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. 2021 Mar 22;11(4):186. doi: 10.1007/s13205-021-02730-x

Cryopreservation and low-temperature storage of seeds of Tillandsia species (Bromeliaceae) with ornamental potential

Rafaelle Souza de Oliveira 1, Fernanda Vidigal Duarte Souza 2, Iasmin Lima dos Santos 1, Sandra de Oliveira Souza 1, Lidyanne Yuriko Saleme Aona 1, Everton Hilo de Souza 1,✉
PMCID: PMC7984166  PMID: 33927977

Abstract

Tillandsia (Bromeliaceae) species have high endemism, and due to their strong ornamental potential, predatory extraction is threatening the extinction or drastic population reduction of many of them. In light of this scenario, it is necessary to find strategies for the conservation of these endangered species. The objective of this study was to evaluate two seed preservation strategies (freezing at − 5 °C and cryopreservation at − 196 °C) for 20 Tillandsia species occurring in the state of Bahia. We initially evaluated the morphometry, thousand-seed weight, and water content, followed by tests of germination and desiccation. After selecting the best result of the germination test (Germitest paper and incubation at 30 °C) and desiccation (3 h on silica gel), we established conservation tests utilizing two temperatures (freezing at − 5 °C and liquid nitrogen at − 196 °C), with storage times of 1, 7, 30, 180 and 450 days. Analysis of variance indicated that the 20 species had different behaviors when submitted to the two temperatures and different storage times. After 450 days there was a reduction in the germination percentage and germination speed index (GSI) of all the species studied when the seeds were preserved in the freezer. The storage in liquid nitrogen was efficient for the preservation of Tillandsia seeds when dried to a moisture content of approximately 7%. Our results support the establishment of a cryobank for Tillandsia to conserve these endemic species.

Keywords: Bromeliads, Dehydration, Endangered species, Endemic species, Liquid nitrogen, Tillandsioideae

Introduction

The genus Tillandsia L. is the most representative among the family Bromeliaceae Juss., with 755 recorded species (Gouda et al. 2021, continuous updated), distributed in seven subgenera (Barfuss et al. 2016), variously with high ornamental potential, ecological value and/or possible uses in the chemical and pharmaceutical industries (Estrella-Parra et al. 2019). In the Brazilian state of Bahia, there are 25 recorded species in four subgenera, Anoplophytum (Beer) Baker, Diaphoranthema (Beer) Baker, Phytarrhiza (Vis.) Baker and Tillandsia (G.Nicholson). These species are found in the Caatinga, Mata Atlântica (Atlantic Forest) and Cerrado (savanna) biomes (Flora do Brasil 2020).

Tillandsia species are in high demand as ornamental plants, both in the domestic and international markets (Flores-Palacios and Valencia-Díaz 2007; Castillo et al. 2013; Toledo-Aceves et al. 2014), mainly in the United States, Europe, and Asia (Dematê 2005; Negrelle et al. 2012). In addition, Tillandsia species are often used in traditional rituals and ceremonies in Latin America (Haeckel 2008; Hornung-Leoni 2011), but they are rarely grown commercially in a sustainable form, instead of being obtained by extractivism (Negrelle et al. 2012). The high degree of endemism, predatory extractivism for illegal trade, and destruction of natural habitats, are threatening the survival of many of these species (Hietz et al. 2002; Flores-Palacios and Valencia-Díaz 2007; Forzza et al. 2013; Toledo-Aceves et al. 2014; NOM-059-Semarnat 2019), and causing a need for methods for their conservation.

The conservation of germplasm allows safeguarding the genetic variability of species with ecological and economic importance and/or that are endangered (Machado et al. 2016; Silva et al. 2016; Malhotra et al. 2019; Rao and Parthasarathy 2019; Dar et al. 2020). Among the conservation strategies, cryopreservation enables long-term conservation of genetic material with small space requirements. This can be achieved by storage in liquid nitrogen (− 196 °C) or cold nitrogen vapor (− 150 °C) of vegetative parts, such as calli, stem tips, buds, pollen grains, and seeds (Souza et al. 2016; Jenderek and Reed 2017; Dinato et al. 2020; Wang et al. 2020). The ultra-low temperatures reduce the metabolism of cells until a level that halts the tissue development but allows regeneration with restoration of ambient temperature (Panis and Lambardi 2006; Jenderek and Reed 2017). In the case of preserving seeds, the most important aspect to be considered is the ideal moisture content, which can be a decisive factor to maintain the viability of seeds after freezing and thawing, and thus influence the final percentage of seed germination (Generoso et al. 2019; Vettorazzi et al. 2019; Silva et al. 2021).

Seeds are ideal reproductive structures to store the genetic variability of a species, assuring the conservation of the gene pool (Gosling 2003; Zotz 2013; Generoso et al. 2019; Silva et al. 2021). Therefore, studying the different methods of preserving seeds contributes to the conservation of species, and at the extreme can rescue a species from the threat of extinction. It is common knowledge that bromeliad seeds lose their viability very quickly (Zotz 2013). Several studies involving the conservation of seeds of Bromeliaceae species at low (Vasak 1969; Fernandez et al. 1989; Wester and Zotz 2011; Zotz 2013) and ultra-low temperatures have been published, such as Tarré et al. (2007), Rodrigues et al. (2014), Ferrari et al. (2016) and Silva et al. (2021).

Zotz (2013), studying seven bromeliad species of the genera Guzmania Ruiz & Pav., Tillandsia and Vriesea Lindl., observed that conservation of seeds at room temperature (20 °C) drastically reduced their viability after 12 months, while when conserved at − 20 °C, they remained viable for 26 months.

Tarré et al. (2007) and Ferrari et al. (2016) investigated cryopreservation of the species Encholirium Mart. ex Schult. and Schult.f. and Dyckia Schult. and Schult.f. and observed germination results similar to or better than the control treatment after one year, thus considering this strategy to be efficient for the two species in question. Rodrigues et al. (2014) mainly studied the optimal moisture content of the seeds of 10 endemic species of bromeliads threatened with extinction of the genera Alcantarea (E.Morren ex Mez) Harms, Nidularium Lem, Pitcairnia L’Hér., Vriesea and Wittrockia Lindm. and concluded that the species had orthodox behavior, so it was necessary to dehydrate the seeds before the conservation procedures. The authors also reported that water content lower than 7% affected the viability of the seeds and that the ideal content for cryopreservation was around 6–7% (dry mass basis, DMB). To the best of our knowledge, there are no studies of cryopreservation of species of the Tillandsia despite all importance of this genus in the Bromeliaceae family. Also, based on our experience with a seed bank maintained at Embrapa Mandioca e Fruticultura in a freezer at − 5 °C, we observed a reduction in viability of the stored seeds of some species of Bromeliaceae, mainly of the subfamily Tillandsioideae. Based on the data in the literature and our observations, we decided to study two storage temperatures: (i) − 5 °C, in a freezer, a technique used for many plant species, with easy repeatability and low cost, and (ii) cryopreservation in liquid nitrogen at − 196 °C, with a greater cost for laboratory structure, but with better results observed for other species.

Therefore, our objective was to evaluate the conservation of seeds in a freezer at − 5 °C and by cryopreservation at − 196 °C in liquid nitrogen of 20 Tillandsia species with ornamental potential occurring in the state of Bahia, Brazil, to develop a protocol that can safeguard the genetic variability of these species.

Materials and methods

Plant material

We studied 20 Tillandsia species occurring in Bahia, Brazil (Figs. 1, 2 and Table 1). The seeds were collected randomly from ripe fruits of plants growing naturally, to maintain the variability among the populations. Voucher specimens of each species were deposited in the Recôncavo da Bahia Herbarium (HURB) (Table 1) and the Bromeliad Germplasm Bank of the Embrapa Cassava and Fruits research unit (Embrapa Mandioca e Fruticultura), located in the municipality of Cruz das Almas, Bahia, Brazil. The seed collection was carried out in line with all Brazilin regulations on the protection of biodiversity.

Fig. 1.

Fig. 1

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Species of Tillandsia with ornamental potential occurring in Bahia, Brazil. a T. bulbosa; b T. juncea; c T. paraensis; d T. polystachia; e T. pruinosa; f T. loliaceae; g T. recurvata; h T. tricholepis; i T. streptocarpa 1; j T. streptocarpa 2; and k T. chapeuensis; l T. gardneri

Fig. 2.

Fig. 2

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Species of Tillandsia with ornamental potential occurring in Bahia, Brazil. a T. geminiflora; b T. globosa; c T. pohliana; d T. stricta 1; e T. stricta 2; f T. stricta subsp. piniformis; g T. tenuifolia; and h T. tenuifolia var. vaginata

Table 1.

Species of Tillandsia that occur in Bahia, Brazil, including subgenus, collection municipality, biome and voucher number of the specimen deposited in the HURB Herbarium

Species Subgenus Collection municipality Biome HURB
T. bulbosa Hook.f. Tillandsia Ilhéus Atlantic Forest/Restinga 25395
T. juncea (Ruiz and Pav.) Poir. Tillandsia Santa Terezinha Caatinga 22220
T. paraensis Mez Tillandsia Entre Rios Atlantic Forest 25754
T. polystachia (L.) L. Tillandsia Milagres Caatinga 22131
T. pruinosa Sw. Tillandsia Brejões Atlantic Forest 22152
T. loliacea Mart. ex Schult. and Schult.f. Diaphoranthema Milagres Caatinga 22130
T. recurvata (L.) L. Diaphoranthema Milagres Caatinga 22149
T. tricholepis Baker Diaphoranthema Ibitiara Caatinga 25753
T. streptocarpa Baker 1 Phyrarrhiza Milagres Caatinga 18591
T. streptocarpa Baker 2 Phyrarrhiza Milagres Caatinga 25006
T. chapeuensis Rauh Anoplophytum Morro do Chapéu Caatinga/Rupestrian grasslands 23983
T. gardneri Lindl. Anoplophytum Ilhéus Atlantic Forest 25393
T. geminiflora Brongn. Anoplophytum Brejões Atlantic Forest 22222
T. globosa Wawra Anoplophytum Muritiba Atlantic Forest 25569
T. pohliana Mez Anoplophytum Morro do Chapéu Caatinga/Rupestrian grasslands 23978
T. stricta Sol. 1 Anoplophytum Ilhéus Atlantic Forest 25384
T. stricta Sol. 2 Anoplophytum Milagres Caatinga 22180
T. stricta subsp. piniformis Rauh ex. Ehers and Heidt Anoplophytum Morro do Chapéu Caatinga/Rupestrian grasslands 23977
T. tenuifolia L. Anoplophytum Milagres Caatinga 15626
T. tenuifolia var. vaginata (Wawra) L.B.Sm. Anoplophytum Cruz das Almas Atlantic Forest 24541
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Morphometry and thousand-seed weight

The plumose appendages were removed with scissors to prevent the proliferation of microorganisms. Then the number of seeds per capsule was counted, using three capsules per species from different plants. The length and width of 25 seeds were measured from images obtained with a stereomicroscope (Leica EZ4 D, Wetzlar, Germany) using the ImageJ 2 program (Rueden et al. 2017). The thousand-seed weight was determined with a precision scale, according to the Seed Analysis Rules (Brasil 2009).

Water content of the seeds

The seed moisture content was determined by examining three samples of 100 recently collected seeds of each species, applying the fresh weight method by heating to 105 °C for 24 h, also according to the Seed Analysis Rules (Brasil 2009).

Germination and desiccation testing

For the germination test, we used seeds of four species, each one representing one of the subgenera of plants occurring in Bahia: T. streptocarpa (subg. Phytarrhiza), T. recurvata (subg. Diaphoranthema), T. stricta 1 (subg. Anophophytum) and T. polystachia (subg. Tillandsia), with two substrates (Germitest® paper and MS culture medium), at three incubation temperatures (20, 25 and 30 °C).

One lot of seeds was placed to germinate in Petri dishes containing two sheets of autoclaved Germitest® paper moistened with 2.5 times their weight (in grams) of autoclaved distilled water. Another lot of seeds was disinfected in a laminar flow cabinet with 70% ethanol for 5 min and a 2% sodium hypochlorite solution for 20 min, followed by rinsing three times with autoclaved distilled water. The seeds were placed in Petri dishes containing MS medium (Murashige and Skoog 1962) containing half the normal salt concentration, supplemented with 3% sucrose, and solidified with 2 g L−1 of Phytagel®. The pH was adjusted to 5.8 before autoclaving.

Next, the dishes containing the seed samples were incubated at three temperatures (20, 25 and 30 °C) with an 8 h photoperiod under four cool daylight fluorescent lamps of 20 W (total flux rate 120 μmol m−2 s−1). The emergence of the embryonic axis (1 mm) was the criterion used for the start of germination. The germination percentage (G %) and germination speed index (GSI) were calculated according to: G%=N/A×100, where N = number of germinated seeds and A = total number of seeds; GSI=∑Gi/ni, where Gi = number of germinated seeds and ni = counting day.

For the desiccation tests, each seed batch was placed in a chamber with activated silica gel for 3, 6, 9 and 12 h. One lot was evaluated for water content according to the method described above and the seeds of the other lot were placed in Petri dishes to germinate on Germitest® paper at a temperature of 30 °C. The evaluation of germination was performed as described above.

The experimental design was completely randomized in a 4 × 2 × 3 factorial scheme (species × environments × temperature) with four repetitions, where each repetition was composed of 25 seeds, for a total of 100 seeds per treatment. The seeds were observed daily for 20 days. The data on germination percentage and GSI were submitted to analysis of variance and the means were compared by the Tukey test (p < 0.01), with the germination data first being transformed by arcsine x/100 . The analyses were carried out with the R program (R Development Core Team 2019).

Conservation of seeds

The seeds’ appendages were removed and then they were separated into lots of 100 (Fig. 3a, b), each of which was placed in a cryogenic tube (3 mL) and desiccated for 3 h on activated silica gel (best desiccation treatment). After selecting the best result of the germination tests, we established the conservation test, utilizing the two treatment methods (freezer at − 5 °C and liquid nitrogen at − 196 °C). For cryopreserved seeds, the tubes containing the seeds were rapidly immersed in liquid nitrogen.

Fig. 3.

Fig. 3

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a Tillandsia gardneri seeds with appendages. b Tillandsia gardneri seeds with appendage removed and separated into lots of 100. c Tillandsia gardneri seeds germinated after cryopreservation for 450 days. ap appendage, se seed

We evaluated the germination and GSI after 1, 7, 30, 180, and 450 days (Fig. 3c). The seeds were slowly thawed at room temperature for 30 min and placed to germinate in a B.O.D. (Biological Oxygen Demand) incubator growth chamber at 30 °C, with 16: 8 h photoperiod (light: dark). The evaluations were performed daily until the 20th day. The control treatment consisted of seeds removed from the desiccator and placed directly in Petri dishes on Germitest® paper in the same conditions as the conservation test.

The experimental design was completely randomized in a 20 × 2 × 5 factorial scheme (species x environments x evaluation times). Each treatment used 4 repetitions, each composed of 25 seeds, for a total of 100 seeds per treatment. The data were submitted to analysis of variance and the means were compared by the Scott-Knott test (p < 0.01) for species, the F test (p < 0.01) for conservation environments and the Tukey test (p < 0.01) for evaluation times. The germination data were first transformed to arcsine x/100. All the analyses were carried out with the R program (R Development Core Team 2019).

Results and discussion

Morphometry, thousand-seed weight and water content

The analysis of variance revealed significant differences among the species for a number of seeds per capsule and length diameter and water content of the recently collected seeds. Table 2 reports the values of each of these traits along with the coefficient of variation.

Table 2.

Morphometry, water content, and thousand-seed weight of seeds of Tillandsia species (Bromeliaceae) occurring in Bahia, Brazil

Species NSC LEM DIA WC TSW
T. bulbosa 63.00 ± 8.19 e 3.13 ± 0.17 h 0.47 ± 0.06 c 11.98 ± 0.12 d 0.35
T. juncea 117.00 ± 26.21 c 3.10 ± 0.32 h 0.46 ± 0.05 c 13.56 ± 0.10 b 0.41
T. paraensis 118.33 ± 17.95 c 3.63 ± 0.29 g 0.51 ± 0.09 b 14.16 ± 0.08 a 0.51
T. polystachia 155.33 ± 11.02 b 3.93 ± 0.25 f 0.39 ± 0.05 d 13.58 ± 0.12 b 0.42
T. pruinosa 60.00 ± 10.58 e 2.94 ± 0.27 i 0.35 ± 0.04 e 11.31 ± 0.21 e 0.21
T. loliacea 44.33 ± 14.47 f 4.79 ± 0.56 d 0.32 ± 0.07 e 10.35 ± 0.10 f 0.21
T. recurvata 52.33 ± 8.39 e 3.51 ± 0.32 g 0.31 ± 0.06 e 11.18 ± 0.12 e 0.21
T. tricholepis 65.00 ± 10.82 e 4.35 ± 0.34 e 0.32 ± 0.07 e 10.16 ± 0.16 f 0.09
T. streptocarpa 1 82.67 ± 15.01 d 5.63 ± 0.51 a 0.33 ± 0.06 e 13.33 ± 0.10 b 0.42
T. streptocarpa 2 34.33 ± 4.16 f 5.68 ± 0.88 a 0.52 ± 0.07 b 14.58 ± 0.16 a 0.51
T. chapeuensis 24.30 ± 9.00 f 5.36 ± 0.48 b 0.56 ± 0.07 a 12.16 ± 0.21 c 0.12
T. gardneri 71.33 ± 9.02 e 4.36 ± 0.54 e 0.47 ± 0.07 c 14.31 ± 0.18 a 0.54
T. geminiflora 57.33 ± 5.03 e 5.11 ± 0.36 c 0.41 ± 0.06 d 11.86 ± 0.05 d 0.25
T. globosa 53.33 ± 4.16 e 4.70 ± 0.74 d 0.33 ± 0.06 e 12.86 ± 0.08 c 0.35
T. pohliana 202.33 ± 14.64 a 3.87 ± 0.25 f 0.50 ± 0.06 b 13.87 ± 0.11 b 0.45
T. stricta 1 66.67 ± 6.11 e 3.75 ± 0.26 g 0.31 ± 0.06 e 12.13 ± 0.12 c 0.35
T. stricta 2 59.00 ± 10.82 e 3.16 ± 0.36 h 0.30 ± 0.06 e 10.48 ± 0.14 f 0.15
T. stricta subsp. piniformis 30.33 ± 2.52 f 3.63 ± 0.18 g 0.38 ± 0.05 d 12.56 ± 0.09 c 0.37
T. tenuifolia 97.33 ± 13.28 d 3.94 ± 0.28 f 0.41 ± 0.05 d 11.67 ± 0.07 d 0.32
T. tenuifolia var. vaginata 35.67 ± 5.51 f 2.74 ± 0.35 i 0.33 ± 0.07 e 11.26 ± 0.11 e 0.23
CV (%) 10.43 15.63 15.70 11.32
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NSC number of seeds per capsule, LEN length of 25 seeds (mm), DIA diameter of 25 seeds (mm), WC water content in recently collected seeds, TSW thousand-seed weight

The greatest number of seeds per capsule was observed for T. pohliana, with a mean of 202.33, while the lowest quantities were observed for T. loliacea (44.33), T. streptocarpa 2 (34.33), T. chapeuensis (24.30), T. stricta subsp. piniformis (30.33), and T. tenuifolia var. vaginata (35.67), without significant pairwise differences among them (Table 2). According to Rodrigues et al. (1999) and Souza et al. (2015), the number of seeds per fruit is directly related to the number of ovules, so that species that produce more ovules have better chances of producing seeds, favoring their perpetuation in the environment. Other important factors related to the number of seeds are the efficiency of the pollination system (Scrok and Varassin 2011; Bastos et al. 2017) and the absence or presence of reproductive barriers (Souza et al. 2017).

With respect to seed length, T. streptocarpa 1 and T. streptocarpa 2 had the highest averages, with 5.63 and 5.68 mm, respectively, and T. pruinosa (2.94 mm) and T. tenuifolia var. vaginata (2.74 mm) had the lowest averages (Table 2). In relation to seed diameter, T. chapeuensis had the greatest mean, with 0.56 mm while T. stricta 2 (0.30 mm) had the smallest (Table 2). In general, all the species produced positive photoblastic seeds having the same morphology, with elongated, narrow, and filiform shape and plumose appendage in the basal region, despite small variations in the length and thickness of each species. These traits permit good seed dispersal by wind of the Tillandsioideae subfamily (Smith and Downs 1977; Magalhães and Mariath 2012; Barfuss et al. 2016). The need for light exposure (positive photoblasticity) for germination of seeds of Bromeliaceae species has been reported by Benzing (2000), Pereira et al. (2008) and Rodrigues et al. (2014). These researchers mainly correlated the size of seeds and the environment where the species normally occur.

In relation to the thousand-seed weight, there was a variation from 0.09 g for T. tricholepis to 0.54 g for T. gardneri (Table 2). Some authors have reported high positive correlations between the germination percentage and the germination speed (Martins et al. 2000; Silva et al. 2010; Socolowski et al. 2011). The results presented here corroborate those findings, because the lighter seeds (T. tricholepis, T. chapeuensis and T. stricta 2) presented the lowest germination percentages and GSI values.

Regarding the water content of the recently collected seeds, the highest values were observed for T. streptocarpa 2 (14.58%), T. gardneri (14.31%) and T. paraensis (14.16%), while the lowest values were for T. loliacea (10.35%), T. tricholepis (10.16%) and T. stricta 2 (10.48%). Naturally, orthodox seeds already have low moisture levels (Ferrari et al. 2016), and according to Stanwood (1985), orthodox or semi-recalcitrant seeds should be stored with moisture levels below 10%, similar to the results found in this study.

Desiccation and germination tests

After obtaining the morphometric, water content, and thousand-seed weight data, we performed tests of desiccation and germination with four Tillandsia species, one of each subgenus, to help support the other conservation studies (Fig. 4, Table 3).

Fig. 4.

Fig. 4

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Germination percentage and water content of seeds of Tillandsia species (Bromeliaceae) occurring in Bahia, Brazil after different desiccation times on activated silica gel

Table 3.

Germination percentage and germination speed index (GSI) of four Tillandsia species (Bromeliaceae) occurring in Bahia, Brazil in function of culture conditions

T. streptocarpa 1 T. recurvata T. stricta 1 T. polystachia
MS GP MS GP MS GP MS GP
Germination (%)
 20 °C 80 aB 76 aB 96 aA 95 aA 86 aA 88 aA 82 aB 79 aB
 25 °C 96 aA 92 aA 92 aA 91 aA 88 aA 81 aA 91 aA 90 aA
 30 °C 92 aA 89 aA 98 aA 94 aA 82 aA 83 aA 94 aA 92 aA
CV (%) 22.38
Germination speed index
 20 °C 1.40 aB 1.15 aB 3.08 aB 2.96 aB 0.44 aB 0.35 aB 2.30 aB 2.60 aB
 25 °C 3.25 aA 3.02 aA 4.34 aA 4.56 aA 2.23 aA 2.33 aA 4.21 aA 4.21 aA
 30 °C 3.06 aA 3.86 aA 4.77 aA 4.80 aA 2.21 aA 2.01 aA 4.11 aA 3.83 aA
CV (%) 16.22
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Means followed by the same lowercase letters in the row and uppercase in the column do not differ at 1% probability by the Tukey test

MS culture medium containing half of the normal concentration of salts (Murashige and Skoog, 1962), GP Germitest® paper

The desiccation of the seeds on activated silica gel for 3 h significantly reduced the water content, from approximately 12–13% to 7–8%, after which the weights stabilized (Fig. 4). This reduction of water content did not influence the germination percentages of the seeds of the four species studied, indicating it is a good treatment for seed conservation.

The data obtained indicated that the seeds of the Tillandsia species had orthodox behavior, remaining viable with low moisture levels. This tolerance to desiccation of Bromeliaceae seeds was previously described by Rodrigues et al. (2014) and Silva et al. (2021), studying different species of various subfamilies, including the Tillandsioideae subfamily, the focus of this study. Those authors managed to reduce the seed water content to very low levels (around 2.2%) without affecting the viability.

To conserve the seeds of any plant species, it is necessary to reduce and then stabilize the water content (Figueiredo et al. 2017), to prevent the formation of ice crystals in the cells during cryopreservation, or tissue degradation when seeds are stored at higher temperatures. In both cases, this can impair the seed's integrity and viability (Pegg 2010; Figueiredo et al. 2017). Besides this, the tolerance to desiccation is an important characteristic for conservation, since a very low water content within the cells can cause the death of the embryo and compromise the viability of the seeds (Mello et al. 2010; Villela and Peres 2004; Masetto and Faria 2019).

With respect to the germination percentage and germination speed index (GSI), there was no significant interaction between the factors MS culture medium and Germitest® paper for the four species studied (Table 3). Regarding temperature, T. streptocarpa 1 and T. polystachia seeds had low germination percentages, irrespective of the substrate used, when cultured at 20 °C.

For the GSI, the temperature of 20 °C promoted slower germination of the four species. In turn, the germination percentages varied from 76% for T. streptocarpa 1 at 20 °C to 98% for T. recurvata at 30 °C (Table 3).

Temperature is a key factor for seed germination of many plant species, including overcoming dormancy and embryo growth, by affecting the water absorption speed and biochemical and physiological reactions (Silva et al. 2002; Castro et al. 2004; Vivian et al. 2008). We did not observe dormancy in any of the species studied, but the higher temperatures promoted higher germination and GSI. The absence of seed dormancy of Bromeliaceae species has been reported for various species of different genera (Pereira 1988; Pereira et al. 2008; Rodrigues et al. 2014). In contrast, Coelho et al. (2011) reported tegumental dormancy in Bromelia balansae Mez., which was broken by immersion in sulfuric acid PA (98%) for 1 min.

In the Bromeliaceae, the ideal temperature for seed germination appears to be strongly related to the species, although a range exists (15–35 °C) where the great majority of species’ seeds germinate. However, to maximize the germinative potential of a determined species, it is often necessary to optimize the temperature (Mercier and Guerreiro Filho 1990; Nara and Webber 2002; Pinheiro and Borghetti 2003; Vieira et al. 2007).

Pereira et al. (2010) reported no difference in the germinative potential of the bromeliad Nidularium innocentii Lem. when submitted to temperatures of 20 °C, 25 °C and an alternating regime of 20 °C/30 °C. Tarré et al. (2007) also found no significant differences in the germination percentages for species of the genera Encholirium Mart. ex Schult. & Schult.f. and Dyckia Schult. & Schult.f. when exposed to temperatures of 20, 25, and 30 °C. In turn, for Dyckia tuberosa (Vell.) Beer, Vieira et al. (2007) analyzed a variation from 10 to 45 °C and observed that these temperatures did not lead to germination and the ideal temperatures were 30–35 °C to maximize the germination and GSI.

Therefore, for a question of practicality and in light of the GSI of the four species studied, we decided to continue the conservation tests on Germitest® paper at a temperature of 30 °C.

Seed conservation

The analysis of variance revealed different behavior among the 20 species regarding the two conservation conditions (freezer at − 5 °C and liquid nitrogen at − 196 °C) and five evaluation times (1, 7, 30, 180 and 450 days). The results of the germination percentage after the different observation periods and conservation conditions are presented in Figs. 5 and 6 and the germination speed index values (GSI) are shown in Figs. 7 and 8.

Fig. 5.

Fig. 5

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Germination percentage of seeds of Tillandsia species (Bromeliaceae) occurring in Bahia, Brazil stored at temperature of − 5 °C or cryopreserved at − 196 °C for 1–450 days. a T. bulbosa; b T. juncea; c T. paraensis; d T. polystachia; e T. pruinosa; f T. loliacea; g T. recurvata; h T. tricholepis; i T. streptocarpa 1; and j T. streptocarpa 2. Means followed by the same lowercase letters (temperature) and uppercase letters (time) do not differ by the F test and Tukey test (p < 0.01), respectively

Fig. 6.

Fig. 6

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Germination percentage of seeds of Tillandsia species (Bromeliaceae) occurring in Bahia, Brazil stored at a temperature of − 5 °C or cryopreserved at − 196 °C for 1–450 days. a T. chapeuensis; b T. gardneri; c T. geminiflora; d T. globosa; e T. pohliana; f T. stricta 1; g T. stricta 2; h T. stricta subsp. piniformis; i T. tenuifolia; and j T. tenuifolia var. vaginata. Means followed by the same lowercase letters (temperature) and uppercase letters (time) do not differ by the F test and Tukey test (p < 0.01), respectively

Fig. 7.

Fig. 7

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Germination speed index (GSI) of seeds of Tillandsia species (Bromeliaceae) occurring in Bahia, Brazil stored at temperature of − 5 °C or cryopreserved at − 196 °C for 1–450 days. a T. bulbosa; b T. juncea; c T. paraensis; d T. polystachia; e T. pruinosa; f T. loliacea; g T. recurvata; h T. tricholepis; i T. streptocarpa 1; and j T. streptocarpa 2. Means followed by the same lowercase letters (temperature) and uppercase letters (time) do not differ by the F test and Tukey test (p < 0.01), respectively

Fig. 8.

Fig. 8

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Germination speed index (GSI) of seeds of Tillandsia species (Bromeliaceae) occurring in Bahia, Brazil stored at temperature of − 5 °C or cryopreserved at − 196 °C for 1–450 days. a) T. chapeuensis; b T. gardneri; c T. geminiflora; d T. globose; e T. pohliana; f T. stricta 1; g T. stricta 2; h T. stricta subsp. piniformis; i T. tenuifolia; and j T. tenuifolia var. vaginata. Means followed by the same lowercase letters (temperature) and uppercase letters (time) do not differ by the F test and Tukey test (p < 0.01), respectively

According to the results found T. bulbosa (Figs. 5a, 7a), T. pruinosa (Figs. 5e, 7e), T. loliacea (Figs. 5f, 7f), T. tricholepis (Figs. 5h, 7h), T. gardneri (Figs. 6b, 8b), T. pohliana (Figs. 6e, 8e), T. stricta 1 (Figs. 6f, 8f), T. stricta subsp. piniformis (Figs. 6h, 8h) and T. tenuifolia (Figs. 6i, 8i) did not differ significantly regarding germination percentage (Figs. 5 and 6) and germination speed index (Figs. 7 and 8) between the two storage temperatures during 180 days of seed conservation. All the species presented germination higher than 75%, with average of 90%, except for T. tricholepis (Fig. 5h), with an average of 45%, and T. tenuifolia, with 60%. Regarding the GSI, the values varied from 3 to 6, with the smallest ones recorded for T. tricholepis and T. tenuifolia, with values of 3 to 3.5 (Figs. 7h, 8i) in this same evaluation period.

However, after 180 days there was an initial reduction of the germination and GSI when the seeds were stored in the freezer (− 5 °C) for the species T. juncea (Figs. 5b, 7b), T. paraensis (Figs. 5c, 7c), T. polystachia (Figs. 5d, 7d), T. streptocarpa (Figs. 5i, 7i), T. streptocarpa (Figs. 5j, 7j), T. chapeuensis (Figs. 6a, 8a), T. geminiflora (Figs. 6c, 8c), T. globosa (Figs. 6d, 8d) and T. tenuifolia var. vaginata (Figs. 6j, 8j), with an absence of germination and hence GSI of zero for T. stricta 2 (Figs. 6g, 8g). In contrast, there was no decrease in viability of the cryopreserved seeds in this same period for all the species.

Nevertheless, in the evaluations of the seeds stored for 450 days, all the species presented significant differences between the two storage temperatures, with drastic declines of the germination and GSI of the seeds kept in the freezer (− 5 °C). For T. paraensis (Fig. 5c), T. polystachia (Fig. 5d), T. tricholepis (Fig. 5h), T. streptocarpa 1 (Fig. 5i), T. streptocarpa 2 (Fig. 5j), T. chapeuensis (Fig. 6a), T. geminiflora (Fig. 6c), T. globosa (Fig. 6d), T. stricta 2 (Fig. 6g). T. tenuifolia (Fig. 6i) and T. tenuifolia var. vaginata (Fig. 6j), the reduction in the germination percentage was greater than 50%, and no T. chapeuensis seed germination of (Fig. 6a), T. globosa (Fig. 6d) and T. stricta 2 (Fig. 6g).

Of the 20 species evaluated, 13 germinated above 80% when preserved in liquid nitrogen for 450 days and five had percentages between 60 and 78%. The lowest germination percentages after cryopreservation were observed for T. stricta 2 (Fig. 6g), with an average of 33%, and T. tricholepis (Fig. 5h), with an average of 38%.

The highest germination speed index (GSI) values after conservation in liquid nitrogen (− 196 °C) for 450 days were observed for T. streptocarpa 2 (6.55) and T. globosa (6.01), and the lowest values were found for T. tricholepsis (2.50) and T. stricta 2 (2.99). For T. chapeuensis (Figs. 6a, 8a), there were increases in the germination and GSI after the seeds were stored in liquid nitrogen when compared to − 5 °C on days 1, 30, 180, and 450 (Figs. 5 and 6). Increases of 10% in germination percentage after cryopreservation were reported for Vriesea bahiana Leme (Silva et al. 2021) and for Encholirum magalhaestii L.B.Sm. and E. subsecundum Mez (Tarré et al. 2007). The increase in germination after cryopreservation of seeds has been reported for various other species (Pence 1991; Salomão 2002; Khanna et al. 2014; Araújo et al. 2019), besides species of Bromeliaceae (Silva et al. 2021). Salomão (2002), analyzing various Cerrado species, reported the influence of cryopreservation on the physical breaking of dormancy by inducing cracks in the endocarp or seed coat immediately surrounding the embryo. Likewise, Khanna et al. (2014), studying several species of Sarracenia, also reported the same observations. However, Araújo et al. (2019) and Silva et al. (2021) reported that the increase is related to the kinetic acceleration of germination of the seeds triggered by their sensitivity to desiccation, thus enabling them to withstand ultralow temperatures.

The longevity of orthodox seeds, when conserved at low temperatures, has been reported for various species, so cryopreservation can be of great importance for long-term storage of orthodox seeds with short lifetimes (Pritchard et al. 1999; Walters et al. 2004; Pritchard 2007; Pritchard and Nadarajan 2008), as we observed for some species of Tillandsia, which after 180 days already presented low viability when stored in a conventional freezer. The longevity of cryopreserved seeds was reported by Walters et al. (2004), who suggested increase of up to 175 times the normal interval when compared to the normal temperature of seed banks. This extended longevity of cryopreserved seeds was previously studied in lettuce by Pritchard (2007), who observed a 74-fold longer period in comparison with storage at temperature of − 18 °C.

The results of storage in a freezer (− 5 °C) made it evident that the decrease in seed viability with longer conservation time can be related to the maintenance of the seeds’ metabolism, leading to the deterioration of the tissue. Voronkova et al. (2018) stated that storage of seeds at room temperature or in places with high humidity, such as freezers, can negatively affect the germination percentage, besides favoring the proliferation of microorganisms. Ultralow temperatures combined with reduced water content depress the metabolism, thus minimizing the deterioration reactions during storage. It is thus necessary to understand the involvement of the process of tolerance to desiccation with the behavior of seeds during storage. According to Li and Pritchard (2009), cryopreservation is particularly important to conserve orthodox seeds with low longevity, which quickly lose viability in conventional germplasm banks.

Conclusion

After conservation for 450 days, there was a reduction of the germination and the GSI of all the species studied when the seeds were preserved in a freezer, with total absence of germination (and thus GSI of zero) for T. chapeuensis, T. globosa and T. stricta 2. Cryopreservation is an efficient strategy to conserve seeds of the 20 Tillandsia species analyzed when desiccated to the water content of approximately 7% (dry mass basis, DMB). Our results support the establishment of a cryobank of Tillandsia that can be used to assure the preservation of endemic species that are vulnerable or in danger of extinction. Based on these results, obtained with a substantial number of species of the Tillandsia genus, including representatives of four subgenera, we believe it would be possible to apply this protocol to the other species of the genus, with emphasis on those that are vulnerable or threatened with extinction, thus preventing loss of important biodiversity.

Acknowledgments

This paper is part of RSO MSc dissertation and this study was financed in part by the Fundação de Amparo a Pesquisa do Estado da Bahia (BOL0177/2018) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior—Brasil (CAPES)—Finance Code 001. The authors would like to thank the support from the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (PROCAD—88887.124186/2014-00). EHS acknowledges the support of CAPES/PNPD/UFRB (88882.315208/2019-01), FVDS currently holds a productivity grant from the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq—304269/2018-2).

Abbreviations

HURB

Herbario do Recôncavo da Bahia

GSI

Germination speed index

MS

Murashige and Skoog (1962) culture medium

Author contributions

RSO, ILS and SOS: data curation, formal analysis, investigation, methodology, visualization, writing—original draft. FVDS and LYSA: conceptualization, funding acquisition, project administration, resources, supervision, writing—review and editing. EHS: conceptualization, formal analysis, funding acquisition, methodology, project administration, resources, supervision, writing—original draft, writing—review and editing.

Funding

There are no financial conflicts of interest to disclose.

Declarations

Conflict of interest

The authors declare that they have no con ict of interest in the publication.

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