Vriesea incurvata Gaudich. (Bromeliads) is a native epiphytic and endemic species from the tropical rain forest - Atlantic Forest, with occurrence recorded in the southeast and south of Brazil (Fontoura, Scudeller & Costa, 2012).

Plant height is between 30-40 cm, have a compact architecture, compact architecture, green, smooth and shiny leaves and without thorns, which contrast with the red inflorescence, confers to V. incurvata strong ornamental appeal. This species is referenced among the ten most commercialized bromeliads in the flower segment in Paraná State (Negrelle & Anacleto, 2012).

However, easy access to the environments where naturally bromeliads grow, high density of this resource in these places, associated to lack of financial resources, land distribution problems, lack of technical and agricultural support and environmental limitation are factors that discourage the implementation of cultivation systems and reinforce the extractive standard (Negrelle & Anacleto, 2012).

Natural vegetative propagation of bromeliads is slow, due to the low number of side shoots produced by the plants after flowering (Alves, Vesco & Guerra, 2006). In addition, the reproduction of bromeliads in the natural environment shows some difficulties related to the low capacity of seed germination. Negrelle & Muraro (2006), reported V. incurvata with low formation of clone shoots (average = 2±0.37) per plant. The same authors characterize V. incurvata as a species with annual flowering, with high level of flower production, low fruit production, but relatively significant seed production. However, Muraro, Negrelle & Anacleto (2014), showed that the percentage of germination (emergence) of this species is relatively low (best result = 40%) under natural conditions.

In this context, there is a permanent demand for the search of alternatives that support the production of V. incurvata as for its reintroduction in environments impacted by extractive as for its implementation in cultivation systems that can adequately meet the commercial demand of ornamental flowers and plants.

Faced with this problem, the in vitro germination of V. incurvata can be considered an option. This technique involves the sowing of seed on culture media, in order to promote the seedlings germination and survival under aseptic conditions, with controlled light and temperature. Among the advantages provided by this technique are the optimization and improvement of the nutritional requirements of the cells and tissues in culture medium, and besides it is a very In vitro germination and growth of Vriesea incurvata Gaudich. (Bromeliaceae) important technique in the commercial and ecological context. Plants produced through this way, may be used in native species reintroduction programs in areas of environmental preservation due to genetic variability generated by explant (Schneiders, Pescador, Booz & Suzuki, 2012).

However, is necessary to evaluate the response of each species to different culture media available for this technique of cultivation, as this response is not homogeneous (Chu, Tavares, Kanashiro, Giampaoli & Yokota, 2010; Zeng, Wu, Silva, Zhang, Chen, Xia & Duan, 2012). These culture media should enriched with supplements that promote the survival, growth and development of the explants.

Given these concerns, in order to support the production of V. incurvata seedlings, results of the assessment of the percentage of seed germination and growth and the initial development of seedlings in vitro conditions in different culture media, are presented.

Data were statistically analyzed with R version 3.3.0, using analysis of variance (ANOVA) to detect significant differences among treatments. Means differing significantly were compared by Tukey test at ρ < 0.05. Data of percentage of normal seedlings, percentage of dead seedlings, percentage of chlorotic, necrotic and dead leaves were V(X+1) transformed before subjecting it to ANOVA.

Figure 1, shows the percentage germination of Vriesea incurvata seeds, which performed high rates of seed germination and was higher than 82.8% and was observed for all culture media.

Figure 1. Means followed by the same letter do not differ at 5% by Tukey test. MS: Murashige and Skoog. KC: Knudson C. AC: Activated carbon

Percentage of seeds germination with KC medium + AC increased in 17.2% when compared to MS medium + AC (Figure 1). The results obtained with V. incurvata also confirm the positive response already observed for other species of Vriesea, concerning to the use of KC medium in the in vitro germination.

Total fresh biomass of the seedling was significantly different in the culture media. MS medium had minimum increment of 52.94% of total fresh biomass, when compared to KCV2 medium + AC, KCV2 medium, KC medium and KC medium + AC, respectively (Table 1).

Table 1

Means followed by different letters within a column are significantly different at ρ < 0.05 by Tukey test. Data of percentage seedlings were V(X+1) transformed before subjecting it to ANOVA. ^n.s. not significant. MS: Murashige and Skoog. KC: Knudson C. AC: Activated carbon.

High percentage of normal seedlings (> 82%) was recorded for all culture media. The highest values were obtained in KCV2 medium and KC medium + AC. The lowest values were observed in MS medium + AC and MSV2 medium + AC (Table 1). The achievement of high percentage values of normal seedlings (> 82%) can be considered a favorable indicator for the employment of in vitro cultivation in the development of V. incurvata propagation and reintroduction programs.

Length of roots of V. incurvata seedlings, was significantly affected by the composition of the culture media. The highest values were observed in KCV2 medium + AC (18 mm), KCV2 medium (12.04 mm) and KC medium + AC (10.92 mm) (Table 1). However, roots production was not significantly affected by the culture media (Table 1). Number and length of roots are important variables in the initial growth of bromeliads due to the fact the roots become the principal plant organ to uptake the nutrients provided by the culture medium under in vitro conditions.

MSV2 medium and MS medium, were also highlighted as the best response in relation to the stem length and leaf production. Shoot length was significantly different among the culture media tested, especially as regards to MSV2 medium (25.29 mm) when compared to KC medium + AC (15.50 mm) (Table 2). Production of leaves, was significantly affected by the culture media. The greatest amount of leaves was observed in MS medium (9 leaves in total) and the lowest amount was recorded in KC medium + AC (6.3 leaves in total) (Table 2).

Percentage of necrotic and dead leaves of V. incurvata seedlings, was significantly different among the culture media tested. However, for the percentage of chlorotic leaves was not significantly different in relation to the culture media (Table 2). The lowest percentages of necrotic leaves were recorded in KCV2 medium (0.48%), KC medium (1.74%) and MSV2 medium (2.56%) and the highest percentages were in MS medium + AC (11.78%) (Table 2).

Table 2

Means followed by different letters within a column are significantly different at ρ < 0.05 by Tukey test. Data of percentage leaves were V(X+1 ) transformed before subjecting it to ANOVA. ^n.s. not significant. MS: Murashige and Skoog. KC: Knudson C. AC: Activated carbon.

Additionally, for the percentage of necrotic leaves, were observed differences between KCV2 medium when compared to MSV2 medium + AC and MS medium +AC. (Table 2). For the percentage of dead leaves, KCV2 medium (0%) and KC medium (0.66%) showed the lowest percentages in relation to MS medium + AC (11.79%) and MS medium (18.23%) (Table 2).

The choice of the culture medium, nutritional supplements, physiological germination conditions, and the conditions of the seeds as the origin and maturity of the capsule influence significantly the in vitro seed germination (Zeng, Zhang, Silva, Wu, Zhang & Duan, 2013).

The highest percentage of seeds germina tion, was obtained with the KC medium + AC(100%) and the lowest (82.8%) was obtained in MS medium + AC (Figure 1). When compared to the results of the ex vitro germination (40% ac cording to Muraro, Negrelle & Anacleto (2014), who was showed that the in vitro condition can optimize in an expressive way the production of V. incurvata seedlings, doubling the percentage of germination. The production of seedlings of this species is therefore, favored by this seed germi nation technique, which provides the nutritional requirements appropriate for the emergence, survival and development of the seedlings under aseptic conditions, with controlled temperature and light (Schneiders, Pescador, Booz & Suzuki, 2012; Zeng, Wu, Silva, Zhang, Chen, Xia & Duan, 2012, among others).

High percentages of germination using KC medium, were also observed by Droste, Silva, Matos & Almeida (2005), in V. gigantea and V. philippocoburgii species with 99% and 89%, re spectively. Mercier & Kerbauy (1994), reported up to 90% of seeds germination in V. hieroglyphica using KCV2 medium.

However, despite the KC medium, has been reported as more effective for the growth of ornamental species such as Orchidaceae (Soares, Araújo, Pasqual, Rodrigues & Assis, 2009); this medium was not suitable to promote the initial growth of V. incurvata. This result could be related to the low concentration of potassium and nitrogen (Knudson, 1946).

MS medium had achieved a higher concen tration of nutrients than the KC medium, is likely due to the high concentration of nutrients in MS medium, which favored the increasing of the total fresh biomass of V. incurvata seedlings. This result was also seen in Nidularium minutum (Bromeliaceae) when cultivated in MS medium and MSV2 medium (Carvalho, Hayashi, Braga & Nievol, 2013).

Root systems with long and numerous roots are important, because they ensure the proper storage of carbohydrate reserves for the growth under in vitro conditions (Chu, Tavares, Ka-nashiro, Giampaoli & Yokota, 2010). Already in advanced stages of the plant, the bromeliad roots have as the main function the nutrients fixing, due to plant nutrition is carried out by leaf trichomes (Vanhoutte, Ceusters & Proft, 2016).

However, the values related to the length of roots detected in V. incurvata were much lower than those reported for other species of bromeli-ads. The representatives of Nidularium minutum (Bromeliaceae) held for six months under in vitro conditions reached maximum roots length of 6 cm using MS medium and MSV2 medium (Carvalho, Hayashi, Braga & Nievol, 2013). On the other hand, Carvalho, Santos & Nievol (2014), using MS medium in Acanthostachys strobilacea (Bromeliaceae) observed that seedlings in vitro with 90 days maintained at temperatures of 20 to 25°C, reached the root length of at least 7.6 and 8 cm, respectively. Additional studies on roots production and length in representatives of V. incurvata produced in natural environment, through sexual propagation, should support the better understanding of this in vitro response.

MS medium shows as its main characteristic high nitrogen concentration, essential macro element in the production of proteins and in the physiological processes that occur in plants as the growth (Murashige & Skoog, 1962).

These results support that the addition of activated carbon was unfavorable to promote this growth. Activated carbon is widely used in in vitro propagation of several plants because it contributes in the root formation process and in the absorption of toxic substances present in the culture medium (Thomas, 2008). However, the addition of activated carbon can also determine inhibitory responses (Nicoloso, Erig, Martins & Russowski, 2001). This seems to be the case of V. incurvata, which determined in some culture me dia at least the lowest accumulation of total fresh biomass, leaf production and higher percentages of necrotic and dead leaves. Additionally, these results may be associated to the relatively high concentration of phosphate in the KC medium (1.84 mmol l^-1) in relation to MS medium (1.25 mmol l^-1) (Knudson, 1946; Murashige & Skoog, 1962). According by Kerbauy (2012), the phos phorus deficiency in plants can lead to premature senescence of leaves, reduction of leaf expansion and retrace in the formation of the reproductive organs.

In vitro cultivation is suitable to promote high rates of V. incurvata germination.

MS and MSV2 are the best culture media to promote the achievement of a greater number of normal seedlings, higher production rates of total fresh biomass, more leaves and better performance of stem growth. MSV2 medium contains half of nutrients concentration related to standard medium (MS), showing a major advantage in terms of cost/benefit.

KC and KCV2 medium, also promote highest percentages of normal seedlings and low percentages of necrotic and dead leaves of V. incurvata. Conversely, the addition of activated carbon in the culture media is unfavorable to promote the growth of V. incurvata