Chemical sterilization in in vitro propagation of Arundina bambusifolia Lindl . and Epidendrum ibaguense Kunth

Há uma grande demanda por técnicas de laboratório mais simples, e de menor custo, e por procedimentos mais acessíveis, por parte de orquidófilos que não têm o embasamento teórico necessário à utilização de métodos usuais de produção, seminífera e clonal, de orquídeas, in vitro. O objetivo deste trabalho foi avaliar a eficiência da aplicação de hipoclorito de sódio (NaClO), para a descontaminação, no processo de inoculação de explantes de orquídeas das espécies Arundina bambusifolia e Epidendrum ibaguenses . As soluções de NaClO (1.200; 2.400; 3.600; 4.800 e 6.000 mg L equivalentes a 50; 100; 150; 200 e 250 mL L de água sanitária comercial ASC) foram borrifadas (1,0 mL) sobre explantes e meio de cultura (GB5), na ausência ou na presença de carvão ativado (2 g L ). Os explantes utilizados foram segmentos nodais de plantas adultas, cultivadas a campo. Os procedimentos para inoculação dos explantes foram


INTRODUCTION
The production of orchid seedlings using tissue cultures has attracted increasing interest from orchidgrowers for multiplying the best genotypes of the progenies of various crosses and, in particular, for increasing the availability of native plant species and varieties, some of which are under threat of extinction, making them more accessible at lower cost (Stacanto et al., 2001).
A number of studies have been conducted to find a way of making decontamination methods feasible for amateur growers and non-specialized people, some of them centered on assessing the need to use autoclaves and laminar flow chambers (LFC).Studies involving the use of sodium hypochlorite (NaClO) have clearly shown how it can be used as diluted solution, often promoting better growth of in vitro tissues, as verified in the germination of rice (Chun et al., 1997), orchid seeds (Alvarez-Pardo et al., 2006;Rodrigues et al., 2011), and the cloning of Eucalyptus (Teixeira et al., 2008).Teixeira et al. (2006) used weak solutions of NaClO (1, 3, 5, 7 and 9 mg L -1 ) added to culture media to control contamination in banana.In the autoclaved control, 25% of jars were contaminated, compared with only 10% using a medium containing 1 mg L -1 NaClO.At higher concentrations, there was no contamination and the tissues were unharmed.Sterilizing the culture medium using NaClO doubled the fresh weight yield (76 mg/jar) in comparison to the autoclaved medium (32 mg/jar), and results were similar regardless of the concentration of NaClO.
Sodium hypochlorite and calcium hypochlorite -Ca(ClO) 2 -are commonly used as sterilizing agents for explants grown in vitro.In experiments on apples, contamination control was improved using NaClO, but explant oxidation was higher by comparison with the use of Ca(ClO) 2 (Erig & Schuch., 2003).
Spraying seeds of Bletilla striata, Cattleya loddigessi, Dendrobium kingianum, Habenaria radiata and Phalaenopsis spp. with NaClO or H 2 O 2 sterilizing solutions at different concentrations, Yanagawa et al. (1995) found 100% germination, even at high hypochlorite concentrations (5,000 mg L -1 active Cl), and seed mortality and medium contamination dropped to zero.They also obtained similar results in transplanted explants using the solutions in spray form, without impairment of seedling growth.The responses to H 2 O 2 were similar when used at concentrations of 0.1 g L -1 (0.01%), both added to the culture medium and using spray application.
The use of activated charcoal in the culture medium, a form of carbon with a high surface area, is known to present elevated capacity to adsorb numerous toxic compounds during in vitro cultivation (Nunes et al., 2008), such as phenolic exudates and excess ethylene produced inside the culture jars (Ribeiro et al., 2000;Chagas et al., 2005;Thomas, 2008).
The aim of this study was to assess the response of inoculated explants in non-sterile culture media (no prior autoclaving) using solutions of NaClO at various concentrations, sprayed on the explants in the culture medium, in the absence or presence of activated charcoal.

MATERIALS AND METHODS
The experiments were conducted in the Plant Cell and Tissue Laboratory of the Departamento de Biologia Vegetal at Universidade Federal de Viçosa, in the state of Minas Gerais (MG), Brazil.The culture medium consisted of Gamborg 5 salts -GB5 (Gamborg et al., 1968), 20 g L -1 sucrose, 6 g L -1 agar and 100 mL L -1 of green coconut water, adjusting the pH to 5.5 in the control treatment, each culture jar contained 25 mL of the medium autoclaved at 121 ºC for 20 min.
All procedures of the sodium hypochlorite treatments were carried out outside the laminar flow chamber (LFC).Each treatment consisted of applying 1 mL of the respective sterilization solution in spray form over the solid (cooled) culture medium and explants.The control treatment consisted of using the conventional micropropagation method, conducting all procedures inside the LFC and not applying NaClO.
The plant material consisted of Arundina bambusifolia nodal segments obtained from a home-grown clump in the municipality of Ouro Preto, Minas Gerais state, and nodal segments of aerial shoots of Epidendrium ibaguenses, grown in the ornamental plants section at Departamento de Fitotecnia, Universidade Federal de Viçosa.The plant ma-terial was washed with neutral detergent in running water, then surface sterilized in 70% alcohol for 1 min, and after discarding the alcohol, in commercial bleach (24 mL L -1 ) for 15 min, and rinsed with autoclaved water.
The experimental unit was a 234 mL jar containing 25 mL of GB5 medium (Gamborg et al., 1968) inoculated with three nodal segments of Arundina bambusifolia Lindl.or Epidendrum ibaguenses Kunth.The culture jars were sealed with PVC-lined polypropylene plugs.The same procedures were used for the control treatment, except that after sterilization in alcohol, the explants were handled inside the LFC.For this trial, all jars containing medium were autoclaved at 121 ºC for 20 min.No chemical agents were added to sterilize the medium at this stage.
After treating with NaClO and inoculating the explants, the jars were kept in a culture room at 27 ± 2 ºC, with a 16/ 8 h (light/dark) photoperiod and irradiance of 48 µmol m -2 s -1 .The experiment was assessed in terms of explant oxidation, contamination by bacteria and/or fungi, and fresh weight yield, height and number of shoots.
The results were subjected to analysis of variance and the regression equations of the variables studied were adjusted according to the treatments (NaClO with or without activated charcoal), using the SAEG 9.0 software.

RESULTS AND DISCUSSION
The growth and development variables evaluated for Arundina bambusifolia and Epidendrum ibaguenses indicated that the best results were obtained in the presence of activated charcoal (AC).The results of number of side shoots, length and fresh weigh yield for A. bambusifolia and E. ibaguenses, are presented on Figure 1. Figure 2 shows the response of Arundina bambusifolia to sprayed solutions of sodium hypochlorite (NaClO) in the absence or presence of activated charcoal.These results are in agreement with those obtained by Kent et al. (2004), who reported the positive effects of adding 0.5 g L -1 AC on fresh weight yield and shoot length for nodal explants of Anoectochilus formosamus.
For Catasetum fimbriatum, the addition of 0.5 g L -1 of AC to the Knudson C medium led to an increase in the number of leaves per explant, in comparison with cultures containing no AC (Morales et al., 2006).In the presence of 0.2 g L -1 AC, green somatic embryos of Rhynchostylis rubrum exhibited strong development from embryogenic calluses in NDM and VW media; embryo development was 80% in the NDM medium and 19% in the VW medium (Te-chato et al., 2010).
Increasing the concentration of NaClO in the spray solution decreased the fresh weight yield, the number of shoots and shoot length for both species (Figure 1).
There was a decrease in medium contamination in jars treated with NaClO.However, in the control, we found an average of five bacterial colonies per jar for both species, as well as fungi, but only in the jars containing A. bambusifolia.Contamination was higher in the presence of AC, possibly due to adsorption of some chlorine, reducing the effect of the NaClO on microorganisms in the explant.In contrast, Ket et al. (2004) mentioned that the presence of AC in the culture medium reduced the effect of phenols released by in vitro cultured plant tissues, which in some cases can kill the explant in only a few hours.
Similar results were obtained for fungus contamination in the control (LFC) and in treatments with NaClO, whereas bacterial contamination was generalized for both species in the control (using the laminar flow chamber and without addition of NaClO to the jar), impairing tissue growth and development (Table 1).

Arundina bambusifolia
Epidendrum ibaguenses Bacterial increased.On the other hand, the lowest NaClO concentration was effective in controlling microbial contamination and promoting explant growth (Figure 2), in contrast to the results obtained in the control with the experimental procedure conducted in the LFC and without NaClO spraying (Table 1).

CONCLUSIONS
Spraying NaClO solution over the culture medium after it has been inoculated with the explant (nodal segments) helps control contamination.
Higher concentrations of NaClO impair plant growth.
In terms of fresh weight yield, number of shoots and shoot length, the best results were obtained in the presence of activated charcoal.However, the charcoal treatment caused increase in bacterial and fungal contamination.

Figure 1 .
Figure 1.Responses of Epidendrum ibaguenses and Arundina bambusifolia to sprayed solutions of sodium hypochlorite, in the absence ( ) or presence ( ) of activated charcoal.

Figure 2 .
Figure 2. Response of Arundina bambusifolia to sprayed solutions of sodium hypochlorite (NaClO) in the absence (A) or presence (B) of activated charcoal, after 60 days in culture.

Table 1 .
Bacterial and fungal contamination in jars containing tissue of Arundina bambusifolia and Epidendrum ibaguenses, in response to doses of sodium hypochlorite (NaClO), in the absence or presence of activated charcoal in the culture medium.Numbers in parentheses refers to percentage of colonies per jar