elocation-id: e3744
Abstract
Eysenhardtia polystachya is a plant prized for the strength of its wood and medicinal use. It is propagated by seed; nevertheless, low germination and attack by insects and fungi in natural conditions limit its availability, which makes it highly vulnerable. In vitro culture allows the number of individuals to increase rapidly. The objective was to evaluate the effect of mineral salts of Murashige and Skoog and Woody Plant Medium culture media, both in concentration of 50, 75 and 100% (macro and micronutrients), in combination with two methods of seed disinfection. E. polystachya seeds collected in November 2021 in Tlayacapan Morelos were used; these were disinfected with silver nanoparticles, applied once or twice, and then seeded in vitro. The experimental design was completely randomized with factorial arrangement of treatments, with 10 replications of five seeds. Double application of silver nanoparticles generated 98.3% of aseptic seeds, compared to applying them once (86.7%). Plant height was also 34.5% higher. The MS medium generated better germination in the three concentrations (90 to 99%). Root length and dry matter were more abundant in the MS medium at 50%. It was concluded that it is convenient to use silver nanoparticles twice to disinfect E. polystachya seeds and to use the 50% MS culture medium for germination and seedling growth.
disinfection method, kidneywood, MS medium, WPM medium.
The species Eysenhardtia polystachya (Ortega) has ecological potential due to its ability to establish itself in eroded soils, from plains to places with irregular topography, such as hills and mountainous ravines (Martín et al., 2021). Its wood is appreciated for its hardness and strength, so it is extracted for the construction of furniture, tools, posts for pens and fences, as well as firewood for homes. In addition, it is used in traditional medicine for the urinary and digestive systems and inflammations, both for human and for veterinary use (Martín et al., 2021; Lorenzo-Barrera et al., 2023).
It has several active ingredients that make it toxic to bacteria and fungi (Bernabé-Antonio et al., 2017). To extract the active ingredients, heartwood and sapwood are used; therefore, it is necessary to cut the stems, which in many cases causes the death of the plant; this has caused the reduction of natural populations (Beltrán-Rodríguez et al., 2017; Beltrán-Rodríguez et al., 2020). In addition to climate change, the current restricted distribution in wild conditions limits the availability of seeds and thus the spread; consequently, this species is highly vulnerable.
Seed quality is affected by several factors, such as purity, health, and germination aspects (Raya-Pérez et al., 2020). In E. polystachya, the seeds have a thin and impermeable testa, which can cause germination to be slow and heterogeneous and a low number of seedlings to be produced, causing natural populations not to regenerate quickly; likewise, under natural conditions, they are attacked by insects and fungi (Martín et al., 2021).
Authors such as Núñez-Cruz et al. (2018) observed germination variations in E. polystachya; they obtained 50% emergence in the germination chamber and 30% under natural conditions, without the effect of the pre-germination treatments applied (soaking and soaking + drying), and they point out that germination could be related to the degree of embryonic maturity, latency break, and the appropriate environmental conditions. Gelviz-Gelvez et al. (2020) evaluated seed germination with low water potentials without applying pre-germination treatments and obtained 66% germination.
Lorenzo-Barrera et al. (2024) studied E. polystachya under greenhouse conditions and obtained 34% germinated seeds, without applying treatment, 40% with soaking for 36 h and 96% with 600 ppm of gibberellic acid for 30 min. The use of micropropagation techniques in ornamental plants, plants of agri-food importance and forest species helps to rapidly increase the number of individuals with genetically stable plant material and free of pests and diseases through the cloning and growing of plants in controlled and aseptic conditions (Campos et al., 2020; Ramírez-Mosqueda et al., 2020; Bello-Bello and Spinoso-Castillo, 2022).
Likewise, through in vitro culture, it is possible to obtain extracts or secondary metabolites of plants of interest with pharmaceutical applications, using cell culture, without having to extract them directly from wild plants to avoid overexploitation and deforestation. In this sense, Bernabé-Antonio et al. (2017) used the hexane fraction of methanolic extracts obtained from cell suspension cultures of E. polystachya to inhibit the mycelial growth of Rhizoctonia solani.
In addition to the above, in vitro plant cell culture is a tool with the potential to accelerate the large-scale production of raw material in less time in a controlled manner at any time of the year, and yields of bioactive compounds can be higher than in wild plants (Haida et al., 2020; Bernabé-Antonio et al., 2021); Bernabé-Antonio et al. (2021) used in vitro culture of Eysenhardtia platycarpa in MS medium to culture suspension cells from internodal segments to obtain extracts and evaluate antifungal activity against R. solani and Sclerotium cepivorum.
Nonetheless, problems associated with contamination of culture media can occur in micropropagation, especially in the establishment phase of the in vitro culture, contamination that affects the growth of explants by competing for water, light, space, and essential nutrients (Campos et al., 2020; Ramírez-Mosqueda et al., 2020; Bello-Bello and Spinoso-Castillo, 2022).
This problem can be controlled by adding fungicides and antibiotics to the culture medium; however, due to the resistance of some strains, the use of such products is not recommended. Another alternative is the application of silver nanoparticles (AgNPs) (Ramírez-Mosqueda et al., 2020; Bello-Bello and Spinoso-Castillo, 2022), these have a broad microbicidal spectrum, do not generate resistance as silver attacks a wide range of targets in microbes, are easy to acquire, are non-toxic when used in the correct dose, are inexpensive compared to other products, and have also been used to induce germination, increase yield, and promote the development of in vitro cultures (Spinoso-Castillo et al., 2017; Chávez-García et al., 2020; Bello-Bello and Spinoso-Castillo, 2022).
Based on the importance of kidneywood and the benefits that can be obtained with the use of in vitro culture techniques and contamination control, this research aimed to evaluate the effect of the concentration of mineral salts (50, 75, and 100%) of two culture media, MS (Murashige and Skoog, 1962) and WPM (McCown and Lloyd, 1981), in combination with two methods of seed disinfection to induce the greatest in vitro seed germination and seedling growth of E. polystachya.
The study was carried out under controlled conditions in a micropropagation laboratory. The seeds that were used were collected from a wild tree in Tlayacapan, Mor. Mexico, 18° 95’ 553” north latitude, 98° 98’ 113” west longitude and altitude 1 640 m, in November 2021, and they were stored in a refrigerator at 4 °C. Three concentrations of mineral salts (50, 75, and 100%) of two culture media, MS (Murashige and Skoog, 1962) and WPM (McCown and Lloyd, 1981), without growth regulators, were evaluated. The media were supplemented with 3% sucrose, myoinositol 100 L, thiamine HCl, glycine, pyridoxine, and nicotinic acid (0.5 mg L-1 of each). The pH was set to 5.7 with a potentiometer (HANNA instruments, model HI8424).
Subsequently, 2 g L-1 of activated charcoal was added to prevent the negative effect of phenol exudation; 7 g L-1 of agar (Merk) was used. Twenty milliliters of culture medium were placed in 300 ml glass bottles. The medium was sterilized in a vertical autoclave (CV250) at 18 psi at 120 °C for 18 min. In the laminar flow hood (Novatech, model CFLH-120E), E. polystachya seeds were disinfected using two methods: 1) Roma detergent 0.2 g 100 ml-1, stirring for 5 min, ethanol 96° at 70% (1 min), sodium hypochlorite (NaClO 0.9%) stirring for 5 min, silver nanoparticles (AgNPs) 75 μl 50 ml-1 (AgROVIT-CP), stirring for 10 min and 2) Roma detergent 0.2 g 100 ml-1, stirring for 5 min, 96° ethanol at 70% (1 min), 75 μl 50 ml-1 AgNPs, stirring for 10 min, sodium hypochlorite (NaClO 0.9%) stirring for 5 min, and again AgNPs 75 μl 50 ml-1, stirring for 10 min.
In each case, the seeds were rinsed three times with sterile distilled water and then established in the bottles with the respective culture medium (5 seeds per jar). The seeded bottles were placed in an incubation room at 24 °C with a photoperiod of 16 h with white fluorescent light (32 μE m-2 s-1) and 8 h of darkness. The experimental design used was a completely randomized design with factorial arrangement of treatments, factor A: culture media at different concentrations; factor B: two ways to disinfect seeds. Ten replications per treatment were established, with five seeds per replication (culture bottle).
The following was evaluated in the initial stage: 1) germination variables: germination start (days), recorded when the radicle emerged, percentage of total germination at 30 days, and seed asepsis (%). 60 days after sowing, the following was recorded and 2) seedling growth variables: plant height (cm), root length, leaves per plant and dry matter per seedling (mg). The data were analyzed by variance (Anova) and Tukey’s means comparison test (p≤ 0.05) was performed with the SAS® v. 9.2 statistical package.
The results of the analysis of variance indicated highly significant differences (p≤ 0.01) in seed asepsis due to the effect of the two factors studied and their interaction. The start of germination was affected only by the interaction of the concentration of mineral salts of culture media with disinfection methods (p≤ 0.05), whereas total germination was affected only by the culture media in which the seeds were established (Table 1).
The use of silver nanoparticles (AgNPs) in the disinfection of E. polystachya seeds allowed the establishment of cultures without microorganisms. Asepsis was 100% when the AgNPs were added twice in the disinfection, with the medium MS at 100, 75 and 50%, WPM at 75 and 50%; however, when the MS was used at 100% and the seeds were disinfected with method 1, the culture was also completely aseptic (Table 1).
With disinfection method 1, 70, 80, 90, and 100% seeds free of contaminating microorganisms were obtained (Table 1). This indicates that using AgNPs twice during seed disinfection was the most appropriate way to establish the aseptic culture of this species.
The onset of germination occurred 3 and 4 days after sowing (das). According to the analysis of variance, there were no significant differences, so all media were adequate for the occurrence of the process of seed imbibition and thus the beginning of germination; in addition, the culture medium provided the necessary nutrients for the development of the plant (Andrade-Rodríguez et al., 2015) since the success of in vitro culture depends a lot on the composition of the culture medium used.
Except for orchids, mineral salts and supplements are not required for seed germination. Nevertheless, the concentration of mineral salts modifies the osmotic potential of the media and thus the availability of water for germination. In the same way, the type of medium and concentration of salts do not affect the contamination or percentage of aseptic cultures.
Total in vitro germination of E. polystachya seeds ranged from 82 to 100%, with an average of 92%. The 75% MS medium with disinfection method 2 generated the highest total germination (100%), which was statistically different only from what was obtained in the 50% MS and 50% WPM media, with the disinfection of method 2 and method 1, respectively (Table 1). Similarly, Bernabé-Antonio et al. (2021) obtained 98% germination of Eysenhardtia platycarpa seeds at 10 days of culture in MS medium; these researchers added four drops of Tween 20® to the sodium hypochlorite solution, and they point out that Tween is used as a surfactant agent, which may have helped to improve imbibition and thus increase the percentage of total germination.
Chávez-García et al. (2020) found that in vitro propagation is an alternative to conventional methods of propagation, as a seed with problems can germinate correctly because it increases the germination rate and the seed will be free of pathogens. The WPM medium generated good results at the concentration of 100% with disinfection 1 and 2, and at 75% with disinfection 2 (98 and 96% germination, respectively); this medium presents a lower concentration of salts, NH4 + (5 mM), NO3 - (9.7 mM), and Cl- (1.3 mM), than the MS medium (Andrade-Rodríguez et al., 2015; Campos et al., 2020), so that at 100%, it generated good total germination without having to dilute it.
The analysis of variance showed a highly significant effect of culture media on the four seedling growth variables; in contrast, the seed disinfection method significantly affected only the height and dry matter of E. polystachya seedlings. There was also a significant effect of the interaction of the levels of the factors in the variables studied, hence the results are presented according to the combination of levels of the factors studied.
Seedling height was higher when seeds were sown in the MS culture medium at 100 and 75% (3.3 cm) in combination with disinfection method 2, in which AgNPs were applied twice, they were statistically equal to those of the MS medium at 50% and WPM at 100 and 50%, with disinfection method 2. The lowest growth in height was recorded in the 50% WPM medium with disinfection method 1 (Table 2), which was 1.3 cm less than the plants of the two best media.
Any of the media used gave better results when combined with disinfection method 2. Pinedo-Panduro et al. (2022) mention that the MS medium is widely used and is suitable for most species, except for those most sensitive to salinity and in those cases, it can be used diluted to 75 and 50%. In kidneywood seeds, the three concentrations of the MS medium had the highest seedling height when combined with disinfection 2, which indicates the beneficial effect of AgNPs, which coincides with Chávez-García et al. (2020); Bello-Bello and Spinoso-Castillo (2022), who point out that these can increase yield and promote the development of in vitro cultures.
Contrary to height, seedling root growth was higher when seeds were grown in 50% MS culture medium in combination with disinfection methods 1 and 2 (8.86 and 7.28, cm respectively) and 50% WPM medium with disinfection method 1. The shortest root length was obtained when 100% MS and 75% WPM media were used, both combined with disinfection method 1, which had 4.63 and 4.42 cm less root length than those with longer roots (Table 2).
In general terms, the media that had plants with lower height originated greater root length. The components of the culture medium along with the application of the disinfectant affected both the height and root growth. In this regard, Bello-Bello and Spinoso-Castillo (2022) mention that the application of silver nanoparticles at low concentrations helps stimulate development, break dormancy, or promote seed germination and growth of some species.
Root size is important during the phase of acclimatization and establishment in soil; on this point, Pinedo-Panduro et al. (2022) report that, in order for micropropagated plants to survive the process of acclimatization and transplantation to the final field, it is necessary for them to have a good number and size of roots. However, it should be noted that seedlings with longer roots hinder the process of transplanting to substrate during the passage to the acclimatization phase.
Plant growth is also assessed by the accumulation of dry matter. In this research, the highest amount of dry matter was produced in seedlings grown in 50% MS medium (58.6 mg) with disinfection method 1, followed by those grown in 100% MS (52.2 mg) with disinfection 2 and by those grown in 100% WPM medium with disinfection method 1. The opposite was observed in the seedlings obtained from seeds grown in the WPM medium at 50% with disinfection method 2, since only 39.6 mg dry matter was obtained, 19 mg less than in the best treatment.
The seedlings of the other treatments generated similar amounts of dry matter (Table 2). Martínez-Villegas et al. (2015) mention that the accumulation of dry matter depends on the composition of the culture medium and the species that is being grown since, in Euphorbia leucocephala, the highest accumulation of dry matter (92.2 mg per plant) was obtained in the plants grown in the modified WPM medium; therefore, it is important to choose the appropriate culture medium for the species of interest and thus avoid any type of physiological or growth problem in plants grown in vitro.
The number of leaves per seedling showed few differences due to the effect of the treatments. The culture media of 100% MS with seeds disinfected with method 2, 75% MS with disinfection 2, and 50% MS with disinfection 1 were the ones that generated the highest number of leaves per seedling (6 and 7 leaves) (Table 2). The concentration of nutrients in the MS medium was adequate to supply water and nutrients to the kidneywood seeds; this medium contains high concentrations of ammonium NH4 + ions (20.6 mM), nitrate NO3 - ions (39.4 mM), chlorine Cl- ions (6 mM), and MoO4 - (1 mM) (Martínez-Villegas et al., 2015) and did not have a toxic effect on plants.
The characteristics that give in vitro plants greater chances of surviving during acclimatization in greenhouses are the obtaining of a good size and foliage because these two parameters are related to greater photosynthetic capacity, which allow the plants to perform better radiation capture and therefore, greater accumulation of photosynthates (Pinedo-Panduro et al., 2022). Therefore, E. polystachya developed a good number of leaves during the 60 days after sowing.
Using the double application of AgNPs in the disinfection of E. polystachya seeds reduced the presence of fungi and bacteria in the culture bottles, which generated the highest asepsis, height, and dry matter of the seedlings. The MS culture medium gave better results in seed germination and seedling growth; it generated greater root length and dry matter in a concentration of 50%.
To the National Council of Science and Technology (CONACYT), for its acronym in Spanish for the scholarship awarded to the first author with scholarship number 665515. To the Faculty of Agricultural Sciences and the Autonomous University of the State of Morelos for their support to conduct the research.
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