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Article

Micropropagation of ‘Manacá-de-Cheiro’ (Brunfelsia uniflora (Pohl) D. Don), an Ornamental Species Native to Brazil

by
Ana Victória Conde van den Broek
1,
Mariana Pelais Leite
1 and
Jean Carlos Cardoso
1,2,*
1
Graduate Program in Plant Production and Associated Bioprocesses, Center of Agricultural Sciences, Federal University of São Carlos (CCA/UFSCar), Rodovia Anhanguera, km 174, CP 153, Araras 13600-970, SP, Brazil
2
Lab of Plant Physiology and Tissue Culture, Department of Biotechnology, Plant and Animal Production, Center of Agricultural Sciences, Federal University of Sao Carlos (CCA/UFSCar), São Carlos 13565-905, SP, Brazil
*
Author to whom correspondence should be addressed.
Int. J. Plant Biol. 2025, 16(2), 69; https://doi.org/10.3390/ijpb16020069
Submission received: 24 April 2025 / Revised: 9 June 2025 / Accepted: 11 June 2025 / Published: 17 June 2025
(This article belongs to the Section Plant Reproduction)
Browse Figures
Versions Notes

Abstract

:
The introduction of new ornamental species and cultivars is one of the hallmarks of innovation in global floriculture. Brunfelsia uniflora, a subshrub native to Brazil, has white, lilac, and blue flowers on the same plant, in addition to a distinctive fragrance. As it is a wild species, technologies such as large-scale clonal propagation of superior genotypes are still scarce, limiting its supply to the flower market. Therefore, a successful micropropagation protocol was developed for B. uniflora using nodal segments and shoot tips as initial explants. In the multiplication phase, the use of 6-benzylaminopurine produced the highest multiplication rates (10.3–10.9 shoots/explant) and the number of leaves in the shoots. In vitro shoot rooting using MS medium with reduced macronutrient concentrations and supplemented with IBA resulted in a 91.7% rooting rate. The greatest difficulty in micropropagating this species was the high percentage of shoots that developed calli. The highest percentage of callus formation occurred with the addition of auxins at high concentrations (1.0 and 1.5 mg L−1). Even so, the shoots and plantlets were acclimatized, demonstrating the effectiveness of this technique for the production of B. uniflora plantlets.

1. Introduction

Brunfelsia uniflora (Pohl) D. Don, commonly known as ‘manacá-de-cheiro’, is a species native to Brazil, belonging to the family Solanaceae and found throughout South America [1]. This shrub has simple, alternate leaves [2] and shows great potential as a potted flower or landscaping shrub, especially because of its flowers, which change from white to lilac and emit a sweet aroma, characteristic of this species.
There is little information regarding its propagation. In observations carried out by the authors regarding the commercial production of ‘manacá-de-cheiro’ pots, it was revealed that sexual propagation is limited by the small number of seeds obtained per plant throughout cultivation. Limitations to the sexual propagation of species such as ‘manacá-de-cheiro’ include the high genetic variability, which makes standardization of production difficult; difficulty producing plantlets on a commercial scale due to recalcitrant or dormant seeds; and irregular production and low seed availability, as well as difficulty defining the ideal seed harvesting time [3,4]. Therefore, micropropagation can be advantageous for increasing and accelerating the production of high-quality plantlets based on superior genetic material for ornamental use, as the lack of quantity and quality discourages their use in the flower market.
Thus, given the growing demand in the ornamental plant market, there is a growing interest in species that are underexplored but have striking ornamental characteristics [5], as is the case with B. uniflora. However, for the commercial exploitation of species like this, a necessary step is the regular and large-scale production of clonal plantlets with high genetic and phytosanitary quality, which can be achieved by developing micropropagation protocols. For manacá-de-cheiro, only in vitro organogenesis using the regeneration of Brunfelsia calycina plants from leaf segments has been reported [6], and the encapsulation of Brunfelsia pauciflora is reported as a conservation protocol [7].
Liberman et al. [6] used different types of explants and combinations of plant growth regulators in B. calycina but reported that only leaf explants resulted in organogenesis and shoot regeneration. They also found that the optimal culture medium for shoot organogenesis was achieved by using 6-benzyladenine (BA or BAP) at 4.44–8.88 µM and indole-3-acetic acid (IAA) at 2.5 µM. Using BAP (2.0 mg/L) combined with IBA (0.4–0.8 mg/L) also resulted in the highest shoot proliferation in B. pauciflora, showing that BAP is an important cytokinin that induces organogenesis and shoot multiplication in Brunfelsia species. Organogenesis is indeed an important in vitro technique, especially when tissues with preformed meristems, such as shoot tips and nodal segments, are not an option. However, organogenesis is more dependent on endogenous factors such as genotype and explant age, and also exogenous factors, which makes its large-scale application for the propagation of multiple elite genotypes difficult [8,9].
Therefore, the development of shoot tips or nodal segments in Brunfelsia could be more interesting for producing clonal plantlets by micropropagation. Thus, the objective of this study was to develop a protocol for the in vitro micropropagation of B. uniflora, including establishment, multiplication, rooting, and acclimatization, using explants of a previously selected genotype with high ornamental potential for use as a potted flower or in landscaping.

2. Results

2.1. In Vitro Establishment

No microbial contamination was visually observed in the explants. Successful in vitro establishment of manacá-de-cheiro was achieved, as demonstrated by the development of shoot apices and nodal segments. This resulted in the formation of new leaves and stems from the in vitro inoculated explants (Figure 1). Therefore, 40% of the explants died due to physiological damage caused by asepsis and/or excessive handling. Using nodal segments as initial explants, multiplication rates in the first and second subcultures, obtained by the number of new shoots for each explant inoculated, were 2.0 and 10.3 shoots/explant, respectively. The shoot apices resulted in multiplication rates of 2.7 and 10.9 shoots/explant. In each subculture, the new shoots were individualized and subcultured in new, fresh culture media, maintaining the original composition of the multiplication culture media, until sufficient shoots were obtained for the experiments.

2.2. Effects of Culture Medium and Plant Growth Regulators on the Shoot Proliferation Phase

Plant growth regulators significantly affected (p < 0.01) plant height, shoot multi-plication rate and the number of leaves in in vitro shoots (Figure 2 and Figure 3). In the multiplication phase, there was no effect of the WPM [10] and Murashige and Skoog [11] ½ saline formulations on the analyzed variables. Adding BAP or TDZ to the culture medium reduced plant height by 2.14–2.30 cm, which differs from the control without plant growth regulators (3.23 cm) (Figure 2 and Figure 3).
On the other hand, mainly in the presence of BAP (1.0–3.0 mg L−1), a considerable increase in the number of leaves per inoculated explant and shoot multiplication was observed, greater than 500% using 1.0 or 2.0 mg L−1 compared to the control (Figure 3B). This significant increase in the number of leaves was due to the development of multiple shoots in the explants (Figure 3 and Figure 2 B1–B3). However, the addition of BAP at high concentrations (>1.0 mg L−1) or TDZ at any concentration resulted in vitrification or hyperhydricity of the shoots, which reduced the quality of the shoots obtained in Brunfelsia (Figure 2 T1 and T2). Interestingly, shoots also formed callus at their bases in the control with no PGR addition (Figure 2 Co).
Another important finding of our study was that transferring new shoots to a new culture medium should be done between 28 and 30 days after inoculation in the medium to avoid leaf senescence.

2.3. Effects of Culture Medium and Plant Growth Regulatorss in the Rooting Phase and Acclimatization

There was an effect of the interaction between the culture medium and auxins on plant height, number of leaves and roots, and percentage of rooting (Figure 4). In all treatments and the control without auxins, adventitious rooting of shoots from the multiplication phase (WPM + 0.5 mg L−1 BAP) was observed at different frequencies. Nonetheless, none of the tested treatments achieved 100% rooting of the shoots (Figure 4). Interestingly, the IBA resulted in a higher number of roots than NAA when using the MS ½ culture media, while NAA showed a better response than IBA when using the WPM culture media. This shows an interaction between the auxins and the formulation of the culture media. The use of MS ½ containing 0.5 mg L−1 IBA resulted in the best rooting percentage and number of roots/explant (Figure 4). For shoot development, WPM without auxins or containing IBA at 0.5 mg L−1 resulted in best plant height and number of leaves per shoot (Figure 4; Figure 5A,B). Furthermore, the lowest rooting percentages (16.7–25.0%) were observed in the control using both media and in the treatment of MS ½ containing 1.0–1.5 mg L−1 NAA.
The formation and excessive growth of calli in the basal region of the explants was the most important physiological abnormality in the micropropagation of Brunfelsia uniflora (Figure 5 and Figure 6)). This abnormality was strongly correlated with the addition of auxins (IBA and NAA) and increases in their concentrations (see Figure 6). Lower calli masses were reported using the MS ½ or WPM culture media in the absence of auxins (Figure 5 and Figure 6).
The result of survival in acclimatization showed that the highest shoot/plantlet survival rates (69–81%) were obtained in the WPM or MS ½ culture media, without auxins or with the addition of 0.5 mg L−1 IBA. In general, increasing the auxin concentration in the culture medium decreased the percentage of plantlet survival during acclimatization, possibly due to greater callus development in the nodal segments (Figure 5 and Figure 6). The number of leaves per plantlet and the chlorophyll content in 120-day-old acclimatized plantlets increased only with the lowest dose (0.5 mg L−1) of auxins used in the in vitro rooting phase (Table 1). The number of leaves/plantlets, as well as the chlorophyll content (A, B, and A + B), increased, while the Chl A/B ratio decreased, when 0.5 mg L⁻¹ of IBA was used. However, the best survival in acclimatization occurred without auxins.
Furthermore, modifications in the previous culture media used for experimentation at the rooting phase reduced abnormalities and improved the quality of the shoots and roots of in vitro Brunfelsia plantlets. The use of a culture medium with the following modifications: (1) reducing the concentrations of NH4NO3 (850 to 620 mg L−1) and KNO3 (950 to 715 mg L−1), and (2) reducing the auxin IBA concentration from 0.5 to 0.1 mg L−1, resulted in shoots with a high percentage of rooting (71.4%) and high rooting quality, without excessive callus formation at the base of the explants (Figure 7).

3. Discussion

3.1. In Vitro Establishment

Shoot apices used as explants showed higher proliferation rates than nodal segments in in vitro Brunfelsia. Similar results were observed in the in vitro cultivation of Cordia verbenaceae [12] and Calendula officinalis [13], with a higher number of shoots from shoot apices compared to nodal segments. However, nodal segments can serve as alternative explants, especially when the number of shoot apices is low in some species [14,15].

3.2. Effects of Culture Medium and Plant Growth Regulators on the Shoot Proliferation Phase

The addition of BAP to the culture media resulted in the highest number of shoots and leaves in the multiplication phase, but drastically reduced shoot size. Similar results were reported in the in vitro cultivation of Valeriana jatamansi, where BAP concentrations up to 2.0 mg L−1 resulted in shoots with a higher number of leaves, though there was a significant reduction in shoot and leaf size, especially at the highest BAP concentration tested (3.0 mg L−1) [16].
Liberman et al. [6] also obtained leaf organogenesis with the production of new buds and adventitious shoots in Brunfelsia calycina using an MS medium supplemented with indole-3-acetic acid (IAA) at 2.85 µM combined with either 4.54 µM TDZ or 4.44 µM BAP. The combination of IAA and BAP at 2.85 µM and 4.44 µM, respectively, promoted the best development of shoots, compared to the use of TDZ. Our results also showed that even low concentrations of TDZ resulted in poor development of in vitro shoots of Brunfelsia uniflora, and increased physiological abnormalities, such as vitrification and excessive callus formation. TDZ is considered a potent in vitro plant growth regulator for developing different species of plants that are important for agriculture [17,18]. However, the number of in vitro abnormalities associated with TDZ use has posed a major challenge to its safe use for cloning by micropropagation [19]. Vitrification and callus formation are the main disorders observed, and generally cause a loss of propagation efficiency and difficulties in acclimatizing plants with these symptoms [19,20].

3.3. Effects of Culture Medium and Auxins on Rooting and Acclimatization

In vitro rooting of woody species is influenced by environmental and culture medium factors, including light conditions, the addition of activated charcoal, variations in the concentration of plant growth regulators, sucrose, and salts [21] and presents challenges due to limitations and specific variations between cultivars [21,22].
Increasing IBA or NAA concentrations in both saline formulations resulted in reduced plant height and number of leaves, and increased rooting and callus formation in the shoots of B. uniflora. Ancasi-Espejo et al. [23] observed similar results with Brazil nut (Bertholletia excelsa Bonpl., Lecythidaceae) and concluded that doses of 0.5 and 1.0 mg L−1 of IBA promoted root development under in vitro conditions. For azalea, the optimal IBA dosage for in vitro rooting was 2 mg L−1 [24]. For stinking juniper (Juniperus foetidissima), a dose of 0.5 mg L−1 IBA resulted in a rooting rate of 88.9% [25]. In contrast, Kaviani et al. [26] achieved better results using IAA (1.0 mg L−1) in a standard MS medium rather than IBA for the in vitro rooting of Pyrodwarf® rootstock for pear trees. Although IAA was not used in this experiment, it could be an interesting alternative for the in vitro cultivation of Brunfelsia, given the symptoms of callogenesis and the decreased quality of the resulting plantlets in the presence of IBA and NAA. Interestingly, a greater average callus diameter was reported in B. uniflora in the WPM medium compared to the MS ½ medium, suggesting that the former saline formulation would be more conducive to callus formation. According to Bhojwani et al. [27], the greater proliferation and diameter of calli are mainly linked to the nitrogen supplied by the culture medium. Reductions of N salts, such as KNO3 and NH4NO3, in the culture media improved the rooting performance of Prunus rootstocks [28].
Excessive callus formation during micropropagation can indicate a physiological disorder. This disorder was observed in our study with B. uniflora, especially during the rooting phase. The disorder was exacerbated by the addition of high concentrations of synthetic auxins to the culture medium. Similar results have been reported for peach in in vitro rooting: higher auxin concentrations reduced the rooting percentage and increased callus formation at the bases of shoots [29]. This phenomenon may be due to the greater sensitivity of in vitro shoots of B. uniflora to auxins or the greater accumulation of ethylene in closed containers with little gas exchange, conditions typical of in vitro cultivation and the methods used in this study. Similar results were obtained with Habanero pepper plants grown in vitro in non-ventilated containers. The presence of ethylene, even at low concentrations, produced negative results in plantlet development, including callus formation and symptoms of vitrification [30], similar to those observed here for manacá-de-cheiro (Figure 3, Figure 5 and Figure 6).
The increased frequency and intensity of callogenesis in explants during the rooting phase further supports this hypothesis because synthetic auxins, at higher concentrations, similar to those added to the culture medium, are known to increase ethylene biosynthesis in plants [31].
Survival during acclimatization decreased with increased auxin concentrations in in vitro rooting culture media. This decrease in survival may be specifically associated with increasing and excessive callus formation (Figure 5 and Figure 6), which results in early leaf senescence under in vitro conditions. Even in the control treatment (without auxins), the percentage of rooted plants was low (16.7–25%) (Figure 4). However, there was good survival during acclimatization (81.3%) (Table 1). This demonstrates that greater plantlet or shoot mortality is not related to the absence of roots under in vitro conditions. Rather, it is related to physiological abnormalities, such as excessive calli, observed in the shoots during the in vitro rooting phase. Chlorophyll content in vitro and its range during acclimatization can be used to measure acclimatization [32,33].
Maintaining B. uniflora shoots in the same vessel for longer periods (>35–40 days) resulted in the loss of plantlet vigor, leaf senescence, and a decreased multiplication rate due to tissue death. These symptoms can be attributed to the presence of ethylene in the in vitro environment and are similar to those observed in in vitro rose shoots [34].
Reducing the concentrations of NH4NO3 (from 850 to 620 mg L−1) and KNO3 (from 950 to 715 mg L−1), as well as the auxin IBA (from 0.5 to 0.1 mg L−1), resulted in plantlets with reduced callus formation at the base of the explants and improved adventitious rooting under in vitro conditions (Figure 7). This confirms our hypothesis regarding the issues observed in the first rooting experiment (Figure 5). Woodward et al. [35] demonstrated that the lowest nitrogen dose (7.5 mM) resulted in the highest percentage of rooting and number of roots per shoot in Eucalyptus marginata compared to 15, 30, and 60 mM. These results demonstrate the need for systematic studies to overcome challenges and improve micropropagation protocols for wild woody species.

4. Materials and Methods

4.1. Plant Material

The plant material came from two adult Brunfelsia uniflora plants (Figure 8), which were propagated by stem cuttings and obtained from a mother plant that was previously selected from a commercial pot producer in the region of Holambra, state of São Paulo, Brazil. The main characteristics of interest are the compact, shrubby size (up to 3 m height) and the large number of flowers, which vary in color from white to lilac and blue on the same plant.

4.2. In Vitro Establishment

To initiate in vitro cultivation, only the apical regions of young, herbaceous shoots, approximately three centimeters long, were used. The shoots were initially washed under running water, followed by asepsis with 70% alcohol for 30 s, and immersion in a 2.0–2.5% sodium hypochlorite solution (Ypê®, Amparo, Brazil) for 20 min. Then, three consecutive washes were carried out with previously autoclaved deionized water.
Young shoots were then divided into two types of explants: the shoot apex, with a length of 0.2–0.3 cm, containing two to three leaf primordia, and the nodal segments of the subapical region, with approximately 0.5 cm in length, containing at least one axillary bud. The explants were inoculated in 30 mL Wood Plant Medium (WPM) [10] containing 1 mg L−1 6-benzylaminopurine (BAP), 20 g L−1 sucrose, and 6.4 g L−1 agar (Agargel®, João Pessoa, Brazil). The agar was added after the pH was adjusted to 5.7, and the culture media were transferred to 265 mL glass bottles. After inoculation into the culture medium, the explants were kept in a growth room, in the dark, at 25 ± 2 °C for seven days. Then, they were transferred to a growth room at 25 ± 2 °C, 16 h photoperiod, and light of 25–30 µmol cm−2 s−1 provided by cold white fluorescent lamps for 60 days. The shoots were subcultured four times, each for 30 days, in the same culture medium containing 0.5 mg L⁻¹ BAP until a sufficient number of plants were obtained for the experiments. In all subcultures, the shoots were cut and individualized for the next subculture cycle.

4.3. Effects of Culture Media and Plant Growth Regulators on Shoot Proliferation

The objective of this experiment was to evaluate the effect of different concentrations of 6-benzylaminopurine (BAP) aiming to improve shoot proliferation and the in vitro development of B. uniflora. In addition to BAP, the most commonly used cytokinin in tissue culture, including Brunfelsia species [6,7], we also tested the use of Thidiazuron (TDZ), a diphenylurea that can act as a cytokinin analog, and is conventionally used at lower concentrations to improve shoot proliferation in certain cultures [36]. The culture media used in the multiplication experiment were Murashige and Skoog [11], with half the concentrations of macronutrients (MS ½) and WPM medium [10] (Duchefa Co., Haarlem, The Netherlands), both of which contained 20 g L−1 sucrose and 6.4 g L−1 agar. The pH of the media was adjusted to 5.75 ± 0.05 before adding the agar and then autoclaved at 121 °C for 20 min. In addition to the culture media, different concentrations of BAP at 0.0, 1.0, 2.0, and 3.0 mg L−1 or TDZ at 0.25, and 0.5 mg L−1 were used as treatments. For each treatment, twelve test tubes (replications) containing a nodal segment 0.5 ± 0.1 cm long with at least one axillary bud were used. The cultivation conditions were similar to those described for in vitro establishment; however, the plants were cultivated under light throughout the cultivation period of 42 days. The experiment was conducted in a 2 × 6 factorial design, with two saline formulations and six treatments with plant growth regulators.

4.4. Effects of Culture Medium and Auxins on Rooting and Plantlets’ Acclimatization

Rooting experiments were conducted with different concentrations of the auxins indole-butyric acid (IBA) and naphthaleneacetic acid (NAA) to evaluate shoot rooting under in vitro conditions. The shoots used in this experiment were derived exclusively from the WPM medium containing 0.5 mg L−1 BAP. Similar to the multiplication phase, two culture media were also used for rooting, WPM and MS (½). The treatments consisted of different types and concentrations of IBA and NAA auxins, applied to the culture media at concentrations of 0.0, 0.5, 1.0, and 1.5 mg L−1. The cultivation conditions at this time were similar to those described in the multiplication period, and the cultivation period was 42 days. The experiment was conducted in a 2 × 7 factorial (rooting phase), with two saline formulations of culture media and two types of auxins at three concentrations and one without auxins.
A total of 12 plantlets or shoots per treatment were acclimatized 42 days after in vitro cultivation in treatments with different saline formulations, types, and concentrations of auxins. The plantlets were acclimatized in plastic trays of 50 cells containing Carolina Soil® as substrate, which is composed of peat, charcoaled rice husk, and fine vermiculite. The plantlets, after being planted in the substrate, were transferred to a greenhouse protected on the upper side with light-diffusing plastic and aluminum shade cloth (65% shading) and the other sides protected with black shade cloth (50% shade). Irrigation was carried out using a microsprinkler system, applying a depth of approximately 7 mm/day, and fertilization was performed with ultra-soluble fertilizer PlantProd® 20-20-20 once a week at a concentration of 1 g L−1 fertilizer. Chlorophyll contents (A, B, and Total) were also determined in mature leaves from 120 days of acclimatization, using CFL1001 Clorofilog (Falker®, Porto Alegre, Brazil).

4.5. Experiment to Improve In Vitro Rooting Performance

This experiment aimed to address the issues observed in vitro in the first rooting experiment, which resulted in shoots with a high percentage of undesirable callus formation. Our hypothesis is that the reduction in the concentration of auxins and nitrogen in the culture medium, in conjunction with the addition of activated charcoal (1.0 g L−1), leads to shoots with less callus formation and a higher rooting rate. To this end, the culture medium was based on the MS ½ medium (control), and the treatments consisted of reducing the amounts of KNO3 (950 to 715 mg L−1) and NH4NO3 (850 to 620 mg L−1) reagents in this medium. Another treatment involved adding or not adding activated charcoal (1.0 g L−1) to the medium. IBA was also used at a lower concentration than in the initial experiment, at 0.1 mg L−1 of IBA in all treatments. The experiment was conducted with two factors (activated charcoal × N and K reagents) in completely randomized blocks. All other procedures, as well as cultivation conditions, were similar to those described in the first rooting experiment.

4.6. Experimental Design and Statistical Analysis

In both the multiplication and rooting experiments, there were 12 replications per treatment, each consisting of a test tube containing 15 mL culture medium and 1 nodal segment.
The plant height (cm), number of leaves per clump, and the shoot multiplication rate were obtained at the end of the multiplication experiment and were subjected to analysis of variance (ANOVA). Shoot multiplication was determined by counting the number of new shoots obtained at the end of the culture period. The data were presented as the number of new shoots divided by the initial explant inoculated (one explant/test tube). The treatments were compared using the Scott–Knott mean test at 5% (*) and 1% (**) probabilities. The analyses were performed using the R 3.5.1 software [37].
The following characteristics were analyzed to evaluate rooting: plant height (cm), number of leaves, and number of roots per inoculated plant in the different treatments. The percentage of survival in acclimatization after 120 days of growing plantlets in the substrate was also estimated to evaluate the efficiency of in vitro rooting in the acclimatization of micropropagated plantlets.
In the in vitro rooting experiment, additional analyses were carried out for the mass and diameter of calli from the bases of the nodal segments. The results were tested for normality and homogeneity of variances. The data on callus mass and diameter were transformed to √(X + 1) to meet the assumptions of analysis of variance.

5. Conclusions

In the present study, we developed a micropropagation protocol for Brunfelsia uniflora, a shrubby plant with significant ornamental value. Both MS ½ and WPM media can be used for in vitro multiplication, and a BAP concentration of up to 2.0 mg L−1 is beneficial. The greatest challenge in micropropagating Brunfelsia was the rooting phase, during which excessive callus formation occurred instead of root development. Reducing the amount of NH4NO3, KNO3, and IBA in the medium resulted in good rooting without the physiological abnormalities previously observed. This method can be used to improve in vitro rooting performance and in vitro plantlet formation for acclimatization.

Author Contributions

Conceptualization, A.V.C.v.d.B. and J.C.C.; methodology, A.V.C.v.d.B. and J.C.C.; validation, J.C.C. and M.P.L.; formal analysis, A.V.C.v.d.B. and M.P.L.; investigation, A.V.C.v.d.B.; resources, J.C.C.; data curation, A.V.C.v.d.B. and M.P.L.; writing—original draft preparation, A.V.C.v.d.B. and J.C.C.; writing—review and editing, J.C.C. and M.P.L.; supervision, J.C.C.; project administration, A.V.C.v.d.B. and J.C.C.; funding acquisition, A.V.C.v.d.B. and J.C.C. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), grant number 2018/02595-5 and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPQ), grant number 311083/2018-8. This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior—Brasil (CAPES)—Finance Code 001.

Data Availability Statement

Research data are available upon reasonable request.

Acknowledgments

We thank CAPES, CNPQ and FAPESP for partially supporting this study.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. In vitro development of shoot apices (A,C) and nodal segments (B) of manacá-de-cheiro (Brunfelsia uniflora) after 30 days of cultivation and shoot proliferation after 90 days of cultivation in WPM medium containing 1.0 mg L−1 of BAP (D).
Figure 1. In vitro development of shoot apices (A,C) and nodal segments (B) of manacá-de-cheiro (Brunfelsia uniflora) after 30 days of cultivation and shoot proliferation after 90 days of cultivation in WPM medium containing 1.0 mg L−1 of BAP (D).
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Figure 2. Shoot development and proliferation of Brunfelsia uniflora in WPM culture medium containing different concentrations of BAP (B1, 2, and 3 = 1.0, 2.0, and 3.0 mg L−1) or TDZ (T1, 0.25 mg L−1 and T2, 0.5 mg L−1) and control without PGRs (Co).
Figure 2. Shoot development and proliferation of Brunfelsia uniflora in WPM culture medium containing different concentrations of BAP (B1, 2, and 3 = 1.0, 2.0, and 3.0 mg L−1) or TDZ (T1, 0.25 mg L−1 and T2, 0.5 mg L−1) and control without PGRs (Co).
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Figure 3. Shoot height (A), shoot multiplication rate (B), and number of leaves per clump (C) in the shoot proliferation phase of micropropagation of B. uniflora using 6-benzylaminopurine (BAP) or Thidiazuron (TDZ) in the culture medium. Means ± standard deviation (SD) followed by the same letter and do not differ from each other using the Scott–Knott test at 1% significance.
Figure 3. Shoot height (A), shoot multiplication rate (B), and number of leaves per clump (C) in the shoot proliferation phase of micropropagation of B. uniflora using 6-benzylaminopurine (BAP) or Thidiazuron (TDZ) in the culture medium. Means ± standard deviation (SD) followed by the same letter and do not differ from each other using the Scott–Knott test at 1% significance.
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Figure 4. Plant height (A), number of leaves per clump (B), rooting percentage (C), and number of roots per explant (D) in the rooting phase of B. uniflora micropropagation. Means followed by the same letter, lowercase (auxin concentrations) or uppercase (culture media) letters, do not differ from each other using the Scott–Knott test at 1% of significance.
Figure 4. Plant height (A), number of leaves per clump (B), rooting percentage (C), and number of roots per explant (D) in the rooting phase of B. uniflora micropropagation. Means followed by the same letter, lowercase (auxin concentrations) or uppercase (culture media) letters, do not differ from each other using the Scott–Knott test at 1% of significance.
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Figure 5. Development of shoot/calli/roots among treatments with culture media and auxins indole-3-butyric acid (IBA) and 1-naphthaleneacetic acid (NAA): (A) WPM control without auxins; WPM containing IBA at 0.5 mg L−1 (B), 1.0 mg L−1 (C) and 1.5 mg L−1 (D); WPM containing NAA at 0.5 mg L−1 (E), 1.0 mg L−1 (F) and 1.5 mg L−1 (G); MS ½ control without auxins (H); MS ½ containing IBA at 0.5 mg L−1 (I), 1.0 mg L−1 (J) and 1.5 mg L−1 (K); MS ½ containing NAA at 0.5 mg L−1 (L), 1.0 mg L−1 (M), and 1.5 mg L−1 (N). Scale bars = 1 cm.
Figure 5. Development of shoot/calli/roots among treatments with culture media and auxins indole-3-butyric acid (IBA) and 1-naphthaleneacetic acid (NAA): (A) WPM control without auxins; WPM containing IBA at 0.5 mg L−1 (B), 1.0 mg L−1 (C) and 1.5 mg L−1 (D); WPM containing NAA at 0.5 mg L−1 (E), 1.0 mg L−1 (F) and 1.5 mg L−1 (G); MS ½ control without auxins (H); MS ½ containing IBA at 0.5 mg L−1 (I), 1.0 mg L−1 (J) and 1.5 mg L−1 (K); MS ½ containing NAA at 0.5 mg L−1 (L), 1.0 mg L−1 (M), and 1.5 mg L−1 (N). Scale bars = 1 cm.
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Figure 6. Effects of auxins on callus growth in in vitro shoots in the elongation/rooting stage of B. uniflora micropropagation.
Figure 6. Effects of auxins on callus growth in in vitro shoots in the elongation/rooting stage of B. uniflora micropropagation.
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Figure 7. Rooting and improvement in shoot quality in a modified MS culture medium by reducing the concentration of NH4NO3 and KNO3 salts and the concentration of IBA from 0.5 to 0.1 mg L−1.
Figure 7. Rooting and improvement in shoot quality in a modified MS culture medium by reducing the concentration of NH4NO3 and KNO3 salts and the concentration of IBA from 0.5 to 0.1 mg L−1.
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Figure 8. Selected genotype of flowering Brunfelsia uniflora used as a source of explants for micropropagation.
Figure 8. Selected genotype of flowering Brunfelsia uniflora used as a source of explants for micropropagation.
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Table 1. Survival, number of leaves, and chlorophyll content index of acclimatized B. uniflora plantlets.
Table 1. Survival, number of leaves, and chlorophyll content index of acclimatized B. uniflora plantlets.
AuxinsSurvival inNumber of LeavesChlorophyll Content Index
(mg L−1)Acclimatization (%)/PlantletChl AChl BChl A + BChl A/B
0.081.35.0 ± 1.428.6 ± 0.65.7 ± 0.234.2 ± 0.85.0 ± 0.4
0.568.87.0 ± 1.731.5 ± 3.87.0 ± 1.738.5 ± 5.34.5 ± 0.1
1.031.34.5 ± 1.428.4 ± 0.55.6 ± 0.234.0 ± 0.65.1 ± 0.3
1.531.35.5 ± 1.425.6 ± 5.94.7 ± 1.830.3 ± 7.85.4 ± 0.7
The values represent the mean ± standard deviation (SD). The results presented correspond to the mean of IBA/NAA treatments in their respective concentrations.
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MDPI and ACS Style

van den Broek, A.V.C.; Leite, M.P.; Cardoso, J.C. Micropropagation of ‘Manacá-de-Cheiro’ (Brunfelsia uniflora (Pohl) D. Don), an Ornamental Species Native to Brazil. Int. J. Plant Biol. 2025, 16, 69. https://doi.org/10.3390/ijpb16020069

AMA Style

van den Broek AVC, Leite MP, Cardoso JC. Micropropagation of ‘Manacá-de-Cheiro’ (Brunfelsia uniflora (Pohl) D. Don), an Ornamental Species Native to Brazil. International Journal of Plant Biology. 2025; 16(2):69. https://doi.org/10.3390/ijpb16020069

Chicago/Turabian Style

van den Broek, Ana Victória Conde, Mariana Pelais Leite, and Jean Carlos Cardoso. 2025. "Micropropagation of ‘Manacá-de-Cheiro’ (Brunfelsia uniflora (Pohl) D. Don), an Ornamental Species Native to Brazil" International Journal of Plant Biology 16, no. 2: 69. https://doi.org/10.3390/ijpb16020069

APA Style

van den Broek, A. V. C., Leite, M. P., & Cardoso, J. C. (2025). Micropropagation of ‘Manacá-de-Cheiro’ (Brunfelsia uniflora (Pohl) D. Don), an Ornamental Species Native to Brazil. International Journal of Plant Biology, 16(2), 69. https://doi.org/10.3390/ijpb16020069

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