Volatile profile and micropropagation conditions of Bauhinia forficata Link

Abstract

Bauhinia forficata Link. is a native South American plant, which possesses volatile compounds with pharmaceutical and medicinal properties such as antidiabetic and anti-inflammatory effects. However, the conservation and propagation of this plant are complicated by its recalcitrant seeds and delayed flowering transition. Hence, tissue culture is employed for the safe and efficient propagation of B. forficata. However, the optimal conditions for the in vitro cultivation of B. forficata remain unknown. Thus, this study aimed to characterize the volatile profile of adult B. forficata field plants and evaluate the effects of different light intensities (43 and 70 μmol m⁻² s⁻¹), gas exchange rates (14 and 25 µL L⁻¹ s⁻¹), and exogenous sucrose concentrations (0, 20, and 30 g L⁻¹) on their in vitro development. The results showed that β-caryophyllene is the major volatile compound produced by B. forficata. Moreover, culturing in a medium containing 30 g L⁻¹ of sucrose and flasks with membranes that allow CO₂ exchange at the rate of 25 µL L⁻¹ s⁻¹ produced vigorous and hardened plants with high survival rates independent of irradiance. This study is the first to report the optimal in vitro culture conditions for B. forficata as a reference for future studies on micropropagation and secondary metabolite production using this species.

Supplementary Information

The online version contains supplementary material available at 10.1007/s13205-023-03634-8.

Introduction

The conservation and propagation of native forest species is challenging (Frick et al. 2020) because of their recalcitrant seeds (Bareke 2018) and delayed flowering transition (Hanke et al. 2007; Wang et al. 2011). Bauhinia forficata Link is a native South American species possessing volatile compounds with pharmaceutical and medicinal properties, such as antidiabetic and anti-inflammatory effects (López and Santos 2015). B. forficata has been used in folk medicine to control cystitis and intestinal parasites (Zaccaron et al. 2014). However, the natural B. forficate population has drastically reduced because of rampant extractivism aggravated by irregular germination due to integumentary dormancy and slow vegetative development under natural conditions.

To overcome these impairments, previous studies have used micropropagation as a tool for year-round clonal propagation of healthy and disease-free plants. Micropropagation is reliable and promising in stimulating productivity and sustainability in cultivations (Kaur et al. 2022; Vidal and Sánchez 2019). The in vitro cultivation of plants is vital for maximizing plant machinery and producing specific secondary metabolites with medicinal importance (Dias et al. 2016; Khan et al. 2021). However, the synthesis and accumulation of secondary metabolites are affected by several factors such as internal developmental genetics and external environmental factors including light, temperature, water, and salinity (Li et al. 2020). In vitro cultures can be manipulated to respond to specific biotic and abiotic stresses to regulate the production of secondary metabolites such as phytochemicals (Khan et al. 2021). Thus, the in vitro cultivation of plants with medicinal properties may provide an opportunity for homogeneous production of secondary metabolites in the absence of environmental stresses (Li et al. 2020).

The hermetically closed flasks that are used in conventional in vitro cultivation produce plants with mixotrophic type metabolism. Notably, these flasks restrict gas exchange with the external environment, causing the accumulation of ethylene and other gases, which leads to low CO₂ concentrations, high exogenous carbohydrate contents (e.g., sucrose) in the culture medium, and low levels of photosynthetically active radiance (PAR). These phenomena directly affect the photosynthetic and biochemical processes in plants (i.e., low photosynthetic potential and transpiration rate) and harm their development in vitro, which consequently reduces their propagation and survival during ex vitro acclimatization (Badr et al. 2011; Batista et al. 2018; Gris et al. 2021; Kozai 2010; Nguyen et al. 2016; Rodrigues et al. 2022; Xiao et al. 2011). Sucrose is often incorporated into the culture medium as the energy source for the development of explants (Kozai 2010). This may cause a partial loss of autotrophy, necessitating supplementation with an external carbon source (Fortini et al. 2021). However, in conventional in vitro conditions this may lead to reduced photosynthetic potential (Nery et al. 2021). Alternative approaches such as increasing ambient CO₂ with gas-permeable membranes and reducing or eliminating sucrose content in the culture medium can mitigate these effects and improve growth performance and photosynthetic competence (Pinheiro et al. 2021). These improvements in in vitro culture protocols reduce direct costs, increase productivity, and ensure practical applications in both research and industrial fronts (Fritsche et al. 2022). Flasks with gas-porous membranes can increase the photosynthetic performance and thus enhance the growth rates of plants by aiding gas exchange (natural ventilation or forced air) between the in vitro and ex vitro environments (Batista et al. 2018; Kozai 2010).

Light is another key factor influencing plant photosynthesis; thus, sufficient light intensity in the growing room is essential to improve biomass production (Kaur et al. 2022). Light-emitting diodes (LEDs) economically and effectively improve light quality (Batista et al. 2018) and intensity (i.e., PARs) during in vitro cultivation, which improves plant growth and development (Hassanpour 2022; Lanoue et al. 2017) and stimulates phytochemical production in medicinal plants (Hassanpour 2022).

At present, the optimal conditions for in vitro cultivation of B. forficata remain unknown. Thus, this study aimed to characterize the volatile profile of adult B. forficata plants and evaluate the effects of light intensity, natural ventilation in flasks with gas-porous membranes, and exogenous sucrose concentrations on in vitro development of B. forficata. To the best of our knowledge this is the first study to report the optimal in vitro culture conditions for B. forficata. We believe that these findings would provide a basis for future studies on micropropagation and secondary metabolite production using this species.

Materials and methods

Plant material

For volatile profiling under field conditions, B. forficata Link leaves and seeds were collected from the natural plant populations in the municipality of Estreito, state of Maranhão, Brazil (geographic coordinates; 6°35ʹ00ʺS and 47°26ʹ40ʺW) at 8 a.m. during the rainy season (March) of 2020. The seeds were refrigerated at 4 °C until in vitro experiments were performed. The plants were collected in compliance with Brazilian biodiversity protection laws (SISGEN n. AD7DF67).

Extraction and composition analysis of volatiles in field plants

The volatiles from the plants were extracted and analyzed to characterize the accession of B. forficata used in this study. For this experiment, fresh leaves (50 g) were added to a 1000-mL round-bottom flask and subjected to extraction through hydrodistillation for 3 h using a Clevenger apparatus (Brazil 2019), followed by drying over anhydrous sodium sulfate (Merck-Millipore, São Paulo, Brazil). The yield was calculated based on the plant dry-weight. Plant water-loss was calculated using an infrared moisture balance (Genaka, São Paulo, Brazil). The moisture content estimation procedure was performed in duplicates (Monteiro et al. 2020).

The oil extract was analyzed on a gas chromatograph (CG-2010) coupled to a QP 5000 mass spectrometer (CG-EM QP2010 Plus, Shimadzu Corp., Japan) using a DB-5 ms (30 m × 0.25 mm; 0.25 μm film thickness) silica capillary column (J.W. Scientific, USA). The analysis conditions were as follows: injector temperature of 250 °C; oven temperature programming of 35–240 °C (10 °C min⁻¹); helium as carrier gas, adjusted to a linear velocity of 30 cm s⁻¹ (1.0 mL min⁻¹); split mode injection for 1 μL of sample (1 μL oil:500 μL hexane); split ratio of 1:30; ionization using electronic impact at 70 eV; ionization source and transfer line temperatures of 200 and 250 °C, respectively; and running time of 36.5 min. The compounds were identified using the NIST 08 Mass Spectral Search Program (National Institute of Standards and Technology, Gaithersburg, MD, USA) and confirmed, if possible, by comparing the retention times and mass spectra with available commercial standards.

In vitro germination

B. forficata seeds were disinfected in an air flow chamber by immersion in 70% ethyl alcohol (v/v) for 3 min and commercial sodium hypochlorite solution (NaOCl; 4% (w/v) active chlorine) for 15 min. The surfactant polysorbate 20 (Tween-20^® 0.03%) (v/v) was added, and the seeds were rinsed four times with distilled and autoclaved water.

The seeds were then placed in glass flasks (350 mL) containing 50 mL of MS culture medium (Murashige and Skoog 1962) supplemented with MS vitamins, 100 mg L⁻¹ myo-inositol, 20 g L⁻¹ sucrose (Sigma-Aldrich^® Co, St. Louis, MO), and 2 g L⁻¹ Phytagel^® (Sigma-Aldrich, MO, USA). The pH was adjusted to 5.7 ± 0.1 prior to autoclaving at 120 °C and 1.1 atm for 15 min. The seeds were maintained in a growth room under the following conditions for 30 days until germination: temperature, 25 ± 1 °C; photoperiod, 16 h of light; and irradiance, 43 μmol m⁻² s⁻¹ (provided by two white LED lamps).

Effects of light intensity, CO₂ exchange rate, and sucrose concentrations on the growth of B. forficata plants

Nodal segments (1 cm long) were obtained from 30-day-old in vitro seedlings. Three segments were transferred to each 350 mL glass flask containing 50 mL of MS medium supplemented with 0.54 μM naphthaleneacetic acid and maintained under the same culture conditions described for germination but with different experimental settings for sucrose concentrations (0, 20, and 30 g L⁻¹), CO₂ exchange rates (14 and 25 µL L⁻¹ s⁻¹) (Batista et al. 2017; Saldanha et al. 2012), and light irradiance levels (43 and 70 μmol m⁻² s⁻¹ simulated using two and four LED lamps, respectively). Different CO₂ exchange rates were maintained using flasks covered with polypropylene lids without membranes (14 µL L⁻¹ s⁻¹) and flasks covered with polypropylene lids with 10 mm orifices covered with 0.05 ± 0.01 mm-thick-porous membranes composed of three layers of microporous tape and one layer of polytetrafluoroethylene (25 µL L⁻¹ s⁻¹).

Experimental design, growth analysis, and statistical analyses

A completely randomized experimental design was followed in a 2 × 2 × 3 factorial scheme (light intensity × gas exchange rate × sucrose concentration) for a total of 12 treatments with five replicates, each of which comprised one flask with five seedlings.

The shoot and root fresh and dry weights (mg), plant length (mm), leaf width and length (mm), and number of leaves (NF) and shoots were determined after 45 days of culture. For dry weight quantifications, the plant materials were oven dried at 70 °C until their weights became constant.

The normality and homogeneity of the data were assessed using Shapiro–Wilk and Bartlett tests, respectively. Statistically significant differences were determined using analysis of variance (ANOVA; F test), and means were compared using Tukey’s test (P ≤ 0.05) with SISVAR software (Ferreira 2011).

Results

Extraction and composition analysis of volatiles in field plants

The extracted essential oil was composed of 12 sesquiterpenes (Online Resource 1, Supplementary Fig. 1), the most abundant of which were β-caryophyllene (30.31%), (Z)-α-bisabolene (10.10%), humulene (9.72%), α-cadinol (8.78%), and caryophyllene oxide (8.04%) (Table 1).

Table 1.

Chemical composition of volatiles extracted from the leaves of Bauhinia forficata Link

Peak	Retention time	Compound	Proportion (%)
1	19.486	β-Caryophyllene	30.31
2	19.953	Humulene	9.72
3	20.011	Aromadendrene	1.68
4	20.270	β-Cubenene	6.04
5	20.450	Ylangene	6.41
6	20.514	β-Bisabolene	4.53
7	20.684	α-Copaene	6.13
8	20.907	(Z)-α-Bisabolene	10.10
9	21.461	Germacrene D-4-ol	4.46
10	21.567	Caryophyllene oxide	8.04
11	22.392	α-Cadinol	8.78
12	22.667	α-Bisabolol	3.80
Total			100.00

Bold value (100) represents the sum of the percentages, which is self-explanatory

Light intensity, CO₂ exchange rate, and sucrose concentrations impact the growth of B. forficata plants

Light intensity, gas exchange rate, and sucrose concentration affected all analyzed variables (Online Resource 1, Supplementary Table 1). The plant length (Fig. 1a–c), shoot fresh weight (Fig. 1d–f), and root fresh weight (Fig. 1g–i) of the cultured plants increased when the growth conditions were 20 or 30 g L⁻¹ sucrose concentration, 70 μmol m⁻² s⁻¹ light intensity, and natural ventilation with 25 µL L⁻¹ s⁻¹ CO₂ exchange rate (25 µL L⁻¹ s⁻¹).

Fig. 1.

a–c Plant length (cm), d–f shoot fresh weight (g), and g–i root fresh weight (g) of Bauhinia forficata plants after 45 days of in vitro cultivation under different light intensities (43 and 70 µmol m⁻² s⁻¹), CO₂ exchange rates (14 and 25 µL L⁻¹ s⁻¹), and sucrose concentrations (0, 20, and 30 g L⁻¹). Means followed by the same letter do not differ by Tukey’s test (P ≤ 0.05)

Compared with the plants grown under the other conditions, the plants grown under 25 µL L⁻¹ s⁻¹ CO₂ exchange rate and 70 µmol m⁻² s⁻¹ light intensity produced a greater number of leaves (Fig. 2a) that were larger (Fig. 2b) and wider (Fig. 2d). The leaf length and width increased under 70 µmol m⁻² s⁻¹ light intensity and 30 g L⁻¹ sucrose (Figs. 2c and e, 3c, and i).

Fig. 2.

a Number of leaves; b–c leaf width (mm); d–e leaf length (mm) of Bauhinia forficata plants after 45 days of in vitro cultivation under different light intensities (43 and 70 µmol m⁻² s⁻¹), CO₂ exchange rates (14 and 25 µL L⁻¹ s⁻¹), sucrose concentrations (0, 20, and 30 g L⁻¹). *a, b, d Means followed by the same letter do not differ based on Tukey’s test (P ≤ 0.05). c, e Means followed by the same capital letters do not differ in light intensity within the same sucrose concentration. Lowercase letters do not differ in sucrose concentration within the same light intensity

Fig. 3.

Bauhinia forficata plants after 45 days of in vitro cultivation under different light intensities (43 and 70 µmol m⁻² s⁻¹ of irradiance), CO₂ exchange rates (14 and 25 µL L⁻¹ s⁻¹), and sucrose concentrations (0, 20, and 30 g L⁻¹). Bars: 1 cm

Meanwhile, 20 g L⁻¹ of sucrose reduced plant development (Fig. 3b, e, h, and k), whereas 0 g L⁻¹ of sucrose inhibited plant development (Fig. 3a, d, g, and j). Shoot biomass reduced drastically when cultured in sealed bottles with a low CO₂ exchange rate of 14 µL L⁻¹ s⁻¹ (Fig. 3f and l).

The shoot dry weight increased at 70 µmol m⁻² s⁻¹ light intensity, 25 µL L⁻¹ s⁻¹ CO₂ exchange rate (Fig. 4a), and 30 g L⁻¹ sucrose concentration in the culture medium (Fig. 4b). Additionally, the root dry weight increased at 25 µL L⁻¹ s⁻¹ CO₂ exchange rate (Fig. 4c) and < 70 µmol m⁻² s⁻¹ light intensity, and 30 g L⁻¹ sucrose concentration (Fig. 4d).

Fig. 4.

Bauhinia forficata plants after 45 days of in vitro cultivation under different light intensities (43 and 70 µmol m⁻² s⁻¹), CO₂ exchange rates (14 and 25 µL L⁻¹ s⁻¹), and sucrose concentrations (0, 20, and 30 g L⁻¹). a, b Shoot dry weight (g) and c–d root dry weight (g). *a Means followed by the same capital letters do not differ in light intensities within the same CO₂ exchange rate. Means followed by the same lowercase letters do not differ in CO₂ exchange rate within the same light intensities. b, d Means followed by the same capital letters do not differ in light intensity within the same sucrose concentration. Means followed by the same lowercase letters do not differ in sucrose concentration within the same light intensity. c Means followed by the same letter do not differ based on Tukey’s test (P ≤ 0.05)

Discussion

The abundant concentration of sesquiterpenoids in the essential oil obtained from the B. forficata leaves validated previous reports regarding the chemical composition of the essential oils in the leaves of other Bauhinia species, including B. acuruana (Gois et al. 2011), B. ungulata (Gramosa et al. 2009; Sousa et al. 2016), B. forficata (Sartorelli and Correa 2007), B. dumosa (Silva et al. 2020), B. petandra (Almeida et al. 2015), B. aculeata, B. brevipes, B. longifolia, B. rufa, and B. variegata (Duarte-Almeida et al. 2004).

In the present study, light intensity in association with natural ventilation and sucrose concentration substantially benefited in vitro growth of B. forficata plants. The plants subjected to photomixotrophic micropropagation conditions of 70 µmol m⁻² s⁻¹ light intensity, 25 µL L⁻¹ s⁻¹ CO₂ exchange rate, and 30 g L⁻¹ sucrose concentration in the culture medium showed more vigorous growth than did the plants subjected to the other conditions (Figs. 2, 4i).

Several studies have demonstrated the benefits of photomixotrophy and natural ventilation in the in vitro morphogenesis, growth, and development of different species, such as Epidendrun fulgens (Fritsche et al. 2022), Guazuma ulmifolia (de Jesus Santana et al. 2022), Capsicum frutescens (Gris et al. 2021), C. annuum (Batista et al. 2017), Etlingera elatior (Pinheiro et al. 2021), and Ananas comosus (Alves et al. 2022). Gas-porous membranes reduce the relative humidity and ethylene gas accumulation inside the culture flasks, thereby eliminating or drastically reducing morphophysiological disorders (Pinheiro et al. 2022). In the present study, the B. forficata plants cultivated in flasks without natural ventilation showed poor development (Fig. 3).

Additionally, the plant length and shoot and root fresh weights (Fig. 1) of B. forficata plants increased at 70 μmol m⁻² s⁻¹ light intensity and natural ventilation with 25 µL L⁻¹ s⁻¹ CO₂ exchange rate, showing that these parameters benefited the cultivation of these plants. Therefore, artificial light, along with other factors such as culture medium composition, gas exchange in culture flasks, temperature, and specific physiological outcomes, plays a crucial role in in vitro plant production (Cavallaro et al. 2022). In fact, light is essential for plant growth and development because it regulates photosynthesis, morphogenesis, flowering, metabolism, gene expression, and other physiological processes (Batista et al. 2018; Cioć et al. 2019; Kepenek 2019).

Determining the adequate spectral quality and luminous intensity necessary for plant growth is crucial for improving in vitro plant production (Souza et al. 2022). Cavallaro et al. (2022) reported that the commonly used light intensity commonly for in vitro cultivation varies between 20 and 80 µmol m⁻² s⁻¹ and that each plant requires a specific photosynthetic photon flux density. High light intensities in in vitro cultivation can stimulate plants to form leaves with greater leaf area, causing plants to seek a more natural state of autotrophic functioning (Cioć et al. 2019). This result agrees with our findings presented in Fig. 3i. Low light intensities satisfy the light conditions required for phytochrome and cryptochrome reactions but not for photosynthesis (Zheng and Van Labeke 2018), which may reduce plant growth (Silva et al. 2017). In addition, plants grown under low light intensities are often more susceptible to photoinhibition than those grown under high light intensities (Yao et al. 2017).

Pinheiro et al. (2021) observed that E. elatior shows better growth under photomixotrophic conditions of natural ventilation through gas exchange and sucrose addition to the culture medium. Similar results were observed in B. forficate plants in the present study. Caetano et al. (2021) found that natural ventilation promotes morphophysiological adjustment in Acca sellowiana, thereby promoting the transition from in vitro to ex vitro cultivation.

Different in vitro cultivation systems have been used to increase the photosynthetic capacity and hardening of plants (Caetano et al. 2021). Natural ventilation through gas exchange benefits plants even in in vitro cultivation. This phenomenon can be ascribed to the fact that the low humidity levels in the flasks with natural ventilation stimulate transpiration, allowing the plants to absorb more nutrients from the culture medium (Fritsche et al. 2022). In the present study, similar results were observed in the plants grown in flasks with natural ventilation provided by gas-permeable membranes.

Therefore, this study may serve as a basis for further studies on B. forficata micropropagation under different light intensities and the association between the morphogenesis and in vitro secondary metabolite production in plants with medicinal properties (Rocha et al. 2022).

Conclusion

β-caryophyllene is the major component of the essential oil produced by B. forficata plants that grow in Northeastern Brazil. The optimal conditions for in vitro cultivation of B. forficata were 30 g L⁻¹ sucrose concentration in the culture medium and flasks with a membrane allowing natural ventilation and CO₂ exchange at the rate of 25 µL L⁻¹ s⁻¹ rate independent of light intensity. To the best of our knowledge, this study is the first to report the optimal conditions for in vitro cultivation of B. forficata, providing a basis for further studies on micropropagation and secondary metabolite production using this species.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

MVMP, MLAS, KVS, JPA, TRSM, GLA, TRC, and DSB: performed the experiments; MVMP, KVS, JPA, TRSM, GLA, TRC, and MLAS: raised the in vitro plants for the experiments; MLAS, FEACJ, and OSM: performed the extraction and analyses of essential oils; MVMP, FEACJ, TRC, OSM, and DSB: analyzed the data; MVMP, TRC, FAMMAF, and DSB: contributed to the design and interpretation of the research and to the writing of the paper. All authors read and approved the final version of the manuscript.

Funding

This study was financed in part by the Fundação de Amparo à Pesquisa e ao Desenvolvimento Científico e Tecnológico do Maranhão—FAPEMA—Brazil and by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, Brasília, DF, Brazil: Grant no. PQ 304214/2022-1 to DSB).

Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Declarations

Conflict of interest

The authors have no competing interests to declare that are relevant to the content of this article.

Research involving human participants and/or animals

Not applicable.

Informed consent

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