elocation-id: e3811
The orchid Laelia autumnalis is endemic to Mexico and is under special protection. In vitro culture has contributed to improving the propagation of orchids from seeds. In order to micropropagate this species, an experiment was established in 2023 to evaluate eight culture media: Murashige and Skoog (MS at 100, 75, 50 and 25%), Knudson C, Dalla Rosa and Laneri, Vacint and Went, and Lindemann; all media were supplemented with 0.5 mg L-1 thiamine HCl, 100 mg L-1 myo-inositol and 3% sucrose to determine the best medium for germination of this species. Seeds were disinfected with 0.3% sodium hypochlorite (NaClO) and sown in the media in a completely randomized design with 10 replications per treatment. Germination start and percentage were recorded; seedling height, leaf length and number, root number and length, and pseudobulb diameter were evaluated at 90 days. The 25 and 50% MS media generated a higher percentage of germination (84.57 and 72.54%, respectively), which began 29 days after seeding. Growth in height (5.6 mm), leaf length (4.54 mm) and root length (5.2 mm) were higher in the MS medium at 50%, where seedlings were green. It was concluded that the best culture medium for germination and growth of L. autumnalis seedlings was the MS at 50% concentration.
day of the dead orchid, MS medium, orchid seedlings.
The Orchidaceae family is one of the most important in Mexico, surpassed only by the Asteraceae and Fabaceae families (Villaseñor, 2016). Mexico ranks eleventh in the world in terms of richness of wild orchids (Hágsater et al., 2005), with around 1 315 species (Soto et al., 2007; Solano et al., 2019).
At the state level, Oaxaca and Chiapas rank first in orchid diversity, both with more than 700 species (Solano et al., 2019), and Veracruz is in third place with 432 taxa (421 species, 4 varieties, 2 hybrids and 5 subspecies), representing 32% of the country’s orchids.
Laelia is a genus of mostly epiphytic orchids, so they are found living on trees; in Mexico, 12 species and 2 subspecies have been classified; one of them is Laelia autumnalis, native to Mexico, whose wild populations have been subject to change in land use and massive and illegal extraction of plants in the reproductive stage to meet market demand.
If this activity continues, it will soon be part of the list of threatened species (Hernández et al., 2013). Due to the magnitude of the extraction of plants of this species, in the update of NOM-059-SEMARNAT-2010, it appears in the category of special protection (Pr) (DOF, 2019).
Orchids are species that are difficult to reproduce naturally because their tiny seeds have little or no nutrient reserves and require symbiosis with mycorrhizal fungi for germination, which are not always present due to the disturbance of their habitat. In addition, not all seeds in a capsule are fully formed or fertile (Verma et al., 2014).
These plants stand out for their beauty and attractive shapes; however, they are one of the families most affected by anthropocentric activities, plundering, and illegal trade (Castillo-Pérez et al., 2018). In vitro culture techniques have contributed to improving the propagation of orchids from seeds, both in native species and in hybrids.
In the genus Laelia, in vitro germination has been achieved in L. speciosa using the Murashige and Skoog (MS) culture medium at 50 and 100% of its components (Murashige and Skoog, 1962) and the Knudson C (KC) medium at 50 and 100% of its components (Knudson, 1946) (Aguilar and López Escamilla, 2013), in L. anceps ssp. Dawsonii, in which the formation of somatic embryos was achieved using the culture media of Knudson C (1946) (KC), Vacin and Went (1949) (VW), Murashige and Skoog (1962) (MS) (Lee et al., 2010), and in L. eyermaniana, where morphological development was observed from germination to ex vitro acclimatization in Murashige and Skoog (MS) culture medium at 50% (Francisco et al., 2011).
For the germination of orchid seeds, some means of widespread use are reported, such as Knudson C (1946), Vacin and Went (1949), Murashige and Skoog (1962), Dalla Rosa and Laneri (1977) (Dutra et al., 2009; De Menezes et al., 2016). The main difference in these is the composition of minerals, the simplest is Knudson C and the richest in nutrients is Murashige and Skoog, which includes macro- and microelements; the latter is the most commonly used for orchid cultivation; Lallana et al. (2020) point out that 50% MS has given good results for the most common genera.
The composition of the culture medium is one of the factors that determine the success of generating and propagating seedlings, every medium is basically composed of macronutrients, micronutrients, vitamins, amino acids and a source of carbon (De Menezes et al., 2016). Therefore, the composition of culture media can vary in content of salts (macro and microelements), sugars, organic compounds, and gelling agents. The requirements for orchids can be diverse, even in species of the same genus (Mayo et al., 2010).
Based on the importance of this orchid species and the situation it has in its habitat, the present research aimed to evaluate the in vitro germination of Laelia autumnalis seeds in eight culture media, as well as the initial growth of seedlings, to determine the most suitable for this species.
This research was conducted in the Plant Propagation Laboratory of the Faculty of Agricultural Sciences, Autonomous University of the State of Morelos, Cuernavaca, Morelos, Mexico, from September 2023 to January 2024.
L. autumnalis capsules were collected from plants under cultivation belonging to a private producer in Tepoztlán, Morelos, Mexico. The seeds were dried at room temperature (29 °C), stored in airtight plastic tubes and kept in a refrigerator at 5 °C for 20 days.
Mineral salts from eight culture media commonly used for the germination of orchid seeds were employed: Murashige and Skoog (1962), in four concentrations, 25 (MS25), 50 (MS50), 75 (MS75), and 100% (MS100) of macro and micronutrient concentration; Knudson C (1946) (KC); Vacin and Went (1949) (VW); Lindemann (1970) (L); Dalla Rosa and Laneri (1977) (DRL) (Table 1).
| Compound (mg L-1) | Murashige and Skoog (1962) | Knudson C (1946) | Vacint and Went (1949) | Lindemann (1970) | Dalla Rosa and Laneri (1977) |
|---|---|---|---|---|---|
| NH4NO3 | 1 650 | ||||
| KNO3 | 1 900 | 525 | |||
| CaCl2 2H2O | 332.2 | 1000 | |||
| MgSO4 | 370 | 250 | 122.1 | 58.62 | 250 |
| KH2PO4 | 170 | 250 | 250 | 135 | 250 |
| (NH4)2 SO4 | 500 | 500 | 1 000 | 500 | |
| Ca(NO3)2 4H2O | 1 000 | 347.2 | |||
| H3BO3 | 6.2 | 1.014 | |||
| MnSO4 4H2O | 22.3 | 5.682 | 5.68 | 0.05 | 7.5 |
| ZnSO4 7H2O | 8.6 | 0.565 | |||
| Na2 MoO4 | 0.25 | ||||
| CuSO4 5H2O | 0.025 | 0.019 | |||
| CoCl2 6H2O | 0.025 | ||||
| Kl | 0.83 | 0.099 | |||
| FeSO4 7H2O | 27.85 | 25 | 27.85 | ||
| Na2EDTA 2H2O | 37.27 | 1 050 | 37.25 | ||
| Ferric tartrate | 28 | ||||
| Tribasic calcium phosphate | 200 | ||||
| Nickel chloride | 0.03 | ||||
| Ferric citrate | 4.4 |
All media were supplemented with 0.5 mg L-1 thiamine HCl, 100 mg L-1 myo-inositol, and 3% sucrose; the pH of the media was adjusted to 5.7. Likewise, 0.7% agar was added. The medium was dosed into 150 ml bottles, placing 15 ml of culture medium and sterilizing for 18 min at 121 °C and 15 lb pressure.
Before sowing, the seeds of L. autumnalis were disinfected in the laminar flow hood, using the syringe method, which consisted of the following: a piece of cotton was placed in the nozzle of a 20 ml syringe, the seeds were put inside the syringe and the disinfectant solution with 0.3% sodium hypochlorite (NaClO) and sterilized distilled water were added, then the plunger was placed and the syringe was shaken vertically for 5 min, three rinses were made with sterile distilled water and finally, 20 ml of sterile distilled water was added to the syringe to keep the seeds suspended.
Each of the bottles with culture medium was added with 250 μl of water with approximately 250 suspended seeds, which were counted with a hemocytometer. The bottles were covered and sealed with adhesive plastic, and finally, they were placed in the incubation area under controlled conditions with a photoperiod of 16 h light and 8 h dark at 24 ±1 °C and luminous intensity of 32 μE m-2 s-1.
A completely randomized design with 10 replications per treatment was used to assess seed germination and seedling growth (10 seedlings per replication). Each replication was a bottle of culture.
Two germination variables were evaluated; the start of germination was considered once the seed showed rupture of the testa; the percentage of germination was also determined, the germinated seeds were counted one month after sowing in four fields by replication with a stereoscopic microscope.
Ten seedlings were taken per replication to assess growth; height (mm) was measured from the base to the part where the leaves open; leaf length (mm) was measured from the base to the leaf tip; root length (mm) was measured from the base to the root tip; these three variables were measured with a piece of millimeter graph paper; number of leaves and number of roots were counted; protocorm diameter (mm) was also measured. The data obtained were studied by analysis of variance (Anova); in the variables where there was an effect of the treatments, Tukey’s comparison of means test (p≤ 0.05) was performed with the SAS v. 9.2 statistical package (SAS, 2023).
The analysis of variance for the start of germination and total germination indicated that there was a highly significant effect of the culture media (p≤ 0.01). The R² values were 0.96 and 0.88, indicating that the statistical model used for the analysis of variance was adequate (Table 2).
| Source of variation | DF | Start of germination (days) | Total germination (%) |
|---|---|---|---|
| Culture media | 7 | 365.53** | 3 328.79** |
| Error | 72 | 1.44 | 93.61 |
| R² | 0.96 | 0.88 | |
| CV (%) | 3.49 | 24 | |
| Average | 34.37 | 40.31 |
The germination of the seeds was observed with the naked eye when they changed from cream to green structures and the size of the embryo increased, later the embryo broke the testa, which could be observed under a microscope. This process varied depending on the culture media; seeds began germinating in less time in the MS media at 25, 50, 75% and the KC medium (at 29 days), whereas in the MS at 100%, DRL, VW and L media, it occurred after 35 days; in DRL, it took 14 days longer (Figure 1).
In this species, seed germination was a little slower compared to what was reported by Aguilar and López Escamilla (2013) for Laelia speciosa, which germinated in 10 and 16 days in 50 and 100% MS media; nevertheless, they indicate that in 50 and 100% KC media, germination occurred at 114 and 178 days, respectively. This indicates that the germination of orchid seeds varies depending on the culture medium and species.
The percentage of germinated seeds of L. autumnalis was higher when the macro- and micronutrients of the MS medium were used at 25 and 50% concentration, with no statistical differences between both culture media (84.57 and 78.63%, respectively); the tendency was to decrease germination as the concentration of salts in the MS medium increased, this happened because there was greater availability of water for the process to occur and as Martínez-Villegas et al. (2015) mention, as salts increase, the osmotic potential becomes more negative.
In the VW, KC, and 75% MS media, germination ranged from 28.41 to 47.71%. The media with the lowest germination values were 100% MS, DRL and L, which presented 59.59, 64.59 and 71.33% less germination than that obtained with the MS medium at 25% concentration (Figure 2).
After the change in green, small protocorms formed in the seeds, from which the first small leaf was observed. Growth was also observed with the emission of new leaves, the increase in height and thickness of stems and the growth of the rhizoid, thus forming a seedling. Seeds germinated in the 25% MS medium generated yellowish-green seedlings, indicating nutrient deficiency due to low macro- and micronutrient concentration.
Seeds germinated in the Vacin and Went (1949) and Lindemann (1970) media did not generate seedlings, so they were excluded from the growth results. The analysis of variance for the growth variables of seedling height, leaf length, number of leaves, number of roots, root length, and diameter of pseudobulbs showed that the composition of the culture media had a highly significant effect (p≤ 0.01).
The coefficients of variation were from 18.13 to 22.42, the values of R² were from 0.88 to 0.94 (Table 3), indicating that the statistical model used for the analysis of variance was adequate. The height of the seedlings was higher in the MS media at concentrations of 25 to 75%; in contrast, the height was lower when any of the other media was used. The length of leaves was longer in the 50 and 75% MS media, and the culture media that led to lower growth were MS at 100% and KC.
The number of leaves per seedling was also affected by the culture medium; seedlings growing in the DRL medium had the highest number of leaves (3.03 mm per seedling), which were statistically different only from seedlings growing in the MS medium at 25%, which had the lowest number.
The diameter of pseudobulbs was statistically the same with most of the media, except for seedlings that grew in the MS medium at 25%, which had the lowest values (Table 4). The average number of roots per seedling ranged from 0.75 to 1.24. The KC, MS75 and MS50 media were statistically equal, with the highest root average (Figure 3). The lowest number of these organs occurred in the MS100 medium. Root length was greater in the 50% MS medium, with no statistical difference from the KC and 25% MS media, and the 100% MS medium had the lowest values (Table 4).
The general analysis of the results obtained for the various variables allows us to point out that, for the in vitro germination of L. autumnalis seeds, the 25, 50 and 75% MS culture media and the KC medium were where germination began in the shortest time; nevertheless, the total percentage was higher in the MS media at 25 and 50%. Regarding seedling growth, most variables indicate that it was better and similar in MS media at 25, 50 and 75%.
Nonetheless, seedlings were green in the 50% MS medium; in contrast, seedlings that grew in the 25 and 75% MS media began to show violet or reddish color after 107 days. Therefore, it is considered that the best culture medium for germination and growth of L. autumnalis seedlings was MS at 50% nutrient concentration. Dalzotto (2013) reported that the plant height, number of roots, and dry matter of Oncidium bifolium were higher in the 50% MS medium, which coincides with what was obtained in the present research.
Murashige and Skoog (1962) medium has been used in the germination of several species of orchids in different proportions and supplemented with growth regulators and other substances. The results obtained regarding the beginning of germination and total germination of this research could be due to the fact that the Murashige and Skoog medium contain the necessary macronutrients and micronutrients compared to the other media.
The study by Aguilar and López Escamilla (2013) used L. speciosa seeds and reported that germination occurred 10 days after sowing in 50% MS medium and 16 days after in 100% MS medium, and germination was 100% in both cases, which coincides with the results obtained in this work since the 50% MS medium was the best for the germination of L. autumnalis seeds.
For their part, Hernández et al. (2017) used capsules of Laelia autumnalis which were irradiated with gamma rays and were later grown in Murashige and Skoog (1962) culture medium and observed that the imbibition process occurred between 5 and 40 days and the formation of photosynthetic protocorms began at 10 days and concluded until 60 days.
Findings of Francisco et al. (2011) did not report the days to germination of Laelia eyermaniana; however, the culture medium used to germinate the seeds was Murashige and Skoog at 50%. Jara et al. (2007) used 50% and 100% MS, KC and Morel media for the germination of Chloraea virescens, Chloraea lamellate, and Gavilea araucana and report that the 50% MS medium gave the best results.
In their research, Ávila et al. (2009) germinated seeds of Laelia speciosa using 100 and 50% Murashige and Skoog (1962) and Knudson C (KC) media added with sucrose, and report that the 100 and 50% MS media generated the best response in seed establishment for this species since the first stages of development were better in the MS medium compared to the KC medium.
Regarding seedling height, leaf length, number of leaves, root length, number of roots, root length and pseudobulb diameter, it was observed that the 25% MS medium was not suitable for seedling growth. It is important that the culture media have the right amount of nutrients so that the seedlings do not present nutritional deficiency and have better development, as occurred in the 25% MS medium (Figure 4).
The Vacin and Went (1949) and Lindemann (1970) media were the least suitable for the germination of L. autumnalis since they took longer to germinate; likewise, in the percentage of germination, they presented the lowest values compared to the other media; on the other hand, the seeds died as time went by so there was no growth of seedlings.
Based on the results of this work, in vitro germination of L. autumnalis seeds was better in the MS culture media at 25 and 50% since the start of germination occurred in less time and in a higher percentage. Seedling growth was best in the 50% MS medium, in which seedlings were green. Therefore, the best growing medium for germination and growth of L. autumnalis seedlings was MS at 50% nutrient concentration.
To the National Council of Humanities, Sciences and Technologies (CONAHCYT), for its acronym in Spanish for the scholarship to the master’s student Marcela Cabañas-Rodríguez.
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