Acta Physiol Plant (2017) 39:230 DOI 10.1007/s11738-017-2536-0

SHORT COMMUNICATION

Stem elongation of ornamental bromeliad in tissue culture depends on the temperature even in the presence of gibberellic acid Daniela Soares dos Santos1

•

Poliana Cardoso-Gustavson2 • Catarina Carvalho Nievola3

Received: 6 March 2017 / Revised: 27 June 2017 / Accepted: 8 September 2017 Ó Franciszek Go´rski Institute of Plant Physiology, Polish Academy of Sciences, Krako´w 2017

Abstract Acanthostachys strobilacea Link, Klotzsch, & Otto is an ornamental bromeliad native to Brazilian Atlantic Forest that naturally exhibits a rosette growth pattern. According to the temperature conditions of the in vitro culture, this species can exhibit stem elongation, facilitating the isolation of the nodal segments to be applied in its micropropagation. The rosette morphology is reestablished when this species is maintained under low temperature, thus allowing the maintenance of a germplasm collection (slow growth storage). Gibberellins (GA) are usually applied to stimulate stem elongation in micropropagated plants. Thus, our aim here was to verify the influence of temperature over the stem elongation of A. strobilacea when GA3 is applied to the medium, thus estimating the use of this phytoregulator in slow growth cultures at low temperatures. Physiological and anatomical studies were performed on plants obtained from nodal segments maintained at 10, 15, 20, and 25 °C. Regardless of the applied treatment, no segments developed at 10 °C. Stem elongation occurred at 25 and 30 °C, and was not seen for plants grown under 15 and 20 °C. The application

Communicated by E. Dziedzic. & Catarina Carvalho Nievola [email protected] 1

Programa de Po´s-Graduac¸a˜o em Biodiversidade Vegetal e Meio Ambiente, Instituto de Botaˆnica, Av. Miguel Stefano ´ gua Funda, SP 04301-902, Brazil 3687, A

2

Centro de Cieˆncias Naturais e Humanas, Universidade Federal do ABC, Rua Arcturus 03, Jardim Antares, Sa˜o Bernardo do Campo 09606-070, Brazil

3

Nu´cleo de Pesquisa em Plantas Ornamentais, Instituto de ´ gua Funda, Botaˆnica, Av. Miguel Stefano 3687, A SP 04301-902, Brazil

of 50 lM of GA3 restored stem elongation in plants at 20 but not at 15 °C. The influence of gibberellins on stem elongation of this tropical bromeliad depends on the cultivation temperature, in which low temperature preponderates over the stem elongation effects of GA3. In addition, the optimum temperature for the slow growth of this species depends on the starting temperature of the explant used in the micropropagation. Keywords Acanthostachys strobilacea  GA3  Nodal segments  Tropical species

Introduction Internode elongation is almost fully inhibited during vegetative development of species that exhibit compact rosette morphology, such as Bromeliaceae (Achard et al. 2008). In the natural environment, the reproduction of the tropical ornamental bromeliad Acanthostachys strobilacea occurs through seeds and lateral shoots. Under in vitro conditions, our group established an efficient protocol to the multiplication of this species from nodal segments (Santos et al. 2010; Carvalho et al. 2014). This technology is an appropriate strategy, since it provides an increment in production and quality to ornamental market, besides reducing the pressure over natural wild remnants of this species. A. strobilacea exhibits stem elongation when cultivated in vitro at 25 °C, photoperiod of 12 h and 14 lmol m-2 s-1 of photosynthetically active radiation (Santos et al. 2010). The induction of stem elongation in this bromeliad facilitates the isolation of the nodal segments, providing explants for in vitro production of this species in a large scale (Santos et al. 2010), conferring morphological uniformity, which is an important feature for the trade of

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ornamental plants. Thus, plants can be maintained under slow growth, reducing expenses with subcultures (Reed et al. 2013). In this context, the rosette pattern of this species is maintained for in vitro short-term storage at 10 °C for 15-day-old plants (Carvalho et al. 2014). The temperature parameter must be considered when the objective is to reestablish plant growth or induce stem elongation to obtain nodal segments after the plant has been maintained under slow growth conditions. In many studies, the induction of stem elongation in rosette plants was attributed to gibberellin (GA3), such as the transition from vegetative to reproductive phase of Arabidopsis thaliana (Obroucheva 2008; Matschi et al. 2013) and Lactuca sativa (Fukuda et al. 2009). Matsumoto (2006) showed that GA3 application induced the same effect on the inflorescence emergence of Miltoniopsis orchid hybrids as cold treatment. The GA3 effects on stem elongation of in vitro cultured tropical species were recently reported by Ravi et al. (2014), in which the application of 0.1 mg l-1 of GA3 in the culture medium resulted in the elongation of Jatropha curcas (Euphorbiaceae) buds, but the elongation was inhibited by concentrations above 0.25 mg l-1. GA3 also resulted in the uniform elongation of Vriesea reitzii (Bromeliaceae) buds at 25 °C (Filho et al. 2005). It has been shown that temperature is a factor that influences stem elongation mostly in temperate species (Berone et al. 2007; Fukuda et al. 2009; Kurepin et al. 2011). Regarding the tropical ones, the studies are mainly focused on the injuries caused by low temperatures responsible for substantial post-harvest losses of commercial species (Wang 1994). Our group has demonstrated that native bromeliad species are tolerant to low temperatures, although they exhibit alterations in the vegetative growth, chlorophyll concentration, water-storage parenchyma thickness, and accumulation of soluble and cell wall carbohydrates (Pedroso et al. 2010; Mollo et al. 2011; Carvalho et al. 2013). The cold tolerance of A. strobilacea was related to the maintenance of its rosette morphology (Carvalho et al. 2014), giving rise to the following question: does the application of GA3 accelerate the restoration of the stem elongation of this species under cold conditions? The aim of this study was to verify the influence of temperature over the stem elongation of A. strobilacea when GA3 is applied to the medium, thus estimating the use of this phytoregulator in slow growth cultures at low temperatures. The responses obtained here will highlight the changes that the temperature may cause over the application of GA3 in the in vitro culture of this tropical species when maintained under slow growth conditions.

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Materials and methods Plant material Seeds of A. strobilacea Link, Klotzsch, & Otto were harvested at the Biological Reserve of Mogi Guac¸u in Sa˜o Paulo, Brazil (22°150 04.200 S and 47°090 56.800 W). Seeds’ surface was sterilized with a solution of sodium hypochlorite (2% of active chlorine) and 0.1% (v/v) Tween 20Ò for 20 min, and transferred to 25% hydrochloric acid for 10 min to remove the mucilage. After rinsing in sterilized water, the seeds were immersed in 70% ethanol for 5 min, rinsed in the fungicide Benomyl 0.1% for 15 min, and then rinsed again in the same sodium hypochlorite/ Tween 20Ò solution for 1 h. The seeds were aseptically transferred to 250 ml flasks containing 40 ml of modified Murashige and Skoog (MS; 1962) consisting in a macronutrient dilution at 1/5, with the addition of the MS micronutrients, sucrose (2%), 100 mg l-1 of myo-inositol and 0.1 mg l-1 of thiamine. The pH was adjusted to 5.8 and agar (6 g l-1) was added to the final solution before autoclaving for 15 min at 121 °C. After 60 days, nodal segments (1 cm long) were isolated from plants with elongated stems and transferred to flasks closed with plastic cap containing 40 ml of culture medium as described by Santos et al. (2010). Temperature treatment Flasks containing nodal segments were distributed in germination chambers (347 CDG, Fanem) adjusted to temperatures of 10, 15, 20, and 25 °C, photoperiod of 12 h and 30 lmol m-2 s-1 of irradiance. After 90 days, plants were evaluated according to growth parameters (number and length of leaves and root) and stem elongation (number of nodes and internodes length). Photosynthetic pigments The concentration of chlorophyll a, b and carotenoid was measured according to the protocol described in Lichtenthaler (1987). Pigments were extracted from fresh leaves with 100% acetone at 4 °C. Extracts were filtered and absorbances measured in a spectrophotometer (Quimis, Brazil) at 662, 645, and 470 nm for chlorophyll a, b and carotenoids, respectively. The pigment concentrations were expressed as mg g dry mass-1. Anatomical analysis To determine whether the stem elongation was caused by cell division or elongation according to the thermal

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treatment applied, plants were fixed in FAA50 (formalin, acetic acid, 50% ethanol, 1:1:18) for 24 h (Johansen 1940) and stored in 70% ethanol. The material was processed using the standard methods for Leica historesinÒ (Heraeus Kulzer, Hanau, Germany), embedded, and serially sectioned at 5 lm thickness. Sections were stained with Toluidine Blue O (Sakai 1973) and mounted in water. Slides were observed and digitally photographed with an Olympus BX53 compound microscope equipped with an Olympus Q-Color 5 digital camera with the Image Pro Express 6.3 software. Gibberellic acid (GA3) plus temperature treatment To examine the effects of GA3 on plant growth from nodal segments, the standard medium was supplemented with 5 or 50 lM of ultrafiltered GA3 (Merck) after sterilization of the standard medium by autoclaving. Cultures were then maintained in growth chambers adjusted at 10, 15, 20, and 25 °C, photoperiod of 12 h and 30 lmol m-2 s-1 of photosynthetically active radiation for 90 days. Experimental design and statistical analysis The design of the experiments was completely randomized. Eight glass flasks containing six nodal segments per flask were used in each treatment described above. Data were submitted to analysis of variance using the statistical software SISVAR 4.6 program (Ferreira 1999). ‘Treatment data’ were submitted to a multivariate analysis applied to the values of each variable obtained by principal component analysis. The data of temperature effects over the plants were submitted to one-way ANOVA and the results regarding the mutual effects of temperature and gibberellin were submitted two-way ANOVA, in which the averages were compared by the Tukey test at p \ 0.05.

Results Temperature treatment The temperature of in vitro cultivation directly influenced the time required to the development of the axillary bud from the isolated nodal segment, as shown in Table 1. The first noticeable feature was the absence of regeneration at 10 °C, herein determined as the thermal limit for the in vitro propagation. However, these segments remained green (Fig. 1a). The delay in the plant regeneration at 15 and 20 °C treatments was also noteworthy, in which the development of the meristems was also slowed compared to the ones at 25 °C, herein determined as the optimal temperature for the in vitro propagation (Table 1).

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Table 1 Percentage of regeneration of nodal segment from in vitro cultures of A. strobilacea maintained at different temperatures Temperature (°C)

Observation time (days) 15

30

45

60

75 0

10

0

0

0

0

15

0

0

0

84

100

20 25

0 0

45 100

72 –

100 –

– –

Acanthostachys strobilacea plants kept at 15 and 20 °C displayed normal rosette (Fig. 1b, c), in contrast to 25 °C plants, where short nodes typical of Bromeliaceae (usually not easily recognized) were clearly observed due to the increase in the internode length (Fig. 1d). Temperature altered plant growth and pigments concentration (Table 2) and significant reductions of all evaluated parameters (p \ 0.05) was observed in plants cultivated at 15 and 20 °C when compared to those at the 25 °C treatment. Exceptions were the number of leaves and pigments concentration, in which no significant differences was observed between 20 and 25 °C plants. All plants maintained at temperatures above 10 °C showed a normal pattern of structural development (i.e., without any anatomical abnormalities), differing only in the internodal region, where cell division or elongation was observed. Plants at 15 and 20 °C exhibited rounded shape cells (Fig. 1e, f), and cell elongation was visualized at 25 °C (Fig. 1g). GA3 treatments According to the data summarized in Table 3, GA3 did not induce stem elongation in plants cultivated at 15 and 25 °C regardless of the concentration applied. Conversely, the application of GA3 in the nutrient medium promoted stem elongation only in plants maintained at 20 °C in media with 50 lM. In addition, only two leaves developed in plants cultivated at this temperature and GA3 concentration, a value significantly lower when compared to the plants maintained under the same condition and without the hormone (four leaves). Moreover, an inhibitory effect on the number of roots of plants maintained at 20 and 25 °C caused by the presence of exogenous GA3 was also observed. Effects of temperature and GA3 on growth and stem elongation The main trends of the biometric parameter variation, different concentrations of GA3, and temperatures can be visualized by principal component analysis (PCA), which summarized 83% of the data variability (Fig. 2).

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b Fig. 1 Acanthostachys strobilacea morphology and anatomy. Plants

in vitro cultivated from isolated nodal segments; nodal segments showed no lateral meristem development when kept at 10 °C (a), but grow differently when maintained for 90 days at 15 °C (b), 20 °C (c), and 25 °C (d). Longisections of the internodal region below shoot apical meristem. Cells with round shape at 15 °C (e) and 20 °C (f), and elongated at 25 °C (g). Scale bars: 1 cm (a–d), 100 lm (e–g)

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vectors related to the number of nodes and length of internodes with the presence of 50 lM of GA3 in the nutrient medium, indicating that the in vitro conditions reversed the inhibitory effect of temperature over stem elongation.

Discussion As observed on the right side of axis 1, 15 °C was the most inhibitory temperature condition even when plants were cultivated at different concentrations of GA3, indicating that the temperature was mandatory to the stem elongation. Conversely, plants maintained under 25 °C were ordered close to the vectors, indicating that this thermal condition was more favorable to plant growth regardless of the application of GA3 into the nutrient medium (left side of the axis). Axis 2 represented 18% of the variability, and divided the sampling units of the plants maintained at 20 °C. This result showed that the main coordinates approached the Table 2 Biometric parameters, biomass, and concentration of photosynthetic pigments of A. strobilacea plants cultivated in vitro at different temperatures during 90 days

We have established the temperature ranges to in vitro growth of A. strobilacea plants to obtain elongated stems or to maintain the rosette pattern of growth typical of this species. The cold tolerance by this tropical species has been demonstrated by our group (Carvalho et al. 2014), showing that 15-day-old plants can be maintained at 10 °C. However, as shown here, the nodal segments grown at this temperature did not originate plants. The absence of axillary bud at 10 °C in 90 days showed that this temperature range was inhibitory to cell division and differentiation of lateral meristems.

Evaluated parameters

Temperature (°C) 15

Leaf number (unit) Leaf length (cm)

6 ± 0.8a

6 ± 1.0a

c

7.6 ± 0.3

b

8.4 ± 0.6a

4 ± 0.5

b

6 ± 0.8a

c

2 ± 0.5

c

Root length (cm) Fresh weight of the aerial part (mg) Dry weight of the aerial part (mg) Root fresh weight (mg) Chlorophyll a (mg g

-1

Chlorophyll b (mg g

-1

-1

b

3.6 ± 1.3 20.9 ± 2.4c

7.2 ± 1.1 143.0 ± 18.8b

9.8 ± 0.5a 272.4 ± 21.5a

2.3 ± 0.6c

9.2 ± 1.5b

16.7 ± 1.9a

c

25.9 ± 4.1

b

46.8 ± 3.1a

4.5 ± 1.0

b

7.8 ± 1.3a

8.9 ± 1.0

a

8.8 ± 0.3a

3.8 ± 0.3

a

3.5 ± 0.1a

4.2 ± 0.5

12.6 ± 1.3

a

12.4 ± 0.4a

4.0 ± 0.3b

12.7 ± 1.0a

12.3 ± 0.3a

8.0 ± 1.1

c

Root dry weight (mg)

1.7 ± 0.5

b

dry mass)

3.0 ± 0.2

b

dry mass)

1.0 ± 0.3

b

dry mass)

Total chlorophyll (mg g-1 dry mass)

25

4 ± 0.9b 1.7 ± 0.8

Root number (unit)

Carotenoids (mg g

20

Each value represents the average of 48 plants ± SD Averages followed by the same letter (lowercase in lines) do not differ by Tukey test at 5% (n = 48)

Table 3 Morphometry of A. strobilacea plants maintained for 90 days in a nutrient medium containing different concentrations of GA3

Morphological parameters

GA3 (lM)

Temperature (°C) 15

Nodal segments number (unit)

Internode length (cm)

20 bA

0

1±0

5

1 ± 0bA

50

bA

0 5 50

1±0 0

bA

0

bA

0

bA

25 bA

5 ± 1.6aA

1 ± 0bA

5 ± 0.5aA

1±0

3 ± 0.4 0

bB

0

bB

bA

0.6 ± 0.2

6 ± 0.6aA 0.4 ± 0.1aB 0.3 ± 0.1aB

aA

0.9 ± 0.5aA

Averages followed by the same letter (lowercase in lines and capital letters in columns) do not differ by Tukey test at 5% (n = 48)

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Fig. 2 PCA biplot diagram showing the different temperatures (15, 20, and 25 °C) in which A. strobilacea plants were in vitro cultivated, based on the values of six parameters: number of leaves (nl), roots (nr) and nodes (nn) and leaf (nl), root (lr), and internode (ln) lengths

Environmental temperature may determinate plant growth patterns in a way that the development of temperate species is reduced when they are kept at a temperature range of 4–10 °C (Orlikowska 1992), while in tropical species, this reduction occurs at 15–18 °C (Banerjee and De Langhe 1985). Considering only the temperature effects on the growth of A. strobilacea, the growth and stem elongation were inhibited below 15 °C when compared to plants at 25 °C. This growth reduction can be related to the cold effects over the metabolic reactions (Sage and Kubien 2007) or to the reduction in the cell division rate as observed in Zea mays plants (Granier et al. 2000). Tropical grasses show little or no growth at 10 and 15 °C, considering 15 °C as the standard temperature to grass development (Moreno et al. 2014), somehow resembling the temperature limit of the species growth analyzed herein, and established as the standard temperature to tropical bromeliads. Stem elongation of A. strobilacea was previously reported by Santos et al. (2010) and referred as the visible nodes resulting from the increase in the internode growth. Our anatomical descriptions showed that the elongation of the internodes observed in plants at 25 °C was caused by an increase in cell length (cell elongation), while the stem of the rosette plants (15 and 20 °C) is constituted of cells that just proliferated, retaining

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their rounded shape. These results are related to the fact that stem elongation consists of cell division and posterior elongation (Kutschera and Niklas 2013) and that low temperatures extend all phases of the cell cycle (Granier et al. 2000), leading to the assumption that the occurrence or absence of elongation is influenced by the permanence or delay in some cell division phase. As examples, divisions and relative rate of cell elongation in maize leaves were higher in plants grown at 25.5 °C and decreased as leaf temperature decreased to 14 and 19 °C (Granier et al. 2000); this modulation of temperature and morphology was also observed in A. thaliana plants that exhibit rosette shape when maintained at 6 °C and elongation at 20 °C (Mazzella et al. 2000). The addition of different GA3 concentrations did not induce stem elongation in plants maintained at 15 °C, occurring only in the ones kept at 20 °C, evidence that the temperature is mandatory in the establishment of the rosette morphology. Responses of GA over stem elongation in relation with temperature vary according to the analyzed species and are well established in cultivated species at temperate environments. As examples, the nodal segments of Avena sativa cultivated at 30 °C exhibited a high response to GA3 that decreased as the temperature was reduced (Jusaitis et al. 1982); the hypocotyl tissues of L. sativa at 9.5 °C showed little growth and higher

Acta Physiol Plant (2017) 39:230

elongation according to the concentration of GA3 applied, but they did not show sensitivity to the variations in the concentration of the gibberellin above 13 °C (Stoddart et al. 1978). Although it has been reported that the stem elongation can be induced by the application of GA3 in temperate species regardless the temperature (Farrell et al. 2006), this inductor effect is restricted by a specific temperature range in tropical plants, as shown here. As shown by our results, the application of GA3 as inductor of stem elongation in A. strobilacea is dose and temperature dependent. Stem elongation can be related to the sensitivity of this species to respond to exogenous GA according to the thermal range. Temperatures can act over growth by inducing the expression of some specific genes, while low temperatures may decrease the levels of gibberellin expression by GA2ox1 CBF1-independent mechanism (Achard et al. 2008). Due to the fact that plants kept at 15 °C did not exhibit stem elongation even when grown with 50 lM gibberellin, we hypothesized that either this phytohormone was degraded in plants kept at 15 °C, or the genes related to gibberellin biosynthesis were inhibited by low temperature. Low temperatures induce the expression of transcription factors related to gibberellin catabolism and restrain plant growth (Achard et al. 2008). In conclusion, our results indicated that the in vitro stem elongation of A. strobilacea occurs at 25 °C, and gibberellin application influences this morphological aspect at 20 and 25 °C. Nodal segments maintained at 10 °C do not originate plants, although the previous studies had demonstrated the viability of growing plants at this temperature. Thus, the optimum temperature for the slow growth of this species depends on the starting temperature of the explant used in the micropropagation. This study opens new perspectives to the knowledge of the physiological effects of the low temperature over this tropical species, about the effects of GA application in its micropropagation, and the effects of GA3 and low temperature over the stem elongation, including if the elongation responses are observed in terms of short or long term. Author contribution statement CCN planned the research and provided lab support and orientation during the development of this study, DSS and PCG carried out the experiments and evaluated the resulting data. All the authors drafted, read, and approved the final manuscript. Acknowledgements This study was supported by Brazilian CAPES (PNADB—23038.000037/2010-96 and AUX-PE—453/2010). Compliance with ethical standards Conflict of interest The authors declare that they have no conflict of interest.

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