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Chapter 23
CRISPR edited floriculture crops:
A revolutionary technique to
increase flower production, their
color and longevity
Talakayala Ashwinia,b,e, Veerapaneni Bindu Prathyushaa, Nihar Sahua,c,
Dhanasekar Divyaa,fand Garladinne Mallikarjunaa,d
aPlant Molecular Biology Laboratory, Agri Biotech Foundation, Hyderabad, Telangana, India, bDepartment of Genetics,
Osmania University, Hyderabad, Telangana, India, cDepartment of Horticulture, Sunchon National University, Suncheon,
South Korea, dDepartment of Molecular Biology and Micropropagation Group, Plantigen Agrisolutions, Ragimanupalle,
APCARL Road, Pulivendula, Andhra Pradesh, India, eInternational crop research Institute for Semi-Arid Tropics,
Hyderabad, Telangana, India, fDepartment of Agricultural Education, Sunchon National University, Suncheon, Jeonnam,
Republic of Korea
23.1 Introduction
CRISPR genome-editing is an applied approach for bio-engineering and to make modications in different genomic
locations in any crop for trait improvement. For a couple of years, huge attention has been paid to this technology due
to its wide advantages, particularly in transgene-free regulation. CRISPR-Cas system possesses gRNA and Cas tools, which
are essential for achieving mutations in dened locations in genes or genome through deletion, insertions, and replacements
(Nishihara et al., 2023). There are three reported major Cas systems, i.e., Cas9, Cas13, and Cpf1. Cas9 or cpf1 has a single-
protein CRISPR effector, which creates a blunt cut in the DNA and can be repaired by either nonhomologous end-joining or
homologous recombination (Talakayala et al., 2022a). With this technology, we can create a site-specic edit belonging to
type II A with the ideal of off-targets to escape. Recently, CRISPR-Cas9 has achieved its progress through Agrobacterium-
mediated or protoplast-based transformation systems, to produce transgenic-free plants that are free from the GE regulations.
There is further research on the way with the implementation of new tools like Cas13a and cpf1.
CRISPR-Cas12a–cpf1 system is more efcient for genome engineering in plants, belonging to type V, class II and it
uses a small guide RNA with trans-activating CRISPR-RNA, specically for T-rich regions that have the ability to generate
double-strand breaks (DSB) with staggered regions. Mostly, cpf1 editing is used in monocots and dicots species. Thus, by
improving its PAM sequences we can target the genes related to owering, i.e., color and longevity. Recently, this technology
has been used in commercial owering crop improvement (Bandyopadhyay et al., 2020).
Objectives:
rTo exploit the CRISPR-Cas approach for developing varieties of owering plants.
rTo edit the color, odor, and longevity-related genes to generate better-quality owers.
rTo enhance economic viability in the commercial markets and production of different ower-based products.
23.2 CRISPR-Cas in floriculture
Flowering and ornamental plant production is one of the big industries. Floriculture plants can be categorized into four
types namely bedding plants, houseplants, ower gardens, and cut owers. Bedding plants consists of owers that can
be grown in pots, hanging buckets, and for gardening purpose example: marigolds, ageratum, pansies, and petunias. A
houseplant is also known as a potted plant or as an indoor plant, which is grown indoors for decorative purposes in places
like residences and ofces, e.g., epiphytes, succulents, or cacti. Around 25 owering plants are used for backyard gardens
CRISPRized Horticulture Crops. DOI: https://doi.org/10.1016/B978-0-443-13229-2.00022-3
Copyright c
2024 Elsevier Inc. All rights are reserved, including those for text and data mining, AI training, and similar technologies. 381
382 SECTION | II CRISPR mediated genome editing in horticultural crops
FIGURE 23.1 CRISPR-Cas9 mechanism in oriculture crops. (A) sgRNA that directs Cas9 endonuclease protein binds to a specic region (crRNA) of
the genomic DNA. (B) Formation of target DNA (crRNA) and CRISPR complex. (C) Cas9 enzyme/guide RNA complex to edit specic genomic regions.
(D) Cleavage of DNA, therefore insertion or deletion takes place by disrupting of open reading frame of a gene.
namely: petunia, dahlia, million bells, fuchsia, African daisy, calendula, canna lily, snapdragon, dwarf sunowers, sarlet
sage, dahlia, geraniums, impatiens, begonia, alyssum, zinnia, marigold, bacopa, lobelia, trailing begonia, etc. Cut owers
are ower buds or owers with stem intact for decorative purposes, namely carnations, chrysanthemums, roses, and tulips.
Earlier many tools were used for gene editing such as zinc-nger nucleases (ZFNs) and transcription activator-like
effector nucleases (TALENs), but clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 has become
more popular than other editing tools due to its high specicity for target genes, low cost, simple design, and user-friendly
(Cong et al., 2013;Feng et al., 2013;Shan et al., 2013;Talakayala et al., 2022b). Flower color, shape, size, and fragrance
play an important role in oriculture research. Till now, many varieties have been developed for different traits by plant
breeders using classical breeding and mutation breeding techniques. Recently, gene editing using the CRISPR-Cas9 system
(Fig. 23.1) has been implemented in oriculture crops such as lilium, chrysanthemum, petunia, torenia, etc. for improving the
fragrance, color, and shelf-life of owers. Zhang et al. (2016b) edited the petunia plants with the phytoene desaturase (PDS)
gene, resulting in an albino phenotype. Using CRISPR has been performed on the hexaploid genome of chrysanthemums
for mutational studies (Kishi-Kaboshiet et al., 2017).
23.3 Economic importance of floriculture plants-floral status in the market
Floricultural crops are economically important among the horticulture production. Mainly, cut owers, such as carnations,
chrysanthemums, roses, and tulips, play a key role in the production and commercialization. Even though, the lifespan of
ornamental owers is much shorter but highly demanded by consumers due to their shape, colors, and fragrances (Shibata,
2008). As per a 2018 survey, the top 10 major ower-producing countries in the world are the Netherlands (52% of global
market share), Columbia (15%), Ecuador (9%), Kenya (7%), Belgium (3%), Ethiopia (2%), Malaysia, Italy, Germany, and
Israel (1% each). Cut ower market showed an annual growth rate of 6% during 2017–2018 and was negatively affected by
to COVID-19 pandemic. From an economical point of view, the owers are used in the bouquet market, essential oil and
perfume industry, and for pigmentation.
23.3.1 Flowers in the bouquet market
Flowers have an eminent place in human life socially, economically, and aesthetically. For any occasion like anniversary,
birthday, etc. ower bouquets are always preferable. The global cut ower market generated an income of approximately
$29.2 billion. Roses are highly preferable and generate an income of $14.3 billion (https://www.soocial.com/ower-industry-
statistics). Some of the important owers are discussed below.
23.3.1.1 Daffodil
Daffodil owers are highly preferred for making a bouquet due to its supreme beauty. These ower species originated from
southern Europe during the 16th century. Later, they were cultivated in the Western Mediterranean, particularly the Iberian
peninsula.There are around 40–200 different species available with four colors (yellow, orange, white, and violet).
CRISPR edited floriculture crops Chapter | 23 383
23.3.1.2 Rose
Roses are plants known for their showy owers used in creating bouquets. Rose owers originated from China and later
to Europe, France, Middle-East, Asian countries, etc. there are over 150 species in the genus and thousands of cultivated
hybrids with ten different colors (red, white, yellow, orange, green, blue, pink, purple, bicolor and black).
23.3.1.3 Gerbera
Gerbera ower is the fth most popular ower in the world which adds an extra charm to the celebrations and makes
relationships stronger. In the early 1920s, this ower was originated in North Africa and cultivated worldwide. The Gerbera,
there are more than 40 species of the plant worldwide. Different shades of yellow, white, pink, red, orange, lavender, salmon,
and bicolored.
23.3.1.4 Orchid
Orchids are one of the most symbolic owers around the world for their exotic appeal, vibrant colors, and various scents. In
the ancient days, these owers were used in medicine in China to cure illnesses of the lungs and pacify coughs. This ower
has 28,000 species with different colors like red, pink, white, blue, green, purple, orange, and yellow that have different
meanings. There are also hybrid orchids (crossbreed orchids), some of which are entirely human-made.
23.3.1.5 Carnation
Carnation owers are also commonly preferred by orists to create a bouquet. The carnation owers have been cultivated
for over 2000 years in Asia and Europe and spread to other countries. There are around 320–600 varieties available in a
wide variety of colors including pink, white, red, yellow, and scarlet.
23.3.1.6 Peony
Peony owers are native to Asia, Europe, and Western North America. There are around 25–40 species available with lobed
leaves and high fragrant, colors ranging from purple and pink to red, white or yellow, blooming in late spring and early
summer.
23.3.1.7 Lilya
Lilies are one of the most desired owers in the world. There are over 100 species in the Lilium genus, encompassing 2000
varieties and many hybrids, with different colors white, yellow, pink, red, and orange. Lilies are grown in temperate and
sub-tropical climates along the Northern Hemisphere—Europe, Asia, and North America.
23.3.2 Essential oil
In recent years, extraction and production of essential oils are trending and booming businesses in the perfume industry
worldwide. Most of the essential oil are extracted from owers like rose, tuberose, jasmine, etc. However, using advanced
technology the volatile compounds and essential oil are even extracted from seeds, roots of herbs, stems, leaves, barks
owers, and fruits (Table 23.1).
23.3.3 Production of the essential oil
The essential oils are used in different areas like aromatherapy, cooking, backing, beauty products, candle and incense sticks,
and perfume industries. The globe trade market growth percentage of essential oil in the several countries are Vietnam (14%
growth per annum), Poland (35% per annum), Nigeria (16%), Turkey (25%), South Africa (14%), Indonesia (14%), Saudi
Arabia (14%), India (19%), Spain (13%), Singapore (35%), Switzerland (14%), and Japan (13%). According to a survey
conducted in 2008, the top 10 exporters of essential oil worldwide are the United States, India, France, Brazil, Indonesia,
the United Kingdom, Germany, China, Argentina, and Switzerland. The income generated in the perfume industry in 2008
was 6.98 billion US$ and is estimated to increase 16% by 2027 to 11.68 billion US$.
Flower color plays an important role in plant sexual hybridization by attracting pollinators and the best product among
the oriculture products (Alkema and Seager, 1982). Thus, the industry produces the pigmentation on a large scale using
different protocols for each pigment (Tanaka et al., 2005;Chandler and Tanaka, 2007;Chandler and Brugliera, 2011). Plants
384 SECTION | II CRISPR mediated genome editing in horticultural crops
TABLE 23.1 List of flowers used for essential oil extractions.
S. no. Family name Scientific name Flower common name Oil name Uses
1 Rosaceae Rosa damascena Damask rose Rose oil Perfume ry
2 Rosaceae Rosa rubiginosa Sweet-brier Rosehip Medicinally seeds are used
3Geraniaceae Pelargonium
graveolens
Sweet-scented geranium Geranium Aromatherapy, hormonal
imbalance
4Oleaceae Jasminum
grandiflorum
Spanish jasmine Jasmine Flowery fragrance
5Lamiaceae Lavendula officinalis English lavender Lavender oil Fragrance medicinally
6Asparagaceace Polianthes tuberosa L. Tuberose Tuberose oil Natural flower oil
7 Magnoliaceae Micheliachampaka L. Champak Champaka or
Champa oil
Perfumery—attars and hair
oils
8 Apocynaceae Plumeria acutifolia Red frangipani Temp l e or
Plumeria oil
Perfumery and medicine
preparation
9 Magnoliaceae Magnolia stellata Star magnolia Magnolia Perf umery
10 Solanaceae Cestrum nocturnum Night-blooming jasmine Cestrum oil Perfumery and preparation
of attars
11 Oleaceae Nyctanthesarbortristis Night-flowering jasmine Parijata oil Perf umery
12 Pandanaceae Pandanus
odorotissimus
Kweda Keora oil Cosmetics
13 Asteraceae Calendula officinalis Pot marigold Marigold oil Medicinally used to treat
burns, cuts, greasy skins, and
rashes
14 Lamiaceae Melissa officinalis Lemon balm Melissa oil Oil has a lemony aroma and
sharp, floral lemon flavor
15 Cupressaceae Juniperus communis Juniper Junniper oil Medicinally-hair loss, oily
complexion, cellulite,
diabetes, and female
reproductive system remedy
16 Lamiaceae Ocimum basilicum Commin basil Basil oil Perfume
have three main pigments responsible compounds namely betalains (betaxanthin and betacyanin), carotenoids (carotenes
and xanthophylls) avonoids (chalcones, anthocyanins, avones, and avonols) (Table 23.2).
Majority of the owering plants have only carotenoids and avonoids for their color pigmentation. However, the
Caryophyllales order of dicotyledonous owering plants have betalains along with carotenoids and avonoids compounds
necessary for color development (Mabry, 1964).
23.3.3.1 Betalains
Betalains compounds are water soluble, stable at acidic pH, and induce nitrogen xation in plants (Steglich and Strack,
1990). The betalain is made up of two structural compounds namely betacyanins and betaxanthins. Betaxanthins structure
produces a color range between yellow–and orange and betacyanins structure produces between violet to red range (Mabry
and Dreiding, 1968). Since it has a spectrum of color range, it acts as a pH indicator in many of the food industry (Azeredo,
2009).
23.3.3.2 Carotenoids
Carotenoids are lipid-soluble and synthesized in plastid and it has antioxidant and pro-oxidant activities (Gross, 1991;
El-Agamey et al., 2004), function as photo-protective in plants, and act as precursors for vitamin A biosynthesis (Frank
and Cogdell, 1996;Goodman et al., 1966). In plants, carotenoids are present more in owers and fruits (Tanaka et al.,
2008). These carotenoids are used in the cosmetics and food industries for colors and are involved in developing new avor
molecules in the pharmaceutical industry (Kumar and Sinha, 2004;Schwab et al., 2008). Carotenoids have a color spectrum
CRISPR edited floriculture crops Chapter | 23 385
TABLE 23.2 List of pigments extracted from flowers.
S. no. Flower name Scientific name Family Flower color Pigments
1Marigold, calendula, rose Tagetes, Calendula officinalis Asteraceae Rosoideae Yellow Carotenoids
2Lily Lilium Liliaceae Orange Carotenoid
3Snapdragon Antirrhium Plantaginaceae Orange Anthocyanin and flavones
4Geranium Crane’s bills Geraniaceae Scarlet Anthocyanin
5Tulip Tulipa Lilioideae Scarlet Anthocyanin and carotenoids
6Pap e r flow er Bougainvillea glabraa Nyctaginaceae Red, purple Betalains
7Japanese camellia Camellia and begonia Begoniaceae Magenta Anthocyanin
8Vervain Verbena Verbenaceae Violet Anthocyanin
9Poppy Meconopsis betonicifolia Papaveraceae Blue Anthocyanin and co-pigments
10 Cornflower Centaurea cyanus Asteraceae Blue Anthocyanin-metal complex
of 450–570 nm, ranging from yellow to orange–red colors. Carotenoids can be seen in the plant tissue along with anthocyanin
and it is termed as co-pigmentation (Mudalige et al., 2003).
23.3.3.3 Flavonoids
Flavonoids are water-soluble, polyphenolic secondary metabolites that have many biological functions in plants. These
secondary metabolites protect the plant against pathogens; and ultraviolet (UV) light; increasing pollen fertility, and seed
germination; act as signal molecule in plant-microbe interactions, and provide pigments to owers, seeds, and fruits (Treutter,
2006;Guo et al., 2008;Kovaleva et al., 2007;Naoumkina et al., 2010;Tanaka et al., 2008). Flavonoids are responsible for
colors including orange, yellow, red, purple, violet, and blue. Generally, these compounds chalcones, aurones, anthocyanins,
avones, and avonoids are involved in ower pigments (Forkmann, 1991;Andersen and Jordheim, 2006;Skaar et al., 2012).
23.4 Biosynthetic pathways
The two classes of volatile compounds namely terpenoids and benzenoids are involved in the biosynthetic pathways.
Terpenoids are derived from the ve-carbon isoprene units which consist of 40,000 structures and belong to the largest
class of volatile compounds. Similarly, benzenoids compound is derived from phenylalanine and categorized as a second
class of volatiles.
23.4.1 Terpenoid biosynthesis
Terpenoids, C5 isoprene classied based on their structure as (hemiterpenes), C10 (monoterpenes), C15 (sesquiterpenes),
C20 (diterpenes), C25 (sesterpenes), C30 (triterpenes), C40 (tetraterpenes), and C40 (polyterpenes) (Muhlemann et al.,
2014;Ramya et al., 2018;Martin et al., 2003;Ashour et al., 2010). Isopentenyl diphosphate (IPP) or dimethylallyl
diphosphate are the main sources of terpenoids (DMAPP). These were further classied into three categories:monoterpenes
(1:1), sesquiterpenes and sterols (2:1), and diterpenes, carotenoids, and polyterpenes (3:1) (Gershenzon and Kreis, 1999).
Isopentenyl diphosphate isomerase converts the IPP to DMAPP inside cytosol and plastid (Phillips et al., 2008;Jin et al.,
2020). The plastids are known as monoterpenes (Dudareva and Pichersky, 2000;Chen et al., 2011). The limonene, (E)-b-
ocimene, myrcene, linalool, and a- and b-pinene are known as monoterpenes (Piechulla and Effmert, 2010;Yoo et al., 2013;
Oyama-Okubo and Tsuji, 2013). Geranyl diphosphate (GPP) and geranyl pyrophosphate are precursors to IPP (GGPP). GPP
(C10) is the source of monoterpenes, which are produced when one IPP and one DMAPP molecule are combined to form
GPP by the enzyme GPP synthase (Poulter and Rilling, 1981;Ogura, 1998). The mevalonic acid (MVA) pathway uses the
cytosol to produce sesquiterpenes. Farnesyl pyrophosphate synthase in this pathway condenses two IPP molecules and one
DMAPP molecule to create farnesyl diphosphate. Finally, farnesyl diphosphate is converted to sesquiterpenes by cytosolic
sesquiterpene synthases (Chen et al., 2009;Monson et al., 2013). Phytoene synthase catalyzes the condensation of two GGPP
molecules to form phytoene, the primary carotenoid, which is the rst step in the carotenoid pathway. Phytoene engages in
several enzymatic processes with carotenoid molecules (Zhou and Pichersky, 2020). The concentration of terpenoid varies
with developmental stage in both owers and fruits. Terpenes, for instance, build up in berries in the initial phases of growth.
386 SECTION | II CRISPR mediated genome editing in horticultural crops
Terpene compounds created during this stage could serve as precursors for the nished goods (Kalua and Boss, 2009). In
lavender (Lavandula angustifolia), terpenoid quantities are closely related to ower maturity (Li et al., 2019). Carotenoids
are also involved in the oral scent of Osmanthus fragrans (Baldermann et al., 2010;Xi et al., 2021). The blooming plants
Rosa moschata, Thymus vulgaris, Viola tricolor, Medicago marina, and Myrtus communis all produce b-ionone, a carotenoid
derivative (Paparella et al., 2021).
23.4.2 Phenylpropanoid/benzenoid biosynthesis
The second largest class of plant volatile organic compounds (VOCs) is composed of phenylpropanoids and benzenoids
(Knudsen and Gershenzon, 2006). The shikimate pathway converts the aromatic amino acid phenylalanine (Phe) into
phenylpropanoids and benzenoids (Maeda and Dudareva, 2012;Yoo et al., 2013). Cinnamate 4-hydroxylase (C4H)
hydroxylates CA to create p-coumarate, which is then converted into phenylpropanoids. As a result, p-coumarate is
transformed into p-coumaroyl-CoA by 4-coumaroyl CoA ligase (Deng and Lu, 2017).
The propyl side chain of CA is reduced by two carbons to create benzenoids. Multiple chain-shortening routes have been
postulated, including two cytosolic nonoxidative pathways (CoA-dependent or CoA-independent) and a peroxisome-based
b-oxidative pathway (Widhalm and Dudareva, 2015). The 4-hydroxycinnamoyl CoA ligase converts CA to cinnamoyl-CoA
as part of the CoA-dependent nonoxidative pathway. Following this, lyase converts 3-hydroxy-3-phenyl-propanoyl-CoA
from which cinnamoyl-CoA is catalyzed by hydratase to get benzaldehyde. The 3-hydroxyphenyl-propionic acid that CA
is transformed into by the enzyme hydratase into the nonoxidative CoA-independent route is then reduced by the enzyme
lyase to benzaldehyde (Widhalm and Dudareva, 2015). The nal step is the synthesis of benzoic acid by benzaldehyde
dehydrogenase, which is evident in Snapdragon blooms (Anthirrhinum majus)(Long et al., 2009) and proposed in petunia
owers (Kim et al., 2019). Petunia (Petunia hybrida) owers and Arabidopsis have been used to dene the b-oxidative
pathway (A. thaliana)(Moerkercke et al., 2009;Qualley et al., 2012). Phenylpropanoid and benzenoid biosynthetic pathways
compete for phenylalanine substrate, for example, Phlox subulata cultivars will produce little to no phenylpropanoid volatiles
when emitting more benzenoids, and vice versa (Majetic and Sinka, 2013). Because phenylpropanoids and their derivatives
are widely distributed in owers (Ramya et al., 2017).
23.5 Current status of genetic engineering in floriculture plants
In the oricultural department breeders with great effort develop many varieties for each trait of plants like ower color,
shape, size, and owering time. However, ower color is one of the most special traits in breeding strategies, for the ower
market business and from the consumers point of view. Genetic engineering (GE) technology made a bloom for molecular
biologists to create new ideas to understand basic research. This technology helped in developing new varieties against any
traits of interest within short time than classical breeding. Advantages for developing different owering varieties with many
colors, especially in ornamental plants. The genes responsible for anthocyanin biosynthesis and other genes that inuence
petal color should be identied and characterized through GE. Genetic engineering can be done by three different methods
they are Agrobacterium transformation, RNAi technology, and CRISPRCas9 technology.
23.5.1 Agrobacterium transformation
It is well known that Agrobacterium tumefaciens genetically mediated gene transfer (GM) to plants through its (Ti) plasmid
was one of the inventive ndings of the twentieth century and a widely used tool in many model crops. Different delivery
systems can be used for Agrobacterium-mediated GM such as direct organogenesis or somatic embryogenesis or indirectly
through de-differentiated cells. During 1997, two genes namely petunia avonoid 3’,5’-hydroxylase (F3’5’H) and petunia
dihydroavonol 4-reductase (DFR) were modied using GM technology and released as the rst commercialized genetically
modied purple color ower in carnation cultivar and named as “Moondust” by Suntry Ltd. (Melbourne, Australia) and
Florigene Ltd. (Melbourne, Australia) (Fukui et al., 2003). Similarly, using the same strategy blue roses were developed by
Suntry Ltd. (Katsumoto et al., 2007). Chrysanthemum cultivar Shinma was developed against heat stress-tolerance at South
Korea. By suppressing the F35H gene in cyclamen through antisense inhibition, resulted to reduced delphinidin content
and elevated cyanidin content, leading to a change in petal color from purple to red to pink (Boase et al., 2010). Similarly,
by regulating the accumulation of ions transporter Vit1 in the vacuole, that made petal cells blue in tulips (Momonoi et al.,
2009). The PH5 gene of the petunia generated the blue color by reducing the acidication in vacuoles (Verweij et al., 2008).
23.5.2 RNAi method
Followed by Agrobacterium genetically modication, RNAi technology is more efcient and has been frequently used in
gene-suppression method in plants and ower color modication. Using hairpin RNA construct the gene was modied in the
CRISPR edited floriculture crops Chapter | 23 387
Torenia hybrid plant and changed from the blue color ower to white and pale color (Fukusaki et al., 2004). By silencing the
anthocyanin 5,3’-aromatic acyltransferase (5/3’ AT) and avonoid 3’,5’-hydroxylase (F3’5’H), two key enzymes involved in
gentiodelphin biosynthesis showed reduced ower color, such as lilac or pale-blue owers (Nishihara and Nakatsuka, 2011).
In orchid Phalaenopsis, PeUFGT3 gene was silenced using hpRNA approach resulting in red color owers to different fading
colors in owers (Chen et al., 2011). When the gene anthocyanin 5,3- aromatic acyltransferase gene (5/3AT) was inhibited
in gentian owers color changed from blue to lilac. In contrast, when 5/3AT and F35H genes were co-suppressed, the petals
turned pale blue (Takashi et al., 2010). Similarly, a mixture of volatile phenylpropanoid/benzenoid compounds responsible
for the ower scents was manipulated in petunia by the elimination of some volatile compounds from the scent bouquet
(Underwood et al., 2005). Similarly, down-regulating the genes such as benzyl alcohol/phenyl-ethanol benzoyl-transferase
(PhBSMT), phenyl-acetaldehyde synthase gene (PhPAAS), benzyl-alcohol/phenyl-ethanol benzoyl-transferase (PhBPBT)
and coniferyl alcohol acyl-transferase (CFAT) selectively altered scent component, with minimal changes in the emission
of other volatiles in petunia (Dexter et al., 2007;Kaminaga et al., 2006).
23.5.3 CRISPR method
CRISPR performance is like molecular scissors, which cut and paste, delete, or replace the DNA sequence very precisely.
CRISPR technology is different from genetically modied organisms (GMO), in CRISPR technology only base pair editing
will be done but in the case of GMO, the complete foreign DNA is inserted in the genetic sequences. Using CRISPR
technology the DFR-B gene responsible for ower color was edited in the morning glory plant in Japan (Watanabe et al.,
2017). In another case avanone 3-hydroxylase (F3H) gene, which encodes a key enzyme involved in avonoid biosynthesis
was edited in the plant Torenia ower changed pale blue color (Nishihara et al., 2018). Xu et al. (2021) edited the genes
PhACO3 and/or PhACO4 involved in ethylene biosynthesis to improve ower longevity in petunia cv. Mirage rose showed
longevity (approximately 8.0 d) compared with 6.0 d for the WT line. The LpPDS gene was successfully edited in two
Lilium species which resulted in completely albino, pale yellow, and albino–green (Yan et al., 2019). Editing the PDS gene
in petunias with a CRISPR-Cas9 showed an albino phenotype (Zhang et al., 2016b). In another case, multiple MADS genes
were targeted in the Phalaenopsis orchid and several MADS null mutants were generated (Tong et al., 2020). Another gene
responsible for uorescence protein disruption gene yellowish-green uorescent (CpYGFP) was edited in Chrysanthemum
morifolium (Kishi-Kaboshi et al., 2017). Carotenoid cleavage dioxygenase (CCD) gene in Ipomoea nil resulted in carotenoid
accumulation regulation (Watanabe et al., 2018). A gene nitrate reductase (PhNR) in Petunia resulted in deciency in nitrate
assimilation and another gene PiSSK1 in Petunia showed self-incompatibility (Subburaj et al., 2016;Sun and Kao, 2018).
23.6 Breeding techniques to enhance floral fragrances
The genes responsible for the scent were identied through the transcriptome analysis in some of the ornamental plants.
Till now, around 700 compounds responsible for the scent have been identied from 60 plant species (Knudsen et al.,
1993). The rose cultivars are categorized into different groups based on their oral aroma and their fragrant elements (Du
et al., 2019). Garden roses are primarily used for their fragrance (Smulders et al., 2019). Genes for germacrene D, alcohol
acetyltransferase 1, and various OMTs are part of the map (Won et al., 2009;Kiralan, 2015;Koksal et al., 2015). In the
Lilium variety “Siberia,” which is signicant commercially and is prized by consumers for its scent, snow-white owers
(Hu et al., 2013,2017). Two monoterpene synthase genes (LoTPS1 and LoTPS3) that produce linalool and -ocimene were
found and functionally described. Both genes are expressed in sepals and petals in response to mechanical damage (Abbas
et al., 2019). Du et al. (2019) categorized different ower scent kinds through the assessment of the volatile emissions from
41 Lilium cvs and discovered monoTPS polymorphisms and alternative splicing products. The oral smell compositions
within the genus Camellia were studied by Fan et al. (2019).
23.7 Enhancing flower scents using genetic engineering
Manipulating scent genes through GE, i.e., through GM, biotechnology provides breeders with new chances to create new
aesthetic types. Even though, some of the genes responsible for the oral scent have been cloned but still their biochemical
pathways and molecular function remains unknown (Dudareva, 2002). Till now, few reports are available on the oral scent
genes modication using GE. A transgenic carnation ower was developed based on a fragrant gene that can alter the
fragrance by controlling volatile synthesis, membrane attachment, and cytoplasm segmentation (Schade et al., 2001). The
oral fragrance genes that are most likely to have originated in roses, which also synthesize 400 and more volatile chemicals
and these are being engineered into owering plants (Lavid et al., 2002;Scalliet et al., 2002;Channeliere et al., 2002;
Guterman et al., 2002). Benzyl alcohol acetyl transferase for benzyl acetate synthesis, or Clarkia breweri BEAT gene, was
388 SECTION | II CRISPR mediated genome editing in horticultural crops
inserted into Lisianthus to induce aroma in the petals (Aranovich et al., 2007). The transgenic P. hybrida plants with Clarkia
S-linalool synthase (LIS) expressed under the CaMV35S promoter showed with the accumulation of nonvolatile compound
S-S-linalyl-beta-D-glucopyranoside (Lucker et al., 2001). To further validate this gene was also transferred into carnation
plants showed similar results (Lavy et al., 2002). Roses produce more than 400 volatile compounds and appear likely to be a
good source of scent-related genes such as those encoding S-adenosylmethionine: orcinol O-methyltransferase (Lavid et al.,
2002;Scalliet et al., 2002;Bohlmann et al., 1997;Lucker et al., 2002). The carnation plants were edited with avonoid3-
hydroxylase genes showed several color modications and more fragrant than its respective control plants (Zuker et al.,
2002). Only a few oral scents and color genes were edited because only less information is available on genes responsible
for different traits and its metabolic process.
23.8 Development of new breeding techniques in floriculture
23.8.1 Plant tissue culture
Floriculture industry has become very prominent as a result of science-based techniques, in which tissue culture techniques
can be standardized for different cultural procedures on the essential basis, for commercial onset (Datta, 2019). In vitro
procedures have been standardized to develop and promoting the generation of new commercial varieties. Vegetative
propagation is low through the older propagation process where as tissue culture has high-quality planting material to
produce uniformity to produce new varieties through biotechnology and rapid multiplication through tissue culture.
23.8.2 Callus mediated
The tissue culture technique has an application of producing callus formation by somatic embryogenesis where it has an
implementation of plant hormones such as auxins, and cytokinins and gibberellins for regeneration of new plants with
a higher percentage of propagation through callus tissues by embryogenesis and organogenesis (Efferth, 2019). Callus
cultures can be either embryogenic or nonembryogenic. Callus regenerate a complete plant with a differentiated cells
by somatic embryogenesis in which a plant or embryo is derived from a single cell. Hossain et al. (2007) described the
selection of a salt-tolerant callus variety of Ramat.cv. Maghi Yellow, i.e., C. morifolium to develop a salt-tolerant line with
increased enzymatic activity of superoxide dismutase, glutathione reductase and ascorbate compared with control, i.e.,
sensitive parental line. Similarly, cultivars of gerbera (Gerbera jamesonii Bolus) was developed for its rapid multiplication
by indirect shoot induction from its callus differentiation to generate novel cultivars (Minerva and Kumar, 2013). Recently, a
protocol was described by somatic as well organogenesis in Lilium pumilum under the moderation of specic phytohormones
(picloram, NAA, and IBA) (Zhang et al., 2016a).
23.8.3 Protoplast
By application of new biotechnological interventions oricultural industry has an immense demand for traits with color and
architecture with implications of micropropagation, somatic hybridization, and genetic transformation by protoplast (Davey
et al., 2006). Protoplast culturing system has a totipotency to generate somatic hybrids and cybrid plants. Site-specic
mutation in petunia owers showed certain ower color modications where by using Cas9-ribonuceoproteins (RNP). Yu
et al. (2021) implemented by targeting duplicated genes of wild Petunia protoplasts for ower color modications, i.e., thus
two F3H coding genes were targeted, i.e., F3HA and F3HB, generated seven mutant lines in either both the genes and one
complete set of mutation in both the genes without possessing any selectable marker genes thus conrming a gene knockout
with a DNA-free CRISPR method thus they resulted its transition from laboratory to led stage.
23.9 Molecular markers
Molecular markers act as a global positioning system (GPS) on genome that may be used as reference to track a gene for the
particular trait of interest. Marker-assisted selection (MAS) refer to the use of DNA markers that are tightly linked to target
loci as a substitute for or to assist phenotyping. Using the DNA marker, we can identify the genotypes carrying different
alleles of a single gene or quantitative trait loci (QTLs) along with its phenotype data. The markers associated with the
particular trait are commonly used in the MAS program. Among the two types of molecular markers, polymerase chain
reaction (PCR) based DNA markers are more suitable for any living organisms (Joshi et al., 1999). The polymorphism can
be seen between two individuals at the DNA level.
CRISPR edited floriculture crops Chapter | 23 389
23.9.1 Genetic diversity
For the crop improvement program, we need to characterize many germplasms and the genetic variation among the
germplasm can be studied through molecular markers. Molecular markers have been used for genetic diversity and
relationships, identication of desirable traits, selection of promising parents in hybrid variety, inbred line development,
as well as registration of variety and protection in oricultural crops (Pejic et al., 1998). In many morphological studies, the
results are not consistent because of genotype ×environmental interactions (G ×E), mainly for quantitative genes. Through
molecular markers genetic diversity and identication of cultivars have been done in many oriculture crops (Baliyan et al.,
2014;Kumar et al., 2016;Kumar et al., 2017;Sirohi et al., 2017;Chaudhary et al., 2018).
For the terrestrial woodland orchid Tipularia discolor, very low genetic diversity was observed ranging from
0.00 (0%) to 0.069 (18.2%) among the ISSR polymorphic markers (Smith and Bateman, 2002). Around 31 Dendrobium
species, were selected to study the genetic diversity using 17 ISSR markers. Using UPGMA software, the phylogenic tree
was constructed and 31 Dendrobium species were grouped into six clusters indicating the polyphyletic nature of the genus
with several well-supported lineages (Wang et al., 2009). Using RAPD markers, radio-mutant treated cultivars and normal
plants in chrysanthemum were characterized and showed low diversity in the case of mutant cultivars (Lema-Ruminska
et al., 2004). Similarly, RAPD markers were used in Bougainvillea species and generated 84.4% polymorphism among the
different verities (Chatterjee et al., 2007). In another study, 45 lily germplasm were used for genetic diversity using RAPD
markers and shortlisted to 28 polymorphic markers out of 300 markers tested (Chen et al., 2009). Li et al. (2014) selected 28
cultivars of marigold (Tagetes patula L.) for genetic diversity by using SRAP (sequence-related amplied polymorphism)
markers resulting in 55.72% polymorphic. ISSR markers were used for genetic diversity in chrysanthemum and showed
differences in the genotypes like SKC-83, Ratlam selection, Gaity, and selection 69 for both morphological character and
molecular analysis (Baliyan et al., 2014). Kumar et al. (2017) also studied population structure and genetic diversity in
38 chrysanthemum genotypes using RAPD markers. Ten RAPD markers showed 94% polymorphic between 38 genotypes
and grouped into two sub-populations and mixed populations. Using 22 genotypes of marigold, genetic diversity study was
carried out by RAPD markers resulting in 98.80% polymorphic (Panwar et al., 2017).
23.9.2 Barcoding
DNA barcoding is used for the characterization and documentation of taxonomic impediment oricultural crops. However,
barcoding especially in plants, is still rmly within the realm of scientic research. DNA barcoding have been used to
determine morphologically looking similar species collected from different locations are the same or different and rapid
assessment of new biodiversity by the Morpho-species approach. DNA barcoding has been successfully implemented as a
molecular tool in the identication and differentiation of new species, cultivars, genotypes, and plant germplasm for plant
taxonomists (Vogler and Monaghan, 2007). Very few oricultural crops have been carried out for DNA barcoding. DNA
barcoding was very efcient in freshly collected material as compared to the preserved herbarium specimens. Some of
the examples are as mentioned below. DNA barcoding has been implemented to identify orchid’s species (Parveen et al.,
2012). Two-loci combination rbcL +matK were used as a molecular marker to differentiate the difference between species,
but not much variation was observed within the species. Later, ITS markers were used as potential barcoding markers
for differentiating Dendrobium species, both inter- and intraspecic divergences (Huang et al., 2010). To identify the best
molecular marker for universal DNA barcoding in plants, six markers namely matK, rbcL, rpoB, rpoC1, trnH-psbA, and ITS
were tested in 36 Dendrobium species. The results suggested that trnH-psbA spacer from the chloroplast genome and ITS,
from the nuclear genome are the efcient markers for species differentiation (Singh et al., 2012). de Vere et al. (2012) used the
rbcL and matK marker for DNA barcoding the nation of Wales (1143 species) and their ndings assembled 97.7% coverage
for rbcL, 90.2% for matK. Eleven ISSR markers were used in 10 tuberose varieties for the barcoding study and constructed
CID diagrams using UVpro software (UVTECH, UK) (Khandagale et al., 2014). Two barcoding markers (ITS2 and psbA-
trnH) were used in 21 cultivars of P. lactiora and their wild species. Results suggested that ITS2 is easy and reliable for
intra-species identication compared to psbA-trnH (Li et al., 2017). Similarly, Elansary et al. (2017) used matK and rbcL
markers for the barcoding of plant species in private and public nurseries of horticulture importance and recommended to
use of the combination of ITS with rbcL+matk to increase the performance of taxa identication.
23.9.3 Tagging and mapping
Tagging of genes responsible for ower color, longevity of owers, ower fragrance, ower size, heat tolerance, drought
tolerance and other important developmental pathway genes is a major target. Such tagged genes can also be used to develop
390 SECTION | II CRISPR mediated genome editing in horticultural crops
a hybrid for desirable traits or for genetic transformation studies. Molecular markers have been used for tagging and mapping
important genes in many oricultural crops. The RPP1 gene was tagged and mapped using 117 diploid rose populations
and identied this RPP1 gene is responsible for resistance against powdery mildew diseases (Linde et al., 2006). In another
study, RAPD markers were associated with carnation against bacterial wilt and three genes are involved in the resistance
against bacterial wilt (Onozaki et al., 2003). Similarly, in another study, a RAPD marker (G-02-980) were co-segregated
with an amplication of 980 bp only in fertile plants but not in sterile plants of Tagetes erecta (Wang et al., 2010). Six
genes are identied and reported to be associated with ray oret type, cultivated type, and ower shape of chrysanthemum
ower and these genes do not have any association with ower color (Chong et al., 2016). Chong et al. (2019) generated
81 SNPs and identied two SNPs that highly correlated with capitulum diameter and owering time and further developed
as cleaved amplied polymorphic sequence (dCAPS) markers and used in future breeding programs. However, Malek
et al. (2000) screened diploid roses tetraploid progeny for segregating against black-spot resistance. In another work to
distinguish double- and single-ower phenotypes at the young seedling stage in Japanese gentian plants PCR-based marker
was developed. This marker clearly co-segregated with double-ower phenotype by inserting a retro-transposable element
(Tgs1)intoGsAG1, a gene that belongs to C-class MADS-box genes. Due to the lack of variation in Japanese gentians ower
shapes and require a long breeding period, the developed DNA marker may be helpful for the efcient breeding program
for the development of double-owered cultivars in the future (Tasaki et al., 2017). Back cross mapping population was
developed between Lagerstroemia fauriei Koehne (standard) and L. indica “Creole” (creeping) and 322 SSR markers were
tested in 174 BC1 populations and tagged two SSR markers namely S364 and LYS12 genetic distances of 23.49 centimorgan
(cM) and 25.86 cM from the loci controlling the plant opening angle trait and the branching angle trait, respectively (Zheng
et al., 2019).
23.10 Genomics
23.10.1 Genetics
Generally, Mimulus lewisii, perennial herb that grows in western North America typically have pink color owers. However,
white owers of the same populations have been reported in the Pacic Northwest. To understand the molecular genetics
responsible for ower color pigmentation a classical cross was made between pink color ower and white color ower
and generated an F2 mapping population to study the color pigmentation, results segregated into 3 (pink): 1 (white) ratio,
suggested that pink color is a dominant character of the gene. The gene involved in the anthocyanin pathway namely DFR
showed complete co-segregation with the mapping population and emerged as a potential candidate gene MlDfr. Further
MlDfr gene alleles from white and pink color owers were cloned and sequenced, resulting in 2 bp insertion in the second
exon only in white colored owers. This 2 bp insertion caused a frameshift mutation with a truncated protein of 106 amino
acids in white owers compared with 464 amino acids in pink owers (Wu et al., 2013). A similar, trend was also observed
in other owers, in purple-owered Ipomoea purpurea, white-owered mutants are observed in the populations in the
southeastern United States. In another case, the mutations are encoded at the W-locus and it is due to a deletion in the
coding region of an R2R3-MYB transcription factor (IpMyb1) in Ipomoea (Chang et al., 2005). In another case, within
purple owers rarely white ower mutation is caused by a cis-regulatory mutation in the Chs gene involved in anthocynin
pathway in an arctic mustard (Parrya nudicaulis)(Dick et al., 2011).
Similarly, the ower doubling trait is an important trait in Petunia and it was reported to be controlled by recessive genes
(Saunders, 1910). Later, to understand the genetics a cross was made between Petunia ×hybrida grandiora and observed
that a single dominant gene is responsible for this trait (Natarella and Sink, 1971;Scott, 1937;Sink, 1973). In another case,
the owering time trait was taken, wild roses ower only in the spring season, and cultivated roses are owering all the
time. This change in the owering was due to the mutation arising in the oral repressor gene RoKSN. In addition, another
new allele RoKSNA181, responsible for week reblooming was also detected (Freslon et al., 2021). Another important trait
in oriculture is ower fragrance has been studied in many owers. In rose owers, the fragrance trait is controlled by
one or two genes involved in the volatile synthesis namely nerol, neryl acetate, and geranyl acetate (Spiller et al., 2010).
In contrast, the Mimulus lewisii ower pollinated by bumblebee and Mimulus cardinalis pollinated by hummingbird differ
in their loci/genes encoding LIMONENE-MYRCENE SYNTHASE and an OCIMENE SYNTHASE (Byers et al., 2014). A
different scenario was reported in Petunia and Antirrhinum that fragrance trait is combined with other oral traits. In the
Petunia ower, the Enhancer of Benzenoid II is regulating both fragrance and ower opening and anthesis (Colquhoun
et al., 2011;Van Moerkercke et al., 2011). In Antirrhinum ower, CompatA gene is involved in both fragrance and petal
size (Manchado-Rojo et al., 2012).
CRISPR edited floriculture crops Chapter | 23 391
23.10.2 Transcriptome data
Flowers characteristics like colors and shapes, etc., vary signicantly among closely related species. In order to understand
the genes involved in the three different stages of ower interaction across species in a group of Neotropical plants native
to Mexico—magic owers (Achimenes,Gesneriaceae), RNA-seq were done (Roberts and Roalson, 2017). Three stages of
ower development such Immature Bud, Stage D, and Pre-Anthesis were used for RNA seq. The results suggest that four
species shared proteins involved in DNA binding and transcription factor activity. For ower development, the transcripts of
MADS-box genes or the orthologs of these genes such as AP1, AP3, PI, and AG are crucial for composing oral organ. Genes
namely ANS, DFR, F3H, and F35H are reported to be involved in ower color transitions. Other than these genes, HDA19
(histone deacetylase 19) is also reported to be involved in regulating the transcriptional factor, especially the suppression
of several A- and E-class MADS-box genes that control sepal and petal identity. In petunia plants, PMADS1, PhGLO2 and
PMADS2/PhDEF are over-expressed in petals and stamens (Zhou et al., 2019). In Pyrola alba plants, is over-expressed only
in male ower buds (Matsunaga et al., 2003). To elucidate the molecular basis involved in the aging process of the ower
opening and closing studied in water lilies using RNA-seq transcriptome data. The samples were collected at different
owering timings like ower opening, ower temporary closure, and ower nal closure. Results revealed that multiple
signaling pathways like including Ca2+, reactive oxygen species, and light signaling pathways, auxin, ethylene, and jasmonic
acid signaling pathways were activated during the nal closure stage of waterlily owers. In the case of cell metabolism,
genes related to hydrolase like protease, phospholipase, and nuclease were upregulated at the nal closure stage. Real-
time polymerase chain reaction validation of 13 genes selected through the analysis revealed that one gene Floral homeotic
protein PMADS 2, showed 3.39 fold change in qRT-PCR and 4.41 fold in RNA-Seq analysis at the T1 stage compared to
T3 stage (Li et al., 2021). Similarly, due to the lack of genetic information in Chrysanthemum dichrum, high throughput
RNA seq, and metabolome were conducted for leaves, bud, and ower (Liu et al., 2021). The metabolic compounds such as
including rafnose, 1-kestose, asparagine, glutamine, and other medicinal compounds in leaves, ower buds, and blooming
owers. The transcriptome and RT-PCR showed similar trend of genes.
23.11 Development of transgenics
Crop improvement through nonconventional methods implements its application of biotechnological approaches, i.e., GE
is applicable to alter the plant characteristics using recombinant DNA technology, which it is used to transfer new specic
traits into the desired plant species. Agrobacterium-mediated gene transformation is an indirect process where two strains
are used for transformation, i.e., A. tumefaciens and A. rhizogenes with a specic vector backbone system for the generation
of genetically modied plants. Transferring DNA to the host plant includes a direct gene transformation, i.e., Particle
bombardment, where it is used to accelerate the DNA-coated microparticles which carries the genes into the cell (Klein
et al., 1988;Sanford, 1990). Another method is microinjection where microcapillaries and microscopic devices are used to
incorporate DNA into target cells thus cells survive and proliferate into mature plants (Neuhaus and Spangenberg, 1990).
Protoplast fusion is to deliver DNA to generate transgenic clones (Miki et al., 1987;Neuhaus et al., 1987). Electroporation
is a method where the temporary opening of plasma lemma takes place by the discharge of a capacitor across cell groups
for efcient transformation (Shillito et al., 1985;Dekeyser et al., 1990).
23.11.1 CRISPR-Cas applications in ornamental flowers
Targeted genome editing of genes that control desirable traits, such as owering promotion, which involves both increasing
the number of owers and changing owering time and longevity, color spectrum, aromas, and creation of innovation in
ower structure, can aid in the development of desirable genotypes that can both satisfy contemporary demands and be
protable for investors or producers.
One of the most important aspects of commercial ower production is ower color, which is mostly made of betalains,
carotenoids, and avonoids (He et al., 2013;Chen et al., 2019). One of the key traits of attractive owering plants is
their ower lifespan (Pandey et al., 2000). Antisense chalcone synthase (CHS) or dihydrofavonol-4-reductase (DFR) gene
insertions into transgenic lines were the rst steps in suppressing ower pigmentation (Zhu et al., 2020). Watanabe et al.
(2018) modied the CCD gene in I. nil using CRISPR Cas9-mediated mutagenesis to alter color in higher plants and
generated pale yellow petals (55.5%) in mutant plants. In L. pumilum (DC) as well Lilium lonfforum (white heaven and
sch), the LpPDS mutants were observed when this gene was knocked out (Zhang et al., 2016a;Yan et al., 2019). Tasaki et al.
(2019) selected genes in the Japanese gentian, such as anthocyanin 5/3-aromatic acyltransferase (Gt5/3AT), anthocyanin
392 SECTION | II CRISPR mediated genome editing in horticultural crops
5-O-glycosyltransferase (Gt5GT), and anthocyanin 3-O-glycosyltransferase (Gt3GT), and discovered that glycosylation
occurs after acylation of the 3-hydroxy B-ring group in del is necessary just the plants with the f3ha-f3hb mutation displayed
obvious alterations, including a pale purplish-pink ower color, while the others, including those with a single-copy gene
knockout, which produced violet-colored owers (Yu et al., 2021). Using the CRISPR-Cas9 system it was demonstrated that
corolla tube venation but not DPL-mediated mutation, indicating that DPL had no inuence on the venation of the corolla
tube (Nitarska et al., 2021). Using CRISPR-Cas9-based technology for EIL1 and EIL2 in petunia were silenced by virus-
induced silencing (Liu et al., 2016). Petunia EOBI (emission of benezoids) is a ower-specic transcription factor that acts
in concert with ODO1 and downstream of EOBII. Silencing of this gene caused down-regulation of several shikimate and
fragrance-related genes (Spitzer-Rimon et al., 2012). Based on the CRISPR-Cas9 system Japanese morning glory (I. nil)
petal senescence is assumed to be signicantly regulated by the NAC transcription factor EPHEMERAL1 (EPH1) (Shibuya
et al., 2018). A ower’s development and initiation are crucial phases in an ornamental plant’s life cycle. The lignocellulose
biosynthesis pathway of the orchid family has four genes called 9C3H, 4CL, C4H, CCR, and IRX these genes were targeted
by Kui et al. (2017) and found that the CRISPR-Cas9 system may result in mutations at a rate of 10%–100% for each target.
Chrysanthemum was the subject of only one study in which the CRISPR-Cas9 technology was used (Kishi-Kaboshi et al.,
2017). Subburaj et al. (2016) showed site-directed mutagenesis in the P. hybrida protoplast system by administering pure
Cas9 protein directly, preassembled with guide RNA, to effectively introduce mutations in NR genes (Table 23.3).
23.11.2 CRISPR-Cas technology for producing higher flowering plants with their color and longevity
The plant breeders are in need to develop ornamental owers with more and more new varieties with elite characters like
early owering, increase in the number of owers, ower longevity, ower shape, size, color, and scent. Among these
parameters, ower colors occupy the top priority due to their commercial value. By altering the pathways involved in the
pigmentation we can develop owers with different colors like yellow cyclamens, blue roses, blue chrysanthemums and red
iris, etc. which do not exist in nature (Tanaka et al., 2009).
23.11.2.1 Flower longevity
Xu et al. (2020) reported that in petunia ower quashing the ethylene production of the vital enzyme, has been targeted,
which is involved in ethylene metabolism, i.e., aminocyclopropaane-1-carboxylate oxidase (ACO) and resulted with
increased oral lifetime predominantly under regular silencing process. 1-aminocyclopropane-1-1carboxylase (PhACO)
genes (PhACO1, PhACO3, and PhACO4) were targeted by CRISPR-Cas9 to obtain a mutated line in PhACO1, targeted
which produced less ethylene with the resulting of longer lifespan (Xu et al., 2020). In another study Xu et al. (2021), altered
the PhACO3 and PhACO4 genes in petunia cv. Mirage rose through CRISPR-Cas9 editing which resulted in extended ower
longevity with reduced ethylene production. Liu et al. (2016) implemented a virus-induced silencing of ethylene signaling
components, i.e., EIL1 and EIL2 in petunia owers, thus these genes are functionally redundant by down-regulating the two
genes that affect their oral bloom lifespan. As a result of silencing the EIL gene, there was an increase in ower longevity
without any phenotypic changes in transgenic owering plants (Tieman et al., 2001). The avonoid 3’-hydroxylase (F3H)
enzyme is reported to be involved in the anthocyanin synthesis that gives red color to the leaves of poinsettia plants. In
order to validate the role of this enzyme, a transgenic plant was developed by inactivating this enzyme using CRISPR-
Cas9 technology. The transgenic plant leaves changed from vivid red to brilliant reddish–orange and strong pelargonidin
accumulation in the poinsettias plants when compared to its wild type (Nitarska et al., 2021).
23.11.2.2 Flower color
Flower color is another major characteristic for commercial ower production, thus, targeting ower pigments that were
suppressed by insertion of an antisense CHS or dihydroavonol-4-reductase (DFR) gene by producing altered ower color
with 0%–89% transgenic lines with extent color with differed lightening (Aida et al., 2000). By application of genetic
modication technique like CRISPR (Watanabe et al., 2018) used to modify the color by mutagenesis with modication of
the CCD gene in I. nil thus it achieved a 55.5% mutation with pale yellow petal plants. In another study a DFR-B gene,
which encodes an anthocyanin biosynthetic enzyme, thus observed stem color in initial stages, later due to biallelic change
at DFR-B site by targeting through CRISPR with 75% transformed plants which are anthocyanin-free white owers, with
single base insertion or deletion of more than two bases (Watanabe et al., 2017)(Fig. 23.1). Nishihara et al. (2018) used
this system to detect color change in the Torenia fournieri from blue to white color variation due to an enzyme avone 3-
hydrolase (F3H) for its avonoid biosynthesis. PDS gene, which is a key enzyme that had been modied for the production
of carotenoids and for chlorophyll biosynthesis (Zhang et al., 2016b) modied the petunia for color using CRISPR-Cas9
CRISPR edited floriculture crops Chapter | 23 393
TABLE 23.3 Applications of CRISPR-Cas9 genome editing in ornamental plants.
S. no. Plant species Target gene Transformation method Material transformed Mutation efficiency (%) References
1Japanese gentian (Albireo) Gt5GT, Gt3’GT, Gt5/3’AT Agrobacterium-mediated Leaf 0.07 Tasaki et al. (2019)
2Petunia hybrida (Mirage rose) ACO1 PEG-mediated Protoplast 31.50 Xu et al. (2020)
3Petunia hybrid (Madness mid-night) F3’H PEG-mediated Protoplast 11.90 Yu et al. (2021)
4Euphorbia pulcher-rima (Poinsettia) F3HAgrobacterium-mediated Internode stem 24 Nitarska et al. (2021)
5Petunia hybrida (Mirage rose) ACO3, ACO4 PEG-mediated Protoplast 34.32 Xu et al. (2021)
6Petunia hybrid (Mitchell diploid) DPL Agrobacterium-mediated NA ND Zhang et al. (2021)
7Petunia hybrid (Mirage rose) ACO1, ACO3, ACO4 PEG-mediated Protoplast ND Xu et al. (2020)
8Chrysanthemum Morifolium (Sei-Marin) CpYGFP Agrobacterium-mediated Leaf 0–28.9 Hossain et al. (2007)
9Petunia hybrid (Madness) NR PEG-mediated Protoplast 5.30–17.83 Subburaj et al. (2016)
10 Petunia hybrid (Mitchell diploid) PDS Agrobacterium-mediated Leaf 55.6–87.5 Nishihara et al. (2018)
11 Dendrobium officinaleC3H, C4H, 4CL, Agrobacterium-mediated Protocorm 10–100 Kui et al. (2017)
CCR, IRX
12 Torenia fournieri L. (Crown violet) F3H Agrobacterium-mediated Leaf 80 Nishihara et al. (2018)
13 Ipomoea nil (Violet) EPH1 Agrobacterium-mediated Immature embryo ND Shibuya et al. (2018)
14 Ipomoea nil (AK77/morning glory) CCD Agrobacterium-mediated Immature embryo 55.50 Watanabe et al. (2017)
15 Lilium longiflorum (White heaven) LlPDS Agrobacterium-mediated Tissue culture 4Zhang et al. (2016b)
Seedling scales
16 Lilium pumilu (DC. Fisch) LpPDS Agrobacterium-mediated Callus 29.17 Zhang et al. (2016b)
394 SECTION | II CRISPR mediated genome editing in horticultural crops
construct that targets the PDS and thus it obtained a 55%–87% albino characterizations in ower. Similarly in L. pumilum
(DC Fish) and Lilium lonorum (white heaven), the PDS gene, i.e., LpPDS gene was knocked out, and therefore it resulted
in three mutants, i.e., albino, albino green and pale yellow with a 69.75% and 63.64% mutation rate respectively. Delivering
a Cas9-ribonucleoprotein creates a mutant line to create a petunia with mutation within F3H genes, i.e., f3ha and f3hb
resulted in a pale purple-pink ower color with a single-copy gene knockout compared to its wild-type (Yu et al., 2021).
The DPL’s (R2R2 MYB transcription factor) was reported to be involved in regulating corolla tube venation in Petunia
plants. The CRISPR-Cas9 edited plants showed the disappearance of veins located above the abaxial surface of the ower
but no change in corolla tube venation. This result suggested that DPL has no role over the corolla tube venation in Petunia
ower development (Zhang et al., 2021).
23.12 Regulation
Genome editing technology is implemented in many research labs and companies for its precise editing, dogmatic
acceptance, and short time for product release comparted to normal breeding techniques (Miao et al., 2013;Bortesi and
Fischer, 2015). Globally, many products are being developed by many companies and are yet to be released to the market.
The edited ornamental plants can escape the strict regulations that are generally followed for the GM plant (Abdallah et al.,
2015). Clustered regularly interspaced short palindromic repeat (CRISPR) associated proteins (CRISPR-Cas) systems. For
all SDNs, their mode of-action is in principle the same: once present in a cell by insertion/expression and or transfection, the
SDN can cut the genome at a targeted site (Li and Xia, 2020). The SDN (SDN-1/2/3) terminology has been adopted by many
countries to legally categorize SDN applications. Especially for CRISPR-Cas-systems many varieties and modications are
already known and new variants are being steadily developed. Thus, genome-editing by using SDNs can be categorized in
three types:
rThe induction of single point mutations or InDels (SDN-1).
rShort insertions or editing of a few base-pairs by an external DNA-template sequence (SDN 2).
rThe insertion of longer strands (SDN-3) of allochtonous (transgenes) or autochtonous sequences (cisgenes).
The SDN (SDN-1/2/3) terminology has been adopted by many countries to legally categorize SDN applications. Besides
SDNs, ODM is another genome editing technique that is most comparable to an SDN-2 event (Sauer et al., 2016).
Although numerous countries are working on the development of market-oriented crops for several years, only a handful
of countries claried their opinion toward genome editing (Sprink et al., 2016;Ishii and Araki, 2017). Nevertheless,
controversial discussions are ongoing both in the European Union (EU) and in New Zealand, as more and more (partnering)
countries promote genome editing and products thereof such as Israel and more recently Japan and Australia which stated not
to regulate plants derived by some types of genome editing. The rst countries that released advices, opinions, or regulations
on genome editing are located on the American continent namely Argentina, Chile, the United States, and Canada. To date,
South Africa and Sudan grow genetically modied crops while South Africa has already begun discussing genome editing
and related regulation. Recently, Burkina Faso, Nigeria, and Ghana started cropping GM plants and Uganda still debates
the establishment of GMO legislation without explicitly naming genome editing (ABBC, 2019). Lately, India also just
released a draft document on Genome Edited Organisms in which they suggested a tiered risk approach for the regulation
of genome-editing products. Interestingly, the most active country in genome editing research, China, has not released any
legal documents so far, the reasons for this are unclear, but it is possible that China wants to have a product in hand before
releasing regulations (Modrzejewski et al., 2019). A legal basis to differentiate “articial” from “natural” is not given and
could explain why most product-based legislations do not regulate SDN-1 based edits. Generally, the outcome of case-by-
case approvals is still difcult to predict, but as more and more products enter the markets, the predictability of whether
new genome-editing developments will have a chance of obtaining marketing approval outside strict GMO guidelines—as
in Europe and New Zealand—will increase.
23.13 Conclusion and future prospects
Genome-editing technology can be used for mutational breeding, especially in oriculture which can develop many colors
with different sizes and shapes. CRISPR, a gene editing tool is very precise to target DNA in editing when compared to other
tools like TALENs, ZFNs, and MNs. Till now, few products developed in many crops through CRISPR were commercialized
and many are in the pipeline. This technology has many benets compared to classical breeding in oriculture. In oriculture,
molecular genetics are still challenging due to its complexity of the traits with unknown genome. With this technology, we
can identify the genes responsible for the different traits like ower color, shape, size, fragrance, owering timing, etc., and
CRISPR edited floriculture crops Chapter | 23 395
can be implemented well in oriculture. In many countries, this technology has been accepted and regulation was cleared.
Flowers are very closely associated with human civilization. There is a saying that “human beings are born, live, and nally
die with owers.”
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