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Seed disinfestation methods for in vitro cultivation of epiphyte orchids from Southern Brazil.
Horticultura Brasileira 24: 217-220.
ABSTRACT RESUMO
Disinfestation of orchid seeds is necessary when the germination Métodos de desinfestação de sementes para o cultivo in vitro
is performed in vitro. In such case, cultures are supplemented by a de orquídeas epífitas do Sul do Brasil
source of sugar and salts in order to obtain energy and other A desinfestação de sementes de orquídeas é necessária quando a
requirements. However, the presence of sugar and salts easily allows germinação é efetuada in vitro. Neste caso, as culturas são
the development of unwanted microorganisms. To avoid such suplementadas por uma fonte de açúcar e de sais, para prover ener-
phenomenon, an efficient procedure must be done as a pretreatment gia e outras necessidades. A presença de açúcares e sais facilmente
of the seeds with liquid or gaseous substances. In this study with permite o desenvolvimento de microorganismos indesejáveis. Para
several Brazilian orchids, the seeds were disinfested with sodium evitar estes microorganismos, um procedimento eficiente deve ser
hypochlorite solutions containing 0.4% or 0.8% active chlorine for usado como pré-tratamento das sementes com substâncias liquidas
five minutes and sodium hypochlorite or formaldehyde fumes up to ou gasosas. Neste ensaio com várias espécies de orquídeas brasilei-
two hours, this procedure being efficient for such purpose and making ras, as sementes foram desinfestadas com solução de hipoclorito de
it possible to be used routinely. sódio 0,4% ou 0,8% de cloro nascente por cinco minutos ou, por
vapores de formol ou hipoclorito de sódio por até duas horas, sendo
eficientes estes procedimentos para desinfestação e possível seu uso
rotineiramente.
Keywords: orchids, in vitro culture, germination, sodium Palavras-chave: orquídeas, cultivo in vitro, germinação, hipoclorito
hypoclorite, formaldehyde. de sódio, formaldeido.
1
Part of PhD dissertation, from the first author (Dept. of Botany, UFRGS, Porto Alegre, Rio Grande do Sul, Brazil)
Table 1. Initial viability and orchid seeds germination percentage, disinfested with two sodium active chlorine) in a small vial or one
hypoclorite concentrations with 0.4% or 0.8% of active chloride for 5; 15; 30 and 60 minutes tablet of formaldehyde (Rioquímica-
periods. Porto Alegre, UFRGS, 2004. Brasil) were utilized as disinfestant. The
Petri dishes were sealed with parafilm
and the disinfestant was applied ranging
from 30 minutes to six hours in each of
them every half hour. The seed viability
was evaluated as done in the first assay,
by germination in Knudson C or by
tetrazolium test. For these tests, the
seeds were water embedded for 24 hours
and then placed in 1% tetrazolium
solution (2,3,5-triphenyl tetrazolium
chloride) for 24 hours at 30°C (Singh,
1981). The seeds were counted through
a stereomicroscope, the red ones being
viable. Randomized design was used,
and the coefficient of correlation was
*Different letters indicate statistical differences among treatments. Tukey test, p<0.05.
calculated from the data, then the
regression curve with best adjustment
was determined.
orchid seeds inoculation, four minutes. The vials were sealed to avoid
procedures were devised: two with the loss of chlorine. After that, the RESULTS AND DISCUSSION
liquid (0.4% and 0.8% of active samples were washed three times with
chloride), and two with gaseous distilled water and placed on a paper The eleven species examined
substances (5.5% sodium hypochlorite filter to dry off the excess water. The showed over 75% of initial viability
and 99% formaldehyde fumes). seed viability was evaluated by (Table 1). The disinfestation, with
germination on Knudson C (Knudson, increased concentration of sodium
MATERIAL AND METHODS 1946) plus micronutrients (Arditi et al., hypochlorite solution, decreased the
1982) and 0.05 mg/L of thiamin and germination, and is related to the
Bifrenaria inodora Lindl., nicotinic acid, 20 g/L of sucrose and 6 exposure time (Table 1). The species B.
Brassavola tuberculata Hook., Cattleya g/L of agar. The pH was adjusted to 5.7 tuberculata, C. intermedia and C.
bicolor Lindl., C. intermedia R.Grah, C. with NaOH (Milanese, 1997). Ten intermedia pallida were the most
intermedia var. pallida Lindl., Encyclia milliliters of medium were autoclaved tolerant to the effects of the disinfestant.
pygmeae (Hook.) Dressler, Epidendrum for 20 minutes at 120°C in glass tubes. This may be related to the size of the
fulgens A.Brongn., Laelia purpurata The tubes were tilted; thus, when the seeds, the structure and ornamentation
Lindl. & Paxt, Miltonia regnelli Reichb., medium cooled down a larger surface of the seed’s wall (Milanese, 1997).
Oncidium pumicum Lindl., Pleurothallis was formed, where the seeds were sown. Overall, the most effective treatment
glumacea Lindl. were collected from The tubes were covered with plastic film was the five-minute with 0.4% sodium
native stands in the Northern and and placed at 25°C ± 2°C in a growth hypochlorite solution (Figure 1), which
Eastern regions of the Rio Grande do room lit for 16 hours (30 µmols-1 m-2). reached an average of 87% germination.
Sul State, Brazil, in 1999 and 2000. Ripe The swollen seeds with green embryos At this concentration and time, the
capsules were dehydrated (Seaton & were observed through a treatment was effective to avoid
Prichard, 1989) and stored at 5°C ± 1°C, stereomicroscope after 30 days of contamination in most of the cases. The
in the laboratory of the Federal incubation and were considered as use of a small package made
University of Rio Grande do Sul. The germinated (Hailes & Seaton, 1989). A disinfestation more efficient, therefore
disinfestation was carried out in two randomized design was used and the for larger quantities of seeds several
ways, by a moisten bath and gaseous data for analysis were transformed in packages should be used.
exposition. arcsen (x + 10)½ before submitted to Several protocols for orchid seed
ANOVA, and followed by Tukey test disinfestation (Thompson, 1980; Arditti
Moisten bath: Twenty milligrams
(p<0.05). et al., 1982; Snow, 1987; Pierik, 1990)
of seeds from each of the eleven species
were packed in 10 centimeters of plastic Gaseous disinfestation: Twenty were carried out using a sodium
net (60 µm mesh) closed by plastic clips. milligrams of Cattleya intermedia seeds hypochlorite solution, which is simpler
On a laminar flowhood using a beaker, were placed on a small piece (2x2cm) and less expensive (Butcher & Marlow,
the material was dipped in 15 ml of of aluminum foil on a nine-centimeter 1989; Seaton & Hailes, 1989; Chu &
Sodium hypochlorite 0.4% or 0.8% of diameter Petri dish. Two milliliters of Mudge, 1994: Vujanonic et al., 2000).
active chlorine for 5; 15; 30 and 60 sodium hypochlorite solution (5.5% of Fresh solution and good reliable
ACKNOWLEDGEMENTS
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