Culture medium and agar substitutes for in vitro growth of orchids

El cultivo in vitro es una técnica que ha permitido la propagación de distintas especies de orquídeas, pero el lento crecimiento de este grupo de plantas, así como el costo de las sales minerales y del agente gelificante (agar) empleados en los medios de cultivo, pueden limitar su aplicación. El objetivo del presente trabajo, fue conocer el efecto de la composición del medio de cultivo y el uso de sustratos como sustitutos del agar en el crecimiento in vitro de Laelia anceps Lindl. y Epidendrum sp. Brotes adventicios de ambas especies se cultivaron in vitro en distintas mezclas de sustratos (perlita, tezontle y fibra de coco) y agar con diferentes concentraciones de las sales basales de Murashige y Skoog (50 y 100%) y ácido giberélico (AG3) (0 y 1 mg L-1) para evaluar su longitud, número de hojas, número de raíces y peso fresco en un diseño experimental con tres factores y 15 repeticiones, cada repetición consistio en un frasco con 5 brotes adventicios. El comportamiento in vitro, estuvo en función del genotipo; L. anceps Lindl. respondió mejor que Epidendrum sp. Los brotes crecidos sobre perlita-tezontle o fibra de coco-tezontle, y medios con sales al 50% y AG3 no mostraron diferencias significativas en cuanto a su longitud, número de hojas, número de raíces y peso fresco, Abstract


Introduction
México has a great diversity in animal and vegetal species, to take care and to preserve such diversity is very important.In this regard, México is home to a remarkable wealth of orchids, which has been recorded in 1 260 species and 170 genera (Soto and Salazar, 2004;Hágsater et al., 2005).Of these, 181 are included in some risk category in the official norm in force NOM-059- ECOL-2001(Diario Oficial de la Federación, 2002), 72 are endemic, 58 are in the threatened category, 107 require special protection, 15 are endangered and one species is already extinct in the wild (Laelia gouldiana Rchb.F.) (Diario Oficial de la Federación, 2002).
Laelia anceps Lindl.And Epidendrum sp. are two orchids species that due to their striking flowers have been subjected to a high collection pressure.This pressure, coupled with habitat destruction has led to the rapid decline in their populations (Halbinger and Soto, 1997;Romero-Tirado et al., 2007).
With respect to its reproduction, germination in orchids represents one of the major limitations to its survival, since the endosperm is reduced in some species, while in others it is absent, so to ensure its germination it is necessary that the seeds are associated with mycorrhizal fungi that provide them with nutrients (Téllez, 2011).
The in vitro culture is a technique which facilitates the germination and propagation of virtually any orchid, as it is done aseptically, in the presence of a controlled source of nutrients and physical conditions, which potentiates its reproduction and growth ability (Zettler et al., 2001;Salazar et al., 2013).However, the cost of the materials used in the preparation of the culture media is high, especially the gelling agent (agar or phytagel) which can represent up to 70% of the cost of the plants.In addition, the agar may reduce the oxygen concentration and disable the nutrient diffusion in the medium (Fujiwara et al., 1993;Ichimura and Oda, 1998).los medios de cultivo es alto, especialmente del agente gelificante (agar o phytagel) que puede representar hasta 70% del costo de las plantas.Además, el agar puede reducir la concentración de oxígeno e inhabilitar la difusión de nutrimentos del medio (Fujiwara et al., 1993;Ichimura y Oda, 1998).
Dada la importancia de cuidar y preservar la diversidad de las orquídeas mexicanas y de proponer alternativas para reducir los costos de producción de su propagación in vitro, el objetivo del presente trabajo fue conocer el efecto de la composición del medio de cultivo y el uso de sustratos como sustitutos del agar, en el crecimiento in vitro de Laelia anceps Lindl.y Epidendrum sp.partiendo de la hipótesis de que los medios de cultivo diluidos suplementados con ácido giberélico, y los sustratos permitirán el crecimiento in vitro de ambas especies.
Furthermore, for some species (Laelia halbigeriana Salazar & Soto Arenas, Agave cocui Trelease) it has been documented that the full concentration of the Murashige and Skoog (1962) medium salts, commonly used in tissue culture, exceeds the tissue nutrimental requirements (Raya-Montaño et al., 2011;González et al., 2012).Also, the slow growth of most orchids, contributes to increased production costs, having to keep plants in vitro longer until they reach a size that allows to transfer them to the greenhouse.
The reduction of production costs could be achieved with the use of hydroponic substrates (perlite, vermiculite, coir), as they may be an alternative to replace expensive gelling agents.In this regard, there is evidence that the use substrates during in vitro phase has allowed growth in species like Limonium latifolium Lindl., Ipomoea batatas L., and Myrtus communis L. (Afreen-Zobayed et al., 1999;Afreen-Zobayed et al., 2000;Lucchesini et al., 2006;Xiao y Kozai, 2006).
Given the importance of taking care and preserve the diversity of Mexican orchids and to propose alternatives to reduce production costs of it in vitro propagation, the objective of this research was to determine the effect of the composition of the culture medium and the use of substrates as substitutes for agar, in the in vitro growth of Laelia anceps Lindl.And Epidendrum sp.Based on the assumption that diluted culture means supplemented with gibberellic acid, and substrates would enable the in vitro growth of both species.

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).
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).

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.
Asimismo, se observó que el número de raíces fue estadísticamente similar en agar, FCT y PT a los 45 y 90 días de cultivo; sin embargo, después de 135 días los brotes cultivados en FCT tuvieron un número de raíces 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  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.
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.significativamente menor que aquellos crecidos en agar o PT (Cuadro 4).Al respecto, Keatmetha y Suksa-Ard ( 2004) encontraron que el uso de vermiculita y peat most durante el cultivo in vitro de Anthurium andraeanum L. no promovieron mayor número de raíces con respecto al uso de phytagel.

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 AG 3 after 90 and 135 days after starting the culture (Table 5).

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.
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)  1.1 b 1.9 ab 1.9 c-g 1.2 b 1.6 ab 1.5 e-g MS100-2FCT 1.
Estos resultados concuerdan con los obtenidos por Martínez-Hernández et al. (2009)  ) 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).
(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).
En el presente trabajo, los mejores tratamientos evaluados podrían formar un protocolo en el que se utilicen medios de cultivo diluidos y sustratos como sustitutos del agar.Este protocolo representa una alternativa para la propagación 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.
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.

Conclusions
The use of less concentrated culture medium without AG3 and substrates as support, allowed the growth of L. anceps Lindl.and Epidendrum sp.plants with the same or greater efficiency than the conventional gelled with agar culture method.Replacing agar with substrata and use of culture medium with diluted salts without AG3, not only allows Cuadro 9. Peso freso de los brotes de Laelia anceps Lindl.y Epidendrum sp.después de 60 días de cultivo in vitro. in vitro de L. anceps Lindl.y Epidendrum sp.eficiente y a un costo menor.Dicha disminución en el costo se atribuye a la reducción en la concentración de las sales nutritivas y AG3, pero sobre todo al uso de sustratos baratos, los cuales a diferencia del agar, sólo contribuyen con 13 a 17% del costo del mismo.

Conclusiones
El uso de medios de cultivo menos concentrados, sin AG3 y sustratos como soporte, permitió el crecimiento de las plantas de L. anceps Lindl.y Epidendrum sp.con la misma o mayor eficiencia que el método de cultivo convencional gelificado con agar.La sustitución del agar por sustratos y el uso de medios de cultivo con sales diluidas sin AG3 , no sólo permite el crecimiento in vitro de los brotes de las especies de orquídea en estudio, sino que puede reducir los costos de producción de las plantas propagadas mediante esta técnica.

Efecto de los tratamientos en el número de hojas de Laelia anceps Lindl. y Epidendrum sp. a los 45, 90 y 135 días después del inicio del experimento. 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.
who evaluated vermiculite as a substitute for agar in Citrus volkameriana Ten & Pasq., Citrumelo swingle