Effect of Red-, Far-Red- and Blue-Light-Emitting Diodes on In Vitro ...

<strong>Effect</strong> <strong>of</strong> <strong>Red</strong>-, <strong>Far</strong>-<strong>Red</strong>- <strong>and</strong> <strong>Blue</strong>-<strong>Light</strong>-<strong>Emitting</strong> <strong>Diodes</strong> on In Vitro
Growth <strong>of</strong> Ficus benjamina
S. Werbrouck <strong>and</strong> H. Buyle
Laboratory for Plant Biotechnology
Ass. Faculty <strong>of</strong> Applied Bioscience
Engineering
University College Ghent
Ass. Ghent University
Schoonmeersstraat 52, 9000 Gent
Belgium
D. Geelen <strong>and</strong> M.C. Van Labeke
Laboratory for In Vitro Biotechnology
Faculty <strong>of</strong> Bioscience Engineering
Ghent University
Coupure links 653, 9000 Gent
Belgium
Keywords: LED, micropropagation, elongation, callus, leaf, Ficus benjamina
Abstract
The effects <strong>of</strong> monochromatic blue, red <strong>and</strong> far-red light from commercial
available light-emitting diode modules on the in vitro growth <strong>of</strong> Ficus benjamina
‘Exotica’ were compared with the effect <strong>of</strong> fluorescent light. The plants were micropropagated
on a basal medium with 0.5 mg/L IAA <strong>and</strong> 2 mg/L BA. Our study
showed that monochromatic blue, red <strong>and</strong> far-red <strong>and</strong> their combinations are
suitable to manipulate the number <strong>of</strong> shoots, shoot length, shoot/callus weight ratio
<strong>and</strong> leaf length/width ratio in Ficus benjamina. In general, the presence <strong>of</strong> blue light
had a stimulating effect on the number <strong>of</strong> shoots, but also reduced shoot length.
Grown under red light, the explants produced less but more elongated shoots. <strong>Blue</strong>
light stimulated callus growth even in the presence <strong>of</strong> red light. <strong>Far</strong>-red had a
negative effect on biomass production in general with a reduction in total number <strong>of</strong>
shoots <strong>and</strong> both shoot cluster <strong>and</strong> callus weight. Under red <strong>and</strong> far-red light
conditions, the morphology <strong>of</strong> the leaves was different from blue light conditions
with an increase in relative leaf length as the most prominent characteristic.
INTRODUCTION
Photosynthesis involves excitation <strong>and</strong> electron transfer, energized by the
absorption <strong>of</strong> photons in the wavelength range <strong>of</strong> 400-700 nm. A number <strong>of</strong>
developmental effects, such as elongation, germination <strong>and</strong> flower induction are
controlled by radiation in a limited range <strong>of</strong> wavelength b<strong>and</strong>s: blue, red <strong>and</strong> far-red. The
trend <strong>of</strong> brighter intensity <strong>and</strong> lower price has made LEDs a promising light source for
plant tissue culture. The currently available blue, red <strong>and</strong> far-red LED technology allows
full control <strong>of</strong> the light quality for tissue culture cultivation <strong>and</strong> facilitate the evaluation <strong>of</strong>
effects <strong>of</strong> high photon flux <strong>and</strong> peak spectral output on in vitro plantlet growth. Control <strong>of</strong>
plantlet morphology is important in micropropagation to obtain desired plantlet quality at
different in vitro stages. For instance, elongated internodes are practical to excise node
cuttings, short, thick shoots <strong>and</strong> large leaves are better for acclimatization. A major
concern for commercial application <strong>of</strong> LED irradiation systems is the quality <strong>of</strong> in vitro
plants grown under LED. We have therefore compared the growth pattern <strong>and</strong>
morphology <strong>of</strong> in vitro Ficus benjamina plantlets grown under fluorescent <strong>and</strong>
(combinations <strong>of</strong>) monochromatic blue, red or far-red LED light.
Before high performing LEDs were available, red <strong>and</strong> blue light enriched spectra
could be obtained by rather impractical combinations <strong>of</strong> fluorescent lamps with red <strong>and</strong>
blue polyester filters. <strong>Far</strong>-red light was provided by inc<strong>and</strong>escent lamps in combination
with several other polyester filters (Muleo et al., 2001). This resulted in quite a number <strong>of</strong>
studies that have confirmed the physiological <strong>and</strong> morphological effects <strong>of</strong> light quality.
Here, only in vitro LED literature will be discussed.
MATERIALS AND METHODS
Ficus benjamina ‘Exotica’ was micropropagated in 380 ml glass vessels with a
Proc. 7 th IS on In Vitro Culture <strong>and</strong> Horticultural Breeding
Ed.: D. Geelen
Acta Hort. 961, ISHS 2012
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screw on polycarbonate lid on a basal medium (BM) containing Murashige <strong>and</strong> Skoog
(1962) macroelements, Nitch <strong>and</strong> Nitch (1969) microelements, 35 mg/L NaFeEDTA,
100 mg/L myo-inositol, 0.4 ml/L thiamine HCl, 120 mg/L adeninesulfate, 170 mg/L
NaH 2 PO 4 , 7 g/L agar, 15 g/L sucrose, 0.5 mg/L IAA, 2 mg/L BA (pH 5.8 before autoclaving).
Each culture vessel contained 100 ml autoclaved medium (120°C, 30 min) with
6 uniform shoots with detached leaves. To allow high transmission, the plastic screw-on
lids <strong>of</strong> the vessels where replaced by polycarbonate petri dishes. Each vessel was packed
in low density polyethylene foil.
The cultures were maintained at 23±2°C under a 16 hour photoperiod. Control
treatment was cool fluorescent light (FL), provided by PHILIPS master TLD 36W 830
Reflex ECO (45 µmol m -2 s -1 PAR) or following combinations <strong>of</strong> blue (B, 454 nm), red
(R 660 nm) <strong>and</strong> far red (FR 745 nm) light emitting diodes (Fig. 1): 100% R (=R), 100% B
(=B), 100% FR (=FR), 50% B + 50% R (=BR), 50% B + 50% FR (=BFR), 50% R + 50%
FR (=RFR). By means <strong>of</strong> dimmers <strong>and</strong> a JAZ light meter (Ocean Optics) total PFD was
adjusted to 40 µmol photons m -2 s -1 . The culture racks were divided in boxes with white
walls <strong>and</strong> a white door (Fig. 1), in which Philips GreenPower LED Research Modules
were mounted. After 10 weeks under these conditions, shoot cluster <strong>and</strong> callus weight,
shoot number <strong>and</strong> length were measured. Shoots were counted in three classes: 2 cm. Per explant, length <strong>and</strong> width <strong>of</strong> all leaves <strong>of</strong> one r<strong>and</strong>om shoot per
vessel were measured by image analysis. The experiment was replicated three times, with
9 vessels per light treatment for each replicate. Data <strong>of</strong> the treatments were subjected to
one way ANOVA, mean separation was done by Tukey’s HSD test (p=0.05).
RESULTS AND DISCUSSION
Shoot Number per Explant <strong>and</strong> Shoot Size
Under R, the explants produced less but more elongated shoots (Fig. 3) then under
B <strong>and</strong> FL (Table 1). The presence <strong>of</strong> blue light had a stimulating effect on the number <strong>of</strong>
shoots. More shoots means smaller shoots. Most shoots were recorded under B, RB <strong>and</strong>
FL, which indicates that, for this parameter, blue light ‘dominates’ red light R when used
together. B also dominates FR. FR, R <strong>and</strong> RFR were disadvantageous for the total number
<strong>of</strong> shoots.
These results are in line with a number <strong>of</strong> recent reports. Moon et al. (2006)
analyzed the effects <strong>of</strong> R <strong>and</strong> B on in vitro Tripterospermum japonicum. The longest
nodes were observed from R-treated plants, the shortest from the B-treatment. Although
plants were tallest under red light, their growth was fragile due to excessive internodal
elongation. Vitis vinifera showed a significantly stimulated shoot elongation under R
whereas B <strong>and</strong> the combination <strong>of</strong> B <strong>and</strong> R were associated with short shoots (Heo et al.,
2006). Chrysanthemum shoots showed largest stem length under R <strong>and</strong> RFR. Also here,
the presence <strong>of</strong> B reduced stem length (Kim et al., 2004). All these reports deal with
plants grown on cytokinin free medium <strong>and</strong> as a consequence no data about shoot
proliferation is available. Poudel et al. (2008) showed that, in combination with 0,5 mg/L
BA <strong>and</strong> 0.01 mg/L NAA, plant height <strong>and</strong> internode length <strong>of</strong> Vitis was significantly
longer in plants cultured under R. Shoot length under B <strong>and</strong> FL were not different. In this
study there was no effect on shoot number, in contrast with the findings <strong>of</strong> Chée (1986)
who found a higher shoot production under B. Li et al. (2010) reported that stem length
<strong>and</strong> second internode length <strong>of</strong> Gossypium hirsitum were largest in plantlets cultured
under the BR <strong>and</strong> B, followed by R <strong>and</strong> FL.
By means <strong>of</strong> phytochrome signaling, plants sense an increase in FR radiation
caused by the reflection <strong>of</strong> FR by neighboring plants <strong>and</strong> respond with increased
elongation (Ballaré, 1999). This effect <strong>of</strong> FR is not observed in our experiments with
Ficus benjamina. FR does not drive photosynthesis, so this could be the reason why
plants grown under pure FR are small, in spite <strong>of</strong> the available sucrose in the medium.
Generally, reduced levels <strong>of</strong> blue light occur when the canopy begins to close <strong>and</strong> this
drop causes a reduction <strong>of</strong> stem elongation (Ballaré, 1999). <strong>Blue</strong> light is perceived by
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cryptochrome. The mode <strong>of</strong> CRY signal transduction towards etiolation has recently been
reviewed by Liu et al. (2011).
Shoot Cluster <strong>and</strong> Callus Weight
The positive effect on shoot cluster weight <strong>of</strong> red light was counteracted by the
overall negative effect <strong>of</strong> blue light (Table 2). The highest callus weight was observed
under B <strong>and</strong> RB, followed by FL. <strong>Blue</strong> light strongly stimulated callus growth <strong>and</strong> this
was not prevented by red light application. The presence <strong>of</strong> FR had a negative effect on
both shoot cluster <strong>and</strong> callus weight. The negative effect <strong>of</strong> B on shoot weight <strong>and</strong> its
positive effect on callus growth is reflected in the low calculated shoot/callus weight ratio
(Table 2). These results show that, in all conditions, blue light had a negative effect on the
weight proportion <strong>of</strong> the shoots. Also FL contains blue light <strong>and</strong> yields a low shoot/callus
weight ratio.
Analogue experiments in Chrysanthemum showed that in vitro plants performed
poorly under BFR <strong>and</strong> produced the lowest fresh <strong>and</strong> dry weight under these light
conditions. In contrast with our results, BR <strong>and</strong> FL gave the highest fresh <strong>and</strong> dry weight
(Kim et al., 2004). In micropropagated Tripterospermum japonicum (Moon et al., 2006),
B gave the lowest fresh weight <strong>and</strong> the fresh weight recorded under BR overtook those
under R <strong>and</strong> FL. The same negative effect <strong>of</strong> B on fresh <strong>and</strong> dry weight <strong>of</strong> B was recorded
in in vitro Vitis by Heo et al. (2006). Callus fraction was not determined in these studies
since cytokinins were not supplemented to the medium.
Leaf Length/Width Ratio
The presence <strong>of</strong> R <strong>and</strong> FR yields narrow leaves, in contrast to B that confers oval
leaves, just as FL (Table 3). When combined with red or far red light, blue light can only
level the effect <strong>of</strong> red light, not that <strong>of</strong> far red. This is illustrated by the smaller ratio for
B, RB <strong>and</strong> FL <strong>and</strong> is reflected in the observations <strong>of</strong> Shin et al. (2008) in micropropagated
Doritaenopsis, where leaf length increased for plants grown under R compared to B, BR
<strong>and</strong> FL.
CONCLUSION
Our study shows that monochromatic blue, red <strong>and</strong> far-red <strong>and</strong> their combinations
are suitable to manipulate the number <strong>of</strong> shoots, shoot length, shoot/callus weight ratio
<strong>and</strong> leaf length/width ratio in Ficus benjamina. In general, the presence <strong>of</strong> blue light had a
stimulating effect on the number <strong>of</strong> shoots but also reduced shoot length. Grown under
red light, the explants produced less but more elongated shoots. <strong>Blue</strong> light obviously
stimulates callus growth <strong>and</strong> this could hardly be levelled by red light. FR had a negative
effect on total number <strong>of</strong> shoots <strong>and</strong> both shoot cluster <strong>and</strong> callus weight. The presence <strong>of</strong>
R <strong>and</strong> FR yields longer leaves, in contrast to the rounder leaves that develop under blue
light.
ACKNOWLEDGEMENTS
This research was supported by the Flemish Government (IWT80122) <strong>and</strong> private
tissue culture companies. The authors also thank the framework <strong>of</strong> the “Associatieonderzoeksgroep
Primaire Plantaardige Productie”.
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Tables
Table 1. <strong>Effect</strong> <strong>of</strong> light spectrum on number <strong>of</strong> small (2 cm) shoots. Means within one column followed by the same letters are not
significant different (Tukey, 95%).
Spectrum
Shoot class
2 cm
Total
B 8.7 ab 0.5 b 0.1 b 9.3 ab
R 3.3 e 1.1 ab 1.4 a 5.8 bc
FR 3.0 e 0.9 ab 0.2 b 4.0 c
RB 11.7 a 0.6 b 0.0 b 12.3 a
BFR 5.6 cd 1.0 ab 0.2 b 6.8 b
RFR 3.9 de 1.7 a 1.0 a 6.6 bc
FL 6.5 bc 0.3 b 0.0 b 6.8 b
Table 2. <strong>Effect</strong> <strong>of</strong> light spectrum on shoot <strong>and</strong> callus weight <strong>and</strong> their ratio. Means within
one column followed by the same letters are not significant different (Tukey, 95%).
Spectrum
Shoot weight Callus weight
(mg) (mg)
Shoot weight/callus weight
B 229 b 313 ab 0.73
R 525 a 171 cd 3.07
FR 184 b 54 d 3.41
RB 235 b 366 a 0.64
BFR 137 b 129 cd 1.06
RFR 512 a 190 c 2.69
FL 170 b 215 bc 0.79
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Table 3. <strong>Effect</strong> <strong>of</strong> light spectrum on leaf length, width <strong>and</strong> ratio as measured on one shoot
per explant. Means within one column followed by the same letters are not significant
different (Tukey, 95%).
Leaf length Leaf width
Spectrum
(mm) (mm)
Leaf length/width ratio
B 7.5 a 5.1 a 1.50 b
R 6.0 b 3.4 de 1.81 a
FR 4.2 c 2.6 e 1.60 ab
RB 7.8 a 5.3 a 1.50 b
BFR 7.4 ab 4.3 bc 1.74 a
RFR 6.8 ab 3.9 cd 1.71 ab
FL 7.3 ab 4.8 ab 1.52 b
Figures
2,50
Energy (µmol/m².s)
2,00
1,50
1,00
0,50
R
B
FR
FL
0,00
300 400 500 600 700 800
wavelength (nm)
Fig. 1. Spectrum <strong>of</strong> fluorescent light, blue, red <strong>and</strong> far-red Philips Research LED.
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Fig. 2. Culture cells with white walls <strong>and</strong> door, containing 18 vessels covered with a petri
dish lid <strong>and</strong> polyethylene foil.
Fig. 3. <strong>Effect</strong> <strong>of</strong> B, R <strong>and</strong> FR light on the development <strong>of</strong> a centrally depicted defoliated
Ficus benjamina explant, visualized by sample plants (arrows: left = B; right = R;
under = FR).
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