Sealing system in culture vessels affects the in vitro development of mother-of-millions (Kalanchoe delagoensis Ecklon and Zeyher)

Sistema de vedação em frascos de cultura afeta o desenvolvimento in vitro de mãe-de-mil (Kalanchoe delagoensis Ecklon e Zeyher)

Abstract

Sealing systems that provide greater permeability to gasses can favor plant development in vitro. This study aimed to evaluate the effectiveness of a low-cost sealing system that allows different CO₂-exchange rates [CO_2-ER] to promote the in vitro growth of mother-of-millions (Kalanchoe delagoensis). Nodal segments of K. delagoensis were surface sterilized with sodium hypochlorite (2.5% active chlorine) and inoculated in culture vessels with Murashige and Skoog (MS) medium, sealed with rigid polypropylene lids with one (code: 1M; CO_2-ER 21 μL L⁻¹s⁻¹) or two (code: 2M; CO_2-ER 25 μL L⁻¹s⁻¹) gas-permeable membranes. In the control, the culture vessels were sealed with rigid polypropylene lids without membrane (code: 0M; CO_2-ER 14 μL L⁻¹s⁻¹). After 45 days of culture, growth parameters were measured. Plants kept in culture vessels with 0M and 1M lids had a significant increase in the total plant length, main root, aerial part, and number of nodes when compared to the 2M sealing system. The number of leaves and plantlets along the leaf margin was 35.52% and 43.69% higher in plants grown in culture vessels with a 1M sealing system. In conclusion, sealing systems that allow gas exchange rates of up to 21 μL L⁻¹s⁻¹ (0M and 1M) provided the greatest in vitro development of mother-of-millions.

Keywords:
chandelier plant; culture vessel lid; gas-permeable membranes; Kalanchoe ; micropropagation

Resumo

Sistemas de vedação que proporcionam maior permeabilidade aos gases podem favorecer o desenvolvimento de plantas cultivadas in vitro. O objetivo deste estudo foi avaliar a eficiência de um sistema de vedação de baixo custo que permite diferentes taxas de trocas gasosas [CO_2-ER] para promover o crescimento in vitro de mãe-de-mil (Kalanchoe delagoensis). Segmentos nodais de K. delagoensis foram desinfetados em hipoclorito de sódio (2.5%) e inoculados em fracos de cultura contendo meio MS, vedados com tampas rígidas de polipropileno com uma (código 1M; CO_2-ER 21 μL L⁻¹ s⁻¹) ou duas (código 2M; CO_2-ER 25 μL L⁻¹ s⁻¹) membranas permeáveis a gases. No controle, os frascos de cultura foram vedados com tampas rígidas de polipropileno sem membrana (código 0M; CO_2-ER 14 μL L⁻¹ s⁻¹). Após 45 dias de cultivo, parâmetros de crescimento foram avaliados. Plantas mantidas em frascos de cultura com tampas 0M e 1M tiveram aumento significativo no comprimento total da planta, raiz principal, parte aérea e número de nós quando comparadas ao sistema de vedação 2M. O número de folhas e plântulas ao longo da margem foliar foi 35,52% e 43,69% maior nas plantas cultivadas em frascos de cultura com sistema de vedação 1M. Concluindo, sistemas de vedação que permitem taxas de troca gasosa de até 21 μL L⁻¹ s ⁻¹ (0M e 1M) proporcionaram o maior desenvolvimento in vitro de mãe-de-mil.

Palavras-chave:
flor-da-fortuna; tampa do recipiente de cultura; membranas permeáveis a gases; Kalanchoë ; micropropagação

Introduction

In recent years, Kalanchoe has become a popular ornamental plant, especially as a potted plant (Kahraman et al., 2022). This genus belongs to the Crassulaceae family and includes approximately 140 species exhibiting semi-shrub, shrub, and rarely small tree habits (Smith et al., 2019). Among them, Kalanchoe delagoensis Ecklon and Zeyher (synonym K. tubiflora or Bryophyllum tubiflorum) is a succulent species originally from Madagascar (Akulova-Barlow, 2009), popularly known as mother-of-millons, chandelier plant, or devil’s backbone (North Carolina, 2024). It is commonly used for ornamental purposes and has great pharmacological potential due to its secondary metabolites, for example, phenolic compounds and bufadienolides (Casanova et al., 2020; Katrucha et al., 2021). K. delagoensis is used in traditional medicine to treat allergies and wounds (Hsieh et al., 2013; García-Pérez et al., 2020). In addition to its anti-inflammatory and antioxidant properties, some studies have also shown that K. delagoensis has antimitotic properties, validating its great anti-cancer potential and reinforcing this species as a source of bioactive compounds of high medicinal value (Hsieh et al., 2013; García-Pérez et al., 2018).

In nature, K. delagoensis spreads through plantlets on the margin of the mother-plant leaves. However, the number of plantlets on the leaves can vary depending on environmental conditions (Akulova-Barlow, 2009). Micropropagation techniques have been proposed to propagate Kalanchoe species (Kertrung et al., 2018; Cui et al., 2019; Lozano-Milo et al., 2020) due to the recognized pivotal role of these applications in providing a large number of plantlets with high-quality standard. Moreover, these features could enhance the exploitation of phytochemical resources (Moraes et al., 2021).

The success of in vitro plant development largely relies on in vitro growth conditions created during the micropropagation process. The conventional in vitro cultivation systems are characterized by limited gas exchange, which determines high humidity, low CO₂ concentrations, and high ethylene levels in the culture vessel. Consequently, the photosynthetic capacity of in vitro shoots is also limited at high photosynthetic photon flux. They also present less ability to absorb nutrients from the medium and poor exchange of water vapor with the surroundings, which may affect the growth and differentiation of plant tissues and a lower survival rate during acclimatization (Xiao et al., 2011; Nguyen et al., 2020). In this conventional in vitro cultivation system, aluminum foil, polyvinyl chloride (PVC) film, and polypropylene or stainless-steel covers are usually used to seal the culture vessels (Saldanha et al., 2012; Oliveira Junior et al., 2022).

To minimize the negative effects caused by low gas exchange rates during the establishment of in vitro plants, sealing systems have been proposed that provide greater gas permeability. The use of gas-permeable membranes reduces the relative humidity and the concentration of ethylene inside the vessels, reduces the occurrence of hyperhydricity in plant tissues, and maintains the concentration of CO₂ in the microenvironment, favoring photosynthesis, which, in turn, promotes better vegetative growth of in vitro shoots (Saldanha et al., 2012; Batista et al., 2017). Porous membranes that promote higher gas exchange rates in in vitro culture have been commercialized, although expensive (Zobayed, 2005; Batista et al., 2017; Mamedes-Rodrigues et al., 2019). However, Saldanha et al. (2012) proposed an efficient and low-cost sealing system using polytetrafluoroethylene film and micropore, which have been demonstrated to be efficient for gas permeability, resulting in the optimization and viability of sealing systems in micropropagation. Several species grown in culture vessels with this low-cost sealing system had an increase in growth rate, and a higher survival rate when transferred to the field (Saldanha et al., 2012; Batista et al., 2017; Fortini et al., 2021; Ferreira et al., 2022; Jesus Santana et al., 2022).

In view of the ornamental and ethnopharmacological potential of K. delagoensis and the lack of information on its in vitro performance, the purpose of this study was to evaluate the influence of three types of sealing systems that allow different gas exchanges on the in vitro development of K. delagoensis. We hypothesize that the sealing system in culture vessels affects the in vitro development of mother-of-millions.

Materials and Methods

Plant material

Kalanchoe delagoensis individuals were collected in Jataí, GO (17º55’24’’ S 51º42’58’’ W) and immersed in water to prevent the plant from dehydrating. Thereafter, the leaves were removed from the stem. Under laminar flow, the stem was immersed in 70% (v v^-1) ethyl alcohol for 2 min, followed by a sodium hypochlorite commercial solution (commercial bleach, Super Global®, 2.5% active chlorine) with the addition of two drops of Tween 20® for 15 min. The explants were then washed four times in autoclaved deionized water.

Plant growth conditions

Nodal segments (2 cm long) were excised and inoculated into 250 mL glass vessels containing 35 mL of semi-solid culture medium consisting of MS salts (Murashige and Skoog, 1962), 3% sucrose (3% w v^-1), myo-inositol (0.001% w v^-1) and 0.8% agar (w v^-1) (Merck^®, Darmstadt, Germany). The pH was adjusted to 5.7 ± 0.1 before autoclaving (20 min at 121°C and 1 × 105 Pa (1.1 kg cm^-2 121)). Five nodal segments were inoculated per vessel with three types of sealing, providing different levels of gas exchange: (1) rigid polypropylene caps (RPC) without a membrane (code 0 M; CO₂ gas exchange rate [CO_2-ER] was 14 μL L^-1s^-1; (2) RPC with one orifice (10 mm) covered by a membrane (code 1M), with CO_2-ER 21 μL L^-1s^-1; (3) RPC covered by two membranes (code 2M) with CO_2-ER 25 μL L^-1s^-1 (Fig. 1). The levels of gas exchange [CO_2-ER] were determined by Batista et al. (2017). The membranes used in this study consisted of a layer of Amanco® polytetrafluoroethylene tape between two layers of Cremer® microporous tape (Saldanha et al., 2012). The plant material was maintained for 45 days in a growth room at 25 °C ± 1 with a 16-hour photoperiod and irradiance of 40 μmol m^-2s ^-1.

Fig 1
Detailed diagram of the sealing system with polypropylene caps without a membrane (0M - [CO_2-ER] 14 μL L^-1s^-1), with one membrane (1M - [CO_2-ER] 21 μL L⁻¹ s⁻¹) and two membranes (2M - [CO_2-ER] 25 μL L⁻¹ s⁻¹). The membranes were composed of polytetrafluoroethylene tape between two layers of microporous tape.

Morphometric analyses

At the end of 45 days, growth parameters such as total plant length (cm), primary root length (cm), shoot length (cm), leaf area per plant (cm²), number of leaves, number of nodes, and plantlets formed on the margin of the mother-plant leaves were measured. The plant length, shoot length, and root were measured using a ruler. For leaf area analysis, the leaves were detached from the stem and photographed. The total leaf area was estimated using ImageJ v. 1.43u software (National Institutes of Health, USA).

Statistical analysis

The experiment was performed in a completely randomized design, and each treatment (0M, 1M, and 2M) consisted of 6 replicates, with 30 plants analyzed for each treatment. For all the results analyzed, the data were subjected to analysis of variance (ANOVA), and the means were compared using Tukey’s test (p ≤ 0.05), using R- Bio® version 171.

Results and Discussion

Gas exchange between the internal and external atmosphere of the in vitro culture vessels affected the development of Kalanchoe delagoensis. After 45 days of in vitro culture, new plants were formed from axillary shoot growth of the nodal segments in all treatments (Fig. 2). However, plants grown in 2M vessels (25 μL L^-1s^-1) were less vigorous when compared to plants grown in 0M (14 μL L^-1s^-1) and 1M (21 μL L^-1s^-1) vessels (Fig. 2). The number of gas-permeable membranes used during in vitro cultivation determines the gas flow rate between the microclimate inside and outside the containers (Batista et al., 2017).

The K. delagoensis plants grown under 0M and 1M conditions showed no difference in total length, reaching averages of 9.5 and 10 cm, respectively, and were considered suitable for acclimatization (Fig. 3a). However, plants grown in the 2M condition had lower total growth. The shoot length of plants grown in 2M was 35% and 40% shorter than in plants grown in 0M, and 1M, respectively (Fig. 3b). This parameter did not differ between the 0M and 1M treatments.

The number of nodes was higher in 0M and 1M compared to 2M (Fig. 3c). It is important to note that a lower number of nodes results in lower plantlet production in the next subcultivation. As observed for the aerial plant organs, root length was around 30% shorter in 2M than in the other treatments (Fig. 3d). The treatments with the lowest gas permeability (0M and 1M) provided the greatest growth in the shoot and the root, which was reflected in the total length of the plants grown under these conditions (Fig.3a, b, d).

Fig. 2
In vitro development of Kalanchoe delagoensisunder different sealing systems. (a) Culture vessels sealed with polypropylene caps without a membrane (code 0M - [CO_2-ER] of 14 μL L^-1 s^-1), with one membrane (code 1M - [CO_2-ER] of 21 μL L⁻¹ s⁻¹) and two membranes (code 2M - [CO_2-ER] 25 μL L⁻¹ s⁻¹). (b) Plants were obtained at 0M, 1M, and 2M sealing conditions after 45 days. Bars: a = 3 cm; b = 2.5 cm.

Fig. 3
Growth parameters of Kalanchoe delagoensis after 45 days in culture vessels sealed with polypropylene caps without a membrane (code 0M - [CO_2-ER] of 14 μL L^-1 s^-1), with one membrane (code 1M - [CO_2-ER] of 21 μL L ⁻¹ s ⁻¹) and two membranes (code 2M - [CO_2-ER] 25 μL L⁻¹ s⁻¹). (a) Total length (shoot and root). (b) Shoot length (cm). (c) Number of nodes. (d) Root length. Means followed by the same letter do not differ by Tukey test (P ≥ 0.05). (n = 6). The error bars describe the standard deviation.

It is important to consider that each species has a CO_2-ER optimum and this is related to its metabolic, physiological and genetic performance. In this study, we observed that K. delagoensis plants exhibited more vigor at CO_2-ER of up to 21 μL L^-1s^-1. In contrast, Vernonia condensata (Fortini et al., 2021) and Pfaffia glomerata (Saldanha et al., 2012; Batista et al., 2017) plants demonstrated greater vigor and biomass increase under two-membrane cultivation conditions (CO_2-ER of 25 μL L^-1s^-1). On the other hand, plantlets of Mentha species showed greater vigor and an increase in height, dry weight, and leaf area in sealing systems with four membranes (Oliveira et al., 2021). The differences in the species responses to the sealing system of culture vessels reinforce the importance of in vitro cultivation in different systems to obtain information and make it possible to modulate plant growth and development (Saldanha et al., 2012; Batista et al., 2017; Fortini et al., 2021; Oliveira et al., 2021).

There are also species, such as potatoes (Solanum tuberosum), in which growing conditions without membranes provide greater shoot length, fresh mass, and number of nodes (Mohamed and Alsadon, 2010). Increased gas flux in the in vitro environment results in reduced relative humidity and plant growth in these conditions has often been linked to the prevention of hyperhydricity and easier absorption of water and nutrients (Xiao et al., 2011). However, excessive water loss in these crops with greater gas exchange can change the nutritional characteristics, water, and osmotic potential of the medium, thus affecting plant development and growth (Gonçalves et al., 2008), as observed here. K. delagoensis is a succulent plant with Crassulacean Acid Metabolism (CAM), whose adaptation to arid climates allows it to survive but limits the speed of its growth. Environments with greater water availability can favor the growth of these species. In species with CAM metabolism, traditional in vitro cultivation, where gas exchange is low and relative humidity is high, promoted greater growth and photosynthetic activity than those cultivated in an ex-vitro environment (Malda et al., 1999).

In the present study, the number of leaves was higher in the 1M sealing system, in comparison to plants grown in 2M condition (Fig. 4a). No difference was observed between 0M and 1M treatments. The leaf area showed no significant difference among the treatments, although the 2M sealing system showed a smaller average (Fig. 4b). Increasing the number of leaves can improve light interception and, consequently, the capture of light energy to be used in the photosynthetic process. In parallel with the functions performed by leaves in primary metabolism, they are also responsible for the synthesis and storage of secondary compounds (Oliveira et al., 2021, García-Pérez et al., 2020). For the use of bioactives from K. delagoensis, it is interesting to use cultivation protocols in which the leaves are well formed but also numerous since one of the limiting factors for the extraction of chemical compounds in medicinal plants is the scarcity of biomass (Moraes et al., 2021).

Fig. 4
Growth parameters of Kalanchoe delagoensis after 45 days in culture vessels sealed with polypropylene caps without a membrane (code 0M - [CO_2-ER] of 14 μL L^-1 s^-1), with one membrane (code 1M - [CO_2-ER] of 21 μL L ⁻¹ s ⁻¹) and two membranes (code 2M - [CO_2-ER] 25 μL L⁻¹ s⁻¹). (a) Number of leaves. (b) Leaf area (cm²). (c) Number of plantlets formed on the leaf margin. Means followed by the same letter do not differ by Tukey’s test at 5% probability; values represent means (n = 6). The error bars describe the standard deviation.

The number of plantlets formed on the leaf margin, which will give rise to another plant, was significantly higher in the 1M sealing system (Fig. 4c), which may be directly associated with the more significant number of leaves observed in this sealing system (Fig. 4a). The more significant number of plantlets formed on the leaf margin may result in higher rates of plant multiplication during subcultivation, exponentially increasing the efficiency of the micropropagation system presented here.

In general, the sealing systems 0M and 1M provided K. delagoensis plants with more significant growth variables than the 2M sealing system. But, considering the leaf parameters analyzed here, the 1M condition may be even more advantageous since plants grown under conventional in vitro cultivation conditions (0M) generally present poorly differentiated and/or dysfunctional tissues, which limits their metabolic and physiological performance (Xiao et al., 2011; Nguyen et al., 2020, Ferreira et al., 2022). In species such as Guazuma ulmifolia and Jacaranda cuspidifolia, the cultivation in vessels with a porous membrane, similar to our 1M treatment, was sufficient to promote increased growth and leaf histodifferentiation (Ferreira et al., 2022; Jesus Santana et al., 2022). In this sense, further studies will be necessary to confirm whether the 1M sealing system promoted changes in the leaf histology of K. delagoensis in comparison to the conventional in vitro culture system (0M) to evaluate this hypothesis.

Conclusions

Sealing systems with gas exchange rates of up to 21 μL L^-1 s^-1 (0M and 1M) provided the greatest growth of K. delagoensis, under in vitro conditions. In addition, gas exchange rates higher than 25 μL L^-1 s^-1 (2M) impaired the growth of this species. We also concluded that the 1M sealing system seems more advantageous for micropropagation of K. delagoensis, since it provides more leaves and plantlets. The information obtained in this study will contribute to constructing efficient and viable micropropagation strategies and systems to obtain more vigorous plants with higher concentrations of bioactive compounds.

Acknowledgments

The authors thank the Fundação de Amparo à Pesquisa do Estado de Minas Gerais (Belo Horizonte, Brazil; Grant No. APQ-02581-21; RED-00225-23); Conselho Nacional de Desenvolvimento Científico e Tecnológico (Brasília, Brazil; Grant No. 311282/2021-0), the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (Brasília, Brazil; Grant No. 001), and the Rede Mineira de Biotecnologia em Multiplicação e Clonagem de Plantas.

Data availability statement

Data will be available upon request.

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Edited by

• Editor:
  Chao Yu (Beijing Forestry University - China)

Publication Dates

• Publication in this collection
  07 Oct 2024
• Date of issue
  2024

History

• Received
  04 Mar 2024
• Accepted
  13 Aug 2024
• Published
  30 Aug 2024