Introduction

Within dioecious plants of commercial importance are those from the genre Dasylirion, which recently has been located within the Asparagaceae family (^{APG III, 2009}) and whose distribution includes the region from the Chihuahuan desert and Arid Region of North America (^{Martorell and Ezcurra, 2002}). The importance of this genus lies in some species such as: D. duranguense, D. wheeleri and D. cedrosanum which are used for the production of an alcoholic drink known as “Sotol” (^{NOM 159-SCFI-2004}; ^{De La Garza et al., 2008}). The craftsmanship of this drink and scarce information about the biology, biochemistry and reproduction characteristics of this genre has contributed to poor management of natural populations; therefore, it is of great importance to provide basic information to improve the handling of the species.

For Dasylirion there are no reports on the biochemical or physiological characteristics of plants, i.e., it lacks basic information that at some point could acquire practical importance for the implementation and management of nurseries that contribute to the conservation of species of this genus. The evaluation of the hormonal composition is essential because of the role it plays in the plant, such as formation, development and body specification, provision of resistance to climate changes or pathological infections, among others (^{Santner et al., 2009}).

Within the most studied plant hormones are gibberellins and cytokinins. Gibberellins are a large group of molecules belonging to the family of tetracyclic diterpenoid, within gibberellins with increased biological activity are GA1, GA3, GA4 and GA7, which play a role in different processes of plant growth including, seed development, organs elongation, and flowering time control (^{Yamaguchi, 2008}; ^{Santner et al., 2009}). Furthermore, cytokinins are mobile phytohormones that play a critical role in plant growth and development through the regulation of leaf senescence (^{Kim et al., 2006}), apical dominance (^{Tanaka et al., 2006}), root proliferation (^{Werner et al., 2001}), phyllotaxis (^{Giulini et al., 2004}), reproductive competition (^{Ashikari et al., 2005}) and nutritional signaling (^{Takei et al., 2002}). Among the bioactive cytokinins are trans zeatin (Z) and trans zeatin riboside (tZR) (^{Neuberg et al., 2011}).

The determination of gibberellin A4 (GA4) and trans zeatin riboside (tZR) content in different organs from pistillate and staminate plants of D. cedrosanum can contribute to knowledge of the biochemical composition and hormonal physiology of the species, considering the great importance that gibberellins and cytokinins have in the differentiation, development and formation of plant structures.

The evaluation of gibberellin A4 (GA4) and trans riboside zeatin (tZR) levels, took place in the months of April and May 2013 in leaves, crown and inflorescence of adult plants with pistillate and staminate flowers of D. cedrosanum, which were collected in the region from General Cepeda, Coahuila, Mexico at coordinates 25° 18’ 43.5” north latitude, 101° 45’ 26.5” west longitude at an altitude of 1986 ± 15.63 masl. The region has an annual average rainfall of 300-400 mm, an average annual temperature of 18 to 20 °C and a semiarid climate (BSh) according to Köppen’s classification. Leaves and crown and inflorescence sampling was made under a scheme of systematic sampling where selected plants met the characteristic of having an inflorescence that initiated emergence. Sampling of these organs was performed in four pistillate and four staminate plants, same that were sampled repeatedly once a week since the beginning of inflorescence emergence until it completely dry, after five weeks from inflorescence emergence.

To minimize damage to plant, tissue samples were obtained using a punch that collected 2 to 5 grams of fresh tissue from crown and inflorescence, taking care that the sample did not exceed more than a third of the diameter of the inflorescence. As for leaves, with the help of a knife were cut from the base two of the youngest leaves with full development that were near the site of the inflorescence emergence, which were cut into pieces of 2 cm to be stored. Each sample was placed in their respective pre-labeled plastic container and was immediately immersed in liquid nitrogen, where it remained until being stored in a deep freezer (SANYO MDF-U53VA) at -80 °C.

Subsequently water from each of the samples was removed through a lyophilizer (LABCONCO Model 2.5L) with a vacuum of 0.25 mBar and -40 °C. Finally, the samples were pulverized in a mortar and stored at room temperature in containers containing silica gel and sealed.

For the extraction of GA4 and tZR, 50 mg of each sample were weighed into a microfuge tube and 1 mL of extraction solution (methanol 20% (v/v) was added, diluted in formic acid 0.1% (v/v). The mixture was subjected to vortex (GENIE1 Tocuh Mixer Model SI-0136) for 30 seconds and sonicated (BRANSON Model 1510R-DTH) for 10 min. Afterwards the samples were centrifuged at 12 000 rpm for 10 min at 4 °C and transferred to a freezer at -20 °C to be left incubating for 12 h. After this time the samples were sonicated again for 10 min, and centrifuged for a second time at 12 000 rpm for 10 min at 4 °C. Finally, the supernatant obtained was filtered through 0.45 µm membrane pore diameter and transferred to vials for its injection into liquid chromatograph.

The identification and quantification of GA4 and tZR was performed on a liquid chromatograph (Varian 920LC), using as mobile phase acetic acid at a concentration of 100 mM (Phase A) and acetonitrile 100% (Phase B). GA4 determination was performed with a C18 column (POLARIS 5 C18-A, 250 mm x 4.5 mm, 5 µm) using phase proportions 50:50 (v/v) (phase A: B) and an isocratic flow 0.8 mL min^-1. To quantify tZR a C18 column (AQUASIL C18, 250 mm x 4.6 mm, 5 µm) was used, with phase ratios 80:20 (v/v) (Phase A: B) and an isocratic flow 0.3 mL min^-1. The injection volume of plant extracts for both quantifications was of 50 µL.

Molecule detection of GA4 was performed at 205 nm, while tZR was detected at 268 nm. The retention time of GA4 was 7.8±0.2 min and of tZR was 15.4±0.25 min. For unit’s conversion of absorbance to ppm, standard curves with standard solutions of GA4 and tZR (SIGMA) were performed. The chromatograms were evaluated through Galaxie Version 1.9.302.530 (VARIAN) software.

For statistical analysis ANOVA was used with repeated measurements for the statistical analysis of data quantification of GA4 and tZR. The least significant difference test (LSD) was used to compare means of the variables that showed significance in the F test from ANOVA with a statistical significance of α≤ 0.05. Statistical packages used INFOSTAT Version 2014 and IBM SPSS version 22.

The test results from LSD (Table 1) showed a statistical difference (p≤ 0.05) only between plant organs of the same sex, highlighting the highest value of GA4 in samples obtained from leaves, while the highest values of tZR were found in the crown of plants of both sexes and in staminate leaves. No significant difference was observed (p≤ 0.05) in concentrations of GA4 and tZR when comparing between the same organ of a pistilate and staminate plant.

LSD test showed a statistical difference (p≤ 0.05) between plants of different sex when analyzing the global average of GA4, denoting the highest concentration of this plant hormone in staminate plants (Table 2). For global values of tZR no differences were observed between sexes. For this reason, the overall differences of GA4 found between pistillate and staminate plants of D. cedrosanum are considered relevant since this can be a starting point for further studies to distinguish plants sex in early growth stages.

The results obtained are different from those reported by ^{Zanewich (1993)}, who found that higher levels of gibberellin A1 (GA1), gibberellin A19 (GA19) and gibberellin A20 (GA20) in outbreaks of Brassica napus with an age of 28 days (values between 10 and 20 ng g^-1 dry weight). A lower concentration of the three gibberellins were found in stems, hypocotyl, and finally, in roots, the latter with the lowest levels of GA1, GA19 and GA20 (values below 2.5 ng g^-1 dry weight). The same author in a sampling at 64 days analyzed other organs, such as inflorescence stalk, stem, leaves, roots and flowers; finding the highest levels in flowers (values between 3 and 6 ng g^-1 dry weight), followed by leaves, stems and inflorescence stalks, while the lowest contents were quantified in roots (values below 1 ng g^-1 dry weight). Gibberellin values found in the different organs of Dasylirion cedrosanum are higher than those reported for Brassica napus, since we can find values from 20 000 ng g^-1 dry weight (0.02 mg g^-1 dw in inflorescences of pistillate plants) to 150 000 ng g^-1 dry weight (0.15 mg g^-1 dry weight leaves from staminate plants).

Furthermore, ^{Battal and Tileklioğlu, (2001)} evaluated cytokinins content in Zea mays L. plants under different growth mediums, in which they found that the highest concentration of zeatin riboside was in stems (5.72 µg g^-1 fresh weight to 7.67 µg g^-1 fresh weight), followed by leaves (3.30 µg g^-1 fresh weight to 4.98 µg g^-1 fresh weight) and finally, female flowers (1.55 µg g^-1 fresh weight to 2.2 µg g^-1 fresh weight), behavior that was similar in all treatments. The values of trans zeatin riboside quantified in crown 0.09 mg g^-1 dry weight and 0.1 mg g^-1 dry weight (equivalent to 9 µg g^-1 dry weight and 100 µg g^-1 dry weight), leaves 0.1 mg g^-1 dry weight (equivalent to 100 µg g^-1 dry weight and 150 µg g^-1 dry weight) and pistillate and staminate inflorescences 60 mg g^-1 dry weight (equivalent to 60 µg g^-1 dry weight, in both cases) of D. cedrosanum were higher than those reported for Zea mays L. in similar organs. It is important to note that although the concentrations of GA4 and tZR in different organs of D. cedrosanum are greater than those found in similar organs from other species, there is a similar trend among species in the accumulation of plant hormones in the different organs.

Conclusions

The highest concentration of GA4 and tZR in Dasylirion cedrosanum was found in leaves and crown, respectively, without observing any difference between staminate and pistillate plants. Global averages of these hormones showed that only GA4 levels were different between sexes. The differences in GA4 concentrations found in the same organ from plants of different sex constitute a starting point for further studies focused to detect plants sex of Dasylirion cedrosanum in early growth stages. Likewise, hormonal differences between the various organs analyzed are basic inputs to determine the sampling organ when conducting a hormonal monitoring of some of the two hormonal species studied in this work (GA4 and tZR).