sciences, Kyoto Prefectural University(Sakyo-ku, Kyoto 606-8042, Japan)* Corresponding author: e-mail kattu@affrc.go.jp
Received 6 February 2015; accepted 7 September 2015.
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K. Sasaki & N. Ohtsubo(Mitsuda et al. 2008, Mitsuda et al. 2011a), although it wasdeveloped for the model plant Arabidopsis (Hiratsu et al.
2003, 2004). Therefore, CRES-T may be effective for alarge variety of monocot and dicot ornamental crops. In addition, a collective transformation (CT) system (Shikata etal. 2011) would accelerate development to keep pace withchanging consumer preferences, while still controlling thecosts of commercialization such as in performing massive
screening using the CT system.

TFs perform crucial functions in floral traits
In plants, TFs control the expression of their downstream genes by upregulating or downregulating the genetranscriptions. In Arabidopsis, there are approximately
2,000 TFs, which account for approximately 5%-10% of
their total genes (Riechmann et al. 2000). The number of
TF subfamilies identified in several databases ranges from51-67 (72 in total), depending on the differences in criteria
applied to their DNA binding domains (Riechmann et al.
2000, Mitsuda & Ohme-Takagi 2009). TFs bind to thepromoters of downstream genes by recognizing a specific
DNA sequence — the “cis-element.” The TFs act as a master switch to upregulate or downregulate the other 90%of the plant genes; therefore, TFs have various importantfunctions in the plant body, such as the floral organs. For
example, floral organ identity is explained by the ABCEmodel (Ma 1994, Theißen 2001, Ó'Maoiléidigh et al. 2014)
in which the A-, B-, C- and E-function genes are TFs having
a MADS-box DNA binding domain. Furthermore, floral zygomorphy that is observed in Antirrhinum majus and Lotus
japonicus is also controlled by TFs, such as CYCLOIDEA,DICHOTOMA, DIVARICATA, and RADIALIS (Preston &Hileman 2009, Feng et al. 2006).

Difficulty in the functional analysis of TFs due togene redundancy
The functional analyses of MADS-box and floral
zygomorphy genes have been mainly conducted using
mutants of Arabidopsis and A. majus (Ma 1994, Preston &Hileman 2009). However, it is difficult to analyze the function of redundant TFs by a single mutation because otherfunctionally redundant TFs complement this mutation. In
some plant species, plant genes frequently show duplication
and/or redundancy, including TFs (De Smet & Van de Peer2012, Moore & Purugganan 2005) and MADS-box TFs(Rijpkema et al. 2007, Soltis et al. 2007, Zhan et al. 2005).
Gene-knockout and/or antisense methods specific for aparticular TF also sometimes fail to show phenotypic change
due to its functional redundancy (Bouché & Bouchez 2001).
For example, the mutant phenotype of Arabidopsis TCP was
not sufficiently observed even in a triple mutant (Koyama et80

al. 2010). Class B MADS-box genes that play important rolesin the formation of petals and stamens are redundantly foundin such horticultural crops as Gerbera hybrida (Broholm etal. 2010), Orchis italica (Salemme et al. 2011), and Petunia
hybrida (Vandenbussche et al. 2004), but not in Arabidopsis
and A. majus. The analysis of such redundant TFs by a single
mutation has limitations, particularly in higher polyploidy
plants. Thus, any important functions of floral traits notfound in the mutant analyses of Arabidopsis and A. majus
would still be present in such horticultural crops.

Development and application of CRES-T in ornamental crops
The development of floral organs is mainly regulated by TFs, and CRES-T has reportedly been an important
breakthrough for the functional analysis of plant TFs(Hiratsu et al. 2003). The strong repression domain (SRDX)
derived from plant TFs converts transcriptional activators tochimeric repressors that dominantly repress the function oftarget TFs, even in the presence of functionally redundant
TFs (Hiratsu et al. 2003, Koyama et al. 2007, Mitsuda et al.
2007, Oshima et al. 2013). Figure 1 shows how simple it isto produce a chimeric repressor by attaching SRDX onto
the C-terminal regions of target TFs. Although this method
was developed in Arabidopsis (Hiratsu et al. 2003, 2004), it
has also been applied to monocot rice (Mitsuda et al. 2006,
Mito et al. 2011, Tanaka et al. 2012). In addition, SRDX iseffective for many ornamental crops, such as Torenia, rose,
gentian, lisianthus, cyclamen, chrysanthemum, and morning
glory (Mitsuda et al. 2008, Mitsuda et al. 2011a). Because
this system functions dominantly, the chimeric repressors
even worked in higher polyploidy plants, such as the hexaploid chrysanthemum (Mitsuda et al. 2011a, Narumi et al.
2011). It is noteworthy that chimeric repressors constructedwith Arabidopsis TFs frequently worked in other ornamental crops for the functional analysis of TFs and the development of novel floral traits without any further modification(Narumi et al. 2011, Shikata et al. 2011, Gion et al. 2011).
Information regarding the floral phenotypes generated in
the CRES-T project is available on the FioreDB website:
Database for Flower Bio-engineering by CRES-T (Mitsuda
et al. 2011a; http://www.cres-t.org/fiore/public_db/index.
shtml). The floral phenotypes shown on the web site may
provide useful ideas for the development of new flowers
in plant species for which it is difficult to isolate new floral
traits by using traditional breeding methods.

Chimeric repressors enable the creation of novel
floral traits
Chimeric repressors have produced various novel
flowers in many ornamental crops, including commercially
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Promotion of Efficient Molecular Breeding Using Chimeric Repressors

valuable transgenic flowers. Cyclamens have two class C
AGAMOUS (AG) orthologs — CpAG1 and CpAG2 (Tanaka
et al. 2011) — and the simultaneous overexpression of
CpAG1-SRDX and CpAG2-SRDX produced a multi-petal
flower phenotype not previously seen in cyclamen (Fig. 2;
Tanaka et al. 2013). The co-overexpression of CpAG1SRDX and CpAG2-SRDX caused the stamens and carpelsof the transgenic cyclamen to develop petal-like organs,and the multi-petal flowers contained a total of more than
40 petals and petal-like organs, as compared with 5 petals

in the wild-type cyclamen. Because the gorgeous rose-likephenotype is one of the most commercially preferredphenotypes, the multi-petal cyclamen would be closest tocommercial viability among the GM flowers produced by
the chimeric repressors.
To generate various floral traits concurrently, a CT
system has been applied in Torenia (Fig. 3; Shikata etal. 2011). In this system, Arabidopsis TFs were used to
produce chimeric repressors, and these mixed chimericrepressor plasmids (42-50 TFs) were co-transformed into

Fig. 1. CRES-T is useful to suppress the function of TFs
CRES-T dominantly suppresses the function of target TFs even in the presence offunctionally redundant TFs. This technology is helpful to create novel floral traits inornamental crops. Detailed protocol for CRES-T was reported by Mitsuda et al. (2011b).

Fig. 2. A multi-petal flower phenotype in cyclamenA) A wild-type (WT) cyclamen. B) The transgenic cyclamen co-overexpressing CpAG1SRDX and CpAG2-SRDX genes with a multi-petal phenotype.
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K. Sasaki & N. Ohtsubo

Agrobacterium in equal concentrations. Torenia was inoculated with a mixture of Agrobacterium generating hundredsof transgenic Torenia plants. In this system, more than 80%
of transgenic Torenia plants had a single transgene. The
CT system efficiently generated novel and varied floral
traits at once, and enabled the selection of desirable traits(Fig. 3B), thereby enabling a reduction of costs requiredfor developing a new variety, while meeting changing customer preferences. Although the CT system was applied in
Torenia (Shikata et al. 2011), it would also be effective forother ornamental crops with established and highly efficienttransformation methods, such as those of Torenia (Aida &Shibata 1995) and Arabidopsis.
The introduction of chimeric repressors could not only

Fig. 3. Simplified flow of the CT systemA) The CT system enabled the selection of target floral traits through massive screening (Shikata et al. 2011).
B) Modified floral traits of transgenic Torenia plants generated by the CT system. Floral traits of these transgenic Torenia plants were confirmed within approximately six months after the start of transformation.
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Promotion of Efficient Molecular Breeding Using Chimeric Repressors

lead to the functional analysis of TFs but also to the generation of new floral traits. In Torenia, there are two orthologsof the class B genes DEFICIENCE (DEF) and GLOBOSA(GLO), called TfDEF and TfGLO, respectively (Sasaki et al.
2010). Simultaneous suppression of these genes by chimericrepressors resulted in sepal-like petals (Sasaki et al. 2014),
indicating that TfDEF and TfGLO synergistically played
important roles in the development of petals. However, theexpression of single TfDEF-SRDX or TfGLO-SRDX caused
phenotypic changes in petals, resulting in novel floral traits
in Torenia (Sasaki et al. 2010), unlike the co-suppression ofthese class B genes. TfDEF-SRDX and TfGLO-SRDX plants
showed partially decolorized petals and serrated petals,
respectively (Fig. 4). In addition, the introduction of thesechimeric repressors into blue colored transgenic Torenia
plants (transgenic line no. 416-20; Fig. 4, right) produced
by antisense dihydroflavonol 4-reductase gene (Aida et al.
2000) resulted in differently colored floral traits (Fig. 4).
The combination of chimeric repressors with an additionaltransgene could further produce novel floral traits. On
the other hand, heavy-ion beam irradiation has also led
to a large variety of colors in Torenia flowers (Sasaki etal. 2008). Thus, combination of chimeric repressors andFig. 4. Floral trait variety generated by CRES-T
A) A wild type (WT; left) and 416-20 (right) transgenic Torenia plants. B) Floral phenotypes generated
by class B chimeric repressors. TfDEF-SRDX and
TfGLO-SRDX were introduced into WT (upper) and
416-20 (lower) Torenia plants, respectively. The
numbers at the lower right in each photo denote thenumber of transgenic lines.

heavy-ion beam irradiation (Ohtsubo et al. 2012) could
possibly enable the creation of a wide variety of colorfulflowers with new floral traits.

Modification of chimeric repressor constructs
In the flower CRES-T project (Ohtsubo 2011), mostornamental crops used the 35S promoter from cauliflower
mosaic virus (CaMV), which usually shows high promoter
activity in the whole plant. However, the 35S promoter
did not work in certain ornamental crops, and transgenes
controlled by the 35S promoter were not expressed in gentian due to the gene silencing of the introduced transgene(Mishiba et al. 2005, Mishiba et al. 2010). Therefore, theAgrobacterium rhizogenes rolC promoter and Arabidopsis
Actin2 promoter were used for the transgene expression ingentian instead of the 35S promoter (Nakatsuka et al. 2008,
Nakatsuka et al. 2011). For utilizing chimeric repressors,
the expression of introduced transgenes should be tested

Fig. 5. Changing the promoter leads to the modification offloral traitsA) WT Torenia plants (upper) and transgenic Torenia plants with 35S pro:AtMYB24-SRDX (lower).
B) A floral organ specific promoter was used for the
expression of AtMYB24-SRDX. WT Torenia plants(upper) and transgenic Torenia plants with AtAP1
pro:AtMYB24-SRDX (lower). Red arrowheads indicate the altered points.
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K. Sasaki & N. Ohtsubo

in target plant species. It is desirable to check promoter
activity in a target ornamental crop before use by using theparticle bombardment method.
Among the chimeric repressors, the 35S promoter
sometimes caused an unfavorable phenotype in unexpectedorgans due to the ectopic expression derived from promoter
activity. Overexpression of the Arabidopsis MYB24-SRDX(AtMYB24-SRDX) resulted in unopened flower buds andglossy dark green leaves with curled margins in Torenia(Sasaki et al. 2011, Fig. 5A). Because the petals inside theflower buds exhibited a different coloration pattern thanthat of wild-type plants, a floral organ-specific promoter
was used to express AtMYB24-SRDX for exhibiting theconcealed petal phenotype in Torenia. Floral organ-specific expression of AtMYB24-SRDX from the Arabidopsis
APETALA1 (AtAP1) promoter resulted in wavy petals(Fig. 5B). Use of the floral organ-specific promoter also
prevented unfavorable phenotypes caused by the 35S
promoter other than in floral organs; hence, the plants exhibited the normal leaf phenotype. The generation of a new
floral phenotype without changes in non-targeted organs
is a favorable feature because cultivation conditions forthe new variety may not differ from those for the parental
variety. It is important to culture the new variety in thealready established manner, so as to ensure a stable supplyof this commodity. Thus, changing the promoter can solve
problems that arise when transgenic plants have unfavorable phenotypes in non-targeted organs.

Perspectives
In this review, we have introduced the utilization of
CRES-T for changing floral traits in ornamental crops.
However, the commercialization of transgenic ornamental
crops requires an evaluation of their impact on biodiversity,in accordance with the Cartagena Protocol on Biosafety
and the laws of each country. Blue carnations and roses
have been commercialized through specific processes of
evaluation in Japan. CRES-T could be applicable for further
uses, such as the regulation of flowering, disease resistance,
environmental tolerance, and improvement of growth characteristics. The combined use of the methods introduced inthis review would have high potential for accelerating thedevelopment of ornamental crops. CRES-T is also expected
to contribute to the commercialization of useful GM flowers in the near future.

Acknowledgments
We wish to thank Mr. Teruhiko Terakawa for providing the photographs of cyclamen, Dr. Hiroyasu Yamaguchi
and Ms. Satoko Ohtawa for generating and maintaining
transgenic Torenia plants, and Ms. Yoshiko Kashiwagi for84

maintaining the Torenia plants showed in this review. The
photographs in Figure 3B and Figure 5 are reprinted fromour previous paper (Shikata et al. 2011 and Sasaki et al.
2011, respectively) with the permission of the journal Plant
Biotechnology (Japanese Society for Plant Cell and Molecular Biology). The studies reported in this review were
supported by the Program for Promotion of Basic and Applied Researches for Innovations in Bio-oriented Industry
from the Bio-oriented Technology Research Advancement
Institution, and also by the Scientific Technique Research
Promotion Program for Agriculture, Forestry, Fisheries andFood Industry.

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