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Bioscience Journal Original Article
Biosci. J., Uberlândia, v. 36, n. 1, p. 42-50, Jan./Feb. 2020
http://dx.doi.org/10.14393/BJ-v36n1a2020-42131
DOES SOAKING TIME DURING DISINFESTATION AFFECT
GERMINATION RATES IN Dendrobium?
O TEMPO DE IMERSÃO DURANTE A DESINFESTAÇÃO INFLUENCIA NAS TAXAS
DE GERMINAÇÃO DE Dendrobium?
José Carlos SORGATO1; Jackeline Schultz SOARES1; Silvana de Paula Quintão SCALON1;
Suzana Targanski Sajovic PEREIRA2; Débora Freitas BROTTO3; Luan Marlon RIBEIRO1
1. Universidade Federal da Grande Dourados, Faculdade de Ciências Agrárias, Dourados, MS, Brasil. josesorgato@ufgd.edu.br; 2.
Universidade Estadual Paulista, Faculdade de Ciências Agrárias e Veterinárias, Jaboticabal, SP, Brasil; 3. Universidade Estadual de
Mato Grosso do Sul, Dourados, MS, Brasil.
ABSTRACT: Asymbiotic germination is considered an efficient and viable technique that can increase
germination rates. The effect of type and concentration of disinfestants, and the exposure time to disinfestants
may differ according to the plant species. Therefore, species-specific standardization of disinfestation agent and
procedure is necessary to achieve optimal germination rates. The objective of this study was to determine a
disinfestation methodology to increase in vitro germination rates and the early development of seedlings of
Dendrobium nobile and Dendrobium phalaenopsis, using different times for seed disinfestation and different
culture media. Seeds were disinfected by soaking in a 0.8% sodium hypochlorite solution for 5 or 15 min under
aseptic conditions, after which seed suspensions were either washed with water or left unwashed. Next, they
were seeded in culture flasks containing four different culture media (MS, ½MS, K, and VW). The flasks were
then transferred to a growth room under controlled photoperiod and temperature, where they remained under an
irradiance of 20 μmol m-2 s-1. Germination rates of the species were evaluated 45 days after placement in the
culture flasks. A higher germination rate was observed when the seeds were triple washed, regardless of the
culture medium or soaking time. Seed soaking disinfestation for 5 min is also recommended. MS and ½MS
media were the most effective culture media in promoting in vitro germination of the species under study.
KEYWORDS: Asymbiotic germination. Orchidaceae. Dendrobium nobile. Dendrobium phalaenopsis.
Sodium hypochlorite. Ornamental species.
INTRODUCTION
The production of flowers and ornamental
plants in Brazil is worth more than R$ 7 billion per
year, with an annual growth rate between 8% and
12% that generates over 200,000 direct jobs
(PAIVA et al., 2016; IBRAFLOR, 2017). Orchid
cultivation is an important part of this business
(JUNQUEIRA; PEETZ, 2014; 2017).
Imports of orchid seedlings in 2013
increased by about 20%, compared to that in 2012
(JUNQUEIRA; PEETZ 2014). The largest exporter
of Dendrobium worldwide is Thailand, while Brazil
is one of the main importers (LEKAWATANA,
2010).
Given the need to supply market demand,
there is a need to improve the procedures for
producing flowers and ornamental plants, including
orchids (PASQUAL et al., 2008; JUNQUEIRA;
PEETZ, 2014; 2017). In vitro culture techniques
deserve special mention among production schemes,
as they are important biotechnological tools to
obtain plants for both research and commercial-
scale production (CARDOSO, 2014).
Orchid seeds have been asymbiotically
germinated since the beginning of the last century
(KNUDSON, 1922). This type of seeding
effectively accelerates the production of uniform
orchid seedlings because it allows higher
germination rates than germination under natural
conditions, which depends on mycorrhizal fungi
(FARIA et al., 2012; ABRÃO et al., 2014).
However, although widely used, available
knowledge on asymbiotic germination – particularly
regarding the nutritional composition of culture
media and seeding protocols to foster germination
and minimize crop contamination – is still limited to
a few species.
The most frequently used culture media for
in vitro seeding of this botanical family are K
(KNUDSON, 1946), VW (VACIN; WENT, 1949)
and MS (MURASHIGE; SKOOG, 1962) (SILVA et
al., 2015). Culture medium formulation is essential
for the seed because it provides the necessary
nutrients (e.g., minerals, vitamins, and growth
regulators) for development of the seedling. The
composition of the culture medium varies depending
Received: 05/05/18
Accepted: 10/02/19
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Biosci. J., Uberlândia, v. 36, n. 1, p. 42-50, Jan./Feb. 2020
http://dx.doi.org/10.14393/BJ-v36n1a2020-42131
on the particular requirements of each species
(FARIA et al., 2012).
The success of asymbiotic germination
depends largely on the absence of microbial
contamination from the culture medium. Infection
of the plant material or the culture medium by fungi
and bacteria can result in poor germination or poor
seedling development. In their review, Silva et al.
(2015) reported that ethanol (EtOH), mercury
chloride (HgCl2), and sodium hypochlorite (NaClO)
were the most commonly used products to disinfect
seeds of Dendrobium under asymbiotic cultivation.
Silva et al. (2015) pointed out that the type,
concentration, and time of exposure to the
disinfecting agent differ considerably and, therefore,
require species-specific standardization.
Although seed disinfestation by sodium
hypochlorite is a standard procedure, some seeds of
the Orchidaceae family are susceptible to this
chemical and use of this agent often results in low
germination rates in an asymbiotic environment.
Sodium hypochlorite can be toxic, depending on the
concentration and time of exposure, thereby
reducing germination rates (CHU; MUDGE, 1994).
Therefore, the objective of this study was to
determine a methodology to increase in vitro
germination rates and favor early development of
seedlings of two orchid species (Dendrobium nobile
Lindl. and Dendrobium phalaenopsis Fitzg.) using
different culture media and different methods for
seed disinfestation.
MATERIAL AND METHODS
Seeds were obtained from mature fruits of
D. nobile and D. phalaenopsis. The plants had been
manually pollinated. The orchids had been
established for over eight years and were grown in a
nursery covered by two overlapping 50% shading
screens, which provided an average daily
photosynthetic radiation of 160 μmol m-2 s-1, at 22.6
± 5°C and 73.9 ± 10% relative humidity (RH). The
RH was maintained by micro-sprinkler irrigation.
Fruits were cut from the mature orchids
with a manual pruning shear and taken to the in
vitro culture laboratory at FCA/UFGD (Faculdade
de Ciências Agrárias, Universidade Federal da
Grande Dourados), where they were disinfested
with 70% ethyl alcohol solution. Next, two fruits of
each species were opened with a scalpel and the
seeds were removed, homogenized, and conditioned
in a desiccator with silica gel (25 ± 2°C, 75% RH)
for 14 days.
Experimental culture media treatments for
seed germination included MS (MURASHIGE;
SKOOG, 1962), ½MS (MS at half the salt
concentration), Knudson C (KNUDSON, 1946), and
VW (VACIN; WENT, 1949). All three culture
media were solidified with 4.0 g L-1 bacteriological
agar (Himedia®, India) and supplemented with
sucrose at 30 g L-1. The pH of the medium was
measured and adjusted to 5.8 using HCl (0.1 M) if
the medium was more alkaline than pH 5.8, or KOH
(0.1M) if the medium was more acidic than pH 5.8.
Prepared media were distributed among 50 mL
polypropylene flasks (height = 5 cm; mouth
diameter = 5 cm) and sealed with a screw cap. Each
flask received 20 mL of culture medium. Next, all
flasks were autoclaved at 120°C for 20 min at a
pressure of 1 atm. After cooling, the flasks were
transferred to a sterile environment.
Seed viability was evaluated by the
tetrazolium test described by Soares et al. (2014),
whereby 5 mg seed portions were placed in test
tubes containing 3 mL of an aqueous solution of
triphenyl tetrazolium chloride (0.5%). Seed
suspensions were conditioned in a dark environment
at room temperature (25 ± 2°C). After 24 h, 7 mL of
sterile distilled water was added to the tetrazolium
suspensions and stirred vigorously. A 1-mL aliquot
was pipetted for identification and counting of
potentially viable seeds in a Peters’ chamber, using
a stereoscopic binocular microscope. Seeds with
dark red embryos were considered viable, whereas
colorless or partially stained seeds, and embryo-free
seeds were considered non-viable.
After confirming the viability of seeds, four
seed samples of 5 mg each were weighed for each
species tested. The samples were taken to an aseptic
environment and disinfected with 15 mL of 0.8%
sodium hypochlorite solution. Two seed samples of
each species remained immersed in the hypochlorite
solution for 5 min (5') and two samples for 15 min
(15'). Next, seed suspensions were diluted to 50 mL
with sterile distilled water. A sample from each
species and each disinfestation time was used
immediately for in vitro seeding. The remaining
samples underwent a triple wash with sterile
distilled water (40 mL per wash). After this
procedure, 50 mL of sterile distilled water was
added to the suspensions for in vitro seeding. Using
an automatic pipettor for in vitro seeding, 1,000 μL
of each seed suspension was inoculated in each
flask.
After inoculation, suspension cultures were
transferred into a growth room and kept at 25 ± 2°C,
and a 16 h photoperiod under an irradiance of 20
μmol m-2 s-1 provided by two 20 W white
fluorescent lamps. Percent germination rate (%G)
was recorded after 45 days. Propagules growing in
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the flasks were washed with 3 mL of sterile distilled
water and packed in squared (0.5 × 0.5 cm) acrylic
plates (2.0 × 2.0 × 0.5 cm); non-germinated seeds
(NGS) and chlorophyll propagules (CP) were
counted under a stereoscopic binocular microscope.
Percent germination rate (%G) was calculated with
the following equation: %G = [CP / (NGS + CP)] ×
100. After counting, treatments were photographed
using a digital camera coupled to a stereoscopic
microscope, using AxionVision software version 3.1
(Zeiss®).
The stage of development of propagule
development was classified according to the stages
described in Suzuki et al. (2009): stage 1
(protocorm-like body); stage 2 (seedling with first
leaf formation); stage 3 (seedling with two leaves);
stage 4 (seedlings with leaves and roots).
The experiment was laid out in a completely
randomized design with treatments arranged in a 2 ×
2 × 4 factorial scheme (2 disinfestation times, 2
types of suspension – with or without triple washing
– and 4 different culture media), with 4 replications,
each consisting of one culture flask. Percent
germination data were transformed √ (x +1) and
then analyzed by analysis of variance. Means of
disinfestation times and suspension types were
compared using Student’s t-test, while the means of
culture media were compared using Tukey’s test at a
probability level of 5%. SISVAR (Statistical
Analysis Program v.5.3. Federal University of
Lavras, MG, Brazil) was used for the statistical
analyses.
RESULTS AND DISCUSSION
Significant effects were observed for
disinfestation time, type of seed suspension washing
and culture media, and their interaction, on
germination rate (%G) of D. nobile (p<0.05) and D.
phalaenopsis (p<0.01) seeds.
Overall, seeds submitted to the triple
washing procedure showed higher %G than
controls, irrespective of culture medium used or
disinfestation time. An exception was observed in
D. nobile when sown in culture medium VW and
disinfested for 5', in which case no statistical
difference in %G was observed (Tables 1 and 2).
For D. nobile, 5' disinfestation time resulted
in the highest %G (overall mean of 47.1% among
washing procedures), while 15' time yielded a mean
%G of 43.3%. There was a 33.5% increase in
germination rate for the 5' treatment upon triple-
wash treatment. Seeds submitted to a triple-wash
showed a mean %G of 63.8%, while unwashed
seeds showed a mean %G of 30.3%. The highest
mean %G values were recorded for ½ MS followed
by K, MS, and VW. The highest mean %G value
(81.2%) was observed for seeds soaked for 5' in
disinfecting solution and submitted to a triple-wash
prior to sowing in ½ MS medium (Table 1).
Table 1. Germination rate (%) of Dendrobium nobile seeds according to the culture medium, type of
suspension washing (TW=triple-wash; NTW=no triple-wash), and disinfestation time of seeds.
Disinfestation time 5'
Washing MS ½MS K VW Overall mean
TW 56.8 cAa 81.2 aAa 68.1 bAa 49.2 dAa 63.8
NTW 11.1 cBb 28.8 bBb 33.4 bBa 48.1 aAa 30.3
Overall M 47.1
Disinfestation time 15'
Washing MS ½MS K VW Overall mean
TW 45.5 cAb 72.5 aAb 56.1 bAb 53.7 bAa 56.9
NTW 17.0 bBa 34.4 aBa 36.9 aBa 31.2 aBb 29.8
Overall M 43.3
Lowercase letters within rows compare the culture media for the same disinfestation time (5' or 15') and type of seed suspension
washing (TW or NTW) (Tukey’s test, p<0.05). Capital letters within rows compare the culture media for the same disinfestation time (5'
or 15') as regards the type of seed suspension washing (Student’s test, p<0.05). Superscript letters within columns compare the culture
medium in the same type of seed suspension washing (TW or NTW) regarding disinfestation time (Student’s test, p<0.05).
The 5' disinfestation time of D.
phalaenopsis seeds resulted in the highest mean %G
rate among all culture media and wash treatments.
There was an 82.4% increase in germination when
these seeds underwent triple wash, resulting in a
mean %G rate of 85.5%. Under these conditions,
MS and ½MS yielded the highest mean %G values,
followed by VW and K. The highest mean %G rate
of D. phalaenopsis (95.5%), was observed when
seeds were soaked for 5ʹ in the disinfecting solution
and then submitted to a triple-wash prior to sowing
in either MS or ½MS medium (Table 2).
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Table 2. Germination rate (%) of Dendrobium phalaenopsis seeds according to the culture medium, type of
suspension washing (TW=triple-wash; NTW=no triple-wash) and disinfestation time of seeds.
Disinfestation time 5'
Washing MS ½ MS K VW Overall mean
TW 95.5 aAa 91.8 aAa 76.8 bAa 77.8 bAa 85.5
NTW 0.6 bBa 3.3 bBa 1.6 bBa 6.7 aBa 3.1
Overall M 44.1
Disinfestation time 15'
Washing MS ½MS K VW Overall mean
TW 85.2 aAb 69.8 bAb 70.9 bAb 73.5 bAb 74.9
NTW 1.5 aBa 0.8 aBa 2.2 aBa 3.0 aBa 1.9
Overall M 40.9
Lowercase letters within rows compare the culture media at the same disinfestation time (5' or 15') and type of seed suspension washing
(TW or NTW) (Tukey’s test, p<0.05). Capital letters within rows compare the culture media at the same time of disinfestation (5' or 15')
in relation to the type of seed suspension washing (Student’s test, p<0.05). Overlapping letters within columns compare the culture
medium in the same type of seed suspension washing (TW or NTW) as regards disinfestation time (Student’s test, p<0.05).
The MS and ½MS media also allowed an
increase in the germination of Cattleya loddigesii
Lindl., Epidendrum fulgens Brong. and Miltonia
flavescens Lindl. (ABRÃO et al., 2014; VOGES et
al., 2014; LEMES, 2015). However, other authors
have observed that, depending on the species, media
such as VW and K provided better germination
results, as described by Dutra et al. (2009) and
Suzuki et al. (2010), while studying in vitro
germination of Cyrtopodium punctatum Lindl. and
Cattleya bicolor Lindl., respectively.
Selection of culture medium is extremely
important for successful germination of orchid
seeds, since the most suitable medium for each
species is directly related to the nutrients supplied to
the plants (SUZUKI et al., 2009). Orchid species
can be divided into two large groups, according to
their basic nutritional needs (STEWART, 1989).
One of the groups includes species that germinate in
culture media with lower nutrient concentrations,
such as K and VW. In contrast, the other group is
composed of orchid species that germinate better in
media with higher nutritional concentration, such as
MS. The results presented here suggest that D.
phalaenopsis belongs to the second group, requiring
media with a broader and greater supply of
nutrients, since these seeds showed the highest
germination rates on MS and ½MS media (Figure
1). However, D. nobile belongs to the first group,
which germinates better on culture media with
lower nutrient concentration (Figure 1).
0
20
40
60
80
100
MS MS ½ K VW
TW
NTW
%
G
D. nobile - 5'
0
20
40
60
80
100
MS MS ½ K VW
TW
NTW
%
G
D. nobile - 15'
0
20
40
60
80
100
MS MS ½ K VW
TW
NTW
%
G
Culture media
D. phalaenopsis - 5'
0
20
40
60
80
100
MS MS ½ K VW
TW
NTW
%
G
D. phalaenopsis - 15'
Culture media
Figure 1. Percent germination rate (%G) of Dendrobium nobile (A and B) and Dendrobium phalaenopsis (C
and D) seeds according to the culture medium, type of suspension washing (TW= with triple-wash,
NTW = no triple-wash) and seed disinfestation time.
(A) (B)
(D) (C)
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In addition to the culture medium, the
disinfecting agent used may also affect in vitro
asymbiotic germination. The reduction in %G of the
seeds without the triple-wash may be related to the
presence of sodium hypochlorite, which may hinder
seed germination depending on the concentration
used and time of exposure (CHU; MUDGE 1994).
The disinfecting action of sodium hypochlorite
results from the loss of Cl-, which activates the
oxidation of ions that capture oxygen molecules and
thus inactivates aerobic microorganisms and fungal
spores responsible for most culture media
contaminations (ALVAREZ-PARDO et al., 2006).
The solution pH is another factor that may
have contributed to %G reduction, since the average
pH of the culture medium recommended for
germination of orchid seeds is between 5.6 and 5.8
(FARIA et al., 2012), while the sodium hypochlorite
solution used for seed disinfestation in these
experiments had an alkaline pH (10.8), when no
triple-wash was involved. Seed exposure to this high
pH solution, along with the high salt concentration
of the media with higher nutritional contents, may
have triggered chemical reactions that hindered the
germination of the seeds of the species studied
(Figure 1).
A reaction of ammonia and nitrate
molecules with sodium hypochlorite probably
occurred in the culture media with higher
concentrations of ammonium and nitrate, such as
MS and ½MS (Table 3), when seeds were not
submitted to a triple-wash. The reactions that may
have occurred when sodium hypochlorite reacted
with ammonia and nitrate ions are presented in
Equations 1, 2 and 3:
NH4
+
(aq) + H2O(l) NH3(aq) + H3O
+
(aq) (Equation 1);
NaClO(aq) + NH3(aq)NaOH(aq) + NH4Cl(aq)
(Equation 2);
2NaClO(aq) + 2NO3(aq) 2NaNO3(aq) + 2ClO
-
(aq)
(Equation 3).
Table 3. Nutrient composition of the culture media used for asymbiotic germination of Dendrobium nobile and
Dendrobium phalaenopsis seeds.
Nutrients
Murashige & Skoog
(MS)
Murashige & Skoog
(½MS)
Knudson (K)
Vacin & Went
(VW)
mM mM mM mM
Ammonium (NH4
+) 20.62 10.31 3.79 3.79
Nitrate (NO3
-) 39.43 19.72 4.24 5.20
Phosphate (PO4
---) 1.25 0.625 1.84 2.48
Potassium (K+) 20.06 10.03 1.84 7.04
Sulfate (SO4
--) 1.50 0.75 4.84 4.80
Calcium (Ca++) 3.01 1.505 4.24 0.65
Magnesium (Mg++) 1.50 0.75 1.02 1.02
Chloride (Cl-) 6.03 3.015 - -
Sucrose 87.72 87.72 87.72 87.72
Total nitrogen 60.05 30.025 8.03 8.99
A high pH value was recorded in the first
chemical reaction due to the OH- originating from
the sodium hydroxide as a result of the ammonia–
sodium hypochlorite reaction (Equation 2), which
inhibited germination. In the nitrate–sodium
hypochlorite reaction (Equation 3), hypochlorite in
solution dissociates as indicated by equation 4:
2ClO- 2Cl- + O2 (Equation 4)
Both Cl- and O2 are highly reactive and
capable of acting directly on other molecules in the
medium. Cl- and O2 act through connections to the
chromophore groups and in the formation of
derivative compounds with ions. In addition, oxygen
oxidizes the component molecules of the medium
(ATKINS; JONES 2012). Thus, the reactions
described explain the low germination in the media
with higher ammonium and nitrate values, when in
contact with sodium hypochlorite.
Potassium concentration in MS and ½MS
media was also high (Table 3). Considering the
release of Cl- (Equation 4), there is a high likelihood
that potassium will react with this anion to form
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KCl. Potassium is a vital nutrient for plant growth
and development, so a higher concentration of K+
ions is expected to provide greater germination rate,
growth, and development, because it is directly
linked to the synthesis of proteins, activation of
several enzymatic systems involved in respiration
and photosynthesis, and mechanism of cell
enlargement (TAIZ; ZEIGER, 2009).
Thus, the importance of the triple-wash is
highlighted. The use of this technique increased
percent germination rate and resulted in more
developed propagules at 45 days after placement.
These propagules could be found, according to the
classification described in Suzuki et al. (2009), in
stage 2 (seedling with first leaf formation) and in
stage 3 (seedling with two leaves). For the
propagules in the no triple-wash treatment, most of
the propagules were found in stage 1 (protocorm-
like body), some were chlorophyll deficient and
several seeds did not germinate (Figure 2) after 45
days.
MS 5’ MS 15’ MS½ 5’ MS½ 15’ K 5’ K 15’ VW 5’ VW 15’
With triple-wash
No triple-wash
D. nobile
MS 5’ MS 15’ MS½ 5’ MS½ 15’ K 5’ K 15’ VW 5’ VW 15’
With triple-wash
1mm
No triple-wash
D. phalaenopsis
Figure 2. In vitro germination of Dendrobium nobile (A) and Dendrobium phalaenopsis (B) with triple-wash
and no triple-wash at 45 days after sowing.
Disinfestation time of orchid seeds for in
vitro sowing varies depending on the type and
concentration of the agent used. According to
Arditti and Ernst (1992), application of sodium
hypochlorite solution from 5' to 30' allows complete
disinfestation of the seeds, thus solving any
contamination problem. Soares et al. (2012) used
sodium hypochlorite at 1.25% for 15' without a
triple-wash to disinfect D. nobile seeds and reported
a %G of less than 50%. Similar results were
observed in this study when the triple-wash
procedure was not performed (Tables 1 and 2).
Faria et al. (2012) recommend using a 0.5%
sodium hypochlorite solution for 15'. However, the
results observed in this study demonstrated that
disinfestation time of D. nobile and D. phalaenopsis
seeds, for sowing in an asymbiotic culture medium,
can be reduced to 5' with the use of 0.8% sodium
hypochlorite, without contamination of the culture
media. This procedure increased %G, compared to
disinfestation for 15'. Thus, disinfestation for 5'
effectively was effective for seeds of the species
studied (Tables 1 and 2). Further, a significant
increase in %G was observed when the seeds
remained for 5' in sodium hypochlorite (0.8%),
followed by a triple-wash with sterilized water.
These results agree with those reported by Alvarez-
Pardo et al. (2006), who observed that disinfestation
of orchid seeds required several washes with
sterilized water after soaking in a sodium
hypochlorite solution. The authors emphasized that
this procedure is time-consuming, especially when
using a large amount of seeds. However, in this
study the use of a triple-wash with sterile distilled
water increased %G of D. nobile from 30.3% to
63.8%, and %G of D. phalaenopsis from 3.1% to
85.5%, showing that the procedure is essential for in
vitro seeding of these species.
(A)
(B)
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CONCLUSION
The 5' disinfestation time, followed by a
triple-wash with sterile distilled water and sowing
on MS or ½MS medium is the most effective
protocol to obtain high in vitro germination rates of
D. nobile and D. phalaenopsis.
ACKNOWLEDGEMENTS
The authors thank the Coordenação de
Aperfeiçoamento de Pessoal de Nível Superior
(CAPES) and Federal University of Grande
Dourados (UFGD) for the scholarship and
assistance.
RESUMO: A germinação assimbiótica é considerada uma técnica eficiente e viável resultando em
elevados percentuais de germinação. Apesar do sucesso dessa técnica, o tipo, a concentração e o tempo de
exposição do agente desinfestante diferem, necessitando padronização para cada espécie. Assim, a
padronização do agente desinfestante e do procedimento são necessários para o aumento das taxas
germinativas. Objetivou-se com este trabalho determinar metodologia que aumente a taxa de germinação in
vitro e favoreça o desenvolvimento inicial de plântulas de Dendrobium nobile e Dendrobium phalaenopsis,
utilizando diferentes métodos de desinfestação de sementes e diferentes meios de cultura. Sementes, sob
condições assépticas, foram desinfestadas em solução de hipoclorito de sódio a 0,8%, por cinco ou quinze
minutos. Após esses períodos, as suspensões de sementes receberam ou não a tríplice lavagem com água. Em
seguida, foram semeadas em frascos de cultivo que continham quatro diferentes meios de cultura (MS, MS½, K
e VW). Posteriormente, foram transferidos para sala de crescimento com fotoperíodo e temperatura
controlados, onde permaneceram sob irradiância de 20 µmol m-2 s-1. Quarenta e cinco dias após a semeadura foi
avaliada a porcentagem de germinação das espécies estudadas. Os resultados neste trabalho indicam que,
independentemente do meio de cultura ou do tempo de desinfestação, as sementes quando submetidas à tríplice
lavagem apresentaram porcentagem de germinação superior a àquelas que não receberam este procedimento.
Recomenda-se a desinfestação das sementes por 5 minutos. Os meios MS e MS½ foram os mais efetivos em
promover a germinação in vitro dessas espécies.
PALAVRAS-CHAVE: Germinação assimbiótica. Orchidaceae. Dendrobium nobile. Dendrobium
phalaenopsis. Hipoclorito de sódio. Espécies ornamentais.
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