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.

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)

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.).

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 71: e52338, enero-diciembre 2023 (Publicado Ago. 04, 2023)

anticlinal divisions of the protoderm cells and

divisions in several planes in the parenchyma

cells, while the suspensor and the basal region

cells began to disappear, and the seed coat

collapsed and was retained in the medium

(Fig. 3D). At 30 DAS, the protoderm became

epidermis, presenting rhizomes and trichomes.

Continuous cell divisions gave the protocorm

a flat shape, with parenchymal cells with large

vacuoles (Fig. 3E). At 60 DAS, some proto-

corms began differentiation at the shoot apex

(Fig. 3F), while in others they already showed

the leaf primordium (Fig. 3G). On the other

hand, the brown protocorm samples showed

widespread cell death (Fig. 3H).

The cells of the embryo (0 DAS) were

rich in lipid and protein bodies allotted in

globoids structures of varying sizes, which,

accompanied by limited early starch granules,

are the primary reserves of C. crispa seeds (Fig.

4A, Fig. 4B). During the imbibition process (7

DAS), some events were observed: mitotic

activity in the epidermal cells; rapid deposition

of polysaccharides all over the embryo (Fig.

4C); and digestion of protein bodies, which are

probably a result of the activation of protease

metabolism. In addition, the starch grains got

bigger (Fig. 4D). The polysaccharide granules

were arranged around the nucleus of the cells

after 15 DAS (Fig. 4E). Due to the rapid diges-

tion of the lipids, glyoxysomes became visible

and likely to be found in proximity to the lipid

bodies. Large amyloplasts containing starch

grains were evident (Fig. 4F). Parenchyma cells

became largely vacuolated by the 30 DAS, and

a gradient of polysaccharide density is visible

Fig. 3. Histological aspects of Cattleya crispa during germination and post-seminal development in vitro. A. 0 DAS:

Cross-section. Embryo composed of protoderm (arrow) and promeristem. Seed coat encloses the air space indicated by

the arrowhead. B. 0 DAS: Longitudinal section. The suspensor is projected through the open micropylar end. C. 7 DAS:

Germination. The shoot apex cells in divisions (arrowhead) and the hypophysis cells (arrow). D. 15 DAS: Lateral expansion

of the protocorm. Degradation of the suspensor and the basal region cells (arrow). E. 30 DAS: Protocorm with large lateral

expansion. Parenchyma with large vacuoles. Trichomes and rhizoids in the basal region. F.-H. 60 DAS. F. Differentiation

of the shoot apex cells (arrow). G. Visible leaf primordia. H. Brown protocorm showing cell death. Arrows indicate altered

cells. Pm promeristem; Sc seed coat; Em embryo; S suspensor; Ch chalaza; Pa parenchyma; Ep epidermis; Rh rhizoids; Tr

trichome; Lp leaf primordia. Bar: 50 µm (A); 200 µm (B); 100 µm (C-H).

8Revista de Biología Tropical, ISSN: 2215-2075 Vol. 71: e52338, enero-diciembre 2023 (Publicado Ago. 04, 2023)

from the basal region of the protocorm to the

upper region (Fig. 4G, Fig. 4H). Polysaccha-

rides at 60 DAS were largely mobilized to the

cells of the shoot apex in differentiation and to

the cell layers close to the epidermis (Fig. 4I).

These cells have dense cytoplasm, which evi-

dence the formation and coalescence of lipidic

bodies (Fig. 4J).

We observed that most of the protocorms

collapsed around 60 DAS. The living pro-

tocorms had completely lost the seed coat,

whereas in the brown protocorms the seed

coat was still attached (Fig. 5). Ultrastructural

alterations in the parenchyma of dying proto-

corms confirmed a generalized cell death (Fig.

5D, Fig. 5H).

Seed cryopreservation: viability (%), ger-

mination (%), survival rate (%) and protocorms

weight (mg) did not show significant differ-

ences between +LN and -LN (Table 2). Despite

high viability and germination rates, most of

the protocorms collapsed (reddish-brown pro-

tocorms), which led to a low survival rate in

both treatments (Table 2).

DISCUSSION

Seed morphology: Considering the five

seed size categories established by Barthlott et

al. (2014) (Very small 100-200 µm; small 200-

500 µm; medium 500-900 µm; large 900-2 000

µm; and very large 2 000-6 000 µm), our data

indicate that C. crispa has medium-sized seeds.

This category includes the seeds most found in

the Orchidaceae family (Barthlott et al., 2014).

Fig. 4. Reserve compounds during germination and post-seminal development in vitro of Cattleya crispa seeds. A.-B. 0 DAS.

A. starch granules did not appear with PAS staining. B. Embryo rich in lipid and protein bodies. Arrows indicate a few early

starch granules that did not appear with PAS staining. C.-D. 7 DAS. C. Germination. Deposition of polysaccharides (arrow).

D. Arrow: cell walls were still thin; arrowheads largely digested protein bodies. E.-F. 15 DAS. E. The polysaccharides tend

to congregate around the nucleus (arrow). F. Presence of glyoxysomes, found in proximity to the lipid bodies in process of

digestion (arrow). G.-H. 30 DAS. G. Increased polysaccharides in the basal region of the protocorm (arrow). H. Parenchyma

cells largely vacuolated with disorganized protein (arrow). I.-J. 60 DAS. I. Polysaccharides mobilized to the cells of the

shoot apex in differentiation and to the cell layers close to the epidermis (arrow). J. Cells with extensive lipidic bodies

formation (arrow). Em embryo; Sc seed coat; Cw Cell wall; L lipid bodie; P protein bodies; S starch grain; N nucleus; Nu

nucleolus; Rh rhizoids; * Middle lamella. Bar: 50 µm (A., E.). 100 µm (C., G., I.). 2 µm (B., D., J.). 1 µm (F. , H.).

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 71: e52338, enero-diciembre 2023 (Publicado Ago. 04, 2023)

On the other hand, the shape (fusiform) and

pigmentation (whitish-yellow) in C. crispa

seeds have also been reported in other epiphytic

orchids, including the Cattleya genus (Diantina

et al., 2020). Ramudu et al. (2020) indicate

that fusiform seeds appear to be the basic form

from which all other seed shapes evolved in

this family.

Some studies suggest a probable relation-

ship between the presence of twisted testa cells

along the longitudinal axis with the epiphytic

habitat (Barthlott et al., 2014; Gamarra et al.,

2018). However, C. crispa presented testa cells

with parallel orientation, which is a feature

shared with terrestrial taxa (Gamarra et al.,

2018). Epiphytic orchids of the genera Ansellia,

Graphorkis and Liparis also presented this type

of orientation (Gamarra et al., 2018). Regard-

ing the long multicellular suspensor found in

C. crispa, previous studies have also reported

it in other Cattleya species and other members

of the Laeliinae subtribe (Bazzicalupo et al.,

2021; Gallo et al., 2016). Bazzicalupo et al.

(2021), indicate that the suspensor dimension

does not influence seed development.

Morphoanatomy of post-seminal devel-

opment: The categorization of development

stages allows the measuring of the seedling’s

Fig. 5. Protocorms of Cattleya crispa collected at 60 DAS. A.-D. Green and alive protocorms without the seed coat.

A.-B. Vegetative apex with the leaf primordium in formation (arrows). C. rhizoid formation. D. TEM image shows intact

parenchyma. E.-H. Dying protocorms. E. Protocorm with brown color (arrow). F.-G. protocorm with the seed coat attached.

H. TEM image shows cell death. Rh rhizoids; Sc seed coat; Pr protocorm. Bar: 2 µm (D., H.). 100 µm (A., B., C., E., F. ,

G.). 200 µm (A.). 500 µm (E.).

Table 2

Effect of cryopreservation of Cattleya crispa seeds by direct immersion in liquid nitrogen.

Variable non-cryopreserved seeds (-LN) cryopreserved seeds (+LN)

Seed viability (TCC) (%) 87.15 ± 1.99 ns 88.04 ± 2.20 ns

Seed germination (%) 78.32 ± 3.37 ns 81.14 ± 2.33 ns

Survival rate (%) 8.48 ± 3.61 ns 11.12 ± 3.92 ns

Protocorms weight (mg) 104.27 ± 1.90 ns 104.85 ± 2.82 ns

Data are the mean value ± standard error. The letters ns indicate not significant differences (ANOVA, P < 0.05) between

+LN and -LN.

10 Revista de Biología Tropical, ISSN: 2215-2075 Vol. 71: e52338, enero-diciembre 2023 (Publicado Ago. 04, 2023)

development and compares the effectiveness

of different germination procedures (Hoang et

al., 2016). Even though some authors consider

the loss of the testa with posterior formation

of a tuberiform structure called protocorm as

the characterizing event of orchid germina-

tion (Arditti, 1967), we do not consider this

as the main factor for C. crispa germination.

We define germination as Salazar-Mercado &

Vega-Contreras (2017) did for Cattleya trianae.

In the germination of C. crispa the testa was

broken, and the embryo was visible as a small

green propagate. This event occurred seven

days after sowing. However, in some species

of orchids the onset of germination occurs two

months after seed sowing (Dolce et al., 2020).

Subsequently, the C. crispa seeds form the

protocorms, which are observed in different

stages of development throughout the evalu-

ated time. According to Nikishina et al. (2007),

orchid seeds are quite heterogeneous, and,

among sown seeds, there is usually a popula-

tion of strong seeds, which largely overcomes

the others in their development. The initial

growth of C. crispa was similar to that report-

ed for other orchids (Abrahám et al., 2012;

Hossain et al., 2010).

Regarding the reserve compounds in orchid

seeds, Yeung (2017) indicate that the embryos

have cells full of storage products: abundant

proteins and lipids, while rarely starch gran-

ules. These compounds were found in C. crispa

and have been reported in other species of the

genus (Schvambach et al., 2022) as well as in

other groups of plants (Corredor-Prado et al.,

2014; Martelo-Solorzano et al., 2022). In C.

crispa, the evident presence of polysaccharides

during post-seminal development, including

starch, is attributed to the mobilization of lipids

and imbibition of the sucrose from the culture

medium. Penfield et al. (2005) reported that this

mobilization leads to the formation of acetyl

Co-A with the subsequent synthesis of sucrose

through gluconeogenesis. If the levels of poly-

saccharides in the cytosol are too high, they can

be stored as transient starch. We believe that

there is a correlation between the accumulation

of starch grains during the germination and the

degradation of primary reserves. The mobiliza-

tion was probably greater than the required for

these initial stages. Therefore, to maintain the

osmotic balance, the excess was converted into

starch reserve.

Seeds of Cattleya do not have an inner

integument that provides physical dorman-

cy, which is commonly present in temperate

orchids (Custódio et al., 2016). Likewise, in

C. crispa, we did not identify integumentary

dormancy once the seed coat allows water pen-

etration and subsequent germination. However,

we observed that in some seeds the cover is

persistent, affecting the development of the

protocorm which initiates an oxidation pro-

cess that ends with cell death. These results

are in accordance with previous findings for

other orchids species. According to Dalzotto

& Lallana (2015), the seed coat of Bipin-

nula pennicillata does not limit the asymbiotic

germination of the seeds; instead, it causes a

delay in obtaining protocorms with apical

bud and rhizoids. Other study with ten spe-

cies of Cattleya showed that, by the 63 DAS,

the protocorms’ quality declined and some of

them became shriveled and brown (Hosomi et

al., 2012). Therefore, we suggest procedures

to increase the survival rate of protocorms,

by softening or breaking down the seed coat.

For this, Zeng et al. (2014) indicate the use of

cold treatments, prolonged imbibition, chemi-

cal scarification of the testa with sodium or cal-

cium hypochlorite, or mechanical damage. In

this way, it is necessary to expand the research

focused on C. crispa seed pretreatments, which

could lead to better results. As Arditti (1967)

suggests, orchid seeds, despite their diminutive

and fragile appearance, can resist and survive

relatively harsh treatments.

Seed cryopreservation: In general, the

germinability of cryopreserved seeds is high

among tropical orchid species (70-100 %)

(Popova et al., 2016). It should be noted that

the water content of the seeds is an important

factor for their successful cryopreservation,

since low water levels in embryo cells reduce

lethal intracellular freezing injuries (Hirano et

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 71: e52338, enero-diciembre 2023 (Publicado Ago. 04, 2023)

al., 2005). Therefore, the high viability and ger-

mination of C. crispa in this study was related

to the initial optimal value of water content of

seeds (5 %), which indicates their orthodox

nature and allows a simple cryopreservation

technique. This confirms that orchid seeds

with a moisture content below 13 % can be

successfully cryopreserved by direct immer-

sion in liquid nitrogen (Popova et al., 2016).

However, for other orchid species, is necessary

to use cryoprotectant solutions (Galdiano et

al., 2017; Merritt et al., 2014; Vettorazzi et al.,

2019). Several studies have successfully cryo-

preserved different orchid species finding that

germination rates after the liquid nitrogen stor-

age remained equal or higher (Nikishina et al.,

2007; Popova et al., 2016). Similarly, our results

demonstrated that seed viability, germination,

survival rate, and the weight of the formed

protocorms were not significantly altered by

direct immersion of seeds in liquid nitrogen.

Therefore, the establishment of cryobanks pres-

ents great potential for long-term storage of C.

crispa seeds. According to Silva et al. (2021),

the development of methods for secure stor-

age of seeds contributes to the preservation of

genetic diversity, which is crucial in the case of

species that are vulnerable or endangered.

On the other hand, the tetrazolium test

in seeds showed to be effective to evaluate

the physiological quality in C. crispa similar

to other studies with orchids, the results of

the tetrazolium test were consistent with the

seed germination data (Galdiano et al., 2017;

Hosomi et al., 2012; Vettorazzi et al., 2019).

This test has been successfully used to evalu-

ate cryopreserved seeds of other orchids (Vet-

torazzi et al., 2019).

To our knowledge, this is the first study

that has developed an abiotic culture of C.

crispa using biotechnological tools, as well as

the first morpho-anatomical and ultrastructural

report during the germination, post-seminal

development, and cryopreservation of this spe-

cies. Through the monitoring of the evaluated

development stages, we identified that the seed

coat is sometimes persistent, affecting the

development of the protocorm. Our hypothesis

was accepted, since we found that the viabil-

ity and germination of the seeds, as well as

the formation of the protocorms, did not vary

significantly after the immersion of the seeds

in liquid nitrogen. The cryocapacity of these

seeds is evidence that cryopreservation can be

a strategy for the long-term conservation of C.

crispa. We recommend the assessment of dif-

ferent seed pretreatments, in order to optimize

the obtention of viable protocorms. 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.

Ethical statement: the authors declare

that they all agree with this publication and

made significant contributions; that there is no

conflict of interest of any kind; and that we fol-

lowed all pertinent ethical and legal procedures

and requirements. All financial sources are

fully and clearly stated in the acknowledgments

section. A signed document has been filed in

the journal archives.

ACKNOWLEDGMENTS

We thank the Coordenação de Aperfeiçoa-

mento de Pessoal de Nível Superior (CAPES)

for fellowships and also the Centro de Biologia

Molecular Estrutural da Universidade Federal

de Santa Catarina (CEBIME/UFSC).

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