Biotechnologies Tools for Germination and Preservation of Butia Eriospatha Embryos (Arecaceae)

Butia eriospatha is an endemic species of Brazilian Atlantic Forest and due to the anthropic intervention, it is in the vulnerable conservation status. In this species, plants are only regenerate by seeds which have dormancy and slow as well low germination. To cope with the concern limitation of seedling establishment we study B. eriospatha embryo desiccation tolerance threshold as well the physiological requirement for in vitro germination and cryopreservation. Fresh embryos and desiccated ones were in vitro germinated using a culture medium with hormones and antioxidants. The embryo desiccation tolerance threshold was 0.14 gH 2 O.gDW -1 with 93.33% of germination. During embryos desiccation was observed a signicant increase in PUT, which resulted in the ratio decrease [(SPD+SPM). PUT -1 ] DW), but the SPD was the most abundant polyamine overall. Signicant increase in POD and APX activity led us to suggest that they are the main enzymes involved in cellular protection during desiccation. An increase of amino acids content, especially glutamic acid (Glu), leucine (Leu), lysine (Lys), glutamine (Gln), which are known as osmoprotectors, also was observed. A specic embryo desiccation stage (0.14 gH 2 O gDW -1 ) associated to its biochemical state were successfully used in the cryopreservation protocol and result in more than 90% of recovery and in vitro germination. The physiological and biochemistry approach of this study associated to an applied protocol for plant genetic resources conservation of B. eriospatha embryo can be potentially used for other Arecaceae species. goal to cryopreserve zygotic embryos. We are facing an alarming conservation status of B. eriospatha and the scientic community is sick to develop ex situ conservation strategies as well found out more about seed physiological behavior that can help to do restoration and preserve the remain plant population of this species. Our hypothesis is that there is specic biochemical status and desiccation tolerance threshold of B. eriospatha embryos which make them responsive to cryopreservation protocol and in vitro germination. This study analyzes and discusses the role and interaction of PAs, amino acids and antioxidant enzymatic defense in the maintenance of B. eriospatha embryo viability during dry. In vitro germination and cryopreservation protocols were for the rst time stablished given an ecient tool for long-term ex situ conservation strategy and high germination seeds.


Introduction
Arecaceae species are worldwide popular for their organoleptic and nutritional fruits as well for the ornamental plant characteristics (Hoffmann et al. 2014). However, is not popular known that around 26% of Arecaceae species are on the Red List -International Union for Conservation of Nature (IUCN) (IUCN, 2021). Unfortunately, some of them, such as Corypha taliera Roxb. and Cryosophila williamsii P.H. Allen are already "extinct in the wild" (EW) (Johnson 1988;IUCN 2021). Butia eriospatha (Martius Ex Drude) Beccari is a native Arecaceae of South Brazilian Atlantic Forest and is also on the IUCN list as "vulnerable" (VU) and on the Brazilian Endangered Species list (Instrução Normativa 06, MMA 2008, IUCN 2021). The worry conservation status of B. eriospatha is due the severe anthropic intervention, including the local and international illegal trade, overexploitation of fruits and habitat replacement by exotic tree and cattle farming (Nazareno and Dos Reis 2012, 2014b). As a consequence the small remnants of the B. eriospatha plant population have low levels of genetic diversity (Nazareno and Dos Reis 2012). On top on that plants are only regenerate by seeds and, because of the dormancy they can take up to a year to germinate only around 50% and this is a concerning limitation about seedling establishment and species conservation (Broschat 1998;Fior et al. 2011;Lopes et al. 2011;Magalhães et al. 2013;Schlindwein et al. 2013). Many article reported the worrying genetic situation of the B. eriospatha species (Nazareno et al. 2011; Nazareno and Dos Reis 2012, 2014b, a) and in vitro culture protocols have been studied to preserve its genetic diversity (Minardi et al. 2011;Waldow et al. 2013). Nonetheless, the knowledge regarding its reproductive biology is still limited, especially related to seed germination and desiccation tolerance physiology, which is essential for species conservation and restoration.
Whether or not a seed can survive drying has a direct in uence on in situ and ex situ plant conservation methods (Walters and Pence 2020). Around 50% of the trees of tropical forest, including Arecaceae species, produce recalcitrant seeds that are not desiccation tolerant (Wyse and Dickie 2017; Royal Botanic Gardens Kew 2021). This seeds are shed with high water content and, if they do not nd the right condition to germinate they die, since soil bank seeds are not observed for most of recalcitrant seeds species (Obroucheva et al. 2016; Gonçalves et al. 2020). In tropical ecosystems, ex situ conservation programs have been more and more important due the climate change and forest reduction. They have been largely use biotechnologies tools to preserve tissues, seeds and embryos from many plant species that cannot be storage in conventional seed bank, are from rare collection or have natural issues to germinate (Engelmann 2004; Walters and Pence 2020). Zygotic embryos are a source of genetic diversity and are a suitable target for cryopreservation protocols since they are young tissues (Normah and Makeen 2008; Wen and Wang 2010; Engelmann 2011). However, embryos from recalcitrant seed do not tolerate desiccation and the primary goal of any cryopreservation procedure is to reduce water content to achieve intracellular vitri cation preventing the intracellular lethal ice crystals formation (Berjak and Pammenter 2013; Walters et al. 2013). Plant cell tissue undergo to desiccation have to cope with cellular stress and the protective mechanisms are determinant to maintain cell viability (Walters 2015). Usually, it was observed chromatin compaction, late embryogenesis abundant proteins (LEAs) and heat shock proteins (HSPs) synthesis, reactive species oxygen (ROS) equilibration, lipids, polyamines (PAs), speci cs amino acid (AA) who act in cell membrane stabilization and deposition of non-reducing sugars as llers that prevent mechanical cell collapse (Kleinwachter et al. 2014;Li et al. 2015; Leprince et al. 2017; Dussert et al. 2018). To cope with the lack of these mechanisms, over the last 30 years the use of plant vitri cation solution (PVS 2 and PVS 3 ) has been applied in protocols of cryopreservation (Sakai et al. 1990; Yamada et al. 1991;Nishizawa et al. 1993 Particularly during the last 40 years many efforts have been made to establish cryopreservation protocols for Arecaceae species tissues, for instance Elaeis guineensis. Jacq. (Grout et al. 1983), Phoenix dactylifera L. (Bagnio and Engelmann 1991), Cocos nucifera L. (Assy-Bah and Engelmann 1992), Sabal spp (Wen and Wang 2010), including Butia capitata (Dias et al. 2015), B. odorata (Fior et al. 2020) and B. yatay (Vargas et al. 2020). However, different species require different cryopreservation protocols and not all the previous Arecaceaes studies have the goal to cryopreserve zygotic embryos. We are facing an alarming conservation status of B. eriospatha and the scienti c community is sick to develop ex situ conservation strategies as well found out more about seed physiological behavior that can help to do restoration and preserve the remain plant population of this species. Our hypothesis is that there is speci c biochemical status and desiccation tolerance threshold of B. eriospatha embryos which make them responsive to cryopreservation protocol and in vitro germination. This study analyzes and discusses the role and interaction of PAs, amino acids and antioxidant enzymatic defense in the maintenance of B. eriospatha embryo viability during dry. In vitro germination and cryopreservation protocols were for the rst time stablished given an e cient tool for long-term ex situ conservation strategy and high germination seeds.

Plant material
Mature fruits were collected from 20 B. eriospatha wild plants in the Serrano Plateau of Santa Catarina State (S 27º 12', W 50º 36'), from January to March 2021. Fruits without insects or microorganisms attack were manually processed to get out seed. The seeds enveloped by the woody endocarp (pyrenes) were kept at 25ºC for 3 days and then storage at 8ºC for no more than 15 days before experiments start.

Seed and embryo morphology and morphometry
Seed and embryos measurements (diameter, length, and width) were performed in 100 seeds using caliper. They were pictured using stereo microscope (Olympus -SZH10) equipped with image capture system (Olympus -DP71) and DP Controller software.
The fresh masses of the embryos were determined in their initial condition and after 24h of immersion in the osmotic solutions and their WC were gravimetrically measured.

Embryos drying
Butia eriospatha embryos were partial desiccated according to Engelmann (2004) with modi cation. Embryos were excised from the hard endocarp seed with forceps and scalpel and directly seat in a Petri dish with lter paper soaked in ascorbic acid (0.2 g l -1 ) and citric acid (0.2 g l -1 ) solution to prevent tissue oxidation. For desiccation, embryos were transferred to a dry lter paper in a laminar air ow cabinet at 25 ± 2°C, 55 ± 5% relative humidity [RH] and an air ow of 0.46 m s -1 . Embryos were desiccate for 0 (control); 15; 30; 60; 120; 180; 240 and 300 min and then the desiccation curve was calculated (Hong and Ellis, 1996). Viability and vigor analyses were performed in embryos samples from all desiccation times, while biochemical and cryopreservation analyses were performed in embryos desiccated for 0; 15; 240 and 300 min. Embryos drying rate index (k) was calculated from the amount of water loss in dry basis (gH 2 O g DW -1 ) divided by correspondent elapsed time in minutes.

Viability and vigor
In vitro germination tests and germination speed index (GSI) Seeds were immersed in ethyl alcohol (70% v v -1 ) for 1 min followed by 15 min in sodium hypochlorite (2.5% v/v -1 ) and then rinsed three times with sterilized distilled water. Embryos were excised according to previous description topic "Embryos drying", and were immersed in a commercial sodium hypochlorite solution (0.5% v/v -1 ) for 10 min and then rinsed three times in sterile distillate water (Magalhães et al. 2013). Immediately after that, embryos were inoculated in Petri dish containing 25 ml of MS (75% v/v -1 ) culture medium (Murashige and Skoog 1962) supplemented with 30 g l -1 sucrose, 0.5 mg l -1 thiamine, 1 mg l -1 pyridoxine, 0.5 mg l -1 nicotinic acid, 3 g l -1 activated charcoal, 8 µM GA 3 and 6 g l -1 agar (Ribeiro et al. 2011). The pH was adjusted to 5.8 and autoclaved at 121ºC, 1.5 atm for 15 min. The germination was performed in growth room (25 ± 2°C; 55% RH) with white LED light (Green Power TLED W; Philips TM; 77 µmol m −2 s −1 ) and 16/8 h photoperiod. Germinated embryos were recorded every day considering radicle protrusion. Germination percentage was calculated by the cumulative number of daily germinated seeds with respect of the total number of seeds evaluated (Ranal and De Santana 2006). The germination speed index (GSI) was calculated according to the Maguire's index (Mangure 1962) as the total number of seeds germinated per day between sowing and germination divided by the number of days of the test.
Results were expressed in percentage of seed with positive reaction to TTC.

Seedling morphometry
Seedling measurements (primary root, cotyledonary petiole and leaf sheath) were performed after eight weeks. Seedlings were pictured in stereo microscope (Olympus -SZH10) equipped with image capture system (Olympus -DP71) and DP Controller software.  (Sakai et al. 1990) and PVS 3 (Nishizawa et al. 1993) solutions on an orbital shaker at 90 rpm at 4°C for 60 min (Kartha et al. 1982; Berjak and Pammenter 2014b) and then droplets (10 -15 µL) were placed to aluminum foil strips (0.5 cm x 3.0 cm) and transferred to a sterile polypropylene cryovials (2 ml) before to be immersed in LN. After 24 h, aluminum foil strips and embryos removed from aluminum foil envelopes were thawed in a sucrose solution (1.2 M) at 45°C for 5 min ) and then embryos were transferred to Petri dishes (90 mm diameter) containing the germination culture medium and kept under incubation condition described in topic "In vitro germination tests and germination speed index (GSI)" and seedlings morphometry were realized following the described in topic "Seedlings morphometry". (2007) with modi cations. Free and conjugated PAs were derivatized with dansyl chloride and quanti ed by HPLC using a 5μm C 18 reverse-phase column (Shimadzu Shin-pack CLC ODS). The gradient of absolute acetonitrile was programmed to 65% over the rst 10 min, from 65 to 100% for 10 to 13 min, and 100 % for 13 to 21 min, using 1 ml min -1 ow rate at 40ºC.

Antioxidant enzyme extraction and assays
PAs concentration was determined using a uorescence detector with a wavelength of 340 nm (excitation) and 510 nm (emission). Peak areas and retention times were measured by comparison with standard PAs: putrescine (PUT), spermidine (SPD) and spermine (SPM). The 1,7-diaminoheptane (DAH) was used as internal standard.

Free amino acids (AA) determination
For free amino acids (AA) determination three embryos samples (100 mg FW, ≅ 100 embryos) of each treatment were ground in 1.5 ml of 80% (v v -1 ) ethanol and concentrated in 'speed vac'. Samples were resuspended in 0.5 ml Milli'Q water type and centrifuged at 20.000 × g for 10 min. The supernatant was ltered through a 20-µm membrane. Amino acids were derivatizated with o-phthaldialdehyde (OPA) and identi ed by high-performance liquid chromatography (HPLC), according to Astarita et al. (2003). The samples using a 5-µm C 18 reversephase column (Shimadzu Shin-pack CLC ODS). The gradient was developed by mixing increasing proportions of 65% methanol to a buffer solution (50 mM sodium a cetate, 50 mM sodium phosphate, 20 ml l -1 methanol, 20 ml l -1 tetrahydrofuran and pH 8.1 adjusted with acetic acid). The gradient of 65% methanol was programmed to 20% over the rst 32 min, from 20% to 100% for between 32 min and 71 min, and 100% for between 71 min and 80 min, at 1 ml min -1 ow at 40°C. Fluorescence excitation and emission wavelengths of 250 nm and 480 nm, respectively, were used for amino acid detection. Peak areas and retention times were measured by comparison with known quantities of standard amino acids

Statistical Analyses
Data on seed morphometry was measured in four replicates of 25 seeds each. For water relations analyses were used ve replicates of 10 embryos/treatment. For viability and vigor analyses and cryopreservation experiments were used four replicates of 25 embryos/treatment. The data normality was evaluated using the Shapiro -Wilk test, and then submitted to analysis of variance in a completely randomized design. In the case of signi cance of the F values, means were compared using SNK test (p < 0.05) (Sokal and Rohlf 1995). For statistical processing, the data rate was log transformed. Statistical analysis was carried out with R 3.4.4 programming environment.

Result
Morphology characterization of seeds and embryos B. eriospatha seeds have elliptic irregular shapes; a brown seed coat (sc) and an operculum (os) forming a small protuberance at its proximal extremity (Fig. 1a). Seed diameter (d) were 10.29 ± 0.84 mm and length (h) were 8.56 ± 0.92 mm (Fig. 1a). The white opaque endosperm (en) was covered by thin seed coat (sc) and have a cavity containing small embryo (Fig. 2b), located immediately under to the operculum (Fig. 2b). In the embryos proximal portion (pp) a cotyledonary petiole (cp) envelops a rudimentary embryonic axis while in the distal portion (dp) it is the haustorium (ha) (Fig. 2c).
Embryos were of 1.1 ± 0.04 mm in length and 0.5 ± 0.008 mm in width.

Viability and vigor
Desiccation time did not have a signi cant effect on B. eriospatha embryos germination since after they were dried for up to 300 min it was observed a germination of 93.33% while the control germinates 100% (Fig. 3a). Germination results validated all the data observed in tetrazolium test indicating an adequate protocol for embryo viability in this species (Fig. 3a).
However, signi cant effects were observed in the embryos germination dynamic. In control and embryos that went through drying for up to 60 min the protrusion of the radicle started at 7th day after inoculation and took 10 days to reach 100% of germination (Fig. 3b). Embryos dried for 120 and 180 min extended germination up to 12 days, and showed a small decreased in the germination percentage (96.67%). Embryos dried for 240 and 300 min showed a decrease in germination speed, obtaining 93.33% of germinated embryos only at 18 days after inoculation (Fig. 3b). Germination dynamic is directly associated to the germination speed index (GSI) which show the lowest value in embryos dried for 240 and 300 min (GSI = 0.43 and GSI = 0.42, respectively) compared to the control and embryos dried up to 180 min (GSI = 0.71 and GSI = 0.59, respectively) (Fig. 3c). Butia eriospatha seedlings morphometric analyses indicated an expressive length of the cotyledonary petiole (cp) (Average: 23.25 ± 0.54 mm) but did not present signi cant difference between drying times (Fig. 3d). However, when embryos were dried for 300 min, we observed a decreased in both the lengths of primary root (rp) and leaf sheath (ls) (Fig. 3d) when compared to the control and embryos dried for 15 min.

Cryopreservation
Embryos soaked in antioxidant solution and dried for 15 min, regardless of having been incubated in PVS 2 and PVS 3 solution, did not germinate when were submitted to a high cooling rate and droplet vitri cation technique (Table. 1). These embryos that did not germinate remained unchanged in size for more than eight weeks, without elongation or callus formation. Embryos dried for 180 and 300 min, followed by incubation in PVS 2 and PVS 3 solution, had an increase in their water content (Table. 1). Despite of the increase in the water content, embryos dried for 180 and 300 min, instead of the PVS 2 and PVS 3 incubation, submitted to a high cooling rate and droplet vitri cation technique presented germination rates above 83.33 ± 7.95% and showed no signi cant difference (Table. 1). Germination speed index (GSI) values for embryos dried for 180 and 300 min with or without incubation in PVS 2 and PVS 3 solution did not show signi cantly difference (Table. 1).
Eight weeks after cryopreservation the presence of normal seedling development were seen in the embryos dried for 180 and 300 min and submitted to high cooling rate and droplet vitri cation technique (PVS 2 and PVS 3 solution) ( Table. 1; Fig. 4a).
Embryos dried (180 and 300 min) incubated in PVS 2 solution and submitted to droplet vitri cation technique showed high number of abnormal seedlings (29.17 ± 0.88 and 37.03 ± 1.05), respectively, following by the incubated in PVS 3 solution (14.81 ± 0.80 and 16.66 ± 0.81). These abnormal features include the absence of primary root (arrow) (Fig. 4b) and leaf sheath (Fig. 4c), as well as the swelling of the cotyledonary petiole (cp). In this treatment it was also observed callus proliferation in the basal region of embryo (Fig. 4d).

Polyamines (PAs) content
In B. eriosptha embryos the total free PAs content decreased when the embryo were desiccated and presented the lowest contents of putrescine (PUT), spermidine (SPD) and spermine (SPM) (Fig. 6a -d). Control had higher contents of SPD and SPM compared to embryos desiccated for 300 min (Fig. 6c-d) and the opposite behavior was observed for PUT which show the highest value at the same desiccation time (Fig. 6b). Spermidine (SPD) was the most abundant polyamine, followed by

Free amino acid content
An interesting relation were observed in embryos dried for 300 min with a signi cant increase in total free amino acids content compared to the ones dried for 0; 15 and 180 min (Fig. 7a). Lysine (Lys) represents 42% of the total free amino acids pro le observed during the whole drying time (Fig. 7b; Table. S3). Gamma-aminobutyric acid (Gaba), citrulline (Cit) and arginine (Arg), were amino acid which did not change signi cantly during drying times while lysine (Lys), leucine (Leu), isoleucine (Ile) glutamine (Gln) and glutamic acid (Glu) practically doubled their contents when the embryo was desiccated for 300 min (Fig. 7b; Tab. S1). These amino acids were approximately 97.85% of the total amino acids detected in embryos that were desiccated for 300 min. These results could suggest that in B. eriospatha embryos theses amino acid can be related to desiccation tolerance mechanisms and play a key role in maintaining viability of embryos, once dried embryos for 300 min presented a germination rate of 93.33% . The ratio of ornithine (Orn) and arginine (Arg) to total amino acids indicate that Orn increase preferentially to Arg with dehydration (Fig. 7c). Undesiccated embryos (control) had higher Arg content, which decreased at 15 min of desiccation and remained constant until 300 min of desiccation. The Orn content was higher in the 15 min of desiccation compared to the control, but did not differ from 180 min and 300 min of desiccation (Fig. 7c). indicate the presence of a mature embryo with high physiological quality. Embryos (control) lost 94.53% of their WC when desiccates for 300 min, reaching 0.14 g H 2 O g DW −1 (11.98%) (-2.2 MPa). Although, embryos dried for 300 min exhibited high viability (germination and TTC test > 90%), the germination speed index (GSI) decreased substantially from 0.71 (control) to 0.42 (300 min), indicating a reduction of vigor. A study with B. capitata showed that whole seeds were able to tolerate moisture loss to 10 and 5%, whereas dehydration to 3.5% moisture content was harmful (Dias et al. 2015).

Discussion
Comparatively to these reports our results indicating that the B. eriospatha species produce high physiological quality seeds and the embryo desiccation tolerance threshold is close to the embryos from orthodox seeds ( In contrast Hevea brasiliensis L. zygotic embryos had a high tolerance to PVS 2 in terms of survival, but are sensitive to osmotic stress induced by PVS 3 (Nakkanong and Nualsri 2018). In our study, embryos of B. eriospatha dried for 180 (0.17 gH 2 O gDW −1 ) and 300 min (0.14 gH 2 O gDW −1 ), regardless of whether or not they were incubated in PVS 2 and PVS 3 solution, showed high germination (>80%), after cryopreservation. However, embryos desiccated for 300 min (0.14 gH 2 O gDW −1 ) followed by immersion in LN (-196ºC) showed an increase of normal seedlings (92.86%), suggesting that PVS 2 and PVS 3 was overall toxic. Our study found out for the rst time the desiccation tolerance threshold and a complete and successful cryopreservation protocol of B. eriospatha embryo allowing more than 90% of germination.
Our results suggest that the ability of B. eriospatha embryo to be desiccation tolerant and maintain high viability is associated to the oxidative stress control by the elimination of reactive oxygen species (ROS). The elevated levels of ROS might inhibits repair processes, which are linked, for the most part, to protein synthesis (Umezawa et al. 2006). In embryos not desiccation tolerant, the metabolic balance is disturbed and the generation of ROS is out of control, which means that protective antioxidant reactions are not able to remove ROS quickly enough ( . This also make sense once we nd out a report showing that in not desiccation tolerant Antiaris toxicaria L. embryos, the H 2 O 2 could not be converted into H 2 O effectively because of the less activities CAT and APX (Cheng and Song 2008). In the present work, CAT activity increases up to 180 min and its activity was dramatically reduced after 300 min of desiccation, achieving less activity than was observed in embryos without desiccation. In contrast, POD and APX showed the highest activities at 300 min of desiccation, which suggests that they play an important role in the desiccation tolerance with dehydration.
Water stress tolerance in plant tissues and seeds were also increased by biosynthesis and accumulation of PAs as was indirectly related to desiccation tolerance, since one of the glycinebetaine (GB) synthesis pathways is through glycine Nmethylation Murata 2002, 2011). Additionally, the putative role of Glu in the oxidative defense mechanism has been found to be due not only to its internal metabolism but also to its exogenous use, in soybean seeds priming, that increased the antioxidant activity of CAT, POD and SOD enzymes (Teixeira et al. 2017).
As a conclusion this study designs a schematic integration of the in vitro germination and cryopreservation protocols of B. eriospatha embryos and its biochemical status associated to plant population remnants as well to an ex situ conservation (germoplasm bank) strategy (Fig. 8). Our study points out for the rst time that B. eriospatha embryos can have more than 90% of in vitro germination in a water content range of 2.56 to 0.14 gH 2 O gDW −1 . This germination protocol using culture medium supplemented with hormones and antioxidants ensures the large-scale seed germination of remnant plant population to guarantee the genetic diversity preservation (Fig. 8). In addition to advances in understanding the physiological behavior of desiccation tolerance, we provide relationships between amino acids, antioxidant activity, and polyamines during desiccation stress. At a speci c embryo desiccation threshold (0.14 gH 2 O gDW −1 ) compare to the previous embryos water content, the increase in the activity of APX and POD suggest they were highly e cient against the oxidative stress caused during desiccation (Fig. 8). Also, the increase in the endogenous contents of PUT and the decrease in the biosynthesis of SPD and PMS, in relation to the control (2.56 gH 2 O gDW −1 ), resulted in a reduction in the ratio [(SPD+SPM). PUT −1 ] DW). This biochemical event combined with the increase in the content of Gln, Glu, Lys, Leu, Orn and Met seems to play a key role in the desiccation tolerance and maintenance of embryo viability. At this speci c embryo WC (0.14 gH 2 O gDW −1 ) and biochemical state they do not only were able to germinate in vitro, but even tolerate low temperature storage. The combination of this embryo stage and rapid cooling in LN, allowed us successfully established a cryopreservation protocol with in vitro germination and normal seedling development above 90% (Fig. 8). In this study it was showed that the precise information about the cell state related to water content and its biochemical composition were essential for the establishment of cryopreservation protocols with the reduction of metabolism without cell damage of B. eriospatha embryos. In this way and known the loss of habitats of B. eriospatha combined with the seed dormancy and low germination is essential to develop accessible technologies that ensure propagation and conservation of this species. Thus, both, in vitro germination and the cryopreservation protocol here established are powerful tools directly applied and useful to preserve a vulnerable tropical species from Brazilian Atlantic tropical forest, which is one of the most important hotspots of biodiversity in the world.  Figure 1 Morphology of Butia eriospatha seed (a-b) and embryo (c). External morphology of whole of seeds showing operculum (os) and seed coat (sc); regions of diameter (d) and length (l) measurements (a). Longitudinally sectioned seed with embryo (em), endosperm (en) and seed coat (sc) (b). Mature embryo with cotyledonary petiole (cp) in the proximal portion (pp) and haustorium (ha) in the distal portion (dp) (c). Bars: a, b = 2.0 mm; c = 500 µm