Micropropagation of Vellozia seubertiana (Velloziaceae)

ABSTRACT Velloziaceae is one of the main families in the floristic composition of the Campos Rupestres Montane Savanna ecoregion of the Chapada Diamantina Highland, Bahia, Brazil, and has species with significant ornamental potential and resistance to climate changes. The species Vellozia seubertiana stands out for its beautiful flowers and stems covered by leaf sheaths that ensure protection when in contact with fire. However, there is no information on its propagation, conservation, and physiology, which justifies this study. The objective of this work was to establish a micropropagation protocol for V. seubertiana. Seeds were disinfected and inoculated in Murashige and Skoog culture medium (MS) with half salt concentration (MS½) for in vitro establishment. Plantlets established in vitro were used to induce sprouting in MS½ supplemented with 6-benzylaminopurine (BAP; 0.00, 4.44, 8.88, and 17.76 µM) and 1-naphthaleneacetic acid (NAA; 0.00 and 2.22 µM). The shoots obtained were inoculated in MS½ containing activated charcoal (0.0 and 1.0 g L−¹) and indole-3-butyric acid (IBA; 0.00 and 2.22 µM) for rooting. The rooted plantlets were acclimated under greenhouse conditions. A mean of 5.7 shoots were generated through organogenesis in medium containing 8.75 µM of BAP and 2.22 µM of NAA. The use of activated charcoal resulted in the highest means for aerial part and root lengths in the in vivo rooting phase. Acclimated plants reached 75% survival at 60 days after transplanting to ex vitro conditions. The results indicate that micropropagation is a promising technique for the production of V. seubertiana seedlings.


INTRODUCTION
Chapada Diamantina Highland is in the state of Bahia, northeastern Brazil, and has a great diversity of plant resources that deserves attention.This region is composed of exuberant landscapes, and its flora has high ornamental potential and other uses already known.It explains the extractive removal of large amounts of plants from nature, bringing risks to local plant genetic resources.
Species of the Velloziaceae family have significant degree of endemism in the Campos Rupestres Montane Savanna ecoregion of the Chapada Diamantina Highland (NEVES, 2009).Vellozia seubertiana Goethart & Henrard. is among the species in this region; it stands out for its beautiful lilac flowers (NEVES, 2009) and stems covered by leaf sheaths that ensure protection when in contact with fire (ALVES; SILVA, 2011).V. seubertiana is endemic to Brazil (MELLO-SILVA, 2015), has herbaceous growth habit, and is found on rocks and in sandy soils (NEVES, 2009).
V. seubertiana is a potential resource for use as ornamental plants; however, the sustainability of this activity requires studies for assisting the management of its natural populations and the development of techniques for ex situ conservation and production of quality seedlings.
Plant tissue culture stands out among strategies for seedling production as an excellent alternative for the ornamental plant sector and ex situ conservation of species.Micropropagation is the most common plant tissue culture technique, which provides production of seedlings with similar characteristics to the parent plants (GRATTAPAGLIA; MACHADO, 1998).It is a technique for asexual reproduction started by small fragments (explants), which enables the production of a large number of healthy seedlings in a small space and short time (GEORGE, 2008), differing from conventional propagation methods.
Only two studies on in vitro propagation of species of the Vellozia genus are found in the literature, one for Vellozia flavicans Mart.Ex Schult f. (FREITAS NETO, 2009) and other for Vellozia pyrantha A. A. Conc.(basionym = Vellozia sincorana), which is endemic to the Chapada Diamantina Highland (BORGES, 2015).
Studies focused on establishing efficient protocols for in vitro propagation of plants have shown that the addition of plant growth regulators, mainly auxins and cytokinins, to the culture medium is determinant for a successful micropropagation, as these hormone classes control specific stages of the cell cycle and contributes to cell division (KERBAUY, 2019).The combination of auxins and cytokinins drives morphogenesis and promotes the growth of organs, calluses, and cell suspensions (GEORGE, 2008).
Considering the importance of conservation of plant resources in the Campos Rupestres Montane Savanna ecoregion of the Chapada Diamantina Highland, the efficiency of applying micropropagation to meet the demand of the ornamental plant market and the lack of studies on V. seubertiana, the objective of this work was to establish a protocol for in vitro propagation of plants of this ornamental species.

MATERIAL AND METHODS
Seeds were obtained from dry capsules of the species Vellozia seubertiana Goethart & Henrard.(Figure 5b) collected in the City Park of Mucuge, Chapada Diamantina Highland region, Bahia,Brazil (12°59'47''S,41°22'11''W). The seeds were washed in running water with a drop of a commercial neutral detergent for 10 minutes.In a laminar flow chamber, they were submerged in 70% alcohol for one minute and, then, immersed in 3% sodium hypochlorite with 3 drops of a commercial neutral detergent for 10 minutes.They were then washed four times in sterilized distilled water.

In vitro establishment
The seeds were inoculated in glass test tubes containing 10 mL of Murashige and Skoog culture medium (MS) (MURASHIGE; SKOOG, 1962) with half salt concentration (MS½), supplemented with 30 g L -¹ of sucrose and solidified with 7 g L -¹ of agar and 1 g L -¹ of activated charcoal (BORGES, 2015).After 75 days, the plantlets germinated in vitro were used to induce sprouting.

In vitro multiplication
Aerial parts of plantlets of V. seubertiana were inoculated in glass test tubes containing 10 mL of MS½ supplemented with 30 g L -¹ of sucrose, 7 g L -¹ of agar, and different synthetic cytokinin concentrations (0.00, 4.44, 8.88, and 17.76 µM of 6-benzylaminopurine -BAP) combined with auxin concentrations (0.00 and 2.22 µM of 1naphthaleneacetic acid -NAA).The aerial part was used as explant due to the absence of responses from other explant types subjected to the same multiplication conditions (data not shown).
A completely randomized experimental design was used, in a 4×2 factorial arrangement consisted of four BAP concentrations (0.00, 4.44, 8.88, and 17.76 µM) and two NAA concentrations (0.00 and 2.22 µM).Each treatment consisted of five four-sample replications, totaling 20 tubes per treatment (one plant per tube).
The following variables were evaluated after 60 days of cultivation: percentage of explants with shoots (%ES), number of shoots per explant (NSE), mean length of shoots (MLS), dry weight of shoots (DWS), and survival of initial explants (%S).

In vitro rooting
Shoots with heights of approximately 2 cm, from in vitro multiplication, were individualized and inoculated in 250-mL flasks containing 50 mL of MS½ supplemented with 30 g L -¹ of sucrose, 7 g L -¹ of agar, and combinations of concentrations of auxin (0.00 and 2.22 µM of indole-3-butyric acid -IBA) and activated charcoal (0.0 and 1.0 g L -¹).
A completely randomized experimental design was used, in a 2×2 factorial arrangement consisted of combinations of IBA (0.00 and 2.22 µM) and activated charcoal concentrations (0.0 and 1.0 g L -¹).Each treatment consisted of five four-sample replications, totaling 20 flasks per treatment.
The following variables were evaluated at 30 days: rooting percentage (%R), number of roots per plant (NR), longest root length (LRL), aerial part length (APL), aerial part dry weight (APDW), number of green leaves (NGL), and number of senescent leaves (NSL).

Acclimation
Plantlets of V. seubertiana were removed from the flasks using tweezers, their roots were carefully washed with distilled water to remove culture medium residues and, subsequently, transplanted to 200-mL polyethylene cups, with five holes in the bottom, containing topsoil and vermiculite at the proportion of 1:1.The cups were covered with a transparent polyethylene cup for 10 days (LIMA; LIMA-BRITO; SANTANA, 2020) and then kept in trays with a water layer, in a greenhouse covered with a 70% shade screen, for 60 days.
Each treatment consisted of five four-sample replications.The following variables were evaluated at 60 days after transplanting: percentage of plant survival (%PS), aerial part length (APL), and number of green leaves (NGL).
All in vitro experiments were conducted in a growth room with temperature of 26±2 °C, 14-hour photoperiod, and active photosynthetic radiation of 60 µmol m -2 s -1 obtained through fluorescent lamps (250V).The pH of all culture media used was adjusted to 5.7 before autoclaving at 121 °C for 15 minutes.The tubes and flasks were sealed with polyvinyl chloride (PVC) film.

Statistical analysis
The results were analyzed for normality, using the Shapiro-Wilk test, and no rejection was found.The data were then subjected to analysis of variance and the means of the quantitative and qualitative data were compared by the Tukey's test at 5% probability or regression analysis, respectively, using the statistical program SISVAR (FERREIRA, 2019).

RESULTS AND DISCUSSION
The germination rate of Vellozia seubertiana seeds was higher than 80%.The use of plants from seed germination enhances the maintenance of genetic variability, which is important for the initial establishment of in vitro collections.A high variability is important when the tissue culture is used as an ex-situ conservation strategy.Moreover, maintaining the genetic stability of the parent plant is essential for producing clone seedlings intended to the market, which can be obtained by a direct in vitro propagation.
V. seubertiana leaves were used as explants in preliminary studies, but did not show morphogenetic responses under the conditions evaluated (unpublished data).Similarly, a study using leaves of the bromeliad Orthophytum mucugense WAND and CONCEIÇAO to start multiplication found no significant responses for percentage of explants with shoots and number of shoots per explant (LIMA et al., 2012).However, leaf explants proved to be promising for in vitro multiplication of the bromeliad Aechmea ramosa Mart.ex Schult.f. (FARIA et al., 2018), denoting the importance of specific studies.
Regarding the vitro multiplication phase, induction of shoots at the base of the extant was found in all treatments.No significant interaction between the plant growth regulators tested was found for percentage of explants with shoots (% ES).The presence of the cytokinin BAP in the culture medium had no effect on %ES.However, although the addition of auxin to the culture medium is not essential for the response of explants in in vitro shoot formation, it significantly affected the %ES; the mean %ES found with the NAA concentration of 2.22 µM was significantly higher (58.75%) than that in the treatment without this auxin (Figure 1a).These results indicate that V. seubertiana has enough endogenous auxin for shoot formation; however, the multiplication can be optimized by exogenous supplementation with the plant growth regulator NAA, resulting in hormonal balances more favorable to multiplication.
Considering that only one NAA concentration (2.22 µM) was evaluated and promoted a mean %ES of 58.75%, higher concentrations should be tested to potentialize in vitro multiplications.Lima et al. (2012) also found means lower than 60% when using NAA at 0.65 and 1.30 μM concentrations combined with 2.22 μM of BAP for O. mucugense.Lima, Lima-Brito, and Santana (2020) found higher %ES (99%) for the bromeliad Sincoraea mucugensis (Wand.& A.A. Conc.)LOUZADA & WAND; however, they used higher NAA concentrations (2.60 and 5.20 µM) than that used in the present study and stems as explants.Lima-Brito et al. (2011) evaluated the species Syngonanthus mucugensis Giul.subsp.mucugensis, an evergreen flower of Mucuge, and found %ES of 58% when using stems as explants and no growth regulator, which indicates that the species has similar satisfactory contents of endogenous hormones to the species V. seubertiana.These two species have similar growth habit and occur in the same region.
The number of shoots per explant (NSE) was significantly affected by the interaction between BAP and NAA concentrations.Regarding the absence of NAA, the data did not fit to a regression model.However, the regression analysis for NSE with presence of NAA resulted in a quadratic polynomial model, with the highest NSE of 5.45 estimated for the combination of 8.87 µM of BAP and of NAA; NSE tends to decrease when using higher BAP concentrations (Figure 2).In vitro multiplication experiments intend to improve the cytokinin-auxin balance to establish a protocol with a high shoot production, and the present study is the first on in vitro multiplication of the species V. seubertiana.The shoot production graph curve decreases after reaching the highest point, which was estimated with the BAP concentration of 8.75 μM, showing that the synthetic cytokinin BAP can have positive effects on in vitro multiplication, but it can also have a toxic or inhibitory effect triggered by the use of higher concentrations than the ideal for each species (Figure 4).Lima, Lima-Brito, and Santana (2020) found similar results for the growth regulator BAP in a study on multiplication of Sincoraea mucugensis, with an NSE of approximately 7 for a BAP concentration of 8.92 µM.
Cytokinins are required for in vitro multiplication because they induce the breaking of apical dominance and emergence of new axillary buds (ILIEV et al., 2010).Auxin and cytokinin hormones are essential for the advance of the cell cycle.Thus, all plants naturally have auxins and cytokinins, which directly participate in the cell cycle regulation process, more precisely in the transition from the G1 phase to the starting of DNA synthesis.Auxins control cyclins, and one of them is modulated by a cytokinin, forming an active complex that enables the cell cycle evolution; in addition, cytokinins alone participate in the regulation from the G2 to the M phase (KERBAUY, 2019).
The highest NSE found was 5.45, which was higher than those found by Borges (2015) for V. pyrantha (basionym = V. sincorana), approximately 0.3 when using kinetin and 0.2 when using BAP.
Freitas Neto ( 2009) evaluated subcultures for in vitro multiplication of V. flavicans and found NSE lower than 2 in the first subculture and between 6 and 7 in the third subculture when using BAP at concentrations of 2.22 and 4.44 µM, which confirms that this cytokinin is efficient for in vitro production of Vellozia shoots.Garcia et al. (2021) found that increases in BAP concentration increase the NSE in the bromeliad species Aechmea miniata (Beer) Baker and Aechmea blanchetiana (Baker) L. B. Smith, with the highest NSE of 14.5 for A. miniata and 10.5 for A. blanchetiana after 225 days when using 13.32 μM of BAP.
Dry weight and length of shoots were not affected by the NAA and BAP concentrations in the in vitro multiplication treatments.
The mean survival of initial explants was higher than 95% in all treatments, denoting that the plant aerial part is a promising initial explant for multiplication of V. seubertiana, as it provides high sprouting rate and can be used for new subcultures, which probable explains its use in other studies evaluating Vellozia species (FREITAS NETO, 2009;BORGES, 2015).Similarly, whole-plant explants from seed germination have been used for rosette species, such as bromeliads (DIAS et al., 2020).
Regarding the in vitro rooting phase, roots were found in all plantlets.No interaction was found between activated charcoal and NAA, which had isolated effects.The highest means of aerial part length (3.65 cm) and longest root length (3.60 cm) were found using activated charcoal, which statistically differed from the treatments without this additive (Figures 3a-b).Similar results were reported by Lima et al. (2012), who found positive effect of charcoal on these variables when studying in vitro rooting of O. mucugense.
The use of activated charcoal in the culture medium for in vitro rooting promotes some benefits.Its color provides a dark environment, with no luminosity, which is a favorable condition for plant rooting (GEORGE, 2008).Another benefit is the adsorption of elements that can inhibit rhizogenesis; such elements may be present in the medium itself or released by the plant during cultivation (GRATTAPAGLIA; MACHADO, 1998).
The variables evaluated in the in vitro rooting treatments were not affected by the auxin IBA, except number of green leaves (NGL).This can be explained by the presence of sufficient endogenous levels for the formation of adventitious roots in the shoots, since all rooted.
The negative effect of this auxin on NGL (Figure 3c) can be attributed to leaf toxicity caused by hormonal unbalance, exceeding the limits tolerated by the species; NGL was 7.82 when using IBA and 11 when it was not used.Additionally, synthesized auxins naturally participate in several processes in plants, including senescence (KERBAUY, 2019), and increases in auxin levels by supplementation may have contributed to increases in senescence of in vitro leaves.The plants free from supplementation with auxin presented higher number of green leaves (Figure 3c) and lower number of senescent leaves (data not showed).The auxin and activated charcoal had no effects on the variables analyzed in the acclimation phase.The percentage of plant survival (%PS) at 60 days after transplanting to the ex -vitro environment varied from 50% to 75% among the treatments tested.It denotes that, although significant differences were found for the variables analyzed in in vivo rooting, these differences did not affect the adaptation of V. seubertiana to the external environment conditions evaluated.It is emphasized that all the plantlets were already rooted when transplanted to the substrates, denoting their capacity for in vitro rooting, which is essential for a successful acclimation.
These results differed from those of Lima, Lima-Brito, and Santana (2020), who found that the culture medium containing 1 g L -1 of activated charcoal had a positive effect on acclimation of Sincoraea mucugensis.
Freitas Neto (2009) found changes in leaf cuticle thickness during acclimation of V. flavicans: very thin in samples grown in vitro, very thick in plants grown in the field, and intermediate in acclimated plantlets, which denotes that this species easily adapts to growing conditions.Variation in leaf cuticle thickness is an anatomical adaptation of plants to avoid water loss and, consequently, dehydration under water deficit conditions.
The good adaptation of V. seubertiana to the acclimation can be attributed to fast mechanisms of adaptation to external environments, as described by Freitas Neto (2009) for V. flavicans, which is connected to the rusticity of these species in their local of natural occurrence, the Campos Rupestres Montane Savanna ecoregion, where they present high tolerance to water stress and, in many cases, protected stomata and an efficient metabolism against dehydration (RAPINI, 2008).
The results found denote that the period for obtaining seedlings of V. serbertiana from seeds through organogenesis is approximately 230 days (Figure 5).The present study is unprecedented for the species Vellozia seubertiana and is the first description of a complete in vitro propagation protocol for the genus Vellozia; therefore, it will be useful to guide future studies for other species of the family Velloziaceae.
D. I. J. G. C. PINTO et al.

Figure 1 .
Figure 1.Effect of auxin NAA on percentage of explants with shoots (%ES) in in vitro multiplication of Vellozia seubertiana.

Figure 2 .
Figure 2. Effect of the interaction between auxin NAA (0.00 and 2.22 µM) and cytokinin BAP on number of shoots per explant (NSE) for in vitro multiplication of Vellozia seubertiana.

Figure 3 .
Figure 3.In vitro rooting of Vellozia seubertiana: isolated effect of activated charcoal on aerial part length -APL (a) and longest root length -LRL (b), and isolated effect of IBA on number of green leaves -NGL (c).