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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