Effect of synthetic auxins on in vitro and ex vitro bromeliad rooting 1

1. Article received in Jul./2012 and accepted for publication in Apr./2013 (Registration number: PAT 19167). 2. Universidade Federal de Lavras (UFLa), Departamento de Biologia, Lavras, MG, Brasil. E-mail: jprmartinss@yahoo.com.br. 3. Universidade Federal do Espírito Santo (UFES), Centro Universitário Norte do Espírito Santo, Departamento de Ciências Agrárias e Biológicas, São Mateus, ES, Brasil. E-mails: e.romais.s@gmail.com, rodrigosobreiraalexandre@gmail.com, gizelemagevski@hotmail.com. 4. Universidade Federal de Alfenas (Unifal), Instituto de Ciências da Natureza, Alfenas, MG, Brasil. E-mail: brenors@yahoo.com.br. The tissue culture can contribute to the propagation of several economic species, such as the bromeliads. This research aimed at evaluating the auxins type and concentration in the in vitro and ex vitro rhizogenesis of Neoregelia concentrica bromeliad. N. concentrica shoots were induced in a growth medium with 15.0 μM of 6-benzylaminopurine, for 80 days, followed by sub-cultivation in phytoregulator-free medium, for 45 days. In the in vitro rhizogenesis, the shoots grew in a medium supplemented with indole-3-butyric acid (IBA) or naphthalene-acetic acid (NAA), at the concentrations of 0.0 μM, 1.0 μM, 2.0 μM, 3.0 μM and 4.0 μM. In the ex vitro rhizogenesis, the bases of shoots were immersed, for 60 minutes, in IBA or NAA solutions, at the concentrations of 0.0 μM, 5.0 μM, 10.0 μM and 15.0 μM. After immersion, the shoots were planted in plastic trays with vermiculite. At the end of each rhizogenesis method, the phytotechnical parameters analysis was carried out. For the in vitro rhizogenesis, a higher number of roots were observed when the shoots were cultivated in concentrations higher than 1.0 μM of NAA, when compared to the IBA. However, the rooting rate differed only at 30 days after the in vitro growth, with a higher root induction in the shoots grown with NAA. At 60 days, the rooting rate was higher than 90% and statistically similar in all treatments. In the ex vitro rhizogenesis, a better formation of the rooting system was observed when 5.0 μM of IBA was applied, with higher rooting averages and number of roots.

The most common multiplication methods for bromeliads include seed propagation and lateral shoot division.However, these traditional methods are not appropriate for mass propagation (Carneiro et al. 1999), being the in vitro technique more suitable for reaching it (Guerra & Vesco 2010, Pickens et al. 2006).
Ex vitro rooting can contribute to the cost reduction of in vitro propagated plants (Phulwaria et al. 2012).Ex vitro rooted seedlings may present potential advantages, when compared to the in vitro ones, such as a better root system, easier adaptation and higher survival rate (Yan et al. 2010, Benmahioul et al. 2012, Phulwaria et al. 2013).
This study aimed at evaluating the effect of synthetic auxins types and concentrations on in vitro and ex vitro rhizogenesis of the Neoregelia concentrica (Vellozo) L. B. Smith bromeliad.

MATERIAL AND METHODS
The experiment was conducted at the Universidade Federal do Espírito Santo, in São Mateus, Espírito Santo State, Brazil, between September and November 2011.
In vitro growth and multiplication N. concentrica plants previously established in vitro through seeds were inoculated in test tubes containing 10.0 mL of stationary liquid MS medium (Murashige & Skoog 1962), without paper support, supplemented with 10.0 mg L -1 of citric acid, 30.0 g L -1 of sucrose and 15.0 µM of 6-benzylaminopurine (BAP).After 80 days of growth, shoots were subcultivated, for 45 days, in 250.0 mL vials containing 20.0 mL of stationary liquid MS medium with no phytoregulators and supplemented with 30.0 g L -1 of sucrose.The pH medium was adjusted to 5.8, before autoclaving at 120ºC and 1 atm, for 20 minutes.After inoculation in laminar flow cabinet, the plant material was kept in a growth room, at 27 ± 2ºC and a 16-hour photoperiod, under fluorescent lamps providing 25.2 μmol m -2 s -1 of photosynthetic photon flux.

In vitro rooting
Shoots with approximately 2.0 cm length, from the in vitro multiplication stage, were individualized with the aid of a scalpel and inoculated in test tubes containing 10.0 mL of MS medium solidified with 3.5 g L -1 of agar and PH adjusted to 5.8, before autoclaving at 120ºC, for 20 minutes.This medium was supplemented with 10 mg L -1 of citric acid and concentrations of 0.0 µM, 1.0 µM, 2.0 µM, 3.0 µM and 4.0 µM of IBA (indole-3-butyric acid) or NAA (naphthalene-acetic acid), totalizing 10 treatments.After inoculation in laminar flow cabinet, the material was kept, for 60 days, in a growth room, at 27 ± 2ºC and a 16-hour photoperiod, under fluorescent lamps providing 25.2 μmol m -2 s -1 of photosynthetic photon flux.
The variables rooting (%) and average number of roots were evaluated at 30 and 60 days and the largest root length (cm) at 60 days.
The experimental design was completely randomized, in a 5x2 factorial scheme (auxin concentrations x types), with four repetitions per treatment and each plot comprising 8 test tubes, totalizing 32 explants per treatment.The obtained data were submitted to variance analysis (Anova) and the auxin-type factor averages were compared by using the Tukey test and the auxin concentration factor by regression analysis.

Ex vitro rooting
Shoots with approximately 2.0 cm length, from the in vitro multiplication stage, were individualized with the aid of a scalpel and washed in running water to remove the adhered growth medium.Afterwards, e-ISSN 1983-4063 -www.agro.ufg.br/pat-Pesq.Agropec.Trop., Goiânia, v. 43, n. 2, p. 138-146, abr./jun.2013 their bases were immersed, for 60 minutes, in NAA or IBA, at concentrations of 0.0 µM, 5.0 µM, 10.0 µM and 15.0 µM, totalizing 8 treatments.Then, the material was planted in plastic trays with individual cells, containing vermiculite substrate, and kept in a greenhouse with intermittent mist (relative humidity of 70% and temperature of 30ºC).Every 20 days, a substrate supplementation was performed with MS ½ medium to provide the nutritional maintenance of the plants.
The evaluation process took place at 40 days of growth and the analyzed variables were rooting (%), largest root length (cm), average number of roots, volume (mm 3 ) and superficial area of the root system (mm 2 ).For the last two variables, millimetered pictures of individual roots were taken and measurements were performed with the aid of the Safira ® software (Jorge & Rodrigues 2008).
The experimental design was completely randomized, in a 5x2 factorial scheme (auxin concentrations x types), with four repetitions per treatment and each plot comprising 8 shoots, totalizing 32 explants per treatment.The obtained data were submitted to variance analysis (Anova) and the auxin-type factor averages were compared by using the Tukey test and the auxin concentration factor by regression analysis.

RESULTS AND DISCUSSION
The in vitro rooting of N. concentrica increased linearly at 30 days, with the concentration raise, independently of the auxin type used, acting positively over root formation.The highest auxin concentration resulted in rooting of 91.45% and its absence in 60.4% (Figure 1).
According to Carneiro et al. (1999), every shoot of N. cruentra (R. Graham) L. B. Smith rooted after transferred to the auxin-free medium.Arrabal et al. (2002) also observed root formation in Cryptanthus sinuosus L. B. Smith grown in auxinfree medium, showing that some species present higher rhizogenic potential than others.The same result was observed in pineapple plants (Ananas comosus L.), with every shoot rooted in vitro, in a medium with no growth regulator (Sripaoraya et al. 2003).
When NAA was employed in the growth media, the rooting rate at 30 days was superior to the one with IBA, regardless of its concentration (Table 1).At 60 days, a high rate of root induction was observed in the explants, for both IBA and NAA, being statistically equal (Table 1).Mendes et al. (2008)  The use of synthetic auxin aims at accelerating root formation and increasing rooting rate (Santos et al. 2010a), in accordance with the results obtained for N. concentrica, with a smaller root induction observed at 30 days of growth, when no exogenous auxin was employed, if compared to the other treatments (Figure 1).
The auxin-free medium presented rooting averages of 60% and 91%, respectively at 30 and 60 days (Table 1).This is probably due to the endogenous concentration of 3-indoleacetic acid (IAA), which managed to ensure the response.This natural auxin, which is naturally produced in the leaves and buds, moves to the low part of the plant, increasing its concentration at the base of the cut, along with other endogenous substances, stimulating root formation (Lone et al. 2010, Hartmann et al. 2011).
The number of roots presented significant interaction between the auxin types and concentrations.At 30 days, the highest number of roots was observed in media with concentrations higher or equal to 2.0 µM of NAA.
In this research, the initial absorption and accumulation of BAP in the shoot-induction medium, as well as its transfering to the rooting medium with 2.0 µM of NAA, may have provided a favorable hormonal balance to the root production.According to Mercier et al. (2003), cytokinins are necessary, together with auxins, to provide cellular division from the G1-S to the G2-S stage.At 60 days, the media containing NAA displayed higher averages than the ones with IBA (Table 2).
When the auxin concentration was evaluated for the number of roots, only the NAA-cultivated plants presented differences with the concentration increment, displaying linear and quadratic models, respectively at 30 and 60 days of in vitro cultivation (Table 2).Similar results were observed for Billbergia rosea Hortus ex Beer (Pardo et al. 2008), with an increase in the number of roots, when the NAA concentration increased during the in vitro cultivation.
No significant difference was verified for the highest average root length, in N. concentrica (Table 3).According to Poornima & Ravishankar (2007), auxins may have a negative effect on in vitro adventitious root growth, when plants remain in their presence all the time.This was not observed for the employed IBA and NAA concentrations in N. concentrica.Galvanese et al. (2007) concluded that the NAA concentration raise in the growth medium induced a smaller length in Aechmea blanchetiana (Baker) L. B. Smith.
Under ex vitro conditions, a survival rate of 100% was obtained, with root emission at the base of every N. concentrica shoot.Pompelli & Gerra (2005) also obtained high ex vitro rooting rates, working with the Dyckia distachya Hassler bromeliad, with rooting reaching more than 79% for shoots transferred directly to the substrate, * Averages followed by the same letter, in the line, for each evaluation date, do not differ between themselves, according to the Tukey test, at 5%. (1)  The vermiculite used in the present experiment, as well as other substrates, such as pearlite, presents high macroporosity, what grants a high aeration and water-retention capacity, providing a better exploration of the root system and consequently a larger growth of the aerial part, uniformity and high seedling survival rate, especially during acclimatization (Costa et al. 2009, Ruta & Fortunato 2010).These substrate characteristics, associated to the endogenous IAA synthesized in the buds and young leaves, have probably ensured root induction in the N. concentrica explants, even in the absence of exogenous auxins.
The number of roots also found in non-treated explants is probably due to the supply of MS ½ medium, every 20 days.According to Santos et al. (2010b), the use of MS nutritive solution to irrigate Acanthostachys strobilaceae (Schult.F.) Klotzsch enabled a better growth rate of the root system, during acclimatization.
Abiotic factors, such as light (spectral quality or amount), also help to explain the root emission in non-treated explants, in the acclimatization process, for instance, the irradiance levels interfere in the accumulation of photoassimilates (Huang et al. 2011b).Some irradiance levels can increase the temperature, what can cause accumulation of metabolites, such as myo-inositol, glucose, fructose and sucrose, and may be related to the plant homeostasis maintenance, mostly in the initial phases of acclimatization (Mollo et al. 2011).Temperature also acts on the carbon fixation (CAM or C 3 ), as verified for in vitro pineapple plants (Nievola et al. 2005).
The number of roots in ex vitro cultivation presented interaction between auxin type and concentration.The explants grown in a medium with 5.0 µM of IBA reached a higher average to this variable (6.19 roots) than for NAA (Table 4).Better results with IBA, concerning ex vitro growth (Table 4), were probably obtained because this phytoregulator is metabolized in IBA aspartate and posteriorly combined with peptides.This combined form serves as this auxin storage, which is later gradually released, keeping the concentrations at the ideal levels, especially in the final stages of root formation (Rodrigues & Leite 2004).
As the concentration increased, the number of roots raised quadratically for IBA and linearly for NAA (Table 4).As observed for the in vitro rooting, ex vitro rooting is also affected by the auxin types and concentrations (Yan et al. 2010). Augusto et al. (2006) stated that the ex vitro rooting directly on substrate presents the advantages of decreasing the survival associated difficulties, during acclimatization and production, and reducing costs.
The auxin application at the base of cuttings or shoots, in raising doses, can produce stimulating effects on root induction to a peak from which auxin addition becomes inhibitory (Hartmann et al. 2011), what is consistent with the results obtained for IBA, with concentrations higher than 7.0 µM being harmful to root formation in N. concentrica (Table 4).
The largest root length in ex vitro cultivation also presented interaction among factors.The highest average was obtained with 5.0 µM of IBA, when compared to the same concentration of NAA.The other concentrations had similar results, regardless of the auxin type used (Table 5).
When the concentrations were evaluated according to the largest root length, only the shoots * Averages followed by the same letter, in the line, for each evaluation date, do not differ among themselves, according to the Tukey test, at 5%.  treated with IBA differed from each other, following a quadratic model, as the concentration raised (Table 5), and the best IBA concentration was 5.13 µM.
According to Zietemann & Roberto (2007), root emission in larger number and length is fundamental, when the aim is seedling production in commercial scale.Moreover, a well formed root system increases the exploited soil area, promoting water and nutrients absorption, what enables a better seedling growth (Fracaro & Pereira 2004, Carvalho Júnior et al. 2009).
The volume and superficial area of the root system presented significant interaction among factors.When shoots were treated with 5.0 µM of IBA, the roots formed reached higher averages for these variables, when compared to the same concentration of NAA (Table 6).
In the concentration analyses for each auxin, the models were similar for volume and superficial area, concerning the root system.The model was quadratic for both increases in the IBA and NAA concentrations.However, the best results were obtained for IBA, with root system volume and area presenting higher values respectively at the concentrations of 4.88 µM and 5.24 µM (Table 6).The higher number of physiologically active roots, together with a larger root system area, promotes an increase in volume, which can be exploited, representing significant advantage, since it provides a greater adaptation of plants to the soil environment under adverse conditions, as well as for the nutrient absorption increase (Nibau et al. 2008, Rima et al. 2011).
For the external morphology of the root system formed in vitro and ex vitro, regardless of auxin type and concentration, different characteristics were verified between the rooting methods.In vitro roots were more fragile and did not present ramifications, while ex vitro roots were more resistant and with secondary roots (Figure 2).These characteristics were also observed in strawberry (Fragaria x Ananassa Duch.) (Borkowska 2001) and in Siratia grosvenorii (Yan et al. 2010).However, this is not common to all plant species, as observed in Dickia distachya (Pompelli & Gerra 2005), with limited and fragile ex vitro roots, as opposed to the more functional and numerous in vitro roots.
CONCLUSIONS 1.The auxin type influences on the rhizogenic response of Neoregelia concentrica shoots, being the NAA more indicated to in vitro rooting and the IBA to ex vitro rooting.2. The growth medium supplementation with 3.0 µM of NAA was the most promising method for the in vitro rooting of Neoregelia concentrica shoots.3. The application of 5.0 µM of IBA at the Neoregelia concentrica shoot base resulted in the best ex vitro radicular growth.4. The ex vitro rooting method can be employed for Neoregelia concentrica shoots propagated in vitro aiming the decrease of costs and cultivation time.

Table 1 .
obtained a higher in vitro rooting induction rate with Vriesea cacuminis L. B. Smith, when NAA was used in the growth media, if compared to IBA, similary to what was verified in N. concetrica at 30 days.Influence of the synthetic auxin type on the in vitro rooting of N. concentrica at 30 and 60 days(São Mateus, ES, 2011).Effect of synthetic auxins on in vitro and ex vitro bromeliad rooting Figure 1.In vitro rooting of N. concentrica at 30 days, according to auxin concentration (São Mateus, ES, 2011).*Averages followed by the same letter, in the line, for each evaluation date, do not differ among themselves, according to the Tukey test, at 5%. e-ISSN 1983-4063 -www.agro.ufg.br/pat-Pesq.Agropec.Trop., Goiânia, v. 43, n. 2, p. 138-146, abr./jun.2013

Table 2 .
In vitro number of roots of N. concentrica at 30 and 60 days, according to synthetic auxin types and concentrations (São Mateus, ES, 2011).Averages followed by the same letter, in the line, for each evaluation date, do not differ among themselves, according to the Tukey test, at 5%.