Culture mediums

The culture mediums tested for the growth of adventitious buds of Laelia anceps Lindl. and Epidendrum sp. of 1.1 ±0.1 cm, previously regenerated in vitro, containing nutrient salts of Murashige and Skoog (MS) 50 and 100%, 30 g L^-1sucrose, 0 and 1 mg L^-1 gibberellic acid (AG₃), agar (7 g L^-1) or 30 ml of substrate mixtures: pearlite-volcanic rock (PT) and coir-volcanic rock (FCT) in 3:1 ratio (0.5 mm particle size). The médium pH was adjusted to 5.7-5.8.

Planting of crops

Adventitious shoots were placed in 250 ml glass vials containing 30 ml of the culture medium described above plus the PT, FCT or agar substrates (Table 1). Previously, the flasks were sterilized in an autoclave for 15 minutes at 120 C before the establishment of the shoots (explants).

MS= Sales de Murashige y Skoog (1962).

Table 1 Tested treatments in the in vitro culture of Laelia anceps Lindl. and Epidendrum sp. 

Cultures were incubated in a growth room at 26 ±2 °C and a photoperiod of 16 h provided by cold white fluorescent lamps (25 µmol m^-2 s^-1 light intensity).

Evaluated variables

After 45, 90 and 135 days of initiating the crop, the length of the shoot (from the beginning of the root to the tip of the longest leaf), number of leaves and number of roots were evaluated; and the fresh weight was evaluated at 60 days using an analytical balance.

Experimental design

The experiment was carried out in the Laboratory of Biotechnology and Pathology of Seeds of Campus Montecillo, Colegio de Postgraduados. A completely randomized experimental design was used with three factors: species (2), substrates (3) and médium culture (4) resulting in 24 treatments. Each treatment had 15 replicates and each replicate consisted of one flask with five adventitious shoots. The data of each variable were analyzed by analysis of variance (ANOVA) and the comparison of means by the Tukey test (≤ 0.05). Both procedures were performed with the SAS v.9.0 statistical program (^{SAS Institute, 2002}).

Effect of the species

The analysis of the results showed that the species had a significant effect on shoot length (45, 90 and 135 days), number of leaves (45 days) and number of roots (90 and 135 days) (Table 2). After 45 days of culture, the Epidendrum sp species, showed better response for shoot length; however, after this time it was Laelia anceps Lindl. which showed the highest values (Table 3). The number of leaves of the shoots of Laelia anceps Lindl. after 45 days was higher than Epidendrum sp. but at 90 and 135 days both species behaved in a similar way.

^**= p≤ 0.01; ^*= p≤ 0.05; CV= coeficiente del variación; E= especie; M= medio; S= sustrato; PF= peso fresco.

Table 2 Squares sum of variance analysis for the effect of the species, substrates and culture medium on shoot length, number of leaves, number of roots and fresh weight of Laelia anceps Lindl. and Epidendrum sp. after 45, 90 and 135 days of culture. 

DSH= diferencia significativa honesta. Letras distintas en una columna indican diferencias estadísticas significativas (Tukey≤ 0.05).

Table 3 Effect of the species on shoot length, number of leaves and number of roots at 45, 90 and 135 days after starting the in vitro cultivation. 

Moreover, for the number of roots there were no statistical differences during the first 45 days between the two species, but after this period L. anceps Lindl. showed significantly higher values tan Epidenderum (Table 3). Regarding these results, the genotype effect on the in vitro growth was also observed in Cattleya aurantiaca Bateman, Encyclia adenocaula La Llave & Tex., Laelia speciosa Kunth., Epidendrum radicans Pav. Ex Lindl., Euchile citrina La Llave ex Lex., Laelia albida Bateman ex Lindl., Laelia autumnalis (Lex.) Lindl., Oncidium cavendishianum Bateman and Oncidium tigrinum La Llave & Lex. (^{Ávila and Salgado-Garciglia, 2006}). Also, ^{Abdoli and Moieni (2003)} found that genotype greatly influenced the induction of organogenesis in sunflower (Helianthus annuus L.).

Effect of substrates

The substrates had a significant effect on shoot length (45 and 135 days) and number of roots at 135 days (Table 2). After 45 days of cultivation, shoot length was statistically higher in perlite-volcanic rock (PT), but at 135 days the highest values were recorded in shoots grown on agar, while no significant differences were found in treatments with coir-volcanic rock (FCT) and PT (Table 4) (Figure 1). With respect to the number of leaves, no significant differences were found between the shoots grown on different substrates and agar during the 135 days when shoots were kept in vitro.

FCT= fibra de coco-tezontle; PT= perlita-tezontle; DSH= diferencia significativa honesta. Letras distintas en una columna indican diferencias estadísticas significativas (Tukey≤ 0.05).

Table 4 Effect of the substrates on shoot length, number of leaves and number of roots at 45, 90 and 135 days after cultivation. 

Figure 1 Adventitious buds of Laelia anceps Lindl. (a, b, c) and Epidendrum sp.(d, e, f) 90 days after initiation of in vitro culture. Agar (a, d), coir-volcanic rock (b,e) and perlita-volcanic rock (c, f). Bar 1 cm.  

It was also observed that the number of roots was statistically similar in agar, FCT and PT at 45 and 90 days of culture; however, after 135 days FCT-grown shoots had significantly fewer root numbers than those grown on agar or PT (Table 4). In this regard, ^{Keatmetha and Suksa-Ard (2004)} found that the use of vermiculite and peat moss during the in vitro culture of Anthurium andraeanum L. did not promote more roots compared to the use of phytagel.

Meanwhile, ^{Afreen-Zobayed et al. (2000)} used various combinations of vermiculite and paper pulp to replace agar for in vitro culture of Ipomoea batatas L. obtaining the best results with vermiculite with 30% paper pulp. Also, ^{Labrousse et al. (2012)} evaluated Sorbarod (buffer cellulose), peat and paper pulp as alternative supports to agar in micropropagation for Nemesia denticulata (Benth.) Grant ex Fourc., they found that substrates promoted better root development and increased acclimation survival. Also, ^{Hazarika (2006)} indicates that the roots of Brassica oleracea L. developed in agar have low functionality, limiting the survival of plants during the acclimatization process. Similarly, ^{Kozai (2010)} and ^{Oh et al. (2012)} found that substrates of vermiculite, paper pulp and perlite increase water conductivity, which favors the absorption of nutrients by plants.

Effect of culture medium

It was possible to observe significant differences in shoot length (45 and 135 days), and number of roots after 90 and 135 days of cultivation by medium effect (Table 2). At 45 days after starting the cultivation the shoots grown on MS medium with 50% AG₃ (MS50-2) showed the greatest length, but at the end of the evaluation this medium and the one containing MS salts at 100% without AG₃ showed no statistically differences (Table 5). In this regard, ^{Coello et al. (2010)} found that gibberellic acid was the factor that promoted shoot elongation of Guarianthe skinneri Bateman. On the other hand, the number of leaves was not significantly different between the different treatments throughout the crop. In contrast, ^{Rodrigues et al. (2009)} found that the use of AG₃ Cattleya loddigesii Lindl. growth promoted a greater number of leaves. Also, only the number of roots of shoots grown in MS at 100% was significantly lower than those remaining in MS medium at 50% and AG₃ after 90 and 135 days after starting the culture (Table 5).

MS= sales de Murashige y Skoog; MS100= MS al 100%; MS50= MS al 50%; MS100-2= MS al 100% con 1 mg L^-1 de ácido giberélico; MS50-2= MS al 50% con 1 mg L^-1 de ácido giberélico; DSH= diferencia significativa honesta. Letras distintas en una columna indican diferencias estadísticas significativas (Tukey ≤ 0.05).

Table 5 Effect of culture medium on shoot length, number of leaves and number of roots after 45, 90 and 135 days of cultivation.  

Meanwhile, ^{Jara et al. (2007)} used four mediums (MS 50%, MS 100%, Morel medium, Knudson medium for in vitro germination of Chloraea virescens (Willd.) Lindl., Chloraea lamellata Lindl. and Gavilea araucana Phil., finding that the MS 50% medium induced the best results in the studied species. Similarly, ^{Dalzotto (2013)} reported that plant height, number of roots and dry matter at 95 days of in vitro culture of Oncidium bifolium (Sims) Dumort. were higher in the MS 50% medium. The results of this paper show that less concentrated media containing MS salts at 50% did not limit growth of shoots of Laelia anceps Lindl. and Epidendrum sp. which is consistent with the results of ^{Pervin (1997)}, who observed that orchids of the genera Vanda, Dendrobium, Aerides, Acampe and Apathoglottis can grow in media with few nutrients (peptone, inositol, banana extract or coconut water).

^{Flores-Escobar et al. (2008)} observed that shoots of Oncidium stramineum Lindl. cultured in MS medium supplemented with organic extracts (coconut water, peptone, activated charcoal, polyvinylpyrrolidone) reached a length of 2.05 cm in 90 days, which is a very close to that obtained in the medium containing 50% of the MS salts and 1 mg L^-1 of AG₃ (MS50-2) in a similar time interval.

Effect of treatments

Significant differences were found for shoot length, number of leaves and number of roots at 45, 90 and 135 days in the interaction factors (Table 2). After 135 days of cultivation, the shoot length in L. anceps Lindl. in the treatments of MS salts at 50%, AG₃ and agar (MS50-2A) and 100% MS salts with agar (MS100-A) was statistically higher than the other treatments. However, at 90 days only statistical differences between treatments MS50-2A and MS50A (MS 50% without AG₃) and MS100-2PT (MS100%, AG₃ and perlite-volcanic rock) (Table 6) were observed.

DSH = diferencia significativa honesta. Letras distintas en una columna indican diferencias estadísticas significativas (Tukey ≤ 0.05).

Table 6 Effect of treatments on shoot length of Laelia anceps Lindl. and Epidendrum sp. at 45, 90 and 135 days of culture. 

This indicates that outbreaks of L. anceps Lindl. can grow properly in both agar and perlite-volcanic rock or vermiculite-perlite, and MS salts at 50 or 100% with or without AG₃ for 90 days, and after this time the growth is better in the media gelled with agar, despite the concentration of salts or AG₃. These results agree with those of ^{Martínez et al. (2006)} who evaluated the germination, multiplication and rooting of Citrus volkameriana Ten & Pasq., Citrumelo Swingle (Citrus Paradise Macfad cv. Duncan x Poncirus Trifolia L.) and Citrange in vermiculite, perlite and volcanic rock, without finding significant statistical differences between these and the treatments with agar.

These authors also mention that survival during acclimatization was greater in plants coming from substrates, since the agar ones suffered stress caused by the rupture of roots, causing their death. ^{Afreen-Zobayed et al. (2000)} found that treatments based on agar in the micropropagation of Ipomoea batatas L. were below the treatments based on vermiculite and paper pulp.

Unlike what was observed in L. anceps Lindl. in Epidendrum sp. no significant differences were found in the length of the shoots submitted to the different treatments tested throughout the growing period (Table 6).

On the other hand, there were no significant differences in the number of leaves formed in shoots of L. anceps Lindl. and Epidendrum sp. after 45 and 90 days, but at 135 days only Epidendrum sp. of the MS50A treatment (50% MS agar) had a statistically different number leaves, while for L. anceps Lindl. the treatment MS50 PT (MS 50% perlita-volcanic rock) induced the formation of a larger number of leaves (Table 7).

DSH= diferencia significativa honesta. Letras distintas en una columna indican diferencias estadísticas significativas (Tukey ≤ 0.05).

Table 7 Effect of treatments on the number of leaves of Laelia anceps Lindl. y Epidendrum sp. at 45, 90 and 135 days after the beginning of the experiment.  

These results agree with those of ^{Martínez-Hernández et al. (2009)} who evaluated vermiculite as a substitute for agar in Citrus volkameriana Ten & Pasq., Citrumelo swingle (Citrus paradise Macfad cv. Duncan × Poncirus trifolia L.) and C-35 and found that the number of leaves did not show statistically significant differences in the first eight weeks of in vitro growth.

On the other hand, ^{Xiao and Kozai (2006)} tested a porous material (Florialite) alternative to agar for Limonium latifolium Lindl. growth and found significant statistical differences in leaf area, dry and fresh weight during the first 25 days of cultivation. In this regard, the results obtained in this research indicate that growing outbreaks of L. anceps Lindl. and Epidendrum sp. in media with 50% of the salts and mixtures of perlite-volcanic rock or perlite-coir without AG₃ promotes a similar number of sheets to those grown in the MS salts at 100% and agar.

After 135 days it was observed that only the shoots of L. anceps Lindl. which were cultivated in the MS50-A treatment formed significantly fewer roots than those in the MS50-FCT treatment (Table 8). For Epidendrum sp. the MS100FCT treatments and MS50-2FCT induced a lower number of roots. At that same time interval, the shoots of Epidendrum sp. cultivated in the treatment MS100A-2A showed to be significantly more efficient to form roots than those of the MS50-PT, MS50-A, MS100-2PT, MS100-2FCT, MS100-FCT y MS100-A treatments (Table 8).

DSH= diferencia significativa honesta. Letras distintas en una columna indican diferencias estadísticas significativas (Tukey ≤ 0.05).

Table 8 Effect of treatments on the number of roots of Laelia anceps Lindl. y Epidendrum sp. after 45, 90 and 135 days of culture.  

In this regard, ^{Xia and Kozai (2006)} found that the shoots of Limonium latifolium Lindl. formed a greater number of roots in Florialite than in agar. On the other hand, ^{Mohan et al. (2004)} used organic sugar cane residues as a substitute for agar in the in vitro rooting of apple trees (Malus prunifolia Borkh.) and obtained a 63% increase in the number of roots.

It has been documented that roots formed in mediums with agar are usually thin and fragile, which are damaged during transplantation, leading to the loss of individuals (^{Debergh and Maene 1981}; ^{Roberts and Smith 1990}).

Table 2 shows significant differences for the fresh weight of shoots at 60 days of cultivation. Also, significant differences between L. anceps Lindl control shoots (MS100A) and treatments MS100-PT, MS100-2PT, MS100A for the fresh weight variable, were found (Table 9). While for Epidendrum sp. the fresh weight of the control shoots was statistically similar to the treatments MS50-FCT, MS50-PT, MS50-2FCT, MS50-2PT. In this regard, when studying the effect of vermiculite as a substitute for agar in Carica papaya L. ^{Kataoka (1994)} it was found that fresh root weight was higher in the explants developed in agar. In this research the use of substrates, diluted mediums and addition of gibberellic acid (AG₃) had no negative effect on the fresh weight of L. anceps Lindl. and Epidendrum sp.

DSH= diferencia significativa honesta. Letras distintas en una columna indican diferencias estadísticas significativas (Tukey ≤ 0.05).

Table 9 Fresh weight of Laelia anceps Lindl. And Epidendrum sp. Shoots after 60 days of in vitro culture.  

In this research, the best evaluated treatments could become a protocol in which diluted culture mediums and substrates are used as substitutes for the agar. This protocol represents an alternative to the in vitro propagation of L. anceps Lindl. and Epidendrum sp. Which is efficient and has a lower cost. This decrease in cost is attributed to the reduction in the concentration of nutrient salts and AG₃, but especially to the use of inexpensive substrates, which unlike agar, represents only 13 to 17% of the cost.