Breeding for Different Flower Forms in Ornamental Crops

IndianAgriculturalResearchInstitute,NewDelhi
Breeding for Different Flower Forms in
Ornamental Crops
Abhay Kumar Gaurav
10459
Ph. D. 1st Year
Seminar: FLA 691
Division of Floriculture and Landscaping
Indian Agricultural Research Institute, New Delhi

Flower shape/form is one of the most important characteristics in
ornamental plants
Creation of new flower shapes in ornamental plants is a major
breeding target
Phenotype with unique forms of flower or, the double flower has
higher ornamental value than the single one
Key transcriptional factors for the identification of floral organs
have been clarified by analyzing model plants (Arabidopsis)

ABCDE Model of Flower Development
This model developed on the basis of
Arabidopsis thaliana mutants. Most of the
genes of ABCDE model are MADS-box
genes.
Class A genes (APETALA1) controls
sepal development & together with class B
genes (e.g. PISTILLATA, and APETALA3),
regulates the formation of petals.
Class B genes, together with class C
genes (e.g., AGAMOUS), mediates stamen
development.
Class C genes determines the formation
of carpel.
The class D genes (e.g., SEEDSTICK,
and SHATTERPROOF) specify the identity
of the ovule
Class E genes (e.g., SEPALLATA),
expressed in the entire floral meristem, &
are necessary
(Dornelas & Dornelas 2005)

c)
Sepals

 Hybridization
 Mutation
 Polyploidy
 Genetic Modification
 RNAi or Gene silencing
 Chimeric REpressor gene-Silencing Technology (CRES-T)
 Micro RNA

 Single, semi-double & double type of flower are genetically controlled
 Based on gene governing, doubleness can be transferred into new
cultivar by hybridizing with suitable parent
No. of Gene Single Multiple
Doubleness is dominant Rose
China Aster
Marigold
Impatience
wallerana
Doubleness is incompletely
dominant
Carnation -
Singleness is partially
dominant
Chrysanthemum -
Singleness is dominant Stock Zinnia

c)
 The morphology of
flowers & inflorescences
can be affected by
mutation
 Mutation induction led to
changes in flower size,
petal shape, petal
numbers
 In Compositae, an
increase in whorls of
ligulate florets as well as
a conversion from
ligulate into tubular
florets was described

Carnation varieties co-developed by Kirin Agribio
and the JAEA using ion beams. The flower on the
upper-left corner is the parent(var. .Vital.) and the
others are mutants. Tanaka et al., 2010

Polyploidy
 Polyploidy breeding : Effective
method to double the chromosome
number
 Genetic variations created can be
further used in breeding
 Main consequences of induced
polyploidy are increase in size and
shape of plants/leaves/ branches,
flower parts, fruits & seeds
(Chopra, 2008)
Fig. Field performance of diploid and
tetraploid Gerbera jamesonii Bolus cv. Sciella.
a. Variation in plant characteristics between
diploid (2x) and tetraploid (4x);
b. variation in stalk length between 2x & 4x;
c. variation in flower dia between 2x & 4x
Gantait et al., 2011

Genetic Modification of Ornamental Plants
 It offer new opportunities for breeders of ornamental plants
 Development of new ornamental varieties through gene transfer is
possible by this technique
 Genetic engineering can introduce traits not be generated by
conventional breeding
 Major traits amenable to manipulation by genetic modification include
flower color, fragrance, abiotic stress resistance, disease resistance,
pest resistance, manipulation of the form and architecture of plants
and/or flowers, modification of flowering time, and post harvest life etc.
 Ex: Chrysanthemum, Torenia: Fringed petal
 Cyclamen, Petunia: Double flower

RNA interference technology
 RNA interference (RNAi)
is a naturally occurring
mechanism that leads to
the “silencing” of genes
 In consequence, the
respective protein is not
synthesized
 This technique can be
used for loss-of-function
studies where a gene is
specifically silenced and
character is not
expressed

Chimeric REpressor Gene-Silencing Technology (CRES-T)
 CRES-T is a recently developed technology
 It induces a dominant loss-of-function phenotype of endogenous plant
Transcription Factor by expression of a chimeric repressor
 Transcription factors (TFs) are key regulators for the control of various
plant phenomena
 Here TF is fused with the plant-specific EAR-motif repression domain,
SRDX (Mitsuda et al., 2011)
 It suppresses target genes of a transcription factor dominantly
 CRES-T has been successfully utilized to modify the shape of torenia
(Shikata et al., 2011), chrysanthemum (Narumi et al., 2011), morning glory
(Sage-Ono et al., 2011), cyclamen (Tanaka et al., 2011) and rose plants
(Gion et al., 2011).

micro RNA
 A microRNA (miRNA) is a small non-coding RNA molecule (about 22nucleotides)
found in Eukaryotes, which functions in RNA silencing and post-
transcriptional regulation of gene expression
 miRNAs are involved in almost all biological and metabolic processes (Khraiwesh
et al., 2012)
 miR156: Plant architecture (Jiao et al. 2010). miR319: Leaf & Petal
morphogenesis in Snapdragon (Carle et al., 2007)
MIR gene
RNA Pol
AGO
RNA Pol
miRNA -
mediated slicing
of mRNA and
translational
repression
mRNA
AGO
AGO
AAAn

MADS-box
 The MADS box is a conserved sequence motif found in genes which
comprise the MADS-box gene family
 The MADS box encodes the DNA-binding MADS domain
 The length of the MADS-box are in the range of 168 to 180 base pairs
 Origin:
 MCM1 from the budding yeast, Saccharomyces cerevisiae,
 AGAMOUS from the thale cress Arabidopsis thaliana,
 DEFICIENS from the snapdragon Antirrhinum majus
 SRF from the human Homo sapiens
In plants, MADS-box genes are involved in controlling all major aspects
of development, including male & female gametophyte development,
embryo and seed development, as well as root, flower and fruit
development, floral organ identity and flowering time determination

CASE STUDIES

c)
Case Study-1
Objectives:
1. Study of the effects of the gene silencing of C-class MADS-box genes by using a
VIGS system on flower phenotypes in petunia cultivars.
2. Comparison between Large petaloid stamens induced by silencing both
pMADS3 and FBP6 with small petaloid stamens induced by silencing only
pMADS3.

c)
 Double flowers enhances the commercial value of Petunia hybrida. As
ornamental plants, double flowers with large petaloid stamens and/or
new flowers at inner whorls are desired
 Double flower formation: Mainly due to conversion of stamen and
carpel into petal and new inflorescence
 C-class genes along with B-class genes, specify stamen identity in whorl
3. A/C to ABC model of floral organ identity (Coen and Meyerowitz,
1991)
 Suppressing C-class genes in whorl 3 results in the conversion of stamen
into petal. C-class genes also specify carpel identity in whorl 4 and
control floral meristem determinacy, their suppression induces the
indeterminate development of flowers in whorl 4
 C-class genes belong to AG-clade of the large MADS-box gene family

Petunia has two genes belonging to the AG-clade:
 euAG- subclade gene PETUNIA MADS-BOX GENE3 (pMADS3) and
 PLENA- subclade gene FLORAL BINDING PROTEIN6 (FBP6)
(Angenent et al., 2009; Tsuchimoto et al., 1993)
 Silencing of either pMADS3 or FBP6 resulted in partial loss of stamen
identity and slightly altered carpel morphology. No double flower
 Flowers with both pMADS3 and FBP6 silenced exhibited near-complete
loss of both stamen and pistil identities . They were completely converted
into large petaloid tissues in whorl 3, new flowers were formed instead of
carpels in whorl 4, and ornamental double flowers were produced

Materials and Methods
Plant materials:
 VIGS treatments of each of the C-class MADS-box genes, pMADS3 and FBP6, and
of pMADS3 & FBP6 conducted in four petunia cultivars, ‘Cutie Blue’, ‘Fantasy
Blue’, ‘Picobella Blue’,and ‘Mambo Purple’
Plasmid construction:
 The tobacco rattle virus (TRV)-based VIGS system (suppression of the anthocyanin
pathway via chalcone synthase silencing as reporter as it produced white flower)
 Vector: pTRV1 and pTRV2 VIGS
 PhCHS was amplified and cloned into the EcoR1 site of pTRV2 vector
 The non-conserved regions of petunia C-class genes, pMADS3 and FBP6, were
amplified using the primers and cloned into the SmaI site of pTRV2 PhCHS vector
individually to generate constructs for silencing pMADS3 and FBP6 separately and
fused to generate a construct for silencing pMADS3 and FBP6 simultaneously

Agroinoculation of TRV vectors:
 Virus infection was carried out by
means of the Agrobacterium-
mediated infection of petunias
 Young leaves of 3-week old
petunia plants were inoculated
Quantitative RT-PCR of C- and A-class MADS-box genes:
Quantitative RT-PCR (qRT-PCR) of C- and A-class MADS-box genes in petals
and stamens of VIGS-untreated control flowers and petaloid stamens of
VIGS-induced flowers was performed.

Results and Discussion
In ‘Picobella Blue’ and ‘Mambo Purple’: No white flower was noted
(Unknown genetic background, Chen et al., 2004)
In ‘CutieBlue’ and ‘Fantasy Blue’: Completely white double flowers were
observed, indicating the strong and complete silencing
In flowers inoculated with either pMADS3-VIGS orFBP6-VIGS,
morphologically significant but small conversions in whorls 3 & 4 were
observed
In flowers of pMADS3-VIGS inoculated petunias, anthers converted into
small petaloid tissues but filaments retained their original struc (Fig. 1c & d)
In flowers of FBP6-VIGS inoculated petunias, the stamens were almost
unaffected
In petunias inoculated with pMADS3/FBP6-VIGS, prominent double flowers
with highly ornamental appearance formed. Complete loss of stamen
identity was observed. Both anthers and filaments were completely
converted into petaloid tissues

Fig. 1. Morphological changes in
flowers of P. hybrida cv ‘Cutie Blue’
inoculated with pTRV2-
PhCHS/pMADS3 (pMADS3-VIGS) and
pTRV2-PhCHS/pMADS3/FBP6
(pMADS3/FBP6-VIGS).
(a) VIGS-untreated control flower;
(b) Stamens and a carpel of non-VIGS
flower;
(c) pMADS3-VIGS flower (white and
blue mixed color);
(d) Petaloid stamens and a carpelof
pMADS3-VIGS flower;
(e) pMADS3/FBP6-VIGS flower
(white);
(f) Petaloid stamens and a carpel of
pMADS3/FBP6-VIGS flower
(white).

Fig. 2. Morphological changes in
flowers of P. hybrida cv
‘Fantasy Blue’, ‘Picobella Blue’,
and ‘Mambo Purple’ inoculated
with pTRV2-
PhCHS/pMADS3/FBP6
(pMADS3/FBP6-VIGS).
(a–c) ‘Fantasy Blue’;
(d–f) ‘Picobella Blue’;
(g–i) ‘Mambo Purple’;
(a, d and g) VIGS-untreated
control flowers;
(b, e and h) pMADS3/FBP6-VIGS
flowers;
(c, f and i) stamens and carpels or
converted new flowers of
pMADS3/FBP6-VIGS flowers.

Fig. 3. New flower formation in whorl 4
and from axil of whorl 3 in a double
flower of P. hybrida cv ‘Mambo Purple’
inoculated with (pMADS3/FBP6-VIGS).
(a) An opened double flower with a
second new flower in whorl 4
(b) An opened second new flower;
(c) Fused corolla (left), a carpel
(center), and petaloid stamens
(right) of the second flower;
(d) An ectopic new flower emerging
from the axil of whorl 3;
(e) An unconverted stamen (left) and
petal-like tissues of the ectopic new
flower.
Flowers inoculated with pMADS3/FBP6-VIGS in whorl 4, carpels converted into new flower
(Cultivar-dependent)
In 50% of the double flowers of ‘Mambo Purple’, a 2nd new flower arose instead of a carpel. This
process was repeated, generating 3rd & 4th new flowers. It exhibited a voluminous and
decorative appearance with a high commercial value.

The surface areas of petaloid
stamens in pMADS3/FBP6-VIGS
plants were more than 10 times
as large as those in pMADS3-
VIGS plants
Upper limb-like region of the
large petaloid stamens in
pMADS3/FBP6-VIGS plants
accounted for > 90% of the total
area, so it was mostly due to the
development of this region
The average sizes of epidermal
cells in plants inoculated with
pMADS3/FBP6-VIGS were only
1.5 times as large as those in
plants inoculated with pMADS3-
VIGS

c)
 Double flowers can be induced by virus-induced gene silencing
(VIGS) of two C-class MADS-box genes, pMADS3 and FBP6
 Large petaloid stamens induced by pMADS3/FBP6-VIGS were
compared with small petaloid stamens induced by pMADS3-VIGS
 New flower formation in the inner whorl of flowers silenced in both
pMADS3 and FBP6 gene is cultivar-dependent
 They are valuable for future breeding of petunia cultivars bearing
decorative double flowers with large petaloid stamens and inner
new secondary flowers

c)
Case Study-2
Objectives:
1. Isolation of flower color and shape mutants
2. Development of in vitro mutation technique to obtain new varieties of
chrysanthemum by using gamma rays

c)
 Chrysanthemum morifolium Ramat: Important ornamental flowers
 For commercial floriculture: Demand for novel varieties
 Radiation used for the development of new flower color/shape mutants
in Chrysanthemums (Misra et al., 2003)
 The selection of ornamentals is easy with visible characters (color,
shape and size, or leaf form and growth habit).
 The main bottleneck is formation of chimera where, the size of the
mutant sector varies from a narrow streak on a petal to the entire flower
 Many new flower color/shape mutants, lost due to the lack of a
regeneration system from small-mutated sectors either in vivo or in vitro
 Therefore, a regeneration system to establish mutant in pure form from
a chimera and production of a wide range of new cultivars with novel
flower colors and shapes is required

 Work was done at China Agricultural University, Beijing, China
Plant materials
 White C. morifolium Ramat cv. Youka plants regenerated from explants
of petals (spoon shape) used in the experiment
Culture
 White petals (from Bud) 4 mm in length of the original cultivar were
excised and cultured on MS basal medium
 The calli was induced on a callus medium is comprised of MS medium
supplemented with 1.0 mg/l NAA, 2.0 mg/l BAP
60CO radiation treatments
 Callus was exposed to gamma radiation using 60Co of gamma chamber
with doses of 0(Control), 10, 15 and 20 Gy and dose rate 1.02 Gy/min.
Materials and Methods

Fig. 1 In vitro regeneration C.
morifolium ‘Youka’ from ray
florets.
A) White flower buds;
B) Callus induction medium;
C) Adventitious shoots formation
after 4 weeks;
D) In vitro Roots formations after
25 days;
E) Plantlets in hardening chamber

Results and Discussion
Table: In vitro callus survival
(%) and number of
shoots(Mean ± SE) of white C.
morifolium ‘‘Youka’’ as
influenced by gamma ray doses
Gamma
ray
dose
(Gy)
Callus
survival
(%)
No. of shoots
0 86.67 7.22 ± 0.11a
10 62.43 7.67 ± 0.33a
15 30.33 7.89 ± 0.29a
20 17.23 3.00 ± 0.29b
Effect of gamma radiation on
chrysanthemum callus
Table: Effect of in vitro treatment of C. morifolium ‘Youka’
with gamma radiation on flowering characteristics of the
generated plantlets
Character Control 10 Gy 15 Gy 20 Gy
Flower No/plant 4.22 ±
0.29a
4.38 ±
0.12a
4.05 ±
0.41a
3.00 ±
0.00b
Flower dia (cm) 6.12 ±
0.23a
6.11 ±
0.15a
5.15 ±
0.18b
4.88 ±
0.06b
Petal length (cm) 3.03 ±
0.20a
3.14 ±
0.19a
3.00 ±
0.01a
3.01 ±
0.01a
Petal width (cm) 0.83 ±
0.07a
0.87 ±
0.12a
0.97 ±
0.03a
1.05 ±
0.05a
Petiole length (cm) 4.50 ±
0.17b
4.72 ±
0.18b
6.52 ±
0.29a
6.51 ±
0.30a
Petiole dia (mm) 2.50 ±
0.11a
1.89 ±
0.04b
1.89 ±
0.04b
1.85 ±
0.04b
Similar letters in the same row indicate that they were not significantly
different from LSD005 test
Effect of gamma radiation on flower
characteristics

Fig. Flower of tissue-raised plants .
A) Control, white colored/spoon shaped petals; B) M1, white colored / tubular
petals; C) M2, yellow colored/spoon shaped petals; D) M3, yellow colored/ flat
shaped petals
The frequencies of flower color and shape mutations increased when the total dose
was increased from 10 to 15 Gy, though it was not observed when the dose was
increased from 15 to 20 Gy.
Figure below illustrates the three mutants obtained in 15 Gy treated plants.

c)
 Gamma radiation with 15 Gy dose can be used for in vitro
induction of flower color and shape mutations of
chrysanthemum cv. Youka
 The isolated mutants on in vitro culture can be multiplied and
rooted in vitro to produce new varieties of chrysanthemum

c)
Creation of new flower shapes in ornamentals plants is a major
breeding target as increase its commercial value
Flower development is controlled by Gene (ABCDE-Model)
Different flower shape including double flower can be
developed by different breeding techniques like Hybridization,
Mutation, Polyploidy, Genetic engineering etc.
Now a days new techniques like RNAi, CRES-T, miRNA and
other gene silencing techniques are being used to developed
altered flower shape
Even though there are many techniques available but, very few
variety has been developed for commercial purpose by Genetic
transformation

c)
 Identification of flower shape mutant and development of
in-vitro protocol for their regeneration
 Most of the genetic modification for flower form is done in
Arabidopsis & Snapdragon only, it has be done in
commercially important crop
 Only few GM crop is released for commercial purpose i.e.,
Rose & Carnation (Flower colour). Effort is to be done to
developed GM crop with modified flower architecture

c)

Breeding for Different Flower Forms in Ornamental Crops

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