Vegetative material

C. atlantica plants of 50 cm height were provided by the Nursery El Solitario from Cuautla, Morelos, Mexico. These plants were kept in quarantine in the nursery from CEPROBI-IPN in Yautepec, State of Morelos, Mexico. The plants were applied with an antifungal and antibacterial solution every two day for 15 days. The solution contained Agri-Mycin^® 1.5 g L^-1 and Benlate^® 0.8 g L^-1.

Establishment of axenic culture

Disinfestation of C. atlantica was conducted by the methodology reported for Pinus pseudostrobus and Pinus jaliscana (Rebolledo et al., 2011); which consisted on using 80 plant explants formed by segments of axillary buds of 1 cm. The material was washed in soapy water for 15 min; Explants were treated with 50% ethyl alcohol for 30 seconds and three rinses with sterile distilled water; afterwards explants were immersed in reagent grade sodium hypochlorite (Hycel, HY57011019000) 10% v/v for 15 min, with a drop of Tween 20. The explants were rinsed three times with sterile distilled water.

The culture medium used to grow the explants was the basal medium McCown’s Woody Plant WPM (^{Lloyd and McCown, 1981}) supplemented with 2.7 g L^-1 phytagel. The pH of the medium was adjusted to 5.7 and poured in Gerber type flasks with 30 ml of the medium. The material was sterilized in an autoclave at 120 °C and 15 lbs for 20 min. In total 25 bottles, each with 3 explants was planted. The cultures were incubated at a temperature of 25 ± 3 ^oC with a photoperiod of 16 h light: 8 h dark with light intensity of 11.14 µmol m^-2 s^-1. After 5 days of culture, the determination of contamination percentage was performed: considering the relationship contaminated explants on total explants planted.In addition oxidation frequency of explants was documented.

Induction of multiple bud outbreaks

To induce multiple outbreaks, apical buds obtained after the disinfestation process were used. Explants were inoculated in the culture medium WPM supplemented with sucrose 3%, activated carbon 1 g L^-1 and L-cysteine 25 mg L^-1, with phytagel 2.7 g L^-1. The effect of the cytokinin benzyl amino purine (BAP) in concentrations of 3 and 5 mg L^-1 was tested. Three explants were cultured per flask with three replicates per treatment. After 21 days, the morphometric parameters measured were: explant growth and number of outbreaks.

Rooting

Shoots obtained from the multiplication stage were used; explants to be rooted were inoculated in the culture medium WPM supplemented with 2.5 and 5 mg L^-1 of auxin indole butyric acid (IBA). 10 bottles per treatment with 3 explants per bottle were cultured.

Statistical analysis

Statistical processing of the experimental data was performed through an analysis of variance. To establish differences between treatments, Tukey test was applied with a significance level of p< 0.05 using the JMP statistical software 10.0.

Disinfection and in vitro establishment

The disinfestation treatment in explants of apical buds from C. atlantica used in this work consisted on washing with sodium hypochlorite and alcohol, allowed to obtain 83% of explants without pollution, only 17% of explants showed fungal contamination after the 5^th day (Figure 1). In the unpolluted apical buds there was no oxidation and only polluted explants showed oxidation. Figure 1 shows the mother plant of C. atlantica used, as well as the different stages of culture establishment.

Figure 1 Establishment of in vitro culture of Cedrus atlantica: mother plant (a) explants of apical buds; (b) in vitro cultures without pollution; and (c) contaminated cultures by fungus (d). 

The disinfestation method of apical buds of C. atlantica can be considered satisfactory, considering that the tissue did not show damage and contamination rate was 17%. This result is consistent with that reported by ^{Rebolledo et al. (2006)}, who tested the same disinfectant agents and immersion times but in seeds of Pinus jaliscana and Pinus pseudostrobus. For other pinus species also reported the use of other disinfecting agents with which obtained satisfactory results such as mercuric chloride (^{Tamta and Palmi 2004}; ^{Oliveira et al., 2011}) and hydrogen peroxide (^{Arya et al., 2000}; ^{Zacarias et al., 2006}; ^{Robledo et al., 2009}).

In micropropagation of woody species, one factor limiting the development of the technique is the cut or tissue injury, since the oxidation thereof is generated. Therefore, during the establishment of their in vitro cultures an aspect to control is the development of the oxidation process. Choosing a suitable culture medium and the use of antioxidants are two important considerations for the survival of apical bud explants of in vitro cultures. For apical buds of C. atlantica, the culture medium WPM along with activated carbon and cysteine, turned adequate to prevent explant oxidation. This result is consistent with that reported for Cedrus deodara (^{Tamta and Palni, 2004}) where explant oxidation was avoided using the WPM medium in combination with ascorbic acid. It is also consistent with that reported by ^{Rebolledo et al. (2006)}, who also used the WPM medium for Pinus pseudostrobus and Pinus jaliscana, in combination with ascorbic and citric acid. Also in other studies using mediums such as ^{Murashige and Skoog MS (1962)} and ^{Schenk and Hildebrandt SH (1972)}, widely used in vitro plant cultures for non-woody species is reported. For Pinus cubensis is reported the used the MS medium without antioxidants agents (^{Cantillo et al., 2011}) and for Picea chihuahuana the SH medium supplemented with ascorbic and citric acid was used (^{López et al., 2000}).

Multiplying by multiple sprouting of Cedrus atlantica

Table 1 show that the presence of BAP in the culture medium stimulated sprouting in C. atlantica in both concentrations evaluated (3 and 5 mg L^-1). However, no significant differences were observed in the number of outbreaks. It was also observed that BAP stimulate shoot elongation, showing up to 1.53 cm in length, but with the two concentrations of BAP used, no significant difference in the buds size were observed (Figure 2).

Figure 2 Effect of benzyl amino purine (BAP) in multiple sprouting from apical buds of Cedrus atlantica. 

The effect of BAP on multiple sprouting obtained in this work is consistent with the reports of other authors in plants of the pinacea family. ^{López et al. (2000)} reported for Picea chihuhuana, that with the addition of 3.5 mg L^-1 of kinetin seven shoots per explant are produced; ^{Oliveira et al. (2011)} reported that 0.4 mg L^-1 of benzyl adenine (BA) can generate three shoots per explant in vitro cultures of Pinus taeda.

However, the number of outbreaks obtained from C. atlantica in this work is below those reported for Pinus maximartinezzi, where it was reported that the addition of BA at a concentration of 16 mg L^-1 produced 29 outbreaks per explant ^{Robledo et al. (2009)}; while BA at a concentration of 1.1 mg L^-1 also produces from 7 to 50 outbreaks per explant of Cedrus deodara (^{Tamta and Palni, 2004}). It is evident the importance of cytokinins in the metabolism of pinacea to induce the multiple sprouting process.

Rooting

In this study the concentrations of indole butyric acid (IBA) were used for C. atlantica were not optimal, because it did not formed root, only the presence of callus was observed in explants cultured (Figure 3). This result is consistent with that reported in the literature, which shows that the induction of in vitro rooting are low compared to ex vitro treatments. In addition, ^{Hartmann et al. (2002)} consider that the rooting stage often represents the critical and difficult phase in micropropagation protocols of most woody species. In this regard Paz et al. (2009) reported that in P. maximartinezii, only 3% succeeded in inducing root formation in shoots with IBA concentration of 3 mg L^-1. An alternative to improve the rooting process of C. atlantica seedlings could be the use of IBA in combination with NAA. This considering the results reported for Pinus cubensis (^{Oliveira et al., 2011}) who report that the combination of 1.8 mg L^-1 naphthaleneacetic acid (NAA) and 2.03 mg L^-1 IBA promotes the formation of 47% of roots, without callus formation.

Figure 3 Formation of callus explants of Cedrus atlantica in presence of indole butyric acid.