Nativa, Sinop, v. 11, n. 1, p. 01-09, 2023.
Pesquisas Agrárias e Ambientais
DOI: https://doi.org/10.31413/nativa.v11i1.14394
ISSN: 2318-7670
Clonal microplant production, morphological evaluation and genetic
stability of
Dendrocalamus asper
(Schult. & Schult.) Backer ex. K. Heyneke
Douglas Santos GONÇALVES1, Denys Matheus Santana Costa SOUZA1,
Letícia Vaz MOLINARI1, Maria Lopes Martins AVELAR1, Dulcinéia de CARVALHO1,
Gustavo Leal TEIXEIRA2, Gilvano Ebling BRONDANI1*
1Laboratory of In Vitro Culture of Forest Species, Department of Forestry Sciences, Federal University of Lavras, Lavras, MG, Brazil.
2Institute of Agricultural Science, Federal University of Minas Gerais, Montes Claros, MG, Brazil.
*E-mail: gilvano.brondani@ufla.br
Submitted on 09/20/2022; Accepted on 01/16/2023; Published on 02/07/2023.
ABSTRACT: Bamboo species have many commercial applications, considering that homogeneous plantations
(formed from clonal plants) are essential to high sustainable biomass production. The cloning of selected plants
on an industrial scale through in vitro cultivation has many advantages, being important for the supply of plants
in sufficient quantity and quality to meet commercial demand. The control of the cloning is the basis for an
industrial scale, and its knowledge can optimize the process. This work aimed to evaluate the cloning of
Dendrocalamus asper selected plant through micropropagation. Morphological features by scanning electron
microscopy and genetic stability with ISSR molecular markers were evaluated. Four times of immersion in
sodium hypochlorite (NaOCl) on in vitro establishment of nodal segments were evaluated. The established
explants were transferred to a culture medium that was supplemented with three concentrations of 6-
benzylaminopurine (BAP). Three concentrations of indole-3-butyric acid (IBA) to the in vitro adventitious
rooting were evaluated. NaOCl application for 10 min resulted in 71.4 % of establishment in 30 d.
Supplementation of the culture medium with 2.0 and 3.0 mg L-1 BAP de resulted in the highest averages for
multiplication and elongation stages. The formation of adventitious roots occurred with 4.0 mg L-1 IBA of
supplementation. Micropropagated plants showed normal morphological features and genetic stability,
confirming the cloning of selected plant.
Keywords: bamboo; micropropagation; vegetative propagation; In vitro culture; ISSR; plant growth regulator.
Produção de microplantas clonais, avaliação morfofisiológica e estabilidade
genética de
Dendrocalamus
asper
(Schult. & Schult.) Backer ex. K. Heyneke
RESUMO: Espécies de bambus apresentam diversas aplicações comerciais, visto que os plantios homogêneos
(formados a partir de plantas clonais) são essenciais para a alta produção de biomassa sustentável. A clonagem
de plantas selecionadas em escala industrial por meio do cultivo in vitro apresenta muitas vantagens, sendo uma
importante ferramenta para o fornecimento de plantas em quantidade e qualidade suficientes para atender a
demanda comercial. O controle da clonagem é a base para escala industrial, e seu conhecimento pode otimizar
os processos. O trabalho teve como objetivo avaliar a clonagem de planta selecionada de Dendrocalamus asper
por meio da técnica de micropropagação. Foram avaliadas as características morfológicas por microscopia
eletrônica de varredura e estabilidade genética por meio de marcadores moleculares ISSR. Além disso, foram
avaliados quatro tempos de imersão em hipoclorito de sódio (NaOCl) no estabelecimento in vitro de segmentos
nodais. Os explantes estabelecidos foram transferidos para um meio de cultura que foi suplementado com três
concentrações de benzilaminopurina (BAP). Por fim, foram avaliadas três concentrações de ácido indolbutírico
(AIB) durante o enraizamento adventício in vitro. A adição de NaOCl por 10 min resultou em 71,4 % de
estabelecimento em 30 d. A suplementação do meio de cultura com 2,0 e 3,0 mg L-1 BAP resultou nas maiores
médias para as fases de multiplicação e alongamento. A formação de raízes adventícias ocorreu com a
suplementação de 4,0 mg L-1 de AIB. Plantas micropropagadas apresentaram características morfológicas
normais e estabilidade genética, confirmando a clonagem da planta selecionada.
Palavras-chave: bambu; micropropagação; propagação vegetativa; cultivo in vitro; ISSR; regulador de
crescimento vegetal.
1. INTRODUCTION
Bamboo species occur naturally in tropical, subtropical,
and temperate regions all over the world (SINGH et al.,
2013a; CANAVAN et al., 2017; RAMAKRISHNAN et al.,
2020; MUSTAFA et al., 2021). More than 4,000 traditional
uses and 1,500 commercial applications with economic
viability for bamboos have been described, besides its
ecological and social usages - mainly, due to its morphological
and silvicultural features (LIESE; KOHL, 2015; ZHAO et
al., 2017; SAWARKAR et al., 2021; TEIXEIRA et al., 2021).
In Brazil the production of bamboo is still on a small
scale, despite its great capacity for silvicultural applications
Clonal microplant production, morphological evaluation and genetic stability of Dendrocalamus asper …
Nativa, Sinop, v. 11, n. 1, p. 01-09, 2023.
2
(INBAR, 2015; ROSA et al., 2016). To encourage the
cultivation of bamboo species in Brazil, on 8 September
2011, the Law Number 12,484 was enacted (PNMCB, 2011),
and it institutes a National Policy for Encouragement,
Sustainable Management, and Bamboo Cultivation and aims
the development of the culture through government actions
and private corporations.
Dendrocalamus asper (Schult. & Schult.) Backer ex K.
Heyneke, known as “giant bamboo” or “bucket bamboo”,
stands out from other species. According to Benton et al.
(2015), its fibers produce a resistant raw material that is
flexible and fast-growing, and is used for house construction
in many rural areas and handicrafts. It is suitable for the
production of cellulose and papermaking; the manufacture of
household utensils and furniture; as food - for its edible
sprouts; fiber; biofuel; for construction and the manufacture
of musical instruments (INBAR, 2015; SINGH et al., 2012).
Homogeneous plantings of the species can provide a rapid
source of renewable biomass (MUSTAFA et al., 2021;
KONZEN et al., 2021a), and the cloning of selected plants
on an industrial scale (e.g., in biofactories) can provide plants
in desirable quality and quantity.
Bamboos can be multiplied sexually or asexually;
however, flowering cycles varies between species (some can
reach up to 120 years), and in many cases plants die after
flowering, and seeds have low viability (BANIK, 2015;
BENTON, 2015; LIN et al., 2019; ZHAO et al., 2017). Thus,
to enhance the value of superior genotypes, the best option
is vegetative propagation. Separating clumps and planting
stalk pieces are laborious techniques and low-efficiency
(COSTA et al., 2017; KONZEN et al., 2021a). Despite the
difficulties and considering some alternatives for the
propagation of bamboo species, in vitro cultivation can be a
viable tool for the production of a large number of clonal
plants (RIBEIRO et al., 2016; FURLAN et al., 2018;
HOSSAIN et al., 2018; RIBEIRO et al., 2020; TEIXEIRA et
al., 2021).
Micropropagation is important technique for cloning and
improving selected phenotypes (BHADRAWALE et al.,
2018; TAMBARUSSI et al., 2017; SILVEIRA et al., 2020;
KONZEN et al., 2021a), as it can generate plants with the
same genetic feature of the parent plant (i.e., selected plant in
adult stage) on industrial scale (HARTMANN et al., 2011;
BRONDANI et al., 2017; KONZEN et al., 2021b).
However, in some cases, this technology can induce genetic
and morphological changes, known as somaclonal variations
(LARKIN; SCOWCROFT, 1981; KONZEN et al., 2021b).
This variation is considered a disadvantage when the aim is
cloning, and for this reason, the development of further
studies on genetic stability through in vitro cultivation is
important for understanding the factors involved in the
clonal production of bamboo species (KONZEN et al.,
2017; RAMAKRISHNAN et al., 2020; KONZEN et al.,
2021b).
This work aimed to evaluate the cloning of Dendrocalamus
asper through micropropagation technique, considering the
morphological features and genetic stability of
micropropagated plants.
2. MATERIAL AND METHODS
2.1.
In vitro
establishment
Seedling (i.e., selected plant) of Dendrocalamus asper
(Schult. & Schult.) Backer ex K. Heyneke was transferred to
four-liter-recipient containing mixture of washed sand and
sifted subsoil - 3 mm mesh (1/1, v/v) and suspended at 1 m
of height. The cultivation was carried out in a greenhouse
without temperature and relative humidity control.
The selected plant was fertigated weekly with a nutrient
solution developed for the growth and shoot induction
(MOLINARI et al., 2020). Weed control was carried out by
manual plucking. Irrigation was carried out once a day,
directly on the substrate, avoiding the contact of water with
the aerial parts of the plant.
Shoots from selected plant cultivated by one-year-old
were collected and transported to the laboratory.
Subsequently, the leaf sheaths were removed, and their
remains were carefully scraped with a stylus for yolk
exposition, facilitating asepsis (RIBEIRO et al., 2020;
TEIXEIRA et al., 2021). The tissues were then washed with
deionized and autoclaved water and neutral detergent. Shoots
were reduced to 1 cm long nodal explants and immersed for
30 s in a 70 % hydroalcoholic solution followed by deionized
and sterilized water. Subsequently, they were immersed in a
sodium hypochlorite (NaOCl) solution (1.00 - 1.25 % of
active chlorine), and four times of exposure to chemical
disinfectant were tested (5 - control, 10, 15, and 20 min).
After this immersion, the explants were transported to a
laminar flow chamber, where they were subjected to four
washes with deionized and sterilized water to eliminate
surface residues of sodium hypochlorite.
The explants were inoculated vertically in test tubes (20
× 150 mm) containing 10 mL of MS culture medium
(MURASHIGE; SKOOG, 1962), which were sealed with
plastic film made with poly(vinyl chloride) (PVC). The
experiment was conducted in a randomized design with four
different times of NaOCl immersion, and 40 replications
(one explant per replication). Percentage of total
contamination [i.e., ∑(fungal + bacterial contamination)],
tissue oxidation, establishment and shoot induction (i.e.,
explant with shoot) were evaluated at 30 d. Explants free of
contamination and tissue oxidation were considered
established.
2.2.
In vitro
multiplication and elongation
Established explants were transferred to glass flasks (72
× 72 × 100 mm) containing 50 mL of MS culture medium
supplemented with three concentrations of 6-
benzylaminopurine (BAP) (1.0 - control, 2.0, and 3.0 mg L-1).
Culture medium of all treatments was supplemented with 0.5
mg L-1 of α-naphthalene acetic acid (NAA). Three
subcultures were performed out every 30 d (Sub 1 - 30 d, Sub
2 - 60 d, and Sub 3 - 90 d). The experiment was conducted in
a completely randomized design in factorial arrangement
with three concentrations of BAP and three subcultures, and
15 replications (one explant per replication). Percentage of
survival, shoot induction, number of shoots per explant, and
shoot length per explant (cm) were evaluated in each
subculture.
2.3.
In vitro
adventitious rooting
Explants with 3-cm-long (from the in vitro multiplication
and elongation stages) were inoculated in glass flasks (72 ×
72 × 100 mm) containing 50 mL of MS culture medium
supplemented with three concentrations of indole-3-butyric
acid (IBA) (2.0 - control, 4.0, and 6.0 mg L-1). The culture
medium was supplemented with 1.0 mg L-1 NAA and 0.5 mg
L-1 BAP in all treatments. The experiment was conducted in
Gonçalves et al.
Nativa, Sinop, v. 11, n. 1, p. 01-09, 2023.
3
a randomized design with three concentrations of IBA, and
15 replications (one explant per replication). Percentage of
adventitious rooting was evaluated at 30 d. Leaf samples of
rooted plants were collected for scanning electron
microscopy (SEM) and genetic stability analysis.
2.4. Preparation of the culture medium and incubation
conditions
Culture medium was prepared with deionized water, 6 g
L-1 of agar and 30 g L-1 of sucrose. The pH of the solution
was adjusted to 5.8 with HCl (0.1 M) and/or NaOH (0.1 M)
before adding the agar to the culture medium, and then
autoclaved at 127 °C (1.5 kgf cm-2) for 20 min. BAP, NAA
and IBA were added to the culture medium before
autoclaving. Explants were cultivated in a grow-room with a
temperature of 24 °C (±1 °C), irradiation of 40 μmol m-2 s-1
(cold white tube lamp), and 16 h of photoperiod.
2.5. Scanning electron microscopy
Leaf samples from in vitro rooted microplants were
collected and sectioned in 0.5 cm² sizes, and placed in
microtubes (1.5 mL) containing Karnovsky (1965) fixative
(2.5 % of glutaraldehyde, 2.0 % of paraformaldehyde, 0.05 M
of cacodylate buffer at pH 7.2, and 0.001 M of CaCl2) for 72
h in a refrigerator (4 °C). Subsequently, the samples were
washed three times in cacodylate buffer (0.05 M) for 10 min.
The dehydration step was carried out with a graduated series
of acetone (25, 50, 75, 90, and 100 %) for 10 min, with 100
% concentration transferred three times. Then, the samples
were placed in porous support containing acetone and sent
to the drying stage at the critical point, where the acetone was
volatized and replaced by carbon dioxide (CO2). The
supporters were wrapped in aluminum foil and fixed in
double-sided carbon tape for the assembly of leaf samples in
the blocks. Finally, the gold metallization stage was carried
out. The observation of images was made in SEM (LEO
EVO-40).
2.6. Genetic stability
Young leaves were collected from the in vitro rooted
microplants and selected plant for the evaluation of genetic
stability. DNA extractions were performed according to an
adapted protocol by Ferreira and Grattapaglia (1998).
Eighteen primers were used (Chris, John, UBC809, UBC811,
UBC814, UBC825, UBC827, UBC834, UBC835, UBC840,
UBC841, UBC842, UBC844, UBC848, UBC857, UBC880,
UBC898, and UBC901). ISSR reactions were prepared in
microplates (PCR-96-Axygen Scientific), with 3 μL DNA
(standardized at 20 ng µL-1 for all samples) and 10 μL of
reaction mix [1.5 mM PCR buffer Phoneutria®, 1.5 mM
dNTP, 1 U of Taq polymerase Phoneutria® (5.0 U μL-1),
Diluent Taq (based on BSA and Tris-HCl), and 0.2 mM of
each primer, completing the final volume with ultrapure
water (4.2 μL)].
2.7. Statistical analysis
Data analysis was performed with the R Core Team
Software (2018), ExpDes package version 1.1.2 (Ferreira et
al. 2013). Collected data were analyzed for Hartley (p > 0.05)
and Shapiro-Wilk (p > 0.05) tests. According to the results,
the data were transformed using the Box-Cox test. After, the
data were submitted to analysis of variance (ANOVA, p <
0.05) and the means were compared using the Tukey's test (p
< 0.05).
3. RESULTS
3.1.
In vitro
establishment
NaOCl application as a disinfectant chemical agent,
significantly influenced the in vitro establishment of
Dendrocalamus asper explants (Figure 1). High percentage of
fungal (93.3 %) and bacterial (6.7 %) contamination in 5 min
of exposure to NaOCl denotes the importance of controlling
in vitro establishment conditions (Figure 1A), because there
was total loss of explants. Increasing the exposure time from
10 to 20 min resulted in low contamination (24.4 to 28.5 %)
(Figure 1A), being considered efficient for asepsis. However,
the in vitro establishment and morphological features were
affected (Figure 1B-D).
Percentage of tissue oxidation varied according to time of
exposure to the chemical agent (Figure 1B), and it is possible
to observe that the longest exposure time (i.e., 15 to 20 min)
there was higher tissue oxidation (17.0 to 40.0 %). There was
no tissue oxidation in 10 min of exposure to NaOCl (Figure
1B), furthermore, the highest percentage of in vitro
establishment (71.4 %) (Figure 1C) and explants that emitted
shoots (62.9 %) (Figure 1D) were observed in this time.
3.2.
In vitro
multiplication and elongation
No significant difference was observed between the BAP
supplementations for explant survival in 90 d of in vitro
culture, with values ranging from 75.0 to 100 % (Table 1).
Thus, it can be considered that the protocol for the in vitro
multiplication and elongation of Dendrocalamus asper was
efficient, according to this parameter, due to the low
percentage of explant mortality and adequate shoot induction
(average of 87.5 % of explant with shoot in 90 d).
Multiplication and elongation stages occurred
simultaneously for Dendrocalamus asper, representing an
important phenomenon when considering in vitro culture to
obtain large-scale plants in a reduced time. There was a
significant difference for the number of shoots, in which the
highest values with the use of 3.0 mg L-1 BAP (2.2 shoots per
explant, Figure 2A) and in the third subculture at 90 d (2.1
shoots per explant, Figure 2B) were observed.
Highest shoot length per explant was observed in 2.0 and
3.0 mg L-1 BAP supplementation to the culture medium,
resulting in shoots with an average length of 4.4 and 5.0 cm,
respectively (Figure 2C). Considering the number of
subcultures (performed every 30 d), the highest mean of
shoot length was found in the second (4.5 cm) and third (5.1
cm) subcultures (Figure 2D).
Table 1. Percentage of in vitro survival of Dendrocalamus asper explants
according to BAP supplementation and subcultures.
Tabela 1. Porcentagem de sobrevivência in vitro de explantes de
Dendrocalamus asper em relação à suplementação de
benzilaminopurina (BAP) e subcultivo.
BAP (mg L
-1
)
S
ub
1 (%)
S
2 (%)
S
ub
3 (%)
1.0 (control)
75.0
a
(±1
5
.0)
75.0
a
(±1
5
.0)
75.0
a
(±1
5
.0)
2.0
100.0
a
(±0.0)
87.0
a
(±10.0)
87.0
a
(±10.0)
3.0
100.0
a
(±0.0)
75.0
a
(±1
5
.0)
75.0
a
(±1
5
.0)
Means with the same letters do not differ significantly by the Tukey's test (p
< 0.05). Data presented as mean ± standard error. Sub = subculture (Sub 1
- 30 d, Sub 2 - 60 d, and Sub 3 - 90 d).
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Nativa, Sinop, v. 11, n. 1, p. 01-09, 2023.
4
Figure 1. Variables evaluated during in vitro establishment of Dendrocalamus asper explants, at 30 d. (A) Percentage of total contamination
[∑(fungal + bacterial contamination)]; (B) Percentage of tissue oxidation; (C) Percentage of in vitro establishment; and (D) Percentage of
explants with shoot (i.e., shoot induction). Means with the same letters do not differ significantly by the Tukey's test (p < 0.05). Data
presented as mean ± standard error.
Figura 1. Variáveis avaliadas durante do estabelecimento in vitro de explantes de Dendrocalamus asper, aos 30 d. (A) Porcentagem de
contaminação total [∑(fúngica + bacteriana)]; (B) Porcentagem de oxidação tecidual; (C) Porcentagem de estabelecimento in vitro; e (D)
Porcentagem de explantes apresentando broto (indução de broto). Médias com a mesma letra não diferem significativamente pelo teste de
Tukey (p < 0.05). Dados apresentados como média ± erro padrão.
Figure 2. Variables evaluated on in vitro multiplication and elongation stages of Dendrocalamus asper explants. (A) Number of shoots per
explant according to BAP supplementation; (B) Number of shoots per explant according to subculture; (C) Shoot length per explant (cm)
according to BAP supplementation; and (D) Shoot length per explant (cm) according to subculture. Means with the same letters do not
differ significantly by the Tukey's test (p < 0.05). Data presented as mean ± standard error. Sub = subculture (Sub 1 - 30 d, Sub 2 - 60 d,
and Sub 3 - 90 d).
Figura 2. Variáveis avaliadas durante a multiplicação e alongamento in vitro of explantes de Dendrocalamus asper. (A) Número de brotações
por explante de acordo com a suplementação de BAP; (B) Número de brotações por explante de acordo com o subcultivo; (C) Comprimento
de broto por explante (cm) de acordo com a suplementação de BAP; e (D) Comprimento de broto por explante (cm) de acordo com o
subcultivo. Médias com a mesma letra não diferem significativamente pelo teste de Tukey (p < 0.05). Dados apresentados como média ±
erro padrão. Sub = subcultivo (Sub 1 - 30 d, Sub 2 - 60 d, e Sub 3 - 90 d).
Gonçalves et al.
Nativa, Sinop, v. 11, n. 1, p. 01-09, 2023.
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3.3.
In vitro
adventitious rooting
Adventitious rooting was only observed when the culture
medium was supplemented with 4.0 mg L-1 IBA, resulting in
60.0 % at 30 d of in vitro culture (Table 2).
Table 2. Percentage of in vitro adventitious rooting of Dendrocalamus
asper explants according to IBA supplementation in 30 days.
Tabela 2. Porcentagem de enraizamento adventício in vitro de
explantes de Dendrocalamus asper de acordo com a suplementação de
ácido indolbutírico (IBA), aos 30 dias.
IBA (mg L-1) Adventitious rooting (%)
2.0 (control) 0.0b (±0.0)
4.0 60.0a (±18.0)
6.0 0.0b (±0.0)
Means with the same letters do not differ significantly by Tukey's test (p <
0.05). Data presented as mean ± standard error.
3.4. Scanning electron microscopy
Morphological features of leaf surface of Dendrocalamus
asper microplants in vitro grown were performed (Figure 3A-
D). The adaxial surface of the leaf blade has normal stomata,
spines, single-celled trichomes, and microtrichomes, both in
small quantities (Figure 3A), and the stomata surrounded by
epidermal papillae and microtrichomes (Figure 3B).
On the abaxial surface (Figure 3C), hook-shaped
trichomes, long single-celled trichomes, stomata arranged in
rows close to the trichomes, and a high papillae density was
observed. Papillae (Figure 3D) is often associated with
stomata, hook-shaped trichomes, microtrichome, spine, and
the distribution pattern of the stomata.
Figure 3. Morphological features of the leaf surface of Dendrocalamus
asper microplant in vitro grown. (A) Paradermal section of the adaxial
face with the presence of unicellular trichomes, stomata, spines and
microtrichomes; (B) details of stomata and presence of
microtrichome; (C) abaxial face, microtrichomes, spines, papillae,
stomata and trichomes; and (D) detail of stomata with papillae and
hook-shaped trichomes. (ep) Thorn; (es) Stomata; (mt)
Microtrichome; (pp) Papilla; (tg) Hook-shaped trichome; and (tu)
Unicellular trichome.
Figura 3. Características morfológicas da superfície foliar de
microplanta de Dendrocalamus asper cultivada in vitro. (A) Corte
paradérmico da face adaxial com presença de tricomas unicelulares,
estômatos, espinhos e microtricomas; (B) Detalhes dos estômatos e
presença de microtricomas; (C) Face abaxial, microtricomas,
espinhos, papilas, estômatos e tricomas; e (D) Detalhe dos
estômatos com papilas e tricomas em forma de gancho. (ep)
Espinho; (es) Estômato; (mt) Microtricoma; (pp) Papila; (tg)
Tricoma em forma de gancho; e (tu) Tricoma unicelular.
3.5. Genetic stability
The microplants were identical clones to the parent plant
of Dendrocalamus asper (i.e., selected plant). All tested primers
showed adequate amplification and discernible bands in the
evaluation of genetic stability. There was no polymorphism,
that is, the bands exhibited the same profile for all
microplants (Table 3). The presence of monomorphism
confirms that there was no somaclonal variation during the
in vitro subculture in 90 d.
Table 3. Primer amplification result of Dendrocalamus asper
micropagated clonal plants at 90 d.
Tabela 3. Resultado da amplificação do primer de plantas clonais
micropagadas de Dendrocalamus asper, aos 90 d.
Primer Sequence
Total
bands
Monomorphic
Polymorphic
Chris (CA)7-YG 3 3 0
John (AG)7-YC 6 6 0
UBC809
(AG)8-G 6 6 0
UBC811
(GA)8-C 6 6 0
UBC814
(CT)8-T 6 6 0
UBC825
(AC)8-T 5 5 0
UBC827
(AC)8-G 4 4 0
UBC834
(AG)8-YT 6 6 0
UBC835
(AG)8-YC 4 4 0
UBC840
(GA)8-YT 6 6 0
UBC841
(GA)8-YC 5 5 0
UBC842
(GA)8-YG 3 3 0
UBC844
(CT)8-RC 4 4 0
UBC848
(CA)8-RG 5 5 0
UBC857
(AC)8-YG 6 6 0
UBC880
(GGAGA)3
6 6 0
UBC898
(CA)6-RY 6 6 0
UBC901
(GT)6-YR 3 3 0
Note: R = purine (A or G) and Y = pyrimidine (C or T).
4. DISCUSSION
4.1.
In vitro
establishment
Biotechnology involves effective tools for bamboos
propagation on a large scale (SINGH et al., 2013a;
ORNELLAS et al., 2017). However, contamination by
phytopathogenic organisms is one significant obstacle to
achieving success in the in vitro establishment (RIBEIRO et
al., 2016; TORRES et al., 2016; FURLAN et al., 2018;
RIBEIRO et al., 2020). Microorganism in vitro contamination
has been reported for several bamboo species, such as
Bambusa ventricosa (WEI et al., 2015), Bambusa vulgaris
(FURLAN et al., 2018; RIBEIRO et al., 2020; TEIXEIRA et
al., 2021), Dendrocalamus asper (SINGH et al., 2012) and
Dendrocalamus strictus (PANDEY; SINGH, 2012), being an
important event to be controlled during the tissue in vitro
introduction. Therefore, defining asepsis protocols with
antimicrobial agents is essential to minimize the
contamination (BRONDANI et al., 2017; ORNELLAS et al.,
2017).
Sodium hypochlorite is an alternative in the
micropropagation of Dendrocalamus asper, as it is considered a
low cost and effective product for tissue disinfestation of
forest species (BRONDANI et al., 2013; BRONDANI et al.,
2017). In the present study, the use of this chemical agent was
effective for the control of fungal and/or bacterial
contamination (Figure 1A). However, the exposure time to
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NaOCl can increase tissue oxidation (Figure 1B), and it is
important to define the better exposure time.
The percentage of tissue oxidation increased according to
use of sodium hypochlorite, and the longer exposure times
resulted in greater oxidation (15 at 20 min), denoting that the
species' tissues may be highly susceptible when exposed to
chemical treatment for longer period of time. Oxidation
events are most often related to tissue healing mechanisms,
which cause exudation of phenolic compounds in response
to lesions in explants during preparation and in vitro
inoculation (MUDOI et al., 2013; KONZEN et al., 2021a),
which can make explants unviable, affecting the next stages
of cultivation (BRONDANI et al., 2017; BHADRAWALE
et al., 2018; RIBEIRO et al., 2020).
High percentage of in vitro establishment (Figure 1C) and
shoot induction (Figure 1D) at 30 d were observed in 10 min
of exposure to NaOCl. The value for the establishment can
be considered adequate, denoting the importance of high
percentages of shoot induction to be used for the other stages
(e.g., multiplication and elongation). Satisfactory values for in
vitro establishment were observed for Dendrocalamus asper
(SINGH et al., 2012) and Dendrocalamus strictus (PANDEY;
SINGH, 2012), using active chlorine as a disinfectant - what
corroborates with the results of the present study.
In addition, with the increased exposure time to the
disinfesting agent, the shoot induction was lower. This factor
may be related to the toxicity that the active chlorine can
cause to the explant tissues. According to Brondani et al.
(2013), multiple factors can interfere in the in vitro
establishment, such as genetic material, type and origin of the
explant, ontogenetic and physiological conditions of the
selected plant, asepsis method, and phytotoxicity caused by
the disinfecting agents.
4.2.
In vitro
multiplication and elongation
A direct relationship between BAP concentration and the
number and length of shoots per explant was observed
(Figure 2A-D), considering that the highest percentage of
multiplication and elongation was observed in higher
concentrations of the plant growth regulator. The addition of
cytokinin and auxin in the culture medium is used to induce
multiplication and shoot elongation (HARTMANN et al.,
2011). Plant growth regulators are applied exogenously and
can be used singly or combined, depending of the objective,
species, and the endogenous levels found in the tissues
(SINGH et al., 2004; BANIK, 2015). Higher concentrations
of cytokinin in the multiplication and elongation were
reported for bamboos (NEGI; SAXENA, 2011; PANDEY;
SINGH, 2012), corroborating with the findings in this study.
BAP supplementation in culture medium is reported to get
high multiplication in Dendrocalamus asper (SINGH et al.,
2012; SINGH et al., 2013b; ORNELLAS et al., 2017) and
Drepanostachyum luodianense (LIN et al., 2019).
Highest values for the number of shoots per explant
occurred in the third subculture (Figure 2B); and shoot length
in the second and third subcultures (Figure 2D), suggesting
that the increase in the number of subcultures favors the
multiplication and elongation of Dendrocalamus asper
simultaneously, showing a high apical dominance.
Nevertheless, the increase in BAP concentration in the
culture medium did not favor growth in length of
Dendrocalamus asper (SINGH et al., 2012), showing that lower
concentrations indicated better results under certain in vitro
culture conditions and should be considered.
According to Santos et al. (2016), defining the ideal
number of subcultures is of great relevance for adapting
explants to the newly established conditions, favoring the
absorption of the exogenous source of cytokinin and auxin,
for increasing the response to the morphogenic stimulus.
Numerous subcultures may be necessary in multiplication
until reaching the desired number of microplants, with the
identical genetic composition of the parent plant
(NOGUEIRA et al., 2017).
4.3.
In vitro
adventitious rooting
There was root formation only in the treatment with 4.0
mg L-1 IBA (60.0 % of adventitious rooting, Table 2).
Rooting stage is recommended for radial system obtention
with standard and functional structure, which may favor
plants' survival and ex vitro growth, avoiding possible losses
during acclimatization (NOGUEIRA et al., 2017; KONZEN
et al., 2021a; TEIXEIRA et al., 2021). However, this stage of
micropropagation is one of the major obstacles during in vitro
cultivation in bamboo species (SINGH et al., 2012).
Adventitious rooting is a fundamental step in any
micropropagation system (RIBEIRO et al., 2016; FURLAN
et al., 2018; HOSSAIN et al., 2018; SILVEIRA et al., 2020).
Commonly, bamboo species do not root easily, and it is
common observing a low number of responsive explants
(MUDOI et al., 2013; SANDHU et al., 2018). However,
Ramanayake et al. (2008) evaluated different IBA
concentrations in the rooting of Dendrocalamus giganteus, and
verified up to 100 % of rooting. Nogueira et al. (2019) found
high rooting in Guadua magna and Guada angustifolia when used
3.0 mg L-1 IBA. These observations reinforce the need to
supplement the culture medium with IBA to induce
adventitious root in Dendrocalamus asper.
Nevertheless, it is important that the selected plants (i.e.,
stock plant) utilized for tissue in vitro inoculation have seminal
origin, which indicates a low ontogenetic age. This feature
can influence cellular ability response to plant growth
regulator stimuli, inducing more adventitious rooting
capacity due to tissue juvenility (HARTMANN et al., 2011;
WENDLING et al., 2014; KUMAR et al., 2022).
4.4. Scanning electron microscopy
Ultrastructural morphological knowledge is important in
several investigations, mainly for species identification
(MONTIEL; SÁNCHEZ, 2006a). Leaf surface morphology
of Dendrocalamus asper microplants in vitro grown (Figure 3A-
D) were compatible with the characterization performed by
Montiel and Sánchez (2006a; 2006b). According to these
authors, the leaf blade surfaces of the clones have broad
trichomes and spines, corroborating with the description for
Dendrocalamus asper, denoting absence of morphological
variation.
On the abaxial surface, a large density of papillae was
found (Figure 3C). Those are important structures in the
taxonomy of bamboo species (OLIVEIRA et al., 2008).
Papillae in greater abundance on the abaxial face in
Dendrocalamus asper were observed, but it could also be found
on the adaxial face. It was also observed that there was a
greater presence of stomata, trichomes and papillae cells on
the abaxial face when compared to the adaxial face (Figure
3A-D).
Gonçalves et al.
Nativa, Sinop, v. 11, n. 1, p. 01-09, 2023.
7
4.5. Genetic stability
Dendrocalamus asper microplants are clones from selected
plant, according to observed results. Even though
somaclonal variation was not observed in the present study,
with the increase in subcultures, this phenomenon may occur
due to several factors, including the addition of high
concentrations of plant growth regulators to the culture
media (VENKATACHALAM et al., 2007; KONZEN et al.,
2017; RAMAKRISHNAN et al., 2020; KONZEN et al.,
2021b). It can also happen due to cell cycle disturbances
caused by exogenous application of plant growth regulators
(PESCHKE; PHILLIPS, 1992; HARTMANN et al., 2011).
According to Singh et al. (2013b), verifying the genetic
stability of micropropagated plants in an early stage can
contribute to the definition of reliable protocols, avoiding
future problems with the plants in the field after planting.
Several types of research have been carried out with
bamboo species to verify the genetic stability of clones when
compared to the parent plants, using ISSR markers, such as
in Dendrocalamus asper (SINGH et al., 2012), Bambusa bamboo
(ANAND et al., 2013), Guadua magna and Guada angustifolia
(NOGUEIRA et al., 2019). In this study, no genetic variation
of microplant in vitro grown was observed even after 6 mth
of in vitro cultivation using plant growth regulators. Thus, it is
possible to suggest that the methodology was efficient for the
clonal microplant production of Dendrocalamus asper, and can
be used for applications in biofactories aiming at the
formation of homogeneous forests.
5. CONCLUSIONS
Sodium hypochlorite (NaOCl) in a concentration of 1.00
- 1.25 % of active chlorine for 10 min favored the in vitro
establishment and shoot induction.
Culture medium supplemented with 2.0 - 3.0 mg L-1 BAP
favored the multiplication and elongation stages
simultaneously, showing a high apical dominance.
Adventitious root formation was confirmed only in 4.0
mg L-1 IBA supplemented at culture medium.
Micropropagated plants have normal leaf morphology
and genetic stability to selected plant, which could favor the
formation of clonal plantations of the species.
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Acknowledgements
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”).
Author Contributions
D.S.G. - conceptualization, methodology, investigation or data
collection, statistical analysis and writing; D.M.S.C.S., L.V.M.,
M.L.M.A. - investigation or data collection, writing. D.C., G.L.T.
validation, review; G.E.B. - conceptualization, acquisition of
financing, methodology, supervision, validation, review. All authors
read and agreed to the published version of the manuscript.
Institutional Review Board Statement
Not applicable.
Informed Consent Statement
Not applicable
Data Availability Statement
Study data can be obtained by request to the corresponding
author, via e-mail.
Conflicts of Interest
The authors declare no conflict of interest. Supporting entities
had no role in the design of the study; in the collection, analyses, or
interpretation of data; in the writing of the manuscript, or in the
decision to publish the results.