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Seeds cryopreservation of Vriesea reitzii Leme & A.F. Costa endemic bromeliad from Atlantic Rainforest1 1 Part of the first Author's Master's Thesis

Criopreservação de sementes de Vriesea reitzii Leme & A.F. Costa bromélia endêmica da Mata Atlântica

Francisco S. Montoya-Serrano Lírio L. Dal Vesco Rosete Pescador About the authors

ABSTRACT

Vriesea reitzii is an endemic bromeliad from the Atlantic Rainforest. The objective of this research was to evaluate the cryopreservation using the method of direct immersion of its seeds, collected from capsules at 120, 135, and 150 days after anthesis (DAA). The water content was determined before cryopreservation, while the germination percentage, germination speed index (GSI), and total soluble carbohydrates were quantified after cryopreservation. The highest percentage of moisture (17.6%) was observed in 120 DAA, while the highest percentage of germination (89.6%) and GSI (17.0) were observed in 150 DAA. Optical and transmission electron microscopy analyses were performed, and no cell damage or changes at the morpho-histological and ultrastructural levels were observed after the cryopreservation process. From these results, V. reitzii seeds can be classified as orthodox seeds and the cryopreservation (+LN) is an efficient tool for an ex situ conservation of this species.

Keywords:
Bromeliaceae; carbohydrate; germination; moisture content; morpho-histology

RESUMO

Vriesea reitzii é uma bromélia endêmica da Mata Atlântica. O objetivo deste trabalho foi avaliar a criopreservação de sementes utilizando o método de imersão direta, coletadas de cápsulas aos 120, 135 e 150 dias após a antese (DAA). O teor de água foi determinado antes da criopreservação. A porcentagem de germinação, o índice de velocidade de germinação (GSI) e os carboidratos solúveis totais foram quantificados após a criopreservação. A maior porcentagem de umidade (17,6%) foi observada aos 120 DAA, enquanto a maior porcentagem de germinação (89,6%) e GSI (17,0) foram observadas aos 150 DAA. Em análises de microscopia ótica e eletrônica de transmissão não foram observados danos celulares ou alterações a nível morfohistológico e ultraestrutural após o processo de criopreservação. A partir desses resultados, as sementes de V. reitzii podem ser classificadas como ortodoxas e a criopreservação (+LN) é uma ferramenta eficiente para a conservação ex situ desta espécie.

Palavras-chave:
Bromeliaceae; carboidratos; germinação; morfohistológia; teor de umidade

Introduction

The Bromeliaceae Juss. Family encompasses 78 genera and 3.336 know species. Also it is considered one of the most diverse in the Atlantic forest (Gouda et al. 2021Gouda, E.J., Butcher, D. & Gouda, C.S. 2021. (cont.updated) Encyclopaedia of Bromeliads, Version 4, Utrecht. Available at: http://bromeliad.nl/encyclopedia/ University Botanic Gardens (access in 09-IV-2021).
http://bromeliad.nl/encyclopedia/...
). The genus Vriesea Lindl. comprises of 231 species, 219 out of a which are endemic in Brazil (Costa et al. 2020Costa, A.F., Moura, R.L., Neves, B., Machado, T.M., Kessous, I.M., Uribbe, F.P., Couto, D.R., Gomes-da-Silva, J. 2020. Vriesea in Flora do Brasil 2020. Jardim Botânico do Rio de Janeiro. Available at: http://floradobrasil.jbrj.gov.br/reflora/floradobrasil/FB6414 (access in 09-IV-2021).
http://floradobrasil.jbrj.gov.br/reflora...
). The genus Vrisea is the richest species in terms of the Atlantic Forest biome, it has 169 species in total. a large number of species (145) are endemic, such as Vriesea reitzii Leme & A.F. Costa, which is currently endangered, as reported in the list of the Rio Grande do Sul State, Brazil (Martinelli et al. 2008Martinelli, G., Vieira, C.M., Gonzalez, M., Leitman, P., Piratininga, A., Costa, A.F.D. & Forzza, R.C. 2008. Bromeliaceae da Mata Atlântica brasileira: Lista de espécies, distribuição e conservação. Rodriguésia 59(1): 209-258.).

Considering the ecological importance of bromeliads and endemism, it is of fundamentally important to establish conservation strategies that ensure the availability of genetic diversity (Costa et al. 2014Costa, A.F., Gomes-Da-Silva, J., Wanderley, M. D.G.L. 2014. Vriesea (Bromeliaceae, Tillandsioideae): taxonomic history, and morphology of the Brazilian lineage. The Journal of the Torrey Botanical Society 141: 338-352., BFG 2015). In this context, available information on ex situ conservation procedures of bromeliad species in Brazil is still incipient, revealing the need to establish effective programs for the conservation of native flora (Rodrigues et al. 2014Rodrigues, A.R.P., Forzza, R.C. & Andrade, A.C.S. 2014. Physiological characteristics underpinning successful cryopreservation of endemic and endangered species of Bromeliaceae from the Brazilian Atlantic Forest. Botanical Journal of the Linnean Society 176(4): 567-578.).

In the effort to protect Bromeliads, several strategies have been used to better understand and support future studies. In V. reitzii, certain studies use biotechnological techniques as conservation tools, such as in vitro cultivation with the nodular cultures (NCs) production. NCs can be considered as a morphogenic response pattern with high regenerative potential since they have the ability to generate more than 5300 micro sprouts g-1 in 10 weeks of cultivation, which can be generated from leaf base (Dal Vesco & Guerra 2010Guerra, M.P. & Dal Vesco, L.L. 2010. Strategies for the micropropagation of bromeliads. In Protocols for in vitro propagation of ornamental plants. Humana Press pp. 47-66., Guerra & Dal Vesco 2010Dal Vesco, L.L. & Guerra, M.P. 2010. In vitro morphogenesis and adventitious shoot mass regeneration of Vriesea reitzii from nodular cultures. Scientia Horticulturae 125(4): 748-755. , Dal Vesco et al. 2014aDal Vesco, L.L., Pescador, R., Corredor-Prado, J.P., Welter, L.J. & Guerra, M.P. 2014a. In vitro propagation of Vriesea reitzii, a native epiphyte bromeliad from the Atlantic rainforest. Acta Scientiarum. Biological Sciences 36(3): 271-278.) or seeds (Dal Vesco et al. 2014bDal Vesco, L.L., Vieira, P.M., Corredor, J.P., Pescador, R., Welter, L.J. & Guerra, M.P. 2014b. Induction and development of nodular cluster cultures in Vriesea reitzii (Leme and Costa), and endangered bromeliad from the Brazilian Atlantic Forest. The Journal of Horticultural Science and Biotechnology 89(5): 542-548.). In addition to the morphological and histochemical characterizations regarding the induction and origin of nodular cultures (Corredor-Prado et al. 2015Corredor-Prado, J.P., Schmidt, E.C., Guerra, M.P., Bouzon, Z.L., Dal Vesco, L.L. & Pescador, R. 2015. Histodifferentiation and ultrastructure of nodular cultures from seeds of Vriesea friburgensis Mez var. paludosa (L.B. Smith) L.B. Smith and leaf explants of Vriesea reitzii Leme e A. Costa (Bromeliaceae). Journal of Microscopy and Ultrastructure 3: 1-10.), proteomic analyzes and identification of differentially expressed proteins involved during the induction and regeneration of nodular cultures (Corredor-Prado et al. 2016Corredor-Prado, J.P., De Conti, D., Cangahuala-Inocente, G.C., Guerra, M.P., Dal Vesco, L.L. & Pescador, R. 2016. Proteomic analysis in the induction of nodular cluster cultures in the bromeliad Vriesea reitzii Leme and Costa. Acta Physiologiae Plantarum 38(5): 130., 2019Corredor-Prado, J.P., DE Conti, D, Júnior, D.R., Cangahuala-Inocent,e G.C., Guerra, M.P., DAL Vesco, L.L. & Pescador, R. 2019. Proteomic identification of differentially altered proteins during regeneration from nodular cluster cultures in Vriesea reitzii (Bromeliaceae). Journal of Plant Growth Regulation 38(2): 586-599.), as well as in the dynamics of changes in the levels of proteins, carbohydrates and global DNA methylation during the induction of NCs. (Corredor-Prado et al. 2020Corredor-Prado, J.P., DE Conti, D., Guerra, M.P., Dal Vesco, L.L. & Pescador, R. 2020. Dynamics of proteins, carbohydrates and global DNA methylation patterns during induction of nodular cluster cultures from seeds of Vriesea reitzii. Acta Scientiarum. Agronomy 42: 42448.).

However, an effective alternative is to establish a cryopreservation protocol, since it allows an effective and safe conservation of materials, suiting the long-term storage system. In these protocols, different materials can be used, for the most diverse plant species, such as seeds, zygotic embryos, apical buds, meristems, and others (Engelmann 2011Engelmann, F. 2011. Use of biotechnologies for the conservation of plant biodiversity. In Vitro Cellular & Developmental Biology-Plant 47(1): 5-16.). Direct cryopreservation is presented as a simple methodology, which consists of direct immersion of fresh or dehydrated plant material in liquid nitrogen (LN). when such material is removal in most cases whit reheating it in water at 40 °C and the subsequent regeneration of the material happens (Popova et al. 2016Popova, E., Kim, H.H., Saxena, P.K., Engelmann, F. & Pritchard, H.W. 2016. Frozen beauty: The cryobiotechnology of orchid diversity. Biotechnology Advances 34(4): 380-403.).

Seed conservation is the most efficient and economical way to store germplasm (Kaviani 2011Kaviani, B. 2011. Conservation of plant genetic resources by cryopreservation. Australian Journal of Crop Science 5(6): 778-800.). This technique has been studied in bromeliads with success in its application. Seeds of six species of the genus Encholirium and two of Dyckia cryopreserved without the use of cryoprotectants maintained the germination percentage after cryopreservation (Tarré et al. 2007Tarré, E., Pires, B.B.M., Guimarães, A.P.M., Carneiro, L.A., Forzza, R.C. & Mansur, E. 2007. Germinability after desiccation, storage and cryopreservation of seeds from endemic Encholirium Mart. ex Schult. and Schult. f. and Dyckia Schult. and Schult. f. species (Bromeliaceae). Acta Botanica Brasilica 21(4): 777-783.), even as for Encholirium spectabile Mart. ex Schult. & Schult.f. seeds, which it was proven that the use of cryoprotectants is not necessary for cryopreservation (Ferrari et al. 2016Ferrari, E.A.P., Colombo, R.C., Faria, R.T. & Takane, R.J. 2016. Cryopreservation of seeds of Encholirium spectabile Martius ex Schultes f. by the vitrification method. Revista Ciência Agronômica 47(1): 172-177.), likewise, seeds of Dyckia brevifolia Baker and Dyckia delicata Larocca & Sobral showed respectively 92% and 79% germination after cryopreservation without cryoprotectants (de Paula et al. 2020De Paula, J.C., Men, G.B., Biz, G., Júnior, W. A. & De Faria, R.T. 2020. Cryopreservation of seeds from endangered Brazilian bromeliads - Dyckia brevifolia Baker and D. delicata Larocca & Sobral. Revista Brasileira de Ciências Agrárias 15(4): 1-8.).

In Bromeliaceae, the use of seeds of some species has shown a high tolerance for drying and freezing with the genera Encholirium Mart. ex Schult. & Schult. f. and Dyckia Schult. & Schult. f. (Tarré et al. 2007Tarré, E., Pires, B.B.M., Guimarães, A.P.M., Carneiro, L.A., Forzza, R.C. & Mansur, E. 2007. Germinability after desiccation, storage and cryopreservation of seeds from endemic Encholirium Mart. ex Schult. and Schult. f. and Dyckia Schult. and Schult. f. species (Bromeliaceae). Acta Botanica Brasilica 21(4): 777-783.), in Pitcairnia albiflos Herbert (Pereira et al. 2010Pereira, A.R., Andrade, A.C.S., Pereira, T.S., Forzza, R.C. & Rodrigues, A.S. 2010. Morphological aspects of seed, germination and storage of Pitcairnia albiflos (Bromeliaceae). Seed Science and Technology 38: 79-87.) and ten endemics and threatened Brazilian species (Rodrigues et al. 2014Rodrigues, A.R.P., Forzza, R.C. & Andrade, A.C.S. 2014. Physiological characteristics underpinning successful cryopreservation of endemic and endangered species of Bromeliaceae from the Brazilian Atlantic Forest. Botanical Journal of the Linnean Society 176(4): 567-578.). This tolerance is related to the content and deposition of reserves, such as carbohydrates, lipids, and proteins, which are constituents of cells and membranes, providing protection during the seed maturation and dehydration phase (Mollo et al. 2011Mollo, L., Martins, M.C., Oliveira, V.F., Nievola, C.C. & Rita De Cássia, L. 2011. Effects of low temperature on growth and non-structural carbohydrates of the imperial bromeliad Alcantarea imperialis cultured in vitro. Plant Cell, Tissue and Organ Culture 107(1): 141-149., Carvalho et al. 2019Carvalho, C.P., Cardoso-Gustavson, P., Rodrigues, E., Braga, MR., Mercier, H. & Nievola, C.C. 2019. Low temperature acclimation and de-acclimation of the subtropical bromeliad Nidularium minutum: Implications of changes in the NO, sugar content and NR activity. Environmental and Experimental Botany 159: 34-43.).

According to Mollo et al. (2011Mollo, L., Martins, M.C., Oliveira, V.F., Nievola, C.C. & Rita De Cássia, L. 2011. Effects of low temperature on growth and non-structural carbohydrates of the imperial bromeliad Alcantarea imperialis cultured in vitro. Plant Cell, Tissue and Organ Culture 107(1): 141-149.), in Alcantarea imperialis (Carrière) Harms (Bromeliaceae), carbohydrates undergo appreciable changes, in adaptations to the cold. The authors describe that the sucrose contents of plants under cold conditions can be exchanged for trehalose, raffinose, and stachyose. They also report that starch concentrations changed with the presence of cold, possibly reduced to carbohydrates associated with tolerance and protection of the plant to cold.

Furthermore, intracellular ice crystal formation is the most damaging event during the cryopreservation process. The damage is irreversible. It can occur during freezing or thawing and may lead to cell wall rupture, epidermal rupture, protoplast egress, anomalous nuclei of surviving explants, or explant death (Kaviani 2011Kaviani, B. 2011. Conservation of plant genetic resources by cryopreservation. Australian Journal of Crop Science 5(6): 778-800.). Anatomical studies, such as the one carried out by Wesley-Smith et al. (2014Wesley-Smith, J., Berjak, P., Pammenter, N. W., & Walters, C. 2014. Intracellular ice and cell survival in cryo-exposed embryonic axes of recalcitrant seeds of Acer saccharinum: an ultrastructural study of factors affecting cell and ice structures. Annals of Botany 113(4): 695-709., 2015Wesley-Smith, J., Walters, C., Pammenter, N. W., & Berjak, P. 2015. Why is intracellular ice lethal? A microscopical study showing evidence of programmed cell death in cryo-exposed embryonic axes of recalcitrant seeds of Acer saccharinum. Annals of Botany 115(6): 991-1000), report the importance of understanding the interactions of water content and ice structure through studies for the atomization of cryogenic processes and cell survival since ice crystal size and cell damage can have late effects on cell death.

In this regard, V. reitzii seeds could be a plausible material for the conservation of germplasm with great genetic diversity and with protocols already established for large-scale propagation. Thus, the objective of the present work was to evaluate the viability, cell integrity, and presence of polysaccharides in V. reitzii cryopreserved and non-cryopreserved seeds using anatomical, biochemical and ultrastructural analyzes.

Material and Methods

Physiologically mature capsules of V. reitzii were collected from a natural population, in the municipality of Curitibanos, Santa Catarina State, Brazil (27º17'02.7"S, 50º32'05.5" W, 900 m altitude The collection was carried out at three different times, established by days after anthesis (DAA), according to the maturation curve, between physiological maturation and dispersion: 1) at 120 DAA, when the seeds reached physiological maturation and at the beginning of the reduction in the content of indoor water; 2) at 135 DAA, intermediate point of the natural dehydration process and; 3) at 150 DAA, beginning of carpel opening for seed release and dispersion. The experiments were conducted in the Laboratory of Developmental Physiology and Plant Genetics (LFDGV), Center of Agricultural Sciences, of the Federal University of Santa Catarina (CCA/UFSC) in Florianópolis, Santa Catarina State, Brazil.

The moisture content (%) of the seeds was obtained from a pool of 1000 seeds of different fruit collection times. The oven method was used at 105 ± 3 ºC for 24 ± 1 hours, according to Brazil (2009Brasil. 2009. Ministério da Agricultura, Pecuária e Abastecimento. Secretaria de Defesa Agropecuária. Regras para análise de sementes. Available at: https://www.gov.br/agricultura/pt-br/assuntos/laboratorios/arquivos-publicacoeslaboratorio/manual-de-sementes-site.pdf/view (access in 20-IV-2020)
https://www.gov.br/agricultura/pt-br/ass...
), using four replications of 200 seeds. The moisture content, expressed on a wet basis, was obtained through the expression:

X%= Iw-FwIwx 100
;

Where: Iw: Initial weight of the sample (g); Fw: Final sample weight (g) and X%: water content in percentage of wet basis (w.b), %.

For the seeds conservation, a 2x3 factorial experimental design was used: Two conservation methods: 1) Cryopreservation (+LN; at -196 °C) and; 2) control, non-cryopreserved (-LN) stored in a refrigerator at 4 °C, combined with three collection times (120; 135 and 150 DAA). Each experimental unit consisted of a cryotube containing 30 seeds and four replicates arranged in random blocks. After 24 h of storage (-196 °C and Control), was performed the immersion in a water bath for two minutes at 42 °C. Afterward, the seeds were disinfected according to the method described by Guerra & Dal Vesco (2010Guerra, M.P. & Dal Vesco, L.L. 2010. Strategies for the micropropagation of bromeliads. In Protocols for in vitro propagation of ornamental plants. Humana Press pp. 47-66.).

The germination test was conducted in vitro and the seeds were placed in Petri dishes (60 x15 mm), on sterilized filter paper, soaked with 2.0 mL of liquid culture medium MS (Murashige & Skoog 1962Murashige, T. & Skoog, F. 1962. A revised medium for rapid growth and biossays with tobacco tissue cultures. Physiologia plantarum 15: 473-497.), and added with Morel vitamins (Morel & Wetmore 1951Morel, G.M. & Wetmore, R.H. 1951. Tissue culture of monocotyledons. American Journal of Botany 38: 138-140.) and 30 g L-1 of sucrose. The pH was adjusted to 5.8 before sterilisation in an autoclave for 20 minutes at 121 °C and 1.3 atm. The cultivations were kept in a BOD chamber at 25 ºC ± 2 ºC for a 16 hours photoperiod of light in a luminous intensity of 55 ± μmol m-2 s-1.

Germination percentage data (%) and germination speed index (GSI) were daily obtained until the 15th day of cultivation. Germinated seeds were considered when they presented embryo swelling, seminal envelope rupture and, light green colour as a sign of active metabolism. The GSI was calculated according to Maguire (1962Maguire, J.D. 1962. Speed of germination-aid selection and evaluation for seedling emergence and vigor. Crop Science 2(2): 176-177.) and the formula:

G S I = ( G i / n i )

where: Gi = number of germinated seeds and; ni = counting day.

The determination of total soluble carbohydrates levels (mg g-1) was based on seed samples (200 mg) of each treatment, previously described, using the phenol-sulfuric method (Dubois et al. 1956Dubois, M., Gilles, K.A., Hamilton, J.K., Rebers, P.T. & Smith, F. 1956. Colorimetric method for determination of sugars and related substances. Analytical chemistry 28(3): 350-356.). The samples, in triplicate, were macerated with liquid nitrogen and transferred to test tubes (15 mL), with an addition of 0.4 mL of ethanol (80% at -22 °C) and kept in a water bath at 70 °C for 5 minutes. The extracts were centrifuged at 3.000 rpm for 10 minutes and after its conclusion, the supernatants were collected. This process was repeated 3 times, followed by filtration with glass wool and had the volume adjusted to 2 mL with ethanol (80% at -22 °C). Afterward, 50 μL of the extract was added to 450 μL of distilled water, 0.5 mL of phenol (5%), and 2.5 mL of sulfuric acid (96%). The total carbohydrate content was estimated from a standard curve, determined based on a standard carbohydrate Glucose and with absorbance measured at 490 nm on a spectrophotometer (Pro-Analysis V-1600)

Representative samples of the seeds were fixed in paraformaldehyde solution in phosphate buffer (2.5%) 0.2M pH 7.2 for 72 hours at 4 °C, for anatomical analysis. The dehydration was in an increasing ethyl series (0-100 °Gl), and soon after the samples were pre-infiltrated in a solution of ethanol (100%) and historesin glycol methacrylate (GMA) (1:1 v v) for 6 hours, infiltrated in Historesin (Leica Historesin, Heidelberg, Germany) and arranged in histo-molds at 35 °C in an oven for 24 hours.

Sections (5 μm) were obtained using a hand-rotating microtome (Leica RM 2135) with tungsten razors. The sections were placed on slides and stained with Toluidine Blue (TBO) to identify acid polysaccharides and Lugol for starch identification. The sections were analysed under an optical microscope (Olympus BX40) and photographed with an image capture system (Olympus Q-Color-3C América Ind.).

Transmission electron microscopy (TEM) analysis was performed from seeds of each treatment, fixed in a glutaraldehyde solution (2.5%) in 0.1 M sodium cacodylate buffer pH 7.2 and 0.2 M sucrose at 4 °C overnight; followed by immersion in 0.3 M sodium cacodylate buffer solution, pH 7.2, 0.6 M sucrose and H2O, five times, reducing the concentration of sucrose in each wash by 25% and in osmium tetroxide (2%) and 0.2 M sodium cacodylate, pH 7.2 (1: 1, v v) for 4 hours at room temperature. The dehydration in an increasing series of acetone (30 - 100 °GL) and pre-infiltration in series of acetone and Spurr resin solutions (3:1, 2:1, 1:1, 1:2, 1:3) and infiltrated in Spurr resin. Sections (60-90 nm) were obtained with a Power Tome XL ultramicrotome diamond razor and contrasted with uranyl acetate (1%) and lead citrate (1%). Images were obtained using a Jeol JEM1011 Transmission Electron Microscope (Tokyo, Japan) at CEML-UFSC, and the relevant events were recorded in micrographs.

Germination percentage data (%) and levels of total soluble carbohydrates (mg g-1MF) were submitted to analysis of variance (ANOVA) and test of comparison of means (Tukey 5%), using the statistical program R (R Development Core Team, 2010R Development Core Team. 2010. R: A language and environment for statistical computing[Computer software]. Vienna, Austria. Available at http://www.R-project.org.
http://www.R-project.org...
).

Results

The different periods of fruit collection, days after floral anthesis (DAA), revealed statistically significant water levels (table 1). The highest (p <0.05) average percentage of water (17.6%) present in the seeds was observed in seeds collected at 120 DAA. Seeds collected at 135 and 150 DAA did not show statistical differences in moisture content.

Table 1.
Moisture content (%) and Effect storage temperature (cryopreserved and non-cryopreserved) and germination (%) from Vriesea reitzii Leme & A.F. Costa seeds collected on different days after anthesis - DAA (120, 135 and 150 days).

The average percentage of seed germination did not present any statistically significant difference when comparing conservation methods, cryopreserved (78.9%), and non-cryopreserved (78.3%) (table 1). Therefore, significant differences in the germination percentages of the seeds were observed in the different DAA. The highest and most significant (p <0.01) average germination percentage (89.6%) was observed with seeds collected at 150 DAA (table 1).

Concerning the germination speed index (GSI), no significant differences were found between cryopreserved (+LN) and non-cryopreserved (-LN) seeds. Seeds submitted to the conservation methods started to germinate on the 7th day of cultivation, and the maximum germination point was observed on the 15th day. The seeds collected at 150 DAA had an average GSI equal to 17.0, being, statistically, the largest and the most significant (p<0,05) when compared to the results of seeds collected at 120 and 135 DAA (table 2).

Table 2.
Germination speed index (GSI) from Vriesea reitzii Leme & A.F. Costa seeds collected on different days after anthesis -DAA (120, 135 and 150 days) and cryopreservation.

The average content of total soluble carbohydrates for V. reitzii seeds did not show a significant difference for the studied factors: collection period, and conservation methodology (table 3).

Table 3.
Average levels of total soluble carbohydrates (mg g-1MF) of Vriesea reitzii Leme & A.F. Costa seeds at three different times of fruit collection (120, 135 and 150 days after anthesis - DAA) and subjected to cryopreservation (+ NL) and non-cryopreserved (-NL).

Anatomical analysis by light microscopy of different days of seed collection (DAA) and exposure to cryopreservation and non-cryopreservation, did not reveal mechanical damage, such as rupture of the cell wall as damage to the membrane system. However, differences were observed in the response to histochemical reactions of the contents of chemical compounds in the regions of the seed coat, the endosperm, the aleurone layer, and the embryo (figure 1).

Figure 1.
Morphological and histological aspects of cryopreserved Vriesea reitzii seeds Leme & A.F. Costa (+LN) and non-cryopreserved (-LN) subjected to double staining of toluidine blue (TBO) and Lugol. a. seed overview, the embryo (arrowhead), plumose appendages (arrow). b. TBO: seed cross section, the cross section of the seed containing the embryo, endosperm structure, aleurone layer (arrowhead) and procambium or nuclei of embryonic stem cells (arrow). c-h. it can observe cell wall of thickened aleurone layer (arrowhead) and compounds starch grains (arrow): c. TBO and Lugol: 120 DAA +LN seed; d. TBO and Lugol: 135 DAA +LN seed; e. TBO and Lugol: 150 DAA +LN seed; f. TBO and Lugol: 120 DAA -LN seeds; g. TBO and Lugol: 135 DAA -LN seeds; h. TBO and Lugol: 150 DAA -LN seeds. Bar: 50 µm. DAA: Days after anthesis. TBO: Toluidine Blue. LN: Liquid nitrogen.

In the micrographs of V. reitzii seeds, seeds with the embryo and plumose appendages at the time of conservation are noteworthy (figure 1a). In the embryo region, a reaction of the acid compounds to the TBO was observed, characterized by the procambium, formed by the nuclei of the embryonic cells, which presented a dark purple colour (figure 1b, see arrow).

The presence of a double seed coat was also observed, with a positive reaction to treatment with TBO, where the external seed coat, called testa, presented a dark green colour indicating the presence of tannic compounds (figure 1b). The internal seed coat, called tegmen, showed a greenish colour indicating phenolic composition and purple coloration in the plumose appendages, indicating rich in pectin composition (figure 1b).

The aleurone layer that surrounds the endosperm and the embryo is presented in a single layer of cells, of a thick wall, with the presence of acid polysaccharides in the cytoplasm and cell wall (figure 1c, see arrow). Concurrent, there was an absence of starch grains in the cytoplasm of the aleurone layer.

It was observed the presence of neutral polysaccharides in the endosperm region, as constituents of the cell wall, indicating the presence of cellulose. And, when stained with Lugol, neutral polysaccharide grains were observed, conferring a high concentration of starch grains distributed in amyloplasts (figure 1d, see arrow). Analyses of 120 DAA seeds, non-cryopreserved (figure 1c) and cryopreserved (figure 1f); 135 DAA non-cryopreserved seeds (figure 1d) and cryopreserved (figure 1g), as well as 150 DAA non-cryopreserved seeds (figure 1e) and cryopreserved (figure 1h), all showed structural integrity after both conservation treatments.

Through TEM, the seed ultrastructure's, observed in cells of the embryo of non-cryopreserved seeds (-LN), were identified with the presence of membranes, organelles, and nucleus in the natural state (figure 2a). Besides, in detail, cells with a large number of reserve structures in lipid bodies (figure 2b) and protein bodies (figure 2c). Additionally, analyses of embryonic cells, from cryopreserved seeds (+LN), presented cell integrity, in other words, the absence of damage, lysis or, injury to membranes, and cell walls (figure 2d). As depicted, in detail, the membranes of the nucleus and cell wall (figure 2e), in addition to the structures of protein and lipid reserves (figure 2f). TEM results added to light microscopy results confirm that the seeds preserved the structural integrity after cryopreservation.

Figure 2.
Transmission electron microscopy (TEM) images of Vriesea reitzii Leme & A.F. Costa seed. a. embryo non-cryopreserved cell structure. Protein bodies (PB), nucleus, and lipid bodies (LB). b. detail of the cell nucleus (N), nucleus membranes (NM) and lipid bodies (LB). c. detail of protein bodies (PB), intercellular space (IS). d. embryo cryopreserved cell structure, nucleus, protein bodies (PB), lipid bodies (LB), intercellular space (IS). e. detail of the cryopreserved cell nucleus, Nucleus (N), nucleus membranes (NM). f. detail of protein bodies (PB) and lipid bodies (LB). PB: protein bodies; LB: lipid bodies; IS, intercellular space; (NM), nucleus membranes; N, nucleus. Bar a,d = 2 µm; b, c, e = 1 µm.

Discussion

The time intervals established in seed collections (DAA), according to the maturation and viability curve, allowed the analyzes entrenched in this work, as well as enabled its use in protocols for multiplication and generation of nodular cultures ( Guerra & Dal Vesco 2010Guerra, M.P. & Dal Vesco, L.L. 2010. Strategies for the micropropagation of bromeliads. In Protocols for in vitro propagation of ornamental plants. Humana Press pp. 47-66., Dal Vesco et al. 2014aDal Vesco, L.L., Pescador, R., Corredor-Prado, J.P., Welter, L.J. & Guerra, M.P. 2014a. In vitro propagation of Vriesea reitzii, a native epiphyte bromeliad from the Atlantic rainforest. Acta Scientiarum. Biological Sciences 36(3): 271-278.). The water content in the seeds of V. reitzii, appraised in the present work, revealed the occurrence of a natural desiccation process, which is directly related to its degree of physiological maturity. Starting with this factor, it can be inferred that the seeds of this species have a characteristic orthodox behaviour. In these species, the seed moisture tends to reduce as the seeds pass through the maturation process in the mother plant, resulting in natural desiccation and conceding tolerance to artificial desiccation (Dousseau et al. 2011Dousseau, S., Alvarenga, A.A., Guimarães, R.M., Lara, T.S., Custódio, T.N. & Chaves, I.S. 2011. Ecophysiology of Campomanesia pubescens seed germination. Ciência Rural 41(8): 1362-1368.). Although the water content can be a variable when classifying orthodox seeds, Engelmann & Dussert (2000) report that the moisture content must remain between 10 to 20%. Walters (2015Walters, C. 2015. Orthodoxy, recalcitrance and in-between: describing variation in seed storage characteristics using threshold responses to water loss. Planta 242(2): 397-406.) indicate that the water content must be below 0.1 g H2O g-1 at the time of conservation, which requires to be achieved by artificial desiccation. Thus, the hypothesis that the seeds of V. reitzii are to be contained in the orthodox category with regards to the percentage of moisture as laid out by these authors, is corroborated.

In this context, it is emphasized the relevance of observing the water content present in the plant material used in the cryopreservation protocols. This content can be decisive when defining strategies to avoid possible damage caused by the crystallization of intracellular water (Vendrame et al. 2014Vendrame, W., De Faria, T., Sorace, M. e Sahyun, S. 2014. Orchid Cryopreservation. Ciência e Agrotecnologia 38(3): 213-229. Available at: https://www.scielo.br/j/cagro/a/vXZWKxQDZH8ZGLjtJXwRsDH/?lang=en (access in 02-X-2019).
https://www.scielo.br/j/cagro/a/vXZWKxQD...
). Factor, particularly, not observed in the current work and, according to ultrastructural analyses, characterized by the absence of lysis or damage to membranes and cell walls. According to González-Arnao & Engelmann (2013González-Arnao, M.T. & Engelmann, F. 2013. Consideraciones teóricas y prácticas para la crioconservación de germoplasma vegetal. América Latina y el Caribe 37.), some low water content in the plant material results in the change of the water phase during the immersion in LN, called the “vitrification” phase. This phase allows the elimination of ice structures by inhibiting the union of water molecules, maintaining the physical properties of an amorphous solid (Kulus & Zalewska 2014Martinelli, G. & Moraes, M.A. 2013. Livro vermelho da flora do Brasil. 1 ed. Rio de Janeiro: Andrea Jakobsson: Instituto de Pesquisas Jardim Botânico do Rio de Janeiro.).

The importance of water content is reported in several studies and for various plant materials, such as seeds of Punica granatum L. (Punicaceae) (Silva et al. 2015Silva, L.M. Mata, M.E. & Duarte, M.E. 2015. Teor de água limite para crioconservação de sementes de romã (Punica granatum L.). Engenharia Agrícola 35(2): 313-321.), seeds of Encholirium spectabile Martius ex Schultes f. (Bromeliaceae) (Ferrari et al. 2016Ferrari, E.A.P., Colombo, R.C., Faria, R.T. & Takane, R.J. 2016. Cryopreservation of seeds of Encholirium spectabile Martius ex Schultes f. by the vitrification method. Revista Ciência Agronômica 47(1): 172-177.), seeds of Pyrostegia venusta (Ker Gawl.) Miers (Salomão et al. 2020Salomão, A., Santos, I.R., José, S.C. 2020. Cryopreservation of Pyrostegia venusta (Ker Gawl.) Miers seeds. Hoehnea 47, e1042019. Available at: https://www.scielo.br/j/hoehnea/a/7fkXJntFZHb4J8CgPztymhy/?format=html⟨=en (access in 25-IV-2021).
https://www.scielo.br/j/hoehnea/a/7fkXJn...
), as well as seeds, pollen, protocorms, structures similar to protocorms, apexes extracted from in vitro plants and meristematic tissues of orchids (Popova et al. 2016Popova, E., Kim, H.H., Saxena, P.K., Engelmann, F. & Pritchard, H.W. 2016. Frozen beauty: The cryobiotechnology of orchid diversity. Biotechnology Advances 34(4): 380-403.).

Cryopreservation has advantages considering the possible storage time, as well as cryopreservation by direct immersion, which is the cheapest in the category by not using cryoprotectants or too many reagents. Concerning the storage of plant material for indefinite periods, cryopreservation in liquid nitrogen (-196 °C) is the only current method (Engelmann 2011Engelmann, F. 2011. Use of biotechnologies for the conservation of plant biodiversity. In Vitro Cellular & Developmental Biology-Plant 47(1): 5-16.). Likewise, cryopreservation has vast advantages when compared to traditional conservation methods (Vendrame et al. 2014Vendrame, W., De Faria, T., Sorace, M. e Sahyun, S. 2014. Orchid Cryopreservation. Ciência e Agrotecnologia 38(3): 213-229. Available at: https://www.scielo.br/j/cagro/a/vXZWKxQDZH8ZGLjtJXwRsDH/?lang=en (access in 02-X-2019).
https://www.scielo.br/j/cagro/a/vXZWKxQD...
).

In the present study, it was observed that the anticipation of seed collection, at 120 DAA, resulted in a lower average germinative percentage (70%), when compared to seeds collected in the desiccation period (89.6%), at 150 DAA (table 1). Another important factor observed was that the cryogenic process did not affect the germinative power of the seeds. Yet, the moisture content was a key factor in survival and germination. Likewise, the cryopreservation of Ipê-Roxo (Tabebuia impetiginosa (Mart. ex DC.) Standl.) seeds, with the moisture content of 12.5%, 8.4%, and 4.2%, maintained vigour and germination capacity. However, at 18.3% of moisture content, there was a decline in germination (Martins et al. 2009Martins, L., Lago, A.A., Andrade, A.C. & Sales, W.R.M. 2009. Conservação de sementes de ipê-roxo (Tabebuia impetiginosa (Mart. ex DC.) Standl.) em nitrogênio líquido. Revista Brasileira de Sementes 31(2): 71-76.).

Furthermore, there are reports of different species works that show gains in the germinative percentage of seeds after cryopreservation, of which, the cooling process at ultra-low temperatures can act to break seed dormancy. As an example, Rocha et al. (2009Rocha, M.D.S., Cavalcanti Mata, M.E., Carvalho, J.M. & Lopes, K.P. 2009. Crioconservação de sementes de algodão. Revista Brasileira de Engenharia Agrícola e Ambiental 13(3): 312-318.) found in their studies that the cryopreservation process of Gossypium hirsutum L. seeds promoted an increase in the percentage of seed germination and seedling vigor, after going through the cryopreservation process. Cryopreservation also advanced a higher germination percentage of E. subsecundum (Baker) Mez seeds with a moisture content of 12.7% (Tarré et al. 2007Tarré, E., Pires, B.B.M., Guimarães, A.P.M., Carneiro, L.A., Forzza, R.C. & Mansur, E. 2007. Germinability after desiccation, storage and cryopreservation of seeds from endemic Encholirium Mart. ex Schult. and Schult. f. and Dyckia Schult. and Schult. f. species (Bromeliaceae). Acta Botanica Brasilica 21(4): 777-783.).

From these results, it can be inferred that the period of fruit collection and seed extraction in V. reitzii is an important factor to observed. Also, the highest percentage of germination and GSI are related to lower water content, greater accumulation of reserves, and embryo maturity. These factors were also observed in the germination in Moringa oleifera Lam. seeds (Agustini et al. 2015). Data that corroborate with those observed by Guimarães & Barbosa (2007Guimarães, D.M. & Barbosa, J.M. 2007. Coloração dos frutos como índice de maturação para sementes de Machaerium brasiliensi Vogel (Leguminosae - Fabaceae). Revista Brasileira de Biociências 5(2): 567-569.), in which the increase in GSI in Machaerium brasiliensi Vogel seeds is related to the fruit maturation process. Therefore, the natural desiccation determines the quality and vigour of the seeds, an important factor to be considered in conservation programs, such as the use in cryopreservation.

Carbohydrates, on the other hand, are associated with the seed's capacity to withstand desiccation and storage at negative temperatures, among other structural and biochemical characteristics. For Mollo et al. (2011Mollo, L., Martins, M.C., Oliveira, V.F., Nievola, C.C. & Rita De Cássia, L. 2011. Effects of low temperature on growth and non-structural carbohydrates of the imperial bromeliad Alcantarea imperialis cultured in vitro. Plant Cell, Tissue and Organ Culture 107(1): 141-149.), the carbohydrates present in the seeds of Alcantarea imperialis (Carrière) Harms (Bromeliaceae), trigger mechanisms of adaptation to the cold, when seeds were submitted to low temperatures in growth chambers (5 ± 2 °C, 15 ± 2 °C, 15 °C dark / 30 °C light and 30 ± 2 °C, photoperiod 12-h). According to these authors, the sucrose contents in plants, at cold conditions, can be exchanged for trehalose, raffinose, and stachyose. Moreover, the starch concentrations changed with the presence of cold, possibly reduced to carbohydrates associated with the plant's tolerance and protection from cold (Mollo et al. 2011Mollo, L., Martins, M.C., Oliveira, V.F., Nievola, C.C. & Rita De Cássia, L. 2011. Effects of low temperature on growth and non-structural carbohydrates of the imperial bromeliad Alcantarea imperialis cultured in vitro. Plant Cell, Tissue and Organ Culture 107(1): 141-149.). Hence, it can be inferred that a longer permanence of the fruits connected to the mother plant, after anthesis, represents a greater accumulation of carbohydrates, since, according to the results of the present study, the higher the DAA for the fruit collection, the greater will be the loss of water in the seeds.

It was observed, in the present study, that cryopreserved seeds, when compared to those non-cryopreserved, do not show structural or biochemical differences, thus, corroborating that cryopreservation did not affect the seeds. Seen in these terms, non-reducing sugars, sucrose, and some oligosaccharides are important for the protection of membranes against the formation of crystals at the moment of freezing, as well as the accumulation of lipid bodies and starch grains (Zaritzky 2015Zaritzky, N.E. 2015. The Role of Water in the Cryopreservation of Seeds. In: Water Stress in Biological, Chemical, Pharmaceutical and Food Systems. New York: Springer, pp. 231-244.). In embryonic corn cells, protein bodies were also observed (Wen 2009Wen, B., Wang, R.L. & Song, S.Q. 2009. Cytological and physiological changes related to cryotolerance in orthodox maize embryos during seed development. Protoplasma 236(1-4): 29-37.), while in the embryonic cells of Livistona chinensis (Jacq.) R.Br. ex Mart., the accumulation of these substances was not detected, probably because they are considered recalcitrant seeds (Wen 2010Wen, B. 2010. Cytological and physiological changes related to cryotolerance in recalcitrant Livistona chinensis embryos during seed development. Protoplasma 248(3): 483-491.). Therefore, it was observed, concerning the present study, that the seeds of V. reitzii have reserve organelles in the form of lipid bodies, protein bodies, and orthamyloplasts, which can confer tolerance to desiccation and cooling and also reaffirm the orthodox behaviour of the seeds.

Cryopreservation is the only technique that guarantees the conservation of genetic material, theoretically for an indefinite period, safely and economically. (Engelmann 2011Engelmann, F. 2011. Use of biotechnologies for the conservation of plant biodiversity. In Vitro Cellular & Developmental Biology-Plant 47(1): 5-16.). In liquid nitrogen storage (-196 °C), all metabolic activity and cell divisions are interrupted, preventing the degradation of the stored material and contamination (Cruz -Cruz et al. 2013Cruz-Cruz, C. A., González-Arnao, M. T., & Engelmann, F. 2013. Biotechnology and conservation of plant biodiversity. Resources 2(2): 73-95.). Direct immersion in liquid nitrogen has the advantage of not requiring the use of cryoprotectants in materials with tolerance to cooling and low moisture content, as seen in the present study, and with several examples, such as the one seen for Dyckia brevifolia Beker and Dyckia delicata Larocca & Sobral seeds (De Paula et al. 2020De Paula, J.C., Men, G.B., Biz, G., Júnior, W. A. & De Faria, R.T. 2020. Cryopreservation of seeds from endangered Brazilian bromeliads - Dyckia brevifolia Baker and D. delicata Larocca & Sobral. Revista Brasileira de Ciências Agrárias 15(4): 1-8.), mature seeds of Cattleya guttata Lindl (Vettorazzi et al. 2019Vettorazzi, R. G., Carvalho, V. S., Teixeira, M. C., Campostrini, E., Da Cunha, M., de Matos, E. M., & Viccini, L. F. 2019. Cryopreservation of immature and mature seeds of Brazilian orchids of the genus Cattleya. Scientia Horticulturae 256: 108603.) and for Passiflora edulis Sims seeds (Generoso et al. 2019Generoso, A. L., Carvalho, V. S., Walter, R., Campbell, G., da Silva Araújo, L., Santana, J. G. S., & da Cunha, M. 2019. Mature-embryo culture in the cryopreservation of passion fruit (Passiflora edulis Sims) seeds. Scientia Horticulturae 256: 108638.). Although the present study hasn't observed a difference between the storage treatments, it is assumed that cryopreservation has more long-term advantages when compared to storage in a refrigerator, in which there may be degradation and contamination of the material in the medium term.

Conclusions

In conclusion, the method of immersing the seeds in liquid nitrogen for a period of 24 hours is sufficient to promote the interruption of metabolism. Hence, one can infer the tolerance potential and its indefinite storage projection. The use of the cryopreservation method for V. reitzii seeds, with direct immersion in liquid nitrogen, revealed to be an effective mechanism for long-term ex situ conservation system. Accentuating, however, the importance of the seed maturity state and water content, as important factors for the success in the conservation process. Moreover, V. reitzii seeds have a natural desiccation process in the mother plant and present the ability to tolerate desiccation and storage, key features to be classified as orthodox seeds.

Acknowledgements

The authors thank Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Fundação de Amparo à Pesquisa e Inovação do Estado de Santa Catarina (FAPESC), for fellowships, research grants, and financial support. We also thank Laboratório Central de Microscopia Eletrônica (LCME-UFSC), where part of the work was developed through technical analysis support.

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  • 1
    Part of the first Author's Master's Thesis

Associate Editor

Nelson Augusto dos Santos Júnior

Publication Dates

  • Publication in this collection
    02 Sept 2022
  • Date of issue
    2022

History

  • Received
    24 Jan 2021
  • Accepted
    09 Dec 2021
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