Seed disinfestation methods for in vitro cultivation of epiphyte orchids from Southern Brazil1

Hortic. bras., v. 24, n. 2, abr.-jun. 2006 E orchids have been multiplied routinely from seeds, using a non-symbiotic culture as a propagation method (Knudson, 1922). The inoculation in vitro is the main technique for micro-propagation without symbiotic mycorrhizae association. In order to properly develop this technique, sugar and salts are required in a sterile culture medium, where seeds are free from microorganisms. Orchid embryos can be cultivated in vitro, using immature seeds (Kerbauy & Handro, 1981). In such case, the seeds are taken from the closed capsule to prevent contamination when no other inhibition of germination is found (Van der Kinderen, 1987; Rasmussen et al., 1990). This procedure is not suitable for a germplasm bank, because seeds must reach maturity and have low water ALVAREZ-PARDO VM; FERREIRA AG; NUNES VF. 2006. Seed disinfestation methods for in vitro cultivation of epiphyte orchids from Southern Brazil. Horticultura Brasileira 24: 217-220.

E piphyte orchids have been multiplied routinely from seeds, using a non-symbiotic culture as a propagation method (Knudson, 1922). The inoculation in vitro is the main technique for micro-propagation without symbiotic mycorrhizae association. In order to properly develop this technique, sugar and salts are required in a sterile culture medium, where seeds are free from microorganisms.
Orchid embryos can be cultivated in vitro, using immature seeds (Kerbauy & Handro, 1981). In such case, the seeds are taken from the closed capsule to prevent contamination when no other inhibition of germination is found (Van der Kinderen, 1987;Rasmussen et al., 1990). This procedure is not suitable for a germplasm bank, because seeds must reach maturity and have low water ALVAREZ-PARDO VM;FERREIRA AG;NUNES VF. 2006. Seed disinfestation methods for in vitro cultivation of epiphyte orchids from Southern Brazil.
Horticultura Brasileira 24: 217-220.  , 1993). For germination, the disinfestation is mandatory before inoculation. The disinfestant liquid should remove or kill all microorganisms from the seeds' surface without damaging the embryos. For the purpose of disinfesting orchid seeds, calcium hypochlorite is the most used one (Wilson, 1915;Thompson, 1980;Arditti et al., 1982), besides hydrogen peroxide (Snow, 1985) and sodium hypochlorite (Pierik, 1990). Calcium hypochlorite needs to be filtered before utilization, and cannot be stored over 12 hours (Arditti et al., 1982). Sodium hypochlorite also needs to be fresh, since both are alkaline and lose chloride, the active oxidizing ion, which captures oxygen, killing the aerobic microorganisms and fungi spores, which in turn, are mainly responsible for most of the contamination. These chemicals can improve somehow the germination in a few species by light scarification of the seeds (Haas-von Schmude et al., 1986;Van Waes & Debergh, 1986).

Seed disinfestation methods for in vitro
Sodium and calcium hypochlorite are prescribed to be used at different concentrations and times of exposure to disinfest seeds (Singh, 1988;Butcher & Marlow, 1989;Seaton & Hailes, 1989;Oddie et al., 1994;Chu & Mudge, 1994). In spite of it, some contamination may appear due to small cracks on the surface of seeds that may keep a few spores. Applying sodium hypochlorite fumes for 5 to 30 minutes can solve this problem, allowing a complete disinfestation (Arditti & Ernst, 1992).
In the attempt to attain an efficient method for several Brazilian epiphyte ABSTRACT Disinfestation of orchid seeds is necessary when the germination is performed in vitro. In such case, cultures are supplemented by a source of sugar and salts in order to obtain energy and other requirements. However, the presence of sugar and salts easily allows the development of unwanted microorganisms. To avoid such phenomenon, an efficient procedure must be done as a pretreatment of the seeds with liquid or gaseous substances. In this study with several Brazilian orchids, the seeds were disinfested with sodium hypochlorite solutions containing 0.4% or 0.8% active chlorine for five minutes and sodium hypochlorite or formaldehyde fumes up to two hours, this procedure being efficient for such purpose and making it possible to be used routinely.

Bifrenaria inodora
Moisten bath: Twenty milligrams of seeds from each of the eleven species were packed in 10 centimeters of plastic net (60 µm mesh) closed by plastic clips. On a laminar flowhood using a beaker, the material was dipped in 15 ml of Sodium hypochlorite 0.4% or 0.8% of active chlorine for 5; 15; 30 and 60 minutes. The vials were sealed to avoid the loss of chlorine. After that, the samples were washed three times with distilled water and placed on a paper filter to dry off the excess water. The seed viability was evaluated by germination on Knudson C (Knudson, 1946) plus micronutrients (Arditi et al., 1982) and 0.05 mg/L of thiamin and nicotinic acid, 20 g/L of sucrose and 6 g/L of agar. The pH was adjusted to 5.7 with NaOH (Milanese, 1997). Ten milliliters of medium were autoclaved for 20 minutes at 120°C in glass tubes. The tubes were tilted; thus, when the medium cooled down a larger surface was formed, where the seeds were sown. The tubes were covered with plastic film and placed at 25°C ± 2°C in a growth room lit for 16 hours (30 µmols -1 m -2 ). The swollen seeds with green embryos were observed through a stereomicroscope after 30 days of incubation and were considered as germinated (Hailes & Seaton, 1989). A randomized design was used and the data for analysis were transformed in arcsen (x + 10) ½ before submitted to ANOVA, and followed by Tukey test (p<0.05).
Gaseous disinfestation: Twenty milligrams of Cattleya intermedia seeds were placed on a small piece (2x2cm) of aluminum foil on a nine-centimeter diameter Petri dish. Two milliliters of sodium hypochlorite solution (5.5% of active chlorine) in a small vial or one tablet of formaldehyde (Rioquímica-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 calculated from the data, then the regression curve with best adjustment was determined.

RESULTS AND DISCUSSION
The eleven species examined showed over 75% of initial viability ( Table 1). The disinfestation, with increased concentration of sodium hypochlorite solution, decreased the germination, and is related to the exposure time (Table 1). The species B. tuberculata, C. intermedia and C. intermedia pallida were the most tolerant to the effects of the disinfestant. This may be related to the size of the seeds, the structure and ornamentation of the seed's wall (Milanese, 1997). Overall, the most effective treatment was the five-minute with 0.4% sodium hypochlorite solution (Figure 1), which reached an average of 87% germination. At this concentration and time, the treatment was effective to avoid contamination in most of the cases. The use of a small package made disinfestation more efficient, therefore for larger quantities of seeds several packages should be used.
Several protocols for orchid seed disinfestation (Thompson, 1980;Arditti et al., 1982;Snow, 1987;Pierik, 1990) were carried out using a sodium hypochlorite solution, which is simpler and less expensive (Butcher & Marlow, 1989;Seaton & Hailes, 1989;Chu & Mudge, 1994: Vujanonic et al., 2000. Fresh solution and good reliable VM Alvarez-Pardo et al. chemicals are required, followed by several rinsing with sterile water. These procedures are time consuming, mainly when a large quantity of seeds should be processed. The utilization of gaseous disinfestation devised in this paper showed good results. The disinfestation period was not as restricted as that of sodium hypochlorite, and besides less manipulation was required. Thirty minutes to four hours can be used without the loss of viability ( Figure.  2A). The tetrazolium test used as a fast viability test could show a decrease of viability after 3 hours in the presence of formaldehyde fumes, probably by acidification of seed surface ( Figure  2B). The use of buffered tetrazolium solution should overcome this trouble. After one hour of treatment, no further contamination was observed. This method is recommended because of its efficacy and ease. Formaldehyde fumes can be applied also when a vial with several explants shows a localized contamination. For rare orchid materials, the recovery of in vitro contaminated explants or plantlets can be very suitable.