Establishment of in vitro culture

On June 2018, 15 capsules (Fig. 1B) of E. hyalinobulbon were collected from a population at the Vasco de Quiroga Biological Station (EBVQ), that is under the auspices of the Faculty of Biology of the Universidad Michoacana de San Nicolás de Hidalgo. The EBVQ is located at the “La Alberca” ranch in the town of Toreo Alto, northwest of the urban area of Uruapan, Michoacán, Mexico, at 19°29'26.3''N latitude and 102°00'24.4''W longitude.

The seeds were disinfected in 15% liquid detergent for 5 min, 70% ethanol for 5 min, 3% hydrogen peroxide for 5 min, 1.2% sodium hypochlorite for 5 min and rinsed with sterile distilled water (modified from ^{Ávila-Díaz et al., 2009}). The aseptic seeds were then transferred to culture media.

In vitro development of E. hyalinobulbon with organic supplements

For the sowing of seeds, the medium Phytamax^TM Orchid Maintenance Medium without Charcoal (Phy) (SIGMA, St. Louis, Missouri, EUA) and MS medium (^{Murashige and Skoog, 1962}) at 30% of its basal salts, added with 8 g bacteriological agar (BD Bioxon, Cuautitlán Izcalli, Mexico) and 20 g sucrose, were used in combination with organic supplements as shown in Table 1. The pineapple and banana were manually ground to puree, they were passed through a kitchen strainer to ensure that the paste was homogeneous; once the coconut was split, the endosperm was ground with the coconut water to create coconut milk. The organic supplements were added to each medium, the pH was adjusted to 5.7 with pH-indicator strips McolorpHast^TM (1.09535.0001, Darmstadt, Germany) and they were sterilized in an autoclave Kitlab (AE-22, USA) for 15 minutes at 121 °C, 1.2 kg cm-2.

The observations were performed every month, with an optical microscope (MG-30PL, Cd. Mx., Mexico). The development of 150 individuals per treatment was registered based on the following categories: seed, protocorm 1 (globular embryo within the seed coat), protocorm 2 (embryo leaving 50% of the seed coat), seedling 1 (with one to two leaves) and seedling 2 (with three or more leaves) (Figs. 1C-G).

Effect of sucrose vs. stevia as a carbon source

Additionally, another Phy treatment was prepared at 30% of its basal salts added with 15 g bacteriological agar and 20 g N’Joy Stevia® (ingredients: dextrose 96.5%, stevia 3.5% and natural flavoring) (PhyS) to evaluate the germination effectiveness. Each treatment consisted of six flasks, with 20 ml medium covered with aluminum caps. All the cultures were maintained under the same light conditions and ambient temperature, with a light intensity of 7.844 µmol m^-2 s^-1 and in a temperature range of 26.5 ± 3 °C.

In vitro seedling development of E. hyalinobulbon

To induce the development of seedlings, the following media were tested: medium Phy (100%) added with 20 g/l sucrose, 8 g/l agar and a pH of 5.7, as a control (treatment Phyt). The second treatment was a Phy culture medium with 30% of its basal salts, plus 1.166 ml/l of a commercial product with Plant Growth Promoters (PGP) (Maxi-grow®) with 20 g/l sucrose, 8 g/l agar and a pH of 5.7 (treatment Maxi). The third treatment was the culture medium described by ^{Chiu and Chang (2018)}, but modified, which was named as subculture medium: 50% MS, supplemented with 0.5 mg/l niacin, 0.5 mg/l pyridoxine HCl, 0.1 mg/l thiamine HCl, 20 mg/l glycine, 100 mg/l myo-inositol, 170 mg/l NaH₂PO₄, 1 g/l activated carbon, 1 g/l peptone, 6 g/l potato infusion, 150 ml/l coconut milk, 20 g/l sucrose and 8 g/l agar; the pH was adjusted to 5.2 (treatment Chiu). The shoots were subcultured each 60 days. Measurements were carried out at the beginning of the experiment, and every 30 days thereafter. In all cases, the increment (∆) was calculated, with the difference of the final measurement minus the initial measurement. The parameters evaluated every 30 days were: number of leaves, size of two leaves (small and intermediate size), number of shoots, the size of two shoots (small and intermediate size), the number of pseudobulbs, the length and width of a pseudobulb, the vigor (percentage of the green tissue without any symptoms of disease or dehydration in relation to the total seedling), the number of roots and the size of a root.

Statistical analysis

The data about in vitro development of E. hyalinobulbon with organic supplements and with reference to the effect of sucrose vs. stevia as a carbon source were analyzed using the program SAS version 8 (^{SAS Institute, 2008}), applying a Pearson ChiSquare test. Subsequently, a correspondence analysis was performed.

The data about in vitro seedling development of E. hyalinobulbon were analyzed using the program R version 3.5.1 (^{R Core Team, 2018}), applying the ANOVA and TukeyHSD tests with 95% significance.

In vitro development of E. hyalinobulbon with organic supplements

After 60 days, significant differences were observed in the development of E. hyalinobulbon between the different treatments (ChiSquare=43.725, DF=18, p=0.0006), noting that the PhyB treatment was the best to promote the development of E. hyalinobulbon, with respect to the control.

At 90 days after sowing, medium PhyB was the best for the development of E. hyalinobulbon (Fig. 2). Significant differences were recorded in the development between the tested treatments (ChiSquare=63.696, DF=18, p=0.0001). The correspondence analysis showed that the only significantly different medium was PhyB with respect to the control.

Figure 2: Germination and development of Erycina hyalinobulbon (La Llave & Lex.) N.H. Williams & M.W. Chase in culture media with organic supplements 90 days after sowing; from left to right: Phy (30% Phytamax^TM), MSB (30% MS supplemented with banana), PhyB (30% Phytamax^TM supplemented with banana), MSP (30% MS supplemented with pineapple), PhyP (30% Phytamax^TM supplemented with pineapple), MSC (30% MS supplemented with coconut) and PhyC (30% Phytamax^TM supplemented with coconut). 

Effect of sucrose vs. stevia as a carbon source

The development of E. hyalinobulbon 90 days after the sowing in the different carbon sources, with sucrose and stevia, showed significant differences, with the ChiSquare test (ChiSquare=12.500, DF=3, p=0.0059) being higher with stevia (Table 2). At 120 days there were no significant differences between using sucrose or stevia as a carbon source.

In vitro seedling development of E. hyalinobulbon

Regarding the size of the roots, there were significant differences in shoots rooting after 150 days, between Phyt vs. Chiu and Phyt vs. Maxi, being the Phyt medium the one that promotes better root development. Between Chiu and Maxi media there were no significant differences (Fig. 3A). The number of leaves, number of shoots and size of shoots, 150 days after sowing, compared with the Tukey test, show that there were no significant differences between the Phyt and Chiu media, while the comparison of the Maxi medium against the previous two shows that there were significant differences, the Phyt and Chiu media being better (Figs. 3B-D). For the size of leaves and the number of roots, the Tukey test at 150 days after sowing showed significant differences between the Phyt and Maxi media, but not between Chiu vs. Phyt and Chiu vs. Maxi (Figs. 3E-F). Regarding the number of pseudobulbs and the length × width of the pseudobulb, there were no significant differences. The graphs show negative numbers due to the fact that in some treatments the plants withered, and various healthy tissues and organs were severely affected. These values were exhibited when subtracting from the initial measurement to the final one.

Figure 3: Development of Erycina hyalinobulbon (La Llave & Lex.) N.H. Williams & M.W. Chase in culture media: Phytamax^TM at 100% of its basal salts (Phyt), the subculture medium described by ^{Chiu and Chang (2018)} (Chiu), Phytamax^TM at 70% of its basal salts, plus 30% of the recommended concentration of Maxigrow (Maxi). A. Δ root size; B. Δ number of leaves; C. Δ number of sprouts; D. Δ bud size; E. Δ leave size; F. Δ number of roots. The different letter indicates significant differences with respect to the other treatments (p <0.05). The increment (∆) was calculated, with the difference of the final measurement minus the initial measurement. 

In vitro development of E. hyalinobulbon with organic supplements

In this study, the better percentage of germination (9.3%) was obtained in the Phytamax™ medium with 30% of its basal salts, added with banana puree as an organic supplement, which represents a much lower cost in the acquisition of inputs and allows greater accessibility to reproduce this species. The addition of organic supplements to the culture media improves the development and lower production costs of orchids (^{Moreno and Menchaca, 2007}; ^{Pakum et al., 2016}; ^{Salazar and Vega, 2017}). In general, it has been reported that adding banana to the growing media improves the development of the plant and the roots (^{Azlan et al., 2016}; ^{Menezes et al., 2016}; ^{Wida and Hariyanto, 2019}). It has been reported that banana pulp contains natural cytokinins, mineral, carbohydrate, amino acids, niacin, vitamins, fatty acids, cellulose, sterols and polyols, which are useful for the development of plantlets (^{Tamura, 1970}; ^{Arditti, 2008}). With respect to the results obtained by ^{Sauno (2015)}, a 100% germination and viability is reported with the use of Phytamax™ containing 200% straight salts, added with 0.05 mg/l benzyladenine (BA). ^{Sauno (2015)} reports at 45 days a 0% viability with the application of 200% Phytamax™ medium without the addition of BA. Comparing this with our results, the decrease of up to 30% of the basal salts of Phytamax™ allows the individuals to remain viable and develop, since in the present study 10% of seedlings with two or more leaves and one or more roots were recorded at 120 days. ^{Aguilar-Morales et al. (2016)} compared the germination of Encyclia adenocaula (La Llave & Lex.) Schltr. in three treatments based on MS, with 1 g/l charcoal, 10% banana extract and 10% coconut water, where the best medium was the one with charcoal, obtaining 56.9% germination at 30 days, and the slowest was the banana extract with 23% germination at 59 days. In contrast, in this research, the best treatment was Phytamax™ to which 8.5% banana was added, and 9.3% germination was recorded at 90 days.

Starting an orchid production project is usually expensive due to the equipment that includes laboratory balance, laminar flow hood among others and supplies like PGR, such as auxins and citokinins, which are needed to propagate these plants. One way to lower production costs is to use organic supplements. Although the percentage of germination with the medium proposed here is low (9.3%), there are many seeds in the orchid capsules. As reported by ^{Damon and Valle-Mora (2008)}, on average 17,709 seeds per capsule for E. crista-galli are found. In this case, the conservation program can be established with the seedlings obtained, these being sufficient.

Effect of sucrose vs. stevia as a carbon source

Few studies have been carried out on the effect of stevia on the in vitro development of plants; one of these is the one carried out by ^{Taha et al. (2016)}, where they compared the implementation of a solution of Stevia rebaudiana Bertoni leaves vs. sucrose and combinations of both. They obtained that with the use of stevia the multiplication and growth of Simmondsia chinensis C.K. Schneid. improved, but the combination of 20 g/l sucrose plus 7 ml stevia turned out to be better for the number of shoots and leaves and for the length of the shoot. ^{Altamirano et al. (2017)} carried out an investigation with the sweetener CANDEREL® stevia, using it as the carbon source for Cuitlauzina pendula Lex., concluding that the inclusion of 10, 15, 20, 25 g/l CANDEREL® stevia or 25 g/l sucrose does not present significant differences for the height measurements of the seedlings. In the present work N’Joy Stevia® was applied as a carbon source during the in vitro development process of E. hyalinobulbon in a concentration of 20 g/l vs. 20 g/l of sucrose, obtaining better development with N’Joy Stevia® at 90 days after sowing. At 120 days after sowing, we observed results similar to those of ^{Altamirano et al. (2017)}, since there are no significant differences in the germination percentages. However, we recommend the use of N´Joy Stevia® as carbon source if the acceleration of the germination process is desired.

In vitro seedling development of E. hyalinobulbon

^{Chiu and Chang (2018)} obtained a good development and the subsequent flowering of Erycina pusilla (L.) N.H. Williams & M.W. Chase. In this research where we compared the Chiu, Maxi and Phyt media, the latter was the medium that favored the development of E. hyalinobulbon roots. In the other variables, there were no significant differences in its growth between the implementation of the Chiu and Phyt medium, so that Phyt medium is recommended for the development of E. hyalinobulbon, for promoting root development, ease of preparation and low cost of inputs compared to Chiu. The Maxi medium was the one that presented the lowest development, so we do not recommend its use.