Revista Ciência Agronômica, v.

54, e20228531, 2023

Centro de Ciências Agrárias - Universidade Federal do Ceará, Fortaleza, CE Scientific Article
www.ccarevista.ufc.br ISSN 1806-6690

Cryopreservation of Genipa americana seeds1

Rafaela Ribeiro de Souza2, Patrícia Duarte de Oliveira Paiva3*, Rodrigo Therezan de Freitas4, Diogo Pedrosa
Corrêa da Silva5, Michele Valquíria dos Reis3, Fernanda Carlota Nery6, Renato Paiva3

ABSTRACT - Genipa americana L. is a forest species of high socioeconomic potential. However, predatory extractivism

actions threaten its existence, making it necessary to adopt conservationist practices. G. americana seeds show sensitivity

to desiccation and cooling, making it unfeasible for conservation by conventional methods. Thus, cryopreservation is an

promising alternative for the long-term conservation of species that produce unorthodox seeds, such as the genipap. In this

sense, the objective was to cryopreservation of seeds of G. americana and to evaluate the effects of desiccation and freezing on

germination and establishment of seedlings. Initially, seeds were dehydrated in silica gel 0, 16, 18, 20, 22 and 24 h, and then

were cryopreserved in liquid nitrogen (LN) (-196 °C) during 24 h. After thawing, the viability and germination were analyzed.

Dehydrated and non-cryopreserved seeds were also analyzed. The silica gel desiccation caused a reduction in viability and

germination of the seeds of G. americana. The initial seed water content was so high (47%) that storage in LN (+LN) without

prior dehydration treatment resulted in seed mortality. It was verified that, the dehydration in silica gel for the minimum time 20 h

(corresponding to 14% water content) provides greater freezing tolerance, allowing the successful cryopreservation. Silica gel

dehydration followed by immersion in LN was shown to be highly efficient for cryopreservation of seeds of G. americana,

besides germinatio after thawing, high survival rates (100%) were obtained, with growth and normal establishment of the

seedlings after acclimatization.

Key words: Conservation. Dehydration. Long-term storage. Rubiaceae.

DOI: 10.5935/1806-6690.20230045
Editor-in-Chief: Prof. Salvador Barros Torres - [email protected]
*Author for correspondence
Received for publication 08/07/2022; approved on 16/12/2022
1
Part of the first author’s PhD thesis
2
Universidade Federal do Vale do São Francisco (UNIVASF), Colegiado de Agronomia, Petrolina-PE, Brazil, [email protected] (ORCID
ID 0000-0002-7706-4390)
3
Universidade Federal de Lavras (UFLA), CEP: 37200-000, Lavras-MG, Brazil, patriciapaiva@ufl a.br (ORCID ID 0000-0001-7997-8420),
michele. [email protected] (ORCID ID 0000-0003-0379-2384), [email protected] (ORCID ID 0000-00015107-0285)
4
University of Amsterdam, Swammerdam Institute for Life Sciences, Netherlands, Amsterdam, Holanda, [email protected] (ORCID
ID 0000-0001-5842-1279)
5
Departamento de Engenharia de Alimentos, Escola de Agronomia, Universidade Federal de Goiás, Goiânia-GO Brazil, [email protected]
(ORCID ID 0000-0002-7787-7500)
6
Departamento de Engenharia de Biossistemas, Universidade Federal de São João Del Rei, São João Del Rei-MG, Brazil, [email protected]
(ORCID ID 0000-0003-0021-3973)
 R. R. Souza et al.

INTRODUCTION The dehydration conferred by the material

exposure to silica gel is advantageous both because it
Genipa americana L. is a forest species native to is an easy and low-cost technique and because it allows
South and Central America, belonging to the Rubiaceae preserving the dried material directly in LN without the
family and shows high socioeconomic potential due use of cryoprotectants that can prevent toxicity to the cells
to its medicinal, ornamental, wood and food attributes (PRADA et al., 2015; STEGANI et al., 2017). Furthermore,
(NASCIMENTO et al., 2020; SOUZA et al., 2016, 2019). it is a technique that has been shown to be effective in
Furthermore, was selected by the World Bank, Global cryopreservation of several seed species (NASCIMENTO
Environment Facility and Ministry of the Environment et al., 2020; PINTO et al., 2016; SILVA et al., 2018).
(MMA) among the highest priority species in the “Plants However, there is little information on the tolerance of
of the Future Program”, and with the greatest potential genipap seeds to freezing (SANTOS; SALOMÃO, 2016)
for immediate use among native fruit trees (BRASIL, and there is no well-established cryopreservation
2016; FAO, 2017). However, predatory extractivism protocol for this species. In this context, the aim was
associated with actions such as deforestation, burning to establish a protocol for cryopreservation of Genipa
and mining compromise the existence of this species, americana L. seeds by dehydration in silica gel and the
thus stimulating the adoption of conservationist evaluation of the effects of desiccation and freezing on
practices (SOUZA et al., 2019). germination and establishment of seedlings.
G. americana is classified as intermediate, therefore,
its seeds tolerate desiccation between 7% and 10% of water
content, and cannot withstand low temperatures. In addition, MATERIAL AND METHODS
they lose viability in a short period (60 days), precluding their
conservation using “conventional” storage practices, such Ripe fruits of G. americana (Figure 1A) were
as seed banks (NASCIMENTO et al., 2020; OLIVEIRA pulped and the seeds were rubbed onto sieve for complete
et al., 2011; SALLA; JOSÉ; FARIA, 2016). Alternatively, mucilage removal. For the quantification of the initial
cryopreservation of biological material at ultra-low water content, three replicates consisting of 25 seeds were
temperatures (-196 ºC) using liquid nitrogen (LN) is a used, and the determination was performed by the oven
promising alternative for the long-term conservation of drying method at 105 °C for 24 h.
unorthodox germplasm (REN et al., 2022). In addition
After the determination of the initial water content,
to storage without damage for an indefinite period,
seeds were dried in a closed container (10.5 x 10.5
it allows the maintenance of genetic stability, low
x 10.5 cm) containing 80 g of silica gel for 16, 18, 20, 22,
space requirements, absence of contaminants and low
and 24 h. Monitoring of water loss was performed at each
maintenance need (FOLGADO et al., 2014).
dehydration time using the formula: MC% (Moisture content)
The use of plant material in the form of = [(wet weight - dry weight) x 100)]/wet weight. In total, 150
zygotic embryos and seeds is preferred and has been seeds were used per treatment, 75 for control treatments
successfully used for cryopreservation of several species (dehydrated at different periods and non-cryopreserved)
that show seeds with unorthodox behavior (FIGUEIREDO and 75 for cryopreservation treatments. Seeds dehydrated
et al., 2021; FREITAS et al., 2016; PINTO et al., 2016). in each period (Control, 16, 18, 20, 22, and 24 h) were
The preference for the use of these structures is attributed separated, placed in cryotubes (2 mL) and immersed
to the fact that it constitutes a more organized system and in LN for 24 h. Subsequently, they were subjected to
allows forming an entire plant, dispensing more complex thawing, placing the cryotubes in a water bath at 40 ºC
stages of in vitro cultivation (BALLESTEROS et al., 2014). for 2 min. After thawing, the seeds were subjected to
However, large seed size, irregular geometry and high viability and germination analyses. Dehydrated and non-
moisture content hinder to prevent intracellular ice cryopreserved seeds were also analyzed.
formation that is the main challenge of cryopreservation
(WESLEY-SMITH et al., 2015). Viability after dehydration and freezing

Success in cryopreservation depends on the Embryo excision and submergence in 0.5%

prevention of lethal damage to cell membranes and solution of 2,3,5-Triphenyl tetrazolium chloride
organelles. Typically, these damages are associated with for 2 h in the absence of light in incubator at 30 °C
water content and expansion characteristics during freezing (CLEMENTE; CARVALHO; GUIMARÃES, 2012)
and formation of ice crystals inside cells (REN et al., 2022). were performed. They were then washed in running water,
Thus, the adjustment of the water content inside cells, individually observed and classified as viable or non-viable
triggered by the explant dehydration before freezing, is according to the formed staining. The data were transformed
fundamental (COELHO et al., 2018; PAULA et al., 2018). into and expressed as a percentage of viable embryos.

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 Cryopreservation of Genipa americana seeds

Figure 1 - Genipa americana L.; (a) ripe fruits, (b) seed germination after 13 days of cultivation, (c) seedlings showing a normal
development appearance at 45 days after cultivation, (d) histological section of embryos excised from non-cryopreservation seeds
(control) medium radicle (20x magnification), (e) histological section of embryos excided from cryopreserved seeds (20x magnification).
PD –protoderm; GM ground meristem; PC – Procambium

Germination Histological analysis

The dehydrated (-LN) and cryopreserved (+LN) Light microscopy was performed on histological
seeds were disinfested in 2.5% (v/v) NaOCl solution slides of excised embryos from non-cryopreserved seeds
for 20 min, inoculated in germination medium [½ MS with initial water content (Control) and cryopreserved
(MURASHIGE; SKOOG, 1962) salts + 15 g L-1 sucrose in the best treatment. After thawing, the cryopreserved
and 0.7% agar] and stratified in the dark for 16 days. seed and control treatment embryos were excised, fixed
Afterwards, they were transferred to the growth room in 70% ethanol and dehydrated in an ethanol gradient
under controlled conditions of temperature (25 °C), (70%, 80%, 90%, and 100%), remaining at each
relative humidity (70%), and photoperiod (16 h) (SOUZA concentration for 1h. After dehydration, the infiltration
et al., 2016). At 45 days after cultivation, germination and with hydroxyethylmethacrylate plastic resin (Leica
normal seedling percentages, shoot length (cm), and root Historesin; Heraeus Kulzer, Hanau, Germany) was
length (cm) were evaluated. Germination was considered performed according to the manufacturer’s instructions. Cross
as the radicle protrusion accompanied by geotropic sections of 5 μm thickness were cut with a rotary microtome
curvature, and the well-developed and morphologically (Leica RM 2045) and subsequently stained in 0.1% toluidine
perfect seedlings were considered as normal. blue (SRIDHARAN; SHANKAR, 2012). Sections were

Rev. Ciênc. Agron., v. 54, e20228531, 2023 3

 R. R. Souza et al.

observed at 20x magnifications and images were the seeds of G. americana, resulting in reduction from 47%
captured digitally using a microscope with a video (0 h of desiccations) to 11% of water content at 24 h of
camera coupled to a computer executing the software desiccation (Table 1).
IM50 (Leica microsystem).
The time of exposure of the seeds to silica gel,
Acclimatization besides causing a decreased in the water content, also
caused a reduction in the viability and germination
At 60 days after cultivation, seedlings regenerated
potential of seeds. This resulted in a reduction of
from non-dehydrated (control) and cryopreserved seeds
approximately 23% in the viability and germination of the
(best treatment) (+LN) were removed from the test tubes
seeds when desiccated for 24 h (Table 1). Thus, the longer
and their roots washed in running water. They were then
the desiccation time on silica the lower the viability and
transferred to polypropylene containers (300 ml) filled
germination of G. americana seeds.
with commercial substrate (Tropstrato hp®) and covered
with clear plastic in order to avoid excessive moisture Similarly, evaluated the behavior of dehydrated
loss from the seedlings after transfer from in vitro to ex G. americana seeds in saturated solution of sodium
vitro environment. Cuts were made every five days in chloride and silica gel, Salla, José and Faria (2016)
each plastic bag until complete withdrawal at 15 days. The verified that desiccation at a water content of
seedlings were kept for 14 days in a growth room with approximately 15% resulted in a reduction in viability
controlled temperature of 25 ± 2 °C and photon irradiance and germination. However, the available information
of 67 μm m-2 s-1. They were then transferred to greenhouse regarding the tolerance to desiccation of G. americana
with a 30% shading screen. At 30 days of acclimatization, seeds is conflicting. For some authors (CARVALHO;
the percentage of surviving plants, shoot and root length NASCIMENTO, 2000; SANTOS; SALOMÃO, 2016), seeds
(cm), and number of leaves were evaluated. tolerate desiccation at low water content (approximately 5%)
without loss of viability and germination potential.
Experimental design and statistical analyses
The variation of responses in the different studies
The experimental design was completely may be related to several factors that directly interfere in
randomized with treatments distributed in a 2x6 factorial the behavior and degree of tolerance to desiccation, such
design related to storage in LN (non-cryopreserved (-LN) as: maturity stage of the seeds, employed method, intensity
and cryopreserved in LN (+LN)) and dehydration periods and duration of drying (NASCIMENTO et al., 2020).
in silica gel (0, 16, 18, 20, 22, and 24 h). The obtained Furthermore, the seed variability and its complex structure
data were submitted to analysis of variance (ANOVA) and with very heterogeneous cell composition can result in
and, when significant, to an “F” test (P < 0.05) and then sensitivity and unequal drying rates within the same seed
averages were compared by Skott-Knott test (p > 0.05). batch (BALLESTEROS et al., 2014; SAHU et al., 2017).
Despite the reduction in the water content caused
decreased in seed viability and seed germination, it is
RESULTS AND DISCUSSION noted that the dehydration process was essential for
survival after thawing (Table 1). Whereas, the initial
The time of exposure of the seeds in silica gel water content (47%) of G. americana seeds was
caused a gradual reduction in the moisture content of so high that storage in LN (+LN) without previous

Table 1 - Effect of dehydration in silica gel on water content (%), viability (% ± SE) and germination (% ± SE) of Genipa
americana L. seeds cryopreserved (+LN) or not (-LN)

Viability (% ± SE) Germination (% ± SE)

Desiccation period Seed water content (% ± SE)
-LN +LN -LN +LN
Control 47 ± 0.57 a 97.6 ± 2.2 aA 0.0 ± 0.0cB 83.3 ± 8.3 aA 0.0±0.0 cB
16 h 17 ± 1.66 b 69 ± 2.7 bA 19.3 ± 10 bB 71.7 ± 4.6 bA 0.4 ± 0.3 cB
18 h 15 ± 1.66 b 60.6 ± 5.5 bA 30.3 ± 2.7 aB 64.3 ± 5.6 bA 0.8 ± 0.1 cB
20 h 14 ± 1.15 b 57 ± 1.6 bA 38.6 ± 5.5 aB 60.1 ± 2.3 bA 35 ± 1.3 aB
22 h 13 ± 0.88 b 57 ± 1.4 bA 30.3 ± 2.7 aB 72.2 ± 2.7 bA 24.0 ± 0.9 bB
24 h 11.3 ± 1.33 b 58 ± 9.6 bA 30.3 ± 2.7 aB 68.0 ± 3.6 bA 21.9 ± 0.3 bB
Data were expressed as average ± standard errors (SE). Averages followed by the same capital letter on the column and lowercase on the line do not
differ among themselves by Scott-Knott test (p < 0.05)

4 Rev. Ciênc. Agron., v. 54, e20228531, 2023

 Cryopreservation of Genipa americana seeds

dehydration treatment resulted in total seed mortality. allowed an increase in growth parameters on seedlings
The high water content in the cells during freezing obtained from cryopreserved seeds (Tabela 2).
favors the formation of ice crystals, causing lethal
Although the seedling obtained from cryopreserved
damage to the membranes and hence leading to cell
seeds showed slow and reduced growth was observed that
death (LIMA; DUTRA; CAMILO, 2014; REN et al.,
after acclimatization the seedlings showed 100% survival
2022). Therefore, to avoid damage caused by freezing
in and no differences were observed in relation to the
and desiccation, the adjustment of the amount of water
growth between regenerated seedlings of cryopreserved
present in explants before immersion in LN is fundamental
and non-cryopreserved (Figure 2).
(FIGUEIREDO et al., 2021; NINAGAWA et al., 2016).
In studies on plant cryopreservation, only the
The maximum desiccation period estimated to
evaluation of cell survival after freezing is performed.
obtain maximum percentages of viability and germination
However, in order to obtain an efficient cryopreservation
after thawing (+LN) was 36 h (Table 1). The results also
protocol, the viability of the material subjected to freezing
demonstrate that the drying of G. americana seeds to
in LN should be referred as the recoverability of the
a maximum content of 10% and a minimum of 14% is
largest amount of living cells that provide the regeneration
essential to obtaining better rates of viability, germination
and recovery of individual’s normal growth. Consequently,
and formation of normal seedlings after storage in LN
higher survival rates of seedlings and the establishment in
(Table 1 and 2). Since the drying provided by the exposure
greenhouses and field are expected, besides maintaining
of seeds to silica contributed to the occurrence of the
genetic integrity (FIGUEIREDO et al., 2021; LI et al., 2016).
vitreous state, resulting from the increase in cytoplasmic
viscosity and low cell mobility, conferring better tolerance to The observation of histological sections of
freezing (BALLESTEROS et al., 2014; REN et al., 2022). embryos excised from cryopreserved seeds revealed
intact protoderm cell contents and without the presence
The percentage of normal seedlings obtained from
of large intercellular spaces, with characteristics similar
cryopreserved seeds (+LN) showed a linear behavior as a
to embryos excised from non-cryopreserved seeds
fuction of the desiccation period (Table 2). Aditionally, the
(Figure 1D-E), indicating high cell recovery capacity
desiccation of seeds in silica gel for 22 h before immersion
after cryopreservation of G. americana seeds.
in LN allowed the successful cryopreservation of
G. americana seeds, contributing to obtain normal Dehydration in silica gel followed by immersion
seedlings with similar rates among cryopreserved and in LN is a simple, easy and low cost technique that
non-cryopreserved seeds (Table 2). proved to be efficient for cryopreservation of G.
americana seeds, besides allowing germination after
Seedlings obtained from cryopreserved seeds
thawing, high survival rates, with growth and normal
(+LN) compared to non-cryopreserved seeds (-LN)
establishment of seedlings after acclimatization.
showed reduced growth with lower height and root length
(Tabela 2). Freezing induces a series of stresses on the The results demonstrate that the cryopreservation
plant material, making it susceptible to modifications in of G. americana seed emerges with an important
the ultrastructural organization of cells and subsequent species conservation strategy, due to the small space
growth (GALDIANO et al., 2012). However, it is required for the technique, low maintenance cost, low
important to note that the pretreatment of dehydration labor costs, long-term storage and high survival rates.

Table 2 - Effect of dehydration in silica gel on the percentage of normal seedlings (% ± SE), height (cm ± SE), and root length (cm ±
SE) of regenerated seedlings of Genipa americana L. cryopreserved (+ LN) or not (-LN)

Normal seedlings (% ± SE) Height of seedlings (cm ± SE) Root length (cm ± SE)
Desiccation period
-LN +LN -LN +LN -LN +LN
Control 100 ± 0.0 aA 0.0 ± 0.0 dB 6.2 ± 0.0 aA 0.0 ± 0.0 cB 4.14 ± 0.005 dA 0.0 ± 0.0 eB
16 h 100 ± 0.0 aA 0.0 ± 0.0 dB 5.4 ± 0.13 bA 0.0 ± 0.0 cB 4.68 ± 0.03 bA 0.0 ± 0.0 eB
18 h 100 ± 0.0 aA 100 ± 0.0 aA 5.1 ± 0.12 bA 2.7 ± 0.04 bB 4.61 ± 0.04 bA 3.97 ± 0.01 cB
20 h 92.3 ± 0.6 bA 94.7 ± 0.5 aA 4.9 ± 0.13 cA 3.0 ± 0.05 aB 4.95 ± 0.01 aA 4.51 ± 0.06 aB
22 h 91.6 ± 3.9 bA 75.0 ± 1.5 bB 5.1 ± 0.10 bA 3.1 ± 0.06 aB 4.96 ± 0.02 aA 3.70 ± 0.02 dB
24 h 87.5 ± 0.5 cA 60 ± 0.7 cB 4.9 ± 0.10 cA 3.1 ± 0.09 aB 4.50 ± 0.02 cA 4.16 ± 0.08 bB
Data are expressed as average ± standard errors (SE). Averages followed by the same capital letter on the column and lowercase on the line do not differ
among themselves by Scott-Knott test (p < 0.05)

Rev. Ciênc. Agron., v. 54, e20228531, 2023 5

 R. R. Souza et al.

Figure 2 - Genipa americana L. seedlings after 60 days of cultivation from cryopreserved (a), non cryopreserved seeds (b), ex vitro
acclimatization (c), seedlings after acclimatization, showing normal appearance and without phenotypic changes from cryopreserved
(d) and nono-cryopreserved (e) seeds. Bar: 1cm

CONCLUSION Foundation of the State of Minas Gerais (FAPEMIG) for

the financial support of this study
The Dehydration in silica gel for the minimum
time 20 h (corresponding to 14% water content)
and a maximum of 36 h (content of 10%) allows G.
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