agronomy

Review
Plant Cell Cultures: Biofactories for the Production of
Bioactive Compounds
Vishwas Anant Bapat 1 , P. B. Kavi Kishor 2 , Naravula Jalaja 3 , Shri Mohan Jain 4, * and Suprasanna Penna 5, *

1 Department of Biotechnology, Shivaji University, Kolhapur 416 004, India; [email protected]

2 Department of Genetics, Osmania University, Hyderabad 500 007, India; [email protected]
3 Department of Biotechnology, Vignan’s Foundation for Science, Technology and Research,
Vadlamudi, Guntur 522 213, India
4 Department of Agricultural Sciences, PL 27, Helsinki University, 00014 Helsinki, Finland
5 Amity Centre for Nuclear Biotechnology, Amity Institute of Biotechnology, Amity University of Maharashtra,
Bhatan, Mumbai 410 206, India
* Correspondence: [email protected] (S.M.J.); [email protected] (S.P.)

Abstract: Plants have long been exploited as a sustainable source of food, flavors, agrochemicals,
colors, therapeutic proteins, bioactive compounds, and stem cell production. However, plant habitats
are being briskly lost due to scores of environmental factors and human disturbances. This necessi-
tates finding a viable alternative technology for the continuous production of compounds that are
utilized in food and healthcare. The high-value natural products and bioactive compounds are often
challenging to synthesize chemically since they accumulate in meager quantities. The isolation and
purification of bioactive compounds from plants is time-consuming, labor-intensive, and involves
cumbersome extraction procedures. This demands alternative options, and the plant cell culture
system offers easy downstream procedures. Retention of the metabolic cues of natural plants, scale-up
facility, use as stem cells in the cosmetics industry, and metabolic engineering (especially the rebuild-
ing of the pathways in microbes) are some of the advantages for the synthesis and accumulation of
the targeted metabolites and creation of high yielding cell factories. In this article, we discuss plant
cell suspension cultures for the in vitro manipulation and production of plant bioactive compounds.
Further, we discuss the new advances in the application of plant cells in the cosmetics and food
industry and bioprinting.

Citation: Bapat, V.A.; Kavi Kishor, Keywords: plant cell suspensions; bioactive compounds; bioprinting; metabolic engineering; cosmetics;
P.B.; Jalaja, N.; Jain, S.M.; Penna, S. plant stem cells
Plant Cell Cultures: Biofactories for
the Production of Bioactive
Compounds. Agronomy 2023, 13, 858.
https://doi.org/10.3390/ 1. Introduction
agronomy13030858
Plants are a potent and inexhaustible source of phytoconstituents. Out of 50,000 plant
Received: 2 February 2023 species spread the world over, only one percent of them produce bioactive compounds,
Revised: 12 March 2023 leading to their overexploitation, destruction of natural habitat, and extinction [1]. Nearly
Accepted: 13 March 2023 25% of modern medicines are derived from natural resources such as algae, fungi, and
Published: 15 March 2023 higher plants [2,3]. Plant-based drugs have been in use since time immemorial, and the
market has been growing steadily and may reach USD 50 billion during the next five years
or so [1]. While some plants are endemic, others are endangered. Many plants produce
Copyright: © 2023 by the authors.
nano quantities of secondary plant products and are often dependent on seasonal vari-
Licensee MDPI, Basel, Switzerland.
ations. Conventional methods of obtaining bioactive compounds are confronted with a
This article is an open access article number of inherent drawbacks and are affected by several internal and external factors.
distributed under the terms and Plant tissue cultures are an excellent source for the extraction of many targeted metabolites.
conditions of the Creative Commons Therefore, plant tissue, organ, and cell suspension cultures are being exploited for the
Attribution (CC BY) license (https:// accumulation and analysis of numerous phytochemicals and for the biotransformation of
creativecommons.org/licenses/by/ physiologically not-so-active compounds into pharmacologically active compounds [4–9].
4.0/). Further, morphogenic cell suspensions are a good source and an exceptional platform for

Agronomy 2023, 13, 858. https://doi.org/10.3390/agronomy13030858 https://www.mdpi.com/journal/agronomy

Agronomy 2023, 13, 858 2 of 20

the examination of metabolomic and proteomic changes that take place during the produc-
tion of various bioactive compounds [10,11]. Such an approach may ultimately lead to the
discovery of biosynthetic pathways of the bioactive compounds. The homogeneity of cell
suspensions, scalability, reasonably good cell doubling time, less space requirement, and
ease of handling the cultures are the key features that offer attractive options for the pro-
duction of many secondary plant products. Further, in vitro cultures are free from seasonal
variations and microbial contamination and, hence, are ideal for the large-scale generation
of bioactive molecules. Biochemical and physiological pathways associated with secondary
plant products have been elucidated by integrating the data generated through multi-omic
techniques in many plant systems, including the model plant A. thaliana [12]. Elicitation,
immobilization, and permeabilization coupled with in situ adsorption of secondary plant
products using resins such as XAD7 are possible using suspensions [12,13]. Elicitation is
one well-known yield enhancement strategy, usually implemented to induce or enhance
secondary metabolite synthesis by the plant cells. In addition to enhancing secondary
metabolite production, elicitation involves the manipulation of various parameters that
can induce stress in biochemical and metabolic pathways and, thus, can be applied to
understanding the role of several stress factors on plants by using plant cells or tissue
culture as model systems [14]. This provides an opportunity for intensive research in the
field of biosciences for the exploitation of plant cells to produce high-value secondary
metabolites. Chodisetti et al. [15] reported the elicitation of gymnemic acid in the cell
suspension cultures of Gymnema sylvestre R. Br. using phytohormones such as salicylic acid
and methyl jasmonate as elicitors. In addition, Chodisetti et al. [16] improved gymnemic
acid production in the suspension cultures using biotic elicitors such as Agrobacterium
rhizogenes, Saccharomyces cerevisiae, Aspergillus niger, Bacillus subtilis, and Escherichia coli.
Veerashree et al. [17] reported the elicitation of gymnemic acid through cell suspension
cultures using biotic elicitors such as methyl jasmonate, yeast extract, pectin, and chitin.
Several reports emphasized the importance of yeast extract in eliciting secondary metabo-
lite production for in vitro cultures, including rosmarinic acid and lithospermic acid B [18],
plumbagin [12], and camptothecin [19]. There are several reports on the use of pectin
as an elicitor, for example, the elicitation of triterpene acids from Uncaria tomentosa cell
suspension cultures [20] and menthol from Mentha piperita cell culture [21]. The production
of high-value compounds from plant cell cultures using biotic and abiotic elicitors has insti-
gated a novel research area that has commercial benefits for the pharmaceutical industry.
Hence, numerous factors, such as the type and concentration of elicitor, age of culture, cell
viability, cell line selected, period of elicitor exposure, nutrient composition, and growth
regulation that affect the process of elicitation, should be taken into consideration while
performing elicitation studies [22]. Methods that can be applied to determine the ideal time
include biomass evaluation, carbohydrate utilization, and analyzing extracellular products
formed. The appropriate quantity of elicitor that effectively enhances the production of
secondary metabolite varies widely for each cell material and must be determined.
Such novel techniques facilitate the exploitation of cell cultures for large-scale produc-
tion of secondary metabolites, pigments, aromas, flavors, dietary supplements, fragrances,
cosmetics, and bio-stimulants [23–26]. In situ adsorption of the compounds aid in the
extraction of the compounds directly and avoids the purification steps in the downstream
process [12]. Therefore, hairy root cultures and suspensions are good alternative sources,
especially in the wake of competition for land use in view of intensive agriculture, climate
change, and dwindling natural resources. Elicitation-enhanced synthesis of secondary
products such as chicoric acid, rosmarinic acid, rutin, isoquercetin, linalool, and estragole
have been reported in Ocimum basilicum [26]. In saffron, cell suspension cultures were used
to establish a system for salicylic acid-elicited production of specialized metabolites such
as crocin and carotenoid [27].
High-value compounds obtained from plant cell suspension cultures that have en-
tered the pharmaceutical industry include carotenoids, rosmarinic acid, polysaccharides,
berberine, podophyllotoxin, docetaxel, paclitaxel, scopolamine, and shikonin [1]. Commer-
Agronomy 2023, 13, 858 3 of 20

cialization of such compounds has further encouraged researchers to exploit cell culture
techniques for the accumulation of other high-value and low-volume products on a large
scale. Because of their high homogeneity and growth rate in liquid cultures, suspensions
are also regarded as model systems for deciphering complex biochemical processes [28,29].
Specific tobacco cell lines Bright Yellow 2 (BY-2) have been utilized as model systems to
investigate molecular mechanisms associated with cell divisions, phytohormone signaling,
intracellular trafficking, and the production of secondary products [30,31]. Bioreactors are
now developed for industrial-scale production of cell suspensions having value-added
compounds [1,32]. Numerous types of bioreactors using plant cells, such as stirred tanks,
bubble beds, rotary bioreactors, membrane bioreactors, and reactors for hairy root cultures,
have been designed [33]. The effects of two-stage culture, initially for proliferation and
then for the production of secondary metabolites, have been studied. Bioreactors for im-
mobilized plant cells are also described. Immobilized cells in the bioreactors are protected
from fluid dynamic stresses and are metabolically stable, but mass transfer limitations and
other factors such as oxygen, substrate, and product transports could be problematic and
challenging. Approaches for enhancing the productivity of plant cultures in bioreactors
are coupled with the design of well-organized systems, and an acceptable method is often
linked to using a combination of different techniques and treatments. A number of studies
and patents related to bioreactor designs and fabrications are revolutionizing the entire
process of plant cell culture technology [1,33].
We used the following keywords and web pages to obtain the crucial information
furnished here. While the important keywords include bioactive compounds, cosmet-
ics, foods and food ingredients, hairy roots, industrial production, phytochemicals, plant
cell cultures, and suspension cultures, the following web pages have also been explored:
(a) https://www.abres.it/echinacoside.php (accessed on 20 January 2022); (b) https://
www.abres.it/teupolioside.php (accessed on 25 February 2022); (c) https://www.abres.it/
verbascoside-acteoside.php (accessed on 6 April 2022); (d) https://mibellebiochemistry.
com/phytocelltectm-nunatakr (accessed on 10 March 2022); (e) https://mibellebiochemistry.
com/phytocelltectm-symphytum (accessed on 15 May 2022). PhytoCellTec™ by Mibelle
Biochemistry used bioreactors for the culture of stem cells in large-scale bioreactors, and
other examples include cell suspension bioreactor cultures, including Rosa damascena, Haber-
lea rhodopensis, and Calendula officinalis. Many cosmetic and pharmaceutical companies are
now using bioreactors for modern pharmaceutical and cosmetic products with exceptional
properties [34]. In addition, an anti-aging or antioxidant composition including compounds
derived from a ginseng cambium-derived plant stem cell line as an active ingredient has
been patented by Unhwa Corp. (Patent number: US9095532B2). The product is designed
to minimize the side effects associated with existing anti-aging agents and antioxidants,
making it safe for the skin. It also has antioxidant properties, inhibiting reactive oxygen
species caused by exposure to UV radiation, which is the main cause of skin aging. Another
example is TEUPOL 10P or 50P, which is an extract from Ajuga repta.
Plant cell suspensions, being sustainable, free from pesticide residues, and indepen-
dent of climate changes, have advantages over field-grown intact plants, which display
developmental stages of growth, age of the plant, and organ-specific variations [5,35]. Plant
cell cultures have the potential to release biologically active chemicals through the cell
membrane and cell walls into the intercellular spaces or into the spent medium; hence, the
process of their extraction and purification is meaningfully simplified [36]. The secreted
substances, after downstream processing from the cells, have a high degree of purity with
minimal losses. Several biochemical and genetic factors play significant roles in product
synthesis in the cell cultures. Genetic background, feedback regulation, channeling, cell
selection and screening, and degradation and adsorption of metabolites determine the
metabolite synthesis and accumulation in the cell cultures [7,36]. Plant cell suspension
cultures are now being employed for recombinant protein expression, scaled-up using
bioreactors, transgenic plant development, in vitro germplasm preservation, generation of
novel mutants, bioprinting, cosmetics, and biotransformation (Figure 1). Suspensions have
 significant roles in product synthesis in the cell cultures. Genetic background, feedback
regulation, channeling, cell selection and screening, and degradation and adsorption of
metabolites determine the metabolite synthesis and accumulation in the cell cultures
[7,36]. Plant cell suspension cultures are now being employed for recombinant protein
Agronomy 2023, 13, 858 expression, scaled-up using bioreactors, transgenic plant development, in vitro 4 of 20
germplasm preservation, generation of novel mutants, bioprinting, cosmetics, and bio-
transformation (Figure 1). Suspensions have certain limitations since some of the com-
pounds
certaincannot be produced
limitations since somein of
suspensions, unlike
the compounds that of
cannot be organ
producedcultures like hairy roots
in suspensions, unlike
(exploited for root-derived chemicals) or shooty teratomas (produced
that of organ cultures like hairy roots (exploited for root-derived chemicals) for the synthesis of
or shooty
secondary produced noticed in aerial parts of plants) [37,38]. Cell suspensions
teratomas (produced for the synthesis of secondary produced noticed in aerial parts of estab-
lished
plants)from the meristematic
[37,38]. cellsestablished
Cell suspensions have certain
fromunique features and
the meristematic arehave
cells generally
certaingenet-
unique
ically stable and, hence, are widely exploited for higher metabolite
features and are generally genetically stable and, hence, are widely exploitedsynthesis [39,40]. Ex-
for higher
tensive worksynthesis
metabolite has been[39,40].
carriedExtensive
out usingwork
meristem-derived
has been carried cellout
suspensions for the pro-
using meristem-derived
duction of specialized biomolecules [41,42].
cell suspensions for the production of specialized biomolecules [41,42].

Figure 1. Potential applications of plant cell cultures. Rare and endangered plants can be conserved
Figure 1. Potential
in vitro applications
in the form of plant cell
of cell suspensions (in cultures. Rare andpreservation),
vitro germplasm endangered plants can the
and also be conserved
cells can be
intreated
vitro inwith
the form of cell suspensions (in vitro germplasm preservation), and also the cells can be
physical and chemical mutagens for generating variations in the secondary metabolite
treated with physical and chemical mutagens for generating variations in the secondary metabolite
content. The rest of the applications are explained in the text.
content. The rest of the applications are explained in the text.
2. Production of Therapeutic Recombinant Proteins from Cell Cultures
2. Production of Therapeutic Recombinant Proteins from Cell Cultures
The biopharmaceutical market has been growing fast; several products are commer-
The biopharmaceutical
cialized, and many more aremarket has been
in the pipeline growing
[23,43]. fast; several
Therapeutic products
recombinant are com-
proteins have
mercialized, and many
become an effective more against
arsenal are in the pipeline
several [23,43].
diseases (TableTherapeutic recombinant
1). Currently, pro-
bacteria, yeasts,
teins
andhave
insectbecome an effectivehost
and mammalian arsenal
cellsagainst
grownseveral diseasesare
in fermenters (Table 1). Currently,
the preferred bac-
platforms
teria, yeasts, andrecombinant
for producing insect and mammalian host cells
proteins. Similar grown in fermenters
to mammalian are the preferred
cells and microbial cultures,
platforms for producing
tobacco BY-2 recombinant
suspensions have beenproteins.
found toSimilar to mammalian
be highly encouragingcells andsynthesis
for the microbialof
cultures, tobacco
therapeutic BY-2 suspensions
recombinant have been
proteins. While found system
a microbial to be highly encouraging
is a simple for the
and economically
synthesis
feasible of therapeutic
system, recombinant
it cannot synthesize proteins.
complex While a microbial
proteins systempost-translational
and perform is a simple and
economically
modifications.feasible system,
Contrarily, it cannot
mammalian cellssynthesize complex
perform precise proteins
folding and perform
of proteins and other
essential modifications,
post-translational but economic
modifications. feasibility
Contrarily, is a question.
mammalian Additionally,
cells perform both
precise platforms
folding of
are prone
proteins and and susceptible
other essentialtomodifications,
unwanted contamination,
but economicposing problems
feasibility for downstream
is a question. Addi-
processing
tionally, bothand obtaining
platforms arethe purified
prone andproteins [23].to unwanted contamination, posing
susceptible
In this regard, plant cells undergo post-translational
problems for downstream processing and obtaining the purified modifications
proteinsand can synthesize
[23].
many therapeutic
In this proteins
regard, plant with
cells properpost-translational
undergo folding. Plant cell suspensions and
modifications or organ cultures
can synthe-
have
size manybeentherapeutic
used for the production
proteins with of recombinant
proper folding.proteins,
Plant cellantibodies,
suspensions and orvaccines
organ cul-[44]
(Table
tures 1). been
have The added benefits
used for are a scale-up
the production possibility under
of recombinant a sterile
proteins, environment,
antibodies, and vac-low
growth costs, production of complex proteins, low risk of contamination
cines [44] (Table 1). The added benefits are a scale-up possibility under a sterile envi- with human
pathogens, and the possibility of optimized growth procedures [45].
Agronomy 2023, 13, 858 5 of 20

Table 1. Compendium on therapeutic proteins produced using plant cell cultures/hairy roots.

Name of the Therapeutic Protein Plant Cells Used as Hosts Yield of Therapeutic Protein Reference
Hyaluronic acid (recombinant human hyaluronic acid
synthase2, rhHAS2) Nicotiana tabacum
65.72 ng/kg fresh weight [46]
(A polysaccharide used in pharmaceutical, biomedical, hairy root cultures
and cosmetic fields)
Daucus carota
Miraculin protein in transgenic carrot callus 0.024 µg/µL (0.98% of TSP) at 6
suspension cultures using air-lift [47]
suspensions using air-lift bioreactors days
bioreactors
Solanum tuberosum 301.27 international units of
L-Asparaginase II [48]
hairy root cultures activity/mg of protein
Nicotiana tabacum
Human α1-antitrypsin 34.7 mg/L medium [49]
BY-2 cell cultures
Anti-CD20scFv-Fc (tumor-targeting antibody)
Nicotiana benthamiana
(enhancing the secretion of a glycoengineered 334 mg/L medium [50]
hairy root cultures
anti-CD20 scFv-Fc antibody)
Arachis hypogaea
Human epidermal growth factor (hEGF) 10.7 µg/g weight [51]
hairy root cultures
Brassica rapa
Human alpha-L-iduronidase (IDUA) Not known [52]
hairy roots
Human gastric lipase (hGL)
Brassica rapa
(therapeutic enzyme used for pancreatic enzyme 528 units of hGL/g dry weight of
subspecies rapa [53]
deficiency, it contributes to fatty acid release from tissue
hairy root cultures
ingested triglycerides)
Virus-like particle harboring recombinant protein
Nicotiana tabacum
shells of Johnson grass chlorotic stripe mosaic virus Not known [54]
hairy roots
(JgCSMV) and their use as drug carriers
Recombinant
human erythropoietin
(rhEPO)
Nicotiana tabacum
(glycoprotein hormone that influences the production 66.75 ng/g of total soluble protein [55]
hairy root cultures
of erythrocytes through
erythropoiesis. EPO is used for the treatment of
anemia)
Oryza sativa 160.7–242.8 mg/kg of transgenic
Bevacizumab monoclonal antibody [56]
callus cultures rice callus
Tumor-targeting antibody with a human-compatible Nicotiana benthamiana
2–3 mg/L medium [57]
glycosylation profile hairy root cultures
MAP30 protein of Momordica charantia (valuable type I
ribosome-inactivating protein (RIP) Nicotiana tabacum
Not known [58]
with anti-tumor and hairy root cultures
anti-HIV activities)
Fragaria x ananassa Duch. 160 ng in 100 mg of tissue (0.14%
Human pro-insulin [59]
hairy root cultures of the total soluble protein)
Oryza sativa
Human serum albumin 45 mg/L in a bioreactor [60]
cell suspension cultures
Nicotiana tabacum
Monoclonal antibody M12 for cancer treatment 5.9 mg/L medium [61]
hairy root cultures
Thaumatin sweetener Nicotiana tabacum hairy root
2.63 mg/L medium [62]
(intensely sweet protein) cultures
Oryza sativa cv. Dongjin cell
Bovine trypsins 15 mg/L at 5 days of incubation [63]
suspension cultures
Nicotiana tabacum
Human growth hormone 5.2% total soluble proteins [64]
BY-2 cell cultures
Fed-batch cultivation of Oryza
Cytotoxic T cells surface antigen (hCTLA4Ig) 76.5 mg/L medium [65]
sativa cells
Hairy root cultures of Nicotiana
Human epidermal growth factor (hEGF) 2 µg/g fresh weight [66]
tabacum
Human interleukin-12 Oryza sativa
31 mg/L [67]
(hIL-12), a heterodimeric cytokine) cell suspensions
Human acetylcholinesterase
Nicotiana benthamiana hairy root
(a bio-scavenger of organophosphate toxins used as 3.3% total soluble proteins [68]
cultures
pesticides and chemical warfare nerve agents)
Oryza sativa cv. Donjin 57 cell
Human growth hormone 57 mg/L [69]
suspensions
Human collagen α1 chain industrially
Hordeum vulgare cell cultures 2–9 mg/L medium [70]
important protein
Nicotiana tabacum cv. BY-2 cells as
Human interferon α-2b 28 mg/L [71]
arabinogalactan-protein fusions
Agronomy 2023, 13, 858 6 of 20

Table 1. Cont.

Name of the Therapeutic Protein Plant Cells Used as Hosts Yield of Therapeutic Protein Reference
Human CTLA4ig
(cytotoxic T-lymphocyte antigen 4 immunoglobulin Oryza sativa
31.4 mg/L suspension cultures [72]
(hCTLA4I g) fusion protein, a novel cell suspensions
immunosuppressive agent)
Nicotiana tabacum cv. NT-1 cell
Human secreted alkaline phosphatase 27 mg/L [73]
suspensions
Nicotiana tabacum
Human antibody 14D9 64.03 µg/mL of medium [74]
hairy root cultures
100–247 mg/L
Oryza sativa
Human α-1-antitrypsin (4–10% of the total extracellular [75]
cell cultures
protein)
Oryza sativa
Human serum albumin 76.4 mg/L in 4-day-old cultures [76]
cell cultures
17.4 ng/mL media and 131.6 ng/g
Oryza sativa
Human interferon gamma cell in culture medium and [77]
cell suspension cultures
intracellularly
Human granulocyte-macrophage Oryza sativa
129 mg/L at day 13 [78]
colony-stimulating factor cell suspension cultures
0.6% of the brown rice weight or
Antimicrobial human lysozyme Oryza sativa cv. Taipie grains [79]
45% of total soluble proteins
Human α-1 antitrypsin Oryza sativa cultured cells 200 mg/L [80]
Oryza sativa callus and suspension
Human 1-antitrypsin 4.6–5.7 mg/g dry cell weight [81]
cultures
Human erythropoietin Nicotiana tabacum cv. BY-2 1 picogram on dry weight basis [82]
Nicotiana tabacum and Solanum
Human serum albumin tuberosum Not known [83]
suspension cultures

Upscaling cell cultures in bioreactors enhances the production of recombinant proteins

similar to bacterial or mammalian systems [45,84]. A comparative account has been made
for the production of the anticancer compound viscumin (a lectin) both in microbes and
plant cell packs [85]. The authors observed that cost savings were more than 80% when
produced in tobacco in comparison with E. coli. Further, the yield was 50% higher in
plant cell packs compared to microbes [85], inferring that plant cells have the potential to
produce complex therapeutic proteins which are otherwise toxic to mammalian cells. For
the production of therapeutic pharmaceuticals, antibodies, and vaccine proteins, cells of
Daucus carota, Oryza sativa, and Nicotiana tabacum BY-2 cells are being exploited [86]. Edible
tubers, fruits, and seeds of many plants act as bioreactors and are good candidates for
the production of edible vaccines. Traditionally, vaccines are stored at low temperatures,
but the need for the cold chain for storing the vaccine can be avoided, especially in rural
areas, if edible vaccines are promoted. If vaccines are produced in edible plant parts, the
advantage is that one can leave out the product purification step and, thus, reduce the cost
of production and avoid storage concerns [87].
Human cytotoxic T-lymphocyte antigen 4-immunoglobulin (hCTLA4I g), alpha1-
antitrypsin besides human growth hormone and interferon-gamma (INF-γ) production has
been demonstrated in transgenic rice suspensions [88]. Likewise, immunoglobulins such
as IgA, IgG4, and IgG, surface hepatitis virus antigen B, and rabies virus surface human
immunodeficiency virus (HIV) have been produced in N. tabacum cv. BY-2 suspensions.
Some examples of vaccines produced in plant cell cultures include lysozyme, DNAase
I hemagglutinin-neuraminidases, hemagglutinin, and thermolabile toxin in E. coli [86].
Yao et al. [89] produced the human monoclonal antibody M12 in a bioreactor. Kumar et al. [90]
reported the secretion of hepatitis B surface antigens in transformed tobacco cell suspension
cultures. Sadoch et al. [91] optimized the induction medium for the production of recom-
binant proteins in BY-2 tobacco cells and, thus, paved the way for large-scale production.
Lico et al. [92] churned out protein antigens as vaccine candidates and also antibodies as
therapeutic proteins on a large scale, demonstrating the power of molecular plant farming
as a potential strategy against COVID-19. Imamura et al. [93] produced ORF8 protein from
SARS-CoV2 in suspension cultures of BY-2 tobacco cells using a virus-mediated expression
Agronomy 2023, 13, 858 7 of 20

system. Recombinant proteins fused to a glycomodule tag, such as that of hydroxyproline

(Hyp)-O-glycosylated peptides (designer HypGP tag composed of an ‘Ala-Pro’ motif of
20 units), have been found to secrete proteins in large quantities in cultured plant cells.
Zhang et al. [94] engineered (AP)20 proteins and increased the secretion of human protease
inhibitor α1-antitrypsin protein (34.7 mg/L) in cultured BY-2 cells with a better glycosyla-
tion module (Table 1). Another interesting example is plant cell-secreted stem cell factor that
stimulated the differentiation of key erythropoietic growth factor hematopoietic stem cells
using a designer hydroxyproline (Hyp)-O-glycosylated peptide (HypGP) containing 20 tan-
dem repeats of the “Ser-Pro” motif or (SP)20 in BY-2 cells. Additionally, the stem cell factor
displayed proliferation of the TF-1 cell line [95]. The US FDA approved the production
of taliglucerase-α enzyme (ElelysoR), the first therapeutic recombinant protein produced
from plant cell cultures [96]. Genome editing technologies such as CRISPR/Cas9 are being
utilized to create plant cells with desired characteristics that can aid in the production of
recombinant therapeutic proteins.

3. Specialty Molecules
Several chemotherapy agents appear to be plant-based [97]. Plants are a rich source
of bioactive molecules such as paclitaxel (used as a source for antineoplastic drugs and
approved by the US FDA), ginseng, artemisinin, and veregen, in addition to neutraceuticals,
herbal medicines, and botanical drugs [98,99]. Paclitaxel, an anticancer chemotherapy drug,
was initially produced from cultured cells of Taxus species [100]. Adventitious roots of
Panax ginseng have been cultivated on a large scale in vitro and sold on a commercial
scale [101,102]. Ginseng has been used in China for thousands of years as a dietary
supplement or as an ingredient in many drinks, including tea [99]. Ginseng has also been
approved as a drug for treating certain diseases in Korea [103]. Cell cultures are being
used for the production of colorants, especially anthocyanins, betacyanins, and shikonin,
in addition to health food ingredients [103–105]. Colorants can now be produced up to
kilograms in 600 Litre bioreactors. Shikonin is being produced at up to 5 kg per bioreactor
run by Mitsui Petrochemicals Industries Ltd., using plant cell and tissue culture [105].
It may be noted that the productivity of shikonin in cultured plant cells is 800 times
higher compared to the traditional methods. Likewise, echinacoside was scaled up to a
bioreactor level by Diversa Gesellschaft fur Bio-und Verfahrenstechnik in Germany [106].
The same company (also known as Phyton Biotech) has also been producing paclitaxel on a
commercial scale [107]. Protalix Biotherapeutics targeted the development of a proprietary
bioreactor technology for the mass production of recombinant human therapeutic proteins
using plant cell suspensions [108].
Other examples include the production of pharmaceuticals using plant cell cultures
and transformed cells [89,108,109]. Plant-made antibodies have been commercialized by
Planet Biotechnology, Inc. (Hayward, CA, USA) (https://www.planetbiotechnology.com/
index.html (accessed on 21 June 2022)) [89]. Transformed tobacco plants produced DPP4-Fc,
an immune adhesin for the treatment of MERS-CoV coronavirus infection, and CMG2- Fc
(PBI-220), an immunoadhesin for the treatment of inhalational anthrax. Transgenic carrot
cell suspensions have also been used to produce ElelysoR by Protalix BioTherapeutics, Inc.
(Carmiel, Israel), for controlling type 1 Gaucher’s disease [89]. Plant cell suspension cultures
have also been used in the production of berberine from Coptis japonica and Thalictrum
minus by Mitsui Chemicals, Inc. (Tokyo, Japan) for their anticancer, antibiotic, and anti-
inflammatory properties, Echinacea polysaccharides from Echinacea purpurea, Echinacea
angustifolia by Diversa (Ahrensburg, Germany) as an immunostimulant, anti-inflammatory
activities, and podophyllotoxin from Podophyllum species by Nippon Oil (Tokyo, Japan) for
anticancer treatment [6]. These examples amply demonstrate that it is possible to produce
specialty chemicals on a commercial scale using plant cell and tissue cultures. However,
challenges such as optimal product titer and regulatory issues have to be resolved before
such products are released into the market.
Agronomy 2023, 13, 858 8 of 20

4. Metabolic Engineering for Enhanced Production

Secondary metabolites extracted from plant sources and efforts to improve them as
cost-effective molecules have largely been impaired by the lack of knowledge of their
metabolic pathways, intermediates, and the enzymes involved. The development and
integration of next-generation sequencing technologies, high-throughput proteomics and
metabolomic analyses, and bioinformatics coupled with systems biology have become
handy in unraveling the metabolic pathways. Such an integrated approach helps us elu-
cidate the pathways and genes associated with the pathways, allowing us to perform
metabolic engineering and subsequently enhance the bioactive compounds [110,111]. Ma-
nipulating plant metabolism begins with the elucidation of the biosynthetic pathways
that lead to a preferred product as well as the precise regulation of discrete enzymes. It
is necessary to redeem the metabolic engineering of plants, including regulatory factors
and precursors that influence the metabolic and physiological networks, to enhance the
bioactive molecules [112]. Metabolic engineering enhances the prospects of boosting the
compounds that are synthesized in low quantities in cells or specific tissues and compounds
that are not originally produced in plants. Plant cell suspension cultures have been suc-
cessfully used for the production of shikonin, ginsenosides, and vinblastine [113] and are
considered efficient platforms for integrating the genomic, metabolomics, proteomic, and
for metabolic engineering of key pathways associated with the production of high-value
compounds of economic importance. Jamil et al. [114] reported metabolic profiles of callus
and cell suspensions of mangosteen (Garcinia mangostana). Metabolic engineering comprises
overexpression or downregulation of a specific metabolic pathway by adding precursors,
enzymes, and regulatory proteins in conjunction with recombinant DNA technology and
genome editing. While unraveling the biosynthetic pathways associated with secondary
plant products is of primary importance, advances in technologies have enabled us to
engineer microbial systems to reconstitute the metabolism, which is becoming a dominant
option in synthetic biology [115,116].
Expression of plant genes or gene clusters associated with biosynthetic capabilities
in microbial systems for the production of bioactive compounds has gained importance
over time to incorporate polyculture and co-culture consortiums [112]. For the expression
of complex biosynthetic pathway genes and for the production of secondary metabolites,
hosts such as E. coli, Saccharomyces cerevisiae, Crynebacterium glutamicum, and Yarrowia lipoly-
tica are being exploited [117,118]. In E. coli, the isoprenoid pathway has been optimized
for taxol precursor overproduction [119]. Metabolic engineering of glucosinolates has
been carried out in Raphanus sativa based on comparative genomics [115]. Notably, beta-
lain color has been achieved in Saccharomyces cerevisiae using metabolic engineering [120].
Resveratrol production in E. coli [121], malonyl-CoA metabolism using synthetic antisense
RNAs for the production of natural products in E. coli [122], remarkable production of
stilbenoid antioxidants [123], and production of a plant-derived cancer chemopreventive
precursor glucoraphanin in E. coli [124] have also been accomplished. More astound-
ingly, Lv et al. [125] unleashed a modular method to construct the flavonoid pathways
(antioxidant and anti-aging agents) in the yeast Yarrowia lipolytica to produce flavonoids and
hydroxylated flavonoids. The authors identified the optimal gene copy number for chalcone
synthase (CHS) and cytochrome P450 reductases (CPR) as 5 and 2, respectively. In shake
flask cultures, the yeast strain was able to produce 252.4 mg/L naringenin, 134.2 mg/L
eriodictol (a bitter-masking flavanone), and 110.5 mg/L taxifolin (also known as dihydro-
quercetin, belonging to the subclass flavanonols in the flavonoids) from glucose. These
findings infer that yeast can serve as a workhorse for the large-scale production of natural
products that are otherwise proprietary of plant systems. However, yeast grows slowly,
and there is a need to enhance the biomass in liquid cultures by manipulating the carbon,
nitrogen, and other nutrients that can improve the biomass dramatically and yet be able to
produce bioactive compounds economically and sustainably.
Mora–Vasquez et al. [126] recently reviewed the metabolic engineering for increas-
ing the content of alkaloids in medicinal plants. It was suggested that co-overexpression
Agronomy 2023, 13, 858 9 of 20

of genes in two or more rate-limiting steps is an effective protocol for genetic manip-
ulation. By overexpressing the endogenous pathway gene encoding secologanin syn-
thase, Rather et al. [127] were able to increase camptothecin (an anticancer indole alkaloid)
biosynthesis in Nothapodytes nimmoniana. Likewise, overexpression of the hyoscyamine
6β-hydroxylase (H6H) gene in Datura innoxia resulted in enhanced hyoscyamine con-
tent [35]. On the other hand, heterologous overexpression of the strictosidine synthase gene
isolated from Nothapodytes foetida increased the camptothecin in Ophiorrhiza rugosa
Agronomy 2023, 13, x FOR PEER REVIEW 10 of 22
[128].
In addition to the biosynthetic pathway genes, transcription factors such as CrERF5 (an
APETALA2/Ethylene Response Factor 5 from Catharanthus roseus) and also OpWRKY2 (iso-
pumila, and Ophiorrhiza
lated from pumila
the transgenic WRKY2)
plants showed have been introduced
enhanced bisindole into C. roseus
alkaloids andand O. pumila,
indole alka-
and the transgenic plants showed enhanced bisindole alkaloids and indole
loid camptothecin [129,130]. Further, silencing of the quinolinic acidphosphoribosyl alkaloid camp-
tothecin [129,130]. Further, silencing of the quinolinic acidphosphoribosyl transferase
transferase (QPT) gene improved the accumulation of scopolamine in hairy root cultures (QPT)
gene improved the accumulation of scopolamine in hairy root cultures of Duboisia
of Duboisia leichhardtii [131]. Interestingly, an engineered combinational module of tran- leich-
hardtii [131].
scription Interestingly,
factors an engineered
has also enhanced the combinational
production of module of transcription
indole alkaloids factors has
in Catharanthus
also enhanced the production of indole alkaloids in Catharanthus roseus [132]. Furthermore,
roseus [132]. Furthermore, overexpression of tryptophan decarboxylase and strictosidine
overexpression of tryptophan decarboxylase and strictosidine synthase has boosted the
synthase has boosted the accumulation of vinblastine alkaloids in Catharanthus roseus
accumulation of vinblastine alkaloids in Catharanthus roseus [133]. Thus, overexpression
[133]. Thus, overexpression of the biosynthetic pathway genes encoding endogenous
of the biosynthetic pathway genes encoding endogenous enzymes, heterologous expres-
enzymes, heterologous expressions, transcription factor overexpression, gene silencing,
sions, transcription factor overexpression, gene silencing, and co-overexpressions have
and co-overexpressions have yielded positive results in boosting the bioactive com-
yielded positive results in boosting the bioactive compounds. However, concerns such as
pounds. However, concerns such as identifying the pathway genes for alkaloids, flavo-
identifying the pathway genes for alkaloids, flavonoids, chalcones, and terpenes, need to
noids, chalcones, and terpenes, need to be addressed for the large-scale production of
be addressed for the large-scale production of many bioactive compounds to make them
many bioactive compounds to make them commercially viable.
commercially viable.
5.5.Biotransformation
Biotransformationand andProduction
Productionof ofPhysiologically
PhysiologicallyActive
ActiveCompounds
Compounds
Suspension-cultured
Suspension-culturedcells cellscan
canbebeused
usedininbiotransformation
biotransformationprocesses
processestotoproduce
produce
new
newchemicals
chemicalsor orknown
knownchemicals
chemicalsmoremoreeconomically,
economically,investigate
investigatethe
themetabolic
metabolicfatefateofof
xenobiotics,
xenobiotics, and elucidate the metabolic pathways [134]. Several advantages of plantcell
and elucidate the metabolic pathways [134]. Several advantages of plant cell
cultures
culturesas asaasource
sourcefor
forbiotransformation
biotransformationand andother
otherpurposes
purposesarearelisted
listedininFigure
Figure2.2.The
The
mechanisms
mechanismsofofbiotransformation
biotransformationhave havebeen
beenused
usedfor
forthe
thecommercialization
commercializationof ofbioactive
bioactive
molecules.
molecules.The The conversion physiologicallynot-so-active
conversion of physiologically not-so-activecompounds
compoundsinto into pharmaco-
pharmacologi-
logically active
cally active compounds
compounds is vital
is vital forfor
thethe pharmaceutical
pharmaceutical industry.
industry.

Advantagesofofplant
Figure2.2.Advantages
Figure plantcell
cellcultures
culturesfor
formetabolic
metabolicengineering.
engineering.

The biotransformation of hyoscyamine into scopolamine was achieved in transgenic

tobacco cell cultures [135], and cell suspension cultures of Digitalis lanata synthesized
three new compounds [136]. Moreover, a specific cell death-inducing cardenolide com-
pound was produced in D. lanata cell cultures [137,138]. New compounds such as eu-
desmane (a major sesquiterpene hydrocarbon of calamondin orange) and guaiane (a
Agronomy 2023, 13, 858 10 of 20

The biotransformation of hyoscyamine into scopolamine was achieved in transgenic

tobacco cell cultures [135], and cell suspension cultures of Digitalis lanata synthesized
three new compounds [136]. Moreover, a specific cell death-inducing cardenolide com-
pound was produced in D. lanata cell cultures [137,138]. New compounds such as eudes-
mane (a major sesquiterpene hydrocarbon of calamondin orange) and guaiane (a sesquiter-
pene that inhibits the growth of human renal cancer cell lines) were synthesized from the
biotransformation of (-)α-santonin (a drug used as anthelminthic) in cell cultures of Catha-
ranthus roseus. Moreover, biotransformation of progesterone to 17α-hydroxyprogesterone
has been reported using the plant cell suspension culture of C. roseus [139]. The biotransfor-
mation of bioactive principles such as dydrogesterone (used as a drug against recurrent
miscarriage in humans) was incubated with Azadirachta indica cells for the production of
20R-hydroxy-9β,10α-pregna-4,6-diene-3-one [140].

6. Plant Stem Cells for the Production of Cosmetics

Plant stem cells help to stimulate and regenerate plants after an injury, similar to
human stem cells. There is a growing interest in the extraction of cell culture-based
cosmetics and food products/neutraceuticals [99,141–143]. The plant stem cell culture
technology is environmentally friendly, and its application permits the generation of prized
compounds, even from rare or hard-to-access plants, without causing any disturbance
to their natural ecosystem. Cosmetic and food formulations of plant cell extracts are
available at reasonable prices [144]. Extracts of cultured apple stem cells that are liposome-
encapsulated have been used in anti-aging products (Table 2).

Table 2. Plant stem cells/callus/suspensions/hairy roots being used for cosmetic purposes by
different companies.

Name of the Plant Stem Cells Produced Use in Pharmacy/Cosmetology Reference

Mix of cells that are Improvement of 50% in skin structure as a
dedifferentiated and cell result of pore reduction and a mattifying
Zingiber officinale cultures that are responsible effect. Reduction in the shininess of the [145]
for controlling the synthesis of skin and sebum with an increase in the
active molecules inside the cell synthesis of elastin fibers
Stem cells containing
antioxidant compounds such Protects skin cells from heavy
Solanum lycopersicon [146]
as rutin, flavonoids, and metal-induced damages
metal-chelating factors
Crithmum maritimum Dedifferentiated cells Dermal repair and epidermal regeneration [147]
Enhances the viability of umbilical cord
blood stem cells, reverses the senescence
Encapsulated extract of
Malus domestica signs in human fibroblast cells, and also [148]
stem cells
increases the lifespan of isolated human
hair follicles
Apium graveolens Callus culture extract Works on regeneration of skin Akott Evolution, SRL, Fizzonasco, Italy
Calendula officinalis Cell suspension culture extract Anti-wrinkle and regeneration of skin Innova BM, Sofia, Bulgaria
Liposomal complex of cell Effective anti-aging, antioxidant, and
Centella asiatica Infinitec, Barcelona, Spain
suspension extract anti-wrinkle
Citrus aurantium Callus culture extract Anti-aging, helps in skin conditioning Provital group, Barcelona, Spain
Coffea species Extracts from cell cultures Energizes cells and acts against wrinkles Vitalab, SRL, Napoli, Italy
Extracts from cell suspension Anti-fat properties, acts against edema,
Coleus forskohlii Vitalab, SRL, Italy
cultures good antioxidant
Radiant Inc., Dongnae-myeon, Republic of
Daucus carota Extracts from callus cultures Good antioxidant, skin nourishing effect
Korea
Good anti-aging, anti-wrinkle,
Gardenia taitensis Callus culture extract Biocosmethic, Bonnelles, France
regenerative, and repairing activities
Reduction in hair loss, stimulation of Mibelle AG Biochemistry, Buchs,
Ocimum basilicum Hairy root culture extract
dermal papilla cells Switzerland
Oryza sativa Extracts of callus cultures Antioxidant and skin whitening Sandream Impact LLC., Fairfield, NJ, USA
Anti-aging, anti-wrinkle, skin repair from
Phaseolus radiatus Meristem cell culture extract Innovacos Corp., Mt Arlington, NJ, USA
damage
Saponaria pumila Extracts from cell suspensions Increases skin elasticity, firmness, density Mibelle AG Biochemistry, Switzerland
Controls excessive sebum secretion, good
Solanum lycopersicum Callus culture extract Sandream Impact LLC., Fairfield, NJ, USA
condition of the skin
Vitis vinifera Cell suspension extract Extends skin viability, anti-photo-aging Mibelle AG Biochemistry, Switzerland
Agronomy 2023, 13, 858 11 of 20

Likewise, cells of Catheranthus roseus are being used now in cosmetics [145]. Interest-
ingly, Rubus cell cultures for anti-inflammatory activity, tobacco for collagen synthesis, and
Coffea cells for epidermal hydration and collagen synthesis in the skin cells are some of
the highly rewarding commercial products seen in today’s market from plant cell cultures.
Plant stem cells from apple, grape, jasmine, pine, lilac, and other plants have been widely
and successfully used in cosmetic preparations used the world over. Tomato cultured stem
cells have demonstrated potential in protecting skin from heavy metal toxicity since tomato
is rich in antioxidant molecules such as lycopene, ascorbic acid, vitamin E, flavonoids,
and phenolic acids [145]. Moreover, tomato stem cell extract contained metal-chelating
compounds such as phytochelatins [146]. Phytochelatins are potent metal-binding proteins,
and new products prepared from tomato neutralize the damage caused by heavy metals on
the degradation of collagen by inhibiting collagenase. It has also been found that Coffea
bengalensis culture-derived stem cells [22] stimulate the regeneration of skin cells by invigo-
rating fibroblasts to synthesize collagen [149]. These studies indicate that plant stem cells
preserve exceptional anti-aging properties and stimulate fibroblasts to synthesize collagen,
stimulate skin regeneration, and repair DNA damage. Plant cells contain kinetin, which
perhaps helps to protect skin cells from various stresses, including oxidative stress [150].

7. Plant Stem Cells for the Production of Food Ingredients

The increasing world population and fluctuating climate changes have been creating
strenuous demands on the food supply. This necessitates the exploration of alternate
agriculture options, including plant cell cultures, since they can be grown under a controlled,
closed environment and have a sustainable process of manufacturing important food
ingredients. The use of plant cell cultures instead of whole plants facilitates the preparation
of products for the cosmetics and food industries with less energy, minimal possible impacts
on the environment, and, importantly, independent of location and season. Plant stem cells
are undifferentiated cells present in the meristematic tissues of plants. Both apical and
lateral meristems are vital sources of stem cells. The stem cells are self-renewed,13doofnot
Agronomy 2023, 13, x FOR PEER REVIEW 22
undergo aging, and supply precursor cells that differentiate into diverse tissues or organs
based on their location. In vitro callus is first initiated, suspension cultures are derived from
callus and scaled up, and then cells are analyzed through different analytical techniques
view by Aggarwal
for secondary et al.
product [151]. There
synthesis. are different
For details, see the factors
review that determine
by Aggarwal et the success
al. [151]. of
There
plant stem cell cultivation, for example, optimized cell suspensions, their synchroniza-
are different factors that determine the success of plant stem cell cultivation, for example,
tion, scale-up
optimized cellfor bioreactor, their
suspensions, monitoring of the biomass
synchronization, scale-upduring large-scalemonitoring
for bioreactor, production,of
quality aspects,
the biomass and large-scale
during metabolic profile (Figure
production, 3). aspects, and metabolic profile (Figure 3).
quality

Flowchart
Figure3.3.Flow
Figure chartof
ofplant
plantstem
stemcell
cellcultivation
cultivationand
andproduct
productsynthesis.
synthesis.

A list of commercial firms that use plant callus, suspension cultures, and hairy roots
A list of commercial firms that use plant callus, suspension cultures, and hairy roots
for the production/extraction of stem cells is shown in Table 2. Accordingly, plant cell
for the production/extraction of stem cells is shown in Table 2. Accordingly, plant cell
cultures have been expanded for food use and are now considered a favorable source with
cultures have been expanded for food use and are now considered a favorable source
high nutritional values [28,151]. Cambium meristematic cell-derived suspensions have
with high nutritional values [28,151]. Cambium meristematic cell-derived suspensions
high potential and represent true stem cells. Stem cell isolation has been reported for Ginko
have high potential and represent true stem cells. Stem cell isolation has been reported
for Ginko biloba [152], Taxus cuspidata [39], and Tripterygium wilfordii [40]. A patent on the
technique for the isolation of stem cells has been granted to Unhwa Corporation [103].
Examples of plant cell cultures for extracting colorants, aromas, and food ingredients
have been amply demonstrated [103]. Nutritional facts and sensory properties of cul-
Agronomy 2023, 13, 858 12 of 20

biloba [152], Taxus cuspidata [39], and Tripterygium wilfordii [40]. A patent on the technique
for the isolation of stem cells has been granted to Unhwa Corporation [103]. Examples of
plant cell cultures for extracting colorants, aromas, and food ingredients have been amply
demonstrated [103]. Nutritional facts and sensory properties of cultured cells of cloudberry,
lingo berry, and stone berry have been investigated [153]. Enhanced production of vanillin
flavor, a prime food constituent metabolite, has been achieved by precursor feeding into
cell suspensions [154]. Theobroma cacao suspensions prepared in a wave-mixed bioreactor
have been demonstrated to be of immense use as an important component in chocolate
products. While the sensory profile of cell culture chocolate displayed a fruity and sour
aroma, chemical analysis exhibited volatile and non-volatile flavor compounds (6.69 g/g
of polyphenol content). However, the cultures grown in a stirred tank bioreactor have
been found to differ in their aroma, with enhanced bitterness [155]. However, there is a
need to improve the biomass without compromising the aroma quality, which should be
comparable to that of intact plants. Additionally, the nutritional qualities (carbohydrates,
proteins, lipids, dietary fiber, etc.) of suspensions of Rubus chamaemorus (cloudberry),
Rubus saxatilis, and Vaccinium vitisidaea (lingonberry or mountain cranberry) cultivated on
an industrial scale have been investigated alongside the sensory properties [153,156,157].
Daucus carota cells grown in shake flasks and bioreactors have been found to have food
applications as a colorant (higher anthocyanins than natural red carrot) and for skin care,
nutraceutical, and food applications [144].

8. Regulatory Issues Related to Food Ingredients or Foods Prepared through Plant

Suspension Cultures
Regulations are meant to ensure the safety of food and food ingredients to the con-
sumers, though they may differ from country to country. Foods derived from plant suspen-
sion cultures are considered and come under the novel food category as per the definition
of the European Union Regulation 2015/2283 [158]. The regulatory landscape, with a
complex framework of plant suspension or hairy root culture-derived novel food and
health-related products, is undeniably a limiting factor for the early commercialization
of the products. Any delays in approval will escalate the production costs. However,
food colorants and flavorings do not fall within the scope of European Union Regulation
2015/2283 [158]. In India, the Food Safety and Standards Act (FSS) 2006 is the law that
regulates all food ingredients. Generally, the Food Safety Standards Act of India (FSSAI)
looks after and enforces the safety standards within India. On the other hand, in the USA,
the addition of any substance to food is considered a food additive and needs approval
from the Food and Drug Administration (FDA). However, if the substance is proven as
safe through scientific data generated under the supervision of qualified scientists or has a
traditional history of safe use for a long period of time, or if it meets the procedures laid
down by the Federal Food, Drug, and Cosmetic Act, then approvals may not be necessary
before such products are marketed. In the case of traditional foods, a long history of safe
usage is taken into consideration, but not in plant tissue culture-derived foods and food
ingredients since these are labeled as “novel”. Novel foods derived from plant suspensions
and hairy root cultures may vary in their constituent profile in comparison with traditional
foods. Therefore, such novel foods need a safety assessment performed by the European
Food Safety Authority (EFSA) as per the guidelines laid down with all the evidence of the
scientific data [159]. Extracts derived from the suspensions of plants such as Ajuga reptans
and Echinaceae angustifolia are considered novel food supplements under EU Regulation
2015/2283. However, food colorants such as anthocyanins and betalains have not been
granted any authorizations by the EFSA for use in food, although they are common food
ingredients in grapes, beetroot, and others [158,160]. Lastly, it is vital to take the sensory
perception of consumers into account since this aspect is also crucial.
Agronomy 2023, 13, 858 13 of 20

9. Bioprinting
Bioprinting is a novel technique for creating tissues, organs, and biological materi-
als [161,162]. The technology is based on bio-ink to generate the bio-structures of relevance
in life science, medicine, and bioengineering [163]. For bio-inks, non-hazardous, natural, or
synthetic biomaterials are used, which mimic the properties of cells [163]. This technique
envisages the establishment of a biocompatible and non-toxic scaffold to be integrated with
biological systems for supporting cellular growth [162]. High-throughput structural and
functional 3D bioprinting strategies used in plant cellular modeling consist of bioprinting
materials with multiple functional, structural, and mechanical properties [162]. Research in
bioprinting of plant cells has been used to develop 3D plant cells to study growth-related
functions such as viability, cell division, and cell identity in Arabidopsis and soybean [163].
The 3D bioprinting could also confer advantages to design and control cellular dynamic
structures in a physiologically accurate manner toward tissue regeneration akin to natu-
ral conditions [164]. Different components of the bioprinting workflow using plant cell
suspensions and applications are presented in Figure 4. This technique opens up avenues
for understanding the cellular and physiological processes involved in mechanical and
molecular interactions between cells and their cellular environment. Growing cells are
surrounded by cellular structures governed by complex biochemical networks and many
inherent cellular factors, and hence, the conventional plant cell culture techniques cannot
reproduce such a complex cellular setting to diverse external factors. The embedded cells
in the 3D scaffold adjust their shapes to fit the adjacent spaces and grow, which can be
monitored more easily than the native plant tissues. A recent study on Arabidopsis root cells
and embryonic soybean cells showed that more than half of the 3D bioprinted cells were
viable and divided over time to form microcalli, or small colonies of cells [163]. The 3D
bioprinting has applications in plant sciences, agriculture, and identification of appropriate
compatibility of 3D bioprinting materials with different components of plant cells [165].
Some of the materials used in bioprinting include alginate nanocellulose, carrageenan,
Agronomy 2023, 13, x FOR PEER REVIEW 15 of 22
agarose, pectin, starch, and fucoidan [162,164]. Being in a 3D environment, plant cellular
behavior and physiology might offer vital clues that help to better understand plant cell dy-
namics, suggesting that 3D bioprinting has considerable potential in plant science research
materials could findmaterials
and the bioprinted applications in wound
could dressings, in
find applications implantable medical devices,
wound dressings, and
implantable
drug delivery systems [162].
medical devices, and drug delivery systems [162].

Different components of bioprinting using plant cell suspensions and its applications.
Figure 4. Different applications.

10. Conclusions
10. Conclusions andand Future
Future Outlook
Outlook
Plants are prime sources for assorted biopharmaceuticals and have been extensively
Plants are prime sources for assorted biopharmaceuticals and have been extensively
and widely exploited as medicines and therapeutic products. Stem cells are now being iso-
and widely exploited as medicines and therapeutic products. Stem cells are now being
lated from cultured plant cells in the gigantic cosmetic industry. The advantages, problems,
isolated from cultured plant cells in the gigantic cosmetic industry. The advantages,
and challenges need to be gauged, and new scientific contributions need to be weighed
problems, and challenges need to be gauged, and new scientific contributions need to be
weighed carefully for further exploitation. Although many plants contain precious me-
tabolites, their content is generally low. This has necessitated enhancing their synthesis
by employing elicitors and precursors for a substantial extraction of the products, and
plant cell suspension cultures/bioreactors would be an ideal platform for the production
Agronomy 2023, 13, 858 14 of 20

carefully for further exploitation. Although many plants contain precious metabolites,
their content is generally low. This has necessitated enhancing their synthesis by em-
ploying elicitors and precursors for a substantial extraction of the products, and plant
cell suspension cultures/bioreactors would be an ideal platform for the production of
high-value metabolites [166,167]. The emerging science of omics and the range of hosts
available in synthetic biology has given new insights for understanding the basic mech-
anisms of synthesis and rebuilding plant bioactive metabolites in microbes and yeasts
on a commercial scale. Examples of metabolically engineered microbes producing plant
natural products have been very well established. Culturing cambial meristematic cells
from plants has also been exploited for the preparations of several cosmetics, primarily skin
care products. Now, this technology has been stretched to isolate useful metabolites using
stem cell suspensions/hairy roots. Genes associated with biosynthetic pathways must be
unraveled. For the technology to be sustained, they must be cloned and expressed for the
large-scale production of plant metabolites in microorganisms. In this context, synthetic
biology is advancing with the deployment of new tools for constructing, controlling, and
optimizing complex metabolic pathways of plants in unicellular organisms. Generally,
experimental procedures unleash the feasibilities, demonstrating the potentialities of cell
cultures for industrial applications. In the foreseeable future, plant cells, together with the
development of high-yielding, stable cell lines, need to be separated out, which can be a
base for industrial-scale production. However, both biomass and secondary plant products
must be refined to enable suspensions, hairy root cultures, or stem cell cultures for the scale-
up and subsequent exploitation by the pharmaceutical industry. Further, the design and
development of home bioreactors and their availability to the household must be cheaper
and should produce in kilogram quantities per day. Plant suspension culture-derived or
stem cell-derived products must undergo rigorous regulatory procedures before they are
released into the market.

Author Contributions: Conceptualization—V.A.B.; writing, original draft preparation—V.A.B., S.P.

and P.B.K.K.; review and editing—V.A.B., S.P., P.B.K.K., N.J. and S.M.J. All authors have read and
agreed to the published version of the manuscript.
Funding: This research received no external funding.
Data Availability Statement: Not applicable.
Acknowledgments: VAB thanks INSA, New Delhi, India, for the Honorary scientist fellowship.
Conflicts of Interest: The authors declare no conflict of interest.

References
1. Motolinia-Alcantara, E.A.; Castillo-Araiza, C.O.; RodríguezMonroy, M.; Román-Guerrero, A.; Cruz-Sosa, F. Engineering consider-
ations to produce bioactive compounds from plant cell suspension culture in bioreactors. Plants 2021, 10, 2762. [CrossRef]
2. Zafar, T.; Shrivastava, V.K.; Shaik, B. Pharmaceutical Biotechnology in Herbal Neuroprotection; Springer: Singapore, 2018; pp. 221–228.
3. Elkordy, A.A.; Haj-Ahmad, R.R.; Awaad, A.S.; Zaki, R.M. An overview on natural product drug formulations from conventional
medicines to nanomedicines: Past, present and future. J. Drug Deliv. Sci. Technol. 2021, 63, 102459. [CrossRef]
4. Shriram, V.; Vinay Kumar Kavi Kishor, P.B.; Suryawanshi, S.B.; Upadhyay, A.K.; Bhat, M.K. Cytotoxic activity of 9,10-dihydro-
2,5-dimethoxyphenanthrene-1,7-diol from Eulophia nuda against human cancer cells. J. Ethnopharmacol. 2010, 128, 251–253.5.
[CrossRef] [PubMed]
5. Madhavi, D.; Abhayankar, G.; Reddy, V.D.; Kavi Kishor, P.B. Carbohydrate and elicitor enhanced withanolide (withaferin A and
withanolide A) accumulation in hairy root cultures of Withania somnifera (L.). Indian J. Exp. Biol. 2012, 50, 484–490.
6. Ochoa-Villarreal, O.V.M.; Howat, S.; Hong, S.M.; Jang, M.O.; Jin, Y.W.; Lee, E.K.; Loake, G.J. Plant cell culture strategies for the
production of natural products. BMB Rep. 2016, 49, 149–158. [CrossRef] [PubMed]
7. Jawahar, G.; Punita, D.L.; Rajasheker, G.; Manoharachary, C.; Venkatachalam, P.; Kavi Kishor, P.B. Feeding elicitors and precursors
enhance colchicine accumulation in morphogenic cultures of Gloriosa superba L. Plant Cell Tiss. Organ Cult. 2018, 135, 235–245.
[CrossRef]
8. Espinosa-Leal, C.A.; Puente-Garza, C.A.; García-Lara, S. In vitro plant tissue culture: Means for production of biological active
compounds. Planta 2018, 248, 1–18. [CrossRef] [PubMed]
9. Arya, S.S.; Rookes, J.E.; Cahill, D.M.; Lenka, S.K. Next-generation metabolic engineering approaches towards development of
plant cell suspension cultures as specialized metabolite producing biofactories. Biotechnol. Adv. 2020, 45, 107635. [CrossRef]
Agronomy 2023, 13, 858 15 of 20

10. Moscatiello, R.; Baldan, B.; Navazio, L. Plant Cell Suspension Cultures. In Plant Mineral Nutrients. Methods in Molecular Biology
(Methods and Protocols); Maathuis, F., Ed.; Humana Press: Totowa, NJ, USA, 2013; p. 953. [CrossRef]
11. Gandra, J.; Patel, H.K.; Kumar, S.A.; Doma, M.; Deepthi, Y.; Bhalothia, P.; Jalaja, N.; Chimakurthy, J.; Polavarapu, R.; Katam,
R.; et al. Metabolomic and proteomic signature of Gloriosa superba leaves treated with mercuric chloride and phenylalanine, a
precursor of colchicine alkaloid. Ind. Crops Prod. 2022, 178, 114557. [CrossRef]
12. Komaraiah, P.; Ramakrishna, S.V.; Reddanna, P.; Kavi Kishor, P.B. Enhanced production of plumbagin in immobilized cells of
Plumbago rosea by elicitation and in situ adsorption. J. Biotechnol. 2003, 101, 181–187. [CrossRef]
13. Cortese, E.; Carraretto, L.; Baldan, B.; Navazio, L. Arabidopsis photosynthetic and heterotrophic cell suspension cultures. Methods
Mol. Biol. 2021, 2200, 167–185. [PubMed]
14. Narayani, M.; Shrivastava, S. Elicitation: A stimulation of stress in in vitro plant cell/tissue cultures for enhancement of secondary
metabolite production. Phytochemical. Rev. 2017, 16, 1227–1252. [CrossRef]
15. Chodisetti, B.; Rao, K.; Gandi, S.; Giri, A. Gymnemic acid enhancement in the suspension cultures of Gymnema sylvestre by using
the signaling molecules—Methyl jasmonate and salicylic acid. Vitr. Cell. Dev. Biol.-Plant 2015, 51, 88–92. [CrossRef]
16. Chodisetti, B.; Rao, K.; Suryakala, G.; Archana, G. Improved Gymnemic acid production in the suspension cultures of Gymnema
sylvestre through biotic elicitation. Plant Biotechnol. Rep. 2013, 7, 519–525. [CrossRef]
17. Veerashree, V.; Anuradha, C.M.; Vadlapudi, K. Elicitor-enhanced production of gymnemic acid in cell suspension cultures of
Gymnema sylvestre R. Br. Plant Cell Tiss Organ Cult. 2012, 108, 27–35. [CrossRef]
18. Chen, H.; Chena, F.; Chiu, F.C.; Lo, C.M. The effect of yeast elicitor on the growth and secondary metabolism of hairy root cultures
of Salvia miltiorrhiza . Enzym. Microb. Technol. 2001, 28, 100–105. [CrossRef]
19. Deepthi, S.; Satheeshkumar, K. Enhanced camptothecin production induced by elicitors in the cell suspension cultures of
Ophiorrhiza mungos Linn. Plant Cell Tissue Organ Cult. 2016, 124, 483–493. [CrossRef]
20. Flores-Sanchez, I.J.; Ortega-Lopez, J.; del Carmen Montes-Horcasitas, M.; Ramos-Valdivia, A.C. Biosynthesis of sterols and
triterpenes in cell suspension cultures of Uncaria tomentosa . Plant Cell Physiol. 2002, 43, 1502–1509. [CrossRef]
21. Chakraborty, A.; Chattopadhyay, S. Stimulation of Menthol Production in Mentha piperita Cell Culture. Vitr. Cell. Dev. Biol. Plant.
2008, 4, 518–524. [CrossRef]
22. Wilson, S.A.; Roberts, S.C. Recent advances towards development and commercialization of plant cell culture processes for the
synthesis of biomolecules. Plant Biotechnol. J. 2012, 10, 249–268. [CrossRef]
23. Santos Rita, B.; Rita, A.; Rainer, F.; Markus, S.; Holland, T.H. Putting the spotlight back on plant suspension cultures. Front. Plant
Sci. 2016, 7, 297. [CrossRef] [PubMed]
24. Eibl, R.; Meier, P.; Stutz, I.; Schildberger, D.; Hühn, T.; Eibl, D. Plant cell culture technology in the cosmetics and food industries:
Current state and future trends. Appl. Microbiol. Biotechnol. 2018, 102, 8661–8675. [CrossRef] [PubMed]
25. Krasteva, G.; Georgiev, V.; Pavlov, A. Recent applications of plant cell culture technology in cosmetics and foods. Eng. Life Sci.
2021, 21, 68–76. [CrossRef] [PubMed]
26. Wu, T.; Kerbler, S.M.; Fernie, A.R.; Zhang, Y. Plant cell cultures as heterologous bio-factories for secondary metabolite production.
Plant Commun. 2021, 2, 100235. [CrossRef] [PubMed]
27. Acikgoz, M.A. Establishment of cell suspension cultures of Ocimum basilicum L. and enhanced production of pharmaceutical
active ingredients. Ind. Crops Prod. 2020, 148, 112278. [CrossRef]
28. Garcia-Perez, P.; Miras-Moreno, B.; Lucini, L.; Gallego, P.P. The metabolomics reveals intraspecies variability of bioactive
compounds in elicited suspension cell cultures of three Bryophyllum species. Ind. Crops Prod. 2021, 163, 113322. [CrossRef]
29. Kreis, W. Exploiting plant cell culture for natural product formation. J. Appl. Bot. Food Qual. 2019, 92, 216–225. [CrossRef]
30. Hakkinen, S.T.; Oksman-Caldentey, K.M. Progress and prospects of hairy root research. In Hairy Roots: An Effective Tool of Plant
Biotechnology; Srivastava, V., Mehrotra, S., Mishra, S., Eds.; Springer: Singapore, 2018; pp. 3–19. [CrossRef]
31. Nielsen, E.; Temporiti, M.E.E.; Cella, R. Improvement of phytochemical production by plant cells and organ culture and by
genetic engineering. Plant Cell Rep. 2019, 38, 1199–1215. [CrossRef]
32. Nourani, A.; Popova, E.; Titova, M. Biotechnology based on cell cultures of higher plants. E3S Web Conf. 2021, 265, 04012.
[CrossRef]
33. Furusaki, S.; Takeda, T. Bioreactors for Plant Cell Culture, Reference Module in Life Sciences; Elsevier: Dordrecht, The Netherlands,
2017. [CrossRef]
34. Werner, S.; Maschke, R.W.; Eibl, D.; Eibl, R. Bioreactor technology for sustainable production of plant cell-derived products. In
Bioprocessing of Plant In Vitro Systems. Reference Series in Phytochemistry; Pavlov, A., Bley, T., Eds.; Springer: Cham, Switzerland,
2018. [CrossRef]
35. Kowalczyk, T.; Merecz-Sadowska, A.; Picot, L.; Brčić Karačonji, I.; Wieczfinska, J.; Śliwiński, T.; Sitarek, P. Genetic manipulation
and bioreactor culture of plants as a tool for industry and its applications. Molecules 2022, 27, 795. [CrossRef]
36. Li, Y.; Kong, D.; Fu, Y.; Sussman, M.R.; Wu, H. The effect of developmental and environmental factors on secondary metabolites
in medicinal plants. Plant Physiol. Biochem. 2020, 148, 80–89. [CrossRef] [PubMed]
37. Zagorskayaa, A.A.; Deinekoa, E.V. Suspension-cultured plant cells as a platform for obtaining recombinant proteins. Russ. J. Plant
Physiol. 2017, 64, 795–807. [CrossRef]
38. Subroto, M.A.; Hamill, J.; Doran, P. Development of shooty teratomas from several solanaceous plants: Growth kinetics,
stoichiometry and alkaloid production. J. Biotechnol. 1996, 45, 45–57. [CrossRef]
Agronomy 2023, 13, 858 16 of 20

39. Fazili, M.A.; Bashir, I.; Ahmad, M.; Yaqoob, U.; Geelani, S.N. In vitro strategies for the enhancement of secondary metabolite
production in plants: A review. Bull. Natl. Res. Cent. 2022, 46, 35. [CrossRef] [PubMed]
40. Lee, E.K.; Jin, Y.W.; Park, J.H.; Yoo, Y.M. Cultured cambial meristematic cells as a source of plant natural products. Nat. Biotechnol.
2010, 28, 1213–1217. [CrossRef] [PubMed]
41. Song, Y.; Chen, S.; Wang, X.; Zhang, R. A novel strategy to enhance terpenoids production using cambial meristematic cells of
Tripterygium wilfordii Hook. f. Plant Methods 2019, 15, 129. [CrossRef]
42. Marchev, A.S.; Yordanova, Z.P.; Georgiev, M.I. Green (cell) factories for advanced production of plant secondary metabolites. Crit.
Rev. Biotechnol. 2020, 40, 443–458. [CrossRef]
43. Moon, S.H.; Pandurangan, M.; Kim, D.H.; Venkatesh, J.; Patel, R.V.; Mistry, B.M. A rich source of potential bioactive compounds
with anticancer activities by Catharanthus roseus cambium meristematic stem cell cultures. J. Ethnopharmacol. 2018, 217, 107–117.
[CrossRef]
44. Kesik-Brodacka, M. Progress in biopharmaceutical development. Biotechnol. Appl. Biochem. 2018, 65, 306–322. [CrossRef]
45. Tak, H.; Negi, S.; Ganapathi, T.R.; Bapat, V.A. Molecular farming: Prospects and limitation. In Banana: Genomics and Transgenic
Approaches for Genetic Improvement; Mohandas, K.V., Ravishankar, G.A., Eds.; Springer: Berlin/Heidelberg, Germany, 2016.
46. Burnett, J.B.M.; Burnett, A.C. Therapeutic recombinant protein production in plants: Challenges and opportunities. Plants People
Planet. 2020, 2, 121–132. [CrossRef]
47. Nazeri, A.; Niazi, A.; Afsharifar, A.; Taghavi, S.V.; Moghadam, A.; Aram, F. Heterologous production of hyaluronic acid in
Nicotiana tabacum hairy roots expressing a human hyaluronan synthase 2. Sci. Rep. 2021, 11, 17966. [CrossRef] [PubMed]
48. Park, Y.J.; Han, J.E.; Lee, H.; Jung, Y.J.; Murthy, H.N.; Park, S.Y. Large-scale production of recombinant miraculin protein in
transgenic carrot callus suspension cultures using air-lift bioreactors. AMB Expr. 2020, 10, 140. [CrossRef] [PubMed]
49. Mohammadi, A.; Niazi, A.; Aram, F.; Hassani, F.; Ghasemi, Y. Transformation of the L-asparaginase II gene to potato hairy roots
for production of recombinant protein. J. Crop. Sci. Biotechnol. 2020, 23, 81–88. [CrossRef]
50. Zhang, N.; Wright, T.; Caraway, P.; Xu, J. Enhanced secretion of human α1-antitrypsin expressed with a novel glycosylation
module in tobacco BY-2 cell culture. Bioengineered 2019, 10, 87–97. [CrossRef]
51. Lonoce, C.; Salem, R.; Marusic, C.; Jutras, P.V.; Scaloni, A.; Salzano, A.M.; Lucretti, S.; Steinkellner, H.; Benvenuto, E.; Donini, M.
Production of a tumour-targeting antibody with a human-compatible glycosylation profile in N. benthamiana hairy root cultures.
Biotechnol. J. 2016, 11, 1209–1220. [CrossRef]
52. Yao, Q.; Yu, Z.; Liu, P.; Zheng, H.; Xu, Y.; Sai, S.; Wu, Y.; Zheng, C. High efficient expression and purification of human epidermal
growth factor in Arachis hypogaea L. Int. J. Mol. Sci. 2019, 20, 2045. [CrossRef]
53. Cardon, F.; Pallisse, R.; Bardor, M.; Caron, A.; Vanier, J.; Ele Ekouna, J.P.; Lerouge, P.; Boitel-Conti, M.; Guillet, M. Brassica rapa
hairy root based expression system leads to the production of highly homogenous and reproducible profiles of recombinant
human alpha-L-iduronidase. Plant. Biotechnol. J. 2019, 17, 505–516. [CrossRef]
54. Ekouna, J.P.E.; Boitel-Conti, M.; Lerouge, P.; Bardor, M.; Guerineau, F. Enhanced production of recombinant human gastric lipase
in turnip hairy roots. Plant Cell Tissue Organ Cult. 2017, 131, 601–610. [CrossRef]
55. Alemzadeh, E.; Izadpanah, K.; Ahmadi, F. Generation of recombinant protein shells of Johnson grass chlorotic stripe mosaic virus
in tobacco plants and their use as drug carrier. J. Virol. Methods 2017, 248, 148–153. [CrossRef]
56. Gurusamy, P.D.; Schäfer, H.; Ramamoorthy, S.; Wink, M. Biologically active recombinant human erythropoietin expressed in
hairy root cultures and regenerated plantlets of Nicotiana tabacum L. PLoS ONE 2017, 12, e0182367. [CrossRef]
57. Chen, L.; Yang, X.; Luo, D.; Yu, W. Efficient production of a bioactive bevacizumab monoclonal antibody using the 2a self-cleavage
peptide in transgenic rice callus. Front. Plant Sci. 2016, 7, 1156. [CrossRef] [PubMed]
58. Zhou, Y.; Yang, D.; Qiang, Z.; Meng, Y.; Li, R.; Fan, X.; Zhao, W.; Meng, Y. Ribosome-inactivating protein MAP30 isolated from
Momordica charantia L. induces apoptosis in hepatocellular carcinoma cells. Recent Pat. Anticancer Drug Discov. 2022. [CrossRef]
[PubMed]
59. Moghadam, A.; Niazi, A.; Afsharifar, A.; Taghavi, S.M. Expression of a recombinant anti-HIV and anti-tumor protein, MAP30, in
Nicotiana tabacum hairy roots: A pH-stable and thermophilic antimicrobial protein. PLoS ONE 2016, 11, e0159653. [CrossRef]
60. Tavizi, A.; Javaran, M.J.; Moieni, A.; Mohammadi-Dehcheshmeh, M.; Mohebodini, M.; Ebrahimie, E. Root and shoot parts of
strawberry: Factories for production of functional human pro-insulin. Mol. Biol. Rep. 2015, 42, 1013–1023. [CrossRef] [PubMed]
61. Liu, Y.K.; Li, Y.T.; Lu, C.F.; Huang, L.F. Enhancement of recombinant human serum albumin in transgenic rice cell culture system
by cultivation strategy. New Biotechnol. 2015, 32, 328–334. [CrossRef]
62. Häkkinen, S.; Raven, N.; Henquet, M.; Laukkanen, M.-L.; Anderlei, T.; Pitkänen, J.-P.; Twyman, R.M.; Bosch, D.; Oksman-
Caldentey, K.-M.; Schillberg, S.; et al. Molecular farming in tobacco hairy roots by triggering the secretion of a pharmaceutical
antibody. Biotechnol. Bioeng. 2014, 111, 336–346. [CrossRef]
63. Pham, N.B.; Schäfer, H.; Wink, M. Production and secretion of recombinant thaumatin in tobacco hairy root cultures. Biotechnol. J.
2012, 7, 537–545. [CrossRef]
64. Kim, N.S.; Yu, H.Y.; Chung, N.D.; Shin, Y.J.; Kwon, T.H.; Yang, M.S. Production of functional recombinant bovine trypsin in
transgenic rice cell suspension cultures. Protein Expr. Purif. 2011, 76, 121–126. [CrossRef]
65. Xu, J.; Okada, S.; Tan, L.; Goodrum, K.J.; Kopchick, J.J.; Kieliszewski, M.J. Human growth hormone expressed in tobacco cells as
an arabinogalactan-protein fusion glycoprotein has a prolonged serum life. Transgenic Res. 2010, 19, 849–867. [CrossRef]
Agronomy 2023, 13, 858 17 of 20

66. Park, C.I.; Lee, S.J.; Kang, S.H.; Jung, H.S.; Kim, D.I.; Lim, S.M. Fed-batch cultivation of transgenic rice cells for the production of
hCTLA4Ig using concentrated amino acids. Process. Biochem. 2010, 45, 67–74. [CrossRef]
67. Parsons, J.; Wirth, S.; Dominguez, M.; Bravo-Almonacid, F.; Giulietti, A.; Talou, J.R. Production of human epidermal growth
factor (hEGF) by In vitro cultures of Nicotiana tabacum: Effect of tissue differentiation and sodium nitroprusside addition. Int. J.
Biotechnol. Biochem. 2010, 6, 131–138.
68. Shin, Y.J.; Lee, N.J.; Kim, J.; An, X.H.; Yang, M.S.; Kwon, T.H. High-level production of bioactive heterodimeric protein human
interleukin-12 in rice. Enzym. Microb. Technol. 2010, 46, 347–351. [CrossRef]
69. Woods, R.R.; Geyer, B.C.; Mor, T.S. Hairy-root organ cultures for the production of human acetylcholinesterase. BMC Biotechnol.
2008, 8, 95. [CrossRef] [PubMed]
70. Kim, T.G.; Baek, M.Y.; Lee, E.K.; Kwon, T.H.; Yang, M.S. Expression of human growth hormone in transgenic rice cell suspension
culture. Plant Cell Rep. 2008, 27, 885–891. [CrossRef] [PubMed]
71. Anneli, R.; Wahlström, E.H.; Holkeri, H.; Anders, H.; Katri, E.; Baez, J.; Kristiina, M.; Anna, N. Production of a recombinant
industrial protein using barley cell cultures. Protein Expr. Purif. 2008, 59, 274–281. [CrossRef]
72. Xu, J.; Tan, L.; Goodrum, K.J.; Kieliszewski, M.J. High-yields and extended serum half-life of human interferon α2b expressed in
tobacco cells as arabinogalactan-protein fusions. Biotechnol. Bioeng. 2007, 97, 997–1008. [CrossRef]
73. Lee, S.J.; Park, C.I.; Park, M.Y.; Jung, H.S.; Ryu, W.S.; Lim, S.M.; Tan, H.K.; Kwon, T.H.; Yang, M.S.; Kim, D.I. Production and
characterization of human CTLA4Ig expressed in transgenic rice cell suspension cultures. Protein Expr. Purif. 2007, 51, 293–302.
[CrossRef]
74. Becerra-Arteaga, A.; Mason, H.S.; Shuler, M.L. Production, secretion, stability and n-glycosylation of human secreted alkaline
phosphatase in tobacco NT1 cell suspension cultures. Biotechnol. Prog. 2006, 22, 1643–1649. [CrossRef]
75. Martinez, C.; Petruccelli, S.; Giulietti, A.M.; Alvarez, M.A. Expression of the antibody 14D9 in Nicotiana tabacum hairy root roots.
Electron. J. Biotechnol. 2005, 8, 170–176. [CrossRef]
76. McDonald, K.A.; Hong, L.M.; Trombly, D.M.; Xie, Q.; Jackman, A.P. Production of human α-1-antitrypsin from transgenic rice cell
culture in a membrane bioreactor. Biotechnol. Prog. 2005, 21, 728–734. [CrossRef]
77. Huang, L.F.; Liu, Y.K.; Lu, C.A.; Hsieh, S.L.; Yu, S.M. Production of human serum albumin by sugar starvation induced promoter
and rice cell culture. Transgenic Res. 2005, 14, 569–581. [CrossRef]
78. Chen, T.L.; Lin, Y.L.; Lee, Y.L.; Yang, N.S.; Chan, M.T. Expression of bioactive human interferon gamma in transgenic rice cell
suspension cultures. Transgenic Res. 2004, 13, 499–510. [CrossRef] [PubMed]
79. Shin, Y.J.; Hong, S.Y.; Kwon, T.H.; Jang, Y.S.; Yang, M.S. High level of expression of recombinant human granulocyte-macrophage
colony stimulating factor in transgenic rice cell suspension culture. Biotechnol. Bioeng. 2003, 82, 778–783. [CrossRef] [PubMed]
80. Huang, J.; Nandi, S.; Wu, L.; Yalda, D.; Bartley, G.; Rodriguez, R.; Lonnerda, L.B.; Huang, N. Expression of natural antimicrobial
human lysozyme in rice grains. Mol. Breeding 2002, 10, 83–94. [CrossRef]
81. Huang, J.; Sutliff, T.D.; Wu, L.; Nandi, S.; Benge, K.; Terashima, M.; Ralston, A.H.; Drohan, W.; Huang, N.; Rodriguez, R.L.
Expression and purification of functional human α-1-antitrypsin from cultured plant cells. Biotechnol. Progr. 2001, 17, 126–133.
[CrossRef]
82. Terashima, M.; Murai, Y.; Kawamura, M.; Nakanishi, S.; Stoltz, T.; Chen, L.; Drohan, W.; Rodriguez, R.L.; Katoh, S. Production of
functional human 1 -antitrypsin by plant cell culture. Appl. Microbiol. Biotechnol. 1999, 52, 516–523. [CrossRef]
83. Matsumoto, S.; Ikura, K.; Ueda, M.; Sasaki, R. Characterization of a human glycoprotein (erythropoietin) produced in cultured
tobacco cells. Plant Mol. Biol. 1995, 27, 1163–1172. [CrossRef]
84. Sijmons, P.C.; Dekker, B.M.M.; Schrammeijer, B.; Verwoerd, T.C.; van den Elzen, P.J.M.; Hoekema, A. Production of correctly
processed human serum albumin in transgenic plants. Nat. Biotechnol. 1990, 8, 217–221. [CrossRef]
85. Huang, T.K.; McDonald, K.A. Bioreactor systems for In vitro production of foreign proteins using plant cell cultures. Biotechnol.
Adv. 2012, 30, 398–409. [CrossRef]
86. Gengenbach, B.B.; Keil, L.L.; Opdensteinen, P.; Muschen, C.R.; Melmer, G.; Lentzen, H.; Buhrmann, J.; Buyel, J.F. Comparison of
microbial and transient expression (tobacco plants and plant cell packs) for the production and purification of the anticancer
mistletoe lectin viscumin. Biotechnol. Bioeng. 2019, 116, 2236–2249. [CrossRef]
87. Rosales-Mendoza, S.; Tello-Olea, M.A. Carrot cells: A pioneering platform for biopharmaceuticals production. Mol. Biotechnol.
2015, 57, 219–232. [CrossRef] [PubMed]
88. Gerszberg, A.; Hnatuszko-Konka, K. Compendium on food crop plants as a platform for pharmaceutical protein production. Int.
J. Mol. Sci. 2022, 23, 3236. [CrossRef]
89. Ghag, S.B.; Adki, V.S.; Ganapathi, T.R.; Bapat, V.A. Plant platforms for efficient heterologous protein production. Biotechnol.
Bioprocess Eng. 2021, 26, 546–567. [CrossRef]
90. Yao, J.; Weng, Y.; Dickey, A.; Wang, K.Y. Plants as factories for human pharmaceuticals: Applications and challenges. Int. J. Mol.
Sci. 2015, 16, 28549–28565. [CrossRef] [PubMed]
91. Kumar, G.B.S.; Ganapathi, T.R.; Srinivas, L.; Revathi, C.J.; Bapat, V.A. Secretion of hepatitis B surface antigen in transformed
tobacco cell suspension cultures. Biotechnol. Lett. 2005, 27, 927–932. [CrossRef] [PubMed]
92. Sadoch, J.; Pyc, M.; Urbanowicz, A.; Iglewski, A.; Pilarski, R. High throughput evolutionary optimization of the induction
medium towards recombinant protein production in BY-2 tobacco. Biotechnol. Bioeng. 2021, 118, 676–689. [CrossRef] [PubMed]
Agronomy 2023, 13, 858 18 of 20

93. Lico, C.; Santi, L.; Baschieri, S.; Noris, E.; Marusic, C.; Donini, M.; Pedrazzini, E.; Maga, G.; Franconi, R.; Di Bonito, P.; et al. Plant
molecular farming as a strategy against COVID19-the Italian perspective. Front. Plant Sci. 2020, 11, 609910. [CrossRef]
94. Imamura, T.; Isozumi, N.; Higashimura, Y.; Ohki, S.; Mori, M. Production of ORF8 protein from SARS-CoV-2 using an inducible
virus-mediated expression system in suspension-cultured tobacco BY-2 cells. Plant Cell Rep. 2021, 40, 433–436. [CrossRef]
95. Musiychuk, K.; Sivalenka, R.; Jaje, J.; Bi, H.; Flores, R.; Shaw, B.; Jones, R.M.; Golovina, T.; Schnipper, J.; Khandker, L.; et al.
Plant-produced human recombinant erythropoietic growth factors support erythroid differentiation in vitro . Stem Cells Dev. 2013,
22, 2326–2340. [CrossRef]
96. Wang, X.; Karki, U.; Abeygunaratne, H.; Unnold Cofre, C.; Xu, J. Plant cell-secreted stem cell factor stimulates expansion and
differentiation of hematopoietic stem cells. Process. Biochem. 2021, 100, 39–48. [CrossRef]
97. Dehelean, C.A.; Marcovici, I.; Soica, C.; Mioc, M.; Coricovac, D.; Iurciuc, S.; Cretu, O.M.; Pinzaru, I. Plant-derived anticancer
compounds as new perspectives in drug discovery and alternative therapy. Molecules 2021, 26, 1109. [CrossRef] [PubMed]
98. Newman, D.J.; Gordon, M.C. Natural products as sources of new drugs over the nearly four decades from 01/1981 to 09/2019. J.
Nat. Prod. 2020, 83, 770–803. [CrossRef] [PubMed]
99. Farrar, M.C.; Jacobs, T.F. Paclitaxel; StatPearls Publishing: Treasure Island, FL, USA, 2020.
100. Dou, J.; Weathers, P. Specialty molecules from plants and In vitro cultures as new drugs: Regulatory considerations from flask to
patient. Plant Cell Tiss. Org. Cult. 2022, 149, 105–111. [CrossRef] [PubMed]
101. Fett-Neto, A.G.; DiCosmo, F.; Reynolds, W.; Sakata, K. Cell culture of Taxus as a source of the antineoplastic drug taxol and related
taxanes. Nat. Biotechnol. 1992, 10, 1572–1575. [CrossRef] [PubMed]
102. Choi, Y.E.; Jeong, J.H.; Shin, C.K. Hormone-independent embryogenic callus production from ginseng cotyledons using high
concentrations of NH4 NO3 and progress towards bioreactor production. Plant Cell Tiss. Org. Cult. 2003, 72, 229–235. [CrossRef]
103. Gantait, S.; Mitra, M.; Chen, J.T. Biotechnological interventions for ginsenosides production. Biomolecules 2020, 10, 538. [CrossRef]
104. Lyu, X.; Lyu, Y.; Yu, H.W.; Chen, W.N.; Ye, L.; Yang, R. Biotechnological advances for improving natural pigment production: A
state-of-the-art review. Bioresour. Bioprocess. 2022, 9, 8. [CrossRef]
105. Kobayashi, Y.; Akita, M.; Sakamoto, K.; Liu, H.; Shigeoka, T.; Koyano, T.; Kawamura, M.; Furuya, T. Large-scale production of
anthocyanin by Aralia cordata cell suspension cultures. Appl. Microbiol. Biotechnol. 1993, 40, 215–218. [CrossRef]
106. Yazaki, K. Lithospermum erythrorhizon cell cultures: Present and future aspects. Plant Biotechnol. J. 2017, 34, 131–142. [CrossRef]
107. Aitken-Christie, J.; Kozai, T.; Smith, M.A.L. (Eds.) Automation and Environmental Control in Plant Tissue Culture; Springer: Dordrecht,
Germany, 1995.
108. Georgiev, M.I.; Weber, J.; Maciuk, A. Bioprocessing of plant cell cultures for mass production of targeted compounds. Appl.
Microbiol. Biotechnol. 2009, 83, 809–823. [CrossRef]
109. Tekoah, Y.; Shulman, A.; Kizhner, T.; Ruderfer, I.; Fux, L.; Nataf, Y.; Bartfeld, D.; Ariel, T.; Gingis-Velitski, S.; Hanania, U.; et al.
Large-scale production of pharmaceutical proteins in plant cell culture-the protalix experience. Plant Biotech. J. 2015, 13, 1199–1208.
[CrossRef]
110. Satish, L.; Rency, A.S.; Muthubharathi, B.C.; Shamili, S.; Rameshkumar, R.; Swamy, M.K.; Ramesh, M. Transgenic plant cell
cultures: A promising approach for secondary metabolite production. In Natural Bio-Active Compounds: Volume 3: Biotechnology,
Bioengineering, and Molecular Approaches; Akhtar, M.S., Swamy, M.K., Eds.; Springer: Singapore, 2019; pp. 79–122.
111. Wawrosch, C.; Zotchev, S.B. Production of bioactive plant secondary metabolites through in vitro technologies—status and
outlook. Appl. Microbiol. Biotechnol. 2021, 105, 6649–6668. [CrossRef] [PubMed]
112. Nett, R.S.; Lau, W.; Sattely, E.S. Discovery and engineering of colchicine alkaloid biosynthesis. Nature 2020, 584, 148–153.
[CrossRef] [PubMed]
113. Birchfield, A.S.; McIntosh, C.A. Metabolic engineering and synthetic biology of plant natural products-A mini review. Curr. Plant
Biol. 2020, 24, 100163. [CrossRef]
114. Chandran, H.; Meena, M.; Barupal, T.; Sharma, K. Plant tissue culture as a perpetual source for production of industrially
important bioactive compounds. Biotechnol. Rep. 2020, 26, e00450. [CrossRef] [PubMed]
115. Jamil, S.; Rohani, E.R.; Baharum, S.N.; Noor, N.M. Metabolite profiles of callus and cell suspension cultures of mangosteen. 3
Biotech 2018, 8, 322. [CrossRef]
116. Wang, J.; Qiu, Y.; Wang, X.; Yue, Z.; Yang, X.; Chen, X.; Zhang, X.; Shen, D.; Wang, H.; Song, J.; et al. Insights into the species-
specific metabolic engineering of glucosinolates in radish (Raphanus sativus L.) based on comparative genomic analysis. Sci. Rep.
2017, 7, 16040. [CrossRef]
117. Pyne, M.E.; Narcross, L.; Martin, V.J.J. Engineering plant secondary metabolism in microbial systems. Plant Physiol. 2019, 179,
844–861. [CrossRef]
118. Pandey, R.P.; Parajuli, P.; Koffas, M.A.G.; Sohng, J.K. Microbial production of natural and non-natural flavonoids: Pathway
engineering, directed evolution and systems/synthetic biology. Biotechnol. Adv. 2016, 34, 634–662. [CrossRef]
119. Turanlı-Yıldız, B.; Hacısalihoğlu, B.; Çakar, Z.P. Advances in metabolic engineering of Saccharomyces cerevisiae for the production
of industrially and clinically important chemicals. In Old Yeasts-New Quest; Lucas, C., Pais, C., Eds.; IntechOpen: London, UK,
2017. [CrossRef]
120. Ajikumar, P.K.; Xiao, W.H.; Tyo, K.E.J.; Wang, Y.; Simeon, F.; Leonard, E.; Mucha, O.; Phon, T.H.; Pfeifer, B.; Stephanopoulos, G.
Isoprenoid pathway optimization for taxol precursor overproduction in Escherichia coli . Science 2010, 330, 70–74. [CrossRef]
Agronomy 2023, 13, 858 19 of 20

121. Grewal, P.S.; Modavi, C.; Russ, Z.N.; Harris, N.C.; Dueber, J.E. Bioproduction of a betalain color palette in Saccharomyces cerevisiae .
Metab. Eng. 2018, 45, 180–188. [CrossRef] [PubMed]
122. Lim, C.G.; Fowler, Z.L.; Hueller, T.; Schaffer, S.; Koffas, M.A.G. High-yield resveratrol production in engineered Escherichia coli .
Appl. Environ. Microbiol. 2011, 77, 3451–3460. [CrossRef] [PubMed]
123. Yang, Y.; Lin, Y.; Li, L.; Linhardt, R.J.; Yan, Y. Regulating malonyl-CoA metabolism via synthetic antisense RNAs for enhanced
biosynthesis of natural products. Metab. Eng. 2015, 29, 217–226. [CrossRef]
124. Li, M.; Schneider, K.; Kristensen, M.; Borodina, I.; Nielsen, J. Engineering yeast for high-level production of stilbenoid antioxidants.
Sci. Rep. 2016, 6, 36827. [CrossRef] [PubMed]
125. Yang, H.; Liu, F.; Li, Y.; Yu, B. Reconstructing biosynthetic pathway of the plant-derived cancer chemopreventive-precursor
glucoraphanin in Escherichia coli . ACS Synth. Biol. 2018, 7, 121–131. [CrossRef]
126. Lv, Y.; Marsafari, M.; Koffas, M.; Zhou, J.; Xu, P. Optimizing oleaginous yeast cell factories for flavonoids and hydroxylated
flavonoids biosynthesis. ACS Synth. Biol. 2019, 8, 2514–2523. [CrossRef]
127. Mora-Vasquez, S.; Wells-Abascal, G.G.; Espinosa-Leal, C.; Cardineau, G.A.; García-Lara, S. Application of metabolic engineering
to enhance the content of alkaloids in medicinal plants. Metabolic Enginer. Commun. 2022, 14, e00194. [CrossRef]
128. Rather, G.A.; Sharma, A.; Misra, P.; Kumar, A.; Kaul, V.; Lattoo, S.K. Molecular characterization and overexpression analyses of
secologanin synthase to understand the regulation of camptothecin biosynthesis in Nothapodytes nimmoniana (Graham.) Mabb.
Protoplasma 2020, 257, 391–405. [CrossRef]
129. Singh, S.; Kamble, S.N.; Satdive, R.K.; Fulzele, D.P. Heterologous overexpression of Nothapodytes foetida strictosidine synthase
enhances levels of anti-cancer compound camptothecin in Ophiorrhiza rugosa . Plant Cell Tiss. Org. Cult. 2020, 141, 67–76.
[CrossRef]
130. Pan, Q.; Wang, C.; Xiong, Z.; Wang, H.; Fu, X.; Shen, Q.; Peng, B.; Ma, Y.; Sun, X.; Tang, K. CrERF5, an AP2/ERF transcription
factor, positively regulates the biosynthesis of bisindole alkaloids and their precursors in Catharanthus roseus . Front. Plant Sci.
2019, 10, 931. [CrossRef]
131. Hao, X.; Xie, C.; Ruan, Q.; Zhang, X.; Wu, C.; Han, B.; Qian, J.; Zhou, W.; Nützmann, H.W.; Kai, G. The transcription factor
OpWRKY2 positively regulates the biosynthesis of the anticancer drug camptothecin in Ophiorrhiza pumila . Hortic. Res. 2021, 8, 7.
[CrossRef]
132. Singh, P.; Prasad, R.; Tewari, R.; Jaidi, M.; Kumar, S.; Rout, P.K.; Rahman, L. Silencing of quinolinic acid phosphoribosyl
transferase (QPT) gene for enhanced production of scopolamine in hairy root culture of Duboisia leichhardtii . Sci. Rep. 2018, 8,
13939. [CrossRef] [PubMed]
133. Schweizer, F.; Colinas, M.; Pollier, J.; Van Moerkercke, A.; Vanden Bossche, R.; de Clercq, R.; Goossens, A. An engineered
combinatorial module of transcription factors boosts production of monoterpenoid indole alkaloids in Catharanthus roseus . Metab.
Eng. 2018, 48, 150–162. [CrossRef] [PubMed]
134. Sharma, A.; Verma, P.; Mathur, A.; Mathur, A.K. Overexpression of tryptophan decarboxylase and strictosidine synthase enhanced
terpenoid indole alkaloid pathway activity and antineoplastic vinblastine biosynthesis in Catharanthus roseus . Protoplasma 2018,
255, 1281–1294. [CrossRef]
135. Georgiev, M.I.; Eibl, R.; Zhong, J.J. Hosting the plant cells In vitro: Recent trends in bioreactors. Appl. Microbiol. Biotechnol. 2013,
97, 3787–3800. [CrossRef]
136. Moyano, E.; Palazón, J.; Bonfill, M.; Osuna, L.; Cusidó, R.M.; Oksman Caldentey, K.M.; Piñol, M.T. Biotransformation of
hyoscyamine into scopolamine in transgenic tobacco cell cultures. J. Plant Physiol. 2007, 164, 521–524. [CrossRef]
137. Pádua, R.M.; Meitinger, N.; Dias De Souza, J.F.; Waibel, R.; Gmeiner, P.; Braga, F.C.; Kreis, W. Biotransformation of 21-O-
acetyl-deoxycorticosterone by cell suspension cultures of Digitalis lanata (strain W.1.4). Steroids 2012, 77, 1373–1380. [CrossRef]
[PubMed]
138. Munkert, J.; Franco, M.S.; Nolte, E.; Silva, I.T.; Castilho, R.O.; Ottoni, F.M.; Schneider, N.F.Z.; Oliveira, M.C.; Taubert, H.;
Bauer, W.; et al. Production of the cytotoxic cardenolide glucoevatromonoside by semisynthesis and biotransformation of
evatromonoside by a Digitalis lanata cell culture. Planta Med. 2017, 83, 1035–1043. [CrossRef]
139. Schneider, N.F.Z.; Cerella, C.; Lee, J.Y.; Mazumder, A.; Kim, K.R.; De Carvalho, A.; Pádua, R.M.; Munkert, J.; Kreis, W.;
Kim, K.W.; et al. Cardiac glycoside glucoevatromonoside induces cancer type-specific cell death. Front. Pharmacol. 2018, 9, 70.
[CrossRef]
140. Asthana, G.S.; Asthana, A. Biotransformation of progesterone to 17a-hydroxyprogesterone by using plant cell suspension culture
of Catharanthus roseus . Int. J. Pharm. Pharm. Sci. 2015, 7, 362–368.
141. Khan, S.; Choudhary, M.I. Biotransformation of dydrogesterone by cell suspension cultures of Azadirachta indica . Turk. J. Chem.
2008, 32, 141–146.
142. Morus, M.; Baran, M.; Rost-roszkowska, M.; Skotnicka-Graca, U. Plant stem cells as innovation in cosmetics. Acta Pol. Pharm.
Drug Res. 2014, 71, 701–707.
143. Barbulova, A.; Apone, F.; Colucci, G. Plant cell cultures as source of cosmetic active ingredients. Cosmetics 2014, 1, 94–104.
[CrossRef]
144. Rischer, H.; Szilvay, G.R.; Oksman-Caldentey, K.M. Cellular agriculture-industrial biotechnology for food and materials. Curr.
Opin. Biotechnol. 2020, 61, 128–134. [CrossRef]
Agronomy 2023, 13, 858 20 of 20

145. Bianconi, M.; Ceriotti, L.; Cuzzocrea, S.; Esposito, E.; Pressi, G.; Sgaravatti, E.; Bertaiola, O.; Guarnerio, C.; Barbieri, E.;
Semenzato, A.; et al. Red carrot cells cultured In vitro are effective, stable, and safe ingredients for skin care, nutraceutical, and
food applications. Front. Bioeng. Biotechnol. 2020, 8, 57507. [CrossRef]
146. Miastkowska, M.; Sikora, E. Anti-aging properties of plant stem cell extracts. Cosmetics 2018, 5, 55. [CrossRef]
147. Tito, A.; Carola, A.; Bimonte, M.; Barbulova, A.; Arciello, S.; De Laurentiis, F.; Monoli, I.; Hill, J.; Gibertoni, S.; Colucci, G.; et al. A
tomato stem cell extract containing antioxidant compounds and metal chelating factors protects skin cells from heavy metal-
induced damages. Int. J. Cosmet. Sci. 2011, 33, 543–552. [CrossRef]
148. Lequeux, C.; Lhoste, A.; Rovere, M.R.; Montastier, C.; Damour, O. Model of In vitro healing to test the influence of dedifferentiated
Crithmum maritimum cells on dermal repair and epidermal regeneration. Skin Pharmacol. Physiol. 2011, 24, 75–80. [CrossRef]
149. Schmid, D.; Schürch, C.; Blum, P.; Belser, E.; Züll, F. Plant stem cell extract for longevity of skin and hair. SÖFW J. 2008, 134, 30–35.
150. Othman, E.M.; Naseem, M.; Awad, E.; Dandekar, T.; Stopper, H. The plant hormone cytokinin confers protection against oxidative
stress in mammalian cells. PLoS ONE 2016, 11, e0168386. [CrossRef]
151. Aggarwal, S.; Sardana, C.; Ozturk, M.; Sarwat, M. Plant stem cells and their applications: Special emphasis on their marketed
products. 3 Biotech 2020, 10, 291. [CrossRef] [PubMed]
152. Loake, V.I.P.; Ochoa-Villarreal, M. Cambial meristematic cells: A sustainable platform for the production of plant-derived
anticancer drugs. In Biotechnology and Production of Anti-Cancer Compounds; Malik, S., Ed.; Springer International Publishing:
Cham, Switzerland, 2017; pp. 143–156.
153. Nordlund, E.; Lille, M.; Silventoinen, P.; Nygren, H.; Seppänen-Laakso, T.; Mikkelson, A.; Marja, A.A.; Nohynek, L.; Puupponen-
Pimiä, R.; Rischer, H. Plant cells as food-a concept taking shape. Food Res. Int. 2018, 107, 297–305. [CrossRef] [PubMed]
154. Matam, P.; Parvatam, G.; Shetty, N.P. Enhanced production of vanillin flavour metabolites by precursor feeding in cell suspension
cultures of Decalepis hamiltonii Wight & Arn in shake flask culture. 3 Biotech 2017, 7, 376. [CrossRef]
155. Gnehm, R. Scale-Up of Growth and Production of a Theobroma cacao Suspension Culture. Bachelor’s Thesis, Zurich University of
Applied Sciences, Zurich, Switzerland, 2018.
156. Nohynek, L.; Bailey, M.; Tähtiharju, J.; Seppänen-Laakso, T.; Rischer, H.; Oksman-Caldentey, K.M.; Puupponen-Pimiä, R.
Cloudberry (Rubus chamaemorus) cell culture with bioacive substances: Establishment and mass propagation for industrial use.
Eng. Life Sci. 2014, 14, 667–675. [CrossRef]
157. Agarwal, S.; Saha, S.; Balla, V.K.; Pal, A.; Barui, A.; Bodhak, S. Current developments in 3D bioprinting for tissue and organ
regeneration-A review. Front. Mech. Eng. 2020, 6, 589171. [CrossRef]
158. Gubser, G.; Vollenweider, S.; Eibl, D.; Eibl, R. Food ingredients and food made with plant cell and tissue cultures: State-of-the art
and future trends. Eng. Life Sci. 2021, 21, 87–98. [CrossRef]
159. Turck, D.; Bresson, J.; Burlingame, B.; Dean, T.; Fairweather-Tait, S.; Heinonen, M.; Hirsch-Ernst, K.I.; Mangelsdorf, I.; McArdle,
H.; EFSA Panel on Dietetic Products, Nutrition and Allergies (NDA); et al. Guidance on the preparation and presentation of an
application for authorisation of a novel food in the context of regulation (EU) 2015/2283. EFSA J. 2016, 14, e04594.
160. Stintzing, F.C.; Carle, R. Functional properties of anthocyanins and betalains in plants, food, and in human nutrition. Trends Food
Sci. Technol. 2004, 15, 19–38. [CrossRef]
161. Jovic, T.H.; Kungwengwe, G.; Mills, A.C.; Whitaker, I.S. Plant-derived biomaterials: A review of 3D bioprinting and biomedical
applications. Front. Mech. Eng. 2019, 5, 19. [CrossRef]
162. Landerneaua, S.; Lemarie, L.; Marquette, C.; Petiot, E. Green 3D bioprinting of plant cells: A new scope for 3D bioprinting.
Bioprinting 2022, 27, e00216. [CrossRef]
163. Broeck, L.V.D.; Schwartz, M.F.; Krishnamoorthy, S.; Tahir, M.A.; Spurney, R.J.; Madison, I.; Melvin, C.; Gobble, M.; Nguyen, T.;
Peters, R.; et al. Establishing a reproducible approach to study cellular functions of plant cells with 3D bioprinting. Sci. Adv. 2022,
8, eabp9906. [CrossRef] [PubMed]
164. Indurkar, A.; Pandit, A.; Jain, R.; Dandekar, P. Plant-based biomaterials in tissue engineering. Bioprinting 2021, 21, e00127.
[CrossRef]
165. Shukla, M.R.; Singh, A.S.; Piunno, K.; Saxena, P.K.; Jones, A.M.P. Application of 3D printing to prototype and develop novel plant
tissue culture systems. Plant Methods 2017, 13, 6. [CrossRef] [PubMed]
166. Xu, J.; Zhang, N. On the way to commercializing plant cell culture platform for biopharmaceuticals: Present status and prospect.
Pharm. Bioprocess. 2014, 2, 499–518. [CrossRef] [PubMed]
167. Muthulakshmi, V.M.; Srinivasan, A.; Srivastava, S. Antioxidant green factories: Toward sustainable production of vitamin e in
plant In vitro cultures. ACS Omega 2023, 8, 3586–3605. [CrossRef]

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