1. business and industrial
2. chemicals industry
3. dyes and pigments

35
Molecular Breeding of Flower Color
Kin-Ying To* • Chen-Kuen Wang
Institute of BioAgricultural Sciences, Academia Sinica, Taipei 115, Taiwan
Corresponding author: * [email protected]
Keywords: anthocyanin, betalain, carotenoid, flavonoid, flower color, genetic engineering, pigmentation
ABSTRACT
Flower color contributes mainly to the market value of an ornamental plant, and coloration of flowers is determined by three classes of pigments:
flavonoids, carotenoids and betalains. Flavonoids and carotenoids are widespread; however, betalains can be found only in plants of several
genera in the order Caryophyllales, which belongs to one small group of angiosperms. Among these pigments, flavonoids (mainly anthocyanins)
are the most common flower pigments contributing to a range of colors from yellow to orange to red to purple. During the past few decades,
flavonoid biosynthetic pathway leading to anthocyanin production has been well established in various plant species, and genetic engineering of
flavonoid/anthocyanin biosynthesis has been used to produce cultivars with novel pigmentation in flowers. Here we summarize the current status
of molecular approaches in breeding flower coloration, and describe our study and prospective regarding flower color modification.
1. INTRODUCTION
Flower color is one of the most attractive characteristics in ornamental plants, contributing to the major value in the floricultural market. In nature,
various patterns regarding to the flower color can be easily observed (Fig. 1); however, most of these phenotypic changes are not transmittable
and thus novel varieties with commercial value cannot be obtained. Furthermore, alteration in flower pigmentation is a visible indicator to study
expression and regulation of floral genes in plant molecular biology. Thus, examination and manipulation of flower color is not only important in
basic research areas, it also has a great benefit in biotechnological applications.
Generally speaking, flower color is predominantly determined by two classes of pigments: flavonoids and carotenoids. Flavonoids (mainly
anthocyanins) are the most common flower pigments contributing to a range of colors from yellow to orange to red to purple; and carotenoids are
the red, orange and yellow lipid-soluble pigments found embedded in the membranes of chloroplasts and chromoplasts (Bartley and Scolnik
Fig. 1 Natural occurrence of different flower colors in azalea (Rhododendron spp.). (A) Two
flowers with different colors were found in a same branch. (B) A small sector with red color was
found in the white background of the petal. (C) A red flower was found in a cluster of white
flowers. (D) A typical chimeric flower composed of two sectors of white and red colors.
Fig. 2 Representative structures of pigments. Betacarotene (βcarotene) belongs to carotenoids, and contributes reddish yellow
color in some flowers. Betacyanin (red to red-violet color) and
betaxanthin (yellow color) are two groups of betalains, which
contribute flower pigmentation in several plants of a small group in
the order Caryophyllales. Flavonoid is the major contributor in
flower color in plant species. Flavonoid consists of two aromatic
rings (A and B), and a heterocyclic C ring with oxygen.
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1995). In addition, a third class of pigments, betalains, can be found only in certain plants from 10 families of the order Caryophyllales (including
beetroot and several important genera such as Amaranthus, Celosia, Gomplrena, Iresine, etc.) and in some higher fungi such as fly agaric
(Amanita muscaria) (Strack et al. 2003, Cai et al. 2005). Representative structures of these pigments are shown in Fig. 2. Betalains are watersoluble alkaloids localized in the cell vacuole; interestingly, betalains and anthocyanins have never been reported in the same plants (Stafford
1994). Betalains can be further divided into two major groups: the red to red-violet betacyanins and the yellow betaxanthins (Cai et al. 2005).
Betalains are located in different plant tissues from roots (e.g., beetroot) to fruits (e.g., cactus fruits). The presence of these pigments in flowers
(e.g., Bougainvillea, Celosia, Gomphrena, Portulaca, Mirabilis) is used as an attractant for some insects and animals (Gandía-Herrero et al.
2005a). Betalains have been used as natural pigments for food coloration for many years (Cai et al. 2005); however, metabolic engineering of
betalains has not been reported yet. Carotenoids are C40-tetraterpenoid compounds that are synthesized from the basic C5-isoprene units
(isopentenyl diphosphate and its isomer dimethylallyl diphosphate) in great varieties by bacteria, fungi, algae and higher plants. Animals appear
to be incapable of biosynthesizing carotenoids, but all kinds of animals use carotenoids for a variety of purposes including coloration and as a
precursor of vitamin A (Bartley and Scolnik 1995, Britton 1998). Carotenoids are lipid-soluble pigments and are located in the plastids,
contributing to the majority of yellow hues in a number of flowers (Forkmann 1991). In some flowers such as roses and chrysanthemums, the
orange/red, bronze and brown colors are contributed from carotenoids, or along with red or magenta anthocyanins (Tanaka et al. 2005).
As mentioned, flavonoids are the most common
of the three types of pigment in flower tissue.
Flavonoids are water-soluble compounds and are
based on a C15 skeleton (Fig. 2). Flavonoids, starting
from the general phenylpropanoid pathway, can be
subdivided into 9 major groups according to their
structures: chalcones, aurones, isoflavonoids,
flavones, flavonols, flavandiols, anthocyanins,
condensed
tannins
(proanthocyanins)
and
phlobaphenes (Winkel-Shirley 2001). Among them,
the major classes of flavonoids which contribute color
to plants are the anthocyanins, flavonols, chalcones
and aurones. Anthocyanins are the largest class of
flavonoids that are responsible for the pink, red, violet,
blue and purple colors of flowers and other tissues.
Anthocyanins are water-soluble pigments that
accumulate in vacuoles or anthocyanoplasts. The
anthocyanin biosynthetic pathways from higher plants
including Petunia, maize, snapdragon and
Arabidopsis have been well established (Holton and
Cornish 1995, Mol et al. 1998, Winkel-Shirley 2001).
Three common anthocyanins are pelargonidin-,
cyanidin-, and delphinidin-based pigments (Fig. 3).
In this chapter, we mainly describe genetic
engineering
of
flower
color
via
the
flavonoid/anthocyanin biosynthetic pathway. In
addition, we also briefly discuss several strategies in
modifying flower color. Biotechnology of floricultural
plants for traits such as vase-life in cut flowers, floral
and plant morphology, flower color, flower
senescence and pathogen resistance have been
discussed from various species (Tanaka et al. 1999
2005). Modification of flavonoid biosynthesis and its
application in crop plants including potato and tomato
have been recently reviewed (Schijlen et al. 2004,
Dixon 2005).
Fig. 3 Flavonoid biosynthetic pathway leading to
produce anthocyanins and some classes of flavonoids
relevant to flower color.
2. BIOSYNTHESIS AND FUNCTION OF FLAVONOIDS
During the past few decades, nearly all enzymes involved in the pathways to the different flavonoid classes have been determined. It should be
kept in mind that some branch/modifying enzymes are limited in certain plant species. All flavonoids are derived from a general phenylpropanoid
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pathway which starts from an aromatic amino acid phenylalanine (Fig. 3). The first committed step is catalyzed by chalcone synthase (CHS).
CHS catalyzes condensation of one molecule of p-coumaroyl-CoA and three molecules of malonyl-CoA, resulting one molecule of 4′,2′,4′,6′tetrahydroxychalcone (chalcone or naringenin chalcone), which is a key intermediate in the formation of flavonoids. Characteristics and
manipulation of CHS will be further discussed later in this chapter. Chalcone is rapidly converted into naringin (a kind of flavanone) by chalcone
isomerase (CHI); however, in the absence of CHI, chalcone also spontaneously forms mixed enantiomers of naringenin in vitro (Jez et al. 2000).
Subsequently, gene encoding flavanone 3-hydroxylase (F3H) of the unbranched flow in the flavonoid biosynthetic pathway catalyzes naringenin
to dihydrokaempferol. Extensive modifications including hydrolation, glycosylation, methylation and acylation are carried out to form various
compounds containing major classes of flavonoids. For simplification, the colored products of anthocyanins, anthocyanidin 3-glucosides, being
the major components in flower color, are emphasized (Fig. 3). These colored anthocyanins can be further modified by glycoslation, acylation or
methylation in a species-specific manner. Genes encoding key enzymes in the branch of the flavonoid biosynthetic pathway such as aureusidin
synthase (AS) and UDP-glucose:tetrahydroxychalcone 2′-O-glucosyltransferase (C2′GT) (Fig. 3) will be discussed later.
The physiological functions of anthocyanin pigments are involved in the attraction of pollinators and in fruit and seed dispersal, they also
have key roles in signaling between plants and microbes, in male fertility of some species, in defense as antimicrobial agents and feeding
deterrents, and in UV protection (Dixon and Steele 1999, Forkmann and Martens 2001, Winkel-Shirley 2001). In addition, anthocyanins often
appear transiently at specific developmental stages and may be induced by a number of environmental factors including visible and UVB
radiation, cold temperature and water stress (Chalker-Scott 1999, Forkmann and Martens 2001, Winkel-Shirley 2002). Flavonoids are also linked
to amelioration of adverse effects of heat stress on fertilization and early seed maturation (Coberly and Rausher 2003); and more recently,
flavonoids have been considered as regulators in developmental processes of plants including auxin transport, induction of pollen-tube
germination, allelochemicals, signals for symbionts, induction of cell death by activating reactive oxygen species and detoxication (Taylor and
Grotewold 2005).
3. MOLECULAR APPROACHES TOWARD TO FLOWER COLOR MODIFICATION
Flavonoids/anthocyanins are major contributors to flower coloration (reviewed by Teixeira da Silva and Nhut 2003). As a result, most studies on
genetic engineering of flower color have been carried out via the flavonoid biosynthetic pathway. One major approach is alteration of anthocyanin
contents/composition, which will be discussed extensively in this chapter. Other factors including copigments, vacular pH value and cell shape
will also be described briefly. Recently, genetic engineering of flavonoids has been reviewed extensively (Forkmann and Martens 2001, WinkelShirley 2001, Schijlen et al. 2004, Tanaka et al. 2005), and here we summarize what we know in this field, and describe the recent progress and
future prospects based on this information.
4. OVEREXPRESSING OR SILENCING THE STRUCTURAL GENE
EXPRESSION IN FLAVONOID BIOSYNTHETIC PATHWAY
4.1. Chalcone synthase
So far, most efforts to modify flower color have focused on the general
phenylpropanoid pathway, which produces p-coumaroyl-CoA and
subsequently leads to the synthesis of anthocyanin pigments. An effective
approach of pigment engineering is overexpressing or silencing the structural
gene in the flavonoid biosynthetic pathway by transgenic technology. As
shown in Fig. 3, chalcone synthase (CHS; EC 2.3.1.74) is the first key
enzyme catalyzing 3 molecules of malonyl-CoA and 1 molecule of pcoumaroyl-CoA into 1 molecule of chalcone, which can be converted into
other classes of flavonoids including anthocyanins. The CHS enzyme, usually
found in plant epidermal cells, has a molecular weight of about 42,000,
requires no co-factors, and has been purified from various plant species. In
addition, three-dimensional structure and functional studies of alfafa CHS
enzyme have shown that four residues (Cys164, Phe215, His303, Asn336) in
the catalytic site are responsible for decarboxylation and condensation
reactions for this enzyme, and are highly conserved among different species
(Ferrer et al. 1999, Jez et al. 2000). Thus, the gene encoding CHS enzyme is
the major target for controlling flower color.
We are interested in genetic engineering of flower color. To achieve this
goal, full-length cDNA clones encoding CHS (accession no. AF233638),
chalcone isomerase (CHI; accession no. AF233637), dihydroflavonol-4reductase (DFR; accession no. 233639) and cytochrome b5 (accession no.
AF233640) have been cloned from a flower cDNA library which was
constructed in λTriplEx2 vector (Clontech) with poly(A) mRNA from different
stages of violet flower in Petunia hybrida cultivar Ultra Blue (Fig. 4A). Here
we report the characterization and molecular breeding of flower color through
the introduction of a Petunia chs cDNA into tobacco. A full-length Petunia chs
cDNA was cloned using a PCR-based method (Koes et al. 1986); it encodes a
polypeptide of 389 amino acids (aa) with a calculated molecular mass of 42.6
Fig. 4 Growth stages of flower development and differential chs
mRNA level in Petunia hybrida. (A) Stages of flower development.
S1, flower bud is less than 1 cm in length; S2, flower bud has reached 1
to 3 cm in length; S3, flower bud is larger than 3 cm, purple color is
observed at the top of petal; S4, flower is completely open; S5,
senescent flower with wilting petals. (B) Differential chs mRNA levels in
Petunia hybrida. Total RNA was isolated from leaf, root and different
stages of flower, and 15 µg of each RNA sample was used for RNA blot
analysis. The blot was hybridized with a non-radioactive DIG probe
encoding full-length Petunia chs cDNA. For a loading control, rRNA
bands were visualized by ethidium bromide staining and the RNA gel
was then photographed before being transferred to the membrane.
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kDa and an estimated pI value of 6.96. Our clone
showed 3 aa alternations at positions 288, 291 and
331 as compared to the published P. hybrida (V30)
CHS sequence (Koes et al. 1986). Different
cultivars (Ultra Blue versus V30) may be the
possible explanation for this observation. The
deduced aa sequence of CHS shows 85-95%
identity to the reported CHS from other higher
plants including tobacco (95%), potato (92%),
tomato (89-91%), garden snapdragon (90%),
morning glory (89-90%), soybean (87-88%), pea
(85-88%), kudzu vine (86%) and alfalfa (85%)
(http://www.ncbi.nlm.nih.gov/BLAST/). The threedimensional structure of the CHS enzyme suggests
that four residues (Cys164, Phe215, His303 and
Asn336) participate in the multiple decarboxylation
and condensation sites (Ferrer et al. 1999), and we
found that these four catalytic residues and the
relevant flanking motifs in our CHS sequence
perfectly matched the proposed model (Ferrer et al.
1999, Jez et al. 2000). Sequence alignment of the
active sites of CHS-related enzymes isolated from
bacteria to higher plants shows that these four
residues as mentioned are without exception all
identical and the relevant flanking motifs are highly
conserved (Jez et al. 2000). Nucleotide and
deduced aa sequences of Petunia cDNA encoding
chalcone synthase are shown in Fig. 5A.
To investigate the tissue specificity and
Fig. 5 Petunia cDNA encoding chalcone synthase and its construction to the expression vector.
developmental regulation of chs mRNA, total RNA
(A) Nucleotide and deduced amino acid sequences of Petunia cDNA encoding chalcone synthase.
from different tissues of Petunia plants were
Four catalytic residues (C164, F215, H303 and N336) are highlighted in gray, and the conserved
flanking motifs are underlined. The asterisk (*) indicates the termination codon. (B) The full-length
isolated, and probed with full-length Petunia chs
cDNA encoding Petunia chalcone synthase was replaced the GUS coding region in the binary vector
cDNA. The chs mRNA was detected at a very early
pBI121 (Chen et al. 2003). Total length of the resulting vector pCHS is 14065 bp.
stage (stage S1) of flowering, gradually accumulating to the maximal level in immature flowers
shortly before opening (stage S3), a dramatic
decrease in chs mRNA was observed in completely
opened flowers (stage S4), and a background level
of mRNA was noted in senescent flowers (stage
S5). No chs mRNA was detected in RNA samples
isolated from leaf and root tissues (Fig. 4B). In P.
hybrida V30, there are at least 6 chs genes per
haploid genome, and only one member (chsA) is
actively transcribed in floral tissue (Koes et al.
1986). Our data clearly show that only one chs
mRNA is highly expressed in developing flowers,
and further demonstrate the tissue specificity and
developmental regulation of chs mRNA in petals.
As a first step in engineering of flower color,
the gus reporter gene in the binary vector pBI121
(Chen et al. 2003) was replaced by the Petunia chs
cDNA, and a sense chs construct (pCHS) was
Fig. 6 Flower phenotypes in transgenic tobacco plants. Untransformed tobacco was indicated as
obtained. The expression vector pCHS (Fig. 5B)
“wild-type (W38)”, which had pink color in flowers. Fainter color was observed in a transgenic line
containing Petunia chs cDNA under the control of
CHS11. Deeper colors with different patterns were observed in a transgenic line CHI4, and a line from
CaMV 35S promoter was then transformed into
the cross of CHS11 and CHI4 (indicated as CHS11xCHI4). Faint red in rim of flower petal was
observed in a line from the cross of CHI4 and CHS7 (indicated as CHI4xCHS7). Flowers with
tobacco via Agrobacterium-mediated method. After
complete white color were observed in a transgenic line CHS10.
antibiotic selection and regeneration, 7 transgenic
tobacco plants were obtained; among them, 4 transformants produced white flowers and 3 transformants produced pink flowers similar to the
wild-type tobacco plants. Extensive data including T1 progeny assay, Southern blot, thin layer chromatography analysis, Northern blot, RT-PCR
as well as plant/T-DNA junction sequence analysis support the hypothesis that cosuppression occurs in transgenic plants with white flowers so
that there is not enough CHS enzyme activity to make pigments (Wang et al. 2006). A typical result of producing complete white flowers in
transgenic line CHS10 due to heterologous chs suppression is shown in Fig. 6.
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Flower color modification
Overexpression of sense or antisense chs constructs has been carried out to modify flower color in various plant species including Petunia,
Torenia, chrysanthemum (Dendranthema grandiflora), lisianthus (Eustoma grandiflorum), model species of tobacco, and one herbal plant
Echinacea (Table 1). Some transformants were scored as white flowers or as different patterns; instead, some transformants possessed as wildtype phenotype (Table 1). In conclusion, our and other studies show that genetic modification of flower color, especially in generating white
flowers, can be accomplished by transforming chimeric chs construct into plants. However, effects on color modification are not easy to predict
and can differ between plant species and varieties.
Table 1 Alteration of flower color by sense or antisense chalcone synthase.
Target plant (Original color)
Nicotiana tabacum var. W38 (pink)
N. tabacum var. Bairihong (pink)
N. tabacum var. SR1 (pink)
Petunia hybrida var. VR (violet)
P. hybrida var. VR (violet)
Ibid.
P. hybrida var. Pink Cascade (pink)
P. hybrida var. R18 (pale pink)
P. hybrida var. V26 (purple)
Ibid.
Dendranthema grandiflora
var.
Moneymaker (pink)
Ibid.
Gerbera hybrida var. Terra Regina
(red)
Echinacea purpurea var. Magnus
(red)
Eustoma grandiflorum var. Grise
(purple)
Torenia fournieri var. Crown mix
(violet)
Ibid.
T. fournieri var. Crown mix (reddish
purple)
Ibid.
T. fournieri var. Common violet
(violet)
Ibid.
T. hybrida var. Summerwave Blue
(blue)
T. hybrida var. T-33 (blue)
Gene construct (Source)
sense chs cDNA (Petunia)
sense chs cDNA (Phalaenopsis)
antisense chs cDNA (Petunia)
sense chs genomic clone VIP106 (Petunia)
sense chs genomic clone VIP76 (Petunia)
sense chs cDNA (Petunia)
sense chs cDNA (Petunia)
sense chs cDNA (Petunia)
sense chs cDNA (Petunia)
antisense chs cDNA (Petunia)
sense chs cDNA (D. grandiflora)
T
7
ca. 100
40
20
15
6
6
14
185
85
133
No. of transgenic plants altering flower color
WC
ER
CW
VP
OP
3
4
ca. 80
ca. 20 (deeper)
36
3
1
19
1
14
1
6
3
3
9
5
46
139
15
80
major
1
2
1
2
3
Reference
Wang et al. 2006
Han et al. 2005 (pers. comm.)
van der Krol et al. 1988
van der Krol et al. 1990
Ibid.
Ibid.
Napoli et al. 1990
Ibid.
Jorgensen et al. 1996
Ibid.
Courtney-Gutterson et al. 1994
antisense chs cDNA (D. grandiflora)
antisense chs cDNA (G. hybrida)
83
4
major
2
Ibid.
Elomaa et al. 1993
sense chs cDNA (Petunia)
5
5
antisense chs cDNA(E. grandiflorum)
53
22
10
10
Deroles et al. 1998
sense chs cDNA (T. fournieri)
47
31
1
15
Aida et al. 2000
antisense chs cDNA (T. fournieri)
sense chs cDNA (T. fournieri)
17
41
2
14
15
8
19
19
Ibid.
Ibid.
antisense chs cDNA (T. fournieri)
sense chs cDNA (T. fournieri)
41
15
18
8
23
1
6
Ibid.
Ibid.
antisense chs cDNA (T. fournieri)
sense chs cDNA (Perilla frutescence)
9
121
1
91
8
19
11
Ibid.
Suzuki et al. 2000
sense chs cDNA (Perilla frutescence)
96
64
1
31
Ibid.
Wang and To 2004
T = Total transgenic plants examined, WC = Without change, ER = Evenly reduced floral color, CW = Complete white, VP = Variegated pattern with white or pale sectors, OP = Other patterns
4.2. Chalcone isomerase
As shown in Fig. 3, chalcone isomerase (CHI) catalyzes yellow-colored chalcone to colorless pigment naringenin (or flavanone naringenin). This
conversion can also occur spontaneously. Thus, most plants do not accumulate chalcones. However, some mutant plants accumulate chalcones
due to mutation in the chi locus. Yellow flowers have been reported in chi mutants of aster and carnation (Schijlen et al. 2004). Transgenic
tomato plants carrying the sense chi gene from Petunia accumulated more yellow-pigmented naringenin chalcone in the fruit peel (Muir et al.
2001), and overexpression of alfalfa chi gene in Arabidopsis tt5 mutant (mutation in the chi locus) increased flavonol accumulation (Liu et al.
2002). However, in comparison with the effectiveness of color modification by the chs transgene (Table 1), production of yellow flowers by
transforming with the chi construct and suppressing the endogenous chi expression has not been reported yet.
4.3. Flavanone hydroxylase/Flavonoid-3′-hydroxylase/Flavonoid-3′,5′-hydroxylase
The hydroxylation in position 3 of the C ring in flavanones, resulting in dihydroflavonols, has been demonstrated in various plant species. The
reaction is carried out by flavanone-3-hydroxylase (F3H), a member of the 2-oxoglutarate-dependent dioxygenase family. In Petunia and
Antirrhinum, mutation in f3h locus caused a loss of F3H activity and, as a result, white flowers are produced (Schijlen et al. 2004). The transgenic
carnation plants carrying f3h antisense exhibited various degrees of flower color modifications, ranging from partial to complete loss of their
original orange/reddish color (Zuker et al. 2002).
Dihydrokaempferol (DHK), the product catalyzed by F3H, can be further hydroxylated by the cytochrome P450 enzyme hydroxylase
flavonoid-3′-hydroxylase (F3′H) at the 3′ position of the B-ring, leading to the formation of dihydroquercetin (DHQ) and subsequently to the
production of cyanidin-based pigments (red to magenta; Fig. 3). On the other hand, DHK can also be further hydroxylated by the cytochrome
P450 enzyme flavonoid-3′,5′-hydroxylase (F3′5′H) at both 3′ and 5′ positions of the B-ring, leading to the formation of dihydromyricetin (DHM)
and subsequently to the production of delphinidin-based pigments (purple; Fig. 3). Since F3′H and F3′5′H are the direct enzymes for
synthesizing colored anthocyanins, various plant species including Petunia, tobacco, lisianthus, torenia and carnation with novel flower colors
have been reported by transgenic approach (Forkmann and Martens 2001, Tanaka et al. 2005).
4.4. Dihydroflavonol-4-reductase
The enzyme dihydroflavonol-4-reductase (DFR) catalyzes the stereo specific reduction of dihydroflavonols to leucoanthocyanidins (flavan-3,4Floriculture, Ornamental and Plant Biotechnology Volume I ©2006 Global Science Books, UK
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diol). These leucoanthocyanidins are the immediate precursors for the synthesis of anthocyanins. Breeding of transgenic orange Petunia
cultivars by expression of the maize dfr gene and further by traditional crossing-over hybridization has been reported (Oud et al. 1995).
Transgenic tobacco plants carrying cranberry dfr gene produced much darker pink flowers than the controls (Polashock et al. 2002), and
transgenic Petunia plants overexpressing sense dfr construct from Petunia caused production of anthocyanins, resulting in a pink flowers as
compared to wild-type white flowers (Davies et al. 2003). Transgenic carnation plants carrying sense dfr and sense f3′5′h from Petunia produced
violet flowers as compared to the wild-type white flowers (Forkmann and Martens 2001), and transgenic forsythia plants carrying sense dfr and
sense ans produced brownish flowers as compared to wild-type yellow flowers (Forkmann and Martens 2001). In addition, transgenic torenia
plants carrying sense or antisense dfr constructs suppressed the endogenous dfr expression and led to the production of flowers with lighter
color (pale blue) or white color (Aida et al. 2000).
4.5. Anthocyanidin synthase
Anthocyanidin synthase (ANS) catalyzes leucoanthocyanidins into anthocyanidin. It is well demonstrated that ANS, FLS (flavonol synthase) and
F3H are involved in the flavonoid biosynthesis in plants and are all members of the family of 2-oxoglutarate- and ferrous iron-dependent
oxygenases; furthermore, ANS, FLS and F3H are closely related by sequence and catalyze oxidation of the C-ring in the flavonoid. They have
been shown to have overlapping substrate and product selectivities (Turnbull et al. 2004). Nucleotide sequences encoding ans have been
isolated in various plant species (Schijlen et al. 2004). More recently, Kim et al. (2005) reported that the ans gene in yellow onions showed a
point mutation in comparison with red onions. However, application of transgenic ans to pigment modification is less reported.
4.6. Anthocyanidin 3-O-glucosyltransferase (Flavonoid 3-O-glucosyltransferase)
The enzyme UDP-glucose:anthocyanidin 3-O-glucosyltransferase [3GT; this enzyme is also known as UDP-glucose:flavonoid 3-Oglucosyltransferase (UFGT)] transfers the glucose moiety from UDP-glucose to C-3 hydroxyl group of the anthocyanidin, resulting the colored
pigments of anthocyanidin 3-O-glucosides (Fig. 3). This enzyme is not only important for generation of colored pigments, it is also essential for
stabilizing anthocyanidins so that they can accumulate as water soluble pigments in the vacuoles (Schijlen et al. 2004). Anthocyanidin 3-Oglucosides can be further modified with sugars, methyl groups, aliphatic acids and aromatic acids. In addition, there are both species- and
variety-specific differences in the extent of modification and the types of glycosyl and acyl groups attached to the anthocyanidin cores (Tanaka et
al. 2005). It is interesting to find that dfr, ans and 3gt, the late genes in anthocyanin biosynthesis pathway, are co-regulated or may exist as a
functional complex, since mutants with decreased DFR and ANS activities also show decreased 3GT activity (Hrazdina and Jensen 1992).
Overexpression of snapdragon 3GT cDNA in lisianthus plants producing novel anthocyanins and modifying flavonoid glycosylation and acylation
have been reported (Markham 1995, Schwinn et al. 1997).
4.7. Other enzymes
In some species such as snapdragon, cosmos, coreopsis and dahlia, the first key product chalcone can be converted into aureusidin (yellow
color) by aureusidin synthase (AS). DNA sequence analysis revealed that AS belongs to the plant polyphenol oxidase family and is responsible
for flower coloration (Nakayama et al. 2000 2001). By contrast, yellow flowers in carnation are due to the presence of isosalipurposide, an end
product catalyzed by UDP-glucose:tetrahydroxychalcone 2′-O-glucosyltransferase (C2′GT) from chalcone. Other classes of flavonoids, such as
flavones can be converted from naringenin by flavone synthase (FNS) or flavonols can be converted from dihydroflavonols (i.e.,
dihydrokaempferol) by flavonol synthase (FLS; Fig. 3), will be discussed later in the "copigments" sub-section of this chapter.
In addition, some species-specific enzymes involved in flavonoid biosynthetic pathway have been proven successful in pigment modification.
One example is chalcone reductase (CHR), which is not shown in Fig. 3. CHR is an enzyme that co-acts with chalcone synthase (CHS),
catalyzing 1 molecule of p-coumaroyl-CoA and 3 molecules of malonyl-CoA to produce 1 molecule of 4,2′,4′-trihydroxychalcone (isoliquiritigenin;
yellow in color), which is a precursor of 5-deoxy-isoflavonoids. Introducing a chr cDNA from Medicago into two different lines of Petunia, flower
color was changed from either white to pale yellow or deep purple to pale purple (Davies et al. 1998), and introducing a chr gene from Pueraria
montana into tobacco plants, flower color was changed from pink to white-to-pink (Joung et al. 2003).
4.8. Transformation with multiple genes
Since the complexity of the flavonoid biosynthetic pathway, introduction of a single gene into plants may not affect the metabolic flow as well as
flower color. Metabolic engineering of flower color by transforming multiple genes has been attempted and few examples are given as follows
(Forkmann and Martens 2001): transgenic Petunia plants carrying sense dfr and sense f3′5′h altered flower color from white to violet, transgenic
Forsythia plants carrying sense dfr from snapdragon and sense ans from Mattiola altered flower color from yellow to brownish, and transgenic
Dianthus plants carrying sense difF and sense f3′5′h from Petunia altered flower color from deep red with pale pink rim to deep purple with pale
purple rim. To generate potato tubers with increased levels of flavonoids and thus modified antioxidant capacity, transgenic potato plants carrying
single (chs, chi, or dfr), double (chi and dfr) and triple (chs, chi and dfr) gene constructs and in either of two orientations, sense or antisense; it
was found that the most effective in anthocyanin production in potato is the single construct containing sense dfr cDNA (Lukaszewicz et al. 2004).
In addition to chs, we have cloned chi and dfr cDNA from Petunia into the expression vectors pBI121 and pCAMBIA1304, respectively, and
then transformed into tobacco by Agrobacterium-mediated method. After selection by suitable antibiotic, stable homozygous T2 transgenic plants
carrying single copy of transgenes, were then self-crossed or out-crossed to different transgenic lines. By using molecular approach (i.e.,
transgenic technology) coupled with traditional method (i.e., crossing-over), we have generated stable T3 transformants carrying various patterns
with regard to flower color as compared to wild-type tobacco (Fig. 6). We truly believe that this strategy is also useful in other plant species.
5. EFFECTS OF REGULATORY GENES ON FLOWER COLOR MODIFICATION
The final concentrations of anthocyanins/flavonoids in plant cells are not only determined by expression levels of enzymes involved in flavonoid
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biosynthetic pathway, it is now demonstrated very clearly that some regulatory genes are also involved in controlling the transcription level of the
flavonoid biosynthesis genes in some plants examined including maize, snapdragon, Petunia, Arabidopsis and tomato. In general, these
regulatory genes in the flavonoid biosynthesis pathway are specific transcription factors. These DNA binding proteins interact with promoter
regions of the target genes and regulate the initiation rate of mRNA synthesis. These regulatory genes relevant to flavonoid biosynthesis can be
divided into 2 classes: one is MYB transcription factors, another is basic-Helix-Loop-Helix (bHLH) transcription factors (Mol et al. 1998). An
additional third class of WD40 proteins may also be important and universal, although the mechanism is not known. In various plant species, the
tissue-specific expression pattern of the structural genes in the flavonoid biosynthetic pathway is controlled by the combination of regulatory
genes from these two classes of transcription factors. Some transcription factors have been expressed ectopically in various transgenic plant
species such as Petunia, tobacco and tomato, indicating the functional conservation of these regulatory genes among different plant species.
Moreover, the final concentration and class of flavonoids in transgenic plants are determined by the following parameters: the binding affinity of
the transcription factor to the promoter regions of the target genes, the ability to cooperate with endogenous transcription factors, and the
functionality of the endogenous transcription factors (Schijlen et al. 2004).
The most characterized regulatory genes affecting anthocyanin production are the maize leaf colour (Lc) gene which belongs to R gene
family and the maize colorless (C1) gene which belongs to MYB-type transcription factor. The predicted proteins of the R genes (R, Sn, B and
Lc) contain a bHLH motif, and for this reason, maize Lc gene is classified as bHLH-type transcription factors. When the expression vector
containing the constitutive CaMV 35S promoter and the maize Lc gene was transformed into tobacco and Arabidopsis, production of
anthocyanins was increased in floral tissues from both plants (Lloyd et al. 1992). Although the maize MYB-type gene C1 alone had no effect on
anthocyanin production in Arabidopsis, expression of both C1 and Lc genes resulted in tissues that normally did not produce anthocyanins
(Lloyd et al. 1992). Transformation of maize Lc gene into tomato enhanced anthocyanin production in all vegetative tissues that normally did not
produce anthocyanins (Goldsbrough et al. 1996). Similarly, by transforming maize Lc gene into Petunia, it was found that both vegetative and
floral tissues in transgenic plants had increased pigmentation (Bradley et al. 1998). The appearance of purple leaves in transgenic Petunia plants,
due to accumulation of anthocyanins activated by maize Lc regulatory gene, may create a novel ornamental plant of commercial value. However,
same approach of transforming maize Lc gene into rose and carnation, no significant increase or even decrease in anthocyanin production was
detected in these transgenic plants (Tanaka et al. 2005).
6. GENERATION OF VARIEGATED FLOWERS BY USING TRANSPOSONS
The third method of molecular breeding new varieties is mediated by transposable elements. Variegation in either flowers or leaves often attracts
attention from consumers and thus variegated plants can create high value in the ornamental market. Variegated flowers have been observed in
natural populations including Petunia, snapdragon, morning glory, azalea and others, and some of this variegation is caused by transposable
elements. Insertion or excision of transposons in flavonoid biosynthetic genes or regulatory genes produces a mosaic or variegated phenotype
whose pattern is dependent on the frequency and timing. In general, insertion of a flavonoid biosynthetic gene or regulatory gene results in white
sectors of a colored background, and excision of such a transposon from a particular gene often leads to produce colored sectors on a white
background; the sizes of sectors are dependent on the timing of excision: early excision produces large sectors, and late excision produces tiny
sectors (Iida et al. 2004, Tanaka et al. 2005). Variegated flowers have been studied in various plant species, including Petunia and morning glory
(van Houwelingen et al. 1998, Iida et al. 1999 2004).
In higher plants, transposons can be classified into 3 groups: the Ac/Ds superfamily, the En/Spm superfamily, and the Mu family (Iida et al.
2004). In Japanese morning glory, two mutants carrying variegated flowers were caused by integration of En/Spm transposable elements into
the dfr or chi gene; another mutant in the common morning glory bearing variegated flowers was caused by insertion of Tip100, belonging to the
Ac/Ds family, into the chi gene (Iida et al. 1999). Insertions of a transposable element dTdic1, belonging to the Ac/Ds superfamily, in both chi and
dfr genes were found in carnation cultivars bearing variegated flowers (Itoh et al. 2002). To evaluate the potential of using transposons as
molecular tools in producing variegated flowers and to ensure the effectiveness of changing color patterns, a new strategy of employing
transposons and regulatory genes was developed (Liu et al. 2001). In brief, the Arabidopsis transposon Tag1 (3.3 kb) was inserted between the
CaMV 35S promoter and the maize R gene of the plant expression vector pAL69, and the resulting expression vector was transformed into
tobacco via Agrobacterium-mediated method. The transposon Tag1 belongs to the Ac family and is an autonomous element active in
Arabidopsis, tobacco and rice. Once the expression cassette is integrated into host plant genome, the regulatory R gene can be actively
transcribed only when Tag1 is excised, as a result, up-regulating the anthocyanin biosynthetic genes and accumulating pigments can be
observed. Half of the transgenic tobacco plants had observable variegated flower patterns; moreover, each line had a different pattern (Liu et al.
2001). It will be interesting to see whether this system can also be applied to other ornamental plants to produce flowers with commercial value.
7. OTHER FACTORS AFFECTING FLOWER COLORATION
Anthocyanins determine predominantly the pigmentation in flowers; however, the final visible color of a flower is also affected by other factors.
Following is a brief discussion regarding these factors.
7.1. Copigments
Flavonols and flavones are two common copigments. Copigments are often associated with anthocyanins, and thus stabilize the colored
pigments. Most flavones and flavonols are colorless; they appear to provide 'body' to white, cream and ivory-colored flowers. The enzyme
flavonol synthase (FLS) introduces a double bond between position 2 and position 3 of the C-ring in the flavonoid skeleton (Fig. 2), and converts
dihydroflavonols (dihydrokaempferol, dihydroquercetin, dihydromyricetin) into flavonols (kaempferol, quercetin, myricetin, respectively; Fig. 3).
The genes encoding FLS have been cloned from various plants. In several plants such as Petroselinum, Chrysanthenum, Dahlia and Gerbera,
naringenin can also be converted into flavones by the enzyme flavone synthase (FNS) (Fig. 3). Two types of FNS, namely dioxygenase type
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(FNSI) and cytochrome P450 type (FNSII), have been cloned from different species. Flavonols and flavones play an important role in
determining flower color since they can form complexes with anthocyanins. In addition, flavones are proposed to have health-promoting effects
(Forkmann and Martens 2001, Schijlen et al. 2004, Tanaka et al. 2005).
Expression levels of copigments can also be achieved by manipulating FLS and FNS genes (Forkmann and Martens 2001, Schijlen et al.
2004, Tanaka et al. 2005). Since flavonols and flavones share common precursors with anthocyanins, down-regulation of flavonols/flavones
often reduces anthocyanin level. For example, cosuppression of the fnsII gene in transgenic torenia lines showed a decrease in flavones and an
increase in flavanones, and produced paler flower color (Ueyama et al. 2002). However, there are few exceptions. Flowers in one Petunia line
V30 had a deeper red color than the wild-type VR, caused by a reduction of FNS activity in line V30 (Mol et al. 1998). Transgenic lisianthus
plants carrying antisense fls produced deeper red (magenta) flowers than the wild-type plant; furthermore, novel red pigmentation (cyanidin) was
observed at the early stages of flower buds in transgenic lines (Nielsen et al. 2002). More dihydroflavonols but less flavonols were detected in
transgenic flowers, and this novel phenotype was able to be inherited in their progenies.
7.2. Transportation of anthocyanins
All enzymes in the anthocyanin biosynthetic pathway, except for the cytochrome P450 mono-oxygenases (F3′H and F3′5′H), are located in the
cytoplasm; however, accumulation of anthocyanins is in the vacuole (Mol et al. 1998), suggests that a process for anthocyanin transportation is
required during the last step of pigment synthesis. In maize, mutations in the bronze-2 gene, encoding a protein similar to glutathione Stransferase (GST), caused no pigment accumulation in kernels (Marrs et al. 1995). GSTs are the most abundant non-photosynthetic enzymes in
plant cells, and consist of a large gene family encoding approximately 25- to 29-kD proteins. Transditionally, GSTs from plants and other
organisms have been involved in detoxication of herbicides and other toxic compounds; moreover, anthocyanins are the few endogenous
substrates of plant GSTs (Alfenito et al. 1998). In maize and snapdragon, GST carries out the last step of the anthocyanin biosynthetic pathway.
This step is the conjugation of glutathione to anthocyanidin 3-O-glucoside (Fig. 3). The glutathionated anthocyanidin 3-O-glucoside is then
transported to the vacuole via a glutathione pump (GS-X) which locates in the vacuolar membrane (Alfenito et al. 1998, Mol et al. 1998).
However, details of this process are still unclear.
7.3. Vacuolar pH
It is well known that the pH value of the vacuole is acidic (around pH 5.5), and this weakly acidic condition is critical to stabilize anthocyanins.
Any small changes of pH may have visible effects on flower color. In general, decrease in pH causes a reddening, and increase in pH causes a
blueing effect.
How a plant cell regulates the vacuolar pH is not clear, even though some genes influencing pH value have been identified. In Petunia, 7
loci (ph1 to ph7) have been identified, and when mutated, causing blueing of the flower (Mol et al. 1998, van Houwelingen et al. 1998). Other
gene sets encoding regulators of anthocyanin biosynthesis, designed as an1, an2 and an11, may also contribute to control pH (Mol et al. 1998).
Molecular mechanisms controlling pH value within a cell need to be addressed before its application.
7.4. Cell shape
Accumulation of anthocyanin pigments is also affected by the shape of the cells (Mol et al. 1998). For example, epidermal cells in petals of wildtype snapdragon are conical, which confers higher light absorption and as a velvet sheen; a mutant with fainter color was found a flattening of
these epidermal cells. In comparison with other factors, the mechanism controlling the cell shape is unclear, and manipulating the cell shape by
molecular approach is not reported yet.
8. CONCLUSION
So far, the most effective way to engineer flower color is to transform a single gene encoding flavonoid biosynthetic enzyme such as CHS into
host plants. Many of these genes are obtained from model species of Petunia or Arabidopsis. To better understanding the function, expression,
regulation and interaction of the structural genes and regulatory genes and to explore more species-specific genes in flavonoid biosynthetic
pathway in those ornamental plants with high commercial value such as rose, carnation, tulip, chrysanthemum, orchid and so on, we need to
clone these genes from high-valued ornamental plants but not from the model species. Here we would like to propose the concepts of "Flower
EST" and "Flower Array" into the flower research area. The concept of "Flower EST (expressed sequence tags)" is simple: create cDNA libraries
from flower tissues of the target plant, randomly pick up cDNA clones from these libraries, and then perform a single sequencing reaction from a
large number of clones. Each sequencing reaction generates 300-500 nucleotides or so of sequences that represents an unique sequence tag
for a particular transcript (To 2000 2004). Using this straight-forward strategy, structural or regulatory genes in flavonoid biosynthetic pathway
can be identified simply by comparing abundant sequence information in the public databases, and full-length cDNA can also be obtained by
primer-walking, 5′ RACE or 3′ RACE technique. Furthermore, up to 10,000 EST clones or so can be amplified by PCR, each PCR product
represents a unique DNA sequence or gene from the libraries, and then spotted onto a glass slide by an arrayer. This home-made DNA array,
so-called "Flower Array", will be very useful in global comparison of expression patterns between two samples. For example, mRNA isolated
from two cultivars carrying different flower colors and then labeled with different fluorescent dyes. A typical microarray experiment for plant
functional genomics has been described elsewhere (To 2004). We believe that we can identify a lot of known/novel genes responsible for
flavonoid biosynthesis in any particular ornamental plant. The newly discovered genes will be important in understanding the flavonoid
biosynthesis and may be useful in metabolic engineering. The "Flower Array" will be an essential tool in functional studies of the target plant, and
this array may have potential in analyzing pigmentation in other plant species.
Another important strategy to engineer flower color is mediated by regulatory genes in the flavonoid biosynthetic pathway. These identified
regulators (such as MYB, bHLH, W40 proteins) have been involved in multiple processes such as signal transduction, plant growth and
development. However, molecular mechanism and regulation between these regulatory genes and their target genes are poorly understood.
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Phytoene (C40 derivative) (colourless)
Fig. 7 Carotenoid biosynthetic pathway emphasizing from geranylgeranyl diphosphate to carotene by several key enzymes as indicated.
Flower color modification by transforming regulatory genes has been attempted; however, only bHLH-type genes (namely, maize Lc and R
genes) had succeeded (Lloyd et al. 1992, Bradley et al. 1998). Exploration of more regulatory genes, using EST and microarray approaches as
previously discussed, and better understanding of molecular mechanisms in these regulatory genes may help to develop novel strategy for
pigmentation modification. For example, Davuluri et al. (2005) reported recently that suppression of an endogenous photomorphogenesis
regulatory gene (DET1) resulted a significant increase of flavovoid and carotenoid contents in transgenic tomato fruits, it thus provides a novel
example of the use of regulatory gene to increase pigmentation by transgenic approach.
Carotenoids are most common in yellow to orange flowers (e.g., rose, chrysanthemum, gerbera, iris, etc.) and fruits (e.g., tomato, pepper,
etc.). The carotenoid biosynthetic pathway in many plants as well as bacteria has been demonstrated (Bartley and Scolnik 1995, Britton 1998)
and the key important steps are described as follows (Fig. 7): Phytoene (colorless), the first carotenoid, is synthesized from two molecules of
geranylgeranyl diphosphate (GGDP) by phytoene synthase (PSY). Phytoene desaturase (PDS) catalyzes phytoene to ξ-carotene (yellow), which
is converted into lycopene (red) by ξ-carotene desaturase (ZDS) and then further converted into β-carotene (orange) by β-lycopene cyclase (βLYC). Lycopene can also be converted into α-carotene in the presence of ε-lycopene cyclase (ε-LCY) and β-LYC. The important key intermediate
β-carotene is a vitamin A precursor. It is now know that, in plants and algae, two similar but distinct enzymes (PDS and ZDS) are required for the
desaturation from phytoene to lycopene; however, only a single gene crtI encoding CRTI enzyme is required for the desaturation process in
bacteria and fungi (To et al. 1994, Römer et al. 2000). Two functional psy genes were found in tobacco; however, overexpression of either psy
gene resulted in severe phenotypic effects including dwarfism, altered leaf morphology, and generation of white flowers in transformed lines in
comparison with pink flowers of control plant (Busch et al. 2002). In canola (Brassica napus), overexpression of psy gene from bacterium Erwinia
uredovora driven by the seed-specific promoter from napin increased total levels of carotenoids in transgenic seeds (Shewmaker et al. 1999).
Moreover, plasmids containing one (bacterial psy gene), two (bacterial psy gene plus bacterial GGDP synthase or crtI or β-lyc) or three (bacterial
psy and crtI and β-lyc) expression cassettes were transformed into carola, transgenic seeds from all the double and triple constructs showed an
increase in the levels of total carotenoids (Ravanello et al. 2003). In potato, overexpression of PSY from E. uredovora increased total levels of
carotenoids in transgenic potato tubers (Ducreux et al. 2005). In tomato, overexpression of Arabidopsis β-lcy gene driven by tomato pds
promoter resulted a significant increase of β-carotene content and displayed different color phenotypes in transgenic tomato fruits; however, leaf
carotenoid content was unaltered in all transformants (Rosati et al. 2000). Similarly, transgenic tomato lines expressing bacterial psy or crtI
showed increased total carotenoids by two folds (Fraser et al. 2001). The most famous study in engineering of carotenoid biosynthesis is socalled "Golden Rice", which contains two carotenoid biosynthesis transgenes of plant psy from daffodil (Narcissus psendonarcissus) and the
bacterial crtI from E. uredovora, is yellow due to the accumulation of β-carotene (provitamin A) and xanthophylls (Ye et al. 2000). More recently,
a better version of "Golden Rice 2" has been developed by introducing the maize psy gene in combination with E. uredovora crtI gene into rice
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endosperm, and an increase in total carotenoids of up to 23-fold was observed as compared to the original "Golden Rice" (Paine et al. 2005).
However, engineering of carotenoid biosynthesis for changing flower color of ornamental plants has not been reported. To evaluate the possibility
of changing flower pigmentation by carotenoid content, we have cloned several key genes from tomato, and constructed to the plant expression
vector. It will be very interesting to examine whether the color patterns will be different in those transformants carrying single or multiple
transgenes as compared to wild-type control.
The betalain biosynthetic pathway has been recently proposed; interestingly, some early and late steps in the pathway are catalyzed by
identified enzymes, while several intermediate steps are assumed to proceed spontaneously (Strack et al. 2003, Gandía-Herrero et al. 2005b).
Betalains are derived from the amino acid tyrosine. Early reactions are catalyzed by the bifunctional tyrosinase and the dopa
(dihydroxyphenylalanin) 4,5- or 2,3-diooxygenase, and the late reactions are catalyzed by glucosyl-, hydroxycinnamoyl- and malonyltransferase.
The critical steps in betalain biosynthesis, i.e., condensation of the betalain chromophore betalamic acid with cyclo-dopa and amino acids or
amines in the respective aldimine formation of the red-violet betacyanins and the yellow betaxanthins, are most likely to be non-enzymatic
(Strack et al. 2003). Although several key genes encoding tyrosinase, dopa dioxygenase, dopa decarboxylase, glucosyltransferases, and
hydroxycinnamoyltransferases have been cloned and charactered from various plant species such as red beet, pokeberry, daisy, cactus, hairy
bougainvillea, and iceplant (Strack et al. 2003), overexpression of betalains by transgenic approach has not been attempted yet, probably due to
absence of efficient systems for plant regeneration and transformation in those plants of most families of the Caryophyllales. More recently,
successful transformation and related regeneration have been reported in most important ornamental plants including orchids (Dendrobium,
Oncidium and Phalaenopsis), chrysanthemum, carnation, lily, rose and others (Tanaka et al. 2005). We thus believe that new varieties with novel
flower color in some common floricultural crops will be produced in the near future. Once tissue culture and regeneration systems are set up in
some plants with commercial value in the Caryophyllales, we may have chance to engineer betalain production via transgenic approach.
ACKNOWLEDGEMENTS
We are grateful to the Institute of Plant and Microbial Biology, Academia Sinica, for providing greenhouse and transgenic core facilities. This work was financially supported
by grants from the Genomics Research Center of Academia Sinica (grant no. 94F007-4) and the National Science and Technology Program for Agricultural Biotechnology
(grant no. 94S-0203) of the Republic of China to Dr. Kin-Ying To.
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