Access to this full-text is provided by Springer Nature.
Content available from Biological Procedures Online
This content is subject to copyright. Terms and conditions apply.
Rajabietal. Biological Procedures Online (2022) 24:3
https://doi.org/10.1186/s12575-022-00164-0
RESEARCH
Genetic engineering ofnovel yellow color
african violet (Saintpaulia ionantha) produced
byaccumulation ofAureusidin 6-O-glucoside
Amir Rajabi1, Leila Fahmideh2* , Mojtaba Keykhasaber3 and Valiollah Ghasemi Omran4
Abstract
Background: Flower color is one of the main characteristics of ornamental plants. Aurones are light yellow flavo-
noids produced in the petals of a limited number of plant species including snapdragon (Antirrhinum majus). As a
commercially-recognized species, African violet can be found in various colors except yellow. This research, aiming
at changing the petals’ color of African violet from white to yellow, was conducted using the simultaneous expres-
sions of chalcone 4’-O-glucosyltransferase (4’CGT ) and aureusidin synthase (AS1) genes without the need for silencing
anthocyanin biosynthesis pathway genes via both transient and stable transfer methods.
Results: The transient gene transfer among transgenic plants led to a clear change of petals’ color from white to light
yellow. This occurs while no change was observed in non-transgenic (Wild type) petals. In total, 15 positive transgenic
plants, produced via stable gene transfer, were detected. Moreover, since their flower color was yellow, both genes
were present. Meanwhile, the corresponding transformation yield was determined 20-30%. The transformation,
expression and integration of genes among T0 transgenic plants were verified using the PCR, qRT-PCR and Southern
blotting techniques, respectively. Furthermore, the probable color change of petals’ cross-section and existence of
Aureusidin 6-O-glucoside (AOG) compound were determined using a light microscope and HPLC-DAD-MSn analysis,
correspondingly.
Conclusions: Generally, the creation of aurones biosynthesis pathway is only viable through the simultaneous
expression of genes which leads to color change of African violet’s petal from white to yellow. This conclusion can
lead to an effective strategy to produce yellow color in ornamental plant species.
Keywords: Anthocyanin, Flower color, Genetic engineering, Ornamental flowers, Pigment biosynthetic pathway
© The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which
permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the
original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or
other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line
to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory
regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this
licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecom-
mons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.
Background
Production of new ornamental cultivars with different
colors of flowers is one of the main goals in floriculture
[1]. Variability in flower color can increase the commer-
cial value and marketing of ornamental plants [2]. e
global use of ornamental plants is more than $300 bil-
lion and the area under cultivation in 2018 is estimated
2,600,000 and 4200ha worldwide and Iran respectively
[2, 3]. Pigments in higher plants are generally grouped
in three major classes: flavonoids, carotenoids, and beta-
lains [4]. Anthocyanins are a class of flavonoids respon-
sible for a range of colors: from orange to red, violet and
blue. Similarly, carotenoids and betalains mainly yield
yellow or red colors. Flavonoids such as chalcone and fla-
vone are pale-yellow and often invisible with human eye
[4].
Aurone is a class of rare flavonoids with brighter yel-
low present flowers in a limited number of species, such
as A. majus, Cosmos bipinnatus (garden cosmos), and
Open Access
*Correspondence: l.fahmideh@gau.ac.ir
2 Department of Plant Breeding and Biotechnology, Gorgan University
of Agricultural Sciences and Natural Resources, Gorgan, Iran
Full list of author information is available at the end of the article
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
Page 2 of 16
Rajabietal. Biological Procedures Online (2022) 24:3
Limonium (sea-lavender) [4–6]. Figure1 shows flavonoids
and a simple diagram of their biosynthetic pathways in A.
majus. Biosynthesis of Aureusidin 6-O-glucoside (yel-
low pigment) and associated enzymes (AS1 and 4’CGT )
have been well characterized in this plant [5]. 2’,4’,6’,4 tet-
rahydroxychalcone (THC) is the first compound in this
pathway, which is the catalytic product of the key enzyme
chalcone synthase (CHS). THC is glucosylated at its
4’-hydroxyl group in the cytoplasm, before transfer to the
vacuole, and later converted to aureusidin 6-O-glucoside
via the catalysis of AS1 [5]. THC can later be converted
to aurones, flavanones, and other classes of flavonoids,
including anthocyanins.
African violet (Saintpaulia ionantha H. Wendl.; Gesne-
riaceae) is a commercial ornamental plant that can be
easily propagated [7]. African violet has more than 22
species and 2000 cultivars with diverse petal color, floral
shape, and color range of white, red, purple, and pink [8].
e Saintpaulia ‘Jolly Diamond’ cultivar has white petals;
the number of petals is much more than its flowers and
the petals hang slightly as they grow inwards [9]. Despite
the variety of colors available among African violets,
however, yellow has not yet been observed in this species
[5, 7].
Transformation technology has potential to produce
novel flower phenotypes that are not available in nature
[1]. Genetic engineering can be used to create novel
flower colors in a variety of ways: (a) by introducing new
genes that encode enzymes to create new biosynthetic
pathways that do not exist, (b) by directing biosynthetic
pathways through up-regulation of existing genes, or (c)
by suppressing biosynthetic pathways through down-reg-
ulation of genes [10]. However, genetic transformation is
a valuable tool that can be exploited in plant functional
genomics, gene discovery, and gene characterization [11].
Transient gene transformation (agroinfiltration system)
using Agrobacterium-mediated Transformation (ATMT)
in different plant tissues allows for rapid and scalable
development of functional genomics assays [12–15]. is
method is an efficient tool aimed at studying the func-
tion of the gene construct and tests it before stable trans-
fer [16]. Stable transfer, on the other hand, is a lengthy
process that often requires tissue culture techniques for
full plant growth from transformed cells or tissues. In
Fig. 1 Simplified diagram of biosynthetic pathways flavonoids in A. majus. The 4’-O-glucosylation of chalcone by cytosolic 4’CGT followed by
oxidative cyclization by vacuolar AS is the biochemical basis of aurone 6-O-glucoside biosynthesis in vivo. Chalcone is the branching point of
aurone pathway from the flavone/anthocyanin pathway. CHS, chalcone synthase; CHI, chalcone isomerase; F3H, flavanone 3-hydroxylase; DFR,
dihydroflavonol 4-reductase; ANS; anthocyanidin synthase; 3GT, anthocyanin 3-O-glucosyltransferase; FNS, flavone synthase; AS, aureusidin synthase;
4’CGT , chalcone 4’-O-glucosyltransferase; THC, tetrahydroxychalcone; PHC, pentahydroxychalcone. Red arrows, the aurone biosynthetic pathway in
vivo reported in this study
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
Page 3 of 16
Rajabietal. Biological Procedures Online (2022) 24:3
this method, T-DNA is artificially integrated into the
genome of host cell using ATMT; therefore, it subse-
quently passed on to the next generation [17, 18]. Trans-
genic yellow flowers of Torenia hybrida are produced by
co-expression of the 4’CGT and AS1 genes along with
RNAi-mediated silencing of flavanone 3-hydroxylase
(F3H) or dihydroflavonol 4-reductase (DFR) genes [5]. In
transformed Ipomoea nil petals, the co-expression of the
4’CGT and AS1 genes induced expression of AOG and
intensified the pale yellow color of the primary petals to
visible yellow [19]. Genetically engineered production
of yellow petals in important ornamental plants such as
Geranium, Lathyrus odoratus, Cyclamen and Saintpaulia
has not been reported [7, 19].
In spite of the presence of various colors among Afri-
can violet ornamental plant cultivars, it lacks the yellow
cultivar. erefore, due to the popularity of the African
violet ornamental plant, the production of this plant with
a new yellow color is very valuable commercially and is of
considerable importance in the market. us, the present
study aimed to change the color of African violet flowers
from white to new yellow color through genetic manipu-
lation (using both transient and stable transformation)
and the simultaneous expression of 4’CGT and AS1 genes
involved in aurone biosynthetic pathway without any
necessity for silencing of anthocyanin biosynthesis genes
(CHI, F3H and DFR).
Results
Transient expression of4’CGT andAS1 genes inAfrican
violet petals
e function of 4’CGT and AS1 in the aurone biosyn-
thesis was verified using the agroinfiltration method
(Fig. 2b, c). ree days after infiltration, the color of
the petals has changed from white to yellow (pale yel-
low), while no change was observed in the control group
and the petals were injected with agroinfiltration solu-
tion without gene structure. Successful infiltration was
later confirmed by light microscope. e results clearly
showed the color change to yellow in surrounding parts
injected with needle (Fig. 2b). Additionally, the pres-
ence both of 4’CGT +AS1 genes in infiltrated petals was
confirmed by PCR analysis and these two genes were
not detected in control plants (non-injected plant and
injected plant without gene construct) (Fig. 2c). is
experiment was repeated twice and similar results were
obtained (data not shown).
Transgenic Plants ofAfrican violet bysimultaneous
expression 4’CGT andAS1
To produce AOG, we inserted 4’CGT and AS1 genes into
leaves through ATMT. Two binary vectors: pBI121 to
express the 4’CGT gene and pCAMBIA1304 to express
the AS1 gene were constructed. A few weeks after the
transfermation of gene constructs, small white calli
with green buds began to grow on some of the inocu-
lated petiole explants. Totally, 15 putative independ-
ent transgenic plants were obtained from a total of 60
transformed explants (Table 1). 20–30% regeneration
efficiency was achieved which enabled us to obtain 53
plantlets from 15 regenerated samples (Table1; Fig.3D).
Regenerated plantlets were screened on Hygromycin
and Kanamycin selection (Fig.3E). Availability of Hygro-
mycin and Kanamycin-resistant gene have successfully
confirmed using PCR in putative transgenic plants. All
putative transgenic plants acclimated and transferred to
greenhouse until maturity (about 16 weeks). In trans-
formants with white-yellow flowers, the area of yellow
sectors increased in the white background of the petals
during the late phase of flowering. With time, the color
of most of the petals turned to yellow in all the late-born
flowers of transgenic plants. In contrast, no change was
observed in the petals of non-transgenic plants. No mor-
phological difference other than the color change was
observed between non-transgenic and transgenic plants
(Fig.4a-f).
Expression of4’CGT andAS1 intransgenic African violet
Flowers
We confirmed the expression of the transgenes with
PCR. e size of PCR products was 1374 and 1689bp for
4’CGT and AS1 genes, respectively (simultaneous expres-
sion 4’CGT and AS1 in T0 transgenic plants), identical
with the positive control. ese genes were not detected
in non-transgenic plants (Fig.5).
Two samples from PCR positive transgenic plants
(Fig.5) were selected for further confirmation by South-
ern blot assay. For blotting, the researchers used specific
1374 and 1689bp probes labeled with digoxin molecules.
A Single signal was detected for each gene in the blotting
of two transgenic African violet genomes, which were
positive in PCR analysis, whereas no hybridization sig-
nal was observed in the non-transformed plant samples
(African violet negative control). e lengths of hybrid-
ized fragments were 4000 and 6000 bp for 4’CGT and
AS1 genes, respectively (Fig.6).
Evaluation oftheexpression pattern of4’CGT andAS1
genes intransgenic petals
e relative expression levels of 4’CGT and AS1 genes in
transgenic plants with high expression levels were fur-
ther confirmed by qRT-PCR. e amplification efficiency
of genes ranged between 98.71% and 99.83% and cor-
relation coefficients varied from 0.977 (4’CGT ) to 0.996
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
Page 4 of 16
Rajabietal. Biological Procedures Online (2022) 24:3
(AS1) (Supplementary Table S1). ese results indicated
the expression of both genes studied in transgenic pet-
als as compared to Non-transgenic petals. e results
showed that the expression level of the studied genes was
not naturally present in African violet white (Non-trans-
genic plant), while in transgenic African violets (yellow),
it expressed significantly and was to some extent equal to
A. majus (yellow) (Fig.7).
Microscopic analysis oftransgenic petals
To further study the expression of 4’CGT +AS1 genes in
petals of transgenic plants, transverse sections of petals
were examined by microscopic analysis (Fig.8). In wild
type, pale-yellow pigments (Chalcones) were localized in
the adaxial epidermis (L1 layer) and the underlying mes-
ophyll (L2 layer) appeared to be white. In contrast, both
L1 and L2 layers were yellow in petals of transgenic plant
(Fig.8). It generally represents the merger of both genes
together in yellow transgenic African violet.
Aureusidin 6‑O glucoside accumulation inthe4’CGT
andAS1 expressing transgenic petals
Flavonoids were extracted from petals of not-trans-
formed African violet, transformed African violet, and A.
majus. ese metabolites were then analyzed by HPLC.
e HPLC chromatogram demonstrated that the yellow
flowers of A. majus contained a compound that was not
present in non-transgenic African violet (Fig. 9e) but
was observed in transgenic African violet petals (Fig.9f).
Retention times (RT) in transgenic African violet and
A. majus flowers were identical, thereby confirming the
presence of AOG (Fig.9a; Table2). e amount of AOG
component (Peak 1ʹ, RT 3.9min) in flowers of the trans-
genic African violet and A. majus was 2.95 and 3.45mg/g,
respectively (Table2). HPLC chromatogram showed that
the components were properly separated. MS⁄MS analysis
of peak 1ʹ, which exhibited an [M + H]- ion at m⁄z 449,
yielded MS2 fragmentation at m⁄z 287 due to the loss
of 162 atomic mass units (amu), corresponding to one
Fig. 2 Restriction maps of transformation vectors and the results of Agrobacterium infiltration in African violet petals. a Binary vectors. The cloning
vector pBI121 and pCAMBIA1304 containing the CaMV 35 S promoter driving the 4’CGT and AS1 genes respectively. b Agroinfiltration steps in
African violet petals (1): The injection of Agrobacterium infiltration solution in the base of the petal by syringe without a needle, blue arrows without
injection, a green arrow without gene construct and orange arrow with gene construct 4’CGT+AS1; (2) a color change from white to yellow in petal
influenced by gene construct 4’CGT+AS1; (3, 4): Microscopes in the petal color injected samples with a gene construct and without construct; Light
microscopes in the sample with a gene construct (5) and without a gene construct (6); (7): Shown are petal color on the adaxial side of sample
injection with a gene construct; (8): Cross-sections arrow show that fluorescence is restricted to the pigmented adaxial epidermis of gene construct
(Scalebar:100). c The results of extraction DNA and PCR detection of agroinfiltration petals; M: GeneRuler DNA Ladder Mix; CK-: Non-transgenic
plants; CK+: cDNA of A. majus with mixture two primers AS1 and 4’CGT ; (1): without injection; (2): injection without gene construct; (3): injection
with gene constructs 4’CGT+AS1
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
Page 5 of 16
Rajabietal. Biological Procedures Online (2022) 24:3
Table 1 Analysis of plant transformation
No.
Experiment Total Number of
regenerations Percentage of
regeneration Number of
meristematic
stems or plantlets
The average
length of
regenerated stems
Phenotype Remarks
Transgenic African
violet experiment 1 20 5 25% 17 5/4 cm White-yellow No seed
Transgenic African
violet experiment 2 20 4 20% 15 7/5 cm yellow No seed
Transgenic African
violet experiment 3 20 6 30% 21 7/0 cm yellow No seed
NT ( Wild type) 20 0 0 0 0 - -
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
Page 6 of 16
Rajabietal. Biological Procedures Online (2022) 24:3
glucose moiety (Fig.9g). Peak 1 is unlikely due to the sat-
uration of peak 1ʹ as the two peaks are well separated and
not tailing. In A. majus, a trace amount of molecular ion
m⁄z 465 was revealed to be co-eluted with broad peak 1ʹ,
which was fragmented at m⁄z 287 (data not shown) and
tentatively identified as bracteatin-6-O-glucoside. Addi-
tionally, the mass spectra and UV⁄V are features of the
peaks 1 and 1ʹ, a compound identified in A. majus and
transgenic African violet (Fig.9d, f) but not in the wild
type (Fig. 9e). ese features possibly correspond to a
structural isomer of AOG.
Discussion
Genetic engineering supports the idea of changing as
well as creating new colors in ornamental plants. is
research has been conducted with the purpose of chang-
ing flower colors and creating new ones in African violet
species. Flower and ornamental plants industry has been
trying to develop new cultivars with specific characteris-
tics such as new colors [20]. e development of orna-
mental cultivars with new flower colors, considering the
major purpose of flower and ornamental plants industry,
will result in increased economic value [21]. Ornamen-
tal cultivars of Petunia, chrysanthemums, Rosa hybrid,
Ipomoea nil, Rosa rugose and Nierembergia species
with respective colors of light pink, blue-violet, purple,
light yellow, yellow and light violet have been produced
using the simultaneous expression or overexpression of
the corresponding genes [19, 22–25]. e anthocyanins
were the principal pigments leading to the development
of new colors including red, pink and blue. is is while
aurone compound has been used to develop yellow-
colored flowers in recent studies [19, 26].
Although African violet can be found in various colors,
no color change has been reported through genetic engi-
neering of this precious species. In this investigation, the
white-colored petals of S. ‘Jolly Diamond’ cultivar have
been used. erefore, no CHI is available to turn chal-
cone into AOG. Chalcone, considered as a key enzyme
in flavonoid biosynthesis of flowering plants including
aurones, is turned into colorless naringenin and plays
an important role in color of ornamental flowers [2, 21].
In most plant species, chalcone is not the final prod-
uct. Combined with other enzymes, it turns into other
classes of flavonoids such as aurones, flavonones, dehy-
droflavonols and finally anthocyanins as a major class
of colors [27]. In a molecular analysis investigation, two
separate genes of chalcone synthesis (i.e., SaCHSA and
SaCHSD) were detected in African violet [28]. It should
be noted that the identification of chalcone in this orna-
mental plant species is highly significant to conduct this
research.
For the first time, the simultaneous transfer of AS1
and 4’CGT genes was successfully conducted in African
violet using Agrobacterium tumefaciens strain LBA4404
with respective plasmids of pCAMBIA1304 and pBI121.
Fig. 3 Generation process of transgenic plants African violet. a Plantlet regeneration in MS basal medium with 1 mg.L−1 6-benzylaminopurine
(BA) and 1 mg.L−1 indole-3-butyric acid (IBA). b Control of African violet leaves. c Control of African violet leaves without gene constructs. d
Agrobacterium-mediated genetic transformation of African violet leaves with gene constructs. e Screening of Agrobacterium-mediated genetic
transformation of leaves. f Induction of screened transforms. g Generation of transgenic seedlings. h Outdoor transplantation
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
Page 7 of 16
Rajabietal. Biological Procedures Online (2022) 24:3
In an investigation carried out on African violet by [29],
two strains including A218 and EHA105 were employed.
e results indicated that only strain A281 led to more
efficient and stable transfer of traits in leafstalk micro-
samples while strain EHA105 caused no regeneration.
In another research by [30], Agrobacterium tumefaciens
strains LBA4404 and EHA101 were used in Pink Veil
cultivar of the African violet. It was found that strain
LBA4404 (pTOK233) had better performance. Accord-
ing to this research and the investigation conducted by
Kushikawa et al. [30], LBA4404 can be considered an
appropriate strain in transfer of genes to African violet.
e transient gene transfer enables investigating the
genetic construct. Besides, the flower color change can
be evaluated without the need for tissue culture. e
transgenic plants can also be selected at lower cost
and time if this technique is applied [12, 31, 32]. is
method, showing high transfer yield in a wide range
of plant species, has been employed to study the Afri-
can violet petals. To this end, agrobacterium contain-
ing gene construct has been injected into the base of
petals. ree days later, the phenotypic evaluation of
color change of petals as well as the existence of 4’CGT
and AS1 genes in the transgenic petals were verified
using light microscope and PCR test. According to the
results, the S. ‘Jolly Diamond’ cultivar, having white
petals, is suitable for genetic modification and transfer
of genes involved in the aurones biosynthesis pathway
with the purpose of changing the flower color to yellow.
Other researchers have also used the transient gene
transfer method. Nazari etal [33] used this method to
transfer monogenic (Viola-F3ʹ5ʹH gene) and constructs
(Viola-F3ʹ5ʹH gene and Iris-DFR gene). After injection
of gene constructs, they conducted to produce delphin-
ine anthocyanin in petals of Gerbera flower (up to 44
and 75%, correspondingly), resulted in color change
of petals into blue. In a similar research, the trans-
fer of gene construct containing F3ʹ5ʹH and DFR gene
from respective Petunia hybrida and Iris to the pet-
als of Gerbera jamesonii caused a change in the level
of anthocyanins among the injected petals and turned
the stigma and pollen into blue [34]. Shang etal [35]
Fig. 4 Alteration of flower color in transgenic African violet plants carrying 4’CGT+AS1 transgene. a Non-transgenic plant. b At the beginning of
flowering, only a few flowers showed yellow regions on white petals. c All the flowers at the late-flowering stage in transgenic plants looked yellow.
d Non-transgenic plant (Wild-type) petals. e to f Flower phenotypes from a spot of yellow color in the white background to complete yellow color
observed in the same transgenic plant. The arrows showed a color change from white to yellow in different parts of 4’CGT+AS1 transgenic petals
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
Page 8 of 16
Rajabietal. Biological Procedures Online (2022) 24:3
Fig. 5 Results of PCR detection of wild-type and transgenic plants. a Extracted solutions from flowers of wild-type and transgenic petals. b W T:
wild-type. No. 1, 2: Transgenic plants
Fig. 6 Results of southern blot hybridization of T0 generation transgenic plants. M: 1 kb ladder, Fermentas: P: pBI121-4’CGT and pCAMBIA304-AS1
vectors, WT: Non-transgenic plants, 1, 2: T0 generation transgenic plants
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
Page 9 of 16
Rajabietal. Biological Procedures Online (2022) 24:3
investigated the effect of RNAi agroinflitration of chal-
cone synthase gene on the A. majus petals. By silencing
this gene, no pigments were found in A. majus pet-
als and their color changed from purple to white. e
silence of chalcone isomerase gene involved in the bio-
synthesis pathway of Petunia hybrida’s pigments pro-
duction led to the accumualation of anthocyanin while
a decrease was observed in the endogenous mRNAs of
the respective targets in the petals’ infiltrated zones of
four colors of this plant species [36]. e production of
transgenic plants is an expensive and time-consuming
process. According to the literature, transient transfer
is a fast, simple and economic method to test and acti-
vate constructs prior to their mass production as gene
constructs are most likely transferred following their
verification.
In the stable gene transfer method, a certain part of
DNA containing a new gene or a combination of mul-
tiple genes is artificially inserted into an organism’s
genome using laboratory methods [37]. Due to the
complex nature of flavonoid biosynthesis pathway, the
mere transfer of a gene to the plants might not have any
specific effects on the biosynthesis pathways of flower’s
pigments. erefore, the genetic engineering of flower
color has been investigated via transferring of multiple
genes [38]. In such research, the white-colored petals
of African violet were turned into yellow using two vec-
tors (pBI121 and pCAMBIA1304 vectors for 4’CGT and
AS1 gene expressions, respectively) without any need for
silencing anthocyanin biosynthesis pathway genes (CHI,
DFR and F3H). e results of this research are similar to
those of To and Wang [26]. In that research, the CHI and
DFR genes corresponding to Petunia’ cDNA pattern were
cloned in pBI121 and pCAMBIA1304 carriers, respec-
tively. By transferring them to tobacco via agrobacterium,
different color patterns were developed compared to
non-transgenic tobacco.
For the first time, the relative expression of both genes
transferred to the petals was observed and verified in
this research. While no analyses (qRT-PCR, South-
ern blotting and light microscope) were carried out to
verify the results observed in previous investigations,
the above-mentioned analyses have been conducted
to demonstrate the transfer and integration of genes
Fig. 7 qRT-PCR confirmation of 4’CGT and AS1 transgenic plants. Relative expression levels/Actin of 4’CGT and AS1 genes in African violet white
(Non-transgenic plant), Transgenic African violet (Yellow), and A. majus (Yellow). plants were investigated by qRT-PCR. Relative expression levels
were normalized to a value of 1
Fig. 8 Light microscope observation of transverse section through petals of Transgenic African violet and wild type. Bars= 100 μm
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
Page 10 of 16
Rajabietal. Biological Procedures Online (2022) 24:3
Fig. 9 Aurone formation in African violet transgenic. a Flower of untransformed A. majus. b untransformed African violet. c transgenic African violet.
d HPLC chromatogram of A. majus flowers. e untransformed African violet. f transgenic African violet. showing a eluting as peaks 1 and 1ʹ at 400 nm.
Mass spectra and MS-MS fragmentations of m⁄z 449 of AOG from A. majus. g mAU, milliabsorbance units
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
Page 11 of 16
Rajabietal. Biological Procedures Online (2022) 24:3
in genetically-modified plants. According to the qRT-
PCR analysis, 4’CGT and AS1 genes were successfully
expressed in the African violet petals. is is while non-
transgenic African violet plants lack these two genes. To
verify the integration process of the transferred genes
to the genome of genetically-modified plants as well as
determining the number of gene transcripts, the South-
ern blotting test was applied. Findings showed that the
genetically-modified plants possessed one or two tran-
scripts of the transferred genes. By investigating the
genetically-modified plants having 4’CGT and AS1 genes
sequencing, the band lengths were determined 4500 and
6000bp, respectively. In a recent research, HPLC analysis
was used to verify and quantify these genes in the trans-
genic Ipomoea nil ornamental plant [19].
In another investigation, the 4’CGT and AS1 genes
were separately transferred from A. majus to the blue-
colored petals of Petunia. According to the results, 4
out of 9 transgenic SRY4’CGT plants developed blue-
white flowers. Moreover, the proportion of white-
colored sectors gradually increased until the petal color
turned completely white. In contrast, 3 out of 13 trans-
genic SRYAS1 developed blue-white sectors. However,
the amount of transgenic flowers did not increase and
no complete white flowers were observed. Besides, the
chalcone synthase significantly increased in the blue-
colored sector while a decrease was observed in the
white-colored Sect.[39]. In the blue-colored petals of
Torenia hybrida ornamental plant, expression of these
genes along with silencing DFR or F3H gene led to the
development of yellow color. In an investigation by the
RNAi technique, no change was reported in the appear-
ance of flower [5]. Recently, the transfer of these genes
to the petals of Ipomoea nil ornamental plant changed
the color of flower from light yellow to yellow. However,
most transgenic plants contained light brown-colored
unbloomed flowers which might have consisted of
necrotic cells [19]. In contrast, this research led to the
production of 15 yellow-colored transgenic plants out
of 60 explants transferred by ATMT. Meanwhile, there
was no need for gene silencing (RNAi) and no other
morphological difference was observed between trans-
genic and non-transgenic plants.
e flowers of yellow-colored A. majus cultivar pro-
duce a considerable amount of AOG and bractea-
tin 6-O-glucoside along with smaller quantities of the
4’-glucosides of THC and PHC [40, 41]. According to
the HPLC analysis conducted on transgenic Torenia
hybrida ornamental plant, the AOG compound was pro-
duced in the transgenic plants (0.422mg/g) while there
was no trace of such compound in non-transgenic [5].
e enzymatic formation of aurones observed in the
yellow-colored A. majus flowers extracts indicated that
the main pigments pathway to produce AOG was open
followed by reduction in PHC-glucoside, bractatin-6 glu-
coside, THC-4 glucoside, respectively. In this research,
the HPLC-DAD-MS chromatogram revealed the exist-
ence of AOG compound in the transgenic African violet
plants containing yellow-colored petals. is is while no
such compound is produced in the white-colored petals
of non-transgenic African violet plants naturally.
Conclusions
For the first time, the genetic engineering of aurone pig-
ment biosynthesis pathway led to the production of yel-
low color in the white-colored African violet petals. is
method was employed by simultaneous expression of
4’CGT and AS1 genes. Furthermore, agroinfiltration sys-
tem was highly effective to evaluate the performance of
genes and color of flowers. In this research, the S. ‘Jolly
Diamond’ cultivar containing white-colored petals was
used. e simultaneous expression of 4’CGT and AS1
Table 2 Flavonoid analysis of transgenic and non-transgenic flowers
UD, undetectable
Flavonoids in all the transgenic lines and nontransformants were identied by HPLC, and the data were summarized. Tentative peak identication: Peaks 1 and 1′, AOG;
Peak 2,3,4, Anthocyanidins: Peak 5,6,7, Flavones (NC: naringenin chalcone and derivatives(
Flavonoid content, mg/g fresh petal weight
(No) Genotype Aurone
at 400
nm
Anthocyanidins at
520 nm Flavones at 360
nm
(NC)
Phenotype
Peak 1 (2.5
min) Peak 1′(3.9
min)
NT S. Jolly Diamond UD UD UD 0.986 White
Transgenic African
violet 4′CGT +AS1 0.528 2.95 UD 1.23 Yellow
A. majus cv. Snap Yellow (Endogenous
Am4′CGT and AmAS1)0.632 3.45 UD 1.5 Yellow
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
Page 12 of 16
Rajabietal. Biological Procedures Online (2022) 24:3
genes led to the formation of new yellow color. is is
while the existence such phenotype was not reported in
the African violet. e results of this investigation indi-
cated that the simultaneous expression of these genes in
the white-colored petals, containing chalcone, contrib-
uted to the accumulation of AOG as the final compound
of aurones. As the African violet petals produce chalcone,
the existence of malonyltransferase caused accumula-
tion of aurones. e phenotypic and molecular evaluation
of integration indicated that these genes, detected in the
genome of transgenic petals, were not in a single locus.
is observation also demonstrated the production of
transgenics by clonal reproduction. Furthermore, yellow
pigments and accumulation of AOG were observed in the
petals of 4’CGT/AS1 transgenic plants while no change
was detected in the petals of wild type. erefore, transfer-
ring these genes to other ornamental plants can lead to the
production of yellow-colored petals provided that white-
colored petals contain chalcone to produce AOG inside the
vacuoles. Moreover, the simultaneous expression of 4’CGT
and AS1 was conducted without the need for silencing
genes to reduce the anthocyanin biosynthesis adjustment.
us, the mere application of these genes is applicable
to change the petals’ color from white to yellow. Besides,
this method can be considered an appropriate strategy to
change the color of white petals as well as producing new
color in the flowers of ornamental plant species.
Materials andmethods
Vectors Construction
To clone the full-length cDNA of 4’CGT and AS1 genes,
RNA from yellow petals of A. majus was isolated by TRI-
zol protocol as previously described [42]. Specific primer
sets P1 and P2 (Supplementary Table S2) were used to
amplify full-length reading frames of 4’CGT (1374 bp)
and AS1 (1689bp), respectively. PCR products were then
analyzed by 1% agarose gel electrophoresis. After purifi-
cation, PCR products were cloned into Pet28a Easy vec-
tor (Promega) and then sequenced by an automatic DNA
sequencer (Sanger Sequencing method, ABI 3730 XL).
e full-length cDNA of the 4’CGT gene was cloned into
the pBI121 binary vector using SacI and BamHI restric-
tion sites. AS1 gene was cloned into pCAMBIA1304
vector by NcoI and BstEII restriction sites, respectively
(Supplementary Table S2; Fig.2a). All these vectors were
verified by restriction enzyme cutting and sequencing.
Agrobacterium‑Mediated Transformation
Agrobacterium tumefaciens (strain LBA4404) provided
from of the Institute ABRII (Agricultural Biotechnology
Research Institute of Iran) was used to carry recombi-
nant binary vectors and Agrobacterium-mediated plant
transformation assay. For transformation, the research-
ers used the electroporation (BTX™ ECM™ 630, USA) at
25 µF, 400 Ω, and 1.25kV. 1 ml of LB medium was then
added immediately into the mixture, incubated at 28˚C
for 3 h. 100 µl of the following bacterial mixture was
transferred onto selective LB medium (containing 50mg.
L−1 Kanamycin and 100mg.L−1 Rifampin) as described
in the manufacture’s protocol.
Plant materials andgrowth conditions
African violet (S. Jolly Diamond), was used in the present
study. All Saintpaulia genotypes were provided from the
genotype collection of the Institute GABIT (Genetics
and Agricultural Biotechnology Institute of Tabarestan)
and the plant material was maintained in greenhouses
under controlled conditions. For plant transformation,
the leaves were sterilized with 1% sodium hypochlorite
for 20 min, washed thoroughly with sterile water, and
cultured to germinate on MS basal medium (pH 5.7) con-
taining MS salts [43] 2% sucrose, 0.8% bacto-agar, 1mg.
L−1 6-benzyl amino purine (BA), and 1 mg.L−1 indole-
3-butyric acid (IBA). e cultures were then incubated in
a growth chamber under the condition of 25±1ºC, a 16-h
florescent light (100 µmol/m2/sec), and a 8-h dark cycle.
8 weeks later, young leaves were collected and used for
the transformation test.
Transient expression viainltration ofpetal
African violet flowers were infiltrated with Agrobac-
terium tumefactions strain LBA4404 harboring both
pBI121 and pCAMBIA1304. For this, the LBA4404 cells
were cultured in 10 ml LB broth with antibiotics over-
night, pelleted and re-suspended in a medium #1003 (AB
media salts + NaH2PO4 240mg.L−1 + glucose 10g/l +
MES 14.693g/l) supplemented with 100 µM acetosyrin-
gone and cultured for 4h [35]. e cells were then pel-
leted and re-suspended to a concentration of A600 = 0.5
in 1% (w/v) glucose solution (PH 5.3) supplemented with
100 µM acetosyringone [35]. Flower buds or opened
flowers were pierced with a needle and infiltrated with
the Agrobacterium culture using a syringe. ree days
after injection and subsequent change in specimen’s con-
ditions, the injected petals (both with and without gene
construct) were separated from non-injected ones. After
DNA extraction, the existence of two genetic parts was
examined using PCR test. To further investigate the color
change of petals, the cross section of the injected petals
(with and without gene construct) was evaluated using a
light microscope.
Stable transformation
A single colony of plasmid-carrying Agrobacterium was
picked up and incubated in 10 ml LB medium containing
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
Page 13 of 16
Rajabietal. Biological Procedures Online (2022) 24:3
50 mg.L−1 Kanamycin (Km) and 100 mg.L−1 Rifampin
(Rif) at 28˚C overnight. Bacteria cells (OD 600 - 0.5 - 0.6)
were then washed and resuspended in AB medium (5g/l
glucose; 1g/l NH4Cl; 0.3g/l MgSO4∙7H2O; 0.15g/l KCl;
10mg.L−1 CaCl2; 2.5mg.L−1 FeSO4∙7H2O; 3g/l K2HPO4;
1.15g/l NaH2PO4∙H2O) without antibiotics. e cultures
were incubated at 28°C for 6h to reach mid-log phase,
followed by the addition of 100µl of acetosyringone (AS;
SigmaAldrich, St. Louis, MO, USA) and further incuba-
tion at 28°C for 4h. e bacterial suspension was then
centrifuged at 3000rpm for 10 min, and the pellet was
dissolved in MS medium (MS salts; 0.9mg.L−1 thiamine;
1mg.L−1 BA; 1mg.L−1 IBA; 200mg.L−1 KH2PO4; pH
5.6) supplemented with 100 µM acetosyringone and 5%
glucose. e suspension was then diluted to final OD600
0.6-0.8. One day before the infection, leaves were excised
from 4- and 6-week-old in vitro grown African violet and
incubated on MS solid medium supplemented with 100
µM acetosyringone. Infection was carried out by adding
15 ml of diluted Agrobacterium suspension to the pre-
cultured explants for 1h. Excess Agrobacterium explants
were blotted on sterile filter papers to remove excess liq-
uid. e infected explants were transferred on MS solid
medium containing 100 µM acetosyringone and 5% glu-
cose, and left to grow for 3 days in a dark condition at
28˚C. en they were washed 2-3 times with shaking
(the first shaking at 220rpm and subsequent shakings at
100rpm; 30min each time) in MS liquid medium with
500mg.L−1 cefotaxime. Explants were dried with sterile
filter papers and transferred to MS medium (MS salts;
Nitsch vitamins; 1% sucrose; 1mg.L−1 BA; 1mg.L−1 IBA
0.7% bacto-agar; pH 5.8) with 250mg.L−1 cefotaxime (for
inhibition of Agrobacterium growth), 50 mg.L−1 Kana-
mycin (for selection of pBI121/4’CGT vector) and 75mg.
L−1 Hygromycin (for selection of pCAMBIA1304/AS1
vector). Plates were then incubated at 28˚C in a growth
chamber with a 16-h florescent light (100 µmol/m2/sec)
and 8-h dark cycle. Explants were regularly subcultured
to new MS medium supplemented with 250mg.L−1 cefo-
taxime and suitable antibiotic (75 mg.L−1 Hygromycin
and 50mg.L−1 Kanamycin) every two weeks. e single
shoot regenerated from inoculated explants was excised
from the calli and transferred onto the MS basal medium
supplemented with 100 mg.L−1 cefotaxime. Rooted
plantlets were then transferred into pots containing 70%
peatmoss; 30% perlite and grown in above-mentioned
conditions.
CAPS analysis
DNA extraction andPCR
Total DNA was extracted from fresh petal of puta-
tive transgenic plants with CTAB method as previously
described [44]. For PCR analysis, p1 and p2 specific
primers were used for 4’CGT and AS1 genes (Supplemen-
tary Table S2). PCR was performed using GoTaq® Green
Master Mix (Promega, Madison, WI, USA) in a T100
thermal cycle (Bio-Rad), with initial denaturation at 95
ºC for 5min, followed by 35 cycles at 95 ºC for 45s, 58
ºC for 30s and 72 ºC for 2min, and a final extension step
at 72 ºC for 10min. e analysis of products was per-
formed via 1% agarose gel electrophoresis and sequenc-
ing (Sanger Sequencing method, ABI 3730 XL).
Southern blot analysis
For Southern blot analysis, genomic DNA of petals was
treated with 10µg L−1 RNase for 4h at 25°C, followed by
phenol-chloroform extraction and ethanol precipitation.
Representative probes were prepared with digoxigenin,
by used DIG DNA labeling and detection kit. 30µg DNA
of each leaves sample was cut with NcoI enzyme for AS1
and BamHI enzyme for 4CGT (ermo Fisher) by incu-
bating for 16h at 37°C. e digested products were sepa-
rated by 1% agarose gel, denatured in 1.5M NaCl, 0.5M
NaOH for 30min each, and transferred to hybridization
membrane (GeneScreen, DuPont, Boston, MA, USA).
Hybridization was performed at 65°C with digoxigenin-
labeled probe (Amersham Rediprim II, GE Healthcare,
Pittsburgh, PA, USA). After 20h of hybridization, the
membrane was washed twice in 2× SSC at room temper-
ature for 15min each, twice in 2× SSC, 1% SDS at 65°C
for 30min each, and finally once in 0.1× SSC at room
temperature for 30min. e membrane was exposed to
an imaging plate at room temperature and the signal was
detected by a phosphor imager (Typhoon FLA 7000, GE
Healthcare, Pittsburgh, PA, USA).
Quantitative realtime PCR (qRTPCR) fortheexpression
analysis of4’CGT andAS1 genes
For real-time quantitative PCR, total RNA was extracted
from African violet white (non-transformation), trans-
genic A. violet, and A. majus petals all treated with
DNase I, as previously described by wang et al. [45].
Next, cDNA was synthesized from total RNA (100 ng)
using the Superscript III First-Strand Synthesis System
(Invitrogen, ermo Fisher Scientific, Waltham, MA,
USA) and oligo (dT) 20. e transcript levels of 4’CGT
and AS1 were analyzed via RT-qPCR (Applied Biosys-
tems 7900HT Fast Real-Time PCR System) using Power
SYBRTM Green PCR Master Mix (Applied Biosystems,
ermo Fisher Scientific) according to the manufac-
turer’s instructions. Transcript levels were calculated
based on ∆∆Cq (formerly ∆∆Ct) method [46] using Actin
(accession number: AB596843.1) gene as references. Sta-
tistical significance of the differential expression levels
was assessed as independent experiments (with mean
centering and autoscaling) [47]. e Tukey–Kramer test
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
Page 14 of 16
Rajabietal. Biological Procedures Online (2022) 24:3
at the 1% level was used for the analysis of aurone bio-
synthesis-related genes. e results are presented as the
standardized mean of SE.
Light Microscopy analysis
e morphology of regenerates transgenic and non-
transgenic petals were examined under a VH-Z75 light
microscope. Petals were cut into 5 × 5 mm pieces and
embedded in 4% agarose. in sections (thickness, 100
mm) were cut with a Micro Slicer DTK-1000 (D.S.K.).
Sections were examined using a VH-Z75 light micro-
scope (Keyence).
Aureusidin 6‑O glucoside identication byHPLC‑DAD‑MSn
e African violet white, transgenic African violet,
and A. majus petals (12 mm) were evaluated sepa-
rately. Aurone analyses were performed using an Agi-
lent HPLC series 1200 equipped with ChemStation
software, a degasser, quaternary pumps, autosampler
with chiller, column oven, and diode array detector.
e guard column operated at a temperature of 35C.
e mobile phase consisted of 0.1% TFA⁄water (elu-
ent A) and 90% acetonitrile in 0.1% TFA⁄water (eluent
B) at a flow of 0.8 mL⁄min using the following gradient
program: 20% B (0–3min); 20–60% B (3–20min); 60%
B isocratic (20–27min); 60–90% B washing step (27–
30 min); and equilibration for 10min. e total run
time was 40min. e injection volume for all samples
was 10L. Specific wavelengths were monitored sepa-
rately at 400 nm for aurone and 360 nm for flavones.
Additionally, UV⁄Vis spectra were recorded at 520 nm
for anthocyanins. e HPLC system was coupled online
to a Bruker (Bremen, Germany) ion trap mass spec-
trometer fitted with an ESI source. Data acquisition and
processing were performed using Bruker software. e
mass spectrometer was operated in positive ion mode
and auto MSn with a scan range from m⁄z 100 to 1000.
HPLC-grade acetonitrile, water, trifluoroacetic acid
(TFA), naringenin, and chalcone standards were pur-
chased from Sigma (St. Louis, MO). All standards were
prepared as stock solutions at 10mg⁄mL in methanol
and diluted in water except for chalcone, which was
prepared in 50% methanol. UV external standard cali-
bration was also used to obtain calibration curves of
cyanidin-3-O-glucose, naringenin-7-O-rutinoside, and
chalcone, which were used to quantify anthocyanins,
flavones, and chalcones, respectively. UV external cali-
bration of maritime was employed for the quantitation
of AOG.
Abbreviations
4’CGT : Chalcone4’-O-glucosyltransferase; AS1: Aureusidinsynthase; ATMT:
Agrobacteriumtumefaciens-mediated transformation; THC: 2’,4’,6’,4tet-
rahydroxychalcone; CHS: Chalconesynthase; CHI: Chalconeisomerase;
AOG: Aureusidin-6-O-glucose; F3H: Flavanone3-hydroxylase; DFR:
Dihydroflavonol4-reductase.
Supplementary Information
The online version contains supplementary material available at https:// doi.
org/ 10. 1186/ s12575- 022- 00164-0.
Additional le1: TableS1. Genes used for RT-qPCR in African violet
white (wild type), transgenic African violet and A. majus (Yellow). TableS2.
List of primers used in the study.
Acknowledgements
The authors would like to appreciate the Ali Dehestani and all members of
Genetic and Agricultural Biotechnology Institute of Tabarestan, University of
Agriculture Science, and Natural Resources Sari; Niranjan Hegde from Plant
Science Department, McGill University for their helpful discussion and techni-
cal assistance.
Authors’ contributions
The research project was carried out by collaboration among all authors. AR
Student and performed the experiments and carried out data analyses; LF
Supervisor and the final manuscript version; VGO: Advisor and supplied the
plant materials; LF and VGO Jointly designed the experiments; MK Advisor and
prepared the initial manuscript draft. All authors have read and approved the
manuscript.
Funding
This study was financially supported by grant No: 980901 of the Biotechnol-
ogy Development Council of the Islamic Republic of Iran.
Availability of data and materials
The data sets supporting the conclusions of this article are included within the
article and its additional files.
Declarations
Ethics approval and consent to participate
Not applicable.
Consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
Author details
1 Department of Plant Breeding and Biotechnology, University of Zabol,
98613-35856 Zabol, Iran. 2 Department of Plant Breeding and Biotechnology,
Gorgan University of Agricultural Sciences and Natural Resources, Gorgan,
Iran. 3 Department of Plant Pathology, University of Zabol, Zabol, Iran. 4 Genetic
and Agricultural Biotechnology Institute of Tabarestan, University of Agricul-
ture Science and Natural Resources, Sari, Iran.
Received: 24 August 2021 Accepted: 25 January 2022
References
1. Noman A, Aqeel M, Deng J, et al. Biotechnological Advancements
for Improving Floral Attributes in Ornamental Plants. Front Plant Sci.
2017;8:530. https:// doi. org/ 10. 3389/ fpls. 2017. 00530.
2. WU Y, ZHU M, Juang Y, et al. Molecular characterization of chalcone
isomerase (CHI) regulating flower color in herbaceous peony (Paeonia
lactiflora Pall.). J of Integrative Agriculture. 2018;17(1):122–9. https:// doi.
org/ 10. 1016/ S2095- 3119(16) 61628-3.
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
Page 15 of 16
Rajabietal. Biological Procedures Online (2022) 24:3
3. Azadi P, Bagheri H, Nalousi AM, et al. Current status and biotechnological
advances in genetic engineering of ornamental plants. J Biotechnology
Advances. 2016;34(6):1073–90. https:// doi. org/ 10. 1016/j. biote chadv. 2016.
06. 006.
4. Tanaka Y, Sasaki N, Ohmiya A. Biosynthesis of plant pigments: anthocya-
nins, betalains and carotenoids. Plant J. 2008;54(4):733–49. https:// doi.
org/ 10. 1111/j. 1365- 313X. 2008. 03447.
5. Ono E, Fukuchi-Mizutani M, Nakamura N, et al. Yellow flowers generated
by expression of the aurone biosynthetic pathway. Proceedings of the
National Academy of Sciences. 2006; 103(29): 11075 LP – 11080. https://
doi. org/ 10. 1073/ pnas. 06042 46103.
6. Venkateswarlu S, Panchagnula GK, Subbaraju GV. Synthesis and anti-
oxidative activity of 3’,4’,6,7-tetrahydroxyaurone, a metabolite of Bidens
frondosa. Bioscience Biotechnology Biochemistry. 2004;68:2183–5.
7. Teixeira da Silva J, Dewir Y, Wicaksono A, et al. African violet (Saintpaulia
ionantha H. Wendl.): Classical breeding and progress in the application of
biotechnological techniques. Folia Horticulturae. 2017;29:99–111. https://
doi. org/ 10. 1515/ fhort- 2017- 0010.
8. Haston E, De Craene LPR. Inflorescence and floral development in
Streptocarpus and Saintpaulia (Gesneriaceae) with particular reference to
the impact of bracteole suppression. Plant Syst Evol. 2007;265(1):13–25.
https:// doi. org/ 10. 1007/ s00606- 006- 0494-x.
9. The Plant List. Saintpaulia. Available online at: http:// www. thepl antli st.
org/ tpl1.1/search?q=Saintpaulia; cited on 20 May 2017. http:// www.
thepl antli st. org/ tpl1.1/ record/ kew- 25888 96.
10. Chandler S, Brugliera F Genetic modification in floriculture. Biotechnol
Lett. 2011; 33:207–214. https:// link. sprin ger. com/ conte nt/ pdf/ 10. 1007/
s10529-010-0424-4.
11. Hayta S, Smedley MA, Li J, Harwood WA, Gilmartin PM. Agrobacterium-
mediated transformation systems of Primula vulgaris. Plant Methods.
2018;14:93. https:// doi. org/ 10. 1186/ s13007- 018- 0360-1.
12. Fister AS, Shi Z, Zhang Y, et al. Protocol: transient expression system for
functional genomics in the tropical tree Theobroma cacao L. Plant Meth-
ods. 2016;12(1):19. https:// doi. org/ 10. 1186/ s13007- 016- 0119-5.
13. Gil-Humanes J, Wang Y, Liang Z, et al. High-efficiency gene targeting
in hexaploid wheat using DNA replicons and CRISPR/Cas9. Plant J.
2017;89(6):1251–62. https:// doi. org/ 10. 1111/ tpj. 13446.
14. Jelly NS, Valat L, Walter B, Maillot P. Transient expression assays in
grapevine: a step towards genetic improvement. J Plant Biotechnology.
2014;12(9):1231–45. https:// doi. org/ 10. 1111/ pbi. 12294.
15. Nanjareddy K, Arthikala MK, Blanco L, et al. Protoplast isolation, transient
transformation of leaf mesophyll protoplasts and improved Agrobacte-
rium-mediated leaf disc infiltration of Phaseolus vulgaris: tools for rapid
gene expression analysis. J BMC Biotechnology. 2016;16(1):53. https:// doi.
org/ 10. 1186/ s12896- 016- 0283-8.
16. Verweij W, Di Sansebastiano GP, Quattrocchio F, Dalessandro G. Agrobac-
terium-mediated transient expression of vacuolar GFPs in Petunia leaves
and petals. Plant Biosyst. 2008;142:343–7.
17. Rivero L, Scholl R, Holomuzki N, et al. Handling Arabidopsis plants: growth,
preservation of seeds, transformation, and genetic crosses. In Arabidopsis
protocol. Humana Press, Totowa, NJ. 2014; 3–25. https:// link. sprin ger. com/
conte nt/ pdf/ 10. 1007/978-1-62703-580-4_1.
18. Tague BW, Mantis J In planta Agrobacterium mediated transformation by
vacuum infiltration. In Arabidopsis protocol. Humana Press. 2006; 215–223.
https:// link. sprin ger. com/ conte nt/ pdf/ 10. 1385/1-59745-003-0:215.
19. Hoshino A, Mizuno T, Shimizu K, et al. Generation of Yellow Flowers of
the Japanese Morning Glory by Engineering Its Flavonoid Biosynthetic
Pathway toward Aurones. Plant Cell Physiol. 2019;60(8):1871–9.
20. Roberts WR, Roalson EH. Comparative transcriptome analyses of flower
development in four species of Achimenes (Gesneriaceae). BMC Genom-
ics. 2017;18(1):240. https:// doi. org/ 10. 1186/ s12864- 017- 3623-8.
21. Kazemian M, Kazemi EM, Kolahi M, Omran VG. Floral ontogeny and
molecular evaluation of anthocyanin biosynthesis pathway in pinwheel
phenotype of Saintpaulia inontha Wendl. periclinal chimera. Sci Hortic.
2020; 263,109142. https:// doi. org/ 10. 1016/j. scien ta. 2019. 109142.
22. Qi Y, Lou Q, Quan Y, et al. Flower-specific expression of the Phalaenopsis fla-
vonoid 3′, 5′-hydoxylase modifies flower color pigmentation in Petunia and
Lilium. Plant Cell, Tissue and Organ Culture (PCTOC). 2013; 115(2):263-273.
23. Noda N, Yoshioka S, Kishimoto S, et al. Generation of blue chrysanthe-
mums by anthocyanin B-ring hydroxylation and glucosylation and its
coloration mechanism. Sci Adv. 2017;3(7):.e1602785.
24. Nakamura N, Katsumoto Y, Brugliera F, et al. Flower color modification
in Rosa hybrida by expressing the S-adenosylmethionine: anthocyanin
3′,5′-O-methyltransferase gene from Torenia hybrida. J Plant Biotechnol-
ogy. 2015;32(2):.109–117.
25. Li Z, Zhao M, Jin J, Zhao L, Xu Z. Anthocyanins and their biosynthetic
genes in three novel-colored Rosa rugosa cultivars and their parents. J
Plant Physiology Biochemistry. 2018;129:421–8. https:// www. scien cedir
ect. com/ scien ce/ artic le/ abs/ pii/ S0981 94281 83027 91? via% 3Dihub.
26. To KY, Wang CK. Molecular Breeding of Flower Color. In Floriculture, Orna-
mental and Plant Biotechnology (Vol. 1). 2006;1: 300-310.
27. Schijlen EG, De Vos CR, van Tunen AJ, Bovy AG. Modification of flavonoid
biosynthesis in crop plants. Phytochemistry. 2004;65(19):2631–48.
28. Caro SE, Stampfle JM, Greene MJ, Kotarski MA. Using a chalcone synthase
Gene to Infer Phylogenies in the Genus Saintpaulia. Bios. 2006;77(3):72–6.
http:// www. jstor. org/ stable/ 46087 76.
29. Mercuri A, De Benedetti L, Burchi G, Schiva T. Agrobacterium-mediated
transformation of African violet. Plant Cell Tissue Organ Cult. 2000;60:39–
46. https:// link. sprin ger. com/ conte nt/ pdf/ 10. 1023/A: 10064 57716 959.
30. Kushikawa S, Hoshino Y, Mii M. Agrobacterium-mediated transformation
of Saintpaulia ionantha Wendl. Plant Sci. 2001;161:953–60. https:// doi.
org/ 10. 1016/ S0168- 9452(01) 00496-4.
31. Manavella PA, Chan RL. Transient transformation of sunflower leaf discs
via an Agrobacterium-mediated method: applications for gene expression
and silencing studies. Nat Protoc. 2009;4(11):1699–707. https:// doi. org/ 10.
1038/ nprot. 2009. 178.
32. Yasmin A, Debener T. Transient gene expression in rose petals via Agro-
bacterium infiltration. Plant Cell, Tissue and Organ Culture (PCTOC). 2010;
102: 245–250. https:// doi. org/ 10. 1007/ s11240- 010- 9728-2.
33. Nazari F, Khoshkhui M, Azadi P. Production of Delphinidin Anthocyanin
in the Flower Petals of Gerbera by Agroinfiltration of Flower Color Gene
Constructs. J of Plant Production Research. 2017;23(4):145–64. https:// doi.
org/ 10. 22069/ jopp. 2017. 10583. 2000.
34. Hussein GM, Abu El-Heba GA, Abdou SM, Abdallah NA. Optimization of
transient gene expression system in Gerbera jemosonii petals. GM Crops
Food. 2013;4(1):50–7. https:// doi. org/ 10. 4161/ gmcr. 23925.
35. Shang Y, Schwinn KE, Bennett MJ, et al. Methods for transient assay of
gene function in floral tissues. Plant Methods. 2007;3(1):1. https:// doi. org/
10. 1186/ 1746- 4811-3-1.
36. Keykha F, Bagheri A, Moshtaghi N, Bahrami AR, Sharifi A. RNAi-induced
silencing in floral tissues of Petunia hybrida by agroinfiltration: A rapid
assay for chalcone isomerase gene function analysis. Cellular Molecular
Biology. 2016;62(10):26–31. https:// doi. org/ 10. 14715/ cmb/ 2016. 62. 10.4.
37. Marion J, Bach L, Bellec Y, Meyer C, Gissot L, Faure JD. Systematic
analysis of protein subcellular localization and interaction using high-
throughput transient transformation of Arabidopsis seedlings. The Plant J.
2008;56:169–79.
38. Forkmann G, Martens S. Metabolic engineering and applications of flavo-
noids. Curr Opin Biotechnol. 2001;12(2):155–60. https:// doi. org/ 10. 1016/
S0958- 1669(00) 00192-0.
39. Wang CK, Chin YC, Lin CY, Chen PY, To KY. Transforming the snap-
dragon aurone biosynthetic genes into petunia alters coloration
patterns in transgenic flowers. Advances in Bioscience Biotechnology.
2015;6(12):702–22.
40. Schwarz-Sommer Z, Davies B, Hudson A. An everlasting pioneer: The
story of Antirrhinum research. Nat Rev Genet. 2003;4:655–64.
41. Sato T, Nakayama T, Kikuchi S, et al. Enzymatic formation of aurones in
the extracts of yellow snapdragon flowers. Plant Sci. 2001;160(2):229–36.
https:// doi. org/ 10. 1016/ S0168- 9452(00) 00385-X.
42. Wang HM, Yin WC, Wang CK, To KY. Isolation of functional RNA from differ-
ent tissues of tomato suitable for developmental profiling by microarray
analysis. Botanical Studies. 2009;50(2):115–25.
43. Murashige T, Skoog FA. Revised Medium for Rapid Growth and Bio Assays
with Tobacco Tissue Cultures. Physiol Plant. 1962;15(3):473–97. https:// doi.
org/ 10. 1111/j. 1399- 3054. 1962. tb080 52.
44. Wang CK, Chen P, Wang HM, To KY. Cosupression of tobacco chalcone
synthase using Petunia chalcone synthase construct results in white flow-
ers. Botanical Studies. 2006;47:71–82.
45. Wilkie S, Clark MS, Leroy P, et al. Genomic DNA, Isolation. Southern
Blotting and Hybridization BT - Plant Molecular Biology — A Laboratory
Manual. Springer Berlin Heidelberg. 1997;pp. 3–53. https:// doi. org/ 10.
1007/ 978-3- 642- 87873-2_1.
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
Page 16 of 16
Rajabietal. Biological Procedures Online (2022) 24:3
•
fast, convenient online submission
•
thorough peer review by experienced researchers in your field
•
rapid publication on acceptance
•
support for research data, including large and complex data types
•
gold Open Access which fosters wider collaboration and increased citations
maximum visibility for your research: over 100M website views per year
•
At BMC, research is always in progress.
Learn more biomedcentral.com/submissions
Ready to submit your research
Ready to submit your research
? Choose BMC and benefit from:
? Choose BMC and benefit from:
46. Livak KJ, Schmittgen TD. Analysis of Relative Gene Expression Data Using
Real-Time Quantitative PCR and the 2 – ∆∆CT Method. Methods. 2001;
25(4): 402–8. https:// doi. org/ 10. 1006/ meth. 2001. 1262.
47. Bustin SA, Benes V, Garson JA, et al. The MIQE Guidelines: Minimum Infor-
mation for Publication of Quantitative Real-Time PCR Experiments. Clin
Chem. 2009;55(4):611–22. https:// doi. org/ 10. 1373/ clinc hem. 2008. 112797.
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in pub-
lished maps and institutional affiliations.
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
1.
2.
3.
4.
5.
6.
Terms and Conditions
Springer Nature journal content, brought to you courtesy of Springer Nature Customer Service Center GmbH (“Springer Nature”).
Springer Nature supports a reasonable amount of sharing of research papers by authors, subscribers and authorised users (“Users”), for small-
scale personal, non-commercial use provided that all copyright, trade and service marks and other proprietary notices are maintained. By
accessing, sharing, receiving or otherwise using the Springer Nature journal content you agree to these terms of use (“Terms”). For these
purposes, Springer Nature considers academic use (by researchers and students) to be non-commercial.
These Terms are supplementary and will apply in addition to any applicable website terms and conditions, a relevant site licence or a personal
subscription. These Terms will prevail over any conflict or ambiguity with regards to the relevant terms, a site licence or a personal subscription
(to the extent of the conflict or ambiguity only). For Creative Commons-licensed articles, the terms of the Creative Commons license used will
apply.
We collect and use personal data to provide access to the Springer Nature journal content. We may also use these personal data internally within
ResearchGate and Springer Nature and as agreed share it, in an anonymised way, for purposes of tracking, analysis and reporting. We will not
otherwise disclose your personal data outside the ResearchGate or the Springer Nature group of companies unless we have your permission as
detailed in the Privacy Policy.
While Users may use the Springer Nature journal content for small scale, personal non-commercial use, it is important to note that Users may
not:
use such content for the purpose of providing other users with access on a regular or large scale basis or as a means to circumvent access
control;
use such content where to do so would be considered a criminal or statutory offence in any jurisdiction, or gives rise to civil liability, or is
otherwise unlawful;
falsely or misleadingly imply or suggest endorsement, approval , sponsorship, or association unless explicitly agreed to by Springer Nature in
writing;
use bots or other automated methods to access the content or redirect messages
override any security feature or exclusionary protocol; or
share the content in order to create substitute for Springer Nature products or services or a systematic database of Springer Nature journal
content.
In line with the restriction against commercial use, Springer Nature does not permit the creation of a product or service that creates revenue,
royalties, rent or income from our content or its inclusion as part of a paid for service or for other commercial gain. Springer Nature journal
content cannot be used for inter-library loans and librarians may not upload Springer Nature journal content on a large scale into their, or any
other, institutional repository.
These terms of use are reviewed regularly and may be amended at any time. Springer Nature is not obligated to publish any information or
content on this website and may remove it or features or functionality at our sole discretion, at any time with or without notice. Springer Nature
may revoke this licence to you at any time and remove access to any copies of the Springer Nature journal content which have been saved.
To the fullest extent permitted by law, Springer Nature makes no warranties, representations or guarantees to Users, either express or implied
with respect to the Springer nature journal content and all parties disclaim and waive any implied warranties or warranties imposed by law,
including merchantability or fitness for any particular purpose.
Please note that these rights do not automatically extend to content, data or other material published by Springer Nature that may be licensed
from third parties.
If you would like to use or distribute our Springer Nature journal content to a wider audience or on a regular basis or in any other manner not
expressly permitted by these Terms, please contact Springer Nature at
onlineservice@springernature.com
Content uploaded by Mojtaba Keykhasaber
Author content
All content in this area was uploaded by Mojtaba Keykhasaber on Feb 10, 2022
Content may be subject to copyright.
