Scientia Horticulturae 211 (2016) 295–304

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Scientia Horticulturae
journal homepage: www.elsevier.com/locate/scihorti

The effect of light quality on seed germination, seedling growth and

selected biochemical properties of Stevia rebaudiana Bertoni
Magdalena Simlat a,∗ , Patrycja Ślezak
˛ a
, Maria Moś a , Marzena Warchoł b , Edyta Skrzypek b ,
a
Agata Ptak
a
Department of Plant Breeding and Seed Science, University of Agriculture in Krakow, Łobzowska 24, 31-140 Krakow, Poland
b
Department of Biotechnology, The Franciszek Górski Institute of Plant Physiology Polish Academy of Sciences, Niezapominajek 21, 30-239 Krakow, Poland

a r t i c l e i n f o a b s t r a c t

Article history: Poor germination of Stevia rebaudiana Bertoni seeds is a phenomenon with unknown reason and the
Received 27 January 2016 research concerning the improving of germination are still in progress. We investigated the influence of
Received in revised form 7 September 2016 light-emitting diodes (LEDs) spectra on Stevia seed germination as well as seedling growth and selected
Accepted 8 September 2016
morphological and biochemical parameters. Blue LED light increased seed germination and affected the
development of the largest number of leaves and roots in 4-week-old Stevia plantlets. It has also the most
Keywords:
favourable effect on the number and opening of stomata. Red LED light however, significantly increased
Antioxidant enzymes
the length of stems and roots, although there was not correlation with the fresh weight (FW). The highest
Germination
Light emitting diodes (LEDs)
FW of Stevia plantlets achieves under combined red and white LED light at temperature of 20 ◦ C as
Photomorphogenesis well as under white fluorescent light at temperature of 25 ◦ C. Blue LED light also positively affected the
Pigments carotenoids concentration, whilst the highest concentration of chlorophyll a and b was found, in plantlets
Stevia grown under white fluorescent light. The less favourable effect on the synthesis of all the examined
pigments was exerted by red LED light. The largest amounts of phenolics and soluble sugars accumulated
plantlets growing in the darkness and irradiated blue LED light. In addition, all LEDs affected the activity
of antioxidant enzymes. The blue LED light increased the activity of catalase (CAT) and peroxidase (POD),
especially at 25 ◦ C. Red LED light significantly increased activity of superoxide dismutase (SOD) whilst
for the activity of CAT and POD opposite effect was observed. The combined red with white LED light
was the most favourable for the activity of CAT at 25 ◦ C. We conclude that the seed germination and the
quality of Stevia plantlets could be improved by controlling light quality.
© 2016 Elsevier B.V. All rights reserved.

1. Introduction
Stevia (Stevia rebaudiana Bertoni) is a perennial herb which
belongs to the Asteraceae family. The leaves of Stevia accumulate
eight steviol glycosides (SGs), among which stevioside and rebau-
dioside A are the major ones. These compounds are more than
200 times sweeter than common sugar (Yadav et al., 2011). Their
Abbreviations: AG, agar gel; B, blue LED light; BSA, bovine serum albumin;
CAT, catalase; D, darkness; dH2 O, distilled water; EDTA, ethylenediaminetetraacetic
acid; FW, fresh weight; H2 O2 , hydrogen peroxide; LEDs, light emitting diodes; MS,
Murashige and Skoog medium; POD, peroxidase; R, red LED light; R+W, red+white
LED light mixed in energy ratio 1:1; ROS, reactive oxygen species; SE, standard error;
SG, ssteviol glycosides; SOD, superoxide dismutase; WF, white fluorescent light.
∗ Corresponding author.
E-mail addresses: m.simlat@ur.krakow.pl (M. Simlat), patty.slezak@gmail.com
˛
(P. Ślezak), rrmos@cyf-kr.edu.pl (M. Moś), m.warchol@ifr-pan.edu.pl (M. Warchoł),
e.skrzypek@ifr-pan.edu.pl (E. Skrzypek), mfptak@cyf-kr.edu.pl (A. Ptak).
low caloric content and resistance to high temperature have con-
tributed to an increased interest in Stevia in recent years and the
use of SGs as sweeteners. Stevia is naturally propagated by seeds
(1000 seeds weigh 0.3–1.0 g), however in some reports (Shock,
1982; Carneiro et al., 1997; Lester, 1999; Raina et al., 2013) poor
fertility of Stevia seeds is noticed. Fertile seeds are usually dark
in colour, whereas infertile seeds are pale or clear (Felippe, 1978;
Monteiro, 1980; Oddone, 1997; Goettemoeller and Ching, 1999;
Yadav et al., 2011; Raina et al., 2013). Although selection of fertile
seeds is possible, the propagation by seeds is not the widespread
method for Stevia commercial production. The main reason for that
is poor germination of seeds (germination percentage is usually
less than 50%) and a long period required for the development of
seedlings suitable for planting in the field (it can last up to 60 days).
Some reports also described the deterioration of seeds during stor-
age (Khalil et al., 2014). Alternatively, Stevia may be propagated by
stem cuttings but this method, limits the large-scale production of
planting material (Yadav et al., 2011). There are also some reports

http://dx.doi.org/10.1016/j.scienta.2016.09.009
0304-4238/© 2016 Elsevier B.V. All rights reserved.
296 M. Simlat et al. / Scientia Horticulturae 211 (2016) 295–304
Table 1
Treatments used for seed germination and plantlets growth experiments.
Experiment Substrate Temperature [◦ C] Light conditions – 24 h day
Germination Filter paper 20 25 LED – Red,
AG 638 nm* (R)
Plantlets growth MS
AG – agar gel.
MS – Murashige and Skoog medium (Murashige and Skoog, 1962).
*
Spectral measurements of LEDs and fluorescent lamps were performed by the producers of the used growth chambers.
concerning the use of tissue culture for Stevia propagation (Sivaram
and Mukundan, 2003; Mitra and Pal, 2007; Jain et al., 2009; Khalil
et al., 2014). However, there is a need to improve the germination
rate and optimize the conditions of seedling growth. Some species,
utilize light to indicate conditions suitable for seed germination
(Chen et al., 2013). Light is also one of the most important environ-
mental factors for plant growth and development (Dong et al., 2014;
Bian et al., 2015). In recent years light-emitting diodes (LEDs) have
been widely used as a potential alternative light source for plants.
LED systems have several unique advantages, including the ability
to control spectra composition, long operating lifetime, wavelength
specificity, relatively cool emitting surfaces, small size and dura-
bility (Massa et al., 2008). It is known that seeds and seedlings
are able to respond not only to the intensity of light but also to
its quality (Bewley and Black, 1994; Yamazaki, 2010). Different
light wavelengths may set off a variety of responses in plants. Red
light may initiate seed germination and root development (Bewley
and Black, 1994; Daud et al., 2013). The application of red light
during plant vegetation can also increase the photosynthesis rate.
Senger (1982) reported that among LEDs, it is blue light is most
important for chloroplast development, chlorophyll biosynthesis
and stomata opening. Blue wavelengths also increase the leaf pho-
tosynthetic pigment content, affect phototropism (Johkan et al.,
2010) and improve the stomatal conductance (Hogewoning et al.,
2010). Johkan et al. (2010) also postulated that amongst various
light spectra, blue and red wavelengths have the strongest influ-
ence for the primary and secondary metabolism. However, plants
exhibit a wide range of morphological and phytochemical plasticity
in response to a given kind and wavelength of light (Hoenecke et al.,
1992; Goins et al., 1997; Yorio et al., 1998; Macedo et al., 2011).
Light quality might also evoke the photoxidative changes in
plants, which lead to produce the different form of reactive oxy-
gen species (ROS). The main source of ROS generation in plants
are chloroplasts. ROS are believed to be the major free radicals
leading to cell damage in plants. They are able to initiate a range
of metabolic changes, such as reduced enzyme activities and a
decrease in the photosynthetic rate. ROS are formed in many cel-
lular processes in a response to a variety of stress factors, such
as, e.g. light stress, temperature stress. If ROS are not removed
immediately, they can induce an oxidative damage at the cellu-
lar and molecular level. To minimize the ROS effects, plants have
evolved the detoxification mechanisms consisting of low molecu-
lar weight substances, named antioxidants and several antioxidant
enzymes. Among them catalase (CAT; EC 1.11.1.6.), peroxidase
(POD; EC 1.11.1.7) and superoxide dismutase (SOD; EC 1.15.1.1)
plays a crucial role in decreasing and eliminating the ROS. It has
been demonstrated that exposure to LEDs radiation can increase
the antioxidant enzyme activity (Kim et al., 2013; Dong et al., 2014;
Manivannan et al., 2015). Also under different temperatures, some
antioxidant enzyme activities are observed to change (Yan et al.,
2013). Thus, the improvement of a temperature together with light
quality is often regarded as being related to the enhanced activities
of antioxidant enzymes in plants.
Although there are some reports on the effect of light on Ste-
via seed germination (Abdullateef and Osman, 2011; Kumar and
LED – Blue, LED − Red + White, Fluorescent Darkness (D)
445 nm* (B) R: W = 1: 1* light – White, – control 2
(R + W) 390–760 nm*
(WF) – control 1
Sharma, 2012), the effects of LEDs have not been analysed yet. LED
wavelengths modify the seed germination, growth and develop-
ment of many plants (Bewley and Black, 2012; Heo et al., 2002; Su
et al., 2014; Poudel et al., 2008; Ryu et al., 2012). Therefore, the main
goal of our research was to evaluate the effect of light quality, using
red, blue and red + white LEDs, on Stevia seed germination and
seedling development so as to obtain high-quality plantlets useful
for transplantation. Good quality plantlets have many character-
istics, including morphological and biochemical properties (Oda,
2007). For this reason we analysed the morphological characteris-
tics, number of stomata and stomatal opening, concentrations of
phenolics, soluble sugars and pigments, as well as the activity of
antioxidant enzymes. To our knowledge, this is the first report for
Stevia rebaudiana showing the effect of LED light on seed germi-
nation and seedling development, both on the morphological and
biochemical level.
2. Materials and methods
2.1. Plant material and germination conditions
Stevia rebaudiana Beroni seeds obtained from W. Legutko Breed-
ing and Seed Company, Poland, were used for our experiment.
Before starting the experiment, the seeds were stored at a tem-
perature of +4 ◦ C for 3 months. For germination, seeds were first
carefully washed under running tap water and then treated with
70% ethanol for 15 s. Surface sterilization was made by soaking
the seeds in 20% Domestos (containing 5% chloride) for 15 min. To
remove the surfactant, the seeds were washed in sterile distilled
water (dH2 O) at least three times for 15 min and finally dried on fil-
ter paper. The disinfected seeds were placed on 7 cm-diameter Petri
dishes containing different substrates. One of them was filter paper
(three layers) moistened with 2 ml of sterile dH2 O and the second
substrate was agar gel (AG) (dH2 O solidified with 0.7% (w/v) Difco
Bacto Agar). Seed germination was performed in controlled plant
growth chambers (MCA 1600, Snijders Scientific, The Netherlands
(standardly equipped with LEDs, Phillips), and Adaptis-A1000TC,
Conviron, Canada (standardly equipped with fluorescent lamps,
Osram)), under various light conditions at an established tempera-
ture 20 ◦ C or 25 ◦ C and 70 ± 5% relative humidity. The germination
experiment included three different light wavelengths using LEDs:
red (R), blue (B), and a combination of red and white (R + W) light
(mixed at a 1:1 energy ratio). White fluorescent light (WF) and dark-
ness (D) were used as control treatments (respectively, control 1
and control 2) to assess the effects of LEDs on germination (Table 1).
In all the light treatments the same light intensity, expressed
as photosynthetic photon flux density (PPFD) of 60 ␮mol m−2 s−1
under 24 h day, was maintained. The experiment consisted of three
replications of 25 seeds per each light and temperature treatment.
During the experiment sterile dH2 O was added to each Petri dish
containing filter paper, as required. The number of germinated
seeds was recorded every day, starting from the first day after the
seeds were initially placed on Petri dishes. After 21 days of incuba-
tion the percentage of germinated seeds was determined.
 M. Simlat et al. / Scientia Horticulturae 211 (2016) 295–304 297

Fig. 1. Percentage of germinated seeds in different light treatments (R – LED red light; B – LED blue light; R + W – LED red + white light, mixed in energy ratio 1:1; WF –
fluorescent light white (control 1), D – darkness (control 2)). Germination conditions: (a) 20 ◦ C, filter paper; (b) 25 ◦ C, filter paper; (c) 20 ◦ C, AG; (d) 25 ◦ C, AG. The results are
averages of three replicates (n = 3).

2.2. Seedling growth conditions
During the germination experiment, ten well-developed
seedlings, obtained from each light/temperature combination and
AG substrate, were transferred to hormone-free MS medium
(Murashige and Skoog, 1962) supplemented with 3% (w/v) sucrose
and 0.7% (w/v) agar. The medium was adjusted to pH 5.8 before
autoclaving. Seedlings were grown in the same growth chambers
as those used for seed germination for a further four weeks under
the same conditions (light, temperature, humidity) (Table 1). After
defined time, morphological observation of the obtained plantlets
and biochemical analyses were made.
2.3. Morphological observations
For measurements of morphological features, all 10 plantlets
derived from each light and temperature combination were taken.
Table 2
Variance analysis for percentage of germinated seeds after 21 days of incubation in
different conditions of light, temperature and substrate.
The FW, height of stems and length of roots were measured. The
number of roots and leaves were also calculated. Each measure-
ment was expressed as a mean from 10 replicates.
2.4. Scanning electron microscopy (SEM)
To observe the stomata, samples were taken from fully
expanded leaves of three plantlets grown under each light regime
at 20 and 25 ◦ C. The samples were fixed with 2.5% (w/v) glutaralde-
hyde in 0.1 M dm−3 phosphatic buffer at pH 7.2–7.4 for 15 min. They
were dehydrated using a graded series of ethanol (30–100% v/v) and
acetate (100%), and dried with liquid CO2 in a critical point dryer
(Type E3100 Industrial LADD, USA). Samples were then coated with
gold using a sputter coater (Jeol JFC-1100E, Japan). Finally, the pre-
pared slides were observed using a scanning electron microscope
(Jeol model JSM 5410, Japan) (Pathan et al., 2008). The number of
stomata was determined by counting them in 10 different areas of
each leaf.

Source df MS F emp 2.5. Determination of total phenolic compounds

Light 4 719.05 9.36**
Temperature 1 1112.24 14.48** For phenolic estimation, 5 mg samples of fresh tissue were
Substrate 1 4194.44 54.64** homogenized in 1 ml of 80% ethanol and centrifuged at 15,000 rpm
Light* temperature 4 116.09 1.51ns for 15 min. The supernatant was mixed with 20% Na2 CO3 and Folin-
Light* substrate 4 91.14 1.18ns
Ciocalteau reagent (Singleton and Rossi, 1965). The absorbance
Temperature* substrate 1 530.03 6.90*
Light* temperature* substrate 4 126.22 1.64ns
(␭ = 760 nm) of the samples was estimated spectrophotometrically
Error 40 76.76 using a micro-plate reader (Synergy 2, Bio-Tek, Winooski, VT, USA).
The total phenolics content was calculated as milligrams of chloro-
ns not significant.
*
Significant at the 0.05 probability. genic acid per 1 g of tissue FW. For this analysis plants grown under
**
Significant at the 0.01 probability. each of the tested light conditions at 20 ◦ C were used.
298 M. Simlat et al. / Scientia Horticulturae 211 (2016) 295–304

Table 3
Effect of light treatment on morphology of Stevia rebaudiana plantlets and the number of stomata after four week growth in in vitro conditions.

Light Total fresh Shoot fresh Stem length [cm] Internode length Number of leaves Root length [cm] Roots number Number of
treatment weight [g] weight [g] [cm] per plantlet stomata per
10,000 ␮m2

20 ◦ C
R 0.13 ± 0.02c 0.08 ± 0.02b 6.30 ± 3.25a 1.77 ± 1.06a 9.60 ± 1.67ab 5.36 ± 2.20a 9.80 ± 3.70ab 3.90 ± 0.84c
B 0.23 ± 0.08ab 0.15 ± 0.05a 1.18 ± 0.49c 0.37 ± 0.13b 11.00 ± 2.00a 1.58 ± 0.43c 13.20 ± 8.93a 8.57 ± 2.59a
R+W 0.17 ± 0.03bc 0.10 ± 0.01b 1.80 ± 0.34c 0.52 ± 0.21b 9.20 ± 1.10ab 3.36 ± 1.00abc 7.40 ± 0.82abc 7.28 ± 2.00b
WF 0.26 ± 0.09a 0.18 ± 0.05a 3.18 ± 1.11bc 0.91 ± 0.32b 8.80 ± 1.10b 4.10 ± 1.52ab 6.60 ± 0.89bc 4.02 ± 1.69c
D 0.01 ± 0.00d 0.01 ± 0.00c 4.92 ± 1.58ab 1.87 ± 0.20a 3.60 ± 0.89c 2.32 ± 1.50bc 2.60 ± 1.14c 1.95 ± 0.95d
25 ◦ C
R 0.12 ± 0.03c 0.08 ± 0.01c 10.26 ± 2.35a 3.78 ± 0.91a 10.40 ± 0.89b 5.34 ± 2.47a 7.00 ± 5.96ab 3.96 ± 0.74c
B 0.37 ± 0.04a 0.22 ± 0.04a 3.14 ± 1.18b 0.60 ± 0.40b 15.40 ± 4.10a 2.94 ± 1.66ab 12.60 ± 6.54a 8.70 ± 1.65a
R+W 0.41 ± 0.09a 0.22 ± 0.03a 5.64 ± 1.65b 1.70 ± 0.76b 13.00 ± 2.45ab 5.32 ± 2.62a 12.40 ± 3.78a 7.81 ± 0.73a
WF 0.28 ± 0.08b 0.16 ± 0.04b 5.95 ± 2.99b 1.72 ± 1.07b 12.50 ± 1.00ab 3.50 ± 1.29ab 11.50 ± 5.57a 5.08 ± 1.45b
D 0.01 ± 0.00d 0.01 ± 0.01d 2.76 ± 2.10b 1.08 ± 0.86b 2.00 ± 0.00c 1.48 ± 0.86b 1.40 ± 0.55b 2.43 ± 0.65d
Significance
Source
** ** ** ** ** ** ** **
Light
** ** ** ** ** ns ns *
Temperature
Light* temperature ** ** ** ** ** * ** ns

R – LED red light; B – LED blue light; R + W – LED red + white light, mixed in energy ratio 1:1, WF − fluorescent light white (control 1), D – darkness (control 2).
Means ± SE (n = 10, for stomata frequency assessment n = 30) followed by different letters are significantly different, based on ANOVA followed by Duncan’s test at P ≤ 0.05.
ns: not significant.
*
Significant at the 0.05 probability.
**
Significant at the 0.01 probability.

2.6. Determination of total soluble sugars
One hundred-␮g samples of fresh tissue were homogenized
and soluble sugars were extracted with 80% ethanol. The amounts
of total sugars were estimated after centrifugation (15,000 rpm,
15 min) by the phenol-sulfuric method (Dubois et al., 1956). The
supernatant was mixed with 5% phenol and 95% sulphuric acid.
The absorbance (␭ = 490 nm) of the samples was estimated spec-
trophotometrically on a micro-plate reader (Synergy 2, Bio-Tek,
Winooski, VT, USA). The total soluble sugars content was calcu-
lated as milligrams of glucose per 1 g of tissue FW. For this analysis
plants grown under each of the tested light conditions at 20 ◦ C were
used.
2.7. Determination of pigments
measured spectrophotometrically (␭ = 595 nm) by the method of
McCord and Fiodovich (1969). One unit was defined as the amount
of enzyme necessary for 50% inhibition of cytochrome c in a coupled
system with xanthine and xanthine oxidase. The reaction kinetics
for all enzymes was examined 60 s after initiation of the reaction.
For this analysis plants grown under all the light and temperature
(20 and 25 ◦ C) conditions except darkness, were used.
2.9. Data analysis
The results were subjected to analysis of variance (ANOVA). For
the comparison of means, Duncan’s multiple test at the significance
level of P ≤ 0.05 implemented in the statistical package STATISTICA
(data analysis software system), version 10.0, www.statsoft.com,
was used. The data are reported as means ± standard error (SE).

Chlorophylls and carotenoids were extracted from fresh tissue 3. Results

samples (100 ␮g) in 1 ml of 80% ethanol for 12 h. After centrifu-
gation (15,000 rpm, 15 min), an aliquot of extract was added to
micro-plate wells and absorbance was measured spectrophoto-
metrically at 470, 648, 664 nm on micro-plate reader (Synergy 2,
Bio-Tek, Winooski, VT, USA). The concentrations of chlorophylls
a, b and carotenoids were determined according to Lichtenthaler
and Wellburn (1983) and expressed per 1 g of tissue FW. For this
analysis plants grown at each light condition at 20 ◦ C were used.
2.8. Antioxidant enzymes analyses
Fresh plant tissue (100 ␮g) was homogenized at 4 ◦ C with phos-
phate buffer (pH 7.8) containing 0.01 M EDTA and 0.5% BSA. The
homogenate was centrifuged at 10,000 rpm for 15 min. Activ-
ity of CAT was measured spectrophotometrically (␭ = 240 nm) by
the method of Aebi (1984). The reaction mixture consisted of
0.05 M dm−3 phosphate buffer (pH 7.0) containing 0.1 mM EDTA
and 0.03 M dm−3 H2 O2 , in this buffer and the supernatant. Activity
of POD was measured by the method of Lűck (1962). The mea-
surement was carried out spectrophotometrically (␭ = 485 nm), by
measuring the amount of the products in 1% p-phenylenediamine
(pPD) and the supernatant, in 0.05 M dm−3 phosphate buffer con-
taining 0.1 mM EDTA, pH 7.0. The reaction was started in the
presence of 0.05 cm3 of 0.03 M dm−3 H2 O2 . Activity of SOD was
3.1. Seed germination
At a temperature of 20 ◦ C, on filter paper as a substrate, the
most favourable effect on germination was exerted by B and R + W
LED light. Throughout the experiment, germination dynamics was
similar, in these two types of light. However, first symptoms of ger-
mination were observed earlier (on the fourth day) under B than
under R + W LED light. For both LEDs light the germination percent-
age calculated on the 21st day of the experiment was significantly
higher compared with the control 1 (WF) (26.7% for B LED, 25.0% for
R + W LED and 15.0% for WF). Considering the types of light used, a
less favourable effect on germination was exerted by R LED light. In
this case the germination percentage calculated on the 21st day of
the experiment was similar to that found for the darkness (control
2) (Fig. 1a).
At a temperature of 25 ◦ C, using filter paper as a substrate, the
most favourable effect on germination was exerted by WF light
(control 1) (germination percentage of 28.3%). Under these con-
ditions the seeds germinated till the 19th day of the experiment.
Similar germination dynamics was observed for seeds incubated
under R + W LED light, although under these conditions first seeds
with symptoms of germination were noted five days later. Under
both R and B LED light germination percentage was 16.7%; however,
 M. Simlat et al. / Scientia Horticulturae 211 (2016) 295–304
Fig. 2. Stevia rebaudiana plantlets grown for four weeks in in vitro conditions (20 ◦ C, MS medium) under different light treatments: (a) R – LED red light, (b) B – LED blue
light, (c) R + W – LED red + white light, mixed in energy ratio 1:1, (d) WF – fluorescent light white (control 1), (e) D – darkness (control 2).
germination energy was higher under blue light. The lowest germi-
nation percentage was found for the darkness (control 2) (11.7%)
(Fig. 1b).
At a temperature of 20 ◦ C, on the AG substrate, a more favourable
effect on seed germination, compared with control 1 (WF), was
found for B LED light. Under these conditions seeds with symp-
toms of germination were observed as early as on the third day of
incubation, and throughout the experiment the percentage of ger-
minating seeds was maintained at the highest level reaching 37.8%
on the 21st day. Under the conditions of R + W and R LED light ger-
mination percentage was 24.4% and 22.2%, respectively, and it was
lower than under WF (control 1). The poorest germination of seeds
was recorded in D (control 2), where the germination percentage
reaching only 17.8% on the 21st day of the experiment (Fig. 1c). At
a temperature of 25 ◦ C, using the AG substrate, the best germina-
tion results were obtained for seeds incubated under B LED light.
Considering all the tested light conditions, in this case both the
percentage and the rate of germination were the highest. Under
the conditions of B LED, the maximum percentage of germination
was observed as early as on the 7th day of incubation (50.0%), and
this value was close to the control 1 (WF) but significantly higher
than that calculated for R + W and R LED light. Under R LED light
and R + W LED light, germination started on the third day of incu-
bation. Under these conditions germination dynamics was similar
throughout the experiment, although in the end more seeds ger-
minated under mixed light than under red light −47.2% and 42.4%,
respectively. Darkness significantly inhibited Stevia seed germina-
tion (germination percentage of 19.4%) (Fig. 1d).
Three-factor analysis of variance showed significant differences
(P ≤ 0.05) in Stevia rebaudiana seed germination in relation to indi-
vidual factors: light, temperature and substrate. However, analysis
of variance (ANOVA) did not confirm that the coaction of all the
three tested factors had a significant effect. It should be noted,
however, that the highest germination percentage was obtained
when seeds were germinated under B LED light with combination
of temperature of 25 ◦ C and AG substrate. It was found that only
the coaction of temperature and substrate had a significant effect
on seed germination (Table 2).
3.2. Plant morphology
At 20 ◦ C, plantlets grown under white fluorescent light (WF –
control 1) attained the greatest FW, the main component of which
299
was the weight of the stem. Similar weight was found for plantlets
grown under B LED light. The least favourable effect on the fresh
weigh had R LED light. B LED light had also a favourable effect on
the number of roots and leaf development. Under these light condi-
tions the plantlets developed twice as many roots as compared with
the control 1 and about five times as many roots as compared with
the control 2 (D). Plantlets grown under B LED light also developed
the greatest number of leaves with large and correctly formed leaf
blade, as compared with other conditions, especially with the dark-
ness. Under B LED light, however, the plantlets attained the smallest
size, which resulted mainly from short internodes and roots. The
highest plantlets were obtained under R LED light. Both, the aver-
age length of the stems with considerably lengthened internodes
and the average length of roots were the greatest in plantlets grown
under this light. The effect of R LED light on the plantlet size was
similar to that observed for the control 2 (Table 3, Fig. 2).
At 25 ◦ C, the results for the impact of light source with different
wavelengths on the FW showed that R + W LED light as well as B
LED were beneficial for the weight of whole plantlets mainly due
to the thickening of the stem and developing the largest number of
leaves and roots. Red LED light alone was beneficial for the length of
stems and roots. The stems of plantlets obtained under R LED were
almost twice as high as the stems of plantlets obtained under WF
light (control 1) and almost thrice as high as the stems of plantlets
grown in D (control 2). However, it should be noted that plantlets
obtained under R LED light had a different habit. Due to consider-
ably lengthened internodes these plantlets, in spite of the greatest
height, did not develop the largest number of leaves. The average
number of leaves per plant made the R LED the less beneficial light
among the tested types of light. Less leaves were developed only
by the plantlets grown in the darkness (control 2) (Table 3).
Considering the tested growth conditions, a temperature of
25 ◦ C had a more favourable effect on the plant growth than 20 ◦ C.
Plant weight, height, and the number of leaves and roots were
greater at a temperature of 25 ◦ C, irrespective of the type of light
used. Only for the darkness opposite relationships were observed.
In turn, at a temperature of 25 ◦ C, B and R + W LED light had a more
favourable effect on plant weight than the light control (WF).
3.3. Stomata characteristics
In darkness, independently of the temperature used, the leaves
of Stevia developed the lowest number of stomata as compared
300 M. Simlat et al. / Scientia Horticulturae 211 (2016) 295–304

Fig. 3. Stomata of Stevia rebaudiana plantlets grown for four weeks in in vitro conditions (25 ◦ C, MS medium) under different light treatments: (a) R – LED red light, (b) B –
LED blue light, (c) R + W – LED red + white light, mixed in energy ratio 1:1, (d) WF – fluorescent light white (control 1), (e) D – darkness (control 2).

Table 4
The content of total phenolics, soluble sugars, chlorophylls and carotenoids in Stevia rebaudiana plantlets grown for four weeks in in vitro conditions under different light
treatments.

Light Phenolics Soluble sugars Chlorophyll a Chlorophyll b Carotenoids

[mg/g FW]
R 8.24 ± 0.66b 167.96 ± 14.26b 2.55 ± 0.41b 1.64 ± 0.14b 0.16 ± 0.05b
B 9.70 ± 0.47a 180.19 ± 5.19b 3.97 ± 0.71ab 2.13 ± 0.24ab 0.31 ± 0.03a
R+W 8.64 ± 0.74b 167.91 ± 8.83b 3.77 ± 0.98ab 2.13 ± 0.42ab 0.29 ± 0.11a
WF 8.96 ± 0.72ab 170.79 ± 11.30b 4.22 ± 0.82a 2.42 ± 0.40a 0.26 ± 0.09ab
D 9.77 ± 0.16a 202.95 ± 0.15a nd nd nd

FW – fresh weight.
R – LED red light; B – LED blue light; R + W – LED red + white light, mixed in energy ratio 1:1, WF – fluorescent light white (control 1), D – darkness (control 2).
nd: not detected.
Means ± SE (n = 3) followed by different letters are significantly different, based on ANOVA followed by Duncan’s test at P ≤ 0.05.

Table 5
Variance analysis for antioxidant enzyme activity in Stevia rebaudiana plantlets grown in in vitro conditions under different light and temperature treatments.

Source CAT activity POD activity SOD activity

df MS F emp MS F emp MS F emp

Light 3 0.03 15.26** 1.14 13.24** 0.00 14.17**

Temperature 1 0.04 17.73** 0.68 7.92* 0.11 2474.41**
Light* temperature 3 0.00 1.37ns 0.29 3.40* 0.00 14.14**
Error 16 0.00 0.09 0.00

ns: not significant.

*
Significant at the 0.05 probability.
**
Significant at the 0.01 probability.

with light treatments. We also observed various effects of light
quality on the number of stomata and stomatal opening. It is worth
noting that the tendency was similar at temperatures of 20 and
25 ◦ C. The highest number of stomata was observed in the leaves
of plantlets grown under B LED light. Similar results were observed
when R + W LED light was applied. Both the obtained values were
higher as compared with the light control (WF). On the contrary,
R LED light reduced the number of stomata as compared with the
light control (Table 3). In D, where plantlets developed fewer leaves
and smaller leaves, many rudimentary stomata, open or closed,
were observed. Different effects were observed under B and R + W
LED light conditions. These types of light showed positive effects
on guard cells, which were well organized, and on stomata open-
ing. Plants grown under R LED light also showed well organized
guard cells but the stomata were observed to be closed. On the
other hand, WF light caused the stomata to open but some of them
were improperly developed (Fig. 3).
3.4. Content of phenolics, soluble sugars, chlorophyll a, b, and
carotenoids
The tested light conditions had slight effect on phenolic, solu-
ble sugars, chlorophyll a, chlorophyll b and carotenoids contents in
Stevia rebaudiana. In plantlets obtained under B LED light, higher
levels of phenolics and soluble sugars were detected compared
to WF (control 1) and other LED treatments. But only the differ-
ences in phenolics content were significant. Darkness stimulated
the synthesis of phenolics to the same level as B LED, but as regards
soluble sugars, darkness had the most favourable effect. In contrast
to plantlets grown under B LED light, the plantlets grown under R
LED light showed the lowest values of all the examined parameters:
phenolics, soluble sugars as well as chlorophylls and carotenoids. It
should be noted that none of the used types of LED light stimulated
the synthesis of chlorophylls to the level that exceeded the control
1 (WF). The highest level of carotenoids was observed in plantlats
grown under B LED light, although the differences in relation to
R + W LED light were insignificant. In turn, plantlets grown in the
darkness (control 2) lacked the tested pigments (Table 4).
3.5. Response of the reactive oxygen defence system to different
treatments
In the present study we found that the activities of CAT, POD
and SOD enzymes altered depending on the light treatment and
temperature. At a temperature of 20 ◦ C the lowest CAT activity
was found in plantlets grown under R LED light (0.18 U/␮g of pro-
teins), and the highest CAT activity was noted under R + W LED light
(0.32 U/␮g of proteins). A similar level of CAT activity was observed
in plantlets grown under B LED light. Also at a temperature of 25 ◦ C
the lowest CAT activity was found in plantlets grown under R LED
 M. Simlat et al. / Scientia Horticulturae 211 (2016) 295–304 301

light (0.06 U/␮g of proteins). In plantlets grown under other light

conditions the activity of this enzyme was four times as high as
compared with R LED light. Statistical analysis showed that plants
grown under B LED light demonstrated the highest CAT activity. It
is also worth noting that at a temperature of 20 ◦ C the activity of
this enzyme was higher than that at a temperature of 25 ◦ C under
all the light conditions (Fig. 4a). Both, light as well as temperature
show significant effect on CAT activity (Table 5).
POD activity, at a temperature of 20 ◦ C was similar in plantlets
grown under WF light (0.63 U/␮g of proteins) and R LED light
(0.56 U/␮g of proteins). However, significantly higher values were
observed under B LED light and R + W LED light (1.11 and 10.01 U/␮g
of proteins, respectively). A similar effect of light on POD activity
was observed at a temperature of 25 ◦ C. The activity of the tested
enzyme, at both temperatures, was similar under all types of light
except for B LED light. Under B light, POD activity at a temperature
of 25 ◦ C was about twice as high as the activity at a temperature
of 20 ◦ C (2.10 and 1.11 U/␮g of proteins, respectively) (Fig. 4b).
Consequently, light, temperature as well as their interaction had
a significant impact on POD activity (Table 5).
At a temperature of 20 ◦ C the highest SOD activity was observed
in plants grown under R LED light. Lower values were noted in
plants grown under other light conditions, although B LED and WF
light significantly, to the greatest extent, inhibited the activity of
this enzyme. However, under all the light conditions the values of
this activity were very low – several thousand times as low as those
observed under a temperature of 25 ◦ C. At a temperature of 25 ◦ C
the activity of the tested enzyme showed, however, similar rela-
tionships. The highest activity of this enzyme was noted in plantlets
grown under R LED light (0.15 U/␮g of proteins), and the lowest
activity was found in plantlets grown under B LED light (0.10 U/␮g
of proteins) (Fig. 4c). The tested light and temperature conditions
showed significant effect on SOD activity. Also their interaction had
strong influence on tested parameter (Table 5).
Antioxidant enzyme activities in plants grown in the dark-
ness were not determined due to a small number of the obtained
plantlets, their weak condition and low weight.

4. Discussion

Light is an important regulator of plant growth and develop-

ment. The application of LEDs to the plant growth system was first
reported by Bula et al. (1991). The studies that have been performed
since that time suggest that plant species differ in their responses
to LED treatments and exhibit a high degree of morphological and
physiological plasticity to changes (Heo et al., 2002; Poudel et al.,
2008; Dong et al., 2014; Su et al., 2014; Manivannan et al., 2015).
Besides, there are some reports on the effect of light on Ste-
via seed germination it is still a phenomenon how different light
wavelength modified Stevia development. Abdullateef and Osman
(2011), Kumar and Sharma (2012) reported that light is a positive
stimulator of Stevia seed germination. Also in our study, germi-
nation of Stevia seeds was greatly inhibited by darkness. These
observations demonstrate that Stevia belongs to the positive pho-
toblastic species. Seeds of that species not only require light to
germinate but also the proper spectral composition (Bewley and
Black, 1994). Abdullateef and Osman (2011) found that germination
potential of Stevia seeds was enhanced by red light, compared to
white light. A different reaction was observed at presented exper-
iment where LEDs light were examined. Red LED light, under the
Fig. 4. Activity of antioxidant enzymes in Stevia rebaudiana plantlets grown in
majority of the tested conditions (temperature, substrate), exhib- in vitro conditions (20 ◦ C and 25 ◦ C, MS medium) under different light treatments:
ited an inhibitory effect on Stevia seed germination compared with R – LED light red, B – LED blue light, R + W – LED red + white light, mixed in energy
other LEDs and WF light. In our study the best effect on germi- ratio 1:1, WF – fluorescent light white (control 1). Activity of catalase (a); activity of
nation was exerted by B LED light (50% of germinated seeds at peroxidase (b); activity of superoxide dismutase (c). The results are averages of three
replicates (n = 3) with error bars representing the SE of the mean. The means with
25 ◦ C, on AG substrate). However, other species from the Asteraceae
different letters are significantly different, based on ANOVA followed by Duncan’s
test at P ≤ 0.05.
302 M. Simlat et al. / Scientia Horticulturae 211 (2016) 295–304
family exhibit a different reaction to light. Seed germination of
Artemisa monosperma and Lactuca sativa was also enhanced under
blue light (Bewley and Black, 2012). On the contrary, Ryu et al.
(2012) reported that red, blue and blue + red LED light treatments
delayed the speed of Taraxacum officinale seed germination, as com-
pared with white fluorescent light. Also the germination rate after
18 days of their experiment was lower in the case of LED light
treatments as compared with the fluorescent light treatment. In
some species, germination was affected not only by light but also
by temperature which turned out to be involved in the regulation
of that process (Heschel et al., 2007; Dechaine et al., 2009; Chen
et al., 2013). Kumar and Sharma (2012) noted that Stevia seed ger-
mination was hampered by low temperature and they concluded
that 20 ◦ C was the optimal temperature for seed germination of
that species. We observed that Stevia seed germination was bet-
ter at 25 ◦ C than at 20 ◦ C what is in agreement with the findings
of Takahashi et al. (1996). We also observed, especially at 25 ◦ C,
that seed germination was better on the AG substrate than on fil-
ter paper. Kumar and Sharma (2012) also noticed that the ability of
Stevia seeds to germinate might by affected by germination surface.
It is also known that plants are able to modulate their mor-
phological features and physiological properties according to light
quality. Scientists have concluded that among LEDs, red and blue
light exert the greatest effects on plant growth. Ryu et al. (2012)
reported that the growth characteristics of T. officinale were greatly
influenced by different supplemental LED light treatments. Among
these treatments, red LED light had the most favourable effect on
plant weight and height, number of leaves per plant and root length,
as compared with fluorescent light. In the study presented by
Poudel et al. (2008) plant height and internode length were also sig-
nificantly longer in plants grown under red LED light. Similar results
were also described by Manivannan et al. (2015) for Rehmannina
glutinosa cultured in vitro. Under red LED light the hypocotyls and
cotyledons of lettuce seedlings also became elongated but the addi-
tion of at least 15 ␮mol m−2 s−1 of blue light prevented this effect
(Hoenecke et al., 1992). However, Goins et al. (1997) and Yorio
et al. (1998) suggested that the addition of blue light to red light
can improve the growth and quality of plants. In turn, Heo et al.
(2002) reported that blue LED light alone had the best stimulation
effect on marigold and salvia plant height compared with red LED
and fluorescent white light. In their research blue LED light also
more effectively stimulated the length of internodes as compared
with red LED and white fluorescent light. The studies performed by
Yanagi et al. (1996) showed that lettuce plants grown under red LED
light had more leaves than plants grown under blue LED light. Su
et al. (2014) also reported that among the tested light treatments,
red LED light had a stimulating effect on leaf number and root
length; however, an opposite effect was observed for plant height,
leaf area, above-ground part and below-ground part weight. How-
ever, Manivannan et al. (2015) found that in R. glutinosa blue LED
light induced the largest number of leaves per plant compared with
other lights. Similar effect of blue fluorescent light was described by
Macedo et al. (2011). In our study the morphological characteristics
of Stevia plantlets grown under in vitro conditions for four weeks
were highly influenced by light. Stevia plantlets grown under B LED
light had shorter stems and roots, which was especially evident at
20 ◦ C; however, leaf formation was enhanced compared with other
light treatments. It was in accordance with the results obtained
for other species (Moreira da Silva and Debergh, 1997; Schuerger
et al., 1997). In our research we found that the exposure of Stevia to
R LED light had a beneficial effect on root length, while B LED light
affected favourably the number of roots. The influence of LED light
on the root system had already been reported. Red LED light stimu-
lated adventitious root growth in in vitro culture of Jatropha curcas.
Daud et al. (2013) reported that red LEDs, compared with blue and
white LEDs, produce more favourable effects on the rate of rooting
and root number. That finding was in agreement with the results of
the research on other species (Gabryszewska and Rudnicki, 1997;
Poudel et al., 2008; Baque et al., 2010; Iacona and Muleo, 2010).
Some reports described the effect of light on the number of
stomata and their size (Poudel et al., 2008; Macedo et al., 2011;
Li et al., 2010). Macedo et al. (2011) reported that blue and red
fluorescent light reduced the number of stomata on the abaxial
face and the adaxial face of Alternanthera brasiliana leaves, respec-
tively. On the contrary, Poudel et al. (2008) found that blue LED light
affected the formation of the largest number of stomata in all the
tested Vitis genotypes. Also Heo et al. (2002) observed that com-
pared with red LED light, blue LED light increased the number of
stomata in Tagetes erecta. In Salvia splendens, however, an opposite
relationship was observed. In our research we also observed the
effect of LEDs on number and opening of stomata. We found that B
LED light affected the formation of the largest number of stomata
and, in contrast to R LED light, it had also a favourable effect on
stomata opening. In some reports also the influence of tempera-
ture on stomata was described (Ghosh et al., 1996; Crawford et al.,
2012). However in our research we did not observe the effect of
temperature on stomatal frequency and opening.
Light is the factor which is also very important for synthesis of
chlorophylls (Hoenecke et al., 1992; Tripathy and Brown, 1995).
Akoyunoglou and Anni (1984) reported that blue LED light could
be effective in inducing chlorophyll formation. In our study all LED
treatments inhibited the chlorophyll synthesis in Stevia plantlets,
as compared with white fluorescent light. We found that consider-
ing the tested LEDs, lower content of chlorophyll a and chlorophyll
b were detected under R LED as compared with B LED light. Sim-
ilar results were obtained by Tanaka et al. (1998) and Li et al.
(2010). In Cymbidium and Gossypium hirsutum plantlets the lev-
els of chlorophyll a, chlorophyll b and their sum were higher under
blue LED light compared with red LED light. Also in the research
conducted by Poudel et al. (2008) the highest chlorophyll content
in all the tested Vitis genotypes was observed under blue LED light,
while the lowest concentration of this pigment occurred when red
LED light was applied. The inhibitory effect of red LED light on
chlorophyll synthesis was also described by Nhut et al. (2003) in
Fragaria × ananassa. However, in the studies conducted by Dong
et al. (2014) and Manivannan et al. (2015) on Triticum aestivum and
R. glutinosa, respectively, the best effect on chlorophyll synthesis
was found for red LED light. Moreover, Su et al. (2014) reported no
significant differences in the chlorophyll content of Cucumis sativus
seedlings when red and white LEDs were used, while blue LED light
exhibited an inhibitory effect.
Under the conditions of our experiment the effect of LED light
on carotenoid synthesis was observed. Under blue and red + white
LED light higher content, and under red LED light – lower content of
carotenoids were detected as compared with the white fluorescent
light. Johkan et al. (2010) reported positive effect of blue fluores-
cent light on carotenoid accumulation in L. sativa. On the contrary,
Wu et al. (2007) who carried out studies on pea seedlings, and Cui
et al. (2009) who conducted research on C. sativus and Lycopersicon
esculentum seedlings showed that carotenoid concentration was
increased when red LED light was used.
Many studies demonstrated that the accumulation of soluble
sugars in plants is also affected by light quality and the response of
plants varies among species and cultivars (Li et al., 2010, 2012; Lin
et al., 2013). However, we did not observe the significant effect of
LEDs on soluble sugar concentration in Stevia plantlets. Considering
all the light treatments, we detected higher values of soluble sugars
only in plants grown under B LED light.
Phenolics function as natural antioxidants and an increase
in their concentrations can protect plants from environmental
stresses, including photodamage. In our research the highest con-
centration of phenolics was found in Stevia plantlets grown in the
 M. Simlat et al. / Scientia Horticulturae 211 (2016) 295–304
darkness and under B LED light, whilst the lowest concentration
of these compounds was observed under R LED light. Also Johkan
et al. (2010) in their research on L. sativa seedlings found a beneficial
effect of blue light on the accumulation of phenolics.
It has been demonstrated that exposure to various LED radiation
can increase not only the antioxidants content but also antiox-
idative enzymes activities. LED radiation exerts also an effect on
metabolic and photooxidative processes in plants. Schmidt et al.
(2006) reported that CAT activity in winter rye leaves was enhanced
in a higher degree by blue light treatment than by red or far-
red light treatments. Similar results were obtained by Manivannan
et al. (2015). Kim et al. (2013) also found that the activity of that
enzyme was increased in blue LED light-treated tomato leaves com-
pared with the control, whereas the red LED treatment significantly
decreased CAT activity. These findings are in agreement with our
research. We also observed that the activity of POD was enhanced
by B LED light, whilst R LED light exerted an opposite effect. As
regards SOD activity, contrary to the above observations, R LED light
enhanced, and B LED light decreased the activity of that enzyme.
Quite different results were described by Manivannan et al. (2015).
In this research the activity of SOD was higher under LED light com-
pared with the control. Significantly higher activity was observed
under blue LED light than under red LED light. Also Kim et al. (2013)
reported enhanced SOD activity in tomato stem under the con-
ditions of blue light compared with the control, while red light
exerted an opposite effect. We also observed that in Stevia plantlets
subjected to LED light treatment, temperature had a strong effect
on the activities of CAT, POD, and especially SOD.
5. Conclusion
From our study it appears that there is a relationship between
light quality and the tested morphological and biochemical param-
eters. If the blue LED light is used, the rate and speed of germination
of Stevia seeds could be improved. By using the blue light it is possi-
ble to obtain good-quality plantlets with the highest fresh weight,
the largest number of leaves and roots and the greatest stomatal fre-
quency as well as high concentration of pigments and high activity
of antioxidant enzymes CAT and POD.
Acknowledgments
The research was financed by the Ministry of Science and Higher
Education of the Republic of Poland (Contract No. DS/3129/KHRiN).
The authors wish to thank the W. Legutko Breeding and Seed Com-
pany, Poland for kindly providing the seeds of Stevia. We also
gratefully acknowledge the technical assistance of Ewa Piotrowska
for help in germination experiment and PhD student Emilia
Morańska for assistance in enzyme activity assay.
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