Seedling production of bromeliad endemic to the semiarid region of Bahia, Brazil

Abstract

Bromeliads are plants that, in addition to marked ecological importance, have increasing ornamental potential in the flower market, being commonly collected in an extractive way, which results in genetic loss of the exploited species, putting them in different degrees of threat. Culture of plant tissues has been shown to be a viable alternative for the propagation of many bromeliads. The aim of this study was to produce seedlings of Neoregelia mucugensis by micropropagation. In vitro multiplication of sprouts via direct organogenesis was obtained in static liquid medium containing 6-benzylaminopurine (BAP) cytokinin. The sprouts were rooted in activated charcoal medium and transferred to the ex vitro condition, and 100% plant survival was obtained. It is concluded, therefore, that the production of seedlings of N. mucugensis is viable by micropropagation.

Key words:
activated charcoal; direct organogenesis; in vitro cultivation; Neoregelia mucugensis

Resumo

As bromélias são plantas que além de acentuada importância ecológica, apresentam crescente potencial ornamental no mercado de flores, sendo comumente coletadas de forma extrativista, o que acarreta perda genética das espécies exploradas, pondo-as em diferentes graus de ameaça. A cultura de tecidos vegetais tem se mostrado uma alternativa viável para a propagação de muitas bromeliáceas. O objetivo deste estudo foi produzir mudas de Neoregelia mucugensis por micropropagação. A multiplicação de brotos via organogênese direta foi obtida em meio líquido estático contendo a citocinina 6-benzilaminopurina (BAP). Os brotos foram enraizados, em meio com carvão ativado, e transferidos para a condição ex vitro, sendo obtidos 100% de sobrevivência das plantas. Conclui-se, portanto que a produção de mudas de N. mucugensis é viável por micropropagação.

Palavras-chave:
carvão ativado; organogênese direta; cultivo in vitro; Neoregelia mucugensis

Bromeliads have been commercially exploited in Brazil since the 1970s, and the demand for these plants in the market is increasing (Anacleto et al. 2008) due to the exuberance of colors and shapes, being widely used as ornamental plants. However, several species are marketed in an extractive way (Anacleto & Negrelle 2009) and, consequently, there is a reduction in the natural population of these plants, with risk to the genetic material. As a result, several bromeliads are included in the red list of threatened species categorized at different levels. Species of the genus Neoregelia are listed in the classifications of less worrying, vulnerable, almost threatened, endangered, and critically endangered (CNCFlora 2012).

Neoregelia mucugensis Leme is a rupicolous herb not yet evaluated for the risk of extinction, but it is native and endemic to Brazil, with confirmed occurrence only in the state of Bahia, in rupestrian grassland vegetation (Flora do Brasil 2020, continuously updated) and exclusively exploited in an extractive way. The predation of bromeliads can interfere with the dynamics of the ecosystem since there are species of invertebrate and vertebrate animals that use them in several ways: aquarium or water source, place of shelter or hunting territory, food and permanent habitat; in addition, other plant species grow in bromeliads (Frank & Lounibos 2009).

In order to preserve these plants and meet the flower market of Brazil, which according to data from Ibraflor (2021) is among the 15 largest flower producers in the world, it is necessary to study the application of sustainable propagation strategies such as the culture of plant tissues. In vitro cultivation can be influenced by luminosity, type and concentration of the culture medium that provide conditions for plant growth and multiplication (Rodrigues et al. 2019), type of explant, and concentrations of plant regulators (Lopes et al. 2017) and other compounds added to the medium, such as activated charcoal. For N. mucugensis, the existing publications address only the stages of in vitro establishment and in vitro multiplication (Bellintani et al. 2007a, 2008), but there are no reports for in vitro rooting and acclimatization. The objective of this study was to produce seedlings of Neoregelia mucugensis, aiming for a commercial scale.

Neoregelia mucugensis seeds collected in Mucugê Municipal Park - Mucugê, Chapada Diamantina, BA, Brazil, were established in vitro (Bellintani et al. 2007a) and used as a source of explants. Whole 3-month-old plants were inserted into test tubes containing 5 mL of MS culture medium (Murashige & Skoog 1962) with half of the salt concentrations (MS 1/2) plus 30 g L^-1 sucrose (static liquid medium) and different 6-benzylaminopurine-BAP concentrations (0.0; 20; 25; 30; 35 μM). After 60 days, the percentage of responsive explants (%ReE) and number of sprouts per explant (NS) were evaluated. The experimental design was completely randomized (CRD) with 5 treatments. Each treatment consisted of 6 replicates with 5 samples.

Sprouts, 2 cm long, were inserted into test tubes containing 15 mL of MS1/2 culture medium plus 30 g L^-1 sucrose, 7 g L^-1 agar and 1 g L^-1 activated charcoal. The experimental design was CRD with two treatments: rooting for 30 and 45 days, with 30 replicates each. After the stipulated period, the percentage of rooted explants (%RoE), the number of roots (NR), and the length of the longest root (LLR) measured in centimeters (cm) were evaluated. Then, these microplants were acclimatized. The microplants rooted in vitro were transplanted to a substrate composed of earth and vermiculite (1:1) (Bellintani et al. 2007b), in 50-mL transparent disposable plastic cups, and kept in a plastic tray with a film of water in a greenhouse with 70% shade net. At 30 days, the percentage of plant survival was evaluated.

The in vitro cultures were kept in a growth room at a temperature of 25 ± 3 ºC, photoperiod of 16 h and active photosynthetic radiation of 60 μmol.m^-2.s^-1. The data were subjected to analysis of variance (ANOVA) and to regression for quantitative data, using the SISVAR 5.6 program (Ferreira 2019).

Regarding the in vitro multiplication of N. mucugensis, the results showed that the addition of BAP concentrations in the culture medium promoted a significant increase in %ReE and NS, which were formed by direct organogenesis pathway (Fig. 1). The results were described by an increasing linear regression model for both variables, with lower mean values in the control, being 11% of %ReE and 0.26 of NS, and incremented with the application of cytokinin, which led to maximum means of 70% and 3.27 for %ReE and NS, respectively (Fig. 1a-b). This attests to the importance of this plant regulator in the process of inducing sprouts, and indicates that higher concentrations of BAP can be tested for this species.

Figure 1
a-b. Percentage of explants responsive to sprout formation (a) and number of sprouts per explant (b) as a function of different 6-benzylaminopurine (BAP) concentrations in Neoregelia mucugensis plants after 60 days of in vitro cultivation.

These results are similar to those obtained by Lima et al. (2021) in a study with Neoregelia mucugensis in which, using BAP and NAA, a maximum rate of 70% for responsive explants and an average of 2.2 shoots in stem explants of plants cultivated in vitro for 365 days were achieved.

The maximum rate of explants that responded to sprout formation, 70%, was higher than that of 54.6% obtained by Bellintani et al. (2008) in stem segments of the same species, also in the presence of BAP, and the average number of sprouts was 4 to 6 per explant (on a measurement scale) in the combination of BAP and naphthalene acetic acid (NAA).

Although the results obtained by these authors for NS are more favorable, the stem segments came from 6-month-old mother plants and the variables were also evaluated at 6 months of inoculation, totaling a period of one year to obtain sprouts. In the present study, 3-month-old seedlings were used as explants and the final evaluation was performed at 60 days, totaling 5 months for the production of sprouts, which represents a reduction of time and costs in the production of seedlings.

The presence of BAP has also had a positive effect on the in vitro multiplication of other bromeliad species, such as the higher number of sprouts for Sincoraea mucugensis (Wand. & A.A. Conc.) & Wand. in a study conducted by Lima et al. (2020), and for Aechmea miniata Beer ex Baker and Aechmea blanchetiana (Baker) L.B.Sm in the study conducted by Garcia et al. (2021). For Ananas comosus L. Merril, the use of BAP also generated an increase in sprouts compared to the absence of this plant regulator, in both liquid and semi-solid medium (Oliveira-Cauduro et al. 2016). Similar behavior was reported by Oliveira et al. (2007) in the in vitro multiplication of Ananas lucidus Miller, as this cytokinin promoted a higher response in the number of sprouts when compared to the control using liquid medium, while in solid medium there was no significant difference between the concentrations used.

BAP efficiency is associated with its ability to break the apical dominance of plants, which stimulates the development of lateral buds (Santa-Rosa et al. 2013), with control of cell division and induction and growth of sprouts (Nelson et al. 2015). On the other hand, when applied at high concentrations, it may cause phytotoxicity, depending on the species (Raposo et al. 2019).

Other bromeliad species have shown a positive effect of the liquid medium on in vitro cultivation, as can be seen in a study conducted by Mengarda et al. (2009), in which the bromeliads Neoregelia cruenta (R. Graham) L. B. Sm, Tilland siastricta Sol., Vriesea gigantea Gaudich., Vriesea guttata Linden & André and Vriesea incurvata Gaudich showed higher average rate of sprout proliferation in in vitro multiplication in static liquid medium compared to semi-solid medium, static liquid medium with paper bridge and liquid medium under agitation. Moreover, the liquid culture medium was also used by Meneghetti et al. (2019) for in vitro multiplication of Neoregelia johannis (Carriére) L.B. Smith. The liquid medium can be beneficial in the in vitro cultivation of plants, as it increases the availability and absorption of nutrients by them (Oliveira et al. 2019), in addition to reducing the cost of production due to the non-use of gelling substance in the culture medium.

After obtaining the sprouts, in vitro rooting was performed, which did not indicate the influence of the tested periods on %RoE, NR and LLR (Tab. 1). However, it is worth noting that high rates of rooted sprouts were generated, 76 to 93%, and that NR and LLR (cm) ranged from 1.86 to 2.03 and from 2.13 to 2.38, respectively (Tab. 1). These results indicate that the 30-day period is sufficient to induce the in vitro formation of N. mucugensis roots and corroborate the efficiency of activated charcoal in this process. The same was observed by Lima et al. (2020) in a study with S. mucugensis in which it was found that the 30-day period is indicated for rooting with activated charcoal. Activated charcoal is commonly added to the culture medium in order to improve morphogenic responses because it reduces or suppresses undesirable compounds due to its adsorption capacity (Sáenz et al. 2010), such as plant exudates, toxic metabolites and phenolic compounds (Rondon et al. 2019; North et al. 2012). The use of activated charcoal in the culture medium to the detriment of the addition of auxins for the in vitro formation of roots is advantageous due to the low cost of this substance compared to the acquisition of plant regulators.

The efficiency of the rooting step is of great importance for acclimatization, and the strategies to be applied vary according to the species. For N. mucugensis, there are no records of previous studies for these steps, and in this study the results indicate that the survival rate after 30 days of acclimatization was 100%, which confirms the success of micropropagation of this species, whose steps are shown in Figure 2. Likewise, for the bromeliad S. mucugensis the study conducted by Lima et al. (2020) concluded that no type of strategy to control water loss needs to be applied and that microplants of this species can be directly exposed to the environment during acclimatization without compromising their survival, whose rate was 95%. Similarly, the transfer to the ex vitro environment of the Nidularium minutum Mez microplants was performed without control of water loss in a greenhouse, and a 100% survival was recorded (Kurita et al. 2013).

Table 1
Percentage of rooted explants (%RoE), the number of roots (NR), and the length of the longest root (LLR) as a function in vitro rooting treatments of Neoregelia mucugensis.

Treatments (1g L-1 de activated charcoal)	%RoE	NR	LLR (cm)
rooting for 30 days	93 A	2,03 A	2,13 A
rooting for 45 days	76 A	1,86 A	2,38 A

The study makes it possible to conclude that BAP stimulation in liquid medium induces the formation of sprouts by direct organogenesis in N. mucugensis seedlings, the 30-day period is sufficient to induce the in vitro formation of roots with activated charcoal, and microplants show a high survival rate after acclimatization; consequently, the production of seedlings by micropropagation is viable for the studied species.

Figure 2
a-h. Micropropagation of Neoregelia mucugensis - a. plant in natural environment; b. capsules containing seeds; c. plants germinated in vitro; d. sprouts obtained after 60 days of in vitro cultivation in liquid medium under the effect of 6-benzylaminopurine (BAP); e. sprout under rooting with activated charcoal; f. rooted microplant; g. plant transferred to substrate, initial time; h. plant after 30 days of acclimatization. Scale bar = 1 cm.

Acknowledgements

This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brazil (CAPES) - Finance Code 001, and the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) (409587/2018-4).

Data availability statement

In accordance with Open Science communication practices, the authors inform that there is no data sharing of this manuscript

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Edited by

Area Editor:

Dr. Diego Rocha

Publication Dates

• Publication in this collection
  09 Sept 2024
• Date of issue
  2024

History

• Received
  14 Dec 2023
• Accepted
  13 May 2024