Biotechnological production of β-carotene using plant in vitro cultures

Abstract

Main conclusion:
β-carotene is biologically active compound widely distributed in plants. The use of plant in vitro cultures and genetic engineering is a promising strategy for its sustainable production. β-carotene is an orange carotenoid often found in leaves as well as in fruits, flowers, and roots. A member of the tetraterpene family, this 40-carbon isoprenoid has a conjugated double-bond structure, which is responsible for some of its most remarkable properties. In plants, β-carotene functions as an antenna pigment and antioxidant, providing protection against photooxidative damage caused by strong UV-B light. In humans, β-carotene acts as a precursor of vitamin A, prevents skin damage by solar radiation, and protects against several types of cancer such as oral, colon and prostate. Due to its wide spectrum of applications, the global market for β-carotene is expanding, and the demand can no longer be met by extraction from plant raw materials. Considerable research has been dedicated to finding more efficient production alternatives based on biotechnological systems. This review provides a detailed overview of the strategies used to increase the production of β-carotene in plant in vitro cultures, with particular focus on culture conditions, precursor feeding and elicitation, and the application of metabolic engineering.

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Vol.:(0123456789)
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Planta (2022) 256:41
https://doi.org/10.1007/s00425-022-03953-9
REVIEW
Biotechnological production ofβ‑carotene using plant invitro cultures
LorenaAlmagro1 · JoséManuelCorrea‑Sabater1· AnaBelénSabater‑Jara1· MaríaÁngelesPedreño1
Received: 6 April 2022 / Accepted: 19 June 2022 / Published online: 14 July 2022
© The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature 2022
Abstract
Main conclusion β-carotene is biologically active compound widely distributed in plants. The use of plant invitro
cultures and genetic engineering is a promising strategy for its sustainable production.
Abstract β-carotene is an orange carotenoid often found in leaves as well as in fruits, flowers, and roots. A member of the
tetraterpene family, this 40-carbon isoprenoid has a conjugated double-bond structure, which is responsible for some of its
most remarkable properties. In plants, β-carotene functions as an antenna pigment and antioxidant, providing protection
against photooxidative damage caused by strong UV-B light. In humans, β-carotene acts as a precursor of vitamin A, prevents
skin damage by solar radiation, and protects against several types of cancer such as oral, colon and prostate. Due to its wide
spectrum of applications, the global market for β-carotene is expanding, and the demand can no longer be met by extraction
from plant raw materials. Considerable research has been dedicated to finding more efficient production alternatives based
on biotechnological systems. This review provides a detailed overview of the strategies used to increase the production of
β-carotene in plant invitro cultures, with particular focus on culture conditions, precursor feeding and elicitation, and the
application of metabolic engineering.
Keywords β-carotene· Biotechnological production· Elicitors· Plant invitro cultures
Introduction
β-carotene is an orange carotenoid often found in leaves with
other pigments, such as chlorophyll or other carotenoids,
as well as in fruits, flowers, and roots (Gao etal. 2011;
Bertínez-García etal. 2014; Miras-Moreno etal. 2019).
Although β-carotene is present in bacteria, fungi, and micro-
and macroalgae, higher plants are the most prolific producers
(Lee etal. 2004).
Biochemically, β-carotene is a 40-carbon isoprenoid com-
posed of eight isoprene units, with a cyclic ring at each end,
and no oxygen. A member of the tetraterpene family, its
conjugated double-bond structure is responsible for some
of β-carotene’s most remarkable properties, endowing the
molecule with stability and high electron resonance. Able to
transfer electrons to other molecules, β-carotene functions as
an antenna pigment in plants (Arab etal. 2001). In addition,
β-carotene has antioxidant properties and protects plants
from photooxidative damage caused by strong UV-B light.
Thus, β-carotene-free plants, which can live in the dark, die
when exposed to normal light conditions (Cazzaniga etal.
2012). Additionally, carotenoids in non-photosynthetic
tissues play a role as pigments in the yellow to red range.
Carotenoids provide the autumn colors of many leaves and
more specifically, β-carotene is responsible for the orange
color of carrots, pumpkin, and oranges, among others. In
addition to being colored attractants for seed dispersal and
pollination in fruits and flowers, carotenoids also are the
precursors to a range of scents and act as photoprotective
compounds (Zhu etal. 2010; Rodriguez-Concepcion etal.
2018).
The last decades the global market value of nutraceutical
with beneficial effects for human health have seen increased
(Keasling 2010).Among the nutraceutical products that have
remarkably increased their commercialization, it is important
Communicated by Gerhard Leubner.
Lorena Almagro and José Manuel Correa-Sabater have equal
contribution.
* Lorena Almagro
lorena.almagro@um.es
1 Department ofPlant Biology, Faculty ofBiology, University
ofMurcia, Campus de Espinardo, 30100Murcia, Spain

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to highlight the use of carotenoids, mainly referring to
β-carotene, canthaxanthin, lutein, astaxanthin and lycopene
(Molino etal. 2019). Indeed, international carotenoid market
gained 1.3 billion euros in 2017 and it is expected to increase
up to 1.8 billion euros by 2022, with a compound annual
growth rate of 5.7% in the next years (Bccresearch Report
2019). Commercially available carotenoids are natural-
based or synthetic, being up to 90% of the total market, the
chemically-synthesized forms. β-carotene is one of the most
important and effective provitamin A, whose use has been
allowed according to Directive 2002/46/EC as food supple-
ment (Directive 2002/46/EC). Vitamin A deficiency is one
of the major micronutrient deficiencies worldwide, and this
deficiency generates serious problems since the provitamin
A is converted into retinol and other related retinoids, which
play important roles in the visual cycle (Grune etal. 2010).
β-carotene is been used as an ingredient for multivitamins
supplements, as additive in cosmetic formulations, as col-
oring agent in animal feeds and above all as antioxidant in
foods. β-carotene is a key ingredient of sunscreens used to
protect skin from the harmful effects of sun radiation, includ-
ing the development of carcinomas (Eggersdorfer and Wyss
2018). There are many oncological and epidemiological
investigations that evidence as a nutritional regimen rich in
carotenoids can reduce the incidence of several degenerative
and cancer diseases including oral, colon and prostate cancer
(Zare etal. 2021). The placing on the market of β-carotene in
Europe has been already approved as food ingredient by the
European Food Safety Authority (EFSA). European multi-
national companies such as BASF and DSM have acquired
some of the main global producers of β-carotene located in
the United States and Australia (Wang etal. 2022).
The expanding global demand for β-carotene cannot
be met by the traditional method of direct extraction from
plant raw material and the human consumption of products
obtained by chemical synthesis is less accepted than those
obtained from natural sources (Berreiro and Barredo 2018).
For this reason, considerable research effort has led to the
development of alternative, more efficient production tech-
niques, such as microalgae and plant invitro cultures (da
Rocha etal. 2015), combined with elicitation treatments
(Miras-Moreno etal. 2019), and metabolic engineering
(Chen etal. 2017). We here provide an overview of different
strategies designed to enhance β-carotene production based
on plant invitro cultures and metabolic engineering.
Biosynthesis ofβ‑carotene
All known carotenes in nature are synthesized from iso-
pentenyl diphosphate (IPP) and its double-bond isomer
dimethylallyl diphosphate (DMADP). IPP is condensed
with DMADP to form C10-geranyl diphosphate (GPP),
which is lengthened to C15-farnesyl diphosphate (FPP) and
C20-geranylgeranyl diphosphate (GGPP), respectively (Liang
etal. 2017).
Algae, photosynthetic cyanobacteria, bacteria, and
higher plants mainly use the methylerythritol 4-phosphate
pathway to biosynthesize IPP and DMADP to form carot-
enoids (Moses etal. 2013). In higher plants and algae, IPP
and DMADP are linked to form GGPP through the action
of geranylgeranyl diphosphate synthase. Then, in the first
limiting step in carotenoid biosynthesis, phytoene synthase
(PSY) catalyzes the conversion of two molecules of GGPP
into phytoene (Torres-Montilla and Rodriguez-Concepcion
2021) (Fig.1). PSY protein sequences in bacteria and fungi
are similar to those of plants, algae, and cyanobacteria, but
some algae and vascular plants have a higher number of
PSY isoforms (Ampomah-Dwamena etal. 2015; Dibari
etal. 2012; Tran etal. 2009). Thus, there are two psy genes
(psy1 and psy2) annotated in eudicots and three paralogous
genes of psy in monocots (psy1, psy2 and psy3) (Dibari etal.
2012). A single psy gene has been detected in Arabidopsis
(Fantini etal. 2013), which is expressed in both photosyn-
thetic and non-photosynthetic tissues (Welsch etal. 2003).
Some PSY isoforms involved in carotenoid biosynthesis
have been detected in non-photosynthetic tissues, including
roots (cassava, maize, and rice PSY3) (Arango etal. 2010;
Li etal. 2008a; Welsch etal. 2008), seed endosperm (maize
PSY1) (Li etal. 2008b) and fruits (tomato PSY1) (Fraser
etal. 2002), whereas other PSY isoforms have been detected
in chloroplast-containing photosynthetic tissues, for exam-
ple, in leaves (tomato PSY2) (Bartley and Scolnik, 1993).
PSY activity can be regulated by different factors such
as light, drought, salinity, or the presence of abscisic acid
(Welsch etal. 2008; Li etal. 2008a, b; Cazzonelli and Pog-
son 2010). It was observed that salt and drought induced an
increase of PSY3 transcript accumulation, which resulted
in higher levels of carotenoid and xanthophyll in maize
roots and rice, respectively (Li etal. 2008a, b; Welsch etal.
2008). Similarly, light was found to enhance the biosynthesis
of carotenoids in Sinapsis alba and Arabidopsis thaliana,
which was correlated with an up-regulation of psy gene tran-
script levels (Von Lintig etal. 1997).
Once phytoene is formed by the action of PSY, the
sequential actions of phytoene desaturase (PDS), ζ-carotene
isomerase (Z-ISO), ζ-carotene desaturase (ZDS), and carot-
enoid isomerase (CRTISO) produce all-trans-lycopene
(Fig.1). Subsequently, in the key step of carotenoid biosyn-
thesis, lycopene ε-cyclase (LYCE) and lycopene β-cyclase
(LYCB) catalyze the formation of α-carotene (introduc-
ing one β- and one ε-ring into lycopene) and β-carotene
(introducing a β-ring at both ends of lycopene), respec-
tively (Fig.1) (Sathasivam and Ki 2018). α- and β-carotene
undergo consecutive hydroxylations catalyzed by HYDB
(non-heme carotene hydroxylases), CYP97A and CYP97C

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(heme hydroxylases), which are involved in zeaxanthin and
lutein biosynthesis, respectively.
Biotechnological approaches
In vitro culture techniques for the production of specialized
metabolites such as β-carotene have considerable advantages
compared to extraction from plant raw material, which is
less efficient and leads to the overexploitation of natural
resources. Dias etal. (2018) performed a quantification of
β-carotene content in different edible vegetables, and the
levels of this compound ranged from 0.5µg/g in banana and
avocado to higher levels in other vegetables such as car-
rots (43.5–88.4µg/g), red peppers (14.4–23.9µg/g), spinach
(31.0–48.1µg/g) or parsley (44.4–46.8µg/g). However, as
will be seen in this review, β-carotene levels can be higher
using some plant invitro cultures with or without genetic
engineering than in raw material. Moreover, invitro cul-
tures allow an easier extraction of metabolites, which can be
carried out under controlled, automated, and pathogen-free
conditions. They also facilitate the isolation of novel com-
pounds with potentially valuable applications (Gonçalves
and Romano 2018). Nevertheless, invitro culture techniques
have some disadvantages, including a requirement for tech-
nological knowledge, specific equipment, and skilled work-
ers (Mohanlall 2020).
An industry closely related to specialized metabolite pro-
duction that has experienced tremendous growth in recent
decades is that of bioreactors, which enable efficient large-
scale processes. Bioreactors are designed to provide the
precise environmental conditions required for cell growth
and bioactive substance harvesting, and different types are
available, depending on the kind of culture and its purpose;
for example, airlift bioreactors constitute a suitable system
for metabolite production by hairy root cultures (Almagro
and Pedreño 2020).
Optimization ofinvitro culture conditions
toimprove β‑carotene production
The efficiency of metabolite production in invitro cultures
depends on factors such as temperature, photoperiod, and the
use of phytohormones in the culture medium, all of which
can provoke alterations in the normal cell behavior (Marchev
etal. 2020). The effect of the culture medium composition
on β-carotene production in Daucus carota cell cultures was
investigated by Hanchinal etal. (2008), who achieved the
maximum production (13.81µg of β-carotene/g dry weight
(DW)) using an optimized liquid Gamborg B5 medium
(Gamborg etal. 1968) containing 0.1mg/L 2,4-dichloro-
phenoxyacetic acid and 0.1mg/L benzyladenine as plant
growth regulators, plus 3% sucrose, 50mM nitrogen (ammo-
nium sulfate) and 1mM phosphorus (sodium di-hydrogen
phosphate). The same authors reported that the inoculum
size greatly affects β-carotene production in D. carota cells
Fig. 1 Schematic view of the
β-carotene biosynthetic pathway
and its transcriptional regulation
in plant cells

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41 Page 4 of 9
(Hanchinal etal. 2008). When using an initial inoculum size
of 8, 20, 40 and 80g of DW/L, the production of β-carotene
was 10.37, 10.83, 9.87 and 9.83µg/g DW, respectively
(Table1). Thus, above the optimum value of 20g DW/L,
the cells competed for nutrients. Shimizu etal. (1979) found
that β-carotene production in D. carota cell cultures was
higher when 2,4-dichlorophenoxyacetic acid was applied
at a concentration of 1mg/L (48.5µg of β-carotene/gDW)
compared to 0.1 or 5mg/L (42.2 and 35µg of β-carotene/
gDW, respectively) (Table1). In a study on carotenoid bio-
synthesis, Oleszkiewicz etal. (2021) used carotenoid-rich
calli of D. carota grown on either modified Murashige and
Skoog basal medium (MS) (Murashige and Skoog 1962)
and/or modified Gamborg B5 basal mineral medium. Finally,
the maximum accumulation of carotenoids (2.19mg of
β-carotene/g DW) was achieved when using a mixture of
both, and the lowest (0.25mg of β-carotene/g DW) with the
modified MS medium (Table1). The results showed that the
production of β-carotene is affected by nitrogen availability,
the composition of nitrogenous salts, and the NO3:NH4 ratio
in the culture medium.
Exposure to light can affect β-carotene production (Rodri-
guez-Concepcion and Stange 2013). Gao etal. (2011) stud-
ied carotenogenesis in citrus calli of four genotypes sub-
jected to different light regimes. In all cases, higher levels
of β-carotene were found when the citrus calli were grown
in the dark for 20days, being highest in Citrus paradisi
(over 3.26µg of β-carotene/g DW) and Citrus sinensis L.
Osbeck (over 3.5µg of β-carotene/g DW). Moreover, the
expression of the psy gene was enhanced in calli of the
two sweet oranges and reduced in Citrus reticulata × C.
sinensis. Expression of the crtiso gene was light-induced,
above all in C. reticulata × C. sinensis, where it increased
up to 12-fold (Table1). Therefore, in citrus calli, light regu-
lates the expression of several genes involved in β-carotene
biosynthesis but may not necessarily result in significant
changes in the production of this bioactive compound.
Studies using whole plants, shoots, calli and cell cultures
of different plant species have demonstrated that the level
of cell differentiation plays an important regulatory role in
the pattern of specialized metabolite production (Efferth
2019). Park etal. (2017) showed that the β-carotene content
(3.6µg/g FW) in invitro-raised shoots was 2.25-fold higher
than shoots of greenhouse-grown plants (1.6µg/g FW),
indicating that the use of invitro culture is a good strategy
to increase the production of β-carotene (Table1). On the
other hand, Engelmann etal. (2010) carried out a screen-
ing and selection of high carotenoid-producing invitro
tomato cell culture lines. These authors demonstrated that
the production of β-carotene was higher in wild-type cal-
lus cultures obtained from tomato seedlings (0.06µg/g FW)
than in callus cultures derived from the ghost phenotype
tomato (0.04µg/g FW), which is deficient in plastid terminal
oxidase (Table1). This enzyme is a plastoquinone-O2 oxi-
doreductase thought to act as a cofactor for dehydrogenases
involved in carotenoid biosynthesis (Shahbazi etal. 2007).
Effect ofelicitors andprecursor feeding
onβ‑carotene production inplant invitro cultures
Widely employed to enhance metabolite production in
invitro cultures, elicitation consists of applying abiotic
or biotic stress factors that induce the expression of key
biosynthetic genes. This strategy can also provide useful
insights into the interactions between metabolic pathways
(Miras-Moreno etal. 2016). Jasmonic acid and methyl
jasmonate (MJ), molecules involved in plant defense reac-
tions, are able to alter the biosynthesis of a wide range of
specialized metabolites (da Rocha etal. 2015), and are the
most frequently used elicitors for increasing the production
of β-carotene (Ho etal. 2020). The effect of elicitation on
secondary metabolism can be modified by factors such as
the duration of treatment or the composition of the culture
medium (Saeed etal. 2014; León etal. 2005; Miclea etal.
2020).
In Cleome rosea callus cultures, elicitation with 60mg/L
MJ improved the total carotenoid production (13.62μg/g
FW) at day 67 of treatment, when the yield was sixfold
higher than in control cultures (da Rocha etal. 2015)
(Table1). In the same study, treatment with different concen-
trations of chitosan or yeast extracts did not have an enhanc-
ing effect. Therefore, MJ-elicited C. rosea in vitro cultures
could constitute a novel source of carotenoids (da Rocha
etal. 2015). In Rosa damascena Mill. cell cultures main-
tained in darkness, elicitation with 1 or 5µMMJ provoked
more than doubled β-carotene content (over 180µg/L) at
21days of culture in comparison with the control (70µg/L)
(Olgunsoy etal. 2017) (Table1). In in vitro-propagated
shoots of Lavandula angustifolia, β-carotene production
was enhanced by the application of 0.5mg/L jasmonic acid
(22.10µg/g FW) or 1.5 mg/L salicylic acid + active car-
bon (14.50µg/g FW), achieving a 5 and 3.3-fold increase,
respectively, compared to control cultures (4.40µg/g FW)
(Miclea etal. 2020) (Table1).
Other elicitors able to enhance carotenoid biosynthesis
are macrocyclic oligosaccharides known as cyclodextrins.
After adding cyclodextrins to the medium of D. carota
cell cultures, Miras-Moreno etal. (2016) observed that
β-carotene extracellular accumulation increased linearly
during elicitation, reaching the maximum concentration
at 21days (over 2.5μg of β-carotene/g DW). In Artemisia
annua L. cell cultures treated with cyclodextrins, β-carotene
extracellular levels were over 0.04μg/g FW at 7day of elici-
tation (Table1) (Rizzello etal. 2014).
An additional methodology to increase β-carotene
biosynthesis consists of supplying invitro cultures with

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Table 1 Strategies to enhance β-carotene production in plant invitro cultures
Material Species Strategy Production Production in
control treat-
ment
Fold increased
respect to control
References
Cell cultures D. carota B5 culture medium
with
0.1mg/L dichlo-
rophenoxyacetic
acid + 0.1mg/L
benzyladenine + 3%
sucrose + 50mM
nitrogen and 1mM
phosphorus
13.81µg/g DW – – Hanchinal etal. (2008)
Cell cultures D. carota Inoculum size:
8g DW/L
20g DW/L
40g DW/L
80g DW/L
10.37µg/g DW
10.83µg/g DW
9.87µg/g DW
9.83µg/g DW
–
–
–
–
– Hanchinal etal. (2008)
Cell cultures D. carota Culture medium with
0.1mg/L dichloro-
phenoxyacetic acid
48.50µg/ g DW – – Shimizu etal. (1979)
Cell cultures D. carota Culture medium
R (modified
Murashige and
Skoog mineral
media)
250µg/g DW – – Oleszkiewicz etal.
(2021)
Cell cultures D. carota Culture medium BI
(modified Gamborg
B5 mineral media)
1637µg/ g DW – – Oleszkiewicz etal.
(2021)
Cell cultures D. carota Culture medium R/
B5 macro
2190µg/g DW – – Oleszkiewicz etal.
(2021)
Callus Citrus sp. Different species
grown in darkness:
C. paradisi
C. sinensis
C. reticulata x C.
sinensis
3.26µg/g DW
3.5µg/g DW
1.4µg/g DW
–
–
–
–
–
–
Gao etal. (2011)
Shoots Levels of cell dif-
ferentiation:
In vitro-raised shoots
Shoots of green-
house-Grown plants
3.6µg/ g FW
1.6µg/g FW
–
–
–
–
Park etal. (2017)
Cell cultures Selection high pro-
ducing cell lines:
Wild-type callus
cultures
Ghost-callus cultures
0.06µg/ g FW
0.04µg/ g FW
–
–
–
–
Engelmann etal.
(2010)
Cell cultures C. rosea Elicitation with
60mg/L MJ
13.62µg/g FW 2.3µg/g FW Sixfold Da Rocha etal. (2015)
Cell cultures R. damascena Elicitation with
5µMMJ
180µg/g FW 70µg/g FW 2.5-fold Olgunsoy etal. (2017)
Shoots L. angustifolia Elicitation with
0.5mg/L jasmonic
acid
22µg/ g FW 4.4µg/g FW Five-fold Miclea etal. (2020)
Shoots L. angustifolia Elicitation with
1.5mg/L salicylic
acid + active carbon
14.5µg/g FW 4.4µg/g FW 3.3-fold Miclea etal. (2020)
Cell cultures D. carota Elicitation with
50mM methylated-
β-cyclodextrins
Over 2.5µg/ g DW ND 2.5-fold Miras-Moreno etal.
(2016)

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41 Page 6 of 9
biosynthetic intermediates such as L-phenylalanine. The
main factors to consider in precursor feeding strategies
are the timing of the treatment and the concentration of
the precursor (Olgunsoy etal. 2017). Thus, the addition
of 500µM L-phenylalanine, which is a precursor of phe-
nylpropanoid compounds, to R. damascena Mill. callus
cultures for 21days improved β-carotene production two-
fold (0.15mg/L) compared to the control (Olgunsoy etal.
2017), but only in conditions of darkness, which is therefore
a limiting factor for obtaining high levels of this bioactive
compound.
Metabolic engineering toenhance β‑carotene
production inplant invitro cultures
By enhancing or reducing the activity of key enzymes in
carotenoid biosynthesis, metabolic engineering can increase
yields of β-carotene and reduce the biosynthesis of unwanted
metabolic products (Saeed etal. 2015; Kim etal. 2012).
Metabolic engineering has been used to increase the pro-
duction of β-carotene in different organisms, including
microalgae, bacteria, and yeasts, but here we focus on its
application in plant invitro cultures. For example, trans-
genic plants were generated from embryogenic calli of Ipo-
moea obscura co-cultured with Agrobacterium tumefaciens
harboring β-carotene 4,4′-ketolase (crtW) and β-carotene
3,3′-hydroxylase (crtZ) genes, which are involved in the
biosynthesis of another pigment, astaxanthin, as well as
isopentenyl diphosphate isomerase and hygromycin resist-
ance genes. The transgenic plants produced less β-carotene
(22.3µg/g FW) than wild-type plants (25.2µg/g FW), but
the levels of astaxanthin increased significantly (Otani etal.
2021) (Table1).
Hypocotyl and cotyledon explants from 15-day-old
seedlings and calli of Morus indica were used to obtain
transgenic plants overexpressing β-carotene hydroxylase1
(Saeed etal. 2015). Under normal conditions, β-carotene
levels were similar between non-transgenic and transgenic
cell lines (approximately 256mg β-carotene/g FW), but
production increased when a range of abiotic stresses were
applied. Consequently, the levels of β-carotene after 4h
of UV treatment (249mg/g FW), 1h of strong light stress
(256mg/g FW) or heat shock treatment (233mg/ g FW)
were higher than in wild-type lines (202, 181 and 196mg
CrtISO carotenoid isomerase, GGPS geranylgeranyl diphosphate synthase, LcyB lycopene β-cyclase, LcyE lycopene ɛ-cyclase, PDS phytoene
desaturase, PSY phytoene synthase, ZDS ζ-carotene desaturase, Z-ISO ζ-carotene isomerase, crtW β-carotene 4,4′-ketolase, crtZ β-carotene
3,3′-hydroxylase, DW dry weight, FW fresh weight, ND not detected, MJ methyl jasmonate, PSY phytoene synthase
Table 1 (continued)
Material Species Strategy Production Production in
control treat-
ment
Fold increased
respect to control
References
Cell cultures A. annua Elicitation with
50mM methylated-
β-cyclodextrins
0.04µg/g FW ND 0.04-fold Rizzello etal. (2014)
Callus R. damascena Feeding with 500µM
phenylalanine
0.15mg/L 0.7mg/L Two-fold Olgunsoy etal. (2017)
Embriogenic calli I. obscura Overexpression
of crtw, crtz and
isopentenyl diphos-
phate isomerase
22.3µg/g FW 25.2µg/g FW Decrease 1.13-fold Otani etal. (2021)
Transgenic plants M. indica Overexpression of
β-carotene hydroxy-
lase1 + 4h UV light
treatment
249mg/g FW 202mg/g FW 1.23-fold Saeed etal. (2015)
Transgenic plants M. indica Overexpression of
β-carotene hydroxy-
lase1 + high light
treatment
256mg/ g FW 181mg/g FW 1.41-fold Saeed etal. (2015)
Transgenic plants M. indica Overexpression of
β-carotene hydroxy-
lase1 + heat shock
treatment
233mg/g FW 196mg/g FW 1.18-fold Saeed etal. (2015)
Callus A. thaliana Overexpression of
psy 650µgα/β-carotene/
g DW
Trace amount 650-fold Maass etal. (2009)
Cell cultures I. batatas Silencing β-carotene
hydroxylase 34.43µg/g DW 0.9µg/g DW 38-fold Kim etal. (2012)

Planta (2022) 256:41
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Page 7 of 9 41
of β-carotene/g FW, respectively) (Table1). These results
indicated that transformation with β-carotene hydroxy-
lase1 alone did not enhance production, but was effective
in combination with appropriate elicitation (Saeed etal.
2015). Likewise, after 16days in darkness, A. thaliana
transgenic calli overexpressing the psy gene from A. thali-
ana were highly effective producers of α/β-carotene (over
650µg/g DW), which constituted about 50% of the total
carotenoid content (Maass etal. 2009) (Table1). In sweet
potato cell cultures, silencing β-carotene hydroxylase in
the carotenoid biosynthetic pathway induced an up-regu-
lation of psy and lycopene β-cyclase genes, whereas the
expression of other biosynthetic genes showed no detect-
able changes (Kim etal. 2012). In the transgenic lines,
the total carotenoid levels were 117µg/g DW, including
34.43µg/g DW β-carotene compared to 0.9µg/g DW in
the non-transgenic lines (Table1). Thus, down-regulation
of the β-carotene hydroxylase from sweet potato gene
increased β-carotene contents up to 38-fold compared to
the control, which improved the antioxidant capacity of
these transgenic lines (Kim etal. 2012).
Conclusions
Due to the health-promoting effects of β-carotene, differ-
ent strategies have been developed to increase its availabil-
ity, among which invitro cultures of plants stand out for
their cost-effectiveness and bio-sustainability, among other
advantages. Importantly, compared to the use of transgenic
microorganisms, plant biotechnological platforms based on
transgenic cells, tissues or organ cultures are more socially
acceptable, as the risk of transgene dissemination is per-
ceived as minimal.
In this review, we have focused on the optimization of
culture conditions, feeding and elicitation strategies, and the
application of metabolic engineering to design β-carotene-
producing biotechnological platforms based on plant invitro
cultures. The literature shows that the most effective sys-
tem to produce β-carotene involves the use of D. carota
cell cultures grown in a modified Murashige and Skoog
basal medium enriched with the macronutrients of Gam-
borg B5 medium, with reported yields reaching 2190µg of
β-carotene/g DW. Metabolic engineering is another effective
approach to improving β-carotene biosynthesis, as the over-
expression of the β-carotene hydroxylase1 gene combined
with exposure to strong light considerably increased the
production of β-carotene in transgenic plants of M. indica
(256mg/g FW). No doubt, new procedures, benefitting from
advancements in metabolic engineering, will continue to be
optimized to establish competitive biological systems to pro-
duce this valuable bioactive compound.
Author contribution statement All authors contributed to the
study conception and design. Material preparation and data
collection were performed by JMC-S, LA, ABS-J, and MAP.
Writing Draft Preparation was carried out by LA, JMC-S,
Ana BS-J, and MAP. All authors read and approved the final
manuscript.
Acknowledgements This research was part of the project
PID2020-113438RB-I00 financed by Ministerio de Ciencia e Inno-
vación (Spain) (MCIN/ AEI/10.13039/501100011033 “Una manera
de hacer Europa”).
Data availability All data analyzed during this study have been already
published. Data sharing does not apply to this article as no datasets
were generated during the current study.
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