Tissue Culture-Plants Casopis

plants
Review
Tissue Culture in Ornamentals: Cultivation Factors,
Propagation Techniques, and Its Application
Hasan Mehbub 1 , Ayasha Akter 2 , Mst. Arjina Akter 3,4 , Mohammad Shamim Hasan Mandal 5 ,
Md. Ashraful Hoque 3 , Monika Tuleja 6 and Hasan Mehraj 4, *
1 The United Graduate School of Agricultural Science, Ehime University, Matsuyama 790-8556, Japan
2 Department of Horticulture, Bangladesh Agricultural University, Mymensingh 2202, Bangladesh
3 Department of Plant Pathology, Bangladesh Agricultural University, Mymensingh 2202, Bangladesh
4 Graduate School of Agricultural Science, Kobe University, Rokkodai, Nada-ku, Kobe 657-8501, Japan
5 Graduate School of Advanced Science and Engineering, Hiroshima University, Hiroshima 739-8511, Japan
6 Department of Plant Cytology and Embryology, Institute of Botany, Faculty of Biology, Jagiellonian University,
Gronostajowa 9, 30-387 Krakow, Poland
* Correspondence: hmehraj02@yahoo.com or hmehraj02@port.kobe-u.ac.jp
Abstract: Ornamentals come in a variety of shapes, sizes, and colors to suit a wide range of climates,
landscapes, and gardening needs. Compared to demand, a shortage of plant materials and diver-
sity force the search for solutions for their constant acquisition and improvement to increase their
commercial value, respectively. In vitro cultures are a suitable solution to meet expectations using
callus culture, somatic embryogenesis, protoplast culture, and the organogenesis of protocorm-like
bodies; many of these techniques are commercially practiced. Factors such as culture media, explants,
carbohydrates, plant growth regulators, and light are associated with the success of in vitro propaga-
tion. Techniques, especially embryo rescue and somatic hybridization, are widely used to improve
ornamentals. The development of synthetic seed allows season-independent seed production and
Citation: Mehbub, H.; Akter, A.;
Akter, M.A.; Mandal, M.S.H.; Hoque, preservation in the long term. Despite the advantages of propagation and the improvement of orna-
M.A.; Tuleja, M.; Mehraj, H. Tissue mentals, many barriers still need to be resolved. In contrast to propagation and crop developmental
Culture in Ornamentals: Cultivation studies, there is also a high scope for molecular studies, especially epigenetic changes caused by
Factors, Propagation Techniques, and plant tissue culture of ornamentals. In this review, we have accumulated and discussed an overall
Its Application. Plants 2022, 11, 3208. update on cultivation factors, propagation techniques in ornamental plant tissue culture, in vitro
https://doi.org/10.3390/ plant improvement techniques, and future perspectives.
plants11233208
Academic Editor: Keywords: in vitro; callus; somatic embryogenesis; hybridization; protoplast fusion; protocorm-like
Iyyakkannu Sivanesan body; synthetic seeds; epigenetic variation
Received: 19 October 2022

Accepted: 17 November 2022
Published: 23 November 2022
1. Introduction
Publisher’s Note: MDPI stays neutral Ornamental horticulture, flowering, and landscape horticulture are economically vi-
with regard to jurisdictional claims in
able industries in the agricultural sector. It deals with producing and commercializing
published maps and institutional affil-
flowers, flowering plants, foliage plants, and landscape plants; plants used in ornamental
iations.
horticulture are collectively called ornamentals. Plant tissue culture, including micropropa-
gation, is the most applicable plant propagation technique in ornamentals. It allows the
production of several exact genetic copies from small pieces of plant tissue (known as ex-
Copyright: © 2022 by the authors.
plants), and the propagation of uniform, season-independent, and seed-borne diseases free
Licensee MDPI, Basel, Switzerland.
seedlings is an additional advantage [1]. Thus, micropropagation techniques are frequently
This article is an open access article used in the commercial production of seedlings in diversified plant species. Explants are
distributed under the terms and usually cultured in a nutrient-supplemented medium under sterile conditions. White’s
conditions of the Creative Commons medium was the first chemically defined nutrient medium [2–5]. Afterward, Murashige
Attribution (CC BY) license (https:// and Skoog (1962) developed a new nutrient supplemented medium, which is known as
creativecommons.org/licenses/by/ Murashige–Skoog (MS) medium [6], and it is the most used nutrient supplemented medium
4.0/). in the world for plant tissue culture. Other nutrient-supplemented media, such as White
Plants 2022, 11, 3208. https://doi.org/10.3390/plants11233208 https://www.mdpi.com/journal/plants

Plants 2022, 11, 3208 2 of 33
(WH) medium [7], Linsmaier and Skoog (LS) medium [8], Gamborg (B5) medium [9], Nitsch
and Nitsch (NN) medium [10], etc., are also widely accepted. These nutrient supplement
media are the basal media that usually contain major salts (plant macronutrients), minor
salts (plant micronutrients), vitamins, and organic compounds. Solidifying agents are used
in the basal medium to support the plantlets in micropropagation and, to some extent, in
liquid culture medium. Different kinds of agars—phytagel, gelrite, gellan gum, etc.—are
used to solidify the nutrient supplement culture media for plant tissue culture that are
available in the commercial market. The pH of the basal nutrient supplement media keeps
changing during preparation, which is required to adjust before autoclaving. The pH in
the media is considered a dynamic variable for in vitro plant growth and development.
MS medium is the most used medium in vitro, and manipulation of MS medium and
culture conditions according to the plant—specific requirements are also practiced [11,12].
The success of plant tissue culture techniques largely depends on sources of carbon, plant
growth regulators (PGRs), culture environment, lights, genotype, type of explant, etc. The
key tool for the success of plant tissue culture technology greatly relies on the proper culture
media composition and their culture condition because of plant—specific response. Other
than propagation, tissue culture technology has been used for plant improvement, somatic
hybrid development, synthetic seed production, and ploidy manipulation. We reviewed
the research findings of plant tissue culture technologies for ornamental plant propagation,
cultivation factors, and their application in ornamentals from a future perspective.
2. In Vitro Cultivation Factors

2.1. Carbohydrate Supplements as Carbon Sources in Culture Media
Plant tissues, cells, and organs usually go through either heterotrophic or mixotrophic
conditions in vitro. Heterotrophic conditions are the primary obstacle to in vitro plant
growth and development where cultured tissue or cell or organ can only synthesize their
required nutrients from the basal media, and mixotrophy conditions where plants depend
on heterotrophy and can produce food by photosynthesis as well [13]. Plants need ex-
ogenous carbohydrates in both phases for proper growth and development due to their
morphogenic effect on nutritional value, osmotic potential, and cell division [13,14]. The
addition of exogenous carbohydrates can easily supply energy to explants when explants
are not ready for photosynthesis. Even though plants become ready for photosynthesis,
they need exogenous carbohydrate supplements because of their lower photosynthesis
efficiency than in vivo conditions [15]. Exogenous carbohydrate supplements assist plant
embryos in increasing cell division by encouraging cell expansion and reserve accumu-
lation [16]. Many forms of carbon sources are available in commercial markets, such as
sucrose, glucose, fructose, maltose, trehalose, lactose, galactose, sorbitol, etc. The specific
sources and requirements vary according to the plant species, stages, tissues for explants,
culture period, culture environment, etc. [13]. Sucrose is superior and cheaper than other
carbon sources, which ensures favorable effects on in vitro plant growth [17–20]. In plants,
phloem sap contains sucrose to control plant growth and developmental processes [21],
while sucrose is highly soluble in water, acts as a molecule transporter, and is transported
by the plasma membrane [22,23]. Therefore, plants efficiently utilize sucrose for their
carbon requirements during the in vitro heterotrophic and mixotrophic phases. About
2–5% sucrose concentrations are generally used in plant tissue culture of ornamentals [24].
Depending on the plant species and culture conditions, other carbon sources showed more
efficiency than sucrose. For example, the wishbone flower (Torenia fournieri) extended twice
its vegetative culture period in a trehalose-based culture medium over a sucrose-based
medium without alteration in plant viability [25]. Trehalose was found to be equally or
sometimes more effective than sucrose for the propagation of protocorm-like bodies (PLBs)
in Phalaenopsis and Doritaenopsos orchids [20,26]. Glucose stimulates the in vitro shoot and
root growth of chrysanthemum, while its intermediate product, fructose, slowly affects
in vitro plant growth and development; however, its efficiency varies according to plant
species and culture conditions [27,28]. On the other hand, slower hydrolysis (20 times
Plants 2022, 11, 3208 3 of 33
slower than sucrose) of maltose is the main oblige [29]. Plants take a long time to absorb
and metabolize maltose, and the requirement is sometimes twice that of sucrose [29]. From
the above discussion, it is clear that exogenous carbohydrate supplements are crucial for
in vitro plant growth and development. Exogenous carbohydrate concentration is also
varied, and concentrations over a threshold level could be toxic, hamper photosynthesis,
and inhibit in vitro plant growth [30–32].
2.2. Plant Growth Regulators, Inhibitors, and Elicitors in Culture Media

The application of PGRs in basal media accelerates the induction of a new plant from
a cell or tissue. Auxins (Au), gibberellins (GA), cytokinins (CK), abscisic acid (ABA), and
ethylene (ET) are the five groups of PGRs; Au and CK are widely used, while GA, ABA, and
ET are less used for vitro micropropagation of ornamentals [33]. The in vitro propagation of
plants is significantly influenced by the addition of auxin to culture media. Auxin triggers
cell division and leaf initiation before lateral root initiation [34–36], and it is crucial for the
formation of meristems [37]. In culture media with auxin, the cells of the explant rapidly
undergo cell division to form calli [38] and start to develop shoots and/or roots from the
calli [39]. A proper concentration of auxin can assist in initiating plant roots; application of
exogenous auxin can stimulate auxin-triggered pathways and GA biosynthesis; meanwhile,
it can suppress ABA and ET biosynthesis [40]. The naturally occurring auxins (indole-3-
acetic acid (IAA), indole-3-butyric acid (IBA), indole-3-propionic acid (IPA), 4-chloroindole-
3-acetic acid, phenylacetic acid, etc.) and synthetic auxins (4-chlorophenoxy acetic acid
(4-CPA), dicamba, picloram, etc.) are used in plant tissue culture [41,42]. Cytokinin is
naturally found in all plant tissues; however, it is enriched in the root tip, shoot apex, and
immature seeds [43]. In vitro micropropagation, cytokinin stimulates cell division and is
usually used to initiate the growth and proliferation of buds and shoots and slow down
root formation [24]. 6-benzyloaminopurine (BAP), 6-(γ,γ-dimethylallylamino)purine (2iP),
kinetin, zeatin, and thidiazuron-N-phenyl-N-1,2,3 thiadiazol-5-ylurea (TDZ) are commonly
used cytokinins in plant tissue culture. A combination of Au–CK, different combinations of
their concentrations, is frequently used in micropropagation, and the effect of both Au and
CK depends on their relative concentrations. A culture medium with high-cytokinin and
low-auxin causes shoot initiation, while a high-auxin and low-cytokinin medium causes
root initiation [44]. Gibberellic acid (GA3 ) is the most used gibberellin, which is used to
accelerate in vitro plant growth, while the function of ABA can be stimulatory or inhibitory
depending on different factors such as media ingredients, light intensity and quality, or
plant species [24]. We have summarized 200 research articles belonging to 52 ornamentals
studied for the effective PGR concentration and/or effective PGR combinations with their
suitable concentrations (Supplementary Table S1).
Different growth retardants, such as paclobutrazol, daminozide, chlorocholine chloride (CCC),
ancymidol, etc., can be beneficial for plants propagated in vitro media (Supplementary Table S2).
Growth retardants act as anti-gibberellin in sucrose supplemented medium; the addition
of growth retardant in culture media activates adenosine diphosphate-glucose pyrophos-
phorylase (ADGPase) and UDP glucose pyrophosphorylase (UDPGPase), which promotes
starch synthesis [45,46]. Similar to tuberization in potatoes, CCC showed a prompting effect
on in vitro PLB regeneration in Phalaenopsis orchid [26,46]. In vitro treatment of growth
retardants, e.g., paclobutrazol, has an additional advantage in increasing in vitro to ex
vitro transfer efficiency [47,48]. The positive regulation of growth retardants in micro-
propagation cannot be ignored. Elicitors, biotic and abiotic, have been widely used for
triggering secondary metabolites in plant tissue culture [49]. Chitosan (Ch), aminolevulinic
acid (ALA), alginate (Ag), N-acetylglucosamine (NAG), salicylic acid (SA), hyaluronic acid
(HA), silver nitrate (AgNO3 ), jasmonic acid (JA), methyl jasmonate (MeJA), phlorogluci-
nol (PG), pectin, casein hydrolysate, and yeast extracts are some common elicitors [50].
Like growth retardants, elicitors are also beneficial for micropropagation. For example,
MeJA, ALA, AgNO3 , HA, Ch, and PG trigger PLBs, root shoots, and flower organogen-
esis in ornamentals (Supplementary Table S2). In tulips, MeJA has been applied in a
tissue culture [49]. Chitosan (Ch), aminolevulinic acid (ALA), alginate (Ag), N-
acetylglucosamine (NAG), salicylic acid (SA), hyaluronic acid (HA), silver nitrate
(AgNO3), jasmonic acid (JA), methyl jasmonate (MeJA), phloroglucinol (PG), pectin,
casein hydrolysate, and yeast extracts are some common elicitors [50]. Like growth
retardants, elicitors are also beneficial for micropropagation. For example, MeJA, ALA,
Plants 2022, 11, 3208 AgNO3, HA, Ch, and PG trigger PLBs, root shoots, and flower organogenesis 4 of 33
in
ornamentals (Supplementary Table S2). In tulips, MeJA has been applied in a combination
of different polyamines for efficient bulb formation, and MeJA-polyamine combinations
significantly of
combination enhanced
different bulb formation
polyamines [51]. Anbulb
for efficient oxidizing biocicide,
formation, chlorine dioxide
and MeJA-polyamine
(ClO2), has been
combinations used in
significantly the culture
enhanced media for[51].
bulb formation in An
vitro plant regeneration
oxidizing biocicide, chlo- in
rine dioxide (ClOand
chrysanthemum 2 ), has been used
gerbera, and itinaccelerates
the cultureplant
media for in
shoot andvitro
rootplant regeneration
regeneration in
[52,53].
chrysanthemum
Additives, organic and gerbera, and it accelerates
and synthetic, can also plant shoot and
influence root regeneration
in vitro plant growth [52,53].
and
Additives,
development organic and synthetic,Table
(Supplementary can also
S3). influence in vitro plant growth and development
(Supplementary Table S3).
2.3. Light-Emitting Diodes over Conventional Light
2.3. Light-Emitting Diodes over Conventional Light
In vitro culture conditions can be manipulated by altering the light color and
In vitro
intensity forculture
effectiveconditions can be manipulated
morphogenesis or organogenesis.by altering
Plantsthecanlight colorwell
respond and to
intensity
a wide
for effective morphogenesis or organogenesis. Plants can respond well
spectrum of light in terms of plant growth and development with wavelengths of <400 to a wide spectrum
of
nm light
(UVinradiation),
terms of 400~700
plant growth and development
nm (visible), and 700~800with wavelengths
nm (far-red) of <400
[54]. White nm (UV
fluorescent
radiation), 400~700 nm (visible), and 700~800 nm (far-red) [54]. White
light with 350~750 nm wavelengths spectral emission is the conventional light sourcefluorescent light with in
350~750 nm wavelengths spectral emission is the conventional light source
vitro culture; however, the consumption of high electricity, high radiant heat, and uneven in vitro culture;
however, the consumption of high electricity, high radiant heat, and uneven radiation are
radiation are the major disadvantages [55]. Monochromatic light-emitting diodes (LEDs)
the major disadvantages [55]. Monochromatic light-emitting diodes (LEDs) with a specific
with a specific range of wavelengths are widely used for in vitro plant propagation (Figure
range of wavelengths are widely used for in vitro plant propagation (Figure 1).
1).
Figure1.1. The
Figure Theapplication
application of
of monochromatic
monochromatic white
white (a),
(a), red
red (b),
(b),blue
blue(c),
(c),and
andgreen
green(d)
(d)LEDs
LEDswith
with
specific wavelengths (white LED; 420–750 nm, red LED; 580–670 nm, blue LED;
specific wavelengths (white LED; 420–750 nm, red LED; 580–670 nm, blue LED; 420–550 nm, 420–550 nm,and
and
green LED; 460–610 nm) for in vitro PLB proliferation.
green LED; 460–610 nm) for in vitro PLB proliferation.
LEDs
LEDsarearethe
themost
mostefficient
efficientover
overwhite
whitefluorescent
fluorescentlight,
light,which
whichovercomes
overcomesthe thestated
stated
disadvantages [56,57]. Red LED showed efficiency for callus proliferation,
disadvantages [56,57]. Red LED showed efficiency for callus proliferation, PLB PLB organogen-
esis, PLB proliferation,
organogenesis, shoot induction,
PLB proliferation, shootshoot multiplications,
induction, and plantlet regeneration
shoot multiplications, and plantlet
in different ornamentals,
regeneration in differentsuch as orchids,such
ornamentals, gerbera, chrysanthemum
as orchids, gerbera, (cv. Kitam Cheonsu),
chrysanthemum (cv.
anthurium (cv. Violeta
Kitam Cheonsu), and Pink
anthurium (cv. Lady),
Violetaheliconia,
and Pinkpeace
Lady),lily, giant protea,
heliconia, peace and
lily, hosta
giant
(Supplementary
protea, and hostaTable S4). Far red was
(Supplementary Tableefficient
S4). Farfor
redthe plant
was growth
efficient forof
thechrysanthemum
plant growth of
(cv. Ellen) (Supplementary Table S4). A higher percentage of red
chrysanthemum (cv. Ellen) (Supplementary Table S4). A higher percentage LED with aoflower per-
red LED
centage of blue LED is suitable for the PLBs and plantlet regeneration of Phalaenopsis,
Rosa × kordesii, chrysanthemum (cv. Ellen), gerbera, anthurium, heliconia, peony, and
spurflower, while some other ratios of red and blue LED mixture were found to be effective
in some ornamentals (Supplementary Table S4).
A mixture of red and blue LEDs, compared with red LED alone, enhanced both plant
growth and development by increasing the net photosynthesis in Cymbidium [58,59] because
the spectral energy distribution of red and blue light coincides with that of chlorophyll
absorption [60]. Red and blue LED combinations were reported as effective for the growth
and development of PLBs in Cymbidium, Doritaenopsis, Phalaenopsis, and Calanthe [26,57]
(Supplementary Table S4). Blue LED increases the shoot formation of PLB cultures in
Dendrobium officinale and D. kingianum [61,62], while PLBs cultured under red and blue
Plants 2022, 11, 3208 5 of 33
LED showed the lowest and highest, respectively, in vitro differentiation rates on Oncidium
and D. officinale [61,63]. Very little information is available on the effect of green LED
on in vitro micropropagation of ornamentals. In recent studies, it was found that green
LED increased PLB regeneration in Dendrobium [31], and Cymbidium [64]; however, PLB
generation was more efficient under green LED when culture media had anti-auxin, PCIB
(p-Chlorophenoxyisobutyric acid, an anti-auxin), in D. okinawense [31]. Additionally, yellow
and orange light spectra have also been reported, respectively, in PLB, shoot, and plantlet
regeneration of Dendrobium and seed germination and rhizoid development of Bletilla
ochracea (Supplementary Table S4). These results suggest the requirement for a diverse
range of light spectra for in vitro micropropagation, which largely depends on plant species
and culture media supplements (Supplementary Table S4; Supplementary Table S5).
3. Standard Techniques Involved in Plantlet Generation In Vitro

3.1. Callus Culture
In the early 20th century, callus formation and its ability to generate independent life
were first noticed [65,66]. The callus is a mass of loosely packed parenchymatous cells
with various degrees of differentiation, which is raised from the in vitro proliferating cells
of plant tissue in response to biotic and abiotic stimuli. It is similar to non-differentiated
meristematic cells but different from differentiated plant cells. Depending on the accumu-
lated compounds, calli may be pale brown, creamish yellow, greenish, or colorless. The
callus is cytologically diverse in shape and type of cells and is genetically heterogeneous.
Under the influence of selected phytohormones, a certain pool of parenchymal callus cells
is dedifferentiated and has dividing activity. Calli lack chloroplasts for photosynthesis and
have a small vacuole, and their culture can generate new plants. Callus can be inducted
from any plant part, such as seeds, leaves, stem, root, flowers, etc.; successful callus in-
duction depends on plant species, explant used for the callus inductions, culture media,
PGR supplements in culture media, and growth conditions [67]. Two major PGR groups,
auxin and cytokinin, are largely used for callus induction [67]. Some plant species induce
callus in day–night conditions, while some need entirely night conditions. Callus induction
gives an idea of the potentiality of in vitro regeneration of any plant species, while it can
also be a good source of materials for other in vitro culture techniques and can be used for
long-term preservation [67]. Callus has been used for the successful plant regeneration and
genetic modification of different ornamental plant species [68].
3.2. Protoplast Culture

Protoplast culture is used for plantlet regeneration (process illustrated in Figure 2), and
protoplast fusion is used for crop improvement, which is known as somatic hybridization
(details in Section 4.2) [69]. The nature of the explant tissue and the thickness of the cell
wall play an important role in high-efficiency protoplast isolation, which is a critical stage
in the process of seedling regeneration or somatic hybridization. However, protoplasts
were successfully isolated and cultured in different ornamentals, such as Dendrobium [70],
lily [71], rose [72], chrysanthemum [73], petunia [74], carnation [75], coneflower [76],
geraniums [77,78], Persian silk tree [79], etc. Pre-plasmolyzing the explant tissue with
osmotic stabilizers, such as mannitol and sorbitol, before enzyme treatment is effective for
protoplast isolation in most plant tissue [80].
However, protoplasts were successfully isolated and cultured in different ornamentals,
such as Dendrobium [70], lily [71], rose [72], chrysanthemum [73], petunia [74], carnation
[75], coneflower [76], geraniums [77,78], Persian silk tree [79], etc. Pre-plasmolyzing the
Plants 2022, 11, 3208 6 of 33
explant tissue with osmotic stabilizers, such as mannitol and sorbitol, before enzyme
treatment is effective for protoplast isolation in most plant tissue [80].
2. A
Figure 2.
Figure Adetailed
detailedscheme of of
scheme protoplast isolation
protoplast and and
isolation establishment of an in
establishment of vitro
an inprotoplast culture.
vitro protoplast
culture.
Sugar concentration is another important factor for high-yield protoplasts, and the
effective
Sugarsugar concentration
concentration rangesimportant
is another from 0.3 factor
to 0.8 for
M in ornamentals
high-yield [69,74,76,81–84].
protoplasts, and the
Factors such as the concentration of enzyme, digestion period, pH of
effective sugar concentration ranges from 0.3 to 0.8 M in ornamentals [69,74,76,81–84]. the enzyme so-
lution, temperature, and agitation during incubation are also important
Factors such as the concentration of enzyme, digestion period, pH of the enzyme solution, for protoplast
isolation in ornamentals
temperature, and agitation [69,73,74,79,83,85,86].
during incubation are In also
orchids, the first
important forprotoplasts were iso-
protoplast isolation
lated in 1978 [87,88], while few studies reported colony formation [89–93]. After
in ornamentals [69,73,74,79,83,85,86]. In orchids, the first protoplasts were isolated in 1978 successful
protoplast
[87,88], isolation,
while therereported
few studies are somecolony
challenges to plantlet
formation regeneration
[89–93]. from an
After successful isolated
protoplast
protoplast.there
isolation, Typesareofsome
culture medium,toculture
challenges medium
plantlet components,
regeneration from an strength
isolatedofprotoplast.
the culture
medium, carbon sources, pH of the culture medium, supplements of
Types of culture medium, culture medium components, strength of the culture medium,the culture medium,
PGRs, and culture conditions have been proven to be vital factors for plantlet generations
carbon sources, pH of the culture medium, supplements of the culture medium, PGRs,
from protoplasts [69]. Considering these factors and despite these limitations, plantlets
and culture conditions have been proven to be vital factors for plantlet generations from
have been generated successfully in several ornamental plant species [69,73,74,78,94,95].
protoplasts [69]. Considering these factors and despite these limitations, plantlets have
been generated
3.3. Somatic successfully in several ornamental plant species [69,73,74,78,94,95].
Embryogenesis
An alternative to root and shoot regeneration from the callus, regeneration of the whole
3.3. Somatic Embryogenesis
plant from the plant cell throughout embryo formation, was identified in 1958 [96,97]. The
An alternative
development to root or
of an embryo and shoot
plant regeneration
from from the callus,
the vegetative/somatic regeneration
cell is of the
known as somatic
whole plant from the plant cell throughout embryo formation, was identified
embryogenesis [98]. The procedure for somatic embryogenesis is illustrated in Figure 3. in 1958
So-
matic embryogenesis is considered more efficient than other propagation techniques. which
guarantees variability. It produces identical genotypes differing from zygotic embryos,
which guarantees variability. The bipolar structure of a somatic embryo consists of apical
(known as plumule) and basal meristem regions (known as radicles), which are responsible
for shoot and root formation, respectively [99]. Cytological and histological studies have
as somatic embryogenesis [98]. The procedure for somatic embryogenesis is illustrated in
Figure 3. Somatic embryogenesis is considered more efficient than other propagation
techniques. which guarantees variability. It produces identical genotypes differing from
zygotic embryos, which guarantees variability. The bipolar structure of a somatic embryo
Plants 2022, 11, 3208 consists of apical (known as plumule) and basal meristem regions (known as radicles), 7 of 33
which are responsible for shoot and root formation, respectively [99]. Cytological and
histological studies have confirmed that PLBs (details in Section 3.4) are also somatic
embryos [99]. Morphogenesis or regeneration of PLBs can be initiated by direct or indirect
confirmed that PLBs (details in Section 3.4) are also somatic embryos [99]. Morphogenesis
embryogenesis. Organogenesis of PLB avoiding the callus phase is known as direct
or regeneration of PLBs can be initiated by direct or indirect embryogenesis. Organogenesis
embryogenesis, and PLB generated from the callus (an intermediate phase) is known as
of PLB avoiding the callus phase is known as direct embryogenesis, and PLB generated
indirect embryogenesis [99].
from the callus (an intermediate phase) is known as indirect embryogenesis [99].
Figure 3. Diagrammatic presentation

3. Diagrammatic presentation of
of the
the steps
steps involved
involved in
in somatic
somatic embryogenesis
embryogenesis for
for mass
mass
propagation
propagation in plants.
In
In somatic embryogenesis, the
somatic embryogenesis, the morphogenic
morphogenicresponse
responsevaries
variesononfactors
factorslike
likeexplants,
explants,
PGRs, hormones, concentrations of PGRs or hormones, light, etc.
PGRs, hormones, concentrations of PGRs or hormones, light, etc. [99–102]. Plantlet [99–102]. Plantlet regener-
ation by somatic embryogenesis has been reported in many genera of
regeneration by somatic embryogenesis has been reported in many genera of orchids; for orchids; for example—
Cymbidium [103–108], Phalaenopsis
example—Cymbidium [103–108],[108–115], Oncidium
Phalaenopsis [28,116–120],
[108–115], Dendrobium
Oncidium [121–124],
[28,116–120],
Rhynchostylis [125], Renanthera [126], Paphiopedilum [127,128], Malaxis
Dendrobium [121–124], Rhynchostylis [125], Renanthera [126], Paphiopedilum [127,128], [129,130], Epipactis ver-
atrifolia [131], Spathoglottis plicata [132], Geodorum densiflorum [133], Anoectochilus
Malaxis [129,130], Epipactis veratrifolia [131], Spathoglottis plicata [132], Geodorum densiflorum elatus [134],
[133],Nothodoritis
and Anoectochilus zhejiangensis
elatus [134],[135]. In additionzhejiangensis
and Nothodoritis to orchids, [135].
it hasInalso been reported
addition to orchids, in
diverse ornamentals, such as rose [136], Rosa × damascena [137], chrysanthemum
it has also been reported in diverse ornamentals, such as rose [136], Rosa ×damascena [137], [138,139],
lilies [140–146], jasmine
chrysanthemum [138,139],[147],
lilieslisianthus
[140–146],[148–151], carnation
jasmine [147], Camellia carnation
[152],[148–151],
lisianthus [153–157],
Cineraria [158], coneflower [159,160], Crocus [161–163], Clematis [164–166];
[152], Camellia [153–157], Cineraria [158], coneflower [159,160], Crocus [161–163], Clematis Sawara cy-
press [167], cyclamen [168], bellflower [169], passion flowers [170], perennial
[164–166]; Sawara cypress [167], cyclamen [168], bellflower [169], passion flowers [170], daisy and false
daisy
perennial[171,172];
daisy tulip [173],daisy
and false periwinkle [174],
[171,172]; peony
tulip [173],[175,176],
periwinkle anthurium [177–181],
[174], peony gen-
[175,176],
tian [182–185], Exacum trinervium [186], gloriosa [187,188], amaryllis
anthurium [177–181], gentian [182–185], Exacum trinervium [186], gloriosa [187,188], [189], phlox [190], Cen-
taurium
amaryllis erythraea [191], Lachenalia
[189], phlox viridifloraerythraea
[190], Centaurium [192], pine [193–196],
[191], Japanese
Lachenalia black[192],
viridiflora pine [197],
pine
agave [198–201], and hosta [202].
[193–196], Japanese black pine [197], agave [198–201], and hosta [202].
3.4. Protocorm-like Body
3.4. Protocorm-like Body
In Cymbidium orchid, protocorm-like bodies (PLBs) were noticed for the first time
duringIn the
Cymbidium
shoot-tiporchid,
culture protocorm-like bodiesProtocorms
by Morel (1960) [203]. (PLBs) werearenoticed for the first
small spherical time
tuber-like
during theformed
structures shoot-tip
in aculture by Morel
germinating seed;(1960) [203]. Protocorms
protocorm-like arewith
structures small spherical
similar tuber-
characteris-
tics generated from somatic cells in tissue culture techniques are known as PLBs [204,205].
PLBs are induced directly from explants and/or indirectly from calluses [206], and the for-
mation, regeneration, and proliferation of PLBs are among the most efficient techniques of
micropropagation, especially for clonal propagation of orchids [207]. Meristemoids in callus
cells initiate polarized growth, and continuous cell division causes the shoot pole (for shoot
initiation) and the base pole (for root initiation) of a protocorm-like body (PLB) [127,204,208].
The induction of PLBs has several advantages over typical shoot and plantlet regeneration,
such as a higher rate of multiplications, long-term preservation, easy differentiation into
shoots, generations of secondary PLBs, etc. The success of efficient PLB induction, regenera-
Plants 2022, 11, 3208 8 of 33
tion, and proliferation depends on multiple factors. Culture media ingredients, such as car-
bohydrate sources, plant growth regulators, elicitors, etc., are also crucial for efficient PLB
organogenesis and regeneration [205]. Growth retardants also stimulate PLB regeneration
in orchids through the inhibition of GA biosynthesis [26]. Setting up the optimum tempera-
ture in the growth chamber is also necessary for PLB proliferation, and a higher or lower
temperature compared to the optimum causes stress in PLB regeneration in orchids [209].
Light quality is another crucial factor for PLB organogenesis and regeneration for photosyn-
thetic and phototropic responses, and many studies have suggested the efficiency of LEDs
over traditional fluorescent light, suggesting the advantages of monochromatic light for
PLB organogenesis and regeneration (Supplementary Table S4) [205]. However, different
factors can work synergistically for better PLB organogenesis and regeneration compared
with their independent applications. However, all these external factors are highly species-
specific (Supplementary Table S5) [205]. We have also reported the manipulation of culture
media and growth conditions for PLB regeneration in Dendrobium [30,209–214] and Pha-
laenopsis [26,215–217]. We found that culture media manipulation and light quality are
highly species-specific in orchid PLB proliferation.
Besides these techniques, seed culture, meristem culture, anther culture, embryo
culture, ovule culture, cell suspension culture, and direct shoot organogenesis are also
practiced for in vitro plantlet generation in ornamentals.
4. Application of In Vitro Techniques in Ornamentals

Plant tissue culture is well known for producing disease-free plantlets by clonal
propagation. In vitro culture offers a wide range of possibilities for manipulating plant
materials to improve their quality. In vitro techniques are used for hybridization with the
assistance of micropropagation, embryo rescue, and somatic hybridization.
4.1. Plant Improvement by the Application of In Vitro Embryo Rescue

The technique of developing a viable plant from an embryo is known as embryo culture
or embryo rescue (Figure 4). The embryo culture technique was introduced by Hannig,
who cultured mature embryos of a few Brassicaceae plants on sugar-supplemented salt
medium [218]. In 1924, Dietrich disclosed that both mature and immature embryos could be
rescued [219]. The first interspecific hybridization by embryo rescue from nonviable seeds
was reported in the perennial flax (Linum perenne L. × Linum austriacum L.) in 1925 [220].
Plants 2022, 11, x FOR PEER REVIEW Since the first report, embryo rescue has been used for interspecific hybridization in9 many
of 25
crops, flowering, ornamentals, medicinals, and woody plants [221,222].
Processof
Figure4.4.Process
Figure ofembryo
embryorescue
rescuefrom
fromimmature
immature(or
(ornon-viable)
non-viable) seed after hybridization.
ItItallows
allowsfor
for the
the culture
culture of
of the
the ovary,
ovary, ovule,
ovule, and
and embryo
embryo [223–225].
[223–225]. The
The success
successof
of
embryo rescue depends on various factors, such as size and age of the embryo,
embryo rescue depends on various factors, such as size and age of the embryo, intactness intact-
ness of embryo, excision procedure, sterilization, culture medium, supplementation in
of embryo, excision procedure, sterilization, culture medium, supplementation in culture
culture medium, light, temperature, etc. [221,222]. It has been used in crop improve-
medium, light, temperature, etc. [221,222]. It has been used in crop improvement by
ment by intraspecific/interspecific/intergeneric hybrid development, haploid/double
intraspecific/interspecific/intergeneric hybrid development, haploid/double haploid
haploid production, overcoming embryo abortion, overcoming seed dormancy, over-
production, overcoming embryo abortion, overcoming seed dormancy, overcoming self-
coming self- and cross-incompatibility, shortening the breeding cycle, propagating rare
and cross-incompatibility, shortening the breeding cycle, propagating rare plants, etc.
plants, etc. [226–228]. For example, breeding cycles were shortened by embryo rescue
[226–228]. For example, breeding cycles were shortened by embryo rescue in rose [229],
in rose [229], and lily [230]. Interspecific hybrids were developed in chrysanthemums
and lily [230]. Interspecific hybrids were developed in chrysanthemums by embryo rescue
by embryo rescue technique for cold-tolerant [224,225,231], heat-tolerant [232], drought-
technique for cold-tolerant [224,225,231], heat-tolerant [232], drought-tolerant [233,234],
salt-tolerant [235], aphid resistance [236], and heterotic [224,232,237] characteristics. A
new flower shape and cold-tolerant intraspecific (Campanula carpatica ‘White’) and
interspecific (C. medium and C. formanekiana) hybrid, respectively, were developed in
bellflowers [238]. Interspecific hybrids, haploids, or double haploids were developed in
Plants 2022, 11, 3208 9 of 33
tolerant [233,234], salt-tolerant [235], aphid resistance [236], and heterotic [224,232,237]
characteristics. A new flower shape and cold-tolerant intraspecific (Campanula carpatica
‘White’) and interspecific (C. medium and C. formanekiana) hybrid, respectively, were de-
veloped in bellflowers [238]. Interspecific hybrids, haploids, or double haploids were
developed in rose [239–241], tulip [242], lisianthus [243], lily [244], day lily [245], calla
lily [246], alstroemeria or peruvian lily [247–250], Primula [251,252], night-blooming cac-
tus [253–255], gentian [256–258], Camellia [259], begonia [260], Christmas bells or golden lily
of the valley [261], carnation [262,263], Gypsophila [264], Rhododendron [265], cyclamen [266],
and ornamental alliums [267,268]. Embry rescue has been widely studied for crop im-
provement, while its current research has been reduced by the rapid evolution of advanced
molecular breeding.
In addition, embryo rescue is generally used to overcome post-fertilization barriers
in plants, while many ornamentals have pre-fertilization barriers [269,270] that can be
overcome by in vitro pollination. In in vitro pollination, plant reproductive cells (stigma
and anther) are isolated and fused under controlled conditions to develop a zygotic embryo.
The in vitro technique has been applied for in vitro flowering and pollination in different
ornamentals [227,271].
4.2. Plant Improvement by Somatic Hybridization and In Vitro Pollination

Somatic hybridization has been proven to be a great source to produce genetic vari-
ability which is known as somaclonal variation. Many somaclones have been considered
superior hybrids. Two methods are usually followed to produce the somatic hybrid, one
is cytoplast-protoplast fusion and the other is the donor-recipient method. In cytoplast–
Plants 2022, 11, x FOR PEER REVIEW 10 of 25
protoplast fusion, protoplasts are allowed to fuse for combining somatic cells either fully or
partially from different cultivars or species or genera (Figure 5).
Illustration of
Figure 5. Illustration
Figure of somatic
somatic hybrid
hybrid or or cybrid
cybrid development
development through
through protoplast
protoplast fusion.
fusion. Here,
Here,
NaNO
NaNO33; ; sodium
sodium nitrate,
nitrate, Ca(NO
Ca(NO33)2);2 ;calcium
calciumnitrate,
nitrate,PA;
PA;polyvinyl
polyvinyl alcohol,
alcohol, DS;
DS; dextran
dextran sulfate,
sulfate,
polyethylene
polyethylene glycol (PEG).
The combination
The combination of the nuclear genome of one parent with the mitochondrial and/or and/or
chloroplast genome
chloroplast genome of of the
the other
other parent
parent proceeds
proceeds in somatic hybridization. An An alternative
alternative
and improved
and improvedsomatic
somaticincompatibility
incompatibilityis the
is donor–recipient
the donor–recipient fusionfusion
method, where specific
method, where
genes or chromosomes can be transferred [272,273]. Chemicals used for protoplast
specific genes or chromosomes can be transferred [272,273]. Chemicals used for protoplast fusions
are known
fusions are as fusogens,
known and sodium
as fusogens, and nitrate
sodium(NaNO ), calcium
nitrate3 (NaNO nitrate (Ca(NO
3), calcium )2 ), dextran
nitrate3(Ca(NO 3)2),
sulfate, polyvinyl
dextran alcohol, alcohol,
sulfate, polyvinyl and polyethylene glycol are
and polyethylene common
glycol fusogensfusogens
are common [274]. Somatic
[274].
hybridization
Somatic by protoplast
hybridization fusion can
by protoplast develop
fusion either symmetric
can develop or asymmetric
either symmetric hybrids,
or asymmetric
which are known as somatic hybrids or cybrids (Figure
hybrids, which are known as somatic hybrids or cybrids (Figure 5). 5).
The first
The first asymmetric
asymmetric hybrid
hybrid was
was found
found in in somatic
somatic hybridization
hybridization through
through fusion
fusion
between Nicotiana tabacum (tobacco) and Petroselium hortense (parsley)
between Nicotiana tabacum (tobacco) and Petroselium hortense (parsley) [275,276]. Many [275,276]. Many
wild plant species have some significant traits, especially disease and pathogen
wild plant species have some significant traits, especially disease and pathogen resistance, resistance,
and these traits can be transferred into cultivated crop species. Somatic hybridization
allows the transfer of desirable traits to increase yield, resistance, tolerance, etc. [277,278].
It allows breeders to create novel hybrids by the asexual process, bypassing conventional
breeding (Figure 5).
Plants 2022, 11, 3208 10 of 33
and these traits can be transferred into cultivated crop species. Somatic hybridization
allows the transfer of desirable traits to increase yield, resistance, tolerance, etc. [277,278].
It allows breeders to create novel hybrids by the asexual process, bypassing conventional
breeding (Figure 5).
Somatic hybridization has been applied for the genetic improvements of various
flowering and ornamentals, such as rose [72], Dendrobium [279], chrysanthemum [95],
dianthus [280], gentin [281,282], iris [283], lily [284], petunia [285], between petunia and
Calibrachoa [286], hydrangea [287], cyclamen [288], coneflower [289], and Saintpaulia [290].
Somaclonal variants or somatic hybrids can be confirmed by morphological, biochem-
ical, protein marker, cytogenetic, and molecular analyses. Restriction fragment length poly-
morphism (RFLP), simple sequence repeat (SSR), amplified fragment length polymorphism
(AFLP), methylation-sensitive amplification polymorphism (MSAP), transposon-based
marker systems, and Next-Generation Sequencing (NGS) have been applied for the vali-
dation of somatic hybrids at the molecular level in several ornamentals [278]. Somaclonal
variation is highly dependent on the PGRs [291]. The main constraints of somatic hy-
bridization are the difficulties in isolating protoplasts (described in Section 3.2), generating
unexpected and useless variations, newly generated variants that are not novel, etc. [278].
4.3. Production of Synthetic Seeds

Any encapsulated plant tissue, somatic embryos, or any other micropropagules is
known as a synthetic seed or artificial seed (Figure 6). Synthetic seeds have several advan-
tages over natural seeds, such as season-independent seed production, genetic uniformity,
maintain hybrid vigor, long-term storage capacity, rapid multiplication, free from vegeta-
tive and seed-borne pathogens, propagation of high volume with low cost, assure quality
plant materials, and shorten the life cycles [292,293]. Somatic embryos, nodal segments,
and shoot tips are mostly used as explants for the development of synthetic seeds in
ornamentals, while callus is rarely used, and PLBs are mainly used in orchids to pro-
duce synthetic seeds. Synthetic seeds have been generated in Caladium bicolor (caladium),
Eustoma grandiflorum (lishianthus), Pinus patula (pine), Genista monosperma (bridal broom),
Hyoscyamus muticus (Egyptian henbane), and Clitoria ternatea (bluepea or bluebellvine) from
the somatic embryo; Gypsophila paniculata (gypsophila), Saintpaulia ionantha (saintpaulia),
Urginea altissima (tall white squill), and Taraxacum pieninicum (Mniszek pieninski) from
shoot tip; Rosa × damascena f. trigintipetala (Damask rose), Syringa vulgaris (lilac), Nerium
oleander (oleander), Centella asiatica (Asiatic pennywort), Eclipta alba (false daisy), Eryth-
rina variegata (tiger’s claw), Photinia fraseri (red tip photinia), Ruta graveolens (rue), Salix
tetrasperma (Indian willow) from axillary buds/nodes, Anthurium andreanum (anthurium)
from callus, Lilium longiflorum (easter lily) from bulb, and different species of orchids from
PLBs (Cymbidium giganteum, Vanda coerulea, Geodorum densiflorum, Coelogyne breviscapa,
Cremastra appendiculata, Flickingeria nodosa, Spathoglottis plicata, etc.) [292,293].
In vitro synthetic seeds in ornamentals allow season-independent seed production,
to preserve for long term, and to supply in time to the growers. Some factors are crucial
for the synthesis of artificial seeds in ornamentals; these are concentrations of sucrose,
sodium alginate (Na-alginate), and calcium chloride (CaCl2 ). A range of 2–3% sucrose,
2–3% Na-alginate, and 50–100 mM CaCl2 was found to be effective concentrations for
synthetic seed development in ornamentals [292,293].
Synthetic seeds have some limitations over the advantages: low efficient root systems,
development of non-synchronous seeds from the somatic embryo (the most effective plant
material for synthetic seed development), deviation from the normal structure, loss of em-
bryogenic potential with time, etc. Synthetic seed technology can be used more effectively
in the commercial ornamental plant propagation sector after resolving these limitations.
nodosa, Spathoglottis plicata, etc.) [292,293].
In vitro synthetic seeds in ornamentals allow season-independent seed production,
to preserve for long term, and to supply in time to the growers. Some factors are crucial
for the synthesis of artificial seeds in ornamentals; these are concentrations of sucrose,
sodium alginate (Na-alginate), and calcium chloride (CaCl 2). A range of 2–3% sucrose, 2–
Plants 2022, 11, 3208 11 of 33
3% Na-alginate, and 50–100 mM CaCl2 was found to be effective concentrations for
synthetic seed development in ornamentals [292,293].
Figure
Figure6.6. Production
Productionandandapplication
applicationofofsynthetic
syntheticseeds.
seeds.The
Thenumbers
numbersininthe thefigure
figurerepresent
representthe
the
ending
ending point ofeach
point of eachstep,
step, such
such as the
as the production
production of synthetic
of synthetic seeds
seeds (1), (1), short-term
short-term storage
storage of of
synthetic
synthetic
seeds (2),seeds (2), synthetic
synthetic seeds for transportation
seeds for transportation (3), storage
(3), long-term long-term storage ofseeds
of synthetic synthetic seeds
(4), and (4),
plantlet
and plantlet generation from synthetic seeds (5).
generation from synthetic seeds (5).
4.4. In Vitro Ploidy Manipulation

In vitro ploidy manipulation is a way of developing genetic diversification by in-
creasing or decreasing chromosome numbers (Figure 7). The induction of polyploidy is
used for crop improvement in ornamentals and can expand breeding opportunities to
expand traits in ornamentals, environmental tolerances, and restore fertility in wide hy-
brids [294]. Two antimitotic agents, colchicine or oryzalin, are mostly used for chromosome
doubling [295]. Two ginger lily lines: Hedychium gardnerianum Shepard ex Ker Gawl. and
H. coronarium J. Koenig were used for chromosome doubling using colchicine or oryza-
lin and successfully developed the tetraploid ginger lily [296]. Forty—eight tetraploids
were developed in ornamental aroid plants using colchicine (Caladium × hortulanum Bird-
sey) that showed variation in leaf shape, color, and thickness compared to the wild
type [297]. Tetraploid anise hyssop (Agastache foeniculum L.) was induced by the application
of colchicine, which showed a wide range of variation compared to diploid plants in their
morphophysiological characteristics [298]. Polyploid has also been inducted in Dendrobium,
Phalaenopsis, Epidendrum, and Odontioda orchids by the application of oryzalin [299]. Diverse
phenotypic variations were observed in the in vitro-generated polyploids in rose, lilies,
phlox, petunia, bellflowers, rhododendron, etc. [295]. Besides the antimitotic agents, ploidy
manipulation also depends on the species, types of explants, antimitotic agent exposure
method, duration of antimitotic agent exposure, etc.
application of colchicine, which showed a wide range of variation compared to diploid
plants in their morphophysiological characteristics [298]. Polyploid has also been
inducted in Dendrobium, Phalaenopsis, Epidendrum, and Odontioda orchids by the
application of oryzalin [299]. Diverse phenotypic variations were observed in the in vitro-
Plants 2022, 11, 3208 generated polyploids in rose, lilies, phlox, petunia, bellflowers, rhododendron, etc. 12
[295].
of 33
Besides the antimitotic agents, ploidy manipulation also depends on the species, types of
explants, antimitotic agent exposure method, duration of antimitotic agent exposure, etc.
Figure7.7.InInvitro
Figure vitrochromosome
chromosomedoubling
doubling(ploidy
(ploidymanipulation)
manipulation) for
for genetic diversification.
Diverse phenotypic variations were observed in the in vitro-generated polyploids in

roses, lilies, phlox, petunia, bellflowers, rhododendron, etc. [295]. Besides the antimitotic
agents, ploidy manipulation also depends on the species, types of explants, antimitotic
agent exposure method, duration of antimitotic agent exposure, etc. The chromosome
doubling technique produces only additional copies of chromosomes and genes, but it
does not generate new genetic materials. However, it may cause morphological changes,
anatomical changes, loss of duplicated genes, changes in gene expression, changes in
epigenome status, and changes in epigenomic alteration-mediated gene expression, which
ultimately lead to superior phenotypes in polyploids compared with diploids. These
changes may also help generate resistance and tolerance capacity to biotic and abiotic stress.
5. Future Perspective
In vitro plant propagation and multiplication offer significant potential for the ad-
vancement of both basic and applied biological sciences. Rapid multiplication and propa-
gation by callus culture, protoplast culture, somatic embryogenesis, PLB organogenesis,
and direct plantlet regeneration allowed for the cheaper and disease-free seedling of a
diverse ornamental plant species. Millions of in vitro plantlets of different ornamentals are
generated worldwide for commercial purposes. However, it is important to put more effort
into reducing the cost of production. In contrast to propagation, it also facilitates plant
improvement following diverse techniques, such as embryo rescue, somatic hybridization,
in vitro pollination, ploidy manipulation, the development of synthetic seeds, etc., and
large numbers of hybrids in various ornamentals have already been developed. In addition,
the in vitro technique is largely used for phytochemicals and secondary metabolite produc-
tion. However, more effort is needed to reduce species-specific and other factor-specific
responses for the efficient regeneration of ornamentals.
In recent years, researchers have started to study at the molecular level, including
genetic transformation, using in vitro technology in ornamentals [300]. About 40 genera
have been reported on creating transgenic ornamental species using Agrobacterium tumefa-
synthetic seeds, etc., and large numbers of hybrids in various ornamentals have already
been developed. In addition, the in vitro technique is largely used for phytochemicals and
secondary metabolite production. However, more effort is needed to reduce species-
specific and other factor-specific responses for the efficient regeneration of ornamentals.
Plants 2022, 11, 3208 In recent years, researchers have started to study at the molecular level, including
13 of 33
genetic transformation, using in vitro technology in ornamentals [300]. About 40 genera
have been reported on creating transgenic ornamental species using Agrobacterium
tumefaciens-mediated transformation [301]; however, only a few ornamentals, such as
ciens-mediated transformation [301]; however, only a few ornamentals, such as Phalaenopsis
Phalaenopsis and petunia, have suitable and efficient transformation techniques. Some
and petunia, have suitable and efficient transformation techniques. Some studies have
studies have revealed that many genes and transcriptions are involved in the in vitro
revealed that many genes and transcriptions are involved in the in vitro organogenic callus,
organogenic callus, shoot, root, somatic embryos, and PLBs, and the transcriptions of
shoot, root, somatic embryos, and PLBs, and the transcriptions of those genes are also
those genes are also regulated by the exogenous application of different growth regulators
regulated by the exogenous application of different growth regulators [302].
[302].
It is believed that plant tissue culture generates genetically identical genotypes or
It is believed that plant tissue culture generates genetically identical genotypes or
somaclonal variants. Recent studies in Arabidopsis and crop plants, such as rice, wheat,
somaclonal variants. Recent studies in Arabidopsis and crop plants, such as rice, wheat,
corn, barley, and rye, have suggested that tissue culture can alter the genetic nature by
corn, barley, and rye, have suggested that tissue culture can alter the genetic nature by
point mutations [303]. In contrast to genetic factors, different epigenetic regulators, such as
point mutations [303]. In contrast to genetic factors, different epigenetic regulators, such
DNA methylation and histone modifications, are also involved in regulating the success
as DNA methylation and histone modifications, are also involved in regulating the
of in vitro plant propagation [302,303]. Most of the genetic and epigenetic studies were
success of in vitro plant propagation [302,303]. Most of the genetic and epigenetic studies
conducted in the model plant Arabidopsis or crop plants, and this suggests the scope of
were conducted in the model plant Arabidopsis or crop plants, and this suggests the scope
future study in genetic and epigenetic aspects (Figure 8).
of future study in genetic and epigenetic aspects (Figure 8).
Figure 8. Prospects for advanced molecular research in plant tissue culture using orchid plants as an
example. Here, Tc; Tissue culture regenerated plants, Vs; traditional vegetatively propagated plants.
Tissue culture alters genome-wide DNA methylation in the CG, CHG, and CHH
contexts (H represents the A, C, or T), and these alterations change the gene expression that
might be regulating factors for in vitro plant growth and development. DNA methylation
was studied in the callus and somatic embryos of Arabidopsis and crop plants, and callus and
somatic embryos are vulnerable to the alteration of DNA methylation, leading to changes in
gene expression [303,304]. Involvement of di-methylated lysine 4 of histone H3 (H3K4me2)
was associated with successful shoot regeneration from callus in Arabidopsis [305], while
H3K4me3 and H3K27me3 histone marks are involved with the callus tissues in rice [306].
These reports suggest the importance of epigenetic regulation of in vitro regenerated plants.
Besides DNA methylation and histone modification, different miRNAs and sRNAs may
also be involved in the success of in vitro plant propagation. The expression of transposable
elements (TEs) can also be epigenetically regulated in vitro environments; for example, TEs
can be activated by the plant tissue culture [307]. However, there has been no significant
advancement in the molecular mechanisms controlling in vitro regeneration in ornamentals.
Studies on Arabidopsis and crop plants provide fundamental knowledge for disclosing
the molecular mechanisms in ornamental plant species. Therefore, it is high time for ad-
vanced study of the genetic and epigenetic mechanisms that would provide a breakthrough
in the commercialization of in vitro propagation of ornamental plant species.
Supplementary Materials: The following supporting information can be downloaded at: https://www.
mdpi.com/article/10.3390/plants11233208/s1, Table S1: Effective plant growth regulators and other
factors for the in vitro culture in ornamentals [38,110,111,113,114,123,173,192,308–507]; Table S2: Effective
elicitors for the in vitro culture in ornamentals [26,51,215–217,340,404,458,470,482,508–517]; Table S3: Effec-
tive additives for the in vitro culture in ornamentals [342,347,350,363,365,504,518–523]; Table S4: Effective
light emitting diodes (LEDs) for the in vitro culture in ornamentals [26,31,55,58,61,210,440,467,524–554];
Table S5: Studies in combination of light emitting diodes (LEDs) and plant growth regulators in vitro
culture in ornamentals [26,31,55,58,61,62,210,317,440,467,524–559].
Author Contributions: Conceptualization, H.M. (Hasan Mehraj); writing—original draft preparation,
H.M. (Hasan Mehbub), A.A., M.A.A., M.S.H.M., M.A.H. and H.M. (Hasan Mehraj); visualization,
Plants 2022, 11, 3208 14 of 33
H.M. (Hasan Mehraj); writing—review and editing, M.T. and H.M. (Hasan Mehraj). All authors have
read and agreed to the published version of the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Acknowledgments: We thank to our colleagues for accessing, non-open access to us, articles.
Conflicts of Interest: The authors declare no conflict of interest.
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Tissue Culture-Plants Casopis

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plants

Received: 19 October 2022

Plants 2022, 11, 3208. https://doi.org/10.3390/plants11233208 https://www.mdpi.com/journal/plants

2. In Vitro Cultivation Factors

2.2. Plant Growth Regulators, Inhibitors, and Elicitors in Culture Media

3. Standard Techniques Involved in Plantlet Generation In Vitro

3.2. Protoplast Culture

Figure 3. Diagrammatic presentation

4. Application of In Vitro Techniques in Ornamentals

4.1. Plant Improvement by the Application of In Vitro Embryo Rescue

4.2. Plant Improvement by Somatic Hybridization and In Vitro Pollination

4.3. Production of Synthetic Seeds

4.4. In Vitro Ploidy Manipulation

Diverse phenotypic variations were observed in the in vitro-generated polyploids in

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