Nativa, Sinop, v. 9, n. 5, p. 582-588, 2021.
Pesquisas Agrárias e Ambientais
DOI: https://doi.org/10.31413/nativa.v9i5.12541 ISSN: 2318-7670
The influence of light quality in the
in vitro
cultivation
of
Cattleya crispata
(Thunb.) Van den Berg
Denys Matheus Santana Costa SOUZA1, Sergio Bruno FERNANDES1, Letícia Vaz MOLINARI1,
Maria Lopes Martins AVELAR1, Douglas Santos GONÇALVES1, Júlio Cezar Tannure FARIA1,
Gustavo Leal TEIXEIRA1, Gilvano Ebling BRONDANI1*
1Universidade Federal de Lavras, Lavras, MG, Brasil.
*E-mail: gilvano.brondani@ufla.br
(Orcid: 0000-0003-4256-7163; 0000-0002-9214-2377; 0000-0002-2543-4628; 0000-0001-6790-685X;
000-0003-2580-8463; 0000-0001-7081-3726; 0000-0001-7293-0790; 0000-0001-8640-5719)
Recebido em 01/12/2021; Aceito em 16/12/2021; Publicado em 21/12/2021.
ABSTRACT: Micropropagation technique is a valuable alternative for high quality genetic preservation of
endemic species such as the orchid Cattleya crispata from “Campo Rupestre Ferruginoso”. This study aims to
evaluate the influence of light quality on in vitro multiplication and elongation phases, offering new insights on
the limiting factors of C. crispata. Seeds extracted from capsules were used for inoculation in the culture medium.
Four light sources were evaluated for in vitro culture, namely: fluorescent lamp, white LEDs, red LEDs and
red/blue LEDs. Data about the number of shoots, shoot length, shooting vigor and pigment content were
assessed at 90 days of in vitro culture. Based on the recorded results, white LEDs are the most suitable ones for
in vitro multiplication and elongation phases of C. crispata. It offers higher quality for seedling production and
increases the chances of genetic conservation of the species.
Keywords: ‘Campo Rupestre Ferruginoso’; in vitro propagation; wavelength; LEDs.
A qualidade da luminosidade influencia o cultivo
in vitro
de
Cattleya crispata
(Thunb.) Van den Berg
RESUMO: A micropropagação é uma alternativa para a conservação genética de espécies endêmicas do
Campo Rupestre Ferruginoso, como a orquídea Cattleya crispata, tendo vantagem a maximização da propagação
em elevada qualidade. Para solucionar os fatores limitantes ao cultivo in vitro de C. crispata, o presente trabalho
teve como objetivo avaliar a influência da qualidade de luz nas fases de multiplicação e alongamento in vitro.
Sementes extraídas de cápsulas foram utilizadas para a inoculação em meio de cultura. Avaliaram-se quatro
fontes de luz (Lâmpada fluorescente, LEDs branco, LEDs vermelho e LEDs vermelho/azul). Dados referentes
ao número, comprimento de brotos, vigor das brotações e conteúdo de pigmentos foram avaliados aos 90 dias
de cultivo. Com base nos resultados obtidos, pode-se inferir que LEDs branco é mais adequada para ser
utilizada nas fases de multiplicação e alongamento in vitro de C. crispata, sendo uma alternativa para a produção
de mudas e conservação genética.
Palavras-chave: Campo Rupestre Ferruginoso; propagação in vitro; comprimento de onda; LEDs.
1. INTRODUCTION
The Orchidaceae family has many Angiosperms that
belong to the Asparagales order, which comprises
approximately 24,500 species distributed within 800 genera
(CHASE et al., 2015). Brazil holds a great diversity of orchids.
It has approximately 239 genera and 2,553 species; 1,636 of
them are endemic (Van den Berg, 2008). Some of those are
treasured commercial flowers, but the downside is that their
culture is based on extractivism, which leads to the
destruction of natural habitats and the loss of many
individuals (BARROS et al., 2015; ANUCHAI et al., 2017).
The genus Cattleya is native to tropical regions in Central
and South America; it encompasses 48 species (Van den
BERG, 2014). This genus contains plants that are found in
trees from humid forests - from sea level to altitudes up to
1,500m. Cattleya crispata is a native and endemic species to the
Minas Gerais State in Brazil; it is mainly found in the Serra
de Ibitipoca region (BARROS et al., 2015), which is a chain
of mountains - part of “Campo Rupestre Ferruginoso”
formation (Van den BERG, 2014).
However, Cattleya crispata culture remains a challenge,
since there are specific demands in the germination, growth,
and developmental phases. The population’s regeneration is
compromised by slow growth and mutualistic interactions
with insects and microorganisms, because of pollination or
germination (MASSARO et al., 2018). Micropropagation is
an alternative to the genetic conservation and multiplication
of Cattleya crispata since thousands of plants can be produced
from a single propagule. Micropropagation allows genetic
gain fixation in clonal populations and maximizes plant high-
quality propagation within a small physical space and in a
short period-of-time, regardless of limiting climatic factors
(TRUEMAN et al., 2018; ABIRI et al., 2020).
There is an obvious need for studies on seedling
production and understanding the limiting factors of each in
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583
vitro culture phase of C. crispata. Innovations in the culture
environment, such as light quality from light emitting diodes
(LEDs), stood out among several technologies that have
been proposed to improve micropropagation processes
(FARIA et al., 2019; RIBEIRO et al., 2019). Studies have
indicated that blue (450 – 495 nm), red (620 – 750 nm), extra
red (750 – 850 nm) and green lights (495 – 570 nm) influence
in vitro morphogenesis and plant development (Batista et al.,
2018). Micropropagation studies and research on the effects
of the different LED wavelengths on the yet non-existing
morphogenesis and growth processes have been carried out.
The development of specific micropropagation methods
is of paramount importance, given the need for genetic
conservation of C. crispata species. Thus, the aim of the
present study was evaluating the effect of light quality on in
vitro multiplication and elongation.
2. MATERIAL AND METHODS
2.1. Study site and experimental material
Experiments were conducted at the Laboratory of Tissue
Culture of the Agriculture Department of the Federal
University of Lavras – UFLA, Lavras – MG, Brazil. The
material used for the generation of explants resulted from
seeds extracted from mature Cattleya crispata capsules, which
were provided by the GERDAU Açominas S.A. company,
Ouro Branco – MG, Brazil.
2.2.
In vitro
establishment
Seeds were washed in running water and immersed in a
solution with 2.4 g L-1 of Orthocide 500® (Captan 50% was
used as active ingredient) for 15 minutes. Next, they were
washed five times in autoclaved deionized water and
immersed in 70% hydroalcoholic solution (v/v) for 20
seconds, under constant stirring, in horizontal laminar flow
chamber. Subsequently, seeds were washed five times in
autoclaved deionized water and inoculated under aseptic
conditions in test tubes (25 × 150 mm) added with 10 mL of
culture medium.
The basic culture medium of the experiments was a MS
saline formulation (MURASHIGE & SKOOG 1962) added
with 30 g L-1 sucrose (Synth Ltda), 6 g L-1 agar (Merck S.A.)
and 1 g L-1 activated charcoal (Merck S.A.). Culture medium
pH was adjusted to 5.8 ± 0.05, prior to agar addition. Culture
medium autoclaving was carried out at 127°C under pressure
of 1.5 kgf cm-2, for 20 minutes.
Seeds were kept for 90 days in a growth room at 24 ±
1°C, under 16-h light photoperiod and irradiance of 40 μmol
m-2 s-1 (which was quantified in radiometer, LI-COR®, LI-
250A Light Meter) under cold white fluorescent lamps.
Subcultures were set for culture renewal every 30 days.
2.3. Multiplication
Four shoots (standardized at 0.5cm) were prepared and
inoculated under aseptic conditions in glass flasks (250 mL
capacity) added with 50 mL of MS basic culture medium
supplemented with 0.5 mg L-1 BAP (6-benzylaminopurine –
Sigma®), 0.05 mg L-1 ANA (α-naphthalene acetic acid –
Sigma®), 6 g L-1 agar and 1 g L-1 activated charcoal after seed
germination and in vitro establishment, at 90 days of culture.
The experiment followed a randomized design, under
four different light sources: fluorescent lamp - F/L (HO
Sylvania T12, 110 W, São Paulo, Brazil), White-LEDs – W/L
(SMD 100, 18 W, Vilux®, Vitória, ES, Brazil), Red LEDs –
R/L (LabPARLL-HR / DB-480, 11.6 W, LabLumens®,
Carapicuíba, SP, Brazil) and 1:1 red/blue LEDs – RB/L
(LabPARLL-HR / DB-480, 11.6 W, LabLumens®,
Carapicuíba, SP, Brazil), with eight repetitions (composed of
four explants, each).
The mean number of shoots per explant (> 0.5 cm),
shoots length (> 0.5 cm), vigor based on the scores scale
proposed by Souza et al. (2020a) (Figure 1A-C) and the
photo-synthetic ink content were assessed at 90 days of
culture.
Figure 1. Assessment of vigor, based on the scores scale applied to
Cattleya crispata. A. 1 = Excellent: shootings induction by active
growth, without apparent nutrition deficit; B. 2 = Good: shootings
induction, but reduced leaf size; C. 3 = Low: absence of shootings
induction and/or senescence and death. Bars = 1.0 cm.
Figure 1. Avaliação do vigor de acordo com a escala de notas em
Cattleya crispata. A. 1 = Ótimo: indução de brotações com
crescimento ativo, sem deficiência nutricional aparente; B. 2 =
Bom: indução de brotações, porém com folhas de tamanho
reduzido; C. 3 = Baixo: ausência de indução das brotações e/ou
senescência e morte. Barras = 1,0 cm.
2.4. Elongation
Shoots produced in the in vitro multiplication phase were
prepared; four shoots (standardized at 0.5cm) were isolated
and inoculated (under aseptic conditions) in glass flasks (250
mL capacity). Each flask was added with 50 mL of MS culture
medium, 30 g L-1 sucrose, 6 g L-1 agar 1 g L-1 activated
charcoal, 0.05 mg L-1 BAP and 0.5 mg L-1 indole-3-butyric
acid (AIB) (Sigma®).
The experiment followed a randomized design, under
four different light sources: Fluorescent lamp F/L, LED
lamp white W/L, LED lamp red R/L and LED lamp
red/blue-RB/L 1:1, with eight repetitions (composed of four
explants, each).
Data about the mean number of shoots per explant (>
0.5 cm), shoot length (> 0.5 cm), vigor based on the scores
scale and photo-synthetic ink content were collected at 90
days of culture.
2.5. Quality of light
A Spectrum radiometer (Ocean Optics Spectra-Suite) was
used for the analyzes of variations in the absolute irradiance
(μW cm-2 nm-1) and light wavelength (nm) of different lamp
types during the in vitro multiplication and elongation phases.
It was done to plot the graphics of different light spectra
(Figure 2).
2.6. Photosynthetic pigment analysis
Leaf discs (25 mg of leaf fresh matter) were sampled from
shooting for in vitro multiplication and elongation assays, after
90 days of culture, under different light sources. Samples
were inoculated with 5 mL of DMSO solution (Sigma
Aldrich®) and stored for 48 hours in the dark
(LICHTENTHALER, 1987). Sample absorbance was
determined in triplicate, in 10 mm optical path quartz cuvette,
in Genesys 10UV spectrophotometer (ThermoScientific®,
EUA).
The influence of light quality in the in vitro cultivation of Cattleya crispata
Nativa, Sinop, v. 9, n. 5, p. 582-588, 2021.
584
Figure 2. Variations in absolute (μW cm-2 nm-1) and light wavelength
(nm) of different lamp types during in vitro culture of Cattleya crispata.
A. Fluorescent lamp; B. White LEDs; C. Red LEDs; D. Red/blue
LEDs 1:1.
Figure 2. Variações da irradiância absoluta (μW cm-2 nm-1) e do
comprimento de onda (nm) de luz emitido pelas diferentes
lâmpadas durante o cultivo in vitro de Cattleya crispata. A. Lâmpada
fluorescente; B. LEDs branco; C. LEDs vermelho; D. LEDs
vermelho/azul 1:1.
2.7. Data analysis
The analyses were carried out in R Core Team software,
2018, with the aid of the ExpDes Package, version 1.1.2
(FERREIRA et al., 2013). Data were subjected to analysis of
variance (ANOVA) with Tukey test at 5% significance level.
3. RESULTS
3.1. Quality of light on
in vitro
multiplication
Different qualities of light influenced the number of
shoots per explant and led to the highest means under
sources W/L (4.0 shoots) and F/L (3.7 shoots); results were
statistically different (p < 0.05) under sources R/L and RB/L
(Figure 3A). Shoots length presented similar behavior: the
best results were recorded for sources W/L (2.74 cm, on
average) and F/L (2.44 cm, on average), which recorded
values different from those recorded under sources R/L and
RB/L (Figure 3B).
Figure 3. Features observed at 90 days of in vitro multiplication phase of Cattleya crispata related to different qualities of light (Fluorescent
lamp (F/L), white LEDs (W/L), red LEDs (R/L), and red/blue LEDs (RB/L)). A. Mean number of shoots per explant; B. Mean shoots
length; C. Vigor; D. Mean pigment content. *Means followed by the same letter did not differ from each other in the Tukey test at 5%
significance level. Bars represent sample standard deviation.
Figure 3. Características observadas aos 90 dias durante a fase de multiplicação in vitro de Cattleya crispata em função das diferentes qualidades
de luz (Lâmpada fluorescente (F/L), LEDs branco (W/L), LEDs vermelho (R/L) e LEDs vermelho/azul (RB/L). A. Número médio de
brotos por explante; B. Comprimento médio de broto; C. Vigor; D. Conteúdo médio de pigmentos. *Médias seguidas de uma mesma letra
não diferem entre si, pelo teste de Tukey à 5% de significância. Barras representam o desvio padrão amostral.
W/L presented the lowest means of explant vigor (1.12)
based on the scores scale; this result was significantly
different (p < 0.05) under light sources R/L and RB/L
(Figure 3C). Regarding the content of photosynthetic
pigments, the amount of chlorophyll a, b, a + b and
carotenoids were influenced by different light qualities
(Figure 3D). The results showed that the light sources W / L
and F / L had the highest levels of photosynthetic ink.
3.2. Quality of light on
in vitro
elongation
A difference in responses between light sources and the
growth pattern of C. crispata plants in the in vitro multiplication
Souza et al.
Nativa, Sinop, v. 9, n. 5, p. 582-588, 2021.
585
and elongation phases at 90 days of culture (Figure 5) was
observed. Based on the quality of the analyzed lights, F/L
stood out for producing the largest number of shoots (3.68
shoots, on average, Figure 4A), which was statistically
different from that recorded for the other treatments (p <
0.05). However, the lowest shoots production mean was
observed under light source RB/L (2.0 shoots).
For the shoots length, the best results of were under light
source W/L (4.43 cm, on average) (Figure 4B). Regarding
explant vigor, the lowest mean values based on the score
scale were observed under W/L (1.12) (Figure 4C), which led
to tissue growth due to lack of nutritional deficit.
The best ink content results were those recorded under
light source W/L (Figure 4D); this outcome evidenced the
importance of using light-emitting diodes (LEDs) that
present greater wavelength specificities.
Based on the evaluated characteristics, it was possible to
observe difference in the growth pattern of C. crispata
explants through different light qualities used for in vitro
multiplication and elongation at 90 days (Figure 5).
Figure 4. Features observed at 90 days of in vitro elongation phase of Cattleya crispata due to different qualities of light (F/L) fluorescent lamp,
white LEDs (W/L), red LEDs (R/L), and red/ blue LEDs (RB/L)). A. Mean number of shoots per explant; (B) Mean shoots length; C.
Vigor; D. Mean ink content. *Means followed by the same letter did not differ from each other in the Tukey test at 5% significance level.
Bars represent the sample standard deviation.
Figure 4. Características observadas aos 90 dias durante a fase de alongamento in vitro de Cattleya crispata em função das diferentes qualidades
de luz (Lâmpada fluorescente (F/L), LEDs branco (W/L), LEDs vermelho (R/L) e LEDs vermelho/azul (RB/L). A. Número médio de
brotos por explante; (B) Comprimento médio das brotações; C. Vigor; D. Conteúdo médio de pigmentos. *Médias seguidas de uma mesma
letra não diferem entre si, pelo teste de Tukey à 5% de significância. Barras representam o desvio padrão amostral.
Figure 5. Explants of orchids from the species Cattleya crispata under different qualities of light at 90 days of culture. A-D. In vitro
multiplication; E-H. In vitro elongation; A and E. Fluorescent lamp; B and F. White LEDs; C and G. Red LEDs; D and H. red/blue
LEDs. Bars = 1.0 cm.
Figure 5. Explantes da orquídea Cattleya crispata em diferentes qualidades de luz, aos 90 dias de cultivo. A-D. Multiplicação in vitro; E-H.
Alongamento in vitro; A e E. Lâmpada fluorescente; B e F. LEDs branco; C e G. LEDs vermelho; D e H. LEDs vermelho/azul. Barra =
1,0 cm.
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Nativa, Sinop, v. 9, n. 5, p. 582-588, 2021.
586
4. DISCUSSION
4.1. Quality of light on
in vitro
multiplication
The improvement of protocols for in vitro multiplication
that influences the development of micropropagation, were
studied with the attempt to establish cultivation conditions
that can maximize the production of clonal seedlings on a
large scale. The results of the morphological characteristics
evaluated with the orchid C. crispata, provided the
optimization of the in vitro multiplication phase, through the
use of light quality.
Treatment with white LED light quality provided the best
results for number of shoots per explant, shoot length and
vigor after 90 days of cultivation. Data in the literature
corroborated the current results, since the use of white LEDs
on the orchid species Microlaelia lundii (FAVETA et al., 2017)
and Corymbia torelliana × C. citriodora (SOUZA et al., 2020b)
favored the largest number of and longest shoots. On the
other hand, the smallest number and shortest length of
explants from Ajuga multiflora were recorded under red/blue
LED light (BYOUNG; IYYAKKANNU, 2018).
These results are also in line with those found by Souza
et al. (2020b), who observed adequate explant vigor of
Corymbia torelliana × C. citriodora. According to Abiri et al.
(2020), the success of the multiplication phase requires
explants for the emission of shoots free from contamination
and with adequate vegetative vigor.
Light quality control on in vitro culture under LED light
sources is essential for photosynthetic rate optimization, a
fact that contributes to the growth and increase of shoots
production (SILVA et al., 2017). However, light can also
influence the concentration of growth regulators by acting as
a morphogenic sign to shoots proliferation during the in vitro
multiplication. The initial phases in the light-induced
signaling involve the activation of cytokinins (ROMAN et al.,
2016), which may be beneficial for shoots production.
As for the content of photosynthetic pigments
(chlorophyll a, b a+b and carotenoids), the highest values were
observed in light of white LEDs. Similar results were
observed in Bixa orellana cultivar UESB74 under white LEDs
(FARIA et al., 2019). The biosynthesis of chlorophyll a, b and
of carotenoids was influenced by the quality and intensity of
the emitted light. The photosynthetic process presented the
highest efficiency, mainly when the light source with the
greatest wavelength specificity was adopted (GUPTA;
KARMAKAR, 2017).
4.2. Quality of light on
in vitro
elongation
The specificity of the light quality fluorescent and white
provided the best results for the morphological
characteristics studied in the in vitro elongation. Similar results
were observed when fluorescent light was the light source
provided to Abies borisii-regis explants (SMIRNAKOU et al.,
2016).
In contrast, it was observed that the use of light quality
red and red blue is inadequate. The blue region of the light
spectrum (450 to 500 nm) can act in growth and productivity
inhibition (Abiri et al., 2020).
Wavelengths in LED light sources have a specific action
in plants, they influence different factors in plants’
photosynthetic processes, morphogenesis, and physiological
processes (HE et al., 2017). LED light sources can be used
separately, or in combination, for the optimization of
morphophysiological processes (SHENGXIN et al., 2016).
However, it is important to highlight that light wavelength
has varying responses according to the genotype
(MIRANDA et al., 2020); therefore, adjustments are
necessary for each species and/or genotype.
As for the content of photosynthetic pigments, the
highest values were also observed for fluorescent light
sources and white LEDs. Ink content in Fagopyrum tataricum
explants exposed to white LED was higher than that
recorded for plants treated with blue and red LEDs (TUAN
et al. 2013).
LEDs performance has increased the amount of
chlorophyll and carotenoids and the photosynthetic growth
ability of certain species (SOUZA et al. 2020a). Carotenoids
play an important role in photosynthesis since they protect
the photosystem from photo-oxidation. However, the effects
of different light wavelengths on carotenoid biosynthesis may
depend on the assessed tissues and plants (TIAN et al. 2019).
Given the above, it is important to note that light emitting
diodes (LEDs) are alternative light sources, given their
wavelength specificities, a moderate amount of thermal
emission, little degradation and long service life. These
characteristics favor in vitro culture and reduce costs
compared to fluorescent lamps (BUGBEE, 2016). Therefore,
LEDs are an excellent alternative in plant tissue culture.
5. CONCLUSION
LEDs presented the best results for in vitro multiplication
and elongation of Catlleya crispata also leading to greater vigor,
longer shoots, a larger mean number of shoots per explant,
and higher photosynthetic ink content. This finding turns
LED into an alternative to seedling production and the
genetic conservation of the species.
6. ACKNOWLEDGEMNTS
We are grateful to “Fundação de Amparo à Pesquisa do
Estado de Minas Gerais (FAPEMIG)” for the granted
scholarships. The present study was carried out with the
support of “Conselho Nacional de Desenvolvimento
Científico e Tecnológico (CNPq)” and “Coordenação de
Aperfeiçoamento de Pessoal de Nível Superior (CAPES)”.
We also thank GERDAU Açominas S. A.
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