Initial in vitro plant establishment of seeds and nodal segments from bromeliad Acanthostachys strobilacea (Schult. & Schult.f.) Klotzsch differs in respiratory rates and shoot formation

Estabelecimento in vitro inicial de plantas de sementes e segmentos nodais da bromélia Acanthostachys strobilacea (Schult. & Schult.f.

ABSTRACT

We aimed to investigate the morphological and respiratory differences during in vitro shoot formation from seeds and nodal segments (NS) of Acanthostachys strobilacea (Schult. & Schult.f.) Klotzsch, due to differences in plants obtained by micropropagation. During 35 days of culture, seeds resulted in full plants 14 days earlier than NS, with longer leaves and more roots. Nevertheless, NS plantlets exhibited shoot multiplication. Peaks in O2 consumption and CO2 release were detected at 7 and 14 days for NS and seeds, respectively, suggesting that initial growth has a high energetic requirement. However, the respiration peak was higher in NS than in seeds, possibly due to high energy consumption required for multiple bud breaks. After peaking, respiration decreased, reaching similar values between propagules by 35 days, indicative of an ongoing increase in photosynthesis in both seed and NS plants, possibly due to shoot growth. In conclusion, the development process of NS plants may affect the energy and respiratory demand differently than in seedlings

Keywords: carbon dioxide; lateral buds; micropropagation; oxygen; respiration

RESUMO

Klotzsch difere em taxas respiratórias e formação de brotos). Buscou-se investigar as diferenças morfológicas e respiratórias durante a formação in vitro de brotos a partir de sementes e segmentos nodais (NS) de Acanthostachys strobilacea (Schult. & Schult.f.) Klotzsch, devido às diferenças entre as plantas obtidas pela micropropagação. Durante 35 dias de cultura, as sementes resultaram em plantas completas 14 dias antes do NS, com folhas mais longas e mais raízes. No entanto, as plantas NS exibiram multiplicação de brotos. Picos no consumo de O2 e liberação de CO2 foram detectados aos 7 e 14 dias para NS e sementes, respectivamente, sugerindo que o crescimento inicial tem alta demanda energética. No entanto, o pico de respiração foi maior em NS do que em sementes, possivelmente devido ao alto consumo de energia necessário para as múltiplas quebras de gemas. Após o pico, a respiração diminuiu, atingindo valores semelhantes entre os propágulos aos 35 dias, indicativo de um aumento contínuo da fotossíntese em plantas de sementes e NS, possivelmente devido ao crescimento de brotos. Em conclusão, o processo de desenvolvimento das plantas de NS pode afetar a demanda energética e respiratória de forma diferente daquelas de sementes

Palavras-chave: dióxido de carbono; gemas laterais; micropropagação; oxigênio; respiração

Introduction

An efficient conservation method for endangered plant species is the development of in vitro germplasm banks, since they allow the storage of a great number of plants in small spaces, without excessive maintenance as required for plants in the field or greenhouses (Imarhiagbe et al. 2016, Oseni et al. 2018). This technique can be applied to seeds or tissue explants, such as nodal segments (NS, Pilatti et al. 2011). The ornamental bromeliad Acanthostachys strobilacea (Schult. & Schult.f.) Klotzsch (Bromeliaceae) can be propagated in vitro by seeds and NS isolated from the stems of elongated plants that are subcultured to obtain new plants (Santos et al. 2010).

Micropropagation has been used in the cultivation of diverse bromeliads species to meet the ornamental plants market demand, which aids in preventing illegal extraction (Mercier & Nievola 2003, Negrelle et al. 2012). Due to the possibility of maintaining cultures in controlled conditions (light, temperature, nutrients, etc.), this technique has been used for studies of basic physiology in diverse species (Narayani & Srivastava 2017, Bakhshipour et al. 2019, Chetty et al. 2020), including bromeliads (Carvalho et al. 2013, Freitas et al. 2015, Andrade & Tamaki 2016, Santos et al. 2017, Silva et al. 2017, Andrade-Santos et al. 2020, Silva et al. 2020).

The cultivation of plants under sealed flasks could lead to restriction of gas exchange between the internal and external environment, causing an accumulation of gases such as CO₂ and O₂ inside the flasks (George et al. 2008). Thus, the gaseous composition of the isolated in vitro atmosphere reflects the respiratory and photosynthetic activities of micropropagated plants (Chen 2006). When plants are cultivated under photomixotrophic conditions, defined by the use of high sugar concentration in the culture medium, low irradiance, high relative humidity and reduced gas exchange due to enclosed vessels, the rate of photosynthesis is diminished while respiration is less affected due to the carbon supply (Lucchesini et al. 2001, George et al. 2008, Ševčíková et al. 2019).

The consumption of gases and respiratory activity of seeds and NS would inevitably differ since shoot development from each propagule undergo very distinct processes: germination involves the imbibition of the seed, followed by increased respiration and metabolic activity that result in root emergence from the seed testa (Taiz et al. 2017).

NS lead to shoot formation from an axillary bud meristem that is stimulated after apical dominance is broken due to the node isolation from the mother plant (George et al. 2008). However, no studies about respiration during in vitro plantlet development from lateral buds and how it compares to seed germination were found.

Considering the developmental differences between seeds and NS, we hypothesize that these propagules of A. strobilacea have distinct rates of respiration during plant formation. Hence, to evaluate our hypothesis, we investigated the morphological development during initial in vitro shoot formation from seeds and NS of A. strobilacea and the progression of respiratory rates during 35 days by assessing CO₂ and O₂ levels inside culture vessels. By evaluating the progression of respiratory rates during in vitro plant formation, it would also be possible to detect differences in photosynthetic activity between propagules. The obtained results may indicate the metabolic state of plants from seeds and NS and provide information on the period that plants initiate photosynthetic activity and thus, have higher capability to endure acclimatization (Ševčíková et al. 2019). Therefore, this study may aid in the improvement of the maintenance of germplasm collections and plant production of A. strobilacea, while also presenting the original method of a simultaneous evaluation of the time course of the initial development of these kinds of explants during micropropagation.

Materials and methods

Seeds of Acanthostachys strobilacea (Schult. & Schult.f.) Klotzsch were harvested at the Reserva Biológica de Mogi Guaçu in São Paulo State, Brazil, and stored under 8°C for approximately 12 months prior to the experiments. NS were obtained as described by Santos et al. (2010). The seeds were surface sterilized in 100% (v/v) commercial sodium hypochlorite (2% of active chlorine) containing 0.1% (v/v) Tween 20^® for 20 min and then transferred to 25% (v/v) of hydrochloric acid for 10 min to remove a large part of the mucilage. Seeds were immersed in 70% (v/v) ethanol for 5 min, in the fungicide Benomyl 0.1% (w/v) for an additional 15 min, and in 100% (v/v) commercial sodium hypochlorite containing 0.1% (v/v) Tween 20^® for 1 h (Santos et al. 2010). The seeds were transferred aseptically to 250 mL flasks containing 40 mL of Murashige & Skoog (1962) medium (MS) with 1/5 of the original macronutrient concentration (MS/5), 100% of MS micronutrients, sucrose 2% (w/v), 100 mg L^-1 of myo-inositol, and 0.1 mg L^-1 of thiamine. The pH was adjusted to 5.8, and agar (5 g L¹) was added before autoclaving for 15 min at 121°C. The seeds were kept in a growth room at 25±2°C, 12-h photoperiod and 14 μmol m^-2 s^-1 to induce elongation of the stem axis of the plants (Santos et al. 2010). After three months, NS were isolated from the elongated plants (figure 1a) and transferred aseptically to flasks containing 20 mL of the same culture media described previously, closed with caps containing a 2-mm hole covered by a rubber septum for the harvesting of air samples (Lamarca & Barbedo 2012). The same was performed for a batch of surface-sterilized seeds. Flasks were kept in a growth room under 12-h photoperiod, 25±2 °C and 30 μmol m^-2 s^-1 irradiance. Plant growth evaluation and air samples were analysed during the light period from five flasks containing five propagules each at 0, 7, 14, 21 and 35 days.

The percentage of oxygen (O₂) and carbon dioxide (CO₂) in the flasks was determined by a gas analyser (model ILL6600, Illinois Instruments, Inc., Johnsburg, IL, USA) as described (Lamarca & Barbedo 2012). These analyses allow an indirect assessment of respiratory activity by the calculation of O₂ consumption and CO₂ release rates. The percentage values of O₂ and CO₂ were converted to partial pressure of gas through the equation: p1 P^-1 = v1 V^-1, where: p1: partial pressure of gas (atm), P: local atmospheric pressure (= 0.9 atm), v1: gas volume (%) and V: total volume (= 100%) (Atkins & De Paula 2001). These values were then converted to μmoles of O₂ and CO₂ through the equation: p1 V = n RT, where: V: total volume of air of flasks (L), n: number of moles of gas, R: gas universal constant (0.082 atm L mol^-1 K^-1), T: temperature (in Kelvin) (Atkins & De Paula 2001).

The following growth parameters were evaluated in 20 plants separated at random from the pool of 25 propagules, per period: number of leaves and roots; longest leaf and root length. Only roots over 0.5 cm and expanded leaves were measured. The selection was performed due to the presence of NS that did not sprout or seeds that did not germinate during the experiment.

Figure 1
a. General aspect of the micropropagation procedure of Acanthostachys strobilacea (Schult. & Schult.f.) Klotzsch, indicating the seed, elongated plants, defoliated stem, and isolated nodal segments (from left to right). Visual aspect of nodal segment plants with multiple shoots at b. 15 days and c. 35 days. Seed plants at d. 15 days and e. 35 days. Bar in figure e applies to figures b-d.

For the evaluation of number of shoots per propagule, a separate batch of NS and seeds were cultivated in 250 mL flasks containing 40 mL of MS/5 culture media and maintained in a growth room under 12-h photoperiod, 25±2 °C and 30 μmol m^-2 s^-1 irradiance. Shoot production was assessed every 15 days of in vitro culture up to 75 days in 15 plants per period (five plants per flask in a total of three flasks).

Results and discussion

Leaf formation was first detected in seeds at 14 days of culture, concomitantly to roots (figures 1d and 2). The same occurred for leaves of NS, although roots were detected at 28 days (figure 2). Accordingly, it was reported that Acanthostachys strobilacea (Schult. & Schult.f.) Klotzsch seedlings develop roots earlier than leaves (Pereira 1988) while in node cultures, shoots are generally formed prior to roots, requiring hormonal addition to the media to induce rooting (George et al. 2008) ‒ although this is not the case for A. strobilacea (Santos et al. 2010, 2017).

NS showed shoot multiplication after two weeks of culture (table 1, figures 1b and c), which was not present in seeds (table 1, figures 1d and 2e). However, seedlings had higher leaf elongation, more and longer roots than NS plants by 35 days of culture (figure 2). The more intense root and leaf growth in seedlings might be the result of reserve mobilization from the seed, which provides nutrients for the plant until it is nutritionally independent (Taiz et al. 2017). Meanwhile, shoot multiplication in NS may be due to multiple bud breaks induced by apical dominance release after node excision from the mother plant (George et al. 2008).

The O₂ consumption and CO₂ release rates in seeds of A. strobilacea peaked at 14 days of culture (figure 3), corresponding to full seedling formation (figure 2). Contrarily, respiratory rates in NS peaked at 7 days (figure 3), being five times higher than seeds and before the detection of leaf emergence at 14 days. Therefore, we may assume that NS showed a more intense and earlier requirement for energy generation through respiration than seeds for initiating plant formation. The isolation of nodes and apical dominance release stimulates cytokinin accumulation in the excised tissue, leading to cell proliferation in the lateral buds and new shoots formation (Souza et al. 2010, Buchanan et al. 2015, Li et al. 2018). Thus, respiratory activity would increase accordingly to provide sufficient energy for cell division during shoot formation and multiplication process (Siqueira et al. 2018). Accordingly, it is reported that sugar accumulates in NS cultivated in vitro as to promote bud outgrowth and provide substrates for respiration to sustain the intense cell division (see review by Schneider et al. 2019). A similar pattern was previously described for the bromeliad Ananas comosus (Souza et al. 2010). Furthermore, the higher respiration rates in NS than in seeds may be due to the multiple bud breaks in the former (table 1, figures 1b and c).

Table 1
Number of shoots of Acanthostachys strobilacea (Schult. & Schult.f.) Klotzsch plants obtained from seeds and nodal segments (NS) cultivated in vitro. Values are means±s.d. (n=15).

Time (days)	Seed		NS
0	0	±	0		0	±	0
15	0	±	0		2	±	1
30	0	±	0		4	±	1
45	0	±	0		4	±	1
60	0	±	0		5	±	1
75	0	±	0		5	±	1

Figure 2
Leaf and root number and length in Acanthostachys strobilacea (Schult. & Schult.f.) Klotzsch plants obtained from seeds and nodal segments cultured in vitro. Values are means±s.e. and asterisks show significant differences according to the t test for samples taken at the same time (P<0.05, n=20).

The peaks in respiration during plant formation may increase the flux of electrons in the mitochondria and consequently, formation of reactive oxygen species (ROS) (Møller 2001). ROS are involved in cell signaling pathways and specific gene expression related to seed germination, seedling formation and bud dormancy release (Bailly et al. 2008, Kranner et al. 2010, Considine & Foyer 2014). Hence, it is possible that the respiration pattern of seeds and NS observed herein is associated with fluctuations in mitochondrial ROS that directly influence seedling and plant formation from NS.

The O₂ consumption and CO₂ release rates progressively decreased in NS and seeds after peaking at 7 and 14 days, respectively, reaching similar values by 35 days (figure 3). This decrease in respiration rate might have derived from an ongoing increase in photosynthetic activity due to the development of leaves and multiple shoots in seeds and NS, respectively, and due to depletion of the carbon source of the media. Overall, in vitro plants cultivated under photomixotrophic conditions as in the present study show photosynthetic activity, although at lower rates than respiration (Ševčíková et al. 2019). It has been reported that protocorm-like bodies of the Cymbidium Melody Fair ‘Marilyn Monroe’ orchid showed an increase in photosynthetic rates concomitant to decreases in respiration between 20 and 60 days of in vitro culture in MS medium containing 2% sucrose (Ogasawara et al. 1995). Studies also indicate that an increase in photosynthesis can lead to higher cytosolic ATP/ADP rates, inhibiting respiration (Krömer 1995; Buchanan et al. 2015). The decrease in O₂ consumption observed herein may also reflect the cellular energy demand, which was possibly lower after seedling establishment and full morphogenesis in NS plants - both highly energetic processes (Yaseen et al. 2012, Taiz et al. 2017). Finally, the occurrence of some photosynthetic capacity observed in seed and NS plants by 35 days would possibly facilitate ex vitro acclimatization at that period (Ševčíková et al. 2019). Indeed, our research group observed a 100% survival rate after transplanting one-month-old A. strobilacea seedlings (De Carvalho et al. 2014) and NS plants (unpublished data) to Pinus bark substrate, under the same environmental conditions described.

Figure 3
a. Oxygen (O₂) consumption and b. carbon dioxide (CO2) release in Acanthostachys strobilacea (Schult. & Schult.f.) Klotzsch plants obtained from nodal segments and seeds cultured in vitro. Values are means±s.e. and asterisks show significant differences according to the t test for samples taken at the same time (P<0.05, n=5).

To our knowledge, the present study is the first to compare the time course of in vitro plant development from distinct propagules simultaneously. This approach allowed us to conclude that the developmental process of NS plants formation may affect the cell energy demand and, thus, respiration activity differently than in seedlings. Nevertheless, both seeds and NS plants of A. strobilacea can form viable plants with leaves and roots in a short period of time, which broadens the applicability of in vitro culture for both conservation and commercial production of this species. Indeed, the use of seeds ensures genetic variability, desirable for germplasm conservation (Pence 2010), while the shoot multiplication in NS enables a large-scale clone production, which is of considerable value for commercial production aiming at the ornamental plants market (Kyte et al. 2013).

Finally, considering the involvement of mitochondrial ROS production and carbohydrate mobilization in seed germination and bud outgrowth (Considine & Foyer 2014, Taiz et al. 2017, Signorelli et al. 2018), it may be valuable to investigate the effects of redox status and carbohydrate composition during shoot generation from seeds and NS of A. strobilacea to provide more insight on the developmental discrepancies between them.

Acknowledgments

This work was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq/PIBIC) via scholarship awarded to V.C. and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) via scholarships awarded to D.S.S. and C.P.C.

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• Associate Editor: Nelson Augusto dos Santos Junior

Publication Dates

• Publication in this collection
  13 Dec 2021
• Date of issue
  2021

History

• Received
  19 Aug 2020
• Accepted
  22 Jan 2021