SPore viability of fern SPecieS of the genera

amauroPelta, blechnum, and PhySematium from

central argentina after low temPerature Storage

viabilidad de eSPoraS de helechoS de loS géneroS amauroPelta,

blechnum y PhySematium del centro de argentina luego del

almacenamiento a baja temPeratura

1

2,3

1

& M. Luján Luna *

M. Luz Martinenco , Marcelo D. Arana

Summary

Background and aims: Ferns are sensitive to environmental changes, inhabiting

ecosystems susceptible to degradation. Spore banks and in vitro spore culture are

useful tools employed in their conservation. The objective was to test the ability of

the spores to germinate after being stored in a freezer and to develop gametophytes

and sporophytes, in native fern species of genera Amauropelta, Blechnum and

Physematium.

1

.

Laboratorio de Anatomía

Comparada, Propagación

y

Conservación de Embriofitas,

Facultad de Ciencias Naturales y

Museo, Universidad Nacional de La

Plata, La Plata, Argentina

M&M: Spores of A. argentina, B. auriculatum and P. montevidensis were stored at

2

. Grupo GIVE, Departamento de

-20 ºC in dry conditions for 6 and 12 months. They were sown in vitro in Dyer liquid

Ciencias Naturales, Facultad de

Ciencias Exactas, Físico-Químicas y

Naturales, Instituto ICBIA (UNRC-

CONICET), Universidad Nacional de

Río Cuarto, Río Cuarto, Córdoba,

Argentina

medium and incubated in a growth chamber. The means of germination percentages

were calculated and a one-way Student’s t-test was employed. Gametophyte and

sporophyte development was registered under light and stereoscopic microscopes.

Results: Depending on the species, statistical diﬀerences were recorded in

germination percentages (viability) between both storage periods. The spores of A.

argentina kept in freezer for 12 months completely lost viability. In B. auriculatum,

viability decreased over time, and in P. montevidensis it remained constant.

Gametophytes and sporophytes developed in all cultures; except in A. argentina

spores, with 12 months in freezer.

3

. Instituto Criptogámico, Área

Botánica, Fundación Miguel Lillo,

San Miguel de Tucumán, Tucumán,

Argentina

Conclusions: The viability of the spores under dry storage at low temperature could be

conditioned by the ecological requirements of the species. Protocols used for spore

storage and culture allowed obtaining gametophytes and sporophytes in trials.

*lujanluna@fcnym.unlp.edu.ar

Citar este artículo

Key wordS

MARTINENCO, M. L., M. D.

ARANA & M. L. LUNA. 2024. Spore

viability of fern species of the

genera Amauropelta, Blechnum

and Physematium from central

Argentina after low temperature

storage. Bol. Soc. Argent. Bot. 59:

Amauropelta, Blechnum, ex-situ conservation, fern, freezing, Physematium, spore

longevity.

reSumen

Introducción y objetivos: Los helechos son sensibles a cambios ambientales,

habitando en ecosistemas susceptibles a degradación. Los bancos de esporas y el

cultivo in vitro son herramientas útiles para su conservación. El objetivo fue probar

la capacidad de las esporas para germinar luego del almacenamiento en freezer

y de desarrollar gametoﬁtos y esporoﬁtos, en especies de helechos nativos de los

géneros Amauropelta, Blechnum y Physematium.

M&M: Esporas de A. argentina, B. auriculatum y P. montevidensis se almacenaron

deshidratadas a -20 ºC durante 6 y 12 meses. Se sembraron in vitro en medio

Dyer líquido y se incubaron en estufa. Se calcularon las medias de los porcentajes

de germinación y se empleó una prueba t Student unidireccional. El desarrollo de

gametoﬁtos y esporoﬁtos fue registrado con microscopio óptico y lupa.

2

99-309.

DOI: https://doi.

org/10.31055/1851.2372.v59.

n3.44658

Resultados:Seregistrarondiferenciasestadísticasenlosporcentajesdegerminación

(viabilidad) entre ambos períodos de almacenamiento. Las esporas de A. argentina

mantenidas durante 12 meses en freezer perdieron completamente la viabilidad.

En B. auriculatum, la viabilidad disminuyó con el tiempo y en P. montevidensis

que permaneció constante. Se desarrollaron gametoﬁtos y esporoﬁtos en todos los

cultivos; excepto en A. argentina almacenadas 12 meses en freezer.

Conclusiones: La viabilidad de esporas almacenadas deshidratadas a baja

temperatura podría estar condicionada por los requerimientos ecológicos de las

especies. Los protocolos utilizados para el almacenamiento y cultivo de esporas

permitieron obtener gametoﬁtos y esporoﬁtos en todos los ensayos.

Recibido: 4 Abr 2024

Aceptado: 7 Jul 2024

Publicado en línea: 30 Sep 2024

Publicado impreso: 30 Sep 2024

Editor: Gonzalo J. Márquez

PalabraS clave

Amauropelta, Blechnum, congelación, conservación ex situ, helecho, longevidad de

espora, Physematium.

ISSN versión impresa 0373-580X

ISSN versión on-line 1851-2372

299

Bol. Soc. Argent. Bot. 59 (3) 2024

introduction

(most common 20 ℃, 5 ℃, -20 ℃ or -196 ℃ in

liquid nitrogen) (Pence, 2002, 2008; Quintanilla

Ferns are one of the oldest lineages of vascular et al., 2002; Aragón & Pangua, 2004; Ballesteros

plants, highly sensitive to environmental changes. et al., 2006, 2012; Gabriel y Galán & Prada, 2011;

They usually inhabit ecosystems susceptible to Mikula et al., 2009, 2015; Vargas & Droste, 2014;

degradation, so their protection and conservation Tomiczak et al., 2023). Usually, the viability of fern

contribute to ensuring the maintenance of these spores is retained when they are stored in dry and

ecosystems and the biodiversity therein (Ballesteros cold conditions (Pence, 2008; Gabriel y Galán &

et al., 2006; Arcand & Ranker, 2008; Mehltreter, Prada, 2010).

2

010). Ferns have diverse ecological roles and many

As part of a project that aims to investigate

species are of economic interest as ornamental, the optimal methods for ex situ conservation

food or medicinal plants (Mannan et al., 2008; of ferns that inhabit Argentina, we began our

Lee & Shin, 2010; Sharpe et al., 2010; Liu et al., studies in the central region of this country, one

2

012; Goswami et al., 2016; Huang et al., 2019). of the four centres of fern diversity in Southern

Furthermore, various fern taxa are considered as Cone (Arana et al., 2013). In this area, fern

indicators of environment disturbance, climate populations are concentrated in the mountain

changes and pollution (Della & Falkenberg, 2019).

systems (called “Sierras Pampeanas centrales”),

Procedures for biodiversity plant conservation which act as islands immersed in pampean plains

can be in situ (protection of natural habitats), or (Arana & Ponce, 2004; Oggero & Arana, 2012;

ex situ (botanic gardens, germplasm banks, in Arana et al., 2021). Despite habitat heterogeneity,

vitro culture), both not mutually exclusive but the biota inhabiting isolated mountain systems is

complementary (Dyer, 1994; Ibars & Estrelles, less likely to survive catastrophic events (Taylor &

2

012; Breman et al., 2021). Fern spore banks Kumar, 2016). In central Argentina, the mountain

(

analogous to seed banks for angiosperms) are systems are frequently disturbed by ﬁres which

an ex-situ conservation tool that allows storing cause strong impacts on species composition,

large quantities of germplasm with high genetic vegetation structure, and biogeochemical and

variation in a small space (Dyer, 1994; Pence, hydrological cycles (Whelan, 1995; Morgan et al.,

2

008; Ibars & Estrelles, 2012; Ballesteros & 2001; Argarañaz et al., 2020).

Pence, 2018). Ballesteros et al. (2006) claimed The objective of the present work was to test

that the spores of ferns that inhabit threatened the ability of the spores to germinate after being

ecosystems should be included in germplasm stored in a freezer and to develop gametophytes

banks for experimental purposes and for long-term and sporophytes, in native fern species of genera

biodiversity conservation.

Amauropelta, Blechnum, and Physematium

The sporophytes of most ferns produce large from central Argentina with diﬀerent ecological

quantities of spores that are readily available every requirements.

year, and their viability plays an important role for

the successful establishment of individuals and

populations in new habitats (Dyer, 1994; Sheﬃeld, materialS and methodS

1

996). Due to their desiccation tolerance, spores

can be stored for long periods of time at low

temperatures maintaining their viability (Dyer, (Hieron.)Salino&T.E.Almeida(Thelypteridaceae),

979; Ballesteros, 2010). The reduction in spore Blechnum auriculatum Cav. (Blechnaceae), and

Three taxa were studied: Amauropelta argentina

1

viability can be measured by the decrease in Physematium montevidensis (Spreng.) Shmakov

germination percentage over time (Gabriel y Galán (Woodsiaceae). Materials were collected in four

&

Prada, 2010). localities of the Chaco serrano district, Chaco

Various works address the analysis of the optimal biogeographic province, Neotropical region: Las

conditions to preserve the viability of the spores Jarillas (31° 32´ 03.16´´ S, 64° 32´ 14.98´´ W), Atos

over time, using variables as wet or dry storage Pampa (31° 57´ 49.34´´ S, 64°40´13.19´´ W), Las

(i.e. spores stored in capsules with mineral agar vs. Albahacas (32° 53´ 59.02´´ S, 64° 50´´ 17.97´´ W),

kept in Eppendorf tubes), or diﬀerent temperatures and Achiras (33° 09´ 07.38´´ S, 64° 59´ 32.78´´W).

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M. L. Martinenco et al. - Viabilidad de esporas de helechos del centro de Argentina

The characteristic vegetation of the district is the 28 µmol m-2 s-1, at 20 ± 2 °C. The cultures were

xerophytic forest alternating with shrublands. The examined every 2-3 days under a stereoscopic

analysed taxa occur in diﬀerent microhabitats: microscope Nikon SMZ 1000.

A. argentina is a hygrophilous species that lives

The diﬀerent stages of gametophyte development

associated with water courses, usually along stream were photographed employing a Nikon E200 light

banks, whereas B. auriculatum and P. montevidensis microscope (LM). For this, samples were placed

grow in xeric microhabitats of deep cracks and rock on microscopic slides and mounted in water. When

crevices (Fig. 1A-D). Voucher specimens were the gametophytes reached the cordiform phase

deposited in the herbarium RCVC of the National they were transferred to Petri dishes containing

University of Río Cuarto, Argentina (Thiers, 2024). previously autoclaved soil extracted from the

natural habitats. Cultures were kept in the growth

Spore storage conditions

chamber until sporophyte emergence.

Portions of fronds with mature closed sporangia

were taken from ten individuals per species and Statistical analysis

transported to the laboratory in paper envelopes.

First, the means of germination percentages were

Materials were air-dried at room temperature (~24 calculated in the three fern species at the two cold

°

C) and ambient RH until spore release. Spores storage conditions and then, a one-way Student’s

were ﬁltered using a mesh with pores 88 µm in t-test was employed to determine if the diﬀerences

diameter, in order to eliminate remnants of indusia, in percentages were statistically signiﬁcant at the

scales, sporangia or leaf material. The spores were distinct storage times. A Student’s test calculator

kept for 2 months in a refrigerator (4 ºC) before (InfoStat) was used to calculate the T-value, which

sowing. To test the viability of the spores before can be employed to compare the variability of data

their storage at low temperature, samples were and to determine whether means were signiﬁcantly

sown in vitro following the methodology described diﬀerent or not. Last, p-values were calculated

below. The spores were considered viable when using the diﬀerence in mean at the 5% signiﬁcance

they germinated (rupture of the spore wall and level, for our data from 6 and 12 months of storage

emergence of the ﬁrst rhizoid).

in each species.

Once the viability of the fresh spores was

veriﬁed, subsamples were placed in Eppendorf

tubes, labelled and stored in a freezer at -20 ºC. reSultS

The viability of the spores was evaluated at two

storage times: 6 and 12 months. Six replicates for

Fresh spores germinated quickly, between 5

each storage period per species were employed. days (Blechnum auriculatum) and 7-9 days after

The ﬁnal spore viability was calculated according sowing (Amauropelta argentina and Physematium

to Quintanilla et al., (2002) and Aragon & Pangua montevidensis) (Table 1). In the frozen materials,

(2004), by analysing the germination percentage a variation in germination time was recorded. The

after 30 days from spore sown (no further spores of B. auriculatum stored for 12 months in

germination was observed after). Germination freezer took longer to germinate than those kept for

percentage was calculated from a random count of 6 months, whereas in P. montevidensis germination

1

00 spores from each dish (following Aragón & occurred in both trials 15 days after sown. The

Pangua, 2004).

spores of A. argentina completely lost viability

after 12 months of storage at -20 ℃.

In vitro spore culture and gametophyte development

There were signiﬁcant diﬀerences in germination

The spores were cultured in previously percentages between the two storage periods

autoclaved (120 °C for 20 minutes) Petri dishes 9 in freezer depending on the species analysed

cm in diameter, containing Dyer liquid medium (Table 2). B. auriculatum showed a greater spore

(Dyer, 1979). Spores were not disinfected before germination percentage after a 6-months of

sowing. The capsules where sealed with Paraﬁlm storage in comparison with A. argentina and P.

and placed in a growth chamber with a photoperiod montevidensis. The storage at low temperature

of 12 hours, under white ﬂuorescent illumination during 12 months damaged or reduced the viability

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Bol. Soc. Argent. Bot. 59 (3) 2024

Fig. 1. Sampling area and individuals in their habitats. A: Typical landscape of the Chaco serrano district. B:

individual of Amauropelta argentina growing on a stream bank. C: population of Blechnum auriculatum growing

among rocks. D: individuals of Physematium montevidensis with rupicolous habit. Scales= B-D: 20 cm.

of the spores (expressed as germination percentage) 2-3 cells long (Fig. 2A-B). During the laminar

in A. argentina and B. auriculatum, meanwhile phase, trichomes developed ﬁrst on the margins

storage period did not affect the germination and then on the gametophyte surface (Fig. 2C-F).

percentage in P. montevidensis.

Both smaller elongated and larger symmetrical

Gametophyte and sporophyte development gametophytes were recorded in the cordiform phase

was registered in all cultures, except for those (Fig. 2G-J). Smaller ones were unisexual male

corresponding to the spores of A. argentina kept (Fig. 2H-I), whereas larger gametophytes were

for 12 months in the freezer. The morphological bisexual (Fig. 2J-K). In some instances, branched

characteristics of the gametophytes, their sexual male gametophytes were observed (Fig. 2L).

expression and the emergence of the sporophytes Sporophytes developed in trials where fresh spores

are described in Table 1 and Figs. 2-4. The and those stored in a freezer for 6 months were

developmental characteristics of gametophytes and used (Fig. 2M-N). In B. auriculatum, the ﬁlaments

sporophytes for each species were similar in all were 3-5 cells long and some of them developed a

trials (i.e. fresh spores and under diﬀerent storage terminal trichome (Fig. 3A-B). The bi-dimensional

periods). Brieﬂy, in A. argentina the ﬁlamentous growth phase showed elongated gametophytes that

gametophyte phase consisted of short ﬁlaments bore trichomes on the margins and on the prothallus

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M. L. Martinenco et al. - Viabilidad de esporas de helechos del centro de Argentina

Table 1. Spore germination, gametophyte and sporophyte development in Amauropelta argentina,

Blechnum auriculatum, and Physematium montevidensis laboratory trials. References= GCP:

gametophyte cordiform phase; GFP: gametophyte ﬁlamentous phase; GLP: gametophyte laminar phase;

SG: spore germination.

Species

Fresh spores

Spores 6 months storage Spores 12 months storage

SG: 8-9 days from sown. TG: 60 %

SG: 10 days from sown.

Spores did not germinate

GFP: 2-5 days from SG.

Filaments 2-3 cells long.

GFP: 4-6 days from SG.

Filaments 2-3 cells long.

GLP: 5 days from SG.

GLP: 6 days from SG.

Trichomes on the margins.

GCP: 24 days from SG.

Trichomes on the margins

and the prothallus surface.

GCP: 20 days from SG.

Trichomes on the margins

and the prothallus surface.

Amauropelta

argentina

Antheridia: 50 days from SG.

Archegonia: 60 days from SG.

Antheridia: 60 days from SG.

Archegonia: 67

days from SG.

Gametophyte sexuality: bisexual

and male unisexual.

Gametophyte sexuality:

bisexual and male unisexual.

Sporophyte: 90-100 days from SG.

Sporophyte: 120

days from SG.

SG: 5 days from sown. TG: 70%

SG: 6 days from sown.

SG: 14 days from sown.

GFP: 2-6 days from SG.

Filaments 3-5 cells long, some

with terminal trichome.

GFP: 2-7 days from SG.

Filaments 3-5 cells long,

some with terminal trichome. some with terminal trichome.

GFP: 2-7 days from SG.

Filaments 3-5 cells long,

GLP: 7 days from SG.

Trichomes on the margins.

GCP: 18 days from SG.

Trichomes on the margins

and the prothallus surface.

GCP: 17 days from SG.

Trichomes on the margins

and the prothallus surface.

GCP: 18 days from SG.

Trichomes on the margins

and the prothallus surface.

Blechnum

auriculatum

Antheridia: 40 days from SG.

Antheridia: 75 days from SG. Antheridia: 120

days from SG.

Archegonia: 48 days from SG.

Archegonia: 78

days from SG.

Archegonia: 140

days from SG.

Gametophyte sexuality: bisexual

and male unisexual.

Gametophyte sexuality:

bisexual and male unisexual. bisexual and male unisexual.

Gametophyte sexuality:

Sporophyte: 60 days from SG.

Sporophyte: 90

days from SG.

Sporophyte: 210

days from SG.

SG: 6-8 days from sown. TG: 90%

SG: 15-26 days from sown.

SG: 15-30 days from sown.

GFP: 2-4 days from SG.

Filaments 3-5 cells long.

GFP: 2-5 days from SG.

Filaments 3-5 cells long.

GFP: 2-7 days from SG.

Filaments 3-5 cells long.

GLP: 10 days from SG.

GLP: 8 days from SG.

Trichomes on the margins.

GCP: 25 days from SG.

Trichomes on the margins

and the prothallus surface.

GCP: 30 days from SG.

Trichomes on the margins

and the prothallus surface.

GCP: 30 days from SG.

Trichomes on the margins

and the prothallus surface.

Physematium

montevidensis

Antheridia: 25 days from SG.

Archegonia: 35 days from SG.

Antheridia: 43 days from SG. Antheridia: 35 days from SG.

Archegonia: 49

days from SG.

Archegonia: 38

days from SG.

Gametophyte sexuality: bisexual

and male unisexual.

Gametophyte sexuality:

bisexual and male unisexual. bisexual and male unisexual.

Gametophyte sexuality:

Sporophyte: 52 days from SG.

Sporophyte: 60

days from SG.

Sporophyte: 65

days from SG.

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Bol. Soc. Argent. Bot. 59 (3) 2024

Table 2. Signiﬁcance of storage time on spore germination percentage using Student’s t-test.

References= n: number of capsules. In each one, 100 spores were counted. Porcentage germination

(mean±standar error). T: t-valor. **: p>0,05.

Storage time

of 6 months

Storage time

of 12 months

Species

Source variation

n

T

p-valor

Amauropelta argenina

Blechnum auriculatum

Physematium montevidensis

% of germination

6

30±7

72±3

40±7

0

10,39

26,37

0,32

0,000

30±3

40±7

0,7537**

surface (Fig. 3C-E). The cordiform gametophytes of viable spores after 12 months of storage, as

were mostly elongated throughout their development occurred in our cultures of B. auriculatum. In the

(Fig. 3F-I). Both unisexual male (Fig. 3G-H) and case of P. montevidensis, this percentage remained

bisexual gametophytes were recorded, the latter constant over storage time.

with a wide notch behind which the gametangia

By other hand, the spores of the hygrophilous

diﬀerentiated (Fig. 3I-K). Sporophytes developed species A. argentina completely lost viability when

in all cultures using fresh spores as well as those they remained 12 months stored in the freezer. Our

stored in freezer (Fig. 3L-N). The ﬁlamentous phase results agree in general with those of Quintanilla et

of P. montevidensis gametophytes consisted of al. (2002), who found in other hygrophilous ferns

ﬁlaments 4-5 cells long, with or without a terminal that dry storage killed the spores after 6 months at

trichome (Fig. 4A). During the laminar phase, more -20 °C. According to these authors, hygrophilous

trichomes developed on the margins and on the species require high levels of soil moisture and

gametophyte surface (Fig. 4B-C). As occurred in relative humidity, thus wet storage seems more

the other analysed species, a sexual dimorphism was appropriate for them. In our study, the spores of

observed being the male gametophytes smaller in A. argentina remained viable for up to 6 months

size and cordiform elongated, whereas bisexual ones of dry storage in the freezer, and gametophytes

were larger and symmetrical cordiform (Fig. 4D-F). and sporophytes developed in these cultures. This

Sporophytes developed in all cultures (Fig. 4G).

indicates that the spores of A. argentina resist

desiccation to a certain period of time. Ballesteros

et al. (2012) also observed in some fern taxa, that

shortest lived spores belonged to species from

riparian or shaded forests of the Iberian Peninsula.

diScuSSion and concluSionS

The spores of Blechnum auriculatum and As Aragón & Pangua (2004) suggested for other

Physematium montevidensis maintained their ferns, the ecological requirements of this species

viability for up to 12 months when they were stored seem to have inﬂuenced to certain extent spore

in the freezer. Both fern species completed the life viability.

cycle in laboratory conditions and sporophytes

Various studies have shown the beneﬁcial eﬀects

developed in all essays (after 6 and 12 months of of preserving spores or tissues in liquid nitrogen,

storage). Quintanilla et al. (2002) analysed spore in particular to preserve endangered fern species

germination in ﬁve threatened ferns after diﬀerent (Mikula et al., 2009; Ballesteros, 2012; Pence,

storage conditions, and stated that dry storage at 2015; Filipin et al., 2016; Pence, 2018; Tomiczak

-20 °C or 5 °C is an eﬀective technique to maintain et al., 2023). However, for other purposes, storage

spore viability, requiring low preparation time and in a freezer is much less expensive and requires less

storage space. Aragón & Pangua (2004) also found preparation time. Our ﬁndings demonstrate that the

dry storage at -20 °C fairly eﬀective to maintain spores of B. auriculatum and P. montevidensis can

spore viability in rupicolous taxa of Asplenium. be stored in freezer (at -20 °C) for at least one year,

These authors reported a decrease in the percentage whereas in the case of A. argentina, new essays

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M. L. Martinenco et al. - Viabilidad de esporas de helechos del centro de Argentina

Fig. 2. Amauropelta argentina gametophyte and sporophyte development in cultures of spores stored 6 months

in freezer. A: Earlier stages of gametophyte development. Some spores have not germinated. B: Three-

celled ﬁlamentous gametophyte. C: Gametophyte at the beginning of the laminar phase. D: Gametophyte

slightly wider after successive mitotic divisions. E: Laminar phase (left) and early cordiform phase (right). F:

Detail of gametophyte margin with a unicellular glandular trichome. G: Cordiform gametophyte phase with

scarce marginal trichomes. H: Elongated cordiform male gametophyte. I: Antheridia in detail. J: Symmetrical

cordiform bisexual gametophytes. K: Gametangia (archegonia and antheridia) in detail. L: Proliferation of

male gametophyte bearing antheridia. M: Sporophyte ﬁrst leaf. N: More developed sporophyte with several

leaves. Abbreviations= an: antheridium; ar: archegonium; gm: gametophyte; pr: proliferation; sp: sporophyte;

tr: trichome. Scales= A, H, J, M: 200 µm; B, F: 50 µm; C-D, I, K: 25 µm; E, G, L: 100 µm; N: 2 cm.

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Bol. Soc. Argent. Bot. 59 (3) 2024

Fig. 3. Blechnum auriculatum gametophyte and sporophyte development in cultures of spores stored

12 months in freezer. A: Filamentous gametophyte phase with terminal trichome. B: Filaments without

terminal trichome. C: Early laminar gametophyte phase. D: Laminar phase after successive mitosis. E: Early

cordiform gametophyte phase. F: Mature cordiform gametophytes. G: Male gametophyte. H: Antheridia in

detail (arrow). I: Symmetrical cordiform bisexual gametophyte. J: Gametangia near the notch (arrow). K:

Magniﬁed imagen of gametangia. L: Young sporophyte with ﬁrst leaf. M: Sporophytes with 2-3 leaves. N:

Sporophytes with several leaves with divided blade. Scales= A-C: 50 µm; D-E, G, I: 200 µm; F: 400 µm; H,

K: 25 µm; J: 100 µm; L: 3 mm; M: 5 mm; N: 1 cm.

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M. L. Martinenco et al. - Viabilidad de esporas de helechos del centro de Argentina

Fig. 4. Physematium montevidensis gametophyte and sporophyte development in cultures of spores stored

12 months in freezer. A: Filamentous gametophyte phase. B: Early laminar phase. C: Early cordiform phase.

D: Unisexual male and bisexual gametophytes. E: An archegonium in detail. F: Magniﬁed image of a male

gametophyte. G: Young sporophyte with ﬁrst leaves. Scales= A-B, E: 50 µm; C-D, F: 200 µm; G: 5 mm.

employing other storage and culture conditions will

be tested to achieve a longer spore viability.

This work constitutes the ﬁrst report on the

viability of spores of fern taxa native to Argentina

Concerning the developmental and morphological conserved at low temperature. We intend to provide

features of B. auriculatum and P. montevidensis baseline information for future propagation and

gametophytes, they were previously described by conservation studies in native ferns, particularly

Durán (1997) and Martinenco et al., (2023), and our those which inhabit areas with severe periodic

observations are in coincidence with them. For A. disturbances, as occurs in the central Mountains

argentina, this information is provided here for the systems of Argentina.

ﬁrst time. The spore germination pattern is Vittaria-

type and the gametophyte development follows the

Aspidium-type (according to Nayar & Kaur, 1971). author contributionS

The gametophytic development of each species was

similar in the diﬀerent trials (i.e. using fresh spores

and those stored at low temperatures).

MLM and MLL made the general

conceptualization of the manuscript. MDAcollected

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Bol. Soc. Argent. Bot. 59 (3) 2024

the materials used in the essays. MLM and MLL BALLESTEROS, D. 2010. Conservation of Fern Spores.

realized the experimental tests and collected the

data. All authors participated in the interpretation

of data and in the preparation and writing of the

manuscript.

En: FERNÁNDEZ, H., A. KUMAR & M. A.

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BALLESTEROS, D. & V. C. PENCE. 2018. Fern

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We thank Dr. Antonia Oggero for her

collaboration during the sampling in the ﬁeld. This

research was supported by the Projects 11N/940 of

the Universidad Nacional de La Plata (MLL), PICT

0

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