Nativa, Sinop, v. 9, n. 4, p. 352-358, 2021.
Pesquisas Agrárias e Ambientais
DOI: https://doi.org/10.31413/nativa.v9i4.12164 ISSN: 2318-7670
Activated charcoal application for the micropropagation of
Cattleya crispata
(Thunb.) Van den Berg
Denys Matheus Santana Costa SOUZA1, Sérgio Bruno FERNANDES1, Letícia Vaz MOLINARI1,
Maria Lopes Martins AVELAR1, Gilvano Ebling BRONDANI1*
1Federal University of Lavras, Lavras, MG, Brazil.
*E-mail: gilvano.brondani@ufla.br
(ORCID: 0000-0003-4256-7163; 0000-0002-9214-2377; 0000-0002-2543-4628; 0000-0001-6790-685X; 0000-0001-8640-5719)
Recebido em 09/04/2021; Aceito em 31/08/2021; Publicado em 20/09/2021.
ABSTRACT: Micropropagation is an alternative for the genetic conservation and propagation of endemic
species from rupestrian grasslands, such as the orchid Cattleya crispata. The aim of the present study is to assess
the influence of activated charcoal on the in vitro germination, multiplication and elongation phases of C. crispata.
Seeds extracted from mature capsules were used for inoculation in the culture medium that was adopted to
assess the effect of supplementation, or not, with activated charcoal. Data about germination speed, number
of shoots per explant, length, vigor, oxidation and contamination (bacterial and/or fungal) were assessed
through these phases. Based on the results obtained, the use of activated charcoal was efficient in the in vitro
germination and multiplication phases of C. crispata, providing greater speed and percentage of germination,
less contamination and oxidation of the tissues, greater number, length and vigor of shoots, being effective for
the genetic conservation and production of plants of the species. Culture medium without the supplementation
of activated charcoal provided the best results for the in vitro elongation, with greater length, vigor and less
oxidation of shoots.
Keywords: antioxidant; orchid; propagation in vitro; rupestrian grasslands.
Aplicação de carvão ativado para micropropagação de
Cattleya crispata
(Thunb.) Van den Berg
RESUMO: A micropropagação é uma alternativa para a conservação genética e propagação de espécies
endêmicas do “Campo Rupestre Ferruginoso”, como a orquídea Cattleya crispata. O objetivo do presente estudo
é avaliar a influência do carvão ativado nas fases de germinação, multiplicação e alongamento in vitro de C.
crispata. Sementes extraídas de cápsulas maduras foram utilizadas para inoculação no meio de cultura adotado
para avaliar o efeito da suplementação, ou não, com carvão ativado. Dados sobre velocidade de germinação,
número de brotos por explante, comprimento, vigor, oxidação e contaminação (bacteriana e / ou fúngica)
foram avaliados por meio dessas fases. Com base nos resultados obtidos, o uso do carvão ativado foi eficiente
nas fases de germinação e multiplicação in vitro de C. crispata, proporcionando maior velocidade e porcentagem
de germinação, menor contaminação e oxidação dos tecidos, maior número, comprimento e vigor dos brotos,
sendo eficaz para a conservação genética e produção de mudas da espécie. O meio de cultura sem a
suplementação de carvão ativado proporcionou os melhores resultados para o alongamento in vitro, com maior
comprimento, vigor e menor oxidação dos brotos.
Palavras-chave: antioxidante; campos rupestres; orquídea; propagação in vitro.
1. INTRODUCTION
Orchidaceae is one of the biggest families belonging to
division Angiospermae, which encompasses approximately
900 genera and 27,135 species (CHASE et al., 2015). Brazil
houses one of the largest orchid floras in the world, and many
of them are epiphytes and endemic; they are represented by
240 genera and 2,443 species – 1,634 are endemic (BARROS
et al., 2015). However, because they have high market value,
these species face great extractivism in their natural habitat
for commercial exploration purposes, which leads to the loss
of many individuals (BARROS et al., 2015; ANUCHAI et al.,
2017).
Cattleya includes more than 113 species distributed in
South and Central America (BERKA et al., 2014); in Brazil,
this genus is represented by 48 species of epiphyte plants
whose geographical distribution covers all states in the
country, except for Amapá and Piauí (BARROS et al., 2015).
Among species belonging to this genus, Cattleya crispata
(Thunb.) Van den Berg is an endemic species native to Minas
Gerais State, mainly distributed in Serra de Ibitipoca
(BARROS et al., 2015), which is part of rupestrian grassland
formation (VAN DEN BERG, 2014).
Nevertheless, tissue culture technique has been helping
to preserve these species, since they allow the management
of a large number of individuals living in reduced spaces,
under aseptic conditions (RIGHETO et al., 2012; VILLA et
al., 2014; POOBATHY et al., 2019). Accordingly,
micropropagation, from germination to the subsequent
phases on in vitro culture, is an efficient propagation and
Souza et al.
Nativa, Sinop, v. 9, n. 4, p. 352-358, 2021.
353
conservation technique applied to the germplasm of orchid
species (GOMES et al., 2015; HUNHOFF et al., 2018).
However, the success of orchid in vitro propagation is
influenced by many factors, such as plant genotype and
culture medium composition (HUNHOFF et al., 2018; KIM
et al., 2019).
The definition of an appropriate culture medium is a
limiting factor for orchid micropropagation (KIM et al.,
2019); therefore, it is essential for studies carried out in each
in vitro cultivation phase of C. crispata. Activated charcoal is
among the components that can be added to culture media
in order to act as antioxidant and to promote the adsorption
of auxins and cytokinins, plant exudates and toxic
metabolites (ANUCHAI et al., 2017; CORBELLINI et al.,
2020).
The development of specific micropropagation is a
strategy to be taken into consideration given the need of
genetically conserving and propagating the species C. crispata.
Therefore, the aim of the study was to assess the effect of
activated charcoal supplemented in the culture medium at the
in vitro germination, multiplication and elongation phases.
2. MATERIAL AND METHODS
2.1. Study site and experimental material
The experiments were carried out for 270 days, at the
Laboratory of In Vitro Culture of Forest Species of the
Department of Forestry Sciences of Federal University of
Lavras, Lavras – MG, Brazil. The material used to generate
the explants resulted from seeds extracted from mature
capsules of Cattleya crispata, which were provided by the
Gerdau’s Unidade de Pesquisa e Inovação em Campos
Rupestres Ferruginosos (GERDAU Açominas S.A), located
in Ouro Branco – MG, Brazil.
2.2.
In vitro
germination
Seeds were washed in running water and immersed in
fungal solution with 2.4 g L-1 of Orthocide 500® (50%
Captan 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 immersed in 2.0-2.5%
NaOCl solution (v/v) Clarix®, for 5 minutes (SOUZA et al.,
2020). Finally, they were washed in deionized water and
autoclaved five times; 50 mg of seeds were inoculated in test
tube (25 × 150 mm) filled with 10 mL of culture medium
under aseptic conditions.
MS (Murashige; Skoog, 1962) was the basic culture
medium used in the experiment. It was added with 30 g L-1
of sucrose (Synth Ltda) and 6 g L-1 of agar (Merck S.A.).
Culture medium pH was adjusted to 5.8 ± 0.05 prior to agar
addition; its autoclaving was carried out at 127°C and
pressure of 1.5 kgf cm-2, for 20 minutes.
Seeds were kept in growth room for 90 days at
temperature of 24 ± 1°C, 16-hour light photoperiod and
irradiance of 40 μmol m-2 s-1 (which was quantified in
radiometer LI-COR®, LI-250A Light Meter) under white
cold fluorescent light bulb.
The experiment followed a completely randomized
design and used MS culture medium added with 1 g L-1 of
activated charcoal (W/AC) (Merck S.A.), or without it
(Wout/AC), with 30 replications (50 mg of seeds per
replication). Data about germination percentage were
collected at 20, 30, 40, 50 and 60 days. Information about
percentage of contamination by fungi and bacteria, oxidation
and non-responsive seeds were collected at 90 days of
subculture.
2.3.
In vitro
multiplication
After seed germination in vitro at 90 days of subculture,
three standardized seedings (0.5 cm in length) were prepared
and inoculated in glass flasks (250 mL capacity) filled with 50
mL of basic MS culture medium, which was supplemented
with 30 g L-1 of sucrose, 6 g L-1 of agar, 0.5 mg L-1 of 6-
benzylaminopurine (BAP – Sigma®) and 0.05 mg L-1 of α-
naphthalene acetic acid (NAA – Sigma®). The explants were
cultivated for 90 days and the subcultures were used to renew
the culture medium every 30 days.
The in vitro multiplication experiment was installed based
on a completely randomized design. Saline MS culture
medium added with 1 g L-1 of activated charcoal (W/AC),
and without it (Wout/AC), was used in the experiment, with
30 replications (3 explants per replication; 90 individuals, in
total). The mean number of shoots per explant (> 0.5 cm),
shoots length (> 0.5 cm), vigor and oxidation based on the
scores’ scale proposed by Souza et al. (2020) (Fig. 1 A-F) were
assessed at 90 days of in vitro culture.
Figure 1. Vigor and oxidation assessments based on the scores’ scale
of tissues of C. crispata. A-C. Explant vigor (1 = Excellent: shoot
induction due to active growth, without apparent nutrition deficit; 2
= Good: shoot induction, but with leaves presenting reduced size;
3 = low: no seedling induction and/or senescence and death). D-F.
Explant oxidation (1 = Null: no oxidation; 2 = Intermediate:
reduced explant oxidation; 3 = High: full explant oxidation). Bar –
1 cm.
2.4.
In vitro
elongation
Shoots generated in the multiplication phase in vitro at 90
days of culture were prepared as follows: three standardized
seedlings (0.5 cm in length) were inoculated in glass flasks
(250 mL capacity). Each flask was added with 50 mL of MS
culture medium, 30 g L-1 of sucrose, 6 g L-1 of agar, 0.05 mg
L-1 of BAP and 0.5 mg L-1 of indole-3-butyric acid (IBA –
Sigma®). Explants were cultivated at 90 days and subcultures
were used for culture medium removal every 30 days.
The in vitro elongation experiment followed a completely
randomized design. It used saline MS culture medium added
with 1 g L-1 of activated charcoal (W/AC), or without it
(Wout/AC), with 30 replications (three explant per
replication); thus totaling 90 individuals. The mean number
of shoots per explant (> 0.5 cm), shoots length (> 0.5 cm),
vigor and oxidation based on the scores’ scale were assessed
at 90 days of culture.
Activated charcoal application for the micropropagation of Cattleya crispata (Thunb.) Van den Berg
Nativa, Sinop, v. 9, n. 4, p. 352-358, 2021.
354
2.5. Data analysis
Analyses were carried out in the R software, version 3.0.3
(R Core Team 2018), with the aid of package ExpDes,
version 1.1.2 (FERREIRA et al., 2013). Variables that did not
present normal distribution in the Shapiro-Wilk test, at 5%
significance level, were transformed into arcsen. Means
recorded for the treatments were subjected to analysis of
variance (ANOVA) through Tukey test, at 5% significance
level; the regression equations were adjusted.
3. RESULTS
3.1. Activated charcoal in the
in vitro
germination
The addition of activated charcoal to culture medium has
influenced germination speed of orchid C. crispata, in vitro.
Seeds subjected to the treatment with activated charcoal
germinated 30 days after inoculation (30% germination, on
average); on the other hand, the treatment without activated
charcoal recorded germination 40 days after inoculation
(20% germination, on average) (Fig. 2A).
Figure 2. Features observed in the in vitro germination phase of C.
crispata, with and without culture medium supplementation with
activated charcoal. A. Germination speed at 20, 30, 40, 50 and 60
days; B. Fungal contamination percentage; C. Bacterial
contamination percentage; D. Oxidation percentage; E. Percentage
of non-responsive seeds. *Means followed by the same letter did not
differ from each other in the Tukey test at 5% significance level. Bars
represent the standard deviation of the sample.
The treatment with activated charcoal led to higher
germination percentage at 60 days of culture (83.3%, on
average), and this result was statistically different from the
treatment without it (50% germination, on average) (Fig. 2A).
With respect to non-responsive seeds, the treatment without
activated charcoal resulted in high mean for this variable
(30%) and in statistical difference from the treatment without
it (6.6%, on average) (Fig. 2E).
There were similar components in the percentage of
contamination by fungal and bacterial, and oxidation of
tisses; the lowest means recorded for these variables were
observed for treatments with activated charcoal (6.6, 0 and
3.3%), but they did not differ from the treatment without
activated charcoal (Figs 2B, 2C and 2D). The best results
given the characteristics evaluated with the presence of
activated charcoal, denote the potential of the antioxidant for
the in vitro germination of C. crispata.
3.2. Activated charcoal in the
in vitro
multiplication
The effect of supplementation with activated charcoal,
significantly inferred the morphological characteristics
studied in the in vitro multiplication of C. crispata.
Supplementation with activated charcoal at 90 days of
culture for in vitro multiplication of orchid C. crispata led to
the largest number and longest length of shooting (5.0
shooting – 1.8 cm, on average). This outcome was statistically
different from the treatment without it (3.9 shooting – 1.3
cm, on average) (Figs 3A and 3B). The addition of activated
charcoal to the culture medium increases the adsorption of
substances that inhibit plant development, as well as
minimizes the toxicity of plant growth regulators or of
exogenous substances with harming effect, which
contributed to greater explant in vitro growth.
According to the scores’ scale, vigor and oxidation
presented the lowest means (1.3) due to the addition of
activated charcoal to the culture medium; therefore, there
was no statistical difference from the treatment without
activated charcoal (Figs. 3C and 3D). The number of buds
per explant, vigor and oxidation are characteristics that stand
out as a tool to evaluate the efficiency of the multiplication
phase, demonstrating to be factors that exert great influence
on in vitro cultivation.
Figure 3. Features observed at 90 days of in vitro culture in the
multiplication of C. crispata, with and without culture medium
supplementation with activated charcoal. A. Number of shooting
per explants; B. Shooting length; C. Vigor; D. Oxidation. *Means
followed by the same letter did not differ from each other in the Tukey test
at 5% significance level. Bars represent the standard deviation of the sample.
Souza et al.
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355
3.3. Activated charcoal in the
in vitro
elongation
Based on the assessed features, was possible observing
difference in the responses to treatment with, or without,
culture medium supplementation with activated charcoal and
in the growth pattern of C. crispata plants in the in vitro
germination, multiplication and elongation phases at 90 days
(Fig. 5).
The use of activated charcoal had direct influence on the
in vitro elongation of C. crispata explants, it was determining
for the largest number of shoots (3.3 shoots, on average) and
for the shortest shoots length (2.3 cm, on average); therefore,
there was statistical difference from treatments without
activated charcoal supplementation (Figs. 4A and 4B). With
the use of activated carbon at a concentration of 1 g L-1,
inhibition of explant growth was observed in in vitro
elongation, when compared to the system without
antioxidant supplementation.
Vigor and oxidation assessments based on the scores’
scale showed the lowest means without activated charcoal
(1.3 and 1.3, on average) (Figs. 4C and 4D); however, there
was no significant different from treatments without it. The
treatment without the use of activated carbon provided the
best results, being fundamental for the in vitro elongation
phase, contradicting the aforementioned results of the in vitro
germination and multiplication phases of C. crispata.
Figure 4. Features observed at 90 days of culture in the in vitro
elongation of C. crispata, with or without culture medium
supplementation with activated charcoal. A. Number of shoots per
explant; B. Shoots length; C. Vigor; D. Oxidation. *Means followed
by the same letter did not differ from each other in the Tukey test at 5%
significance level. Bars represent the standard deviation of the sample.
4. DISCUSSION
4.1. Activated charcoal in the
in vitro
germination
The improvement of protocols for in vitro establishment
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 germination phase, through the
use of activated charcoal.
The treatment with the use of activated charcoal provided
the highest germination percentage at 60 days of cultivation
and the lowest percentages of contamination, oxidation and
unresponsive explants. These outcomes are similar to those
found by Kim et al. (2019), who used 1 g L-1 of activated
charcoal in the in vitro germination and regeneration of Pecteilis
radiate. The addition of activated charcoal to the culture
medium favored the maturation and germination of somatic
embryos of Acrocomia aculeata (GRANJA et al., 2018), and it
has pointed out the importance of culture medium
supplementation with it.
The response of orchid seeds in in vitro germination is
influenced by many factors, such as seed age, culture medium
composition (carbohydrate source, antioxidants, plant
growth regulators), culture condition and genotype (PARK
et al., 2018; SEON et al., 2018). However, contamination by
microorganisms (fungal and/or bacterial) is an issue
associated with micropropagation, because these
microorganisms establish themselves in the culture medium
and/or in the vegetal material and cause toxicity due to the
high production of toxic metabolites, as well as to pH
reduction in the culture medium, which, consequently, leads
to lower absorption of macro and micronutrients by plants
and, unfortunately, to death (LEONE et al., 2016).
Data in the literature corroborated findings in the current
study, since culture medium supplementation with 1 g L-1 of
activated charcoal reduced oxidation and contamination in
comparison to the treatment without it, during the
micropropagation of Cattleya walkeriana (SOUZA et al., 2007)
and Eugenia pyriformis (ASSIS et al., 2018). Futhermore, the
germination and elongation phases, in vitro, also demand the
control of oxidation caused by phenolic exudation in the
region where explants are cut; oxidation was also responsible
for explant darkening due to the activity of enzymes such as
peroxidase and polyphenol oxidase (WILLADINO et al.,
2013; CORBELLINI et al., 2020).
Activated charcoal acts as antioxidant and promotes the
adsorption of plant exudates and toxic metabolites
(HUNHOFF et al., 2018) due to the presence of a network
of pores in the structure of activated charcoal, which allows
many inhibitory substances in the medium, or released by the
explants, to remain adsorbed (AGUILAR et al., 2016). Thus,
the methodology used in the study with the supplementation
of activated charcoal was observed the best results, providing
greater development of explants, in which are important
strategies to be adopted in the propagation system.
4.2. Activated charcoal in the
in vitro
multiplication
Supplementation with the activated charcoal significantly
affected the morphological characteristics studied in in vitro
multiplication, when compared without its supplementation.
The use of antioxidants (polyvinylpyrrolidone - PVP and
activated charcoal) allowed lower oxidation and greater
development of Terminalia amazonia explants during
micropropagation (MÉNDEZ et al., 2014). The use of
activated charcoal in the intergeneric hybrid of orchid
Brassocattleya pastoral × Laeliocattleya amber accounted for the
largest number of shoots (VILLA et al., 2014).
The addition of activated charcoal to the culture medium
increases the adsorption of substances - such as phenols and
ethylene - that inhibit plant development, as well as
minimizes the toxicity of plant growth regulators or of
exogenous substances with harming effect, which
contributes to greater shoots proliferation and explant in vitro
growth (SARTOR et al., 2013; ABIRI et al., 2020), as
observed in the present study.
Activated charcoal application for the micropropagation of Cattleya crispata (Thunb.) Van den Berg
Nativa, Sinop, v. 9, n. 4, p. 352-358, 2021.
356
According to the scores’ scale, vigor and oxidation
presented the lowest means, due to the addition of activated
charcoal to the culture medium. These results are close to the
ones found by Souza et al. (2020), who observed low
phenolic oxidation and good vigor in the explants of Corymbia
citriodora × C. torelliana in vitro cultivated. The use of
antioxidants (PVP and activated charcoal) in Abelmoschus
esculentus (IRSHAD et al., 2017), Psidium guajava (AGUILAR
et al., 2016) and Lupinus mutabilis (MAMANI et al., 2014),
explants reduced the effects of phenolic exudation and
promoted better development in micropropagation.
4.3. Activated charcoal in the
in vitro
elongation
The supplementation of activated charcoal in in vitro
elongation, showed different responses in relation to the
germination and multiplication phases of C. crispata in vitro.
Depending on the species, explant type or on the cultivation
phase to be propagated, the addition of an antioxidant
(activated charcoal and PVP) to the culture medium can have
positive effects (PANKAJ et al., 2014), as observed in the in
vitro culture of the species under study.
With respect to the negative effects of activated charcoal
supplementation, it is possible highlighting the adsorption of
growth regulators (auxins and cytokinins), vitamins and of
other organic substances important for explant in vitro
development and growth. Activated charcoal also naturally
releases substances that are found on, or adsorbed by, it
(ABIRI et al., 2020; CORBELLINI et al., 2020).
In vitro conditions are often stressing for plant growth and
high concentrations of exogenous antioxidants can affect
development during culture (TISARUM et al., 2018). Kim et
al. (2019), report que culture medium supplementation with
activated charcoal at concentration higher than 3% can
promote and/or inhibit in vitro growth depending on the used
species and tissues, as it was observed in the present study
with the species of orchid C. crispata.
Figure 5. C. crispata explants subjected, or not, to the treatment with
activated charcoal at 90 days after inoculation in each phase. A and
B. In vitro germination; C and D. In vitro multiplication; E and F. In
vitro elongation; A, C and E. without activated charcoal; B, D and F.
with activated charcoal. Bar = 1.0cm.
5. CONCLUSIONS
Supplementation of MS culture medium with activated
charcoal recorded the best results for in vitro germination and
multiplication of C. crispata.
Germination speed and percentage were higher under
these conditions, there was lower contamination and tissue
oxidation, as well as larger number, longer length and better
vigor of explants.
The activated charcoal treatment was effective in the
genetic conservation and production of plants belonging to
this species. For the elongation phase, the MS culture
medium without activated carbon showed the best results:
longer shoots length, better vigor and less oxidation.
6. ACKNOWLEDGEMNTS
We thank the National Council for Scientific and
Technological Development, Brazil (Conselho Nacional de
Desenvolvimento Científico e Tecnológico – CNPq),
Coordination for Improvement of Higher Education
Personnel, Brazil (Coordenação de Aperfeiçoamento de
Pessoal de Nível Superior – CAPES – Código de
Financiamento 001), and Foundation for Research of the
State of Minas Gerais, Brazil (Fundação de Amparo a
Pesquisa do Estado de Minas Gerais - FAPEMIG). We also
thank Gerdau’s Unidade de Pesquisa e Inovação em Campos
Rupestres (GERDAU Açominas S.A.) for the partnership in
the execution of this study.
7. REFERENCES
ABIRI, R.; ATABAKI, N.; ABDUL-HAMID, H.; SANUSI,
R.; SHUKOR, N. A. N.; SHAHARUDDIN, N. A.;
AHMAD, S. A.; MALIK, R. The prospect of
physiological events associated with the
micropropagation of Eucalyptus sp. Forests, v. 11, n. 1, p.
1-29, 2020. https://doi.org/10.3390/f11111211
AGUILAR, L. P.; ESPINO, H. S.; MONTEIRO, L. L. V.;
SEGOVIA, C. P.; BENÍTEZ, S. F. Propagación in vitro
de guayaba (Psidium guajava L.) a partir de segmentos
nodales. Revista Mexicana de Ciencias Agrícolas, v.
7, n. 2, p. 375-386, 2016.
ANUCHAI, V.; HSTEH, C. H. Effect of change in light
quality on physiological transformation of in vitro
Phalaenopsis ‘Fortune Saltzman’ seedlings during the
growth period. The Horticultural Journal, v. 86, n. 3, p.
395-402, 2017. https://doi.org/10.2503/hortj.MI-151
ASSIS, F. A.; RODRIGUES, F. A.; PASQUAL, M.; ASSIS,
G. A.; LUZ, J. M. Q.; JANONI, F.; COSTA, I. J. S.;
COSTA, B. N. S.; SOARES, J. D. R. Antioxidants in the
control of microorganism contamination and phenol
oxidation in Eugenia pyriformis. Bioscience Journal,
Uberlândia, v. 34, n. 1, p. 49-58, 2018.
https://doi.org/10.14393/BJ-v34n1a2018-36311
BARROS, F.; VINHOS, F.; RODRIGUES, V. T.;
BARBERENA, F. F. V. A.; FRAGA, C. N.; PESSOA, E.
M.; FORSTER, W.; MENINI NETO, L.; FURTADO,
S. G.; NARDY, C.; AZEVEDO, C. O.; GUIMARÃES,
L. R. S. Orchidaceae na Lista de Espécies da Flora
do Brasil. Jardim Botânico do Rio de Janeiro, 2015.
Available from: http://www.floradobrasil.jbrj.gov.br
BERKA, M. G.; VENTURIERI, G. A.; TEIXEIRA, T. N.
Development of Cattleya amethystoglossa × nobilior in
simplified culture media. Acta Scientiarum. Agronomy,
Souza et al.
Nativa, Sinop, v. 9, n. 4, p. 352-358, 2021.
357
Maringá, v. 36, n. 4, p. 425-428, 2014.
http://dx.doi.org/10.4025/actasciagron.v36i4.15492
CHASE, M. W.; CAMERON, K. M.; FREUDENSTEIN, J.
V.; PRIDGEON, A. M.; SALAZAR, G.; VAN DEN
BERG, C.; SCHUITEMAN, A. An update classification
of Orchidaceae. Botanical Journal of the Linnean
Society, v. 177, n. 2, p. 151˗174, 2015.
http://dx.doi.org/10.1590/2175-7860201869115
CORBELLINI, J. R.; RIBAS, L. L. F.; MAIA, F. R.;
CORREIA, D. O.; NOSEDA, M. D.; SUZUKI, R. M.;
AMANO, E. Effect of microalgae Messastrum gracile and
Chlorella vulgaris on the in vitro propagation of orchid
Cattleya labiate. Journal of Applied Phycology, v. 32, n.
1, p. 4013-4027, 2020.
http://dx.doi.org/10.1007/s10811-020-02251-9
FERREIRA, E. B.; CAVALCANTI, P. P.; NOGUEIRA, D.
A. ExpDes: Experimental Designs Package. R
packageversion 1.1.2, 2013.
GOMES, L. R. P.; FRANCESCHI, C. R. B.; RIBAS, L. L. F.
Micropropagation of Brasilidium forbesii (Orchidaceae)
through transverse and longitudinal thin cell layer culture.
Acta Scientiarum. Biological Science, Maringá, v. 37,
n. 2, p. 143-149, 2015.
https://doi.org/10.4025/ACTASCIBIOLSCI.V37I2.27
276
GRANJA, M. M. C.; MOTOIKE, S. Y.; ANDRADE, A. P.
S.; CORREA, T. C.; PICOLI, E. A. T.; KUKIB, K. N.
Explant origin and culture media factors drive the
somatic embryogenesis response in Acrocomia aculeata
(Jacq.) Lodd. ex Mart., an emerging oil crop in the tropics.
Industrial Crops and Products, v. 117, p. 1-12, 2018.
https://doi.org/10.1016/j.indcrop.2018.02.074
HUNHOFF, V. L.; LAGE, L. A.; PALÚ, E. G.; KRAUSE,
W.; SILVA, C. A. Nutritional requirements for
germination and in vitro development of three
Orchidaceceae species in the southern Brazilian Amazon.
Ornamental Horticulture, v. 24, n. 2, p. 87-94, 2018.
http://dx.doi.org/10.14295/oh.v24i2.1130
IRSHAD, M.; HE, B.; LIU, S.; MITRA, S.; DEBNATH, B.;
LI, M.; RIZWAN, H. M.; QIU, D. In vitro regeneration of
Abelmoschus esculentus L. cv. Wufu: influence of anti-
browning additives on phenolic secretion and callus
formation frequency in explants. Horticulture
Environment and Biotechnology, v. 58, p. 503-513,
2017. https://doi.org/10.1007/s13580-017-0301-3
LEONE, G. F.; ALMEIDA, C. V.; ABREU-TAZARI, M.
F.; BATAGNIN-PIOTTO, K. D.; ARTIOLI, F. A, C.;
ALMEIDA, M. Antibioticoterapia em microplantas de
abacaxizeiro (Ananas comosus). Ciência Rural, v. 46, n. 1,
p. 89-94, 2016. https://doi.org/10.1590/0103-
8478cr20130958
KIM, D. H.; KANG, K. W.; ENKHTAIVAN, G.; JAN, U.;
SIVANESAN, I. Impact of activated charcoal, culture
medium strength and thidiazuron on non-symbiotic in
vitro seed germination of Pecteilis radiate (Thunb.) Raf.
South African Journal of Botany, v. 124, n. 1, p. 144-
150, 2019. https://doi.org/10.1016/j.sajb.2019.04.015
MAMANI, I.; AVERANGA, K.; ESPINOZA, B.;
TERRAZAS, E. Establecimiento del sistema de
generación in vitro de callos de Lupinus mutabilis Sweet
(Tarwi) a partir de hipocótilos. Revista de Ciencias
Farmacéuticas y Bioquímicas, v. 2, n. 1, p. 25-36, 2014.
MÉNDEZ-ÁLVAREZ, D.; ABDELNOUR-ESQUIVEL,
A. Establecimiento in vitro de Terminalia amazonia (Gmel.)
Excell. Revista Florestal Mesoamericana Kurú, v. 11,
n. 27, p. 07-21, 2014.
https://doi.org/10.18845/rfmk.v11i27.1774
MURASHIGE, T.; SKOOG, F. A revised medium for rapid
growth and bioassays with tobacco tissue cultures.
Physiologia Plantarum, v. 15, n. 3, p. 473-497, 1962.
https://doi.org/10.1111/j.1399-3054.1962.tb08052.x
PANKAJ, K.; PANDEY, S.; ROY, K. Ascorbic and citric
acids in combination resolve the problems encountered
in micro-propagation of litchi from shoot tips. Cell and
Tissue Research, v. 14, n. 1, p. 41-59, 2014.
PARK, H. Y.; KANG, K. W.; KIM, D. H.; SIVANESAN, I.
In vitro propagation of Cymbidium goeringii Reichenbach fil.
through direct adventitious shoot regeneration.
Physiology and Molecular Biology of Plants, v. 24, n.
1, p. 307-313, 2018. https://doi.org/10.1007/s12298-
017-0503-2
RIGHETO, M. V. L.; ALMEIDA, L. V.; BRONDANI, G.
E.; AMARAL, A. F. C.; ALMEIDA, M. Morfofisiologia
de plântulas de Cattleya labiata Lindley e Cattleya eldorado
Linden cultivadas in vitro sob influência de paclobutrazol.
Revista Brasileira de Biociências, v. 10, n. 1, p. 20-25,
2012.
SARTOR, F. R.; ZANOTTI, R. F.; PÔSSA, K. F.; PILON,
A. M.; FUKUSHIMA, C. H. Diferentes meios de cultura
e antioxidantes no estabelecimento in vitro do jacarandá
da Bahia. Bioscience Journal, v. 29, n. 2, p. 408-411,
2013.
SEON, K. M.; KIM, D. H.; KANG, K. W.; SIVANESAN,
I. Highly competent in vitro propagation of Thrixspermum
japonicum (Miq.) Rchb.f., a rare epiphytic orchid. In Vitro
Cellular & Developmental Biology - Plant, v. 54, n. 1,
p. 302-308, 2018. https://doi.org/10.1007/s11627-018-
9890-5
SOUZA, G. C.; CLEMENTE, P. L.; ISAAC, V. L. R.;
FARIA, S. P.; CAMPOS, M. R. C. Contaminação
microbiana na propagação in vitro de Cattleya walkeriana e
Schomburgkia crispa. Revista Brasileira de Biociências,
v. 5, n. 1, p. 405-407, 2007.
SOUZA, D. M. S. C.; XAVIER, A.; MIRANDA, N. A;
GALLO, R.; OTONI, W. C. Light quality, 6-
benzyladenine and number of subcultives for in vitro
multiplication of hybrid clones of Corymbia. Scientia
Forestalis, v. 48, n. 128, p. e3282, 2020.
https://doi.org/10.18671/scifor.v48n128.03
TISARUM, R.; SAMPHUMPHUNG, T.;
THEERAWITAYA, C.; PTOMMEE, W.; CHA, S. In
vitro photoautotrophic acclimatization direct
transplantation and ex vitro adaptation of rubber tree
(Hevea brasiliensis). Plant Cell, Tissue and Organ
Culture, v. 133, n. 2, p. 215-230, 2018.
https://doi.org/10.1007/s11240-017-1374-5
VAN DEN BERG, C. Reaching a compromise between
conflicting nuclear and plastid phylogenetic trees: a new
classification for the genus Cattleya (Epidendreae;
Epidendroideae; Orchidaceae). Phytotaxa, v. 186, n. 2,
p. 75˗86, 2014.
http://dx.doi.org/10.11646/phytotaxa.186.2.2
VILLA, F.; PASQUAL, M. E SILVA, E. F. Micropropagação
de híbridos de orquídeas em meio knudson com adição
de vitaminas do meio MS, benzilaminopurina e carvão
ativado. Semina: Ciências Agrárias, v. 35, n. 2, p. 683-
694, 2014. https://doi.org/10.5433/1679-
0359.2014v35n2p683
Activated charcoal application for the micropropagation of Cattleya crispata (Thunb.) Van den Berg
Nativa, Sinop, v. 9, n. 4, p. 352-358, 2021.
358
WILLADINO, L.; SOUTO, N. F. C.; ULISSES, C.;
PORTO, A. L. F.; CAMARA, T.; SILVA, M. M. A.
Embryos and lateral buds culture of Tapeinochilos
Ananassae (Hassk). K. Schum. International Journal of
Sciences, v. 2, p. 19-25, 2013.
