Nativa, Sinop, v. 10, n. 1, p. 117-124, 2022.
Pesquisas Agrárias e Ambientais
DOI: https://doi.org/10.31413/nativa.v10i1.13351 ISSN: 2318-7670
Seedling production of
Mimosa
calodendron
Mart. ex Benth.
in a temporary immersion bioreactor
Denys Matheus Santana Costa SOUZA1, Andressa Rosa MARTINS1,
Sérgio Bruno FERNANDES1, Juscelina Arcanjo dos SANTOS1, Gilvano Ebling BRONDANI1*
1 Laboratory of In Vitro Culture of Forest Species, Department of Forestry Sciences, Federal University of Lavras, Lavras, MG, Brazil.
*E-mail: gilvano.brondani@ufla.br
(ORCID: 0000-0003-4356-7163; 0000-0002-7578-0916; 0000-0001-8685-1268; 0000-0003-4731-2610; 0000-0001-8640-5719)
Received in 24/01/2022; Accepted in 08/03/2022; Published in 26/03/2022.
ABSTRACT: Micropropagation is one technology to propagate endemic species of the Ferruginous
Rupestrian Grasslands when in vitro genetic conservation is sought. The present study aimed to assess the
breaking of dormancy, in vitro establishment, multiplication, elongation, rooting, and acclimatization of Mimosa
calodendron from culture in a temporary immersion bioreactor system. The seeds used for the experiments were
from plants originating from the Ferruginous Rupestrian Grasslands. The percentage of contamination,
oxidation, unresponsive seeds, germination, number of buds per explant, shoot length, senescence, percentage
of adventitious rooting, and acclimatization were assessed. The breaking of dormancy was most successful by
mechanical scarification (80% germination). Immersion in sodium hypochlorite for 5 minutes was the most
efficient treatment for in vitro establishment (90%). For the in vitro multiplication and elongation phase, the use
of liquid culture medium from cultivation in a temporary immersion bioreactor was the most suitable for the
characteristics number of buds per explant (2.55 buds), vigor (1.1), oxidation (1.3) and senescence (1.3)
according to the score’s scale. Regardless of the in vitro cultivation method, the percentages of rooting and
acclimatization were satisfactory, and it was possible to obtain complete plants in 190 days.
Palavras-chave: in vitro propagation; Ferruginous Rupestrian Grasslands; endemic species.
Produção de mudas de
Mimosa
calodendron
Mart. ex Benth. em biorreator
de imersão temporária
RESUMO: A micropropagação é uma alternativa para a propagação de espécies endêmicas do Campo
Rupestre Ferruginoso, quando se busca a conservação genética in vitro. O trabalho teve como objetivo avaliar a
superação de dormência, estabelecimento in vitro, multiplicação, alongamento, enraizamento e aclimatização de
Mimosa calodendron a partir do cultivo em sistema de biorreator de imersão temporária (BIT). As sementes
utilizadas para os experimentos foram provenientes de plantas oriundas do Campo Rupestre Ferruginoso. A
porcentagem de contaminação, oxidação, sementes não responsivas, germinação, número de gemas por
explante, comprimento de brotos, senescência, porcentagem de enraizamento e aclimatização foram avaliados.
A superação de dormência por escarificação mecânica (80% de germinação) foi a mais adequada para a
superação de dormência. A imersão em hipoclorito de sódio por 5 minutos foi o tratamento mais eficiente para
o estabelecimento in vitro (90%). Para a fase de multiplicação e alongamento in vitro, o uso do meio de cultivo
líquido a partir do cultivo em biorreator de imersão temporária foi o mais adequado para as características
número de gemas por explante (2,55 gemas), vigor (1,1), oxidação (1,3) e senescência (1,3) de acordo à escala
de notas. Independentemente do método de cultivo in vitro, a porcentagem de enraizamento e aclimatização
foram satisfatórios, sendo possível obter plantas completas em 190 dias.
Keywords: propagação in vitro; Campo Rupestre Ferruginoso; espécie endêmica.
1. INTRODUCTION
Mimosa calodendron Mart. ex Benth. is species endemic to
Brazil that belongs to the family Leguminosae. It has a
restricted geographic distribution in southeastern Minas
Gerais associated with the Cerrado domain over rocky
outcrops in the Iron Quadrangle (altitudes between 1,300 and
1,700 m) and the formation of the ferruginous rupestrian
grassland (DUTRA; GARCIA, 2014). Its natural population
has shrunk (DAYRELL et al., 2015) due to mining activities,
parasitism, and seed predation, in addition to difficulties in
seedling production due to physical dormancy and slow
germination (DUTRA et al., 2022).
Given these problems, the micropropagation technique
has numerous advantages, such as the possibility of mass
propagation from a single propagule, the fixation of genetic
gains in clonal populations and the propagation of high-
quality plants in a small physical space in a short time,
independent of climatic factors limiting seed production
(ABIRI et al., 2020). However, the in vitro culture of Mimosa
calodendron is still a challenge because there is a need for
specific management in the stages of seed dormancy
breaking, germination, establishment, multiplication,
elongation, and rooting to obtain a complete plant.
Several technologies have been proposed to automate the
micropropagation, such as adjustments to germination and
multiplication protocols, as well as breaking physical
dormancy through mechanical scarification, chemical
sterilization to reduce contamination, and adoption of
Seedling production of Mimosa calodendron Mart. ex Benth. in a temporary immersion bioreactor
Nativa, Sinop, v. 10, n. 1, p. 117-124, 2022.
118
protocols in culture systems and routine procedures using a
temporary immersion bioreactor (TIB) (SOUZA et al.,
2020a; MOLINARI et al., 2021; RAMÍREZ-MOSQUEDA;
BELLO-BELLO, 2021).
The physical dormancy of Mimosa calodendron seeds is one
of the greatest difficulties hindering its germination
(DAYRELL et al., 2015). Many species that present
dormancy do not germinate under adequate conditions,
preventing their propagation and thus the production of
seedlings for use in genetic recovery and conservation
projects (ARAÚJO et al., 2020). Among the methods for
breaking dormancy, mechanical scarification and thermal
shock are often used to reduce physical impediments, which
usually takes the form of a rigid tegument that prevents the
imbibition process (ARAÚJO et al., 2020).
Aseptic control is also essential for the in vitro
introduction (SOUZA et al., 2020a; MOLINARI et al., 2021),
and it is important to sterilize the culture medium of
microorganisms that hinder the development and growth of
tissues (MEDJEMEM et al., 2016). Recent studies on the
adequate exposure time of tissues to sterilizing agents have
suggested ways to reduce contamination (MOLINARI et al.,
2021), but such chemical agents added to the nutrient
medium can cause phytotoxicity due to tissue oxidation and
growth inhibition (TEIXEIRA et al., 2021), so it is important
to determine the time of exposure to the chemical agent and
its concentration (MOLINARI et al., 2021).
The multiplication, elongation, and rooting phases can be
optimized using TIB technology. A TIB is an automated
system that can improve nutrient supply and gas transfer,
supporting fuller development of micropropagated cultures
(CARVALHO et al., 2019). TIBs yield biomass gains,
reducing the time required for propagation (COSTA et al.,
2021) and increasing plant production per unit area
(RIBEIRO et al., 2016; ALVES et al., 2021).
The present study aimed to assess the dormancy
breaking, in vitro establishment, multiplication, elongation,
rooting, and acclimatization of Mimosa calodendron from
culture in a TIB system.
2. MATERIAL AND METHODS
2.1. Source of seeds
Seeds from adult plants of Mimosa calodendron Mart. ex
Benth. in the natural environment of Ferruginous Rupestrian
Grassland were collected in March 2017, Research and
Innovation Unit belonging to Gerdau (GERDAU Açominas
S.A.), located in Ouro Branco, Minas Gerais, Brazil
(20°31’17.43”S, 43°44’18.89”W). In total, 2,500 seeds were
collected from 50 plants of the species. Mean annual rainfall
is 2,056 mm and mean temperature is 25.5°C. The climate of
the region is classified as Aw (tropical) according to Köppen-
Geiger, with the rainy season from November to March and
dry winters.
2.2.
In vitro
establishment
Seeds were washed under running water and immersed in
an antifungal solution containing 2.4 mg L-1 of orthocide
500® (50% of captan as the active ingredient) for 15 minutes.
Then, the seeds were washed five times in autoclaved
deionized water and immersed in sodium hypochlorite
solution (NaOCl, 2.0-2.5% of active chlorine, Clarix®) –
according to asepsis treatment (exposure time) – under
constant agitation inside a horizontal laminar flow hood.
Finally, the seeds were washed in deionized water and
autoclaved five times. One seed was inoculated in each test
tube (25 × 150 mm) containing 10 mL of culture medium
(Figure 1A).
The basic culture medium used in the experiment was MS
medium (MURASHIGE; SKOOG, 1962) supplemented
with 30 g L-1 of sucrose (Synth Ltda) and 6 g L-1 of agar
(Merck SA). The pH of the culture medium was adjusted to
5.8 (± 0.05) before the addition of agar. The culture medium
was autoclaved at a temperature of 121°C and pressure of
approximately 1.0 kgf cm-2 for 20 minutes. The seeds were
kept for 30 days in a growroom at a temperature of 24°C (±
1°C) under a 16-hour photoperiod and 40 μmol m-2 s-1 of
irradiance (quantified by radiometer, LI -COR®, LI-250A
Light Metre) emitted by a cold-white fluorescent lamp.
2.3. Dormancy breaking
The experiment was arranged in a completely randomized
design (CRD) with 30 replicates of each dormancy-breaking
treatment (mechanical scarification and thermal shock) and
the control (no dormancy breaking) and one seed per
replicate. In the mechanical process, the seeds were scarified
in the region opposite the hilum using sandpaper (water
sandpaper, 225 mm × 275 mm). The thermal shock method
was performed in hot water for 1 minute (temperature of
60°C) and then immersed in water at room temperature at
24°C, both controlled by a digital thermometer. Data on the
percentage of in vitro germination were collected at 30 days.
2.4. Asepsis
Based on the best results of the dormancy breaking
experiment, an experiment on asepsis was conducted. The
experiment was arranged in a CRD to test three immersion
times in NaOCl solution (2.0-2.5% of active chlorine,
Clarix®): 5 (control), 10, and 15 minutes. Each treatment had
30 replicates, consisting of plots containing one seed. Data
on the percentage of contamination, oxidation, unresponsive
seeds, and in vitro germination (Figure 1B-C) were collected
at 30 days.
2.5.
In vitro
multiplication and elongation
After seed germination and in vitro establishment (Figure
1D) at 30 days, three shoots were standardized to 0.5 cm in
length and grown by two methods: 1) in semisolid culture
medium in a 250-mL glass flask with 50 mL of MS culture
medium, 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®); and
2) in liquid culture medium in the TIB (250-mL glass flask
containing 50 mL of MS culture medium, supplemented with
30 g L-1 of sucrose, 0.5 mg L-1 BAP, and 0.05 mg L-1 NAA)
(Figure 1E).
Over the 90 days of culture, tissue immersion in the
bioreactor occurred for 30 seconds at 3-hour intervals.
Subculturing with renewal of the culture medium was
performed every 30 days. The semisolid and liquid culture
media were made with deionized water, and the pH was
adjusted to 5.8 (± 0.05) with NaOH (0.1 M) and/or HCl (0.1
M) before autoclaving. Autoclaving of the culture medium
and the bioreactor equipment was performed at a
temperature of 121°C and pressure of approximately 1.0 kgf
cm-2 for 20 minutes.
Souza et al.
Nativa, Sinop, v. 10, n. 1, p. 117-124, 2022.
119
Figure 1. In vitro germination and multiplication of Mimosa calodendron. (A) Detail of in vitro inoculated seed. (B) Seed germinated with shoot-tip
initiation. (C) Seed germinated with radicle initiation. (D) Explant considered established. (E) In vitro multiplication in the TIB. (F) Explant multiplied
and elongated in the TIB system for 90 days. Bar = 1.0 cm (Figure A - D) or 5.0 cm (Figure F and E).
Figura 1. Germinação e multiplicação in vitro de Mimosa calodendron. (A) Detalhe da semente inoculada in vitro. (B) Semente germinada com iniciação
da brotação apical. (C) Semente germinada com iniciação da radícula. (D) Explante considerado estabelecido. (E) Multiplicação in vitro em biorreator
de imersão temporária (BIT). (F) Explante multiplicado e alongado em BIT aos 90 dias. Barra = 1.0 cm (Figura A-D) ou 5.0 cm (Figura F e E).
At 90 days, the mean number of shoots per explant (>
0.5 cm), shoot length (> 0.5 cm), vigor (Figure 2A-C),
oxidation (Figure 2D-F), and senescence (Figure 2G-I) were
determined according to the scale proposed by Souza et al.
(2020b). The experiment was arranged in a CRD, with 20
replicates composed of five explants each.
2.6.
In vitro
adventitious rooting and acclimatization
Shoots produced in the multiplication and elongation
phases were standardized by isolating four shoots to 3.0 cm
in length with adequate vegetative vigor and inoculating them
in test tubes (25 × 150 mm) containing 10 mL of MS culture
medium supplemented with 30 g L-1 of sucrose, 6 g L-1 of
agar, 0.5 mg L-1 NAA, and 0.05 mg L-1 BAP. The experiment
was arranged in a CRD with 20 replicates composed of four
explants each. At 30 days, the adventitious rooting
percentage was assessed.
In vitro–rooted plants 5 cm in length and three fully
expanded leaves were subjected to the acclimatization.
Seedlings were transferred to a plastic container containing
50 mL of commercial substrate based on decomposed pine
bark and vermiculite, with moisture controlled daily. The
containers were isolated with plastic film for 5 days with
gradual opening. The efficiency of acclimatization was
verified through survival at 40 days.
2.7. Data analysis
The variables that did not have a normal distribution
according to the Shapiro-Wilk test (p > 0.05) were arcsin-
transformed. Hartley test (p > 0.05) was used to verify the
homogeneity of variances. The groups were compared by
analysis of variance (p < 0.05), and the means were compared
by Tukey’s test (p < 0.05). The analyses were processed in the
software R version 3.0.3 (R CORE TEAM, 2018).
Figure 2. Assessments of vigor, oxidation, and senescence according to
the scores scale of Mimosa calodendron explant. (A-C) Vigor of shoots (1
= Excellent: emission of shoots with active growth, without apparent
nutritional deficiency; 2 = Good: emission of shoots, but with reduced
leaves; 3 = Low: no emission of shoots and/or senescence and death).
(D-F) Oxidation of shoots (1 = Null: no oxidation; 2 = Medium:
reduced oxidation of explants; 3 = High: complete oxidation of
explants). (G-I) Senescence of shoots (1 = Null: no leaf senescence; 2 =
Medium: reduced leaf senescence of the explants; 3 = High: complete
leaf senescence of the explants). Bar = 1 cm.
Figura 2. Avaliações de vigor, oxidação e senescência de acordo com a
escala de notas. (A-C) Vigor das brotações (1 = Ótimo: emissão de
brotações com crescimento ativo, sem deficiência nutricional aparente;
2 = Bom: emissão de brotações, porém com folhas de tamanho
reduzido; 3 = Baixo: ausência de emissão de brotações e, ou, senescência
e morte). (D-F) Oxidação das brotações (1 = Nula: sem oxidação; 2 =
Média: reduzida oxidação dos explantes; 3 = Alta: oxidação completa
dos explantes). (G-I) Senescência das brotações (1= Nula: sem
senescência foliar; 2 = Média: reduzida senescência foliar dos explantes;
3 = Alta: senescência foliar completa dos explantes. Barra = 1 cm.
Seedling production of Mimosa calodendron Mart. ex Benth. in a temporary immersion bioreactor
Nativa, Sinop, v. 10, n. 1, p. 117-124, 2022.
120
3. RESULTS
3.1.
In vitro
establishment
There was a significant difference between the treatments
tested for breaking dormancy. Mechanical scarification
resulted in 80% of germination, thermal shock only 40%
(Figure 3A). There was no seedling germination in the
control treatment without dormancy breaking (Figure 3A).
Figure 3. Characteristics assessed during the in vitro establishment phase
of Mimosa calodendron according to the dormancy breaking method and
exposure time to the chemical agent. (A) Percentage of in vitro
germination according to dormancy breaking method. (B) In vitro
contamination according to exposure time to the chemical agent. (C)
Tissue oxidation according to exposure time to the chemical agent. (D)
Unresponsive seeds according to exposure time to the chemical agent.
(E) Percentage of in vitro germination according to exposure time to the
chemical agent. *Mean values followed by the same letters do not differ
significantly according to the Tukey’s test (p < 0.05). Bars represent the
standard deviation relative to the mean value.
Figura 3. Características avaliadas durante a fase de estabelecimento in
vitro de Mimosa calodendron conforme o método de quebra de dormência
e tempo de exposição ao agente quimico. (A) Porcentagem de
germinação in vitro de acordo com o método de quebra de dormência.
(B) Contaminação in vitro de acordo com o tempo de exposição ao
agente químico. (C) Oxidação dos tecidos de acordo com o tempo de
exposição ao agente químico. (D) Sementes não responsivas de acordo
com o tempo de exposição ao agente químico. (E) Porcentagem de
germinação in vitro de acordo com o tempo de exposição ao agente
químico. *Médias seguidas por letras iguais não diferem entre si, pelo
teste de Tukey (p < 0.05). Barras representam o desvio padrão em
relação ao valor médio.
In vitro establishment significantly differed between
treatments for all assessed traits (Figure 3B-E). The
treatments of 5 and 10 minutes resulted in the highest
contamination averages (10%), differing from the immersion
time in NaOCl for 15 minutes (5%), which showed lower in
vitro contamination of Mimosa calodendron seeds (Figure 3B). In
contrast, the percentage of phenolic oxidation of tissues was
significantly higher under the 15-minute treatment (15%)
than under the 5-minute (0%) and 10-minute treatments
(5%) (Figure 3C).
Unresponsive seeds were lowest with the 5-minute
treatment (0%), differing significantly from the 10-minute
(5%) and 15-minute treatments (10%) (Figure 3D). The best
results of germination percentage were also observed in the
5-minute group (90%), followed by the 10-minute (80%) and
15-minute immersion groups (70%) (Figure 3E).
3.2.
In vitro
multiplication and elongation
At 90 days of in vitro cultivation of Mimosa calodendron
explants in the semisolid and TIB cultivation systems, the
number of buds per explant, shoot length, vigor, oxidation,
and senescence were assessed (Figure 4A-E). There was a
significant difference between treatments (semisolid and
liquid culture systems in the TIB) for all morphological
characteristics assessed except for shoot length (Figure 4B).
Figure 4. Characteristics assessed during the in vitro multiplication and
elongation phases of Mimosa calodendron in semisolid and TIB cultivation
systems. (A) Number of buds per explant. (B) Shoot length (cm). (C)
Vigor. (D) Tissue oxidation. (E) Tissue senescence. *Mean values
followed by the same letters do not differ significantly according to the
Tukey’s test (p < 0.05). Bars represent the standard deviation relative to
the mean value.
Figura 4. Características avaliadas durante as fases de multiplicação e
alongamento in vitro de Mimosa calodendron em sistemas de cultivo
semisólido e BIT. (A) Número de gemas por explante. (B)
Comprimento de brotos (cm). (C) Vigor. (D) Oxidação dos tecidos. (E)
Senescência dos tecidos. *Médias seguidas por letras iguais não diferem
entre si, pelo teste de Tukey (p < 0.05). Barras representam o desvio
padrão em relação ao valor médio.
Considering the number of buds per explant (2.55 buds
per explant, Figure 4A), vigor (1.1, Figure 4C), oxidation (1.3,
Figure 4D), and senescence (1.3, Figure 4E), according to a
Souza et al.
Nativa, Sinop, v. 10, n. 1, p. 117-124, 2022.
121
grading scale (Figure 2), the best results were observed with
the culture in TIB liquid medium, differing significantly from
the culture in semisolid culture medium (1.85 buds per
explant, Figure 4A; 1.8 of vigor, Figure 4C; 1.7 oxidation,
Figure 4D; 1.8 senescence, Figure 4E).
Shoot length was similar between the liquid medium
treatments in the TIB and the semi-solid treatment with no
significant difference (Figure 4B).
3.3.
In vitro
adventitious rooting and acclimatization
The percentage of in vitro rooting was similar between the
semisolid (85% of rooting) and TIB (90% of rooting) systems
at 30 days of cultivation (Figure 5). The rooted plants from
the TIB and semisolid systems showed no significant
difference in acclimatization, which had an overall mean of
77.8% at 40 days.
Figure 5. Percentage of in vitro adventitious rooting of Mimosa
calodendron in semisolid and TIB cultivation systems. *Means
followed by the same letters do not differ according to the Tukey’s
test (p < 0.05). Bars represent the standard deviation relative to the
mean value.
Figura 5. Porcentagem de enraizamento adventício in vitro de Mimosa
calodendron em sistema de cultivo semissólido e BIT. *Médias
seguidas por letras iguais não diferem entre si, pelo teste de Tukey
(p < 0.05). Barras representam o desvio padrão em relação ao valor
médio.
4. DISCUSSION
4.1.
In vitro
establishment
Seed dormancy and slow germination are factors that
hinder the production of Mimosa calodendron seedlings, and
studies that investigate dormancy-breaking and germination
mechanisms are important to provide support for the
propagation of the species (DAYRELL et al., 2015). This in
vitro germination experiment comparing two treatments of
breaking dormancy, mechanical scarification and thermal
shock, showed promising results for obtaining seedlings
through in vitro cultivation.
Methods for breaking dormancy by mechanical
scarification in Mimosa calodendron seeds showed efficiency,
with 80% of germination (Figure 3A). The seeds that did not
germinate in the control treatment showed that the species
has physical dormancy. In a study by Dayrell et al. (2015),
assessing the effect of mechanical scarification, lighting, and
different incubation temperatures on seed germination
showed the need for pre-treatment to break physical
dormancy, and scarification was a highly effective method.
Other studies have shown a positive effect of scarification
treatment on germination in other species of Mimosa
(OROZCO-ALMANZA et al., 2003; CHAUHAN;
JOHNSON, 2009; ROSA et al., 2012).
Mechanical scarification is a simple, low-cost technique
that is highly efficient at breaking tegumentary or physical
dormancy, promoting rapid and uniform germination
(SANTOS et al., 2004). This type of dormancy results from
the impermeability of the integument, which may arise due to
the presence of a cuticle and a developed layer of cells in the
palisade, which prevents water absorption and gas exchange
and imposes a mechanical restriction on the growth of the
embryo, delaying the germination process (SANTOS et al.,
2004).
For the asepsis experiment performed with the
germinated material, the best results for most of the assessed
characteristics were observed with immersion times of 5
minutes and 10 minutes in NaOCl solution at 2.0-2.5% of
active chlorine. Although the time of 15 minutes reduced the
percentage of contamination in the culture medium, this
treatment also promoted the highest percentage of oxidation,
unresponsive seeds, and lower germination. Prolonged
exposure to NaOCl for disinfection can hinder seed
germination due to long exposure of cellular tissues,
increased permeability of the tegument, and leaching of plant
hormones that are needed for germination (SILVA et al.,
2019).
Therefore, the determination of the exposure time to the
NaOCl disinfectant is important because it helps reduce the
cytotoxicity and genotoxicity of tissues (SANTOS et al.,
2020). In seedlings of Melanoxylon brauna, the use of NaOCl
at 2.5% of active chlorine reduced contamination and thus
favoured a higher percentage of healthy seedlings under a
maximum exposure time of 25 minutes (SILVA et al., 2019).
Seeds of Dalbergia nigra, after more than 14 minutes of
exposure to NaOCl (2.0-2.5%), showed the development of
seedlings with physiological and genetic disorders, attributed
to phytotoxic, cytotoxic, and genotoxic effects (SANTOS et
al., 2020). NaOCl solution at 5% of active chlorine allowed
lower contamination (63%) than the concentration of 2.5%
of active chlorine (83% of contamination) for explant of
Lychnophora pohlii. However, immersion time (5, 10, 15, 20, or
25 minutes) did not influenced the contamination
(GONZAGA et al., 2021).
Given the above, as the time of immersion in NaOCl
increases, more residues can be adsorbed by the seed,
reacting with the amino acids and generating a high
concentration of ammonium chloride (NH4Cl) and carbon
dioxide (CO2) in the test tube (SANTOS et al., 2020). In
addition, the hydrolysis of NaOCl produces hypochlorous
acid (HClO), a toxic compound that causes cellular and
photosynthetic changes, for example, which negatively affect
growth and cause abnormalities in seedlings (GAMAGE et
al., 2018; SILVA et al., 2019; SANTOS et al., 2020).
Optimal exposure time to the disinfecting agent NaOCl
should be assessed individually and carefully for each species.
Our data showed that Mimosa calodendron showed high
sensitivity to immersion time in NaOCl for almost all
assessed traits, so 15-minute immersion is not recommended.
Five and 10-minutes treatments are the most promising for
the asepsis of Mimosa calodendron, as they provide a lower
percentage of oxidation, more responsive seeds, and thus
better germination.
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4.2.
In vitro
multiplication and elongation
The improvement of protocols for vegetative
propagation that supports plant development was studied in
an attempt to establish the most appropriate cultivation
system to maximize the micropropagation of Mimosa
calodendron. The results of the morphological characteristics
indicate the optimization of the multiplication and in vitro
elongation in TIB systems.
Genetic material used in this study presented different
responses depending on the crop system used. The TIB led
to better results for all assessed traits (number of buds per
explant, shoot length, vigor, oxidation, and senescence) than
the semisolid cultivation system. As in the present study, the
use of TIB has also been more efficient in the multiplication
of buds and elongation of shoots in Bambusa vulgaris
(RIBEIRO et al., 2016), Corema album (ALVES et al., 2021),
and Agave guiengola (CHÁVEZ-ORTIZ et al., 2021).
According to Nogueira et al. (2017), when there is greater
contact between the surface of the explants and the culture
medium, the growth rate and vigor are higher due to greater
absorption of water and nutrients. This relationship was
observed in the present study, as the highest mean number
of shoots per explant, length, and vigor of shoots were
observed in explants subjected to TIB. Therefore, the
number of buds per explant, length, and vigor of shoots are
some of the best characteristics to assess the efficiency of the
in vitro multiplication and elongation (SILVA et al., 2016;
SOUZA et al., 2020b).
Phenolic oxidation and senescence of explants are
problems associated with micropropagation that may
influence crop development. These results may be related to
cultivation factors, such as the use of semisolid medium in
flasks with lower gas exchange, which tend to have low
concentrations of carbon dioxide and high ethylene
concentrations (CHÁVEZ-ORTIZ et al., 2021). In this
context, TIBs can improve the supply of nutrients and the
transfer of gases, making it possible to minimize
physiological disturbances, which will result in greater
development of micropropagated cultures (CARVALHO et
al., 2019). Thus, methods aimed at breaking or reducing
phenolic oxidation and senescence in tissues and organs are
important strategies to be adopted in propagation systems, as
observed during the use of TIB.
Given the above, it is of utmost importance to consider
the physical state of the culture medium during in vitro culture,
which can be semisolid or liquid and can directly interfere
with the development of explants due to the different forms
of contact of the plant with the culture medium (SOUZA et
al., 2020a). Therefore, it is necessary to define the
composition and type of culture medium most suitable for
the growth and development of cultured tissues (SOTA et al.,
2021), as this is a factor that exerts a great influence on in vitro
culture.
4.3.
In vitro
adventitious rooting and acclimatization
In the process of adventitious rooting and
acclimatization, several factors underlie the ability to form
roots in microplants. Among these factors are plant
hormones, cultivation environment (light, temperature, and
gas exchange), and cultivation system, all of which play a
predominant role in rhizogenesis (LIMA et al., 2022). The
difficulty of propagation through adventitious rooting of
microplants in vitro is one of the main problems encountered
in the production of clonal plants of many native and woody
species (ABIRI et al., 2020).
In the present study, regardless of the cultivation system,
the percentages of rooting (90%) and acclimatization (77.8%
survival) were satisfactory, and it was possible to obtain
complete plants in 190 days. Although adventitious rooting
and acclimatization through different cultivation systems are
important factors in micropropagation, there are still few
studies of their effects on Mimosa calodendron. Knowledge
about the most appropriate cultivation system for the growth
and development of plantlets would provide a basis for
making protocols more efficient, thus allowing large-scale
planning and propagation regardless of the season.
5. CONCLUSIONS
Breaking the dormancy of Mimosa calodendron seeds by
mechanical scarification resulted in the highest percentage of
in vitro germination.
Immersion time of 5 minutes in NaOCl (2.0-2.5% of
active chlorine) resulted in better asepsis in seeds.
Liquid culture system with the use of a TIB proved to be
the most appropriate technology for in vitro multiplication
and elongation.
Adventitious rooting and acclimatization were
satisfactory, and it was possible to obtain complete plants in
190 days.
6. AKNOWLEDGEMENTS
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 à
Pesquisa do Estado de Minas Gerais – FAPEMIG’). We also
thank GERDAU Açominas S.A.
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