Introduction

The indigenous peoples and natives americans have used several species of agave for seven thousand years ago. Agaves are monocots of those who obtain various goods and services, such as substances for medicinal use, fuel, shelter, ornamental, fertilizer, materials for housing construction and development of agricultural implements (^{García-Mendoza, 2007}) species. The Agave potatorum Zucc is a very wild species of deciduous forest which is exploited in unplanned manner, so that their populations have been reduced in the central valleys, Mixteca highlands and southern state of Oaxaca. It is used mainly for the craftsmanship of an alcoholic drink distilled called mezcal (^{Pérez and Casas, 2007}), with a flavor and aroma due to the proportion of volatile aromatic compounds (^{Molina et al., 2007}; ^{Vera et al., 2009}; ^{Vera-Guzmán et al., 2010}).

Three to five months before mature plants from collecting, farmers prevent develop the floral stem and therefore seed production, vital for propagation, so prevents the populations of this species have declined dramatically (^{Enríquez, 2008}). The history not documented since the late 1990 's groups of farmers in the southern highlands of Oaxaca have implemented their propagation by seed and have established plantations. However, the number of plants produced even not compensate the amount of adult plants harvested, so propagation in vitro as complementary to conventional methods.

The micropropagation, is the asexual propagation of plants using the techniques of cell culture, tissue or plant organs, with which you can get mass production in a short time and would help to increase in the medium term availability of raw material of this species. To increase the production of plants of this species, protocols need to be generated when the growing conditions are defined at each stage: selection of plants to propagate, in vitro propagation, acclimatization in the greenhouse, growth in nursery, field crop.

In a process of micropropagation the risk of death of plants produced in vitro occurs it is when they are transferred to the ground and must adapt to new environmental conditions ex vitro (^{Domínguez and Donayre, 2006}). This can be overcome by subjecting plants to a process of acclimatization in greenhouse and nursery during which micropropagated plants develop resistant structures to environmental conditions ex vitro, in which conditions of low relative humidity are present, high light intensity fluctuations temperature constant stress and disease resistance.

The successful acclimatization increases the survival rate of the plants when they are set in nursery and field. The history of micropropagation and acclimatization of various species of agaves are taken: A. tequilana (^{Valenzuela-Sánchez et al., 2006}); A. cocui (^{Salazar et al., 2009}); A. fourcroydes (^{Garriga et al., 2010}); A. americana (^{Enríquez et al., 2013}). In the acclimatization of micropropagated greenhouse plants A. angustifolia ^{Enríquez et al. (2012)} showed that the level of nutrient supply had a positive effect on the size that these plants have reached the end of 70 days of this stage.

Several environmental factors affect the productivity of plants: temperature, water availability, CO₂, solar radiation, nutrient supply. For the proper development of plants needed the contribution of nutrients, such as nitrogen, phosphorus, potassium, sulfur, calcium and magnesium and iron micronutrients, copper, manganese, zinc, molybdenum, chlorine, boron and nickel. When a floor does not provide any of these nutrients in sufficient quantity than required by the plants, it is necessary to apply through a fertilizer containing that nutrient. Among the essential nutrients, nitrogen is the most important, because although the plant has adequate supplies of other minerals, its availability in less than optimal amounts manifests with growth, development and deficient production (^{Martínez et al., 2013}). The plants Agave potatorum field, its supply influenced the size of leaf area, chlorophyll concentration, net photosynthesis which affects productivity (^{Moya, 2002}; ^{Martínez-Ramírez et al., 2012}).

Because of the few studies of in vitro production of Agave potatorum Zucc, you need to research through experimentation, suitable means for propagation, so the objective of this study was to evaluate the development nursery plants Agave potatorum micropropagatedacclimatized, which fertigation for five months with different doses of nutrients.

Materials and methods

This research was conducted in the laboratory of plant tissue culture, greenhouse acclimatization and in the nursery of Technological Institute of Oaxaca Valley, in the town of Nazareth, the municipality of Santa Cruz Xoxocotlan, to 12 km from the city of Oaxaca. It is located at coordinates 96° 44’ west longitude, 17° 02’ north latitude and altitude of 1 530 msnm (^{INEGI, 2010}).

The 300 plants Agave potatorum Zucc were obtained in vitro from the proliferation of adventitious shoots and rooted back thereof. Plants, homogeneous characteristics, withdrew from the in vitro condition, rinsed thoroughly to remove the impregnated agar on the roots and individually transferred to pots of 300 cm³, with a substrate was a 1:1 by volume perlite and peat; and they placed under greenhouse conditions acclimatization.

The plants for acclimatization were 60 days in the greenhouse with high relative humidity (80- 90%) created by an irrigation system running intermittent mist 10 s every 12 min from 10:00 to 15:00 h; diffuse solar radiation 500700 µmol m^-2 s^-1. The frequency of watering is controlled by timers. After the period misting plants fertigation level substrate with the universal solution ^{Steiner (1984)} to 50% concentration of nutrients. From day 61 until day 120, they were in nursery plants, with concrete floor, exposed to solar radiation decreased 40% mesh shade. In the nursery plants no longer received nebulized risks, but if fertigation level substrate. After 120 days 210 homogeneous plants were selected in size and separated into six groups of 35 floors each, so that each group received during the days 121-270, fertigation with a different concentration of nutrients, 10 mL per pot with some aliquot (1, 20, 40, 60, 80 and 100%) of the universal solution ^{Steiner (1984)}. Fertigation was applied for 150 days, from day 121 to day 270. The formulation contains 100% Steiner in mg L^-1: 166.01 N, 31.35 P, 277.38 K, 182.06 Ca, 49.08 Mg, 110.898 S; 1.33 Fe, 0.00065 Cu, 0.201 Mn, 0.0375 Zn, 0.077 B, 0.019 Mo.

On day 270 were randomly ten plants per treatment, in which the number of sheets was counted; leaf area (cm²) was measured by placing the blade assembly in an acetate, of which a copy was obtained; images of the leaves were cut and weighed on an analytical balance with capacity of 160 g and 0.1 mg accuracy and this weight was compared with the corresponding piece of paper of the same quality and known area. The stem diameter (cm) was measured by a vernier; number of primary roots; for the root volume (cm³) it was separated from the rest of the plant, immersed in a known volume of water in a 250 ml graduated cylinder, determining the volume by water displacement. To determine in each plant its leaf dry weight (g), stem dry weight (g) and dry root weight (g) these were placed separately in paper bags, which were placed for 72 h in oven dried at 70 °C for after each weigh on analytical balance with capacity of 160 g and 0.1 mg accuracy.

The experiment was established as a completely randomized design. The experimental unit was a plant in a pot of 300 cm³ and 10 repetitions per treatment. Data were subjected to analysis of variance, Tukey test for comparison of means (α= 0.05) and also correlation analysis were performed.

Results and discussion

By applying a plant micropropagation protocol, interested get lots of plants and normal morphology large proportion of them successfully adapt to greenhouse conditions, nursery and field. Background micropropagation of A. potatorum (^{Domínguez et al., 2008}) that the proliferation of outbreaks was achieved in a culture medium with cytokinins have, and incubated for 30 to 45 days under continuous light at 54 mol m^-2 s^-1. The shoots obtained were transferred to a rooting medium without cytokinins, under similar incubation conditions as above the stage and after 30 days; 87% of the outbreaks had formed adventitious roots. The plants were transferred to pots and greenhouse conditions, and after 45 days 73% of these plants had adapted. In this work it was considered that the adaptation of plants had been successful when they showed resumption of growth. Do not have a history of performance in nursery plants Agave potatorum micropropagated-acclimatized. ^{Abreu et al. (2007)} reported that micro-propagated plants of A. fourcroydes when 45- 60 days had elapsed greenhouse acclimation lost their older leaf formed during culture in vitro, as these leaves were not able to take the anatomical changes needed for adaptation.

In this paper, by subjecting micropropagated-acclimatized plants Agave potatorum to fertigation during the days 121 to 270, analyzes of variance (Table 1) showed that fertilization (1, 20, 40, 60, 80 and 100 %) had highly significant different effect (Pr≤ 0.01) on the development of plants in the number of sheets (NH), dry weight of leaves (PSH), leaf area (AF), stem diameter (DT), weight dry stem (PST), number of primary roots (NRP), root volume (VR) and the dry weight of the roots (PSR).

The plants Agave potatorum Zucc, who underwent the nutrient solution with 100% concentration and plants were subjected to the nutrient solution 1% (Table 2) showed 6.5 and 2.79 g of leaf dry weight, 392.35 and 126.53 cm² leaf area, 2.84 and 2.22 cm diameter of the stem; 7.4 and 4.0 cm³ volume root, 0.92 and 0.6 g dry weight of roots, magnitudes that in each case were significantly different (Tukey, 0.05). In the correlation analysis variable number of leaves it had highly significant positive correlation (Pr≤ 0.01) with the dry weight of the leaves (r= 0.76), leaf area (r= 0.88), stem diameter (r= 0.63), foliar volume (r= 0.84) and dry weight of roots (r= 0.48). ^{Teixeira et al. (2005)}, mentioned in the acclimatization plants micropropagated banana, include high humidity, a temperature ranging from 18 to 28 °C, solar radiation decreased by 50% by using shadows, the substrate with sufficient porosity to adequately root aeration and drainage of excess water. Furthermore, nutrient supply has an effect on the size of the plant because ^{Enríquez et al. (2009)} reported that plants Agave angustifolia Haw. micropropagated-acclimatized for 70 days in a greenhouse in containers with perlite substrate and subsequently with mixtures of sand-compost nursery. It was found that plants receiving nutrient solution had higher growth than fertigation not, even though both types of plants had been in substrates high in organic matter.

As the amount of nutrients increased Steiner nutrient solution plants Agave potatorum Zucc. micropropagatedacclimatized, reaching larger sizes, so that larger plants were as fertigation with the nutrient solution 100%. After five months of the plants were fertigation with different concentrations of nutrients, it was observed that all plants subject to supply various nutritional levels increased in the number of sheets (Figure 1), but the increase was in positive relationship with fertigation doses they received. Plants that fertigation with the nutrient solution to 100% concentration of nutrients developed on average 9.75 sheets, significantly amount (Tukey, 0.05) higher than 2.55, 5.35, 6.65, 7.45 and 8.65 sheets that had plants were fertigation to 1, 20, 40, 60 and 80% concentration of nutrients. Correlation analysis showed that doses of fertigation had a highly significant positive correlation (r= 0.82; Pr≤ 0.01) with the number of sheets that formed the plants. ^{Medrano et al. (2010)}, micropropagated Agave durangensis and assessed acclimation of several groups of plants to set them on various substrates that consisted of soil mixtures, volcanic gravel, and sheep manure. After the acclimatization plants grown in substrate with gravel and organic matter, they achieved larger plants established in only gravel substrate, which does not provide nutrients. ^{Uvalle and Velez (2007)} nutrition experiment of Agave tequilana Wever var. Blue mention that the plants that are supplied with nutrients through fertilization, increased photosynthetic capacity for CO₂ fixation and biomass accumulation. The micropropagated plants Agave cocui the effect of ammonium nitrate was evaluated in three treatments (0, 0.5 and 1 g L^-1 respectively), and determined that the plants fertilized with 0.5 g L^-1 formed more leaves that unfertilized plants (^{Díaz et al., 2011}).

Figure 1 Number of sheets that had plants Agave potatorum Zucc, micropropagated-acclimatized, after five months in the nursery, during which received different concentrations of the nutrient solution Steiner.  

The plants accumulated more dry matter as were increased supply of nutrients, such that the fertigation plants with concentrations of 1, 20, 40, 60, 80 and 100% nutrients, their stems were 0.18, 0.25, 0.33, 0.32 , 0.37 and 0.37 g of dry matter (Figure 2), and total biomass was 3.57, 4.74, 6.37, 6.39, 7.28 and 7.79 g, respectively. Between 76.8 and 83.4% of the total biomass concentrated in the leaves, between 4.7 and 5.2% concentrated in the stem and between 11.8 and 17.9% concentrated in the root. The plants that fertigation 100% concentration of nutrients had 2.05 times the amount of dry matter in the stem compared to plants fertigation 1% concentration of nutrients. Plants subjected to the treatment of 100% nutrient concentration, showed no additional accumulation of dry matter on the stem, or root, but increased their foliar dry matter, and therefore in biomass compared to fertigation plants to 80% concentration of nutrients. From the above it is obtained plants fertigation 1, 20, 40, 60, 80 and 100% concentration of nutrients their relationship aerial part/ root was 4.95, 4.47, 5.56, 5.79, 6.82 and 7.46.

In the graph, values with the same letter are not significantly different (Tukey, 0.05).

Figure 2 Dry weight of the stalk, after five months using different concentrations of the nutrient solution Steiner.  

^{Martínez-Ramírez et al. (2013)} evaluated the growth of plants Agave angustifolia Haw and A. potatorum Zucc propagated by conventional methods, to be achieved in three plots differing in soil types, and the total number of plants in each plot were separated into four groups to submit four doses of fertilization. It was reported that plants fertilized with 30-20-15 kg of N-P-K ha^-1 had 58.3% more dry biomass unfertilized seedlings. The average positive correlation was also found, highly significant (r= 0.59; Pr≤ 0.01) between the dose of fertigation and stem dry weight.

The data showed that the plants formed higher number of roots in response to increased nutrient supply, up to 80%, since plants fertigation with concentrations of 1, 20, 40, 60 and 80% of nutrient solution ^{Steiner (1984)} were on average 10.5, 13.4, 14.6, 15.3 and 17.1 taproots. In the case of plants fertigation 100% of nutrients, they no longer formed significantly more roots compared to plants fertigation 80% (Figure 3). Correlation analysis showed that the number of primary roots that formed the plants had a highly significant positive moderate correlation (r= 0.66; Pr≤ 0.01) with fertigation doses they received. In this study it was determined that the appropriate dose of fertigation for 121-270 days ex vitro growth of plants Agave potatorum Zucc, micropropagation, was 80% of the Steiner nutrient formulation (1984). Handling a fertilization is important because if micropropagated plants Agave cocui Trelease, ^{Díaz et al. (2011)} it is reported that some plants receiving too high doses of nitrogen decreased their amount and maximum root length, due to the toxic effect of fertilizer.

In the graph, values with the same letter are not significantly different (Tukey, 0.05).

Figure 3 Number of primary roots, after five months applying different concentrations of the nutrient solution Steiner. 

Conclusions

The plants Agave potatorum Zucc micropropagatedacclimatized, showed further development and growth in the number of leaves, leaf dry weight, leaf area, stem diameter, stem dry weight, number of primary roots, root volume and dry weight roots, in response to increasing doses of nutrient solution ^{Steiner (1984)} during the five months that fertigation in nursery.