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Revista de Biología Tropical, ISSN: 2215-2075, Vol. 71: e52338, enero-diciembre 2023 (Publicado Ago. 04, 2023)
Morpho-anatomy of in vitro germination and cryopreservation
of the orchid Cattleya crispa (Orchidaceae)
Bruna Vargas Andriolli1; https://orcid.org/0000-0002-9643-9995
Jenny Paola Corredor-Prado2; https://orcid.org/0000-0002-4605-9740
Rosete Pescador1*; https://orcid.org/0000-0002-4667-9894
Francisco Sebastian Montoya-Serrano1; https://orcid.org/0000-0002-9976-9770
Lírio Luiz Dal Vesco3; https://orcid.org/0000-0002-4545-2081
Rogério Mamoru Suzuki4; https://orcid.org/0000-0003-1124-9875
1. Programa de Pós-Graduação em Recursos Genéticos Vegetais, Universidade Federal de Santa Catarina, Rodovia
Admar Gonzaga, 1346, Itacorubi, 88034-001, Florianópolis, SC, Brasil; bruandriolli@hotmail.com, rosete.pescador@
ufsc.br (*Correspondence), sebast.montoya@gmail.com
2. Departamento de Biología y Química, Universidad de Sucre, Puerta Roja, 28 # 5-267, Sincelejo, Sucre, Colombia;
jenny.corredor@unisucre.edu.co
3. Departamento de Ciências Naturais e Sociais, Universidade Federal de Santa Catarina, Rod. Ulysses Gaboardi, Km 3,
Curitibanos, SC, 89520-000, Brasil; lirio.luiz@ufsc.br
4. Instituto de Botânica, Núcleo de Pesquisa-Orquidário do Estado, P.O. Box 04301-012, 04301-902 São Paulo, SP,
Brasil; rogeriosuzuki@gmail.com
Received 30-IX-2022. Corrected 12-IV-2023. Accepted 26-VII-2023.
ABSTRACT
Introduction: Cattleya crispa is an ornamental epiphytic orchid with geographic distribution restricted to the
Brazilian Atlantic Forest. Due to predatory extractivism and human-induced habitat loss, this species appears on
the Red List of Brazilian Flora.
Objective: To characterize morpho-anatomical aspects regarding germination and post-seminal development
from C. crispa seeds; as well as studying the effect of cryopreservation on these seeds.
Methods: We used light microscopy and electron microscopy to describe the microstructure of a 100 ripe seeds.
We evaluated seed viability, seed germination, survival rate and protocorm weight in cryopreserved and non-
cryopreserved material, with four replicas per treatment using 20 mg of plant material.
Results: The seeds are fusiform, whitish yellow with a length from 700 to 900 µm and a water content of 5
%. Germination began seven days after sowing, the formation of the globular protocorm at 30 days and the
formation of the seedling occurred 150 days. The persistent seed coat can compress the protocorm and cause
it to collapse. The cryopreserved seeds presented 87.15 % viability, 78.32 % germination, 8.48 % survival and
protocorms with 104.27 mg five months after sowing. Data wasn’t different to non-cryopreserved seeds.
Conclusions: The cryocapability of the seeds shows that cryopreservation can be used for long-term conserva-
tion. The results of this work contribute to the overall biology of C. crispa and to the propagation and storage of
genetic material for conservation purposes.
Key words: embryo; Orchidaceae; ornamental; protocorm; viability.
https://doi.org/10.15517/rev.biol.trop..v71i1.52338
BOTANY & MYCOLOGY
2Revista de Biología Tropical, ISSN: 2215-2075 Vol. 71: e52338, enero-diciembre 2023 (Publicado Ago. 04, 2023)
INTRODUCCIÓN
Orchidaceae represents one of the most
diverse families of flowering plants, consisting
of about 35 000 species, which have fascinated
botanists and plant enthusiasts over centuries
(Barthlott et al., 2014), due to its extensive
horticultural, medicinal, and culinary uses. The
Cattleya genus is one of the most popular and
widely cultivated in this family; the high orna-
mental value of its members and large ability
for genetic recombination are attractive to the
market (Galdiano et al., 2017). Cattleya crispa
Lindl. is an ornamental epiphyte, endemic to
the Brazilian Atlantic Forest (Van Den Berg,
2020); it grows slowly, and its generation
time is estimated at about ten years (CNC-
Flora, 2022). Due to predatory extractivism and
human-induced habitat loss this species appears
on the Red List of Brazilian Flora (CNCFlora,
2022). As a species threatened with extinction,
C. crispa is protected internationally under the
appendix II of the Convention on International
Trade in Endangered Species of Wild Fauna
and Flora (UNEP-WCMC, 2022).
Programs for ex situ conservation of wild
plant germplasm are fundamental to preserving
the world’s declining biodiversity (Merritt et
al., 2014). To achieve this goal, in vitro culture
techniques provide important tools, however it
is necessary to understand the biology of the
seeds, as well as the structure and function of
the protocorm, to optimize methodologies for
the of orchid seedlings (Yeung, 2017). Accord-
ingly, morphoanatomical research on seeds
and on post-seminal development of Cattleya
plants in vitro has been carried out (Bazzica-
lupo et al., 2021; Gallo et al., 2016; Hosomi et
al., 2012; Salazar-Mercado & Vega-Contreras,
2017). This type of studies contributes to the
understanding of physiological processes, to
the interpretation of germination tests and to
the development of efficient propagation tech-
niques and conservation programs (Corredor-
Prado et al., 2014; Gallo et al., 2016).
For long-term conservation, cryopreser-
vation is a recommended technique for plant
with non-orthodox seeds, vegetatively propa-
gated plants, and rare and endangered species
RESUMEN
Morfoanatomía de la germinación in vitro y criopreservación
de la orquídea Cattleya crispa (Orchidaceae)
Introducción: Cattleya crispa es una orquídea epífita ornamental con distribución geográfica restringida a la
Mata Atlántica brasileña. Debido al extractivismo depredador y a la pérdida de hábitat inducida por el hombre,
esta especie aparece en la Lista Roja de la Flora Brasileña.
Objetivo: Caracterizar aspectos morfoanatómicos de la germinación y desarrollo inicial de semillas de C. crispa;
así como estudiar el efecto de la criopreservación de estas semillas.
Métodos: Utilizamos microscopía óptica, microscopía electrónica de barrido y microscopía electrónica de
transmisión para describir la microestructura en 100 semillas maduras. Evaluamos la viabilidad de la semilla, la
germinación de la semilla, la tasa de supervivencia y el peso de los protocormos en el material criopreservado y
no criopreservado, con cuatro réplicas por tratamiento de 20 mg de material vegetal.
Resultados: Las semillas son fusiformes, amarillo blanquecinas, con una longitud de 700 a 900 µm y un conte-
nido de agua del 5 %. La germinación comenzó siete días después de la siembra, la formación del protocormo
globular a los 30 días y la formación de la plántula a los 150 días. La cubierta de semilla persistente puede
comprimir el protocormo y provocar su colapso. Las semillas criopreservadas presentaron 87.15 % de viabilidad,
78.32 % de germinación, 8.48 % de supervivencia y protocormos con 104.27 mg a los cinco meses de la siembra.
Los datos no fueron diferentes a las semillas no criopreservadas.
Conclusiones: La capacidad criogénica de las semillas muestra que la crioconservación puede utilizarse para la
conservación a largo plazo. Los resultados de este trabajo contribuyen a la biología general de C. crispa y a la
propagación y almacenamiento de material genético con fines de conservación.
Palabras clave: embrión; Orchidaceae; ornamental; protocormo; viabilidad.
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Revista de Biología Tropical, ISSN: 2215-2075, Vol. 71: e52338, enero-diciembre 2023 (Publicado Ago. 04, 2023)
(Engelmann, 2011). Adequate protocols can
provide high plant regrowth after thawing,
thus facilitating the establishment of cryobanks
of plant genetic resources, in an organized
and strategic manner (Benelli, 2021). The use
of seeds allows the maintenance of a wider
genetic basis and is appropriate for endangered
species, since the parent plant does not have
to be destroyed to obtain the seeds (Kulus &
Zalewska, 2014). In orchids, the cryopreserva-
tion of seeds of some species has been suc-
cessful (Kaur, 2019). However, complexities
in the behavior of low-temperature storage still
require explanation and resolution (Merritt et
al.,2014).
Considering that the advancement of
knowledge on reproductive structures and use-
ful for conservation programs (Gallo et al.,
2016), this study aimed to characterize mor-
pho-anatomical aspects regarding germination
and post-seminal development from C. crispa
seeds; as well as studying the effect of cryo-
preservation on these seeds. We hypothesized
that the immersion of C. crispa seeds in liquid
nitrogen does not cause a negative effect on
plant development processes.
MATERIAL AND METHODS
Seed material: Seeds of C. crispa come
from the ex situ conservation collection of the
Orquidário Frederico Carlos Hoehne - Institute
of Botany, located in Água Funda, São Paulo-
Brazil. Seven months after cross-pollination,
six mature capsules were collected from three
different individuals. All capsules were in pre-
dehiscence stage. Seed moisture content (MC)
was determined by the low-constant tempera-
ture oven method (ISTA, 1985), at 103 ± 2 ºC
for 17 h. Three seeds replicate of 10 mg were
used, and the moisture content was expressed
as a percentage:
W1 = weight of aluminum boat, W2 = weight
of aluminum boat + seeds before drying, W3 =
weight of aluminum boat + seeds after drying.
In vitro culture: The seeds were soaked
in sterile-distilled water with a drop of surfac-
tant detergent (Tween™ 20) during 10 min., in
constant agitation. Then, they were sterilized
with 0.5 % sodium hypochlorite (NaClO) for
10 minutes and rinsed three times with sterile
distilled water. Solution changes were made
using a sterilized Pasteur pipette. The seeds
were sown on Murashige & Skoog (1962)
medium supplemented with 30 g/l sucrose (P.A.
Sigma™), solidified with 2 g/l gelling agent
(Phytagel: Sigma™), and set into pH 5.5 before
being sterilized at 120 ºC for 15 minutes. About
1 000 seeds were cultured in polystyrene Petri
dishes (150 mm × 15 mm) with 20 ml of cul-
ture medium (four replicates were made). The
cultures were maintained in growth room at 25
± 2 ºC and photoperiod of 16 h with luminous
intensity 50-60 µmol m−2 s−1 by clear fluores-
cent light.
Morpho-anatomical description: The
material was analyzed under stereomicroscope,
light microscope, and electron microscopy. The
characters analyzed were coloration, shape,
length, and the presence of polysaccharides.
For biometric description 100 ripe seeds were
randomly selected and had their length record-
ed by Image J version 1.8.0 software (Nation-
al Institutes of Health, Bethesda, Maryland,
USA). We consider mature seeds those devel-
oped 7 months after the cross, before being
inoculated in vitro. Then, seeds inoculated
in vitro as indicated above, were examined
weekly under a stereoscopic microscope (SZH
10: Olympus™), to evaluate developmental
stages from seed to seedling formation. We
classify these stages according to an adapta-
tion of Arditti (1967) (Table 1). Collections for
microscopic analyses were performed 0, 7, 15,
30 and 60 days after sowing (DAS).
Light microscopy (LM). The fresh sam-
ples were removed from the culture medium
and dabbed dry on filter paper. The material
was fixed in 2.5 % glutaraldehyde and 0.1M
phosphate-buffered saline (PBS) (1:1, v/v)
followed by dehydration in series of ethanol
aqueous solutions. The samples were infiltrated
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with hydroxyethyl-methacrylate (Historesin:
Leica™), according to the manufacturer’s
instructions. For sectioning (5 µm), we used a
manual microtome (Slee Cut 4055: Mainz™).
The histochemical tests performed were peri-
odic acid-Schiff reagent (PAS) for polysaccha-
rides (Feder & O’Brien, 1968), and toluidine
blue O (TB-O) for metachromasy (O’Brien et
al., 1964). The images were obtained using a
light microscope (BX40: Olympus™) with a
high-resolution color digital camera (DP71:
Olympus) and Capture Pro 5.1 Image Software.
Scanning electron microscopy (SEM).
After fixation and dehydration as described
above, the samples were placed on strips of
carbon tape, and affixed on the sample stub to
continue dehydration, by the low surface ten-
sion solvent 1,1,1,3,3,3-hexamethyldisilazane
(HMDS). The dried samples were covered with
20 nm of gold in metallizer (EM SCD 500:
Leica™), to be studied under a scanning elec-
tron microscope (JSM-6390LV: Jeol™).
Transmission electron microscopy (TEM).
The material was fixed with 2.5 % glutaral-
dehyde, 0.1 M sodium cacodylate buffer (pH
7.2) and 0.2 M sucrose. The samples were
then post-fixed in 1 % osmium tetroxide for
6 h, dehydrated in graded acetone series and
embedded in Spurr’s resin (Leica™). After
sectioning, the material was stained with aque-
ous uranyl acetate followed by lead citrate. The
sections were examined under a transmission
electron microscope (JEM-1011: Jeol™).
Cryopreservation: Two treatments
were designed: cryopreserved seeds in liquid
nitrogen, referred to as +LN, and non-cryopre-
served seeds in liquid nitrogen, referred to as
-LN. The cryopreservation of the seeds (+LN)
was carried out by ultra-rapid freezing by direct
immersion in liquid nitrogen (-196 ºC). Four
1.5 ml plastic cryotubes containing 20 mg of
seeds were used. After 48 h, the cryotubes
were thawed in a water bath at 40 ºC for 2 min.
Then, the seeds were sterilized and cultured
as previously described. Non-cryopreserved
seeds (-LN) were sterilized and cultured imme-
diately. Four replicates per treatment were
considered. The protocorms formed from seeds
(+LN and -LN) were sub-cultured on the same
medium type.
A subset of seeds from both treatments
was used to assess viability using the 2,3,5-tri-
phenyltetrazolium chloride (TTC) test. The
seeds were soaked in distilled water for 17 h
at 25 ± 2 °C. The water was removed, and the
material was soaked in 0.5 % TTC solution in
total darkness for 15 h. After staining, viability
(%) was determined by counting the number
of seeds that showed red coloration (viable).
A lack of red coloration and/or pale pink col-
ors would indicate the death of the embryo
(Salazar-Mercado et al., 2020), and therefore
the non-viable seed (Fig. 1). Four replications
of 300 seeds were analyzed using a microscope
(BX40: Olympus™).
Seed germination (%), survival rate (%)
and protocorms weight (mg) were evaluated
5 months after sowing. Swollen and green
embryo with ruptured testa was the criteria
used to define germination. The germination
was calculated by dividing the germinated
Table 1
Stages initial development in vitro of Cattleya crispa seeds adapted from Arditti (1967)
Stage Description
0Unviable seeds
1Swollen and green embryo with ruptured testa (= germination)
2Early globular protocorm (bodies formed by the continued embryo enlargement after germination= protocorm)
3Protocorm showing a pointed vegetative apex and rhizoids
4Protocorm with one emerged leaf
5Protocorm with two spreading leaves
6Seedling showing two or more leaves with root presence (= seedling)
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Revista de Biología Tropical, ISSN: 2215-2075, Vol. 71: e52338, enero-diciembre 2023 (Publicado Ago. 04, 2023)
seeds per total amount of seeds inoculated in
each Petri dish. The survival rate was evaluated
by counting as live the green protocorms and
seedlings among the germinated ones. Lastly,
weight of the protocorms (mg) was obtained
from 30 protocorms of each Petri dish. To
identify if immersion in liquid nitrogen influ-
ences the development of C. crispa, data were
subjected to analysis of variance (t-test, P <
0.05) using Statistica® software (Statsoft Inc.,
Tulsa, OK, USA).
RESULTS
Moisture content and seed morphol-
ogy: Cattleya crispa seeds presented 5 %
water content. They presented whitish-yellow
coloration, fusiform shape and length varying
in the range of 700-900 µm (excluding the
suspensor). The seeds were relatively undif-
ferentiated and characterized by the presence
of one ellipsoidal shaped embryo at the center
and a slightly pigmented seed coat without any
visible endosperm or cotyledon (Fig. 2A). The
chalaza extremity was tapered and closed, and
the basal region had an overture at the micropy-
lar end, and in some seeds, the suspensor pro-
jected through the micropyle (Fig. 2B, Fig. 2C,
Fig. 2D). The suspensor is multicellular and
consists of 2-3 cell layers. The seed coat cells
were elongated, rounded at the end and without
intercellular gaps. The orientation of the testa
cells was parallel to each other.
Morphoanatomy of post-seminal devel-
opment: Seeds at the 5 DAS on the culture
medium presented the swollen embryo due
to the imbibition process, however, it was not
possible to differentiate viable from non-viable
seeds. The germination process started 7 DAS
(stage 1), when the embryo was swollen and
green, visibly different from unviable seeds,
which showed white color and no alterations
in the embryo/seed coat ratio (stage 0) (Fig.
2E, Fig. 2F). The seeds presented continuous
growth of the chlorophyll structure, filling
and stretching the central zone of the ruptured
seed coat (15 DAS) (Fig. 2G). By the 30 DAS,
the globular protocorm is identified (stage 2).
An early oxidation with reddish-brown color
appearance was also visible in some proto-
corms (Fig. 2H). The vegetative organs started
to develop at 60 DAS (stage 3). The upper part
of the protocorms showed a vegetative apex
containing the leaf primordium in formation,
whereas at the base the rhizoids were observed
(Fig. 2I, Fig. 2J). At 70-80 DAS, the first
leaf emerged in some protocorms (stage 4)
(Fig. 2K).
Although the germination process showed
to be morphologically homogeneous, proto-
corms were observed at different development
stages over time. With 100-110 DAS, the proto-
corms showed a well-spread second leaf (stage
5), while the first leaf gradually became wider
and thicker. The roots were observed at 150
DAS (stage 6); we define this morphological
change as the ending mark of the protocorm
stage and the beginning of the seedling phase
(Fig. 2L, Fig. 2M).
The embryo was formed by a protoderm
that delimited the promeristem (0 DAS) (Fig.
3A). The longitudinal sections showed an
embryo with elliptic shape and the discernible
chalazal-micropylar axis. The chalaza end was
closed and composed of smaller and denser
cells, while the micropylar region contained
Fig. 1. Cattleya crispa seeds submitted to the tetrazolium
viability test. A. Viable embryo has red color. B. Unviable
embryo, non-colored. Em embryo; Sc seed coat. Bar:
200 µm.
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larger cells, with a suspensor projecting from
the open micropylar end (Fig. 3B). At the
beginning of germination (7 DAS), the swelling
embryo became more globous due to imbibi-
tion and increased cell number. Changes in
shape and volume were accompanied by histo-
logical differentiation, evidencing the embryo
bipolarity (Fig. 3C). Within 15 DAS, the pro-
tocorm showed lateral expansion. The shoot
apex showed intense meristematic activity, with
Fig. 2. Seed, germination, and post-seminal development in vitro of Cattleya crispa. A.-D. 0 DAS: General aspect of external
seed morphology. A. Slightly pigmented seed coat (arrow) with the embryo inside. B. Arrow suspensor projected through
the micropylar end; arrowhead: closed chalaza extremity. C.-D. Close view of the micropylar end. C. Arrow: opening in the
seed coat. D. Arrow: suspensor. E.-F. 7 DAS. E. Seed germination. Ruptured seed coat. F. Arrowhead: unviable seed (Stage
0); arrow: swollen and germinated embryo (Stage 1). G. 15 DAS: Continuous growth of the chlorophyll structure. H. 30
DAS: arrowhead: protocorm with signs of oxidation; arrow: globular-shaped protocorm (Stage 2). I.-J. 60 DAS: I. Exposed
protocorm (arrow), with broken seed coat. J. Protocorms with leaf primordia (arrow) and rhizoids (Stage 3). K. 80 DAS:
Protocorm with the first leaf emerged (Stage 4). L. 110 DAS: Seedling with two leaves and the presence of root (Stage 6).
M. Gathering of the development process, from the protocorm with leaf primordia until complete seedling stage. Arrow:
protocorm with two spreading leaves and absence of root (Stage 5). Arrowhead: marks the end of the protocorm stage (Stage
6) with complete seedlings forward. Em embryo; Rh rhizoids; Fl first leaf; Sl second leaf; Rt root; Sc seed coat; Lp leaf
primordia. Bar: 50 µm (C., D., E.); 100 µm (A., B., F., G., H., I.); 500 µm (J.); 1 mm (K.-M.).