In Vitro Cellular & Developmental Biology - Plant

https://doi.org/10.1007/s11627-018-9902-5

INVITED REVIEW

Light quality in plant tissue culture: does it matter?

Diego Silva Batista 1 & Sergio Heitor Sousa Felipe 1 & Tatiane Dulcineia Silva 1 & Kamila Motta de Castro 1 &
Talita Cristina Mamedes-Rodrigues 1 & Natane Amaral Miranda 2 & Anyela Marcela Ríos-Ríos 3 & Daniele Vidal Faria 1 &
Evandro Alexandre Fortini 1 & Kristhiano Chagas 1 & Gabriela Torres-Silva 1 & Aloisio Xavier 2 & Ariel Domingo Arencibia 4 &
Wagner Campos Otoni 1

Received: 20 September 2017 / Accepted: 27 March 2018 / Editor: John Finer

# The Society for In Vitro Biology 2018

Abstract
The primary issues regarding the lack of protocol reproducibility among laboratories are environmental factors. Light (quantity
and particularly quality), is one of those main factors, and studies seldom present the spectral quality of the light sources used.
With the advent of light-emitting diode (LED) technology, impressive progress has been made in environmental controls and
morphogenetic responses, as directed by the light used in the culture shelves. A wide array of LED lights with different spectra are
currently available and light is important in large-scale propagation, especially liquid bioreactor systems. LED technology
continues to evolve rapidly and has created additional possibilities. This laboratory has dedicated extensive efforts to implement
photoautotrophic propagation, and light is a key component of the system. This review presents relevant topics on the influence
of light in various plant tissue culture-based techniques.

Keywords Light-emitting diodes . Morphogenetic responses . In vitro culture . Gene expression . Secondary metabolites

Introduction The light reaching the plant is reflected, transmitted, or

Light perception and photomorphogenesis and circadian
rhythm in plants Plants are sessile organisms, so their growth
and development are modified by environmental factors that
influence morphogenesis. Light is one of the main signals
perceived by plants because it is a source of energy for pho-
tosynthesis and physiological processes such as the produc-
tion of secondary metabolites (Hart 2012; Kozai 2016).
* Wagner Campos Otoni
wcotoni@gmail.com
1
Departamento de Biologia Vegetal/Laboratório de Cultura de
Tecidos/BIOAGRO, Universidade Federal de Viçosa, Viçosa, Minas
Gerais, Brazil
2
Departamento de Engenharia Florestal/Laboratório de Cultura de
Tecidos/BIOAGRO, Universidade Federal de Viçosa, Viçosa, Minas
Gerais, Brazil
3 phytochromes (phyA–E), cryptochromes (Cry1–3), and
Departamento de Química/Laboratório de Cultura de Tecidos/
BIOAGRO, Universidade Federal de Viçosa, Viçosa, Minas Gerais,
Brazil
4
Center of Biotechnology in Natural Resources, Faculty of Agronomy
and Forestry Sciences, University Catholic of Maule, Talca, Chile
absorbed. Part of the photosynthetic photons is converted into
chemical energy, which is used in the morphogenetic process,
and part is lost in the form of heat (Kozai 2013). The photo-
receptors are responsible for identifying the quantity and qual-
ity of light, the direction, and day length (Parihar et al. 2016).
Different types of photoreceptors, auxin carriers, and other
signaling components are involved in the perception of light
by plants (Vierstra and Zhang 2011; Li et al. 2012).
Photomorphogenesis is the physiological process that allows
for the development and growth of plants under the control of
light (Kendrick and Kronenberg 2012).
Phytochromes are chromoproteins that absorb light in
the far red to red region and have a linear tetrapyrrole
chromophore linked through a cysteine residue to their
N-terminal domain (Vierstra and Zhang 2011). This pho-
toreceptor is capable of reversibly interconverting the
chromophore into two states: active by absorbing red light
(Pr) and inactive by absorbing far red light (Pfr) (Vierstra
and Zhang 2011). In Arabidopsis thaliana (L.) Heynh.,
phototropins (phot1–2), can be classified according to their
function, chromophore type, cell location, spectrum
range, structure, and cellular signaling (Gomelsky and
Klug 2002; Casal et al. 2013; Parihar et al. 2016).
 BATISTA ET AL.
Cryptochromes are responsible for UV-A to blue light per-
ception and contain flavin adenine dinucleotide (FAD) as a
chromophore. Three cryptochrome genes are found in the
A. thaliana genome. CRY1 is located in the nucleus during
the dark and in the cytoplasm in the presence of light
(Cashmore et al. 1999; Yang et al. 2000). Cry2 is located in
the nucleus (Kleiner et al. 1999), and Cry3 is located in the
mitochondria and chloroplasts (Kleine et al. 2003).
The photoreceptors responsible for absorbing light in the
region of UV-A to blue are the phototropins. These receptors
control phototropism, stomatal opening, and chloroplast move-
ments (Jarillo et al. 2001; Briggs and Christie 2002; Celaya
and Liscum 2005). The two types of phototropins known as
phot1 and phot2, have the N-terminal domains LOV1 and
LOV2, respectively, and the chromophore flavin mononucle-
otide (FAM) (Christie et al. 1999). In addition, in A. thaliana,
there are ZTL-type receptors that absorb blue to UV-A light,
and a UVR8 receptor that absorbs UV-B light (Chory 2010;
Casal et al. 2013). This set of photoreceptors is responsible for
the input of light into the circadian clock of plants (Devlin and
Kay 2000). The circadian clock is an endogenous oscillator
with a duration of approximately 24 h. Present in almost all
individuals, the circadian clock synchronizes physiology with
the environment. There are a few components of the circadian
clock and they are products of convergent evolution, with well-
conserved domains (Loudon 2012).
Several plant physiological processes are controlled by the
circadian clock, such as photosynthesis (Dodd et al. 2005) and
stomatal movements (Salomé and McClung 2005). The met-
abolic pathways affected by the circadian clock in plants in-
cludes chromatin-level regulation in a complex, broad regula-
tory network that involves a set of genes, which act at distinct
times throughout the day and night (Harmer et al. 2000;
Barneche et al. 2014; Hsu and Harmer 2014). Time informa-
tion, by the circadian rhythm in organisms, is understood
through the model based on transcription–translation feed-
back loops (TTFLs) that allow the expression or repression
of rhythmic genes, such as circadian clock associated 1
(CCA1), late elongated hypocotyl (LHY), pseudo response
regulator (PRR), and timing of cab expression 1 (TOC1) in
A. thaliana (Huang et al. 2012; Haydon et al. 2013). In the
morning, high levels of CCA1 and LHY transcripts, together
with PRR5, PRR7, and PRR9 are observed. Similarly, high
expression of TOC1, gigantea (GI), early flowering 3 and 4,
and luxarrhythmo (LUX) genes are identified at night (Lu
et al. 2009; Nusinow et al. 2011).
The central oscillators of the circadian clock are coordinat-
ed by external factors such as temperature and light.
Temperature variation can alternatively affect the splicing in
clock genes such as LHY and PRR7, which decreases the ex-
pression of non-functional transcripts (James et al. 2012).
Several studies mention the influence of light on the regula-
tion of the circadian rhythm, by altering the transcription and
translation and the stability of mRNA (Staiger and Green
2011). In addition, the central oscillators and light actively
participate in the photomorphogenesis, with a nocturnal pro-
tein complex acting in the elongation phase of the hypocotyl
cells (Nusinow et al. 2011).
The knowledge of light perception by plants, circadian
rhythm mechanisms, and their genetic regulation as regulated
by light, needs to be better understood regarding the morpho-
genic responses in plants (Kim et al. 2005; Kami et al. 2010;
Kozai 2016). In addition, the use of LED lamps in in vitro
culture systems is a useful tool to study these factors in plants,
because it allows the irradiance to be controlled and to emit
specific spectral patterns (Gupta and Jatothu 2013).
Light-emitting diodes—an historical perspective The basic
principle of light-emitting diodes (LED) was born in the early
twentieth century with the physical effect of the electrolumi-
nescence described by Round (1907). Subsequently, Oleg
Vladimirovich Lósev developed a crystalline diode with zinc
oxide and silicon carbide, and demonstrated the first LED,
albeit of low-brightness (Zheludev 2007). Nick Holonyak Jr.
(Holonyak and Bevacqua 1962) developed the first practical
red LED and LED lights started to be marketed. In subsequent
years, green, orange, and yellow LED bulbs also appeared. In
1993, Shuji Nakamura introduced the blue LED (Nakamura
et al. 1994) and from that, it was possible to generate white
LED by the fusion of the three basic colors: red, green, and
blue, which allowed great advances with varied applications,
industrial and horticultural (Yeh and Chung 2009; Shchekin
and Craford 2017) (Fig. 1).
LED lighting can play a variety of roles in horticul-
ture and plant tissue culture. Plant growth and develop-
mental processes are regulated by light quality, quantity,
and duration (photoperiod) (Higuchi and Hisamatsu
2016). Studies using LED lights in plant cultivation by
Bula et al. (1991) reported that the leaf shape, color,
and texture of Lactuca sativa plants under red LED,
supplemented with blue fluorescent lamps, were not dif-
ferent from those found in plants grown under cool
white fluorescent plus incandescent lamps. In recent
years, several studies have applied the use of LED tech-
nology in plant tissue culture with the objective of eval-
uating their effects on the morphogenic pathway
(Table 1). LED lights are increasingly used by commer-
cial plant tissue culture laboratories because of the ad-
vantages over conventional lighting systems (fluorescent
light, halide metal, high-pressure solid, and incandes-
cent), such as wavelength specificity, durability, small
size, long operating lifetime, relatively cool emitting
surface, a photon output that is linear with the electrical
input current, and the ability to control spectral compo-
sition (Agarwal and Gupta 2016; Bello-Bello et al.
2017; Shukla et al. 2017).
 LIGHT QUALITY IN PLANT TISSUE CULTURE: DOES IT MATTER?
Fig. 1 Summarized historical
evolution of light-emitting diodes
(LED)
With the advancement of the technology and the reduction
of LED prices, there is a tendency to replace fluorescent lamps
with LED lamps, as different studies show more vigorous
in vitro plants cultivated under LED lighting conditions
(Hahn et al. 2000; Moon et al. 2006; Lin et al. 2011; Hung
et al. 2015; Batista et al. 2016; Ferreira et al. 2017).
Consequently, it is essential to develop research using LED
technology to evaluate the impact on morphogenesis of dif-
ferent plant species for protocol optimization.
Light and in vitro competence for photosynthesis Light is the
primary energy source for the growth and development of
plants and the LED lamp represent a relevant artificial lighting
source (Darko et al. 2014), which can influence plant photo-
synthesis, morphogenesis, and physiological processes (Wang
et al. 2016; He et al. 2017). LED lamps with different wave-
lengths (< 400 ultraviolet; 400–450 violet; 450–500 blue;
500–570 green; 570–590 yellow; 590–610 orange/amber;
610–760 red; and > 760 nm infrared color), can be used alone
or combined to optimize photosynthesis (Singh et al. 2015;
Shengxin et al. 2016). However, to select an appropriate color
LED to increase in vitro photosynthesis, the different photo-
receptors present in plants should be considered, which in-
clude phytochromes that absorb red and far-red light, and
cryptochromes and phototropins that absorb blue light
(Higuchi and Hisamatsu 2016).
Photosynthesis is a photobiochemical process that uses
light energy to convert carbon dioxide into several organic
compounds. Functionally, the chlorophylls a and b and carot-
enoid molecules that form the light harvesting antenna of pho-
tosystems, initiate photosynthesis by capturing light energy
and converting it into chemical energy (Caffarri et al. 2014).
Photosynthesis is strongly influenced by red, blue, and red
plus blue colors of LED lamps (Wang et al. 2016;
Manivannan et al. 2017), because they are the major energy
sources for photosynthetic CO2 assimilation in plants (wave-
lengths between 400 and 700 nm) (McCree 1971). The differ-
ent spectra of red (Labpar LL HR/dB 480, LabLumens®,
Barueri, Brazil); blue (Labpar LL HR/dB 480,
LabLumens®); red plus dark red (Led Tec-LUX, Tecnal®,
Piracicaba, Brazil); red plus blue (Led Tec- Lamp, Tecnal®);
and white LED (18 W, LLTV03 VILUX®, Novo Horizonte,
Brazil), compared to the fluorescent light (20 W, HO,
Osram®, São Paulo, Brazil) are shown below (Fig. 2). The
differences in spectral quality between LEDs and fluorescent
lamps directly influence the photosynthesis, growth, and de-
velopment of plants in vitro.
In micropropagation, plants are often exposed to conditions
characterized by high relative humidity inside the vessels,
reduced gas exchange, poor photosynthetic active radiances
(PARs), low CO2 concentration during the photoperiod, and
high carbohydrate, nitrogen, and growth regulator concentra-
tions (Kozai 2010; Badr et al. 2011; Xiao et al. 2011; Nguyen
et al. 2016). These factors directly affect several physiological
and biochemical processes, such as photosynthesis in plants
in vitro. In contrast, increased gas exchange (natural or forced
ventilation), and improvement of light quality such as the use
of LED lights, can be exploited to improve photosynthetic
performance.
In previous studies, the in vitro photosynthetic rate of cul-
tures was assessed by measuring CO2 exchange (Fujiwara
et al. 1987; Supaibulwattana et al. 2011; Wu and Lin 2013),
and the O2 evolution rate (Chen 2006; Hopfner et al. 2014),
with infrared gas analyzers (IRGA) (Iarema et al. 2012; Badr
et al. 2015; Schmildt et al. 2015; Sáez et al. 2016). However,
these measurements by conventional methods may not accu-
rately represent the conditions in vitro. In addition, to date,
there are few studies that aim to optimize the gas exchange
analysis techniques in in vitro systems, as observed in Vitis
vinifera (Düring 1988; Düring and Harst 1996), Solanum
tuberosum (Kubota and Kozai 1992), Fragaria ananassa
(Fujiwara et al. 1988), Hyptis marrubioides, and Hancornia
speciosa (Costa et al. 2014). It is essential to advance im-
provements in the photosynthesis measurement techniques
so that the results of the photosynthetic rates and in vitro en-
vironment conditions are not underestimated.
Few studies are reported on in vitro cultivation of plants
under LED lamps. Dendranthema grandiflorum and Withania
Table 1 Summary of the historical use of LED lighting in plant cell, tissue and organ culture

Species Morphogenic Aim of the Light type and quality of LED Conclusion Reference
pathway work

Dianthus caryophyllus
‘Green
Beauty’, ‘Purple Beauty’,
and ‘Inca Magic’
Solanum tuberosum
Stevia rebaudiana
Saccharum spp. ‘RB98710’
Vaccinium corymbosum
Organogenesis Effect of light on the reduction of
hyperhydricity
Organogenesis Test whether pre-cryopreservation
light spectral quality affects
cryopreservation success
Organogenesis Effect of light on morphogenesis
and growth
Embryogenesis and Effect of LEDs on morphogenic
organogenesis processes following somatic
embryogenesis, the formation
of shoots, rooting and changes
in redox metabolism during
acclimatization
Organogenesis Effects of light on shoot
proliferation
in medium with or without
hormone, and ex vitro rooting
of in vitro shoots
Red and blue
White, blue, red, and 90%
red + 10% blue
White, red, blue,
and blue + red (1:1)
82% red + 18% blue
Red, blue, 50% red +50%
blue, and 80% red +20% blue
Red and blue light LEDs reduced hyperhydricity Muneer et al. (2017)
Blue light may increase the cryopreservation success, Edesi et al. (2017)
but red light alone is not favorable
for pre-cryopreservation cultivation
Red light induced root formation; however, Ramírez-Mosqueda
blue + red produced longer shoots et al. (2017)
with a higher number of leaves and stimulated
chlorophyll content, which favored
the acclimation of in vitro plantlets
The generation of plants through SE was most effective Ferreira et al. (2017)
when the explants were cultivated
under FL lighting; However,
the remaining micropropagation
process stages were more favored under LED
Mixed LEDs red/blue (1:1) light-improved Hung et al. (2016)
plant growth with respect to notably
increased shoot and root biomass
BATISTA ET AL.

Phoenix dactylifera
Populus euramericana
‘I-476’ and ‘Dorskamp’
Alternanthera sessilis,
Alternanthera
philoxeroides,
Alternanthera tenella,
and Alternanthera
brasiliana
Curculigo orchioides
Musa acuminata
Musa spp.
Organogenesis Effects of light on the induction
of shoot tip adventitious buds
and shoot multiplication
Organogenesis Effects of LEDs on shoot
regeneration, wood formation,
and growth
Organogenesis Evaluate light quality on growth
and production of secondary
metabolites
Organogenesis Effect of light on antioxidative
enzymes and shoot
organogenesis
Organogenesis Effect of light on the stomata
formation and chlorophyll
levels
Organogenesis To determine whether LED or
white
fluorescent light was optimal
Red/blue (18:2)
Red, blue, 50% red + 50% blue,
70% red + 30% blue, 70%
red + 20% blue + 10% green
Red, blue, and white
Red, blue, and red/blue (1:1)
White and deep red/white
White
The combination of red and blue LEDs Al-Mayahi (2016)
substantially increased soluble carbohydrates,
starch, and free amino acids in shoots; induced
the synthesis of new bands of proteins
and improved the tissue of the palm
The ratio of red and blue LEDs Kwon et al. (2015)
improved shoot quality and secondary xylem
formation
The control of light quality with appropriate Reis et al. (2015)
rates of white, blue, or red can improve
the secondary metabolite content
The LED light induced changes in oxidative events Gupta and Sahoo
and influenced shoot organogenesis (2015)
LED lighting increased chlorophyll levels Vieira et al. (2015)
and number of stomata
The LEDs in TIS system located above the Wilken et al. (2014)
culture vessels and employment of additional
Table 1 (continued)
Species Morphogenic Aim of the
pathway work
illumination for banana
propagation in a standard TIS
Cymbidium hybrid Organogenesis Effects of N-acetylglucosamine
(NAG) under different
LED lights (red, green, and
blue) on organogenesis
Platycodon grandiflorum Organogenesis Effect of light spectra
on leaf morphology,
anatomy and chemical
composition using LEDs
Jatropha curcas Organogenesis Improve rooting
of regenerated shoots
Brassica napus Organogenesis The effects of different light
qualities on growth
and morphogenesis
Rubus idaeus Organogenesis To evaluate the use of LEDs
in multiplication and rooting
of raspberry ‘Batum’
and ‘Dorman Red’
Protea cynaroides Organogenesis Effect of light on growth
and morphogenesis,
and concentrations
of endogenous
phenolic compounds
Dendrobium officinale Organogenesis Effect of light on the production
of shoot, growth, and
development
Gossypium hirsutum Organogenesis Effect of light on the growth
and morphogenesis
Hybrid grapes Organogenesis Effect of light on shoot
and root growth
Doritaenopsis hort. Organogenesis Effect of light on growth
and carbohydrate
and chlorophyll contents
Tripterospermum japonicum Organogenesis Effect of light on shoot growth
Zantedeschia jucunda Organogenesis Effect of light on
‘Black Magic’ photomixotrophic
growth
Strawberry ‘Akihime’ Organogenesis Effect of light
on plantlet growth
Light type and quality of LED
Green, red, and blue
Blue, 75% blue + 25% red,
50% blue + 50% red, 25%
blue + 75% red, and red
Blue, red, 50% red + 50% blue
Blue, 75% blue + 25% red, 50%
blue + 50% red, 25% blue +
75% red, and red
Blue, green, and red and lamps grow The red LEDs contribute to increased multiplication
lux
Red, blue, and red + blue
Red, blue, 50% red + 50% blue,
67% red + 33% blue, and 33%
red + 67% blue
Red, blue, and red + blue
Red and blue
Red, blue, and red + blue
Red, 70% red + 30% blue,
red + 50% blue, and blue
Red and blue
Red, blue, and red + blue
Conclusion Reference
gassing without immersion is a recommended
setup for the micropropagation of banana
Red and green LEDs in the presence of NAG Kamal et al. (2014)
in the media enhanced PLBs growth of a
Cymbidium hybrid in vitro cultures
Blue and blue/red (3:1) light to induce higher Liu et al. (2014)
ETR, PSII, qp, and lower NPQ and produced
higher whole plant dry mass. This suggests
that blue light is probably necessary for
in vitro plantlet growth
Red LED light had additional benefits to shoot growth Daud et al. (2013)
and improved plant regeneration capacity
The use of a LED light source was good at promoting Li et al. (2013)
the differentiation, proliferation and growth;
Light B:R = 3:1 is the most suitable for in vitro rapeseed
planting growth
Rocha et al. (2013)
and rooting of shoots of raspberry ‘Batum’
and ‘Dorman Red’
The highest rooting percentage was observed in plantlets Wu and Lin (2012)
cultured under red LEDs; however, they resulted
in the lowest concentrations of endogenous phenolic
compounds
Blue or 33% red + 67% blue significantly promote shoot Lin et al. (2011)
production and increases the dry matter and the
accumulation of shoot dry matter in vitro
Red + blue light produced larger, healthier plantlets Li et al. (2010)
LIGHT QUALITY IN PLANT TISSUE CULTURE: DOES IT MATTER?
and greater biomass. Blue and red LED
was the most suitable light for growth
Plants produced the longest shoots with longer internodes Poudel et al. (2008)
and higher rooting percentage. Blue light was also
responsible for a higher number of stomata
Carbohydrate and leaf pigment biosynthesis of the plants Shin et al. (2008)
increased in plants grown under red plus blue. The
blue plus red light sources for plant production is
suggested
Plant growth was generally healthier for cultures Moon et al. (2006)
irradiated with mixed LEDs
Blue light is involved in plant height and chlorophyll Jao et al. (2005)
development control mechanism
Plantlet growth it is best at 70% red + 30% blue Nhut et al. (2003)
 BATISTA ET AL.

Red light affected the morphology rather than the growth Miyashita et al. (1997)
somnifera, showed higher chlorophyll content and higher

Tanaka et al. (1998)

Lian et al. (2002)
photosynthetic rates under blue plus red LED, when compared
to fluorescent light, or LED light of solely blue, red, distant
Reference

blue, or distant red wavelengths (Kim et al. 2004; Lee et al.

2007). Oncidium plantlets showed higher pigment content
under blue LED and higher starch content under red LED,
chlorophyll content; however, this phenotype was when compared to yellow and green LED, or fluorescent
lamps (Mengxi et al. 2011). Eucalyptus, Musa, and
Red plus blue LED is suitable for bulblet growth

Spathiphyllum showed higher growth of plantlets grown

Red light promoted leaf growth but decreased

in vitro under a 4:1 red/blue LED ratio, compared to white

light (Nhut and Nam 2010). Brassica napus showed a higher
concentration of soluble sugar and chlorophyll under a 3:1
blue/red LED ratio, compared to red LED or fluorescent
lamps (Li et al. 2013). These results show that the effects of
reversed by blue light

spectral quality on photosynthetic competence vary according

to the plant species, with LED lights being more efficient
compared to fluorescent lights in in vitro culture. In addition,
Conclusion

the combination of different color LEDs can overcome the

limitations of individual colors. New light systems are being
rate

developed to increase plant quality and productivity. For in-

stance, Shukla et al. (2017), by means of 3D printing, devel-
oped prototypes of culture pots with LED-based light sources
Light type and quality of LED

to control the color of light independently.

Finally, it should be considered that the spectral irradiance
Red, blue, and red + blue

is significantly affected by the culture vessel format and its

material characteristics. Depending on the thickness and com-
position of the glass or plastic, the vessel can transmit and/or
Red and blue

reflect more or less photosynthetic flux density (Huang and

Chen 2005).
Red

Light and gene expression in in vitro culture systems Plant

morphology of plants cultured

tissue culture is a tool with high potential for rapid multipli-

Effect of light on growth and

photoautotrophically and

cation and conservation of clean and indexed plant material. It

on bulblet regeneration

photomixotrophically

also allows for better control of several abiotic factors, such as

on plantlet growth

light quality, irradiance and photoperiod (Gupta and Jatothu

2013; Sáez et al. 2013).
Effect of light

Effect of light
Aim of the

The use of LED lights with different spectra of the

visible region, from a blue light (450–495 nm) to distant
work

red (750–850 nm), has attracted the attention of several

research groups trying to optimize growth, development,
and metabolism in plants (Li et al. 2010; Lin et al. 2013;
Golovatskaya and Karnachuk 2015). As with fluorescent
Organogenesis

Cymbidium Sleepmg BeauTy Organogenesis

Organogenesis
Morphogenic

lamps, LED light bulbs have been used for growing plants
pathway

in controlled environments, such as in vitro culture. The

more uniform distribution of light, energy efficiency, du-
rability, and spectrum control capabilities of LED lights
are some of the benefits of using those lighting devices
(Yeh and Chung 2009; Gupta and Jatothu 2013).
Table 1 (continued)

Lilium oriental hybrid

Solanum tuberosum

In recent years, several studies have confirmed the effects

‘Golden Bird’

of light quality on the production of secondary metabolites in

‘Benimaru’
‘Pesaro’

different plant species (Table 2). The light quality regulates

Species

plant growth and development by different mechanisms in-

cluding the selective activation of light receptors, such as of
 LIGHT QUALITY IN PLANT TISSUE CULTURE: DOES IT MATTER?
Fig. 2 Spectral distribution of
light-emitting diodes (LED) (a)
and fluorescent lamps (b). Dashed
lines and arrows indicate the
absorbance points of chlorophyll
a and b and carotenoids. The
absorption spectra at the bench
were recorded with a
spectroradiometer and Ocean
Optics Spectra-Suite data
acquisition software system
(Ocean Optics®, Dunedin, FL)
phytochromes by red and far-red light, cryptochromes and
phototropins by blue light, and UV-B receptors by ultraviolet
light (Chen 2006; Pedmale et al. 2016; Li et al. 2017).
Photochemical control of light quality affects in vitro gene
expression in various metabolic pathways, affecting the syn-
thesis of nucleic acids, amino acids, organic acids, sugars, and
lipid peroxidation, which are essential for cell maintenance,
cell division, respiration, and reproduction (Eckstein et al.
2012; Cepaukas et al. 2015; Li et al. 2017). In vitro changes
in explants to stress, and reorientation of their developmental
program results in the expression of protein kinases, transcrip-
tion factors, and structural genes that contribute to the adapta-
tion (Neelakandan and Wang 2012).
Many studies report on the role of light in morphogenesis
in vitro, but little is specified on the programs of gene expres-
sion during the development of shoots, roots, callus, and
somatic embryos. Raspor et al. (2012) investigated how al-
tered levels of endogenous cytokinins in transgenic potato
affected the tuberization process. Continuous darkness is a
strong inducer of tubers in potatoes and was used as an induc-
er treatment, and a 16-h photoperiod with fluorescent light
was the non-inducing treatment. The effect of the light quality
in order to modulate endogenous cytokinins levels is well-
known and has been demonstrated in several species such as
Rosa hybrida and Chlorella minutissima (Stirk et al. 2014;
Roman et al. 2016).
Eckstein et al. (2012) investigated the influence of irradi-
ance and sugar content on the growth and photosynthetic ap-
paratus of A. thaliana. The authors used 1 or 3% (w/v) sucrose
or glucose, along three photosynthetic flux densities (PFDs):
25, 100, and 250 μmol m−2 s−1 provided by fluorescent lamps.
Plants without added sugar were used as controls. The expres-
sion of genes related to photosynthesis, chloroplast
reallocations induced by light, and xanthophyll cycle activity
were analyzed. Different culture conditions (irradiance and
sugar concentration) led to varied phenotypes. The presence
of sugar was considered essential for adequate development.
The sugar concentrations tested (1 or 3%) did not have a
significant effect. However, the plants developed different
phenotypes depending on the irradiance. Plants grown under
low irradiance had smaller leaves, with long petioles extended
towards the light. Plants grown both in medium and high
irradiance were well-developed and with higher growth. A
strong positive regulation of sucrose and light was observed
in the CAB genes encoding chlorophyll a/b binding proteins.
The highest expression was achieved with 1% (w/v) sucrose
Table 2 Secondary metabolites stimulated by different qualities of light provided by LED lamps in in vitro plant tissue culture

Species Tissue/explant Light conditions tested Compound(s) stimulated Light quality that acted as elicitor Reference

Hyptis suaveolens Nodal segments Blue, red, yellow, white, green, Volatile compounds The major compounds sabinene, Andrade et al. (2017)
and red/blue (4:10; 10:4, and 7:7) LEDs β-pinene, β-phellandrene, E-caryophyllene,
germacrene and bicyclogermacrene increased
or decreased depending on the light spectral
Plectranthus amboinicus Apical segments Red, blue, and blue plus red Carvacrol Blue LED Silva et al. (2017)
(1:2.5, 2.5:1, and 1:1) LEDs and white γ-Terpinene Red, blue plus red (1:2.5, 2.5:1, and 1:1) LED
fluorescent p-Cymene White fluorescent
Swertia chirata Shoot Blue, red, blue/red (1:1), blue/red Total phenolics, flavonoids Blue LED Gupta and Karmakar
(3:1), blue/red (1:3) LEDs and white fluorescent and flavonols (2017)
Lippia alba Plantlets (aerial part) White, blue/red LEDs, and white fluorescent Essential oil profile The use of white or blue/red LEDs changed Batista et al. (2016)
the essential oil profile of Lippia alba
chemotypes
even shifting the major compound for the three
chemotypes analyzed
Peucedanum japonicum Callus and tissue Blue/green/red (28:43:29); blue/green/red Chlorogenic acid Blue/red/far-red LED (57:43:37) Chen et al. (2016)
culture plants (8:46:46); blue/red (16:84); blue/green/red
(17:9:74); blue/red/far-red (57:43:37);
red (100)/blue (100); far-red (100).
The numbers in parentheses correspond
to LED chips used for a particular lid
Schisandra chinensis Shoot-differentiating Far-red/red/blue/UV-A/white light Dibenzocyclooctadiene Blue LED Szopa and Ekiert (2016)
callus cultures Lignans
Phenolic acids
Achillea millefolium Leaves Blue, red, green, white LEDs Monoterpenes Red LED Alvarenga et al. (2015)
BATISTA ET AL.

and white fluorescent Sesquiterpenes Green LED

Photosynthetic flux density: Monoterpenes 47 and 69 μmol m−2 s−1
13, 27, 35, 47, 69 μmol m−2 s−1 Sesquiterpenes 13 μmol m−2 s−1
Aquilaria agallocha Shoots Red and far-red LEDs Cucurbitacins Red LED (cucurbitacin content increased Kuo et al. (2015)
as red light exposure increased)
Panax vietnamensis Plantlets Blue, green, yellow, red, white, red plus blue Ginsenoside Rg1 and Rb1 White fluorescent Nhut et al. (2015)
(90:10; 80:20; 70:30; 60:40; 50:50; Ginsenoside MR2 Red plus blue LED (20:80)
40:60; 30:70; 20:80; 10:90) LEDs
and white 3 U compact fluorescent and
fluorescent
Rehmannia glutinosa Shoots Red, blue LEDs, and white fluorescent Total phenols Blue LED Manivannan et al. (2015)
Scrophularia Adventitious shoot Red, blue LEDs, and white fluorescent Harpagoside, (−)-epicatechin, Blue LED Jeong and Sivanesan
takesimensis caffeic acid, sinapic acid, (2015)
ferulic acid
Trans-cinnamic acid Blue and red LED
(−)-gallocatechin Red LED
Ellagic acid Red LED and White fluorescent
Chicoric acid White fluorescent
Oryza sativa cv. Ilmi Leaves Blue, green, red, white (mixture of blue, Amino acids, organic acids, Blue LED Jung et al. (2013)
green, and red lights) LEDs, and a shade fatty acids, and flavonoid
condition (no light treatment) glycosides
Panax ginseng Adventitious roots Red, blue, and white fluorescent Blue LED Park et al. (2013a)
 LIGHT QUALITY IN PLANT TISSUE CULTURE: DOES IT MATTER?

and in high irradiance, leading to an increased CAB expression

Shohael et al. (2006)

in conjunction with increased photosynthetic activity.

Park et al. (2013b)

Shin et al. (2003)

The expression of eight genes that encode NADPH oxidase

Yu et al. (2005)
enzymes were studied in vitro during shoot development of
Reference

Malus domestica Borkh. cv. Golden Delicious and Gala

grown on MS medium (Murashige and Skoog 1962) under
fluorescent lamps for 42 d (Cepauskas et al. 2016). The work
demonstrated an increase in lipid peroxidation, a symptom of

decreased with white fluorescent, red and

reactive oxygen species (ROS) that correlated to some

Blue plus far-red LED (betalain production

NADPH oxidase expression patterns.
Light quality that acted as elicitor

Li et al. (2017) analyzed the differential gene expression of

Blue LED and white fluorescent

in vitro grown plantlets of V. vinifera ‘Manicure Finger’ sub-

Red LED and white fluorescent

Red LED and white fluorescent

jected to white, blue, red, and green LED light. Specific light
treatments were associated with the expression of a large num-
Blue/red/white LED

ber of genes, including those involved in glucose, starch, and

White fluorescent

White fluorescent

red plus LED) sucrose synthesis. These genes were regulated in a manner
that could explain the increase in starch and soluble carbohy-
Red LED

drate content in plants. The blue light positively regulated

microtubule synthesis-related genes, serine carboxypeptidase,
chlorophyll synthesis, sugar degradation, and a large number
of resistance-related genes in grape. Blue light promoted leaf
Compound(s) stimulated

Betalain (betacyanin and

growth, stimulated chlorophyll synthesis and chloroplast de-

velopment, increased the chlorophyll a/b ratio, and decreased
Total phenolic acids,

Total flavonoids and

Eleutheroside E1

proportion of carbohydrate to proteins, and inhibited stem and

and β-amyrin

Eleutheroside B
Eleutheroside E
α-tocopherol

betaxanthin)
chlorogenic
Total phenols
Anthocyanin

root elongation. The red and green light seems to impose

Ginsenoside

“shade stress” on the plantlets and high regulation of defense

acid

gene expression. Interestingly, 1601 genes were differentially

expressed, and the number of genes expressed in the green
light were more than double the genes expressed in blue and
red light.

Light and cell wall biosynthesis The cell wall is composed of

red plus far-red (1:1 PFD) LEDs
(1:1 photon flux density, PFD),
white fluorescent and darkness
(1:1) LEDs, white fluorescents

an abundance of polysaccharides such as cellulose, hemicel-

blue plus far-red (1:1 PFD),

lulose, lignins, and pectins. In several cell types, after the

Red, blue, blue plus far-red
Red, blue, and white LEDs
Light conditions tested

growth and extensibility phase, the process of lignin deposi-

and white fluorescent
Red, blue, red plus blue

Red, blue, red plus blue

tion is initiated (Carpita and Gibeaut 1993; Didi et al. 2015).

(1:1 PFD) LEDs,

Biosynthesis of the cell wall requires the action of several

and darkness

cellular compartments with functions not fully understood.

Cellulose, for example, is synthesized in the plasma mem-
brane, whereas hemicelluloses, pectins, proteins, and glyco-
proteins, are synthesized in Golgi bodies and transported to
the cell wall (Oikawa et al. 2013). Lignin is synthesized in the
Somatic embryos
Tissue/explant

cell wall by the polymerization of the monolignols catalyzed

Hairy roots

Hairy roots

by peroxidases and laccases (Wang et al. 2016). The develop-

Plantlets

ment of the cell wall is complex and heterogeneous, and in-

fluenced by interactions with symbionts, pathogens, and the
environment (Carpita and Gibeaut 1993; Scheller and
Beta vulgaris cv. Detroit

Ulvskov 2010; Didi et al. 2015).

Table 2 (continued)

The biosynthesis, chemistry, mechanics, and cell wall

Perilla frutescens
Eleutherococcus

Panax ginseng

genes have attracted extensive research. In vitro culture may

senticosus

dark red

be a tool to study lignification. For instance, Tobimatsu et al.

Species

(2011) analyzed the copolymerization of lignin monomers by

peroxidases in Zea mays L. (corn) suspension cells and
 BATISTA ET AL.
extended the opportunity to study the transport and deposition
of monolignols in other biological systems, in a variety of
in vitro studies intended to understand lignification using
fluorescence-tagged monolignols.
Spectral quality of light affects growth and morphogenesis
(Poudel et al. 2008; Ouzounis et al. 2014; Alvarenga et al.
2015; Ahmad et al. 2016; Hung et al. 2016; Li et al. 2017;
Manivannan et al. 2017), and consequently light regulates the
gene expression of cell wall biosynthesis. Ma et al. (2001)
cultivating A. thaliana in vitro in different light qualities, in-
cluding far-red light, red, and blue, reported more than 26
cellular pathways that were regulated in a coordinated way
by light. Among the light-regulated pathways, the cell wall
biosynthetic and phenylpropanoid pathways were upregulat-
ed. In this study, genes encoding hydrolytic cell wall enzymes
were downregulated under white light. The enzymes
xyloglucan endotransglycosylase, expansin, and endo-1,4-β-
D-glucanase were correlated with cell wall loss (Ma et al.
2001). Therefore, the proportion of lignocellulosic biomass
varies in composition, and may vary in response to different
abiotic factors, such as light quality (Zhong and Ye 2015).
Light effects occur via the phenylpropanoid pathway
(OuYang et al. 2015; Ahmad et al. 2016), and lignins are
derived from multiple branches of the phenylpropanoid bio-
synthesis pathways (OuYang et al. 2015). Responses to light
quality or intensity occur at the transcriptional level of genes
and enzymes directly involved with the cell wall, such as
phenylalanine ammonia lyase (PAL), which is a key enzyme
in the phenylpropanoid pathway. For Prunus sp. grown in vitro
under fluorescent light, PAL expression increased, which led
to the increase of soluble phenolic compounds, and a reduc-
tion in lignification (Pina and Errea 2008). For Picea abies
(L.) Karst, the PAL gene was upregulated under blue LED
(OuYang et al. 2015). In Dianthus caryophyllus cultured
in vitro, red LED significantly increased the activity of the
PAL enzyme and lower activity levels were found using fluo-
rescent light (Manivannan et al. 2017). The authors did not
evaluate effects on cell wall chemistry, but it is possible to
speculate that it affected lignin and/or lignin monomers caus-
ing pleiotropic effects in the other lignin biosynthetic pathway
genes (Ralph et al. 2004). In Triticum aestivum, red LED
increased the total lignin content (Dong et al. 2014b).
Light regulates the modulation of cellulose, the largest
structural component of the cell wall. Light acts on the activa-
tion of cellulose synthase complexes (CSCs) (composed of
multiple domains in the form of rosettes). Cellulose synthesis
is performed on the plasma membrane as well as mechanism
of callose synthesis. The CSC complex is regulated by phyto-
chrome B (phyB). In the dark, the activity of the subunits of
CSC are reduced, and the process is reversed through the
return to light and photochemical activation of phyB, and
the interaction rate between CSCs and microtubules is main-
tained in a fine-tuning (Bischoff et al. 2011). As demonstrated
in Gossypium hirsutum in vitro, the fibers of an ovule cultured
under light presented higher concentrations of carbohydrates,
cellulose, and expression of cellulose biosynthesis genes com-
pared to cultures grown in the dark (Qian et al. 2016a).
However, the combination of red and white LEDs, with low
luminous intensity during the grain filling stage increased cel-
lulose and hemicellulose, and reduced lignin in T. aestivum
(Dong et al. 2014a). Directly or indirectly, several studies
identify the effects of light quality on morphogenesis and cell
wall modulation (Table 3).
Light and production of pigments and bioactive compounds
Light influences plant development, and defense against her-
bivores, pathogens, and oxidative stress through regulation of
secondary metabolites biosynthesis (Bian et al. 2015; Shimizu
2016; Legris et al. 2017; Viczián et al. 2017). Light quality
has been involved in the regulation of biosynthetic pathways
of pigments (carotenoids such as lutein and β-carotene), and
bioactive compounds such as phenols (phenolic acids, antho-
cyanins, and flavonoids), glucosinolates, and terpenoids
(Ballaré 2014; Darko et al. 2014; Huché-Thélier et al. 2016;
Demotes-Mainard et al. 2016; Taulavuori et al. 2017). This
regulation is made through the selective activation of different
photoreceptors (Ballaré 2014; Huché-Thélier et al. 2016;
Demotes-Mainard et al. 2016), gene modulation, transcription
factors, and enzyme expression (Maier et al. 2013; Leivar and
Monte 2014; Bou-Torrent et al. 2015; Seiler et al. 2017).
Many studies analyzed the influence of light quality on the
production of secondary metabolites in plants (Arena et al.
2016; Szopa and Ekiert 2016; Gupta and Karmakar 2017).
These studies focused on enhancing the nutritional value of
leafy greens (Kopsell et al. 2014; Chen et al. 2016; Amoozgar
et al. 2017), sprouts (Carvalho and Folta 2014; Liu et al. 2016;
Qian et al. 2016b), and on increasing the medicinal potential
(Alvarenga et al. 2015; Ellins et al. 2015; Lowe et al. 2015;
Batista et al. 2016). However, the majority of these studies are
still restricted to ex vitro culture conditions, but in vitro culti-
vation has many advantages, such as fewer external factors,
control of the cultivation environment, and the possibility to
use elicitors for the metabolic pathway of interest (Hussain
et al. 2012; Murthy et al. 2014).
One of the difficulties in the literature available on light
quality in in vitro plant cultivation is the unclear methodology,
or the lack of specifications of the type of lamp used (fluores-
cent or LED), as observed by Ahmad et al. (2016), Kawka
et al. (2017), and Pedroso et al. (2017). Methodological in-
consistency hinders the reproducibility and application of that
methodology to other species that produce similar
metabolites.
There has been an increase in the number of studies that
explore the advantages of the in vitro culture method for the
production of secondary metabolites and most of them used
fluorescent lamps (Shahzad et al. 2017; Shukla et al. 2017).
Table 3 The effect of light (quality and/or quantity) on the formation of the cell wall

Species Effect of light (quality and/or quantity) Effect on cell wall Genes involved In vitro Evaluation assay Reference
or ex
vitro

White fluorescent tubes, light intensity of Rescued the biosynthesis of syringyl lignin-units in the COMT In vitro Maüle reagent and the determination of Ho-Yue-Kuang
Arabido- 110 mE m−2 s−1 comt-1 mutant in vitro the monomers lignin (H, G, S, and et al. (2016)
psis 5-OH G)
Gossypium Light intensity of 100 μmol m−2 s−1 (12 h Under light, there were higher concentrations of EXP1, SuSy, In vitro Cellulose contents Qian et al.
hirsutum luz) carbohydrates, cellulose, and expression of cellulose β-1,3-glucana- (2016a)
biosynthesis genes se, CelA1, and
CelA2
Ultraviolet (UV)-B radiation Reduction of total PAL activity and lignin content PAL Ex vitro Total lignin content—acetyl-bromide Zhang et al.
Arabido- method (2015)
psis
Triticum LEDs with different intermittent spectra of Continuous lighting promoted lignin content increase; – Ex vitro Acid detergent lignin Dong et al.
aestivum red and white however, the light/dark cycle is involved with the lig- (2015)
L. cv. nin content decrease
‘Dwarf’
Long-day conditions (16/8 h light/dark 6% reduction in total lignin amount as compared to the ATR2 (CYP450 Ex vitro Wiesner and Mäule staining, Sundin et al.
Arabido- cycle, 120 μE s−1 m−2) wild type gene) saccharification, lignin content via (2014)
psis acetyl bromide, monomers lignin and
cellulose
Triticum LED devices with different light intensities The decrease in light intensity during grain filling – Ex vitro Hemicellulose, cellulose, and lignin Dong et al.
aestivum spectra of red and white increased cellulose and hemicellulose and reduced (2014a)
lignin
Triticum Combinations between blue and red LEDs Red LED increased the lignin content, and when – Ex vitro Hemicellulose, cellulose, and lignin Dong et al.
aestivum combined, caused a reduction in lignin content (2014b)
LIGHT QUALITY IN PLANT TISSUE CULTURE: DOES IT MATTER?

Nicotiana Ultraviolet (UV)-B radiation
attenuata
Prunus spp. White fluorescent tubes, photosynthetic
flux density of 17 μmol m−2 s−1
Nicotiana Under varying luminous intensity and
tabacum photoperiod (3.000 lx photoperiod
16/8 h) and (14.000 lx photoperiod
14/10 h)
Lower total lignin content and lower S/G ratio of trans- CAD Ex vitro Lignin content (Klason) and composi- Kaur et al.
genic to wild type tion (thioacidolysis and NMR) (2012)
During grafting cicatrization, there was an increase in PAL In vitro Phloroglucinol-HCl (Wiesner) Staining Pina and Errea
PAL expression, which led to the increase of soluble (2008)
phenolic compounds and reduction in lignification
Caused variations in total lignin content and S /G ratio, CCR and COMT Ex vitro Wiesner and Mäule staining, Lignin Pinçon et al.
both in the control and transgenic plants content (Klason) and composition (2001)
(thioacidolysis)
 BATISTA ET AL.
Different light qualities provided by fluorescent tubes have
been used to stimulate the biosynthesis of antioxidant second-
ary metabolites in callus and shoot cultures of various species
(Tariq et al. 2014; Fazal et al. 2016; Szopa and Ekiert 2016;
Gazolla et al. 2017; Kawka et al. 2017). However, the spec-
trum of light emitted by fluorescent lamps was not specific,
cannot be controlled and, like intensity, its stability decreased
over time (Darko et al. 2014; Taulavuori et al. 2017).
Currently, LED lights have been used to circumvent prob-
lems related to fluorescent lamps and to optimize in vitro plant
development (Gupta and Jatothu 2013; Shukla et al. 2017).
However, the influence on secondary metabolism is still
underexplored.
The use of monochromatic or composite LED lights with
different spectral qualities may be a tool for the induction of
bioactive metabolites and pigments of interest in in vitro cul-
ture (Table 2). Studies have shown that the use of LED lights
was more effective than fluorescent lamps in the growth
rooms. Blue LED lights were most efficient in the elicitation
of phenolic compounds (Jung et al. 2013; Jeong and
Sivanesan 2015; Manivannan et al. 2015; Gupta and
Karmakar 2017; Silva et al. 2017). These compounds also
increased with the use of red LED lights (Jeong and
Sivanesan 2015; Manivannan et al. 2015; Shohael et al.
2006). The use of LED lamps also interfered in the composi-
tion of the essential oils of medicinal plants such as Achillea
millefolium L. (Alvarenga et al. 2015), Lippia alba (Batista
et al. 2016), and Hyptis suaveolens (Andrade et al. 2017).
However, phytochemical biosynthesis under the influence of
light quality may differ according to the species and metabo-
lite studied (Shimizu 2016). For example, some ginsenosides
had a higher production in white fluorescent light (Yu et al.
2005; Nhut et al. 2015). The majority of in vitro studies using
LED light primarily impacted the production of phenolic com-
pounds and terpenoids (Table 2). However, light quality also
influenced the production of alkaloids (Zheng et al. 2012; Liu
et al. 2015; Matsuura et al. 2016) and pigments (Tuan et al.
2013; Reis et al. 2015; Qian et al. 2016b). Additional studies
that explore the potential of LED lighting and its influence on
secondary metabolism are necessary because LED properties
provide more control of in vitro light conditions. This type of
approach will contribute to understanding the biosynthesis
pathways of compounds of interest and will enable the devel-
opment of new biotechnological techniques, and commercial
and pharmacological products.
Light and in vitro propagation of woody species Light qual-
ity, quantity, and photoperiod can affect woody plant growth
and development. There is extensive data on the effect of light
quality on in vitro herbaceous species, while there is less data
concerning woody species. One reason may be the reduced
effects of light quality on woody species compared to that of
herbaceous species (Morini and Muleo 2003). Many studies
of woody plant growth are based on conventional light
sources such as fluorescent light, and especially cool white
light. Cool white fluorescent light has been reported in the
in vitro propagation of Eucalyptus (Oberschelp and
Gonçalves 2016; Oliveira et al. 2017), Quercus ilex
(Martínez et al. 2017), Castanea hybrid (Cuenca et al.
2017), Chamaecyparis thyoides (Ahn et al. 2017), and
Fraxinus nigra (Lee and Pijut 2017). Cool white fluorescent
lamps have been widely used in tissue culture rooms.
In most experiments, light quality studies revealed that the
application of a light spectrum more active than white light
increased micropropagation efficiency (Gupta and Jatothu
2013). In some studies with woody species, the manipulation
of light quality was applied with fluorescent tubes in combi-
nation with filters, to provide the various qualities of light
(Muleo and Morini 2006; Iacona and Muleo 2010; Ruedell
et al. 2013; Ruedell et al. 2015; Gazolla et al. 2017).
However, there may be some imprecision in the control of
light quality (Tisserat et al. 2000). The use of LED lights
allows specific spectra to regulate the photosynthetically and
photomorphogenic responses required for the cultivation of
each species in vitro (Gupta and Jatothu 2013). Experiments
comparing LED lights with fluorescent tubes must be done to
verify the reproducibility of the anatomical and morphological
changes (Macedo et al. 2011). Studies using LED lamps as
light sources during ex vitro cultivation of tree species resulted
in improved plant growth and metabolic activity (Arena et al.
2016; Smirnakou et al. 2016, 2017). It is possible that similar
results could be observed with in vitro cultivation of these
species.
In order to reduce costs and to improve production efficien-
cy and quality of the micropropagated woody plants, there is a
need for an efficient light source. The utilization of a more
versatile and efficient light source for in vitro plant regenera-
tion and growth can offer new and important possibilities to
achieve success in commercial micropropagation (Monteuuis
2015). The in vitro propagation is still a stressful procedure for
plants and it has some limitations. The high cost of produc-
tion, the need for adaptation of protocols, and little investment
for development and innovation in the in vitro production
process are some factors that hinder large-scale application
(Kaur and Sandhu 2015; Shukla et al. 2017; Nunes et al.
2018). In addition, plants with limited survival capacity are
often produced after their transfer to ex vitro conditions
(Chandra et al. 2010; Dias et al. 2014). Thus, the development
of new in vitro environmental control systems is necessary to
overcome the shortcomings for large-scale in vitro production
of high quality plants (Gago et al. 2014; Arencibia et al. 2017;
Shukla et al. 2017). A great number of quality woody plants
needs to be produced at a low cost to make the
micropropagation viable.
The LED for plant growth in a controlled environment has
emerged as an attractive alternative technology, with
 LIGHT QUALITY IN PLANT TISSUE CULTURE: DOES IT MATTER?
expectations to increase productivity and is attracting consid-
erable interest because of its vast potential for commercial
application (Massa et al. 2008; Yeh and Chung 2009; Bian
et al. 2015; Yeh et al. 2015). However, even with the recent
technological advancement of LED lights, few studies have
been carried out with woody species to investigate their effects
on in vitro culture (Table 4).
In Populus euramericana, a combination of red and blue
light produced the highest percentage of shoot regeneration
compared to monochromic or fluorescent light. However,
light influenced morphogenesis in a genotype-dependent
manner (Kwon et al. 2015). Red wavelengths provided by
LED lamps during the germination steps of Pinus taeda so-
matic embryos, resulted in higher frequencies of somatic em-
bryo germination and conversion than white light (Merkle
et al. 2005). Positive effects of red LED light were also dem-
onstrated in germination of Pinus densiflora somatic embryos.
Intensity, spectral range, photoperiod, and type of light source,
including LED lights, can be explored for some woody plants
in the future for tissue culture, secondary metabolite produc-
tion, and other goals (Silva et al. 2017).
The use of technology for these plants is still at a nascent
phase of development and there is still a tremendous opportu-
nity for future exploration of the great majority of woody
species. This initial research for LED applications is a stimu-
lus for the development of LED-based lighting systems for
physiology and plant development experiments. Therefore,
individual experiments, evaluating the effects of LED lighting
Table 4 Summary of the use of LED lighting in in vitro propagation of woody species
Species Light quality, quantity, and photoperiod/spectrum Regeneration system/
Acer saccharum No light (dark); red and blue LED (14% blue +
7% green-yellow + 79% red LED (450, 660,
and 625 nm); Full spectrum light LED (25%
blue + 37% green-yellow + 38% red)
Vaccinium corymbosum Cool white fluorescent; 100% red LEDs
(660 nm); 80% red + 20% blue LEDs; 50%
red + 50% blue LEDs; 100% blue LEDs
(460 nm).
Populus euramericana Fluorescent light; 100% red LED (650 nm); 70%
red + 30% blue (440 nm) LEDs; 50% red +
50% blue LED; 70%
red + 20% blue + 10% green (510 nm) LEDs
Pinus Fluorescent light; 100% red LED (660 nm);
densiflora 100%
blue LED (450 nm), 50% red + 50% blue;
50% red + 50% far-red (730 nm)
Pinus densiflora Fluorescent light;
100% red LED (660 nm); 100% blue LED
(450 nm); 50% red + 50% blue LEDs; 50% red
+ 50% far-red LEDs (730 nm)
Pinus taeda, White fluorescent; red LED (600–700 nm); blue
P. elliottii and LED (400–500 nm)
P. palustris
are necessary to predict the large-scale applicability in in vitro
propagation.
Light quality in photomixotrophic and photoautothrophic
systems, and natural illumination in in vitro propagation
Photosynthesis is the key metabolic pathway for plant growth
and development but may be limited during many in vitro
plant culture procedures. The optimization of an in vitro envi-
ronment includes balanced control of physical, chemical, and
biological parameters to create artificial conditions for cells
and organs (Kania and Giacomelli 2000).
The standard use of closed plant culture vessels to control
microbial contamination creates a limitation on the CO2 avail-
ability in vitro. However, it was not until about 20 yr ago that
Kozai et al. (1992, 1997) stated that the heterotrophic or
photomixotrophic characteristics of conventional in vitro plant
cultures is the main cause for a deficient photosynthetic abil-
ity, and low or negative net photosynthetic rates.
For more sustainable inputs and to increase the economic
efficiency of traditional tissue cultures, the advance of new
technologies for plant in vitro micropropagation have been
more focused on physical environmental factors than on bio-
logical factors, to increase plantlet growth and multiplication,
and exploit their genetic background (Kirdmanee and
Mosaleeyanon 2000). The advent of liquid-based systems
and automatic-based control to monitor the environmental pa-
rameters such as light, temperature, CO2, and ethylene levels,
among others, within vessels, have significantly contributed to
yield improvements in in vitro propagation. Bioengineering of
Morphogenetic Reference
pathway response
Organogenesis (axillary Shoot growth Singh et al. (2017)
bud proliferation)
Organogenesis (axillary In vitro shoot proliferation Hung et al. (2016)
bud proliferation) and ex vitro root
formation
Organogenesis (axillary Shoot regeneration and Kwon et al. (2015)
bud proliferation) elongation
Somatic embryogenesis Germination of somatic Kim et al. (2015)
embryos
Somatic embryogenesis Germination of somatic Kim and Moon (2014)
embryos
Somatic embryogenesis Germination and Merkle et al. (2005)
conversion of
somatic embryos
and rooting
Table 5 Summary of trials related to the improvement of in vitro photosynthesis by means of a more efficient use of light sources

Plant species Tissue culture support Air quality (CO2) Light quality Sucrose Approach Reference

Populus spp. Temporary immersion bioreactors 0.4 MPa Natural light/fluorescent lamps Reduced Micropropagation Arencibia et al. (2017)
100 μmol m−2 s−1
Vaccinum corymbosum Temporary immersion bioreactors 0.4 MPa Natural light/fluorescent lamps Reduced Micropropagation Arencibia et al. (2013a)
60 μmol m−2 s−1
Rubus spp. Temporary immersion bioreactors 0.4 MPa Natural light/fluorescent lamps Reduced Micropropagation Arencibia et al. (2013b)
80 μmol m−2 s−1
Saccharum spp. Temporary immersion bioreactors 0.4 MPa Natural light/fluorescent lamps Reduced Secondary Yang et al. (2010)
110 μmol m−2 s−1 metabolites
Phalaenopsis Semisolid medium. Glass vessel 1000 μmol mol−1 Fluorescent lamps Free Micropropagation Yoon et al. (2009)
60 μmol m−2 s−1
Saccharum spp. Temporary immersion bioreactors 0.4 MPa Natural light/fluorescent lamps 30 g L−1 Secondary Arencibia et al. (2008, 2012)
110 μmol m−2 s−1 metabolites
Ipomoea batatas Liquid medium, Florialite 1.8 × 10−3 (v/v) Fluorescent lamps Free Micropropagation Yulan and Kozai (2006)
Magenta-type vessel 200 μmol m−2 s−1
Limonium latifolium Semisolid medium, Florialite 1500 μmol mol−1 Fluorescent lamps Free Micropropagation Xiao and Kozai (2006)
Magenta-type vessel 100 μmol m−2 s−1
Cocos nucifera Semisolid medium. Magenta-type vessel – Natural light 45 g L−1 Micropropagation Talavera et al. (2005)
250 μmol m−2 s−1
Ananas comosus Temporary immersion bioreactors 2000 μmol mol−1 Fluorescent lamps Reduced Micropropagation González-Olmedo et al.
BATISTA ET AL.

275 μmol m−2 s−1 (2005)

Musa AAB Temporary immersion bioreactors During acclimatization a. Fluorescent lamps a. 30 g L−1 a. Multiplication Aragón et al. (2005)
375 μmol mol−1 30–40 μmol m−2 s−1
b. 2000 μmol m−2 s− b. Free b. Acclimatization
Samanea saman Vermiculite and liquid medium. 1000 mmol mol−1 Fluorescent lamps Reduced Micropropagation Mosaleeyanon et al. (2004)
Glass vessel 100 μmol m−2 s−1
Solanum Semisolid medium. Magenta-type vessel 4000 μmol mol−1 Fluorescent lamps Reduced Organogenesis Van Le et al. (2001)
lycopersicum 500 μmol m−2 s−1
Ananas comosus Temporary immersion bioreactors – Fluorescent lamps 30 g L−1 Elongation, Escalona et al. (2003)
225 μmol m−2 s−1 micropropagation
Paulownia fortunei Semisolid medium Magenta-type vessel 1400–1500 μmol mol−1 Fluorescent lamps Free Micropropagation Khan et al. (2003)
125 μmol m−2 s−1
Eucalyptus Semisolid medium, Florialite 1400–1500 μmol mol−1 Fluorescent lamps Free Micropropagation, Khan et al. (2002)
tereticornis Magenta-type vessel 125 μmol m−2 s−1 failed rooting
Musa spp. Semisolid medium. Magenta-type vessel 1340 μmol mol−1 Fluorescent lamps Free Micropropagation Nguyen and Kozai (2001)
200 μmol m−2 s−1
Myrtus communis a. Semisolid medium; b. Vermiculite and 600 cm3 m−3 Fluorescent lamps 30 g L−1; Micropropagation Lucchesini et al. (2001)
liquid medium. Glass vessel 150 μmol m−2 s−1 15 g L−1, free
 LIGHT QUALITY IN PLANT TISSUE CULTURE: DOES IT MATTER?
processes and environmental controls, such as light quality
and quantity, should be incorporated into the technological
developments related to high-throughput plant
micropropagation systems (Kania and Giacomelli 2000), no-
tably those liquid-based systems such as temporary immersion
bioreactors (TIBs).
In parallel, the design and construction of the plant growth
and multiplication chambers may use sunlight during the year,
depending on the geographical site, resulting in an improve-
ment or increase of in vitro photosynthesis, which has been
demonstrated in Saccharum spp., Vaccinium corymbosum,
Robus spp., and Populus spp. (see Table 5).
In this sense, biotechnologists could efficiently handle and/
or control all the variables that are related to photosynthesis,
that is, carbon dioxide, water, and sunlight as a source of
energy. As a consequence of the integrated management of
these environmental variables, plants can improve the process
of in vitro photosynthesis by inducing a more efficient
photomixotrophic stage.
Concluding Remarks and Future Directions
Light conditions affect in vitro morphogenetic responses of
cultured cells and tissues, and irradiance used in the protocols
and the spectral quality are not well specified. LED technolo-
gy has evolved rapidly and LED lights for plant growth in a
controlled environment have emerged as useful technology to
increase productivity for commercial application. In plant cell,
tissue, and organ culture, a growing demand for using these
light devices is expected because they not only last longer and
offer better lighting conditions, but also generate fewer waste
products. The environmental issues and concerns regarding
fluorescent bulbs, which generate a lot of waste products from
their auxiliary devices such as starters and ballasts, are well-
known, in addition to the presence of phosphor and toxic
mercury. The implementation of scaled-up mass propagation
in liquid systems in bioreactors, and in vitro photoautotrophic
systems are well supported by LED technology that provides
adequate PARs in a wide range of choices for spectra qualities.
The potential of LED technology for secondary metabolite
production is tremendous and this approach will contribute
to a better understanding of the biosynthesis pathways of com-
mercially important bioactive compounds.
Acknowledgements The co-authors are acknowledged for promptness in
accepting the invitation to collaborate in this review. The Brazilian spon-
soring Agencies, namely FAPEMIG, CNPq, and CAPES are acknowl-
edged for financial support with grant proposals and scholarships for
post-docs and post-graduate students from Agrochemistry, Forestry,
Botany, and Plant Physiology Postgraduate Programs from Federal
University of Viçosa. Also, “Innovation and Competitiveness Fund”
(FIC), Regional Government of Maule, Chile, is acknowledged for finan-
cial support for Dr. A. D. Arencibia.
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