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African violet (Saintpaulia ionantha H. Wendl.): Classical breeding and progress in the application of biotechnological techniques

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As a result of its domestication, breeding and subsequent commercialization, African violet (Saintpaulia ionantha H. Wendl.) has become the most famous and popular Saintpaulia species. There is interest in producing cultivars that have increased resistance to pests and low temperature, the introduction of novel horticultural characteristics such as leaf shape, flower colour, size, and form, improved productivity and enhanced flower duration in planta. In African violet, techniques such as the application of chemical mutagens (ethylmethanesulfonate, N-nitroso-N-methylurea), radiation (gamma (γ)-rays, X-rays, carbon ion beams) and colchicine have been successfully applied to induce mutants. Among these techniques, γ radiation and colchicine have been the most commonly applied mutagens. This review offers a short synthesis of the advances made in African violet breeding, including studies on mutation and somaclonal variation caused by physical and chemical factors, as well as transgenic strategies using Agrobacterium-mediated transformation and particle bombardment. In African violet, Agrobacterium-mediated transformation is affected by the Agrobacterium strain, selection marker, and cutting-induced wounding stress. Somaclonal variation, which arises in tissue cultures, can be problematic in maintaining true-to-type clonal material, but may be a useful tool for obtaining variation in flower colour. The only transgenic African violet plants generated to date with horticulturally useful traits are tolerant to boron (heavy metal) stress, or bear a glucanase-chitinase gene.
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Published by the Polish Society
for Horticultural Science since 1989
Folia Hort. 29/2 (2017): 99-111
Folia
Horticulturae
DOI: 10.1515/fhort-2017-0010
http://www.foliahort.ogr.ur.krakow.plREVIEW Open access
ABSTRACT
As a result of its domestication, breeding and subsequent commercialization, African violet (Saintpaulia
ionantha H. Wendl.) has become the most famous and popular Saintpaulia species. There is interest in
producing cultivars that have increased resistance to pests and low temperature, in the introduction of novel
horticultural characteristics such as leaf shape, ower colour, size and form, and in improved productivity
and enhanced ower duration in planta. In African violet, techniques such as the application of chemical
mutagens (ethylmethanesulfonate, N-nitroso-N-methylurea), radiation (gamma (γ)-rays, X-rays, carbon ion
beams) and colchicine have been successfully applied to induce mutants. Among these techniques, γ radiation
and colchicine have been the most commonly applied mutagens. This review offers a short synthesis of the
advances made in African violet breeding, including studies on mutation and somaclonal variation caused by
physical and chemical factors, as well as transgenic strategies using Agrobacterium-mediated transformation
and particle bombardment. In African violet, Agrobacterium-mediated transformation is affected by the
Agrobacterium strain, selection marker, and cutting-induced wounding stress. Somaclonal variation, which
arises in tissue cultures, can be problematic in maintaining true-to-type clonal material, but may be a useful
tool for obtaining variation in ower colour. The only transgenic African violet plants generated to date with
horticulturally useful traits are tolerant to boron (heavy metal) stress, or bear a glucanase-chitinase gene.
Key words: Agrobacterium-mediated transformation, Gesneriaceae, mutation, particle bombardment,
somaclonal variation, transgenic
African violet (Saintpaulia ionantha H. Wendl.):
classical breeding and progress in the application
of biotechnological techniques
Jaime A. Teixeira da Silva1, Yaser Hassan Dewir2,3, Adhityo Wicaksono4,
Leela Sahijram5, Haenghoon Kim6, Songjun Zeng7,
Stephen F. Chandler8, Munetaka Hosokawa9
1 P.O. Box 7, Miki-Cho Post Ofce, Ikenobe 3011-2, Kagawa-Ken, 761-0799, Japan
2 Plant Production Department, P.O. Box 2460, College of Food & Agriculture Sciences
King Saud University, Riyadh 11451, Saudi Arabia
3 Department of Horticulture, Faculty of Agriculture, Kafrelsheikh University, Kafr El-Sheikh 33516, Egypt
4 Laboratory of Paper Coating and Converting, Centre for Functional Material
Åbo Akademi University, Porthaninkatu 3, 20500 Turku, Finland
5 Division of Biotechnology, Indian Council of Agricultural Research (ICAR) – Indian Institute of Horticultural
Research, Hessaraghatta Lake Post, Bangalore, Karnataka, 560 089, India
6 Department of Well-being Resources, Sunchon National University, Suncheon, 540-742, South Korea
7 Guangdong Provincial Key Laboratory of Applied Botany, South China
Botanical Garden, the Chinese Academy of Sciences, Guangzhou, 510650, P.R. China
8 School of Applied Sciences, RMIT University, Bundoora, Victoria, VIC 3083, Australia
9 Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan
*Corresponding authors.
E-mail: 1jaimetex@yahoo.com, 2ydewir@hotmail.com, 4adhityo.wicaksono@gmail.com, 5leelas@iihr.res.in,
6cryohkim@sunchon.ac.kr, 7zengsongjun@scib.ac.cn, 8schandler230@gmail.com, 9mune@kais.kyoto-u.ac.jp.
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100 African violet breeding and progress in biotechnological techniques
INTRODUCTION
African violet (Saintpaulia ionantha H. Wendl.;
Gesneriaceae) is the most famous and popular
Saintpaulia species. It has been domesticated,
bred and commercialized, and can be vegetatively
propagated fairly easily (Teixeira da Silva et
al. 2016a). A historical text by Tsukamoto et al.
(1982) indicates how the cultivation of Saintpaulia
originally started in Europe, but its development
as a horticultural plant took place in the United
States after World War II. Tsukamoto et al. (1982)
also noted that by 1981 there were about 20 species
in the genus Saintpaulia, although The Plant List
(2017) indicates that there are currently 25 species,
including accepted, synonymic and unresolved
species.
This review offers a short synthesis of advances
made in mutation and somaclonal variation
caused by physical and chemical factors, as well
as transgenic strategies using Agrobacterium-
mediated t ransformation and pa rticle bomba rdment.
Classical breeding has the ability to transfer
a desired trait into different cultivars of any plant
species, but if one or more of the parent cultivars
used in cross-breeding are poorly adapted, then
a back-crossing strategy needs to be implemented
to recover the elite type. Moreover, poor combining
ability of some parental genotypes may occur and
doubled (i.e., multi-whorled) owers do not produce
male or female organs. For example, aromatic rice
varieties have poor combining ability, and cross-
breeding with non-aromatic varieties will lead to
a decrease in aroma and quality (Bourgis et al.
2008). Under such circumstances, the induction of
mutations can be advantageous to produce cultivars
with desired traits within dened germplasm
pools. Mutation breeding thus offers a solution to
difculties encountered in classical breeding. For
example, if genes conferring undesirable characters
are tightly linked to genes controlling desirable
traits, then induction of mutations may result in
a cross-over event or isolation of an independent
mutation for the desired trait (van Harten 2007).
Mutation breeding may be the only acceptable
way of classically increasing variability in plant
species that do not produce seeds (Ahloowalia
and Maluszynski 2001), and to develop novel
colours and variations in vegetatively propagated
ornamental plant species (Broertjes and van Harten
1988, Datta and Teixeira da Silva 2006, Kondo et
al. 2009). Thus, mutation breeding is considered to
be a valuable conventional breeding strategy.
Mutation techniques have made signicant
contributions in ornamental crop improvement.
Hundreds of mutant cultivars have been ofcially
released for various traits in horticulture, including
the colour and shape of owers and fruits, and
esh colour (Datta and Teixeira da Silva 2006).
However, plant breeders are under continuous
pressure to improve existing cultivars or to develop
new ones. Therefore, there is a need for newer
alternatives or technologies that, when combined
with conventional breeding methods, can help
create greater variability with desirable novel traits,
while reducing the time taken to do so. In vitro
culture of plant cells and organs generates genetic
variability (‘somaclonal variation’) resulting in
‘somaclones’ (regenerated plants that are not true-
to-type) which can be used in sexual hybridisation
for introgression of desirable alien genes into crop
species, or to generate variants of a commercial
cultivar at a high frequency without hybridizing it
to other genotypes (Larkin and Scowcroft 1981).
Ever since the rst formal report of morphological
variants in sugarcane plants produced in vitro in
1971, numerous somaclonal variations have been
reported in several horticultural crops (Krishna
et al. 2016). Somaclonal variation manifests itself
as qualitative or quantitative phenotypic changes,
sequence changes, and gene activation/silencing
(Kaeppler et al. 2000). Epigenetic changes occur via
changes in DNA methylation patterns, activation
of quiescent transposable elements (TEs) or
retrotransposons (Duncan 1997, Huang et al. 2009).
In important horticultural crops, DNA methylation
patterns are highly variable among regenerants
and polymorphisms exist in somaclones (Sahijram
2014, 2015). In African violet, 47% of somaclonal
variants from in vitro cultures did not ower. No
variation was seen in ower colour in the remaining
owering plants (Jain 1993a, 1993b, 1997a, 1997b),
although these studies did not assess genetic
alterations.
It is possible to induce variation in ower
colour and introduce several other novel traits to
ornamental plants using somaclonal variation.
Often, a plant regeneration system, particularly
callus-mediated plant regeneration, introduces
variations that may be heritable (Krishna et al.
2016). The concentration and combination of plant
growth regulators (PGRs), as well as subculture
frequency and duration, also result in a higher
frequency of variation (Matsuda et al. 2014). TE
transposition and a combination of factors such as
colchicine, gamma (γ)-rays, ion-beams, and PGRs,
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J.A. Teixeira da Silva, Y.H. Dewir, A. Wicaksono, L. Sahijram, H. Kim, S. Zeng, S.F. Chandler, M. Hosokawa 101
may serve as a good set of tools for expanding
variations in ower colour in African violet.
Classical breeding
1. Self-pollination
Saintpaulia owers are zygomorphic with two
upper and three lower lobes. The upper two lobes
are smaller than the lower ones. Saintpaulia has
a very short corolla tube and yellow protruding
anthers that are probably associated with buzz
pollination (Harrison et al. 1999). The anthers of
Saintpaulia have only one chamber, the owers have
a distinct disk, the style and stigma usually have the
same colour as the petals, and the ovary is exserted
slightly to the left or right of the center of the corolla
(Harrison et al. 1999). All Saintpaulia species are
enantiostylous (the style is strongly deected to
the left or right of the main oral axis), a feature
often linked to buzz pollination S. ionantha is
a protandrous species, and the anthers appear to be
full of pollen, even during the pistillate phase. The
anthers do not wilt, remain yellow and have thecae
and stiffened walls (Vogel 1978). Most Saintpaulia
species can hybridize with their congenerics and
produce fertile hybrids (Arisumi 1964).
The diploid chromosome number in African
violet was indicated by Vazquez et al. (1977),
Espino and Vazquez (1981) and Sun et al. (2010)
to be 2n = 28. Farjadi-Shakib et al. (2012) were
of the opinion that since African violet had tiny
chromosomes with a propensity for sticking
together and a centromere difcult to distinguish,
the diploid chromosome number in this species
was incorrectly described as 2n = 2x = 28 (Sugiura
1936, Adisorn 2004). More detailed cytological
analyses indicated that the diploid chromosome
number was 2n = 2x = 30 (Ehrlich 1956, Farjadi-
Shakib et al. 2012), and 2n = 60 for the tetraploid
hort. var. ‘Ionantha Amazon’ (Ehrlich 1958), with
a total genome length of 29.995 µm, and a ratio
of the longest to the smallest chromosome of 2.77
(Farjadi-Shakib et al. 2012). Using ow cytometry,
Loureiro et al. (2007) determined that the nuclear
DNA content (i.e., genome size) of diploid
S. ionantha was 2C = 1.5 pg.
Spontaneous self-pollination has been reported
to occur frequently in some commercial African
violet cultivars through an abnormal mode of ower
development in which the stigma grows into the
anther (Anonymous 2002). Self-fertilization and
mating between close relatives in small populations
may lead to inbreeding depression (Charlesworth
and Charlesworth 1987, Kolehmainen and
Mutikainen 2006). Kolehmainen et al. (2010)
investigated inbreeding depression in 12 populations
of a threatened, endemic African violet, S. ionantha
ssp. grotei, using one microsatellite locus, and
concluded that inbreeding occurred frequently and
led to signicant inbreeding depression.
2. Interspecic crosses
Saintpaulia is likely to predominantly outcross
because it is pollinated by ying insects and
because the owers have two different stylar
morphs (i.e., enantiostyly), which has been shown
to promote cross pollination (Jensson and Barrett
2002). Crossing experiments have shown that the
majority of Saintpaulia taxa can hybridize and that
hybrid offspring are fertile (Clayberg 1961, Arisumi
1964). However, Afkhami-Sarvestani et al. (2012a)
were unable to produce viable progeny, even using
embryo rescue, from intergeneric crosses between
Streptocarpus sub-genus Streptocarpella and
S. ionantha. Af khami-Sarvestani et al. (2012b)
were able to induce callus after fusing protoplasts.
Arisumi (1967) conducted a large breeding
experiment in which he selfed and crossed
several clones of Saintpaulia species that had no
anthocyanin in their leaf blades, namely, according
to the Interagency Taxonomic Information System
& World Checklist of Gesneriaceae, S. amaniensis
E. Roberts [now accepted as S. ionantha ssp. grotei
(Engler) I. Darbysh, taxonomic serial No. (TSN)
832566], S. confusa B. L. Burtt. [now accepted as
S. ionantha ssp. grotei (Engler) I. Darbysh, TSN
832554], S. difcilis B. L. Burtt. [now accepted as
S. ionantha ssp. grotei (Engler) I. Darbysh, TSN
832556], S. diplotricha B. L. Burtt. [now accepted
as S. ionantha var. diplotricha (B.L. Burtt) I.
Darbysh., TSN 832560], S. grandifolia B. L. Burtt.
[now accepted as S. ionantha ssp. grandifolia (B.L.
Burtt) I. Darbysh, TSN 832561], S. grotei Engl.
[now accepted as S. ionantha ssp. grotei (Engler)
I. Darbysh., TSN 832562], and S. magungensis E.
Roberts [now accepted as S. ionantha ssp. grotei
(Engler) I. Darbysh., TSN 832616] (Skog and Boggan
2010, ITIS available online at: https://www.
itis.gov). After germinating seeds and growing
seedlings according to Arisumi (1964), Arisumi
(1967) determined genetic ratios after assigning
the symbols A and a, respectively, to dominant
and recessive genes for anthocyanin. Arisumi
(1967) found that: a) A and a segregated in classic
Mendelian ratios; b) S. diplotricha (syn. S. ionantha
var. diplotricha) was homozygous recessive (aa),
whereas all the other species were heterozygous
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102 African violet breeding and progress in biotechnological techniques
(Aa); c) the A gene might control anthocyanin
production in L-I, L-II and L-III layers; d) S.
ionantha ‘Snow Prince’ and ‘Northern Snowake’
had a recessive gene for white ower colour which
prevented the formation of anthocyanin anywhere
in the plant. Arisumi and Frazier (1968) induced
a single chimeric polyploid plant from 29 regenerants
after treating leaf cuttings with colchicine.
After intensive breeding over the past century,
thousands of cultivated Saintpaulia varieties are
mass propagated by the horticulture industry
(Baatvik 1993). These cultivars have mainly been
bred from two natural species, S. ionantha and
S. confusa (S. ionantha ssp. grotei) (Baatvik 1993,
Eastwood et al. 1998).
3. Ploidy and polyploidization
Espino and Vazquez (1981) regenerated polyploid
and mixoploid plants from leaf explants using
caffeine or colchicine, but no polyploidy was
detected in regenerants grown on control medium.
More specically, exposure of leaf explants to basal
Murashige and Skoog medium containing 500 or
1000 mg l-1 caffeine for 4-16 days resulted in 2-8%
polyploid plants (including chimeric plants), whereas
exposure to 100-200 mg l-1 colchicine resulted in
22-46% polyploids. Their results were in contrast
to the low level of mixoploidy (1-2%) observed by
Broertjes (1974) when petioles and leaves were
exposed to colchicine. Winkelmann and Grunewaldt
(1995) conrmed that 16% of regenerants derived
from protoplast culture were polyploid (mostly
tetraploid). Seneviratne and Wijesundara (2003)
applied 0.05% colchicine for 18 h to leaves of
four unspecied African violet varieties, and
succeeded in producing dwarf plants with small,
succulent leaves with a short petiole, suitable for
use as table-top miniature plants. Bhaskaran et al.
(1983) obtained anther-derived plants that were not
haploid, but diploid or tetraploid, possibly due to the
endomitosis during tissue culture.
4. Mutation breeding and chimeras
Periclinal chimeras were observed in pinwheel
owering African violet varieties (Lineberger and
Druckenbrod 1985) and several breeding-oriented
mutations have been reported in Saintpaulia. Several
techniques are available to induce mutations in
ornamental plants, including chemical and radiation
(γ- or X-ray irradiation, ion-beam treatment) or
TE activation (Datta and Teixeira da Silva 2006).
Sparrow et al. (1960) suggested that chimeras
originating from a low percentage of S. ionantha
mutants (0.7%) might be of multicellular origin.
4.1. Chemical mutagenesis
Wareld (1973) produced 13% mutations
after treating petioles of leaf cuttings of
S. ionantha (cultivar not specied) with 0.5 M
ethylmethanesulfonate (EMS) for 1 h, including
leaf-patterning mutants, leaf colour variants, and
dwarf plants. Except at a lethal dose, Kelly and
Lineberger (1981) found that thermal neutron
irradiation (250, 1000 and 5000 Rad of thermal
neutrons) of cuttings reduced root emergence and
induced changes in peroxidase proles, but did not
induce any morphological mutants. By applying
400 mg l-1 N-nitroso-N-methylurea (NMU) to in
vitro leaves (Geier 1983), leaf albinism and mottling
could be induced; this could be increased to 50%
when 0.1-1.0% dimethyl sulfoxide was added. In
the same study, exposure to 97.45 or 292.36 mM
EMS produced different levels of shoot inhibition
and chlorophyll-decient shoots relative to controls
and NMU-treated leaves (Tab. 1). Grunewaldt
(1983) observed that as many as 40% of regenerants
from NMU-treated leaf explants showed altered
leaf pigmentation. Gaj and Gaj (1996) induced
chlorophyll chimerism (variegated leaves) in
100% of explants when leaves were treated with
5 nM NMU for 1.5 or 2 h. In all of these studies,
stable transmission of mutations from one clonal
generation to the next was not possible. Leaf
blade and petiole cuttings of S. ionantha ‘Ulery’
exposed to thermal neutron irradiation (250-5000
Rad) did not generate any morphologically distinct
mutants, although variation in peroxidase levels
was observed.
4.2. Physical mutagenesis (radiation)
Using an unnamed cultivar, Seneviratne and
Wijesundara (2007) reported a change in ower
colour pattern (white petals with wide, pink
margins) by coupling 15 Gy of γ irradiation with
a dip treatment of leaves bearing 2.5 cm long
petioles in 0.06% colchicine for 22.5 h. The same
treatment resulted in a change in plant architecture,
measured as reduced petiole length (3.9 cm vs
8 cm in the control), reduced leaf surface area
(14.7 cm 2 vs 34.8 cm2 in the control), smaller ower
diameter (2.1 cm vs 3.8 cm in the control) and
shorter inorescence height (3.7 cm vs 6.4 cm in
the control). In their experiment, all the plants died
after γ irradiation of 20 Gy. Wongpiyasatid et al.
(2007a, 2007b) tested γ-ray (0-100 Gy) treatment of
leaf cuttings of two unnamed cultivars of African
violet (Wongpiyasatid et al. 2007a) or ‘Optima
Hawaii’ (Wongpiyasatid et al. 2007b). The LD50(60) ,
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J.A. Teixeira da Silva, Y.H. Dewir, A. Wicaksono, L. Sahijram, H. Kim, S. Zeng, S.F. Chandler, M. Hosokawa 103
which indicates 50% dead leaves at 60 days after
irradiation, was 56 Gy, and the highest frequency
of mutation (18.33%) occurred when 60 Gy was
applied; irradiation exceeding 80 Gy caused the
death of all leaves. Vegetative and oral mutations
induced changes in plant canopy width, number
of leaves, number of inorescences per plant,
number of owers per inorescence, and ower
form, colour and size. For example, at 60 Gy, 24.9
leaves/plant were formed versus 20.5 in the control.
In a few cases (Wongpiyasatid et al. 2007a), there
was conversion from a vegetative meristem into
a oral meristem and subsequent formation of
an inorescence in place of leaves. According
to Wongpiyasatid et al. (2007a), detached leaves
exposed to X-rays and fast neutrons also showed
dose-dependent mutagenesis (Broertjes 1968,
1971, 1972). Zhou et al. (2006a, 2006b) irradiated
adventitious shoots in vitro with carbon ion beams
and X-rays, and found that 49.6% and 43.3% of
‘Mauve and ‘Indikon’ shoots, respectively, were
malformed in response to carbon ion beams, but
only 3.7% and 11.3% following X-ray irradiation.
Somaclonal variation as a result of tissue culture
In vitro leaf and petiole homogenate cell clusters of
S. ionantha ‘Crimson Frost’ were used for studies
on somaclonal variation (Paek and Hahn 1999). The
resulting plants showed variations in leaf colour
(67% of variants) and leaf shape (19% of variants),
but no difference in protein proles was observed
using SDS-PAGE. Semi-double and double ower
types were more common than the single ower
type, showing 81% normal (mixed) colour vs. pink,
red or white (19% of variants) (Paek and Hahn 1999).
Their results indicate that the use of homogenate
cell clusters was not a proper method for true-to-
type propagation of African violet. In Saintpaulia
‘Thamires’ (Saintpaulia sp.), ower colour variants
in tissue culture-derived regenerants were formed
due to the excision of a TE (Sato et al. 2011a, 2011b).
Much of the variability seen in micropropagated
plants may be either the result of, or may be related
to, oxidative-stress damage in plant tissues during
in vitro culture (Matsuda et al. 2014). Simply
by culturing leaf explants in vitro, Shajiee et al.
(2006) were able to induce variegated leaves in a
maximum of 0.78% of regenerants in one of the
studied genotypes.
In Saintpaulia, somaclonal variation is
considered problematic when clonal (true-to-type)
material is desired, as indicated by the studies
above, but is also very useful for improving traits.
Sato et al. (2011b) identied a hAT superfamily TE
(VGs1) in the avonoid 3', 5' hydroxylase (F3'5'H )
promoter region and found that when the TE was
Table 1. Key results from Geier (1983) (modied) showing the impact of 1 h exposure of two chemical mutagens,
N-nitroso-N-methylurea (NMU) and ethylmethanesulfonate (EMS), on shoot inhibition and production of chlorophyll-
decient shoots in ‘Rhapsodie 26’ at 73 days of treatment
Treatments Rinse (H2O)
(min.)
Number of shoots per
explant
Inhibition of shoot
formation (%)
Chlorophyll-decient
mutants (%)
EMS
Control* 43.3 0.00 0.00
1% (97.45 mM) 3 × 2 17.6 59.35 1.14
3 × 20 23.8 45.03 0.84
3% (292.36 mM) 3 × 2 5.6 87.99 1.92
3 × 20 20.1 53.58 1.49
NMU
Control 38.0 0.00 0.00
400 mg l-1 (3.88 mM) 3 × 2 10.9 71.91 12.84
3 × 20 19.6 49.48 8.67
800 mg l-1 (7.76 mM) 3 × 2 3.5 90.98 20.00
3 × 20 7.6 80.41 18.42
Control 51.6 0.00 0.00
200 mg l-1 (1.94 mM) Undened 38.3 25.78 4.44
400 mg l-1 (3.88 mM) Undened 24.0 46.51 8.33
600 mg l-1 (5.82 mM) Undened 9.8 81.00 14.29
800 mg l-1 (7.76 mM) Undened 6.0 88.37 20.00
*No chemical mutagen treatment
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104 African violet breeding and progress in biotechnological techniques
excised from this region, ower colour changed from
pink to purple (Fig. 1). In Saintpaulia T h am ire s’,
Sato et al. (2011a) concluded that the origin of
somaclonal variation could be mainly attributed to
‘newly induced mutations’. Matsuda et al. (2014)
tried to identify factors involved in the induction of
transposition of VGs1, and plant growth regulators
added to the culture medium were candidate factors
for the induction of somaclonal variation.
Molecular breeding
1. Molecular and biochemical regulation
of owering
The genetic control of ower initiation and ower
development, and the molecular mechanisms that
are responsible for the regulation of these processes
have been studied in detai l (reviewed in Stewar t et al.
2016). Saintpaulia has been included in some of these
studies. Wang et al. (2006) isolated two CYC-like
genes, SiCYC1A and SiCYC1B, from zygomorphic
and actinomorphic cultivars respectively, of
S. ionantha, using mTAIL-PCR. The two SiCYC1A
genes contained the entire regulatory domains
(i.e., TCP and R domains) that were functional
in establishing oral symmetry, and these were
homologous with the Antirrhinum majus CYC gene.
Unexpectedly, the two SiCYC1B genes from the
actinomorphic cultivar had a sequence identical to
genes from the zygomorphic cultivar. Comparative
analysis of molecular alterations in CYC-like genes
responsible for morphological transformation from
zygomorphy to actinomorphy indicated that the
two closely related SiCYC1A and SiCYC1B genes
were perhaps regulated by a common, upstream
regulator. Change in this regulator could result in
the silencing of both SiCYC1A and SiCYC1B, thus
L* 27.7
a* 72.7
b* -41.3
L* 34.0
a* 69.7
b* -33.4
L* 50.4
a* 39.7
b* -18.8
L* 57.8
a* 39.1
b* -9.8
(a)
(b)
(c)
(d)
(e)
Figure 1. Schematic representation of F3'5'H and its promoter region (not to scale). Variegated individuals (a) have
two types of F3'5'H (avonoid 3',5'-hydroxylase) sequences: one has a 3844-bp insertion and the other appears to
have a post-excision sequence (see c). Solid-pink mutants (b) also have two types of sequences: one has imperfect
transposon insertion and the other has erratic post-excision sequences varying from 58 to 70 bp. Solid-blue mutants
(c) have a sequence with an 8-bp footprint. Deep-purple (d) and solid-purple (e) mutants have sequences lacking 21-
and 24-bp regions, respectively, compared to the solid blue mutant (see: c). Accession numbers (GenBank): transposon
VGs1AB596833, solid pink – AB596835-AB596837, solid blue – AB596834, deep purple – AB596838, solid purple
– AB596839. Figure modied from Sato et al. (2011a,b) and Matsuda et al. (2014). L*, a*, b* – refers to the Hunter
colour scale (Hunter 1948)
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J.A. Teixeira da Silva, Y.H. Dewir, A. Wicaksono, L. Sahijram, H. Kim, S. Zeng, S.F. Chandler, M. Hosokawa 105
controlling the development of the adaxial and
lateral organs in a ower.
Pei et al. (2012) reported that the range of pH
values in cellular sap of the linguoid petal in blue
and white ower S. ionantha cultivars was 3.0 to
7.0. The pH value (6.21) of the blue ower cultivar
was higher than that of the white ower cultivar
(pH 3.32) on the rst day of owering and increased
when the owering period was extended from 1 to
20 days. The changes were greater in the white
cultivar than in the blue cultivar. On the 20th day of
owering the pH value was 6.64 in the blue cultivar
and 4.21 in the white cultivar.
2. Genetic modication
Genetic modication of ornamental plants is an
important means of introducing new characters
such as modied ower colour, leaf shape or plant
architecture (Teixeira da Silva et al. 2016b). The
technology for genome editing has progressed
considerably in recent years, and using techniques
such as CRISPR/Cas9 (Samanta et al. 2016) it
is now possible to knock out a specic gene or
to target a specic position on the genome for
genetic modication. Now in its infancy, articial
chromosome technology offers an opportunity
for multiple gene transfer in the future as part of
a genetic engineering strategy (Yu et al. 2016).
It seems that genetic transformation could also
help create plants resistant to Melodogyne arenaria
Thamesii, a root-knot nematode known to infect
the roots of potted S. ionantha plants (Goidanich
and Garavini 1959).
2.1. Transformation of Saintpaulia
Several researchers have reported the production
of transgenic Saintpaulia plants. Kushikawa et
al. (2001) succeeded in Agrobacterium-mediated
transformation of S. ionantha. After testing
Agrobacterium strains LBA4404 (plasmid
pTOK233), EHA101 (pIG121 hygromycin) and
LBA4404 (pIG121 hygromycin), positive results
were obtained for LBA4404. In their experiment,
a suspension culture of Saintpaulia ‘Pink Veil’
was exposed to LBA4404 for 48 h, and after
4 months of culture on selection medium containing
50 mg l-1 hygromycin, hygromycin-resistant callus
was obtained. Transgenic plants harbouring the
gusA gene were conrmed by PCR and Southern
blot analysis, although the number of transgenic
plants produced and the transformation efciency
were not indicated. No transformants were derived
directly from leaves but only via callus. Using the
same plasmid constructs as in their 2001 study,
Kushikawa et al. (2002) were able to increase the
number of GUS foci by co-cultivating leaf explants
with 300 µM acetosyringone. Mercuri et al. (2000)
obtained transgenic Saintpaulia ‘Rhapsody’ plants
using A. tumefaciens oncogenic strain A281 after
infecting petiole slices (3-10 mm thick), but no
plants were recovered from co-cultivated leaves.
No success was obtained when the disarmed strain
EHA105 was used. In their protocol, 30-min.
co-culture with Agrobacterium followed by the
culture of explants in the dark on selection medium
containing 100 mg l-1 kanamycin resulted in high
(90% of explants) transient GUS gene expression
after 3 days, reduced expression (30% of explants)
after 15 days, and no expression after 25 days.
The GUS gene was not detected by PCR in all the
GUS-positive plants tested, but the nptII gene was
detected in Southern blot analysis, suggesting either
loss of the transgene, or transgene silencing. Ram
and Mohandas (2003) used LBA4404 harbouring
pBINAR carrying a glucanase-chitinase gene to
transform Saintpaulia ‘Sailors Delight’ leaves after
co-cultivation for 5 min., and following culture in
the dark in the presence of 70 mg l-1 kanamycin.
A high percentage (75%) of putative transformants
showed a signal for the glucanase gene in Southern
blot analysis. Ohki et al. (2009) suggested
that Saintpaulia varieties ‘Heavens A-calling’,
‘Kris’, and ‘New Mexico’ leaf explants dipped in
a solution of A. tumefaciens (pIG121HM) should be
cultured at a minimum of 30 mg l-1 hygromycin for
selection. Ye et al. (2014) genetically transformed
the AtTIP5;1 gene (a highly expressed Arabidopsis
pollen-specic gene related to aquaporin) into
the leaves of an unspecied S. ionantha cultivar
using A. tumefaciens strain GV3101. The authors
found that pre-culture for 2 d on non-selective
shoot induction medium, infection for 15 min., co-
culture for 2 d with 100 µmol l-1 acetosyringone
and selection with 40 g l-1 hygromycin were the
optimal transformation conditions. After screening
with PCR and RT-PCR, 17 transformed plants
containing the AtTIP5;1 transgene were obtained.
Tra nsfor med AtTIP5;1 plants showed improved
tolerance to boron stress.
In addition to the Agrobacterium strain and
the selection marker used, the stress caused by
cutting the explants is important for Agrobacterium
infection since S. ionantha plants are very sensitive
to local wounding of leaves (Yang et al. 2002).
Wounding of leaves induces a hypersensitive
state in explants, such as the leaves of the species
of the Acanthaceae and Gesneriaceae, including
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106 African violet breeding and progress in biotechnological techniques
Saintpaulia sp., resulting in the browning of
unwounded areas within 30 min. as a result of
secondary wounding (Yang et al. 2002, 2003,
2006). Therefore, leaf wounding, such as that
caused during the preparation of leaf segments, may
have a negative effect on Agrobacterium infection.
Ghorbanzade and Ahmadabadi (2015) used particle
bombardment to genetically transform a local
Iranian cultivar of African violet with the GUS
gene. An endochitinase gene was then introduced
and stable integration was conrmed by PCR and
RT-PCR in 4 out of 7 lines.
2.2. Flower colour modication in transgenic
Saintpaulia
Depending on the variety, Saintpaulia owers
produce delphinidin-, cyanidin- and/or pelargonidin
-based anthocyanins (Griesbach 1998, Tatsuzawa et
al. 2012, Tat su z awa and Hos o k awa 2016 ). Cyan id in -
based anthocyanins accumulate in the leaves of
some varieties, imparting a deeper, but less green
colour to the leaves (Arisumi 1967). Chalcone-
based avonoids have been shown to be the yellow
pigment present in yellow owers (Deguchi et al.
2016).
Molecular analysis has shown that there are
two distinct chalcone synthase (CHS) genes in
the Saintpaulia genus, SaCHSA and SaCHSD
(Caro et al. 2006). CHS codes for the rst enzyme
in the avonoid production pathway. Using
phylogenetic analysis, the CHS genes have been
used to differentiate several Saintpaulia species
[S. grandifolia (S. ionantha ssp. grandifolia),
S. grotei (S. ionantha ssp. grotei), S. intermedia
(S. ionantha ssp. pendula (B.L. Burtt) I. Darbysh.
TSN 832528), S. ionantha and S. orbicularis
(S. ionantha ssp. orbicularis (B.L. Burtt) I. Darbysh.
TSN 832596] (Caro et al. 2006). Jiao et al. (2014)
cloned the avonoid 3',5'-hydroxylase (F3′5′H ) gene
from S. ionantha (cultivar not indicated) and found
maximum homology among S. ionantha cultivars,
and also with Antirrhinum kelleggii and Torenia
hybrida. Sense F3′5′H expression vectors were
transformed into tobacco using A. tumefaciens
strain LBA4404. In transgenic tobacco, the
avonoid content detected using HPLC was 4.0-
16.3% higher than that in the wild type (1.76%), and
owers were light purple.
Despite the large palette of colours available
amongst African violet varieties, there are still
colour modications that would be very valuable
PAL, 4CL, CHS
CHI
F3’H
F3’5’H
F3H
F3’5’H
In sertion of auron e
bi osynthesis genes
RNA i o r co-s uppres sion to
bl ock anthocyanin
bi os ynthesis
D o wn regu la tion o f F 3’5 ’H to
l ead to 100% cya nidin-base d
a nt ho cyani ns
Phenylalanine 2’,4’,6’,4-tetrahydroxychalcone
Yellow flowers
Naringenin
Cyanidin Pelargonidin Delphinidin
Pink flowers
Peonidin Petunidin, malvidin
Red flowers Purp le violet flowers
Figure 2. Schematic representation of the ower pigment biosynthesis pathways in Saintpaulia showing (lled text
boxes with grey-light blue) the points at which genetic modication could be applied. In this simplied diagram, key
enzymes are abbreviated as follows: PAL Phenylalanine ammonia lyase, 4CL 4-Coumarate-CoA ligase, CHS
– chalcone synthase, CHI – chalcone isomerase, F3H – avanone 3β-hydroxylase, F3'H – avonoid 3’-hydroxylase,
F3'5'H – avonoid 3’,5’-hydroxylase
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J.A. Teixeira da Silva, Y.H. Dewir, A. Wicaksono, L. Sahijram, H. Kim, S. Zeng, S.F. Chandler, M. Hosokawa 107
in Saintpaulia and which can only be achieved
through genetic engineering. Broadly, the rst
strategy would be to increase the colour range in
a single, very high quality variety, as was done
using genetic engineering in torenia (Nishihara
et al. 2013). The second strategy would be to use
genetic engineering for the generation of colours
not found in the species, such as a true red (van
Schaik and Newlands 1963) or deep yellow. Aside
from an undetailed report of a European eld trial
in 2001 (Cadic and Widehem 2001), ower colour
modication by genetic engineering has not yet
been demonstrated in Saintpaulia, although there
are multiple theoretical avenues for ower colour
modication in this genus, as illustrated in Figure
2. In summary, avenues that may be explored alone
or in combination are:
In white-owered varieties, including
coloured varieties in which the anthocyanin
pathway has been down-regulated, transfer
of betalain pigment biosynthesis genes for
the production of novel yellow aurone-based
pigments (Ono et al. 2006).
Up and/or down regulation of the avonoid
hydroxylase genes on the existing
anthocyanin pathway to alter the type and/
or ratio of anthocyanins accumulated. As
an example of such a strategy, to produce
a ‘true red’ variety, the anthocyanin pathway
could be manipulated to produce varieties
that only accumulate red cyanidin-based
anthocyanins.
Manipulation (through methylation or
glycosylation) of the secondary structure
moieties of existing anthocyanins to alter
spectral qualities and thus ower colour
(Tatsuzawa and Hosokawa 2016).
Down-regulation of endogenous chalcone
synthase genes to block anthocyanin
biosynthesis. With suitable promoters this
strategy could also be used to alter leaf colour
by inhibiting anthocyanin accumulation in
the leaves of affected varieties.
CONCLUSIONS
Radiation- or colchicine-induced mutagenesis has
served as a useful tool to broaden genetic diversity
in African violet, a vegetatively propagated
ornamental species. As described in several papers
in this review, at the research level mutation breeding
and tissue-culture induced somaclonal variation
have led to several changes in African violet plant
architecture, including petiole length, surface area,
ower diameter, inorescence height and number
of leaves and inorescences per plant. Several
economically desirable traits such as ower colour
pattern, chlorophyll chimerism and dwarng have
also been observed in African violet using radiation
and chemical mutagens. These traits can be stably
propagated through vegetative means, including
leaf culture (Teixeira da Silva et al. 2016a). African
violet plants are very sensitive to alteration in light
intensity, temperature, and humidity, and minor
changes can lead to the development of leaf spot
disease (Yun et al. 1997, Yang et al. 2001, Chen and
Henny 2009). Plants resistant to biotic and abiotic
stresses will need to be developed using transgenic
strategies, and this requires robust and reproducible
genetic engineering and tissue culture protocols
to regenerate transformants. These techniques
are now available. Agrobacterium-mediated
genetic transformation has been shown to alter
Saintpaulia genetic makeup by the introduction of
a glucanase-chitinase gene (Ram and Mohandas
2003). Transformants with improved tolerance
to boron stress have also been obtained (Ye et
al. 2014). Genetic modication is also a tool that
can be used to generate novel ower colours in
Saintpaulia. However, to date, transgenic varieties
of African violet have not been commercialised.
Given the complexity of the regulatory processes
associated with the release of transgenic plants,
our expectation is that conventional breeding
methods, including mutagenesis, will thus continue
to dominate the product pipeline for African violet.
The conservation of wild Saintpaulia species will
continue to be a priority, and the best way to achieve
this is through in vitro propagation (Teixeira da
Silva et al. 2017). Molecular methods to classify
germplasm (Teixeira da Silva et al. 2007) will need
to be developed for African violet. To date, the
use of 5S nuclear ribosomal DNA non-transcribed
spacer sequences or internal transcribed spacer
sequences to clearly differentiate Saintpaulia
species in conservation studies (Möller and Cronk
1997, Lindqvist and Albert 1999, 2001) allows
germplasm to be accurately classied, propagated
and preserved.
ACKNOWLEDGEMENT
We are thankful to Ravi R. Sonani and Mafatlal
M. Kher, Sardar Patel University, and Sylvia
DeMar, Managing Editor, American Society for
Horticultural Science, for providing difcult-to-
access literature. We also thank Mafatlal M. Kher
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108 African violet breeding and progress in biotechnological techniques
for some useful suggestions about the review
structure in an earlier draft.
AUTHOR CONTRIBUTIONS
The authors contributed equally to all aspects of
review development and writing.
CONFLICT OF INTEREST
Authors declare no conict of interest.
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Received January 13, 2017; accepted May 23, 2017
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... African violet (Saintpaulia ionantha H. Wendl.; Gesneriaceae) 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]. ...
... The 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]. ...
... 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]. ...
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Background Flower color is one of the main characteristics of ornamental plants. Aurones are light yellow flavonoids 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 expressions 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.
... Expression of the CHS gene increased at the early stage of Petunia floral development and eventually decreased at the late stages (Saito et al., 2006). Studies have shown that CHS gene silencing blocked the biosynthetic pathway (Teixeira da Silva et al., 2017). Sun et al. (2015) indicated that in the transgenic Petunia, inhibition of CHS gene expression resulted in petal coloration (pink-to-white color). ...
... White flowers can be created by reducing the expression of genes controlling the anthocyanin biosynthesis pathway (Tai et al., 2014). Genetic regulation of F3H and F3'5′H genes in the anthocyanin biosynthesis pathway will lead to the accumulation of different anthocyanidin and eventually different colors (Han et al., 2017;Teixeira da Silva et al., 2017). Transcription factors are regulators of the anthocyanin biosynthesis pathway that modulate this pathway (Li, 2014). ...
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Adventitious buds from in vitro leaf explants of two different Saintpaulia ionahta (Mauve and Indikon) cultivars were irradiated with X-rays or 9 MeV electron beams, the tissue increase, buds differentiation and leaf morphology changes were studied. The results showed that the fresh weight of leaf explants of Mauve and Indikon irradiated to 40 Gy with the X-rays increased by 27.3 and 26.3 times, whereas they increased by 49.7 and 27.4 times with the E-beam irradiation of the same dose. Changes of fresh weight increase of explants irradiated to less than 20 Gy by both X-rays and electron beams demonstrated a tendency of going down first and then increasing, which differed from those irradiated to higher than 20 Gy. The percentage of bud formation of Mauve and Indikon irradiated to 100 Gy with the electron beams were 3.7% and 11.3 %, respectively, while they were 7.5% and 64.1% with the X-ray irradiation of the same dose. The percentage of plantlets with malformed leaves in Mauve irradiated with 60 Gy E-beam irradiation was 22.2%, while it was 14.8 % with X-ray irradiation of the same dose. For Saintpaulia ionahta Indikon with 40 Gy E-beam irradiation, the percentage was 35.2 %, whereas it was 5.8 % for X-ray irradiation of the same dose. The results show that the effect of mutation induction by the electron beam irradiation on Saintpaulia ionahta is better than by the X-ray irradiation. An optimal mutagenic treatment is 40-60 Gy irradiation with the electron beams.