.
Acosta Fernández, ontes Marrero & Martínez-Montero /
INGE CUC, vol. 17 no. 2, pp. –10. Julio - Diciembre, 2021
Liquid Nitrogen as promotor of seeds germination and seedling growth in tropical legumes
El nitrógeno líquido como promotor de la germinación de las semillas y del crecimiento de las plántulas en las leguminosas tropicales
DOI: http://doi.org/10.17981/ingecuc.17.2.2021.01
Artículo de Investigación Científica. Fecha de Recepción: 31/10/2020. Fecha de Aceptación: 22/02/2021
Yanier Acosta Fernández 
University of Ciego de Ávila. Ciego de Ávila (Cuba)
yacfdez@gmail.com
Dayami Fontes Marrero
University of Ciego de Ávila. Ciego de Ávila (Cuba)
Marcos E. Martínez-Montero
University of Ciego de Ávila. Ciego de Ávila (Cuba)
Para citar este artículo:
Y. Acosta Fernández, D. Fontes Marrero & M. E. Martínez-Montero, “Liquid Nitrogen as promotor of seeds germination and seedling growth in tropical legumes”, INGE CUC, vol. 17, no. 2, pp. –10. DOI: http://doi.org/10.17981/ingecuc.17.2.2021.01
Abstract
Introduction— The hard seed is the main cause of dormancy in most of the Leguminoseae species. Seed scarification methods, where physical damage is sought to break hard seed coat without diminishing quality, have been modified over time to make them more effective. The most commonly used seed scarification methods include heat, mechanical scarification, and freeze-thaw. Some methods for freeze-thaw scarification include ultra-low temperature immersion in Liquid Nitrogen (LN, –196°C).
Objective— Determine the effectiveness use of Liquid Nitrogen (LN) as a scarification method to overcome dormancy in seeds of species of the Leguminoseae family.
Methodology— The physiological quality of all freshly harvested seeds was determined and scarified by direct immersion in LN for 30 minutes. Total germination was determined under laboratory conditions, as well as the time required for the seeds to reach 50% germination (T50) and the total number of seeds that remained hard at the end of the experiment. The percentage of emerged seedlings and their vegetative growth was evaluated for 21 days after sowing.
Results— The seeds of all species evaluated showed a high physiological quality at the time of harvest. Scarification with LN improved germination, emergence and vegetative growth in the species Desmodium scorpiorus, Teramnus labialis, Neonotonia wigthii and Phueraria phaseoloides.
Conclusions— Dormancy was effectively overcome in the seeds of the species D. scorpiorus, T. labialis, N. wigthii and P. phaseoloides. It was possible to increase the percentage and speed of germination and emergence, managing to obtain plants with greater vegetative growth during the first 21 days after sowing.
Keywords— Legumes; seed dormancy; germination; liquid nitrogen
Resumen
Introducción— La semilla dura es la principal causa de latencia en la mayoría de las especies de Leguminoseae. Los métodos de escarificación de la semilla, en los que se busca un daño físico para romper la cubierta dura de la semilla sin disminuir su calidad, se han modificado a lo largo del tiempo para hacerlos más efectivos. Los métodos de escarificación de semillas más utilizados son el calor, la escarificación mecánica y la congelación-descongelación. Algunos métodos de escarificación por congelación-descongelación incluyen la inmersión a muy baja temperatura en Nitrógeno Líquido (LN, –196°C).
Objetivo— Determinar la efectividad del uso de Nitrógeno Líquido (LN) como método de escarificación para superar la latencia en semillas de especies de la familia Leguminoseae.
Metodología— Se determinó la calidad fisiológica de todas las semillas recién cosechadas y se escarificaron por inmersión directa en LN durante 30 minutos. Se determinó la germinación total en condiciones de laboratorio, así como el tiempo necesario para que las semillas alcanzaran el 50% de germinación (T50) y el número total de semillas que permanecieron duras al final del experimento. Se evaluó el porcentaje de plántulas emergidas y su crecimiento vegetativo durante 21 días después de la siembra.
Resultados— Las semillas de todas las especies evaluadas mostraron una alta calidad fisiológica en el momento de la cosecha. La escarificación con LN mejoró la germinación, emergencia y crecimiento vegetativo en las especies Desmodium scorpiorus, Teramnus labialis, Neonotonia wigthii y Phueraria phaseoloides.
Conclusiones— La dormancia fue superada efectivamente en las semillas de las especies D. scorpiorus, T. labialis, N. wigthii y P. phaseoloides. Se logró aumentar el porcentaje y la velocidad de germinación y emergencia, logrando obtener plantas con mayor crecimiento vegetativo durante los primeros 21 días después de la siembra.
Palabras clave— Leguminosas; latencia de semillas; germinación; nitrógeno líquido
I. Introduction
The legume family (Leguminoseae) is one of the most numerous within the flowering plants and has been one of the most economically important for humans for 3000 years [1]. This taxa, is made up of 700 genera and approximately 20000 species [2]. It is found throughout the planet, except polar regions and deserts with extreme temperatures, and in the tropical region there is a wide biodiversity, where the genera Neonotonia, Macroptilium, Clitoria, Desmodium, Phueraria and Teramnus stand out [3].
Legumes are important in animal nutrition, both for the nutritional value of fruits and seeds, and for the rest of the plant used as forage or grazing. In this sense, the biomass of trees, shrubs and especially creeping legumes have played a leading role due to their considerable protein content and acceptable nutritional value [4]. In addition, legumes have a generalized characteristic that is to harbor atmospheric nitrogen-fixing bacteria in their roots to provide it to the plant when necessary [5]. However, the use and management of the different species is affected, almost entirely, by the low percentages of germination of their seeds [6].
Hard seed (that is, the presence of a hard seed coat that blocks the germination process by not allowing water to pass to embryo) is the main cause of dormancy in most species of Leguminoseae [7]. There is considerable scientific and technical literature on scarification methods to overcome dormancy in the seeds of legume species [1]. Seed scarification methods, where physical damage is sought to break the hard seed coat without diminishing quality, have been modified over time to make them more effective.
The most commonly used seed scarification methods include heat, mechanical scarification, and freeze-thaw [1]. Freeze-thaw scarification methods allow breaking the seed coat by exposing them to alternating low and high temperatures. Some methods for freeze-thaw scarification include ultra-low temperature by immersion in Liquid Nitrogen (LN, –196°C) [8].
II. Theoretical Foundation
LN is mainly used for long-term safe storage in seeds of species of agricultural and forestry interest [9]. However, since 1930 the first works began where LN was used to overcome dormancy in seeds [10]. Some researchers [11] compiled the results obtained in seeds of Melilotus alba, Lotus corniculata, Trifolium repens, Medicago sativa, Trifolium hybridum, and Coronilla varia, when the exposure time, as well as the number of occasions that the seeds are introduced into the LN were the elements that were the object of experimentation.
Rapid temperature changes cause tensions between the different tissues that make up the seeds, which generate expansion and contraction that cause the appearance of cracks in the seed coat [10]. Other studies [12] found cracks in the coat and lens of T. arvense seeds after immersion in LN. These cracks, in the seeds of other species, are associated with the entry of water and with this the beginning of germination [13].
For their part, publications from University Cambridge (USA) [14] considered that the size of the seed has a marked influence on the number of cracks and the place that these appear in the coat, while Institute of Biology and Soil Science (Russia) [15] demonstrated that small seeds present a greater germination after immersion in LN compared to larger seeds. These authors remarked that the species and the hardness of the seed are elements that determine whether or not germination is promoted after immersion in LN.
Studies carried out to date have shown that the use of LN as a seed scarification method can be effective for many species but not for others [11]. For this reason, this research aims to determine the effectiveness use of LN as a scarification method to overcome dormancy in seeds of species of the Leguminoseae family.
III. Materials and Methods
A. Species and seeds material
The species under study in this research (Desmodium scorpiurus (Sw.) Desv., Macroptilium atropurpureum (DC.) Urb., Teramnus labialis (Lf) Spreng., Neonotonia wigthii (cv.) Copper, Clitoria ternatea, Phueraria phaseoloides) were found in the germplasm bank belonging to the Bioplant Center of the University of Ciego de Ávila Máximo Gómez Báez, located at (21˚53`05.63” N, 78˚41`30.35” W), in Cuba. The seeds were manually separated from the ripe fruits harvested in 2018, and were placed in an amber glass container in a conventional refrigerator at 4°C until use.
B. Seeds moisture content (%, H2O mass: fresh mass)
To completely remove the internal moisture from the seeds, the oven-drying method was used, together with dielectric water content sensors using the LCR (HP.2358) equipment [16]. Three samples of 50 seeds were taken for each species, placed in a porcelain container and weighed on an analytical balance (SARTORIUS, BL 1500). The containers were placed in an oven (HS62A) at a temperature of 130°C for up to constant mass. Subsequently, the containers were placed in a desiccator with silica gel and allowed to cool for 45 min. Next, the samples were weighed again and the internal moisture content of the seeds was calculated based on the fresh mass, using the following formula (1):
(1)
Where:
SMC: Seeds Moisture Content.
Fresh mass: Mass of the seeds before placing in the oven.
Dry mass: Mass of the seeds after drying in the oven.
C. Seed viability
Three samples of 50 seeds were taken for each species to determine viability by means of the topographic tetrazolium test [17]. In the coat of all the seeds a small cut with a scalpel was made in the region opposite the thread to facilitate imbibition. Each sample was placed in a Petri dish with felt paper previously moistened with 10 mL of distilled water for 24 h. After that, the coat of each seed was removed to expose the embryo. The embryos were placed back in the Petri dishes, but this time 10 mL of a 1% solution of 2, 3, 5 Triphenyl-2H-Tetrazolium Chloride (TTC) was added. After 6h in the dark, the embryos were classified according to their coloration as: 1) Viable, when they were totally stained deep red, pale red or with discolored sections; and 2) Non-viable, when they remained with their original color; and the result was expressed as a percentage of live embryos out of the total evaluated [18].
D. Scarification by immersion in LN
Two hundred seeds of each species were taken and placed in cryo-vials that were placed directly in a tank with LN (–196ºC) for 30 minutes [19]. The cryo-vials were recovered and placed in a tray in the open air until the seeds reached equilibrium with room temperature [20].
E. Germination
Five samples were taken from 20 seeds previously scarified with LN and 5 samples from 20 seeds without scarification (control). Each seed sample was placed in a Petri dish with felt paper previously moistened with 5 mL of distilled water. The Petri dishes were placed in a germination chamber (Model, RTOP-D series) for 21 days at 30ºC, 80% relative humidity and a photoperiod of 14 h light / 10 h darkness, under fluorescent light tubes with a flow of photosynthetic photons (FFF) of 50 μmol m-2s-1.
Germinated seeds were counted daily taking the emergence of the radicle (≥2 mm) as a criterion. On the last day (day 21) the ungerminated seeds which that were apparently viable, were counted and designated hard seeds. With the total number of germinated seeds, the time required for reaching 50% of germinated seeds (T50) was calculated [21].
F. Emergence and plant development
Five samples were taken from 20 seeds previously scarified with LN and 5 samples from 20 seeds without scarification (control). The seeds were placed to germinate in polyethylene trays 60 cm long and 30 cm wide, with 20 alveoli each. The trays were filled with a substrate based on typical Fersialytic Brown-Reddish soil (50%) and humus (50%) and one seed was sown for each socket at a depth of 2.0 cm. The trays were placed in a controlled environment pre-germination chamber (Model, RTOP-D Series) for 21 days. Simulating natural conditions, the temperature 30°C/25°C and the photoperiod 14 h light/10 h darkness was alternated, under fluorescent light tubes with a Photosynthetic Photons Flux (PPF) of 50 μmol m–2s–1.
(2)
At 21 days, the total number of plants was counted and 10 plants were taken for each species and treatment (control and LN) and the length of the stem (cm), the length of the radicle (cm) and the total of true leaves were measured per plant.
G. Statistical analysis of the results
The Statistical Package for Social Sciences utility [40] was used for the statistical processing of the data. The adjustment to the normal distribution of the data of each treatment (Kolmogorov-Smirnov) and the homogeneity of the variances (Levene) were checked. The analysis for the different variables were carried out through the t-Student test, p ≤ 0.05). In some cases, it was necessary to transform the data to achieve the assumptions of the parametric tests carried out using the formula:
III. Results and Discussion
A. Seed moisture content (%, H2O mass: fresh mass)
The internal moisture content of the seeds was less than 10% in all the species studied with the exception of D. scorpiorus, which was 10.08 ± 0.01% (Table 1). The moisture content of the seed at the time of harvest depends considerably on the environmental conditions experienced during the last phase of its development [22], and it is essential that it be as low as possible (2% - 12%) to guarantee a high survival of the seed after scarification with LN [23], [24]. The moisture contents of all studied seeds correspond to a safe degree of hydration that allows survival at the low temperatures caused by the scarification with LN.
Table 1. Physiological seeds quality.
|
Species |
SMC (%) |
Viability (%) |
|
D. scorpiorus |
10.08 ± 0.01 |
98.33 ± 3.88 |
|
M. atropurpureum |
8.75 ± 0.12 |
94 ± 2.35 |
|
T. labialis |
7.71 ± 0.07 |
95 ± 1.52 |
|
N. wigthii |
8.28 ± 0.12 |
95.33 ± 1.66 |
|
C. ternatea |
6.55 ± 0.16 |
91 ± 3.15 |
|
P. phaseoloides |
9.51 ± 0.02 |
95.66 ± 3.22 |
SMC (Seeds Moisture contents).
Source: Authors.
B. Seed viability
Seed viability was greater than 90% in all species and in D. scorpiorus, T. labialis, N. wigthii and P. phaseoloides it exceeded 95% (Table 1). This result is a sample of the good physiological quality of the seeds of these species harvested in the edaphoclimatic conditions of Ciego de Ávila (Cuba). The high viability of a seed translates into its ability to complete germination and generate a new plant, as long as it does not present any kind of dormancy [11].
C. Germination
The seed germination percentage without scarifying (control), in all species, was less than 50%, only N. wigthii exceeded 45% with 46 ± 3.8% (Fig. 1). The lowest values were collected for M. atropurpureum (24 ± ١.٥٤٪), P. phaseoloides (23 ± 1.39%) and D. scorpiorus (7 ± 0.52%). The results achieved in germination show a strong dormant state in all studied species, which was previously reported by several authors.
Fig. 1. Seeds germinated at 21 days. Ds) Desmodium scorpiurus (Sw.) Desv., Ma) Macroptilium atropurpureum (DC.) Urb., Tl) Teramnus labialis (L.f.) Spreng., Nw) Neonotonia wigthii (cv.) Copper, Ct) Clitoria ternatea, Pp) Phueraria phaseoloides. Seeds without scarifying (Control), Seeds scarifying with Liquid Nitrogen (LN). Results with same letter, for each spices, are not statistically different; t-Student, p > 0.05, n = 5.
Source: Authors.
Dormancy has a hereditary component and it has been shown that it can be controlled by the maternal and paternal genotypes [25]. However, the environment in which the seed develops also has an important influence on the acquisition of dormancy [26]. In the seeds of many species of the Leguminosae family, various authors have systematically reported the presence of dormancy [11].
Scarification with LN allowed to effectively overcome the dormancy present in the seeds of the species D. scorpiorus, T. labialis, N. wigthii and P. phaseoloides (Fig. 1), showing statistically significant differences in relation to the control treatment. Scarification with LN causes an increase in the rate of seed imbibition, which translates into increased germination [14]. The increase in the capacity for imbibition in the seeds is due to the occurrence of morphological changes in the seeds coat with the appearance of cracks [27].
In dry seeds, the reserve tissues are made up of organic substances such as proteins, lipids, carbohydrates and sugars that are in a very flexible glassy or rubbery state and presumably very heat resistant [28]. On the contrary, the seed coat is made up of tissues with dead cells and thick lignified secondary walls, generally impregnated with hydrophobic organic substances such as cutin, lignin, quinones, suberin and wax. The stresses caused by changes in temperatures in the different tissues of the seeds cause the coat to break or crack [9], [27].
A similar result to that achieved in this research was described in seeds with physical dormancy of some species of the Leguminoseae family by [11]. Other results, where scarification with LN allowed to overcome dormancy, was described for seeds of Medicago orbicularis, Medicago truncatula and Phillyrea angustifolia [8], [29]. Also in the species Lotus mascaensis [30] and Robinia pseudoacacia [31] the dormancy present in the seeds after direct exposure to LN was exceeded.
For the species M. atropurpureum and C. ternatea, the scarification treatment with LN was not enough to overcome the dormancy present in the seeds (Fig. 1). The loss of impermeability of the seeds when they are scarified with LN occurs mainly during the cooling process [32]. Factors such as coat thickness, seed size, chemical composition and internal seed moisture content provoke different responses to scarification with LN [33]. The size of the seeds, larger in these two species than in the rest of those evaluated, was presumably the factor that caused the used scarification treatment to be ineffective.
Two indicators associated with germination, such as the time required to reach 50% of the germinated seeds (T50) (Table 2) and the percentage of seeds that remained hard at the end of the experiment (Fig. 2) were statistically higher in the species where LN favored overcoming dormancy (D. scorpiorus, T. labialis, N. wigthii and P. phaseoloides).
Table 2. Time required for reaching 50% of germinated seeds (T50) (days).
|
Species |
Control |
LN |
|
D. scorpiorus |
11 ± 0.91 b |
6.8 ± 0.12 a |
|
M. atropurpureum |
11.2 ± 0.66 b |
8 ± 0.31 a |
|
T. labialis |
7 ± 0.44 b |
4.4 ± 0.24 a |
|
N. wigthii |
4.8 ± 0.2 b |
3.1 ± 0.19 a |
|
C. ternatea |
5 ± 0.25 a |
5.1 ± 0.33 a |
|
P. phaseoloides |
5.2 ± 0.31 b |
3.1 ± 0.2 a |
Results with same letter, for each species, are not statistically different; t-Student, p > 0.05, n = 5.
Source: Authors.
Fig. 2. Hard seeds at 21 days. Ds) Desmodium scorpiurus (Sw.) Desv., Ma) Macroptilium atropurpureum (DC.) Urb., Tl) Teramnus labialis (L.f.) Spreng., Nw) Neonotonia wigthii (cv.) Copper, Ct) Clitoria ternatea, Pp) Phueraria phaseoloides. Seeds without scarifying (Control), Seeds scarifying with Liquid Nitrogen (LN). Results with same letter, for each spices, are not statistically different; t-Student, p > 0.05, n = 5.
Source: Authors.
In these species, not only was the final germination percentage improved, but homogenization and its speed were also favored. In addition, it was possible to significantly reduce the percentage of seeds that remain hard at the end of the experiment. The results achieved in these species are similar to those obtained previously for other members of the Leguminoseae family [34].
D. Emergence and plant development
The percentage of emerged plants was significantly higher in the seeds scarified with LN of the species D. scorpiorus, T. labialis, N. wigthii and P. phaseoloides (Fig. 3), which corresponds to the germination results obtained for these same species.
Fig. 3. Emerged plants at 21 days. Ds) Desmodium scorpiurus (Sw.) Desv., Ma) Macroptilium atropurpureum (DC.) Urb., Tl) Teramnus labialis (L.f.) Spreng., Nw) Neonotonia wigthii (cv.) Copper, Ct) Clitoria ternatea, Pp) Phueraria phaseoloides. Seeds without scarifying (Control), Seeds scarifying with Liquid Nitrogen (LN). Results with same letter, for each spices, are not statistically different; t-Student, p > 0.05, n = 5.
Source: Authors.
T. labialis and P. phaseoloides obtained the highest percentages of emergence with 70 ± 2.63 and 72 ± 3.53 respectively. As in germination, the species M. atropurpureum and C. ternatea did not find statistical differences between the seeds of the control treatment and those scarified with LN.
Emergence is probably the most important phenological event that influences the success of a plantation and represents the moment in which a plant begins photosynthetic autotrophism [35].
The results achieved in the emergence of plants obtained from seeds scarified with LN show that this treatment not only favors germination under laboratory conditions, but also emergence under field conditions. Previous results, similar to those obtained in this research, were published for the species T. labialis and N. wightii [20], [36].
The plants that emerged and developed for 21 days showed greater growth (Table 3) in those obtained from seeds scarified with LN for the species D. scorpiorus, T. labialis, N. wigthii and P. phaseoloides (Fig. 4). This result is due to the overcoming of the dormancy present in these seeds through scarification with LN, which allowed a higher emergence speed.
The rapid emergence in the field is related to the development of plants of many species; and it is an advantage for the establishment of the culture [37]. Some Cuban studies [38] define the speed of germination and emergence as one of the most important properties for the establishment of a species.
The time of emergence many times determines whether a plant can compete successfully with its neighbors, flowers, reproduces and matures properly at the end of its growth stage, or is consumed by herbivores [39].
Table 3. Indicators evaluated in plant at 21 days of growth.
|
Species |
Stems length (cm) |
Roots length (cm) |
Leaves per plant |
|||
|
Control |
NL |
Control |
NL |
Control |
NL |
|
|
D. scorpiorus |
2.6 ± 0.10 b |
5.5 ± 0.15 a |
2.2 ± 0.12 b |
2.9 ± 0.18 a |
2 ± 0.0 a |
2.2 ± 0.2 a |
|
M. atropurpureum |
10 ± 0.87 a |
11.2 ± 0.71 a |
4.3 ± 0.12 a |
4.3 ± 0.2 a |
3 ± 0.21 a |
3.4 ± 0.24 a |
|
T. labialis |
3 ± 0.15 b |
3.3 ± 0.11 a |
2.1 ± 0.1 a |
2.2 ± 0.12 a |
2 ± 0.1 b |
3 ± 0.1 a |
|
N. wigthii |
3.2 ± 0.2 b |
5.3 ± 0.2 a |
2.3 ± 0.12 b |
4.8 ± 0.12 a |
2.4 ± 0.24 b |
3 ± 0.1 a |
|
C. ternatea |
5.3 ± 0.35 a |
5.7 ± 0.21 a |
3.1 ± 0.18 a |
3.4 ± 0.15 a |
2.4 ± 0.24 a |
2.6 ± 0.24 a |
|
P. phaseoloides |
5.7 ± 0.3 b |
10 ± 0.35 a |
3.1 ± 0.24 b |
6 ± 0.15 a |
2 ± 0.1 b |
2.8 ± 0.2 a |
Results with same letter, for each species and indicator, are not statistically different; t-Student, p > 0.05, n = 10.
Source: Authors.
Fig. 4. Plant size at 21 days after shown. A) Desmodium scorpiurus (Sw.) Desv., B) Macroptilium atropurpureum (DC.) Urb., C) Teramnus labialis (L.f.) Spreng., D) Neonotonia wigthii (cv.) Copper, E) Clitoria ternatea, F) Phueraria phaseoloides. T) (Control); NL) Liquid Nitrogen. Bar represents 5 cm.
Source: Authors.
IV. Conclusions
The results obtained in this investigation demonstrate the potential of the use of LN as a scarification method for some species of the Leguminoseae family. Dormancy was exceeded in the seeds of the species D. scorpiorus, T. labialis, N. wigthii and P. phaseoloides. It was possible to increase the percentage and speed of germination and emergence, achieving plants with greater vegetative development during the first 21 days after sowing. Only in the species M. atropurpureum and C. ternatea it was not possible to overcome the existing dormancy in the seeds.
References
[1] E. Kimura & M. A. Islam, “Seed scarification methods and their use in forage legumes,” Res J Seed Sci, vol. 5, no. 2, pp. 38–50, Feb. 2012. https://doi.org/10.3923/rjss.2012.38.50
[2] N. Azani, M. Babineau, C. D. Bailey, H. Banks, A. R. Barbosa, R. Barbosa, J. S. Boatwright, L. M. Borges, G. K. Brown, A. Bruneau et al., “A new subfamily classification of the Leguminosae based on a taxonomically comprehensive phylogeny – The Legume Phylogeny Working Group (LPWG),” Taxon, vol. 66, no. 1, pp. 44–77, Feb. 2017. https://doi.org/10.12705/661.3
[3] A. Díaz, E. Castillo, P. C. Martín & J. L. Hernández, “Pre-fattening of crossbred dairy bulls under grazing with glycine (Neonotonia wightii) and tropical grasses with rumen activator supplement,” Cuba J Agric Sci, vol. 47, no. 1, pp. 23–26, 2013. Available: http://cjascience.com/index.php/CJAS/article/view/250
[4] C. A. Mazorra-Calero, D. Fontes-Marrero, L. H. Donis-García, J. Martínez-Melo, Y. Acosta-Fernández, I. Espinosa-Alemán, C. Lavinge, P. Fernandes & A. González-Morales, “Diagnóstico tecnológico y socioeconómico del establecimiento de Psidium guajava L. y Teramnus labialis en Ciego de Ávila, Cuba,” Past y forr, vol. 39, no. 4, pp. 259–264, 2016. Disponible en https://payfo.ihatuey.cu/index.php?journal=pasto&page=article&op=view&path%5B%5D=1919
[5] F. Llamas & C. Acedo, “Las leguminosas (Leguminosae o Fabaceae): una síntesis de las clasificaciones, taxonomía y filogenia de la familia a lo largo del tiempo,” Ambioc, vol. 14, pp. 5–18, 2016. https://doi.org/10.18002/ambioc.v0i14.5542
[6] D. Fontes, R. Machado, N. Cubillas, C. Mazorra, A. Borroto, L. Pulido, Y. Lezcano, N. Hernández & J. Martínez. , “Selección de leguminosas herbáceas para el fomento de cobertura en plantaciones de naranja Valencia late,” Past y forr, vol. 32, no. 1, pp. 1–11, Mar. 2009. Disponible en https://payfo.ihatuey.cu/index.php?journal=pasto&page=article&op=view&path%5B%5D=655
[7] E. Can & N. Çelikta, “Breaking seed dormancy of some annual Medicago and Trifolium species by different treatments,” Turkish J Field Crops, vol. 14, no. 2, pp. 72–78, 2009. https://doi.org/10.17557/TJFC.23426
[8] C. Patanè & F. Gresta, “Germination of Astragalus hamosus and Medicago orbicularis as affected by seed-coat dormancy breaking techniques,” J Arid Env, vol. 67, no. 1, pp. 165–173, Oct. 2006. https://doi.org/10.1016/j.jaridenv.2006.02.001
[9] B. M. Reed, “Plant cryopreservation: a continuing requirement for food and ecosystem security,” In Vitro Cell Dev Biol-Plant, vol. 53, no. 4, pp. 285–288, Ago. 2017. https://doi.org/10.1007/s11627-017-9851-4
[10] B. M. Reed,Cryopreservation of orthodox (Desiccation Tolerant) seeds, H. W. Pritchard & J. Nadarajan,Plant Cryopreservation: A Practical Guide. NY, USA: Springer, pp. 485–501, 2008. https://doi.org/10.1007/978-0-387-72276-4_19
[11] C. C. Baskin & J. M. Baskin, “Seeds: ecology, biogeography, and evolution of dormancy and germination, 2 Ed., AMS, NL: Elsevier/AP, 2014. https://doi.org/10.1016/C2013-0-00597-X
[12] H. W. Pritchard, K. R. Manger & F. G. Prendergast, “Changes in Trifolium arvense seed quality following alternating temperature treatment using Liquid nitrogen,” AOB, vol. 62, no. 1, pp. 1–11, Jul. 1988. https://doi.org/10.1093/oxfordjournals.aob.a087626
[13] L. W. Zeng, P. S. Cocks, S. G. Kailis & J. Kuo, “The role of fractures and lipids in the seed coat in the loss of hardseededness of six Mediterranean legume species,” J Agric Sci, vol. 143, no. 1, pp. 43–55, Jun. 2005. https://doi.org/10.1017/S0021859605005034
[14] X. W. Hu, Y. R. Wang, Y. P. Wu & C. C. Baskin, “Role of the lens in controlling water uptake in seeds of two Fabaceae ( Papilionoideae ) species treated with sulphuric acid and hot water,” Seed Sci Res, vol. 19, no. 2, pp. 73–80, 1 Jun. 2009. https://doi.org/10.1017/S0960258509301099
[15] N. M. Voronkova & A. B. Kholina, “Conservation of endemic species from the Russian far east using seed cryopreservation,” Biol Bull, vol. 37, no. 5, pp. 496–501, Oct. 2010. https://doi.org/10.1134/S1062359010050092
[16] R. F. García, “Determination of the stability of the ethanol mixture with naphtha through the measurement of his dielectric and refractometric properties,” INGE CUC, vol. 16, no. 2, pp. 141–150, 2020. https://doi.org/10.17981/ingecuc.16.2.2020.10
[17] M. Altare, S. Trione, J. C. Guevara & M. Cony, “Stimulation and promotion of germination in Opuntia ficus-indica seeds,” J Prof Assoc Cactus Dev, vol. 8, pp. 91–100, 2006. Available: https://www.jpacd.org/jpacd/article/view/281
[18] M. A. Maldonado-Peralta, G. De Los Santos, J. R. García-Nava, C. Ramírez-Herrera, A. Hernández-Livera, J. M. Valdez-Carrazco, T. Corona-Torres & V. M. Cetina-Alcalá, “Seed viability and vigour of two nanche species (Malpighia mexicana and Byrsonima crassifolia),” Seed Sci Technol, vol. 44, no. 1, pp. 168–176, Abr. 2016. https://doi.org/10.15258/sst.2016.44.1.03
[19] Y. Acosta, Lianny Pérez, C. Linares, L. Hernández, D. Escalante, A. Pérez, B. E. Zevallos, L. Yabor, M. E. Martínez-Montero, I. Cejas, D. Fontes, Sershen & J. C. Lorenzo, “Effects of Teramnus labialis (L.f.) Spreng seed cryopreservation on subsequent seed and seedling growth and biochemistry,” Acta Physiol Plant, vol. 42, no. 1, pp. 1–7, Jan. 2020. https://doi.org/10.1007/s11738-020-3012-9
[20] Y. Acosta, L. Hernandez, C. Mazorra, N. Quintana, B. E. Zevallos, I. Cejas, EmptyYN Y Sershen, J. C. Lorenzo, M. E. Martínez-Montero & D. Fontes, “Seed cryostorage enhances subsequent plant productivity in the forraje species Teramnus labialis (L.f.) Spreng,” Cryo, vol. 40, no. 1, pp. 36–43, Jan. 2019. Available: https://pubmed.ncbi.nlm.nih.gov/30955029/
[21] E. Enriquez-Peña, H. Suzán-Azpiri & G. Malda-Barrera, “Viabilidad y germinación de semillas de Taxodium mucronatum (Ten.) en el estado de Querétaro, México,” Agrocienc, vol. 38, no. 3, pp. 375–381, 2004. Available: https://agrociencia-colpos.mx/index.php/agrociencia/article/view/329
[22] G. K. Jaganathan, K. J. Yule & M. Biddick, “Determination of the water gap and the germination ecology of Adenanthera pavonina (Fabaceae, Mimosoideae); the adaptive role of physical dormancy in mimetic seeds,” AoB PLANTS, vol. 10, no. 5, pp. 1–11, Oct. 2018. https://doi.org/10.1093/aobpla/ply048
[23] M. T. Gonzalez-Arnao & F. Engelmann,. Crioconservación de plantas en América Latina y el Caribe, SJ, CR: IICA, 2013. Available: https://horizon.documentation.ird.fr/exl-doc/pleins_textes/divers14-02/010060280.pdf
[24] S. N. Acharya, D. G. Stout, B. Brooke & D. Thompson, “Cultivar and storage effects on germination and hard seed content of alfalfa,” Can J Plant Sci, vol. 79, no. 2, pp. 201–208, Apr. 1999. https://doi.org/10.4141/P98-043
[25] H. W. M. Hilhorst, “Standardizing Seed Dormancy Research,” Seed Dormancy, vol. 773, pp. 43–52, Jul. 2011. https://doi.org/10.1007/978-1-61779-231-1_3
[26] A. E. Ferreras, S. R. Zeballos & G. Funes, “Inter- and intra-population variability in physical dormancy along a precipitation gradient,” Acta Bot Bras, vol. 31, no. 1, pp. 141–146, 2017. https://doi.org/10.1590/0102-33062016abb0406
[27] Y. Acosta, L. Pérez, D. Escalante, L. Nápoles, O. Concepción, A. Pérez, L. S. Pérez, M. E. Martínez-Montero, D. Fontes, Sershen & J. C. Lorenzo, “Dormancy breaking in Teramnus labialis (L.f.) Spreng seeds through liquid nitrogen exposure is based on the modification of the hilar region, cuticle, and macrosclereid,” Acta Physiol Plant, vol. 42, no. 9, pp. 1–7, Sep. 2020. https://doi.org/10.1007/s11738-020-03134-9
[28] J. D. Bewley, K. J. Bradford, H. W. M. Hilhorst & H. Nonogaki,. Synthesis of Storage Reserves. NY, USA: Springer, pp. 85–131, 2013. https://doi.org/10.1007/978-1-4614-4693-4_3
[29] F. Alane, R. Chabaca, L. Ouafi, M. Abdelguerfi-laouar & A. Abdelguerfi, “Break dormancy, germination capacity of medics after different techniques of scarification (Physical, Chemical and Mechanical),” AJAR, vol. 11, no. 5, pp. 340–351, Feb. 2016. https://doi.org/10.5897/AJAR2015.10303
[30] F. Pérez-García, “Effect of cryopreservation, gibberellic acid and mechanical scarification on the seed germination of eight endemic species from the Canary Islands,” Seed Sci Technol, vol. 36, no. 1, pp. 237–242, Apr. 2008. https://doi.org/10.15258/sst.2008.36.1.29
[31] S. Jastrzębowski, J. Ukalska, W. Kantorowicz, M. Klisz, T. Wojda & M. Sułkowska, “Effects of thermal-time artificial scarification on the germination dynamics of black locust (Robinia pseudoacacia L.) seeds,” Eur J Forest Res, vol. 136, no. 3, pp. 471–479, Jun. 2017. https://doi.org/10.1007/s10342-017-1046-3
[32] I. Martin & C. De la Cuadra, “Evaluation of different scarification methods to remove hardseededness in Trifolium subterraneum and Medicago polymorpha accessions of the Spanish base genebank,” Seed Sci Technol, vol. 32, no. 3, pp. 671–681, Oct. 2004. https://doi.org/10.15258/sst.2004.32.3.03
[33] K. Endoh, M. Matsushita, M. K. Kimura, S. Hanaoka, Y. Kurita, E. Hanawa, S. Kinoshita, N. Abe, H. Yamada & M. Ubukata, “Cryopreservation of Fagus crenata seeds: estimation of optimum moisture content for maintenance of seed viability by Bayesian modeling,” Can J For Res, vol. 48, no. 2, pp. 192–196, Feb. 2018. https://doi.org/10.1139/cjfr-2017-0286
[34] S. Mira, A. Arnal & F. Pérez-García, “Seed germination of Phillyrea angustifolia L., a species of difficult propagation,” FS, vol. 26, no. 1, pp. 1–10, May. 2017. https://doi.org/10.5424/fs/2017261-10216
[35] M. Navarro, “Desempeño fisiológico de las semillas de árboles leguminosos de uso múltiple en el trópico,” Pastos y Forrajes, vol. 26, no. 2, pp. 97–114, 2003. Available: https://payfo.ihatuey.cu/index.php?journal=pasto&page=article&op=view&path%5B%5D=831
[36] Y. Acosta, F. Santiago, D. Escalante, C. Mazorra, I. Cejas, M. E. Martínez-Montero, A. Escobar, Sershen, E. Hajari, J. C. Lorenzo & D. Fontes, “Cryo-exposure of Neonotonia wightii Wigth and Am seeds enhances field performance of plants,” Acta Physiol Plant, vol. 42, no. 1, pp. 1–6, Ene. 2020. https://doi.org/10.1007/s11738-019-3010-y
[37] M. Navarro, G. Febles & V. Torres, “Utilization of the index of efficiency for estimating the influence of seed vigor on growth and seedling development,” Cuba J Agric Sci, vol. 50, no. 4, pp. 593–605, 2016. Available: https://www.cjascience.com/index.php/CJAS/article/view/664
[38] M. Santorum, L. H. P. Nóbrega, E. G. de Souza, D. D. Santos, W. Boller & M. M. Mauli, “Comparison of tests for the analysis of vigor and viability in soybean seeds and their relationship to field emergence,” Acta Sci Agron, vol. 35, no. 1, pp. 83–92, Dic. 2013. https://doi.org/10.4025/actasciagron.v35i1.14955
[39] F. Forcella, R. L. Benech Arnold, R. Sanchez & C. M. Ghersa, “Modeling seedling emergence,” Field Crops Res, vol. 67, no. 2, pp. 123–139, Jul. 2000. https://doi.org/10.1016/S0378-4290(00)00088-5
[40] SPSS. (version 12.0). IBM [Online]. Available: https://www.ibm.com/mx-es/analytics/spss-statistics-software