Revista Mexicana de Ciencias Forestales Vol. 13 (69)

Enero – Febrero (2022)

1Instituto Nacional de Investigaciones Forestales Agrícolas y Pecuarias, Cenid-Conservación y Mejoramiento de Ecosistemas Forestales. México.

La crioconservación de semillas representa una alternativa para la conservación a largo plazo de germoplasma forestal, sobre todo de especies tropicales intermedias o recalcitrantes, cuya viabilidad decae rápidamente bajo condiciones estándares de almacenamiento. Por ello, se evaluó en semillas de cedro rojo el impacto del congelamiento rápido en nitrógeno líquido (N2L) sobre la germinación sensu stricto y desarrollo temprano de la plántula. Para este fin, semillas deshidratadas o sin deshidratar se sometieron a pretratamientos de encapsulación/deshidratación en presencia de agentes osmoprotectores (LS, PVS2 y PVS3), previo a su congelación en N2L. Después de la descongelación a temperatura ambiente, se compararon la capacidad y velocidad de germinación de las semillas de los distintos tratamientos, así como el establecimiento, supervivencia y crecimiento de las plántulas después de cuatro meses en vivero. Los resultados muestran que las semillas de cedro rojo tienen la capacidad de sobrevivir el congelamiento rápido, aunque esto tuvo en general un efecto perjudicial durante las etapas de germinación y emergencia temprana, el cual fue menos severo en semillas sin encapsular. No obstante, los pretratamientos favorecieron la supervivencia de la plántula en vivero; el sistema radicular tuvo mayores afectaciones que la parte aérea en todos los tratamientos de congelación, lo cual incidió en una alta relación PSA/PSR. Se concluye que las semillas de Cedrela odorata sometidas al congelamiento rápido tienen el potencial de sobrevivir, germinar y producir plántulas para trasplante en vivero, aunque es necesario afinar el protocolo para optimizar la respuesta.

Palabras clave: Cedro rojo, conservación ex situ, germinación, germoplasma, nitrógeno líquido, osmoprotector.

Seed cryopreservation represents an alternative for the long-term conservation of forest germplasm, especially of intermediate or recalcitrant tropical species, whose viability declines rapidly under standard storage conditions. The impact of rapid freezing of Spanish cedar seeds in liquid nitrogen (L2N), on sensu stricto germination, and on the early stages of seedling development was evaluated. To this end, both dehydrated and non-dehydrated seeds were subjected to encapsulation/desiccation pre-treatments in the presence of various anti-vitrifying agents (LS, PSV2, and PSV3), prior to freezing. After thawing at room temperature, germination speed and capacity were compared, as were plantlet establishment, growth and survival after transplanting to the nursery. Results show that Spanish cedar seeds have the potential to survive fast freezing, although, in general, this had a damaging effect during the germination and early stages of plantlet establishment, being less severe in seeds that had not been encapsulated. However, the pretreatments favored plantlet survival in the nursery. The roots were the most affected organ, which led to a high shoot root dry weight ratio (S:R). It is concluded that Cedrela odorata seeds subject to rapid freezing have the potential to survive, germinate and produce plantlets that can be transferred to nurseries, although further fine tuning of the protocol is required to optimize response.

Key words: Red cedar, ex situ conservation, germination, germplasm, liquid nitrogen, osmoprotectant.

Introduction

Cedrela odorata L. is the second most commercially important precious wood species in the tropical and subtropical regions of Mexico and Latin America (Pennington and Sarukhán, 2005); therefore, it is considered a priority species for reforestation and commercial forest plantation programs. However, due to the destruction of its habitat by deforestation and excessive use, it was included in Appendix III of the 2013 Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES) in order to regulate its trade and prevent its unsustainable exploitation; is also part of the Red List of the International Union for Conservation of Nature (IUCN) as a vulnerable species (Mark and Rivers, 2017); in Mexico, it is listed in the NOM-059-SEMARNAT-2019 norm as a species subject to special protection (Semarnat, 2019). It is essential to establish strategies for seed management and conservation in order to preserve this genetic resource in germplasm banks, as well as to meet the growing demand for seeds for plantation and reforestation programs.

Based on their storage characteristics, C. odorata seeds are classified as intermediate, as they are relatively tolerant to dehydration, although sensitive to chilling (Andrés et al., 2011). However, their viability declines a few months after collection, which limits the possibility of mid-to-long term maintenance under ex situ conditions (García and Abdelnour, 2013) and reinforces the need to seek alternatives for their preservation.

Cryopreservation, or storage of cells, tissues or organisms at ultra-low temperatures, in liquid nitrogen (N2L, below -150 °C), has been successfully implemented for the conservation of agricultural (Hor et al., 2005; Acosta et al., 2020), ornamental, (Hirano et al., 2009) and forest species (Pita et al., 1998; Michalak et al., 2015). In addition, it represents an option for endangered, as well as tropical species with intermediate or recalcitrant seeds, particularly those that can withstand partial dehydration (Hor et al., 2005), such as Spanish cedar. The technique is intended to halt metabolic processes and cell division, and, thereby, to prevent deterioration (Engelmann and Dussert, 2013).

However, the freezing process has consequences as it can induce crystal formation and osmotic shock, which is lethal to the tissue; therefore, success depends on the proper management of cellular moisture content (Dussert et al., 2001; Hor et al., 2005). Because the water and macromolecule content differs in seeds of different species, standardized protocols are required to limit damage during freezing and subsequent thawing. On the other hand, it is necessary to evaluate the effectiveness of the process, both to preserve the viability and germinability of the seeds, as well as their potential to produce good quality plants.

Research on tissue cryopreservation in the Cedrela genus is scarce, but promising. Studies on C. fissilis Vell. (Da Costa et al., 2003) show that seeds tolerate freezing with N2L and recover to produce vigorous seedlings, as long as the moisture content remains between 6 and 7 %. Similarly, in C. odorata, vitrification and freezing of apices and seeds, as well as subsequent recovery under in vitro conditions promotes high survival and efficient germination, even after several months of freezing (García and Abdelnour, 2013). However, the impact of different seed management strategies prior to N2L treatment on germination, survival, rooting and overall quality of the seedlings obtained is still unknown. Therefore, the objectives of this work were: 1) to determine the efficiency of different cryopreservation strategies on freezing tolerance in seeds of C. odorata, and 2) to test the rooting ability, survival and growth of seedlings 16 weeks after transplanting to the nursery.

A composite mixture of C. odorata seeds collected in 2018 from the states of Chiapas, Tabasco, and Veracruz, Mexico, was used. From this mixture, a working sample of 1 100 seeds was obtained without physical damage or evidence of contamination by pests or diseases. The moisture content (MC) of the lot was 8 % and was determined prior to the establishment of the experiments, according to ISTA guidelines (2016).

The effect of freezing in N2L was compared in intact seeds that did not receive any handling (naked - T0), with seeds subjected to different pretreatments (Table 1), in which the effect of dehydration (T1) and encapsulation/dehydration (T2 and T3) was tested; as well as the additional application of different osmoprotectants, alone (T4, T5 and T6) or in combination (T7 and T8). Five replications of 20 seeds were used in each treatment, for a total of 100 seeds. Untreated and unfrozen seed was used as a germination control (TP).

For treatments that included encapsulation/dehydration, the C. odorata seeds were placed in a 2 % sodium alginate solution (Sigma); then, they were individually transferred to a 0.1 M calcium chloride polymerization solution (Sigma) during 15 minutes. Finally, they were placed in a desiccation chamber with activated silica gel until reaching constant weight (5.13 % CH in the seed without encapsulation and 5.24 % in the encapsulated seed, after 2.5 hours of dehydration). This procedure was repeated with the rest of the treatments.

In the osmoprotectant treatments, the encapsulated/dehydrated seeds were immersed for 10 minutes in LS (Loading solution: Murashige and Skoog (1962) medium, 2.0 M glycerol and 0.4 M sucrose) (Nishizawa et al., 1993); then left for 20 min, in PVS2 (glycerol 30 %, ethylene glycol 15 %, dimethyl sulfoxide 15 %, sucrose 0.4 M) (Sakai et al., 1990), or in PVS3 (glycerol 50 %, sucrose 1.5 M) (Nishizawa et al., 1993).

The seeds of the different treatments were immersed in N2L for 20 minutes, and then thawed at room temperature.

After applying the different treatments, the seeds were placed in germination boxes (SeedburoTM) on filter paper moistened with sterile double distilled water. Incubation was carried out in a Memmert HPP750 climatic chamber (Memmert GmbH + Co.Kg, Germany), at 28 °C (day/night) and room relative humidity. The seeds were checked daily for 22 days and were considered as germinated when the root protruded from the testa by a length of approximately 3 mm. Germination capacity (GC) was evaluated as the percentage of germination at the end of the test; germination energy (GE) was evaluated as the number of days in which 50 % germination was achieved (higher values indicated lower GE) (Juárez-Agis et al., 2006); the germination value (GV) was estimated obtained using the Czabator equation (1962):

Where: ADG (average daily germination) resulted from dividing the final percentage of germinated seeds by the number of days of testing (Viveros-Viveros et al., 2015); and the peak value (PV) corresponds to the cumulative percentage at the inflection point of the germination curve (González-Zertuche and Orozco-Segovia, 1996).

Once germinated, the seeds were placed on moist, sterilized substrate (35 % of peat moss, 35 % of perlite, and 30 % of vermiculite) under greenhouse conditions. The design was completely randomized in accordance with the ten treatments and four replications. After 15 days, the percentage of seeds with main root growth, generation of secondary roots, hypocotyl growth, and the appearance of true leaves were counted.

15 rooted seedlings were randomly selected from each of the treatments and transplanted into 250 mL tubes with sterilized substrate (35 % of peat moss, 35 % of perlite, and 30 % of vermiculite) and 4.2 g L-1 of MulticoteⓇ for growth in the nursery during 16 weeks. A randomized complete block experimental design was applied, with 10 treatments, five replications (blocks) and three plants per experimental unit. Survival (%), hypocotyl height (mm), total height (mm), basal diameter (root collar) (mm), crown diameter (mm), main root length (cm), with a Truper® digital vernier caliper. Root fresh weight (RFW) (g), leaf fresh weight (LFW) (g), aerial dry weight (ADW) (g) and root dry weight (RWW) (g) were also evaluated, which were determined with an analytical scale (OHANUS, model GalaxyⓇ 200) after dehydration of the plants in an electric oven (Riossa, OHF-125 model) at 96 °C, until constant weight was obtained; the total dry weight (TWD) (g) and the S:R ratio (g) were also estimated (Ruano, 2003).

The assumptions of normality and homogeneity of variances of the data for all variables (germination, rooting, survival, and growth) were verified with the Shapiro-Wilk and Levene tests, respectively. Only the percentage of established seedlings met the assumptions (p ≥ 0.0893), for which a Tukey's analysis of variance and comparison of means was performed (p ≤ 0.05). The rest of the variables did not meet the assumptions (p ≤ 0.0433); therefore, they were analyzed using nonparametric tests of variance and multiple comparisons of ranks (Kruskal and Wallis, 1952; Conover, 2012). For survival, growth variables and S:R ratio (p ≤ 0.0335), analyses of variance, and RT-3 nonparametric mean comparisons were performed (Conover, 2012), while for root length data (p ≥ 0.2479) a parametric analysis of variance and Tukey's mean comparison was performed. Finally, the differences between treatments were determined by considering the joint effect of germination and growth variables (survival, growth and S:R ratio); two separate principal component analyses were performed, and the comparison of means was obtained for principal component one (CP1). All analyses were carried out using the SAS V. 9.3.1 statistical analysis software (http://support.sas.com/software/93/).

Seed freezing in N2L reduced the final germination percentage (GC) between 15 and 60 % with respect to the unfrozen control (TP), depending on the treatment. The best results for this stage were obtained for seeds that were frozen without encapsulation (naked), without dehydration (T0) or dehydrated (T1), which reached similar values among themselves and were comparable to the control for final germination (GC), germination vigor (GV) and germination energy (GE) (Figure 1, Table 2). Seed encapsulation (T2 and T3) caused a significant decrease in all germination parameters evaluated (p < 0.0001).

Días después de la siembra = Days after sowing; Capacidad germinativa = Germination capacity.

Each point represents the average percentage (%) of germinated seeds from five replications and the vertical bars represent the standard error.

Figure 1. Germination curve of Cedrela odorata L. seeds subjected to different pretreatments and to freezing in liquid nitrogen.

Table 2. Germination parameters. Average values and comparison of means are shown for the ten cryopreservation treatments of seeds of Cedrela odorata L.

Values with different letters in the column are statistically different Tukey (p ≤ 0.05).

The addition of the osmoprotective agents LS, PVS2, and PVS3 to the encapsulated/dehydrated material, alone (T4, T5 and T6) or in combination (T7 and T8) did not significantly reverse the detrimental effect of encapsulation; only the PVS2-only treatment (T5) significantly increased GC, although the onset of germination was late, which caused GE and GV values to remain low. In general, the lowest values for all parameters were obtained when PVS3 was incorporated (T6 and T8). An important feature at this stage was that freezing, in any of the treatments, significantly increased the variance of germination capacity (TP = 15.8, rest of treatments = 76.5 - 309.2).

In the Principal Component Analysis, principal component one (PC1) of the germination parameters explained 76.82 % of the variance. GC and GV contributed similar values (0.608 and 0.655, respectively) to explain the variability, while GE had a lower contribution (-0.449). CP1 of germination parameters showed statistical differences (p < 0.0001) among treatments, with values ranging from -2.03 (T8) to 3.56 (TP). CP1 showed three groups of treatments: the first group consisted of treatments T0, T1 and the control treatment (TP); the second group consisted of T2, T3, T4, T5 and T7; while T6 and T8 formed the third group (Table 2).

The ability of germinated seeds to form complete seedlings suitable for transplanting was statistically different between cryopreservation treatments (p < 0.049). In contrast to what was observed during sensu stricto germination, the freezing of uncapsulated seeds (T0 and T1) negatively affected subsequent development, since the number of emerged seedlings was significantly reduced. Encapsulation allowed a partial recovery of the response when used in combination with dehydration (T2), or depending on whether the osmoprotective agent PVS2 was applied alone (T7) or in combination with LS (T5); this achieved statistically similar results to the unfrozen control (TP) (Table 2).

The seedling survival after 16 weeks of greenhouse growth, as well as the shoot/root dry weight ratio (S:R) and all the growth variables exhibited statistical differences (p < 0.05) between cryopreservation treatments, with the exception of the basal (root collar) diameter (p = 0.2094) (Table 3).

Table 3. Survival and growth variables of seedlings under cryopreservation treatments for Cedrela odorata L.

Mean values and comparison of means (different letters in the same column indicate significant differences, p less than or equal to 0.05).

Seedlings from uncapsulated seeds (T0 and T1) had similar survival to unfrozen seeds (TP), as well as for practically all growth variables, except for root length and S:R ratio in which seedlings from dry seeds (T0) had significantly lower values than seedlings from unfrozen seeds. The above indicates a positive effect of seed dehydration at this stage, and in particular on root quality, which may have impacted survival. As in the establishment stage, encapsulation, in the absence (T2 and T3) or in the presence (T4 to T8) of osmoprotective agents, promoted survival, particularly in treatments including PVS2 (T5 and T7), which reached 100 % and surpassed even unfrozen seeds (TP). However, the hypocotyl length of seedlings from encapsulated seeds was significantly shorter than that obtained for uncapsulated seeds.

The CP1 of survival, growth variables, and S:R ratio accounted for 41.82 % of the variance. Basal diameter, root length, total height and crown diameter had the greatest contribution to the total variance (0.4103 to 0.5267), followed by hypocotyl height (0.2830); on the other hand, the survival and S:R ratio registered lower contributions to the total variance (0.007 and 0.0374). CP1 showed statistical differences between treatments (p = 0.0111), with values ranging from -1.060 (T7) to 1.139 (TP). In general, CP1 determined three groups of treatments, the first group was made up of treatments (TP, T0, T1, T6) with high values; the second, with intermediate values (T2, T4, T5), and the third (T3, T7, T8), with low values (Table 3).

The implementation of cryopreservation of C. odorata seeds offers an alternative for its long-term management in germplasm banks. Coupling deep freezing with osmoprotective pretreatments is a widely used strategy to maintain seed viability during freeze/thawing. However, the impact of these management practices, as a whole, on the development of the future plant must be explored in its different stages ―from germination to establishment to subsequent growth― in order to assess their feasibility as a conservation strategy.

Under the conditions proposed in the documented trials, it was determined that the effect of the different pretreatments varied at different stages of development. Between 20 and 70 % of the germplasm was found to be lost during sensu stricto germination and the early stages of seedling emergence, depending on the pretreatment; therefore, these represent the stages that are most sensitive to freezing. In particular, it was observed that the best germination response was obtained in the treatments of dehydration and freezing of naked seeds, with or without dehydration (T0 and T1), with respect to encapsulated/dehydrated seeds, without (T2 and T3) or with osmoprotective solutions (T4 to T8). The incorporation of an encapsulation/dehydration and vitrification step with osmoprotective agents prior to freezing was expected to maintain the germination capacity and overall viability of the seeds. However, this was not the case, and these manipulations had no impact (dehydration) or even a detrimental impact (encapsulation/dehydration/vitrification), since the final percentage, as well as the speed and energy of germination, decreased. These strategies are widely used because they favor cell water control by limiting the formation of ice crystals that can damage membranes during freezing and thawing (Gantait et al., 2017).

For example, glycerol behaves as an antifreeze that replaces intracellular water, making it very useful as a cryoprotectant; however, it is highly toxic, and, therefore, its use must be carefully adjusted (Kim et al., 2009). The seeds of C. odorata may be sensitive to the chemical toxicity of glycerol, which is why its use was counterproductive. In addition, the PVS2 and PVS3 formulations, although highly recommended (Sakai et al., 1990; Nishizawa et al., 1993), have high sucrose concentrations, especially PVS3 (treatments T6 and T8), which can generate an osmotic shock.

This reflects the limitations of the tested treatments, which must be addressed in order to succeed in using deep-freezing as a tool for the long-term preservation of C. odorata seeds. However, the fact that the germination response was maintained suggests that, despite its cited sensitivity to cold, it is possible to adapt a cryopreservation strategy for its seeds, which contrasts with what has been observed for the seeds of other tropical tree species such as Pithecellobium saman (Jacq.) Benth. and Gliridicidia sepium (Jacq.) Kunth ex Walp (Abdelnour et al., 2007).

One aspect to consider is that seed viability and subsequent seedling development were possibly affected by the thawing at room temperature used in the present study, since work with the same taxon (García and Abdelnour, 2013) and for C. fissilis (Da Costa et al., 2003), indicated that seeds stored directly in N2L maintain 100 % of their germination capacity, when thawing occurs at 40 ºC. While room temperature thawing is widely used, short incubation in heat may be favorable for certain species, especially those with high lipid content, as it appears to promote thawing of triacylglycerols before they interact with water during imbibition, which limits intracellular structural damage (Volk et al., 2006).

In contrast to what was observed during sensu stricto germination, in the initial establishment of the seedling, and during its subsequent development, a positive effect was observed in plants originating from seeds treated by encapsulation/dehydration, in particular with the use of PVS2 as an osmoprotectant (T5 and T7). This contrasts with plants whose seed received no treatment (T0 and T1), or was encapsulated without osmoprotectants (T2 and T3), or in seeds treated with PVS3, in which less than 50 % continued their development, indicating that components such as ethylene glycol or DMSO, present in PVS2, may exert a protective effect (Kim et al., 2009) and, therefore, should be optimized for the species.

Finally, the negative effect of freezing with N2L decreased with the progress of seedling development, which probably indicates a gradual recovery from the stress thus provoked, as has been observed in sorghum, bean, corn, and tomato (Cejas et al., 2012; Arguedas et al., 2018; Acosta et al., 2020). In particular, seed management by means of some pretreatment (T2 to T8) favored seedling survival once transplanted in the nursery. This may be a reflection of the selection pressure exerted by the treatments in previous stages, which results in only the most vigorous seeds being able to produce seedlings with the potential to establish (see, for example, T6). However, seedlings grown from encapsulated/dehydrated seeds treated with PVS2 (T5 and T7) reached higher values even than the unfrozen control (TP), suggesting that the combination of compounds present in this solution effectively provides a benefit that is expressed after germination. When analyzing the growth variables of the aerial part (shoot) of the seedling, the treatments with dry (T0) and dehydrated (T1) seeds were similar to the unfrozen control (TP), which contrasts with the treatments with PVS2 (T5 and T7) that presented maximum survival but slow growth. Although height is related to the photosynthetic capacity of the seedlings and transpiration surface, as well as to a greater likelihood of competing for resources, an adequate root system is required to support the growth of the seedlings (Rodríguez, 2008; Prieto and Sáenz, 2011).

Root development was affected in all freezing treatments ―more than the development of the shoot― and influenced the high values of the S:R ratio observed, whose optimum value is expected to be within the 1.5 and 2.5 range (Rodríguez, 2008). The smaller shoot size observed in T5 and T7 generated a more balanced S:R ratio; this could benefit the growth of the seedlings in restrictive sites, since it has been demonstrated that plants with lower height and a smaller diameter maintain a better water status, with a moderate water consumption in situations of water deficiency (Leiva and Fernández, 1998).

Given the results of this study, an ultrafreezing protocol for Spanish cedar seeds will require further exploration of thawing alternatives, germination conditions, and the incorporation of PVS2 in order to enhance the survival and vigor of the different developmental stages.

Seeds of C. odorata subjected to flash freezing have the potential to survive, germinate and produce vigorous seedlings for nursery transplanting. The critical phases for the success of a cryopreservation protocol are sensu stricto germination and early seedling emergence. The evaluation of different pretreatments of encapsulation, dehydration, as well as the use of osmoprotectants showed that freezing seeds directly, without the application of any pretreatment, is the most adequate protocol to maintain germination capacity, although it is necessary to optimize the thawing conditions in order to obtain a better yield. The use of the osmoprotective solution PVS2 has a beneficial effect on early emergence and survival of seedlings in the nursery. This suggests a route to follow for optimizing a protocol that combines these factors and provides a viable alternative for the long-term conservation of C. odorata seeds.

The authors thank the individuals and institutions that donated the seeds. The results are part of the project "Transcriptomic analysis of germination in Cedrela odorata L. for the identification of molecular markers for seed conservation", number 941634436, financed by the INIFAP Fiscal Fund.

Florencia García Campusano, Mario Valerio Velasco-García and Liliana Muñoz-Gutiérrez declare that they did not participate in any editorial activity related to this document.

Mario Valerio Velasco-García and Liliana Muñoz-Gutiérrez: design of nursery experiments, nursery data collection, data analysis, and drafting of the manuscript; Dalia Grisel Hernández Arroyo: execution of cryopreservation experiments and data collection; Carlos Castillo Martínez: design and execution of cryopreservation experiments; Miguel Ángel Vallejo Reyna: design of experiments, discussion of results, review and editing of the manuscript; Florencia García Campusano: interpretation of the results and drafting of the manuscript. All authors gave their approval to the final version.

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Treatment	Germination capacity (GC) (%)	Germination value (GV)	Germination energy (GE)	Principal Component One	Emergence of seedlings (%)
TP	92.00 a	200.59 a	4.00 a	2.527 a	76.84 a
T0	74.00 ab	67.42 a	5.00 ab	1.683 ab	38.86 b
T1	75.00 ab	62.12 a	6.40 bc	1.400 b	34.93 b
T3	34.00 de	7.45 bcd	8.00 c	-0.881 cd	30.00 b
T4	44.00 cd	10.56 bc	8.40 d	-0.589 cd	33.33 b
T7	47.00 cd	11.54 b	9.20 d	-0.443 cd	60.56 ab
T5	55.00 bc	15.50 b	9.80 de	-0.390 cd	46.00 ab
T8	26.00 e	3.17 d	11.80 e	-2.060 e	42.87 b
T2	29.00 e	12.19 bc	4.60 a	-0.134 c	50.03 ab
T6	27.00 e	6.01 cd	7.80 c	-1.113 de	33.33 b

Treatment	Survival (%)	Hypocotyl height (mm)	Basal diameter (mm)	Total height (mm)	Crown diameter (mm)	Root length (mm)	S:R Ratio	Principal component 1
TP	73.33 b	36.31 a	3.80 a	113.81 abc	169.29 a	11.32 a	3.83 c	1.139 a
T0	46.67 b	38.14 a	5.08 a	103.49 abc	156.22 a	8.47 bc	2.84 ab	0.768 ab
T1	66.67 ab	39.79 a	4.94 a	116.04 a	139.64 abcd	10.11 ab	4.25 c	0.714 ab
T3	80.00 ab	28.99 b	3.67 a	85.83 c	113.65 d	6.79 c	3.06 abc	-0.987 c
T4	86.67 ab	28.54 b	4.05 a	112.50 a	132.85 abcd	8.42 bc	2.24 a	0.008 abc
T7	100.00 a	24.53 b	3.63 a	91.52 bc	120.27 bcd	7.39 c	3.40 bc	-1.060 c
T5	100.00 a	27.55 b	4.83 a	99.93 abc	133.80 abcd	8.06 bc	3.71 c	-0.208 abc
T8	91.67 ab	27.00 b	3.56 a	85.20 c	116.37 cd	8.42 bc	3.56 bc	-0.853 bc
T2	80.00 ab	29.34 b	3.96 a	111.78 ab	150.54 abc	9.75 ab	3.96 bc	0.406 abc
T6	72.73 ab	28.51 b	4.48 a	126.69 a	154.21 ab	9.30 bc	5.04 c	0.739 ab

Preconditioning						Exposure in N2L
Treatment	Dehydratation (%)	Encapsulation	Vitrification
Treatment	Dehydratation (%)	Encapsulation	LS	PVS2	PVS3
TP	No	No	No	No	No	No
T0	No	No	No	No	No	Yes
T1	5	No	No	No	No	Yes
T2	No	Yes	No	No	No	Yes
T3	5	Yes	No	No	No	Yes
T4	5	Yes	Yes	No	No	Yes
T5	5	Yes	No	Yes	No	Yes
T6	5	Yes	No	No	Yes	Yes
T7	5	Yes	Yes	Yes	No	Yes
T8	5	Yes	Yes	No	Yes	Yes

Crioconservación de semillas de Cedrela odorata L.: germinación y establecimiento temprano en vivero

Introduction