Vernonia condensata Baker: an alternative for large-scale seedling production

ABSTRACT: The increasing use of Vernonia condensata Baker highlights the importance of developing strategies to reduce the impact of exploitation on nature reserves. The aim of this study was to establish a micropropagation protocol to produce homogenous plants with high phytosanitary quality. Apical, nodal, and internodal segments of plants grown in the field were used for in vitro growth. The segments were disinfected in sodium hypochlorite solution (1.0 and 2.0%) for 15 and 30 minutes and then transferred to Petri dishes containing MS culture medium for 30 days. A completely randomized factorial experiment (3 x 2 x 2) with five replicates was designed. After this period, a completely randomized in vitro multiplication experiment was carried out with six treatments (BAP - 0.0; 0.5; 1.0; 1.5; 2.0; 2.5 mg L-1) and six replicates. The shoots obtained in the best treatment were transferred to flasks with rooting medium (MS, MS/2 or MS/4). The experiment was completely randomized with 12 replicates. Microplants were acclimatized in polyethylene terephthalate (PET) bottles filled with autoclaved topsoil. Our results showed that 40.0% of the nodal segments (immersed in 1.0% sodium hypochlorite for 30 minutes) were adequately disinfected and survived. In the in vitro multiplication experiment, the 0.5 mg L-1 concentration of BAP yielded the highest number of shoots and the best vegetative growth. With regard to the assessed characteristics, MS/4 was the best rooting medium, with 100% survival during acclimatization. This study showed that V. condensata in vitro culture might produce 32,000 seedlings in 7 months.


INTRODUCTION
The use of plants with therapeutic properties has been increasingly recognized by society due to the search for healthier practices and the challenges encountered in treating some diseases, such as microbial resistance (BUGNO et al., 2005).
Phytotherapy has adapted to the reality and the need of many municipalities for basic health care. The reasons for the expansion of this type of alternative medicine included consumers' preference for natural treatments, scientific support of the pharmacological properties of medicinal plants, and low cost (MELO et al., 2007). Almeida et al. The National List of Medicinal Plants of Interest to the Public Health System (Relação Nacional de Plantas Medicinais de Interesse ao Sistema Único de Saúde -RENISUS) currently contains 71 species, including Vernonia condensata Baker (BRASIL, 2010). This plant is commonly known in Brazil as alumã (VICENTE et al., 2009) and boldo-da-bahia (AFONSO et al., 2015), among other names. It belongs to the family Asteraceae and has been used since colonial times to treat gastrointestinal problems and because of its analgesic properties (BOORHEM, 1999).
There is little information related to the market, consumption, and use of medicinal plants in Brazil, aspects that reflect the current scenario of production of poor quality plant material in the country (VEIGA-JUNIOR, 2008). Therefore, the use of biotechnological tools, such as tissue culture, in plants with medicinal properties will contribute to the generation of competitive and adapted germplasm, as well as to the development of species that supply biologically active compounds of interest to the pharmaceutical industry. In this sense, tissue culture tools can facilitate the commercial propagation of plants and the in vitro industrial production of secondary compounds (MORAIS et al., 2012;CARVALHO et al., 2013).
The increased demand for products derived from medicinal plants justifies the need for the application of biotechnological tools for large-scale seedling production. Thus, the use of micropropagation techniques to define protocols for in vitro propagation of medicinal species of interest to the Brazilian Public Health System (SUS) and to the pharmaceutical industry is of great relevance (SALES, 2015). However, several factors may influence the establishment of micropropagation methods. The genotype, type of explant, and culture medium are the main factors that control the development of plants grown in vitro (COSTA et al., 2006).
Given the above information, the aim of this study was to establish an efficient micropropagation protocol for V. condensata plants to provide plants for future pharmacological studies, for commercial production, and/or for the establishment of a collection or germplasm bank of this species.

MATERIALS AND METHODS
The study was conducted at the Laboratory of Biotechnology Applied to Health of Maria Milza School (Faculdade Maria Milza -FAMAM). Plants grown in the field, collected in the Recôncavo da Bahia region, were used as sources of explants.
Explants consisted of a) nodal segments, b) internodal segments, and c) apical segments, all approximately 0.5 cm long.

Disinfection and in vitro establishment
An experiment was conducted combining explant types with sodium hypochlorite concentrations (1.0 and 2.0%) and immersion times (15 and 30 minutes). The explants were washed three times in autoclaved distilled water after the disinfection treatment. They were then transferred to Petri dishes (90 x 15 mm) containing 20 mL of MS culture medium (MURASHIGE & SKOOG, 1962) supplemented with 30 g L -1 of sucrose and solidified with 8 g L -1 of agar, and the pH was adjusted to 5.8 before autoclaving (121 ºC for 20 minutes).
The Petri dishes were kept in a growth room with a controlled temperature set at 25 ºC ± 2 ºC, 16-hour photoperiod, and light intensity of 40 μM m -2 s -1 , for 30 days. A completely randomized 3 x 2 x 2 factorial experiment (three explant types, two sodium hypochlorite concentrations, and two immersion times) with five replicates was conducted, with each replicate consisting of a Petri dish containing four segments. After 30 days of culture, the percentage of contaminated and surviving explants was evaluated.

In vitro multiplication
The surviving explants (shoots of nodal segments) were placed in 268-mL flasks containing 20 mL of MS culture medium supplemented with 1.0 mg L -1 of BAP (6-benzylaminopurine), according to VICENTE et al. (2009), under the previously described growth room conditions, to increase the number of shoots over another 30 days.
For the induction of in vitro multiplication, an experiment was set up using the previously obtained shoots, which were cut into segments of ± 1.0 cm in length containing one node and one leaf. These segments were inserted vertically into 268-mL flasks containing 20 mL of MS nutrient medium plus BAP (0.0; 0.5; 1.0; 1.5; 2.0 and 2.5 mg L -1 ) and 30 g L -1 sucrose, solidified with 8 g L -1 agar and pH adjusted to 5.8, to induce maximum shoot proliferation.
A completely randomized experiment with six treatments and six replicates was conducted, with each replicate consisting of a vial containing five segments of ± 1.0 cm. The flasks were stored in a growth room under the same conditions as described above. After 30 days of cultivation, the following characteristics were evaluated: a) number of responsive explants, b) number of shoots per responsive explant, c) number of leaves per shoot, and d) shoot height in cm.

In vitro rooting and acclimatization
The shoots obtained in the treatment with the best induction of multiplication were used in the rooting step. In this step, the shoots were placed in flasks containing the rooting medium, which consisted of MS, MS/2 (50% of the mineral salts in the MS medium), and MS/4 (25% of the mineral salts in the MS medium). All media were supplemented with 30 g L -1 of sucrose and solidified with 8 g L -1 of agar, and the pH was adjusted to 5.8.
The flasks were kept in a growth room under the same conditions as in the previous steps. The experimental design was completely randomized with three treatments and twelve replicates, each consisting of a flask containing one shoot. After 30 days, the following characteristics were evaluated: a) percentage of rooted shoots (microplants), b) number of roots per shoot, and c) length of the longest root in cm.
After the rooting step, the microplants were washed in running water to remove the excess culture medium for evaluation of the aforementioned characteristics. They were then used in the acclimatization step, which consisted of placing the plants in polyethylene terephthalate (PET) soda bottles (2,000 mL) containing substrate (autoclaved topsoil), according to the method described by VICENTE et al. (2009). They were kept in an area covered with a shade cloth (70% light reduction), and the bottle tops were progressively removed for 10 minutes on the first day, 20 minutes on the second day, and so on, gradually increasing until complete removal of the bottle tops. The seedlings were irrigated daily with the aid of a spray bottle. The experimental design of the rooting step was maintained, and after 30 days, the percentage of surviving plants was evaluated. Statistical analysis was performed using SAS (Statistical Analysis System; SAS Institute, 2004).

Explant disinfection and establishment
Results obtained in this experiment showed that, among the explants used, only the nodal segment showed survival. According to BRONDANI et al. (2013), the type of explant, asepsis method, and physiological conditions of the mother plant are factors that influence in vitro establishment. Results obtained herein reinforce the assertion of these authors because different types of explants from the same plant presented different responses to the asepsis treatments. This result might be attributed to the different amounts of microorganisms present in these plant parts, as well as to the possibility that the disinfection process did not effectively target some microorganisms. According to EVERT (2013), apical segments, for example, have leaf primordia that cover the meristems. Therefore, these primordia can protect microorganisms, not allowing the effective action of the decontaminating agent. The internodal segments are more exposed due to the absence of these structures; however, they may contain a greater amount of microorganisms on their surfaces, thus requiring a more rigorous disinfection processes.
According to table 1, there were significant differences among the tested treatments for the nodal segment explants. The combination of the concentration of 2.0% sodium hypochlorite with the immersion time of 30 minutes demonstrated the lowest percentage of contamination (30%). However, the non contaminated explants showed necrosis, marked darkening (oxidation), and did not survive ( Figure 1A). It is possible that immersion of the explants at the 2.0% sodium hypochlorite concentration for 30 minutes was an important cause of the death of 70% of the microorganisms present but also presented a phytotoxic effect, leading to cell death by oxidation (Table 1). VICENTE et al. (2009) reported that oxidation, observed in 2 to 14%, of their plants, affected the morphogenetic response in the disinfection of V. condensata explants. Both the concentration and immersion time in sodium hypochlorite can, in addition to eliminating microorganisms, cause cell death due to excess chlorine or a long exposure to the solution (SILVA et al., 2015). These authors disinfected seeds of the medicinal plant Byrsonima intermedia A. Juss. using a solution containing 1.0% active chlorine and varied the immersion time to up to 10 minutes to avoid phytotoxic effects. SOUZA et al. (2011) reported that the oxidation caused by sodium hypochlorite may be due to changes in cell membranes as well as in the oxygen supply to the explants. Therefore, it is possible that the supply of oxygen, released by sodium hypochlorite, contributed to enzymatic reactions involving phenolic compounds, which led to the death of the explants by oxidation.
With regard to the contaminated explants, it is important to mention that only fungal contamination was observed. In addition, the contaminated explants did not survive; the surviving explants were those that were not contaminated (Table 1). Additionally, according to table 1, the 1.0% sodium hypochlorite concentration combined with the 30 minutes immersion time was less effective for disinfection compared with the 2.0% concentration combined with the same immersion time, but it ensured the survival of the explants ( Figure 1B). This result indicated that the immersion time of 30 minutes was not the main factor causing phytotoxicity to the explants, because when combined with the lowest concentration (1.0%), it ensured the survival of 40% of the explants (Table 1). It is possible that subdividing the sodium hypochlorite concentrations between 1.0 and 2.0% combined with different immersion times between 15 and 30 minutes may increase the efficiency of this process in future experiments. In addition to subdivision of the concentration and immersion time, other disinfection agents should also be tested.

In vitro multiplication
The results for the responsive explants and number of shoots are shown in table 2 and illustrated in figure 2, as it was not possible to fit equations with biological meaning and a high R² (coefficient of determination) to show the variation observed in these characteristics as a function of the BAP concentrations. Figure 2A shows small shoots established in vitro, which were transferred to flasks to ensure sufficient development for use as a source of explants ( Figure  2B and 2C). Figure 2D shows explants from elongated shoots (nodal segments measuring ± 1.0 cm in length) containing one leaf, which were vertically introduced into the culture medium, representing an innovation of the method used by VICENTE et al. (2009) in the micropropagation of V. condensata Baker. Table  2 shows that the addition of BAP was essential for   The addition of BAP was also essential for the induction of adventitious buds, increasing the number of shoots compared with the treatment without this growth regulator (Table 2). It was also observed that the 0.5 mg L -1 BAP concentration resulted in, on average, 4.0 shoots/explant (Table 2 and Figure 2E); although, it did not differ from the other treatments, except for the absence of BAP. However, this result might be associated with the residual effect of the cytokinin present in the culture medium (1.0 mg L -1 BAP) after the in vitro establishment step due to the need to increase the number of shoots for use in the in vitro multiplication experiment. In the low-cost micropropagation of banana cv. Maçã, BERNARDI et al. (2004) increased the BAP concentration in the nutrient medium after the in vitro establishment step, also with the aim of increasing the number of shoots.
The residual effect of BAP in the preculture medium was observed by JESUS et al. observing a phytotoxic effect at BAP concentrations greater than 2.0 mg L -1 . For this reason, concentrations lower than those evaluated in that study were tested in the present study. However, a phytotoxic effect was observed at the highest concentrations used due to the reduction in the number of shoots and the production of hyperhydric shoots. Although, the concentration of 2.5 mg L -1 of BAP resulted in 4.3 shoots per explant, the vegetative growth of these shoots was negatively affected ( Figure 2F). As the BAP concentration increased, there was lower shoot vegetative growth (Figure 3).
With the increase in BAP concentration, there was a decrease in the number of leaves ( Figure  3A) and in shoot height ( Figure 3B). The phytotoxic effect of the highest BAP concentrations was evidenced at the concentration of 2.5 mg L -1 of BAP, where many shoots showed hyperhydricity, a phenomenon known as vitrification. In addition, some studies have emphasized that shoots obtained from nutrient medium with lower concentrations of BAP exhibit greater vegetative   et al. (2009) reported vitrified shoots at BAP concentrations above 2.0 mg L -1 (starting at 3.0 mg L -1 ) for V. condensata Baker, corroborating the data found in the present study. Even at the lowest BAP concentrations, those authors observed the beneficial effect of adding 1.0 mg L -1 GA 3 (gibberellic acid) to induce shoot elongation. Gibberellins are important in cell elongation and are synthesized mainly in young tissues of shoots, leaves, fruits, and seeds (TAIZ & ZEIGER, 2004). Therefore, it is possible that the vertical placement of the explants (young stem branch) and the presence of a young leaf favored the endogenous synthesis of gibberellins in the present study because the shoots elongated without the need for exogenous addition of GA 3 , which is an economic advantage for the V. condensata Baker micropropagation protocol. The use of 0.5 mg L -1 of BAP in the induction of shoots in vitro in V. condensata Baker, even with a preculture of 30 days in culture medium with 1 mg L -1 of BAP after the establishment step, was found to be the best condition, not only due to the reduced cost but also the absence of hyperhydric shoots and satisfactory in vitro elongation. Furthermore, it is estimated that 32,000 shoots can be produced with three subcultures in the in vitro multiplication step, as shown in figure 4.

In vitro rooting and acclimatization
The in vitro rooting and acclimatization data are shown in figure 5. Reducing the amount of salts in the MS medium resulted in 100% rooting of the shoots, demonstrating a significant difference from the MS medium, which showed only 58% rooting ( Figure 5A). Regarding the number of roots and length of the longest root, reduction of the salts to 25% (MS/4) showed the best response, with a mean of 16 roots and 6.15 cm, respectively, demonstrating a significant difference from the other treatments, as shown in figures 5B and 5C. The performance of the culture media used in the rooting of the shoots is also illustrated in figure 6A. A lower rooting performance was observed in plants grown in MS and MS/2 culture media compared with MS/4 ( Figure 6A).
Some studies have shown that the use of only basic MS medium is sufficient for the in vitro rooting of medicinal plants (OLIVEIRA et al., 2011;MONFORT et al. 2012). In addition, other studies have demonstrated the need to use plant growth regulators (especially auxin) or activated charcoal (NAVROSKI et al., 2014;RESENDE et al., 2015;HOULLOU et al., 2015). Reducing the amount of salts in the nutrient medium has been a strategy that is frequently used to induce in vitro rooting (DINIZ, 2014). For example, the use of MS/4 culture medium has provided promising responses for obtaining in vitro adventitious roots in some species, as observed in studies conducted with Azadirachta indica, Lychnophora pinaster, and Pluchea sagittalis (CHATURVEDI et al., 2004;SOUZA et al., 2004;ROSSATO et al., 2015). According to WAREING & PHILLIPS (1982), rooting is favored in more diluted culture media, which may be explained by the cumulative synthesis of endogenous auxins due to the low availability of salts in the culture medium, increasing the metabolic activity of the tissue and, consequently, root induction.   The results of the acclimatization step are shown in figure 5D. Microplants from MS/4 medium showed 100% survival, which did not differ significantly from those from the MS/2 culture medium, with 75% survival. In turn, microplants from MS medium showed only 58% survival. These results can be explained by the higher percentage, number, and length of roots provided by the MS/4 culture medium ( Figure 6A), which might have been essential for ensuring the survival of all evaluated microplants ( Figure 6B). Plants from standard MS medium presented only 58% rooting at the rooting step ( Figure 5A) and this same percentage of survival in the acclimatization step ( Figure 5D), whereas plants from MS/2 and MS/4 media presented 75 and 100% acclimatized plants, respectively ( Figure 5D). Based on these results, the rooting step is crucial for successful acclimatization of plants from in vitro culture, as well as for their transfer to field conditions, as observed in another study conducted with V. condensata Baker (VICENTE et al., 2009). Thus, the nonsurvival of shoots from MS medium might be explained by the absence of roots. Similar results were observed by RESENDE et al. (2015) in the micropropagation of Lippia rotundifolia and by HOULLOU et al. (2015) in the micropropagation of Azadirachta indica (neem).

CONCLUSION
The concentration of 1.0% sodium hypochlorite combined with 30 minutes of immersion ensures the survival and development of explants.
A concentration of 0.5 mg L -1 of BAP allows the proliferation of shoots with the best vegetative development conditions. The best conditions for rooting are provided in MS/4 culture medium. The developed acclimatization system enables 100% survival of microplants originating from MS/4 culture medium. It is possible to estimate a production of 32,000 V. condensata Baker seedlings with three subcultures.

ACKNOWLEDGMENTS
The authors thank Faculdade Maria Milza for providing the physical infrastructure and financial support essential for this study and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for partial finance support, Brasil -Finance code 001.