Initial growth during acclimatization

When he finished the stage of invitro cultivation of plants of Agave americana these, were on average 4.6 leaves, 10.1 cm in the longest leaf, 8.5 cm in diameter crown, and contained 20.1 g cm^-2 of chlorophyll sheet foliar. After five weeks the plants showed statistically significant differences produced by the main factors. Both the substrate and the fertilization and the interaction of both influenced significantly (p≤ 0.01) on increasing the height of the longest leaf (AHML), the number of sheets (NH) and the diameter of the crown (DC). While chlorophyll content (CL) showed only significant (p≤ 0.01) dose fertilization (Table 1).

Table 1 Mean squares and significance of increased variables at the end of the first five weeks of acclimatization of micropropagated plants Agave americana var. Oaxacensis L. 

This may be due to the natural process of acclimatization, since plants are obliged to respond in a stressful environment, even if they are under some protection where nutrient availability has more restrictions than in the in vitro environment, which responds better dose fertilization, using minerals are provided and diluted in chemical forms usable for them to be diluted in irrigation water. Given that has little ability to change their morphology, the only way to increase their photosynthetic capacity it is increasing its light gathering area, so the chlorophyll content remains unchanged. About ^{Salazar et al. (2009)} mention that achieved adapting Agave cocui Trelease and plants had normal morphology after one week.

As for the effect of the substrate it was observed that the plants established in the sand-peat substrate without fertilization showed greater increases in height of the longest leafand number of leaves (Table 2). The foregoing suggests that the content of organic matter in the soil promotes plant nutrition as it stabilizes the pH, increased water retention, drainage, cation exchange capacity and availability of nutrients, which is reflected in growing dedicated to photosynthesis organs to ensure their survival, on the other hand it is important to consider that possibly net photosynthesis is zero or may even have negative values due to the lack of control experienced by vitro plants not being prepared to face conditions outside lab. However, nutrients available are not sufficient to trigger further accumulation of reserve substances and dry matter accumulation.

Table 2 Means of treatments increases for the first five weeks of acclimatization of Agave americana var. Oaxacensis L. 

It is noteworthy that the highest value for the diameter of the crown, was observed when plants were established in a sand substrate:- vermiculite and also received irrigation with a 25% solution of Steiner, the accumulation of dry matter can due to the vermiculite it contains potassium (K), magnesium (Mg) and calcium (Ca), although such plants relatively decreased increased height and number of leaves as the highest value for the last variable mentioned was recorded in the vermiculite substrate sand- plus 25% of nutrient solution. These results coincide with ^{Enriquez et al. (2005)}, who mentioned that micropropagated plants of Dendranthema grandiglora not require the application of some mineral solution to show growth, draws attention to the results above seem to be influenced by the combination of sand and peat in some proportion in the substrate. This behavior seems logical considering that the tested plants are subject to some degree of stress caused by the sudden change in environmental conditions, because although apparently they change gradually to establish plants outside the lab, suffer a total lack of control, because they do not have mechanisms that allow them to regulate basic functions such as gas exchange, which also affects their water status.

It was observed that the number of sheets, up to the largest sheet, crown diameter and chlorophyll content were continuously increased during the first five weeks later the plants began to lose the older leaves during the period between 35 and 70 days of acclimatization. These results agree with those reported by ^{Abreu et al. (2007)}, who mentioned that fourcroydes Agave plants micropropagated had a positive response in terms of growth and development after 30 days of acclimatization. Moreover ^{Monja-Mio et al.(2015)} mention that plants Agave angustifolia cultured in vitro, experience severe changes in morphology and leaf surface during acclimatization, the more noticeable are the development of stomatal, deposition epiticulares waxes, formation protuberances and calcium oxide crystals in the leaf epidermis, which achieved after three months. Which was partially coincides with the results of this study because despite the effort made by the plants to acclimatize, showed growth in AHM and NH, and the dry matter accumulation by increasing the DC during the first five weeks. Moreover, some authors like ^{Pospisilova et al. (1999)}; ^{Obledo et al. (2004)}; ^{Abreu et al. (2007)}; ^{Enriquez (2008)} and ^{Monja-Mio et al. (2015)} agree that plants lose leaves during the process of acclimatization due to stress they suffer when they are transferred from the in vitro conditions to greenhouse conditions or field, as these plants do not have the anatomical and physiological features operate efficiently, preventing showing significant increases in growth and development, so that their efforts are channeled to the minimum processes to survival during this very critical stage. Regarding the acclimatization stage, ^{Morales et al. (2009)}, indicate that is a period of time during which the plants are more susceptible to environmental stress. ^{Preece and Sutter (1991)}, agree that should be adapted to have a continuous growth and development, to new environmental conditions such as lower relative humidity, with more light and septic substrates.

In many species of plants, leaves formed in vitro are unable to be further developed under greenhouse conditions and are replaced by new leaves (^{Pospisilova et al., 1999}). ^{Enriquez (2008)} mentions that plants Agave angustifolia replaced the leaves that had formed in the laboratory by new leaves in the acclimatization stage. This phenomenon occurs in plants generated in vitro, they are explained by ^{Taiz and Zeiger (2002)}, as a specific response that plants have to get rid of non-essential parts; that is, taking into account that the leaves were formed in vitro under controlled conditions where it is not necessary to develop any adaptation strategy, so they are not able to respond to changes involving their acclimatization, so it is functionally preferable translocate useful components and form new leaves with morphological and functional characteristics that replace the previous ones.

Such plants require a process of adaptation to the new environment which, during the period of time that are more susceptible to environmental stress to have a continuous growth and development face (^{Morales et al., 2009}). ^{Abreu et al. (2007)} mentions that plants grown in vitro, alter their morphology, anatomy and physiology as a result of the environment in vitro, so during the acclimation present inability to control various processes, among which water loss, so their rate of photosynthesis is reduced, so that a specific response of plants is to get rid of non-essential parts and recover the nutrients present in these parts, of which melts to essential organs for them.

Growth at the end of the adaptation stage

Plants with age 210 to 245 days showed that the factor fertilization had no significant effect on the growth of the height of the longest leaf (AHML), however influenced the increase in crown diameter, number of leaves and chlorophyll content (p≥ 0.05). Plants that were established in the sand-peat substrate and fertigation to 50% concentration of nutrients of the Steiner solution showed greater increases in height of the longest leaf and number of leaves that plants were established in other substrates and were applied to other doses of fertilizers in regard to the number of sheets, they had higher concentrations increased with 75% solution. Furthermore, the plants established vermiculite substrate and irrigated with water only showed greater increases in crown diameter. These results agree with those reported by ^{Enriquez et al. (2005)}, which mentioned that during the acclimation of plants Dendranthema grandiglora micropropagated and settled on a substrate with high content of organic matter not required to be applied mineral solution to show growth, while plants fertigation 50% had further increases of chlorophyll in the leaves (Table 3). This means that the more nutrients has the blade, will have more concentration, but without excess nutrients from the substrate as mentioned ^{Enriquez et al. (2005)}.

Table 3 Increase in the characteristics of plants during the 210-245 days of acclimatization levels according main factors. 

Established plants in peat substrate showed greater increases in height of the longest and crown diameter blade, this is because the peat has characteristics that are close to an ideal substrate: aeration capacity, 10 to 20%; water retention, 55 to 70%; granulometry 2.25 mm 0.25; bulk density <0.4 g cm^-3; total pore water readily available 48% and 93% space (^{Urresatarazu, 2000}, ^{Iskander, 2002}). While established plants in vermiculite substrate, showed that the number of irrigated leaves with 75% Steiner solution and the content of irrigated chlorophyll with 50% had greater increases, therefore neither the substrate had a high concentration of nutrients nor solution dose was very high. Which can be attributed to the characteristics of vermiculite, and that has a high cation exchange capacity which facilitates mobility and absorption of ions in chemical forms to keep available.

In the period of 210-245 days after transplantation, the plants no longer showed senescence of leaves as occurred during the period between 40 to 60 days, because at this age already had new leaves that had developed in the ex vitro environment (^{Martinez et al., 2005}). Therefore, also it increased crown diameter, the height of the largest sheet and therefore chlorophyll content was constant.

Gonzalez et al. (2007), they worked with fourcroydes Agave and report that the 42 days of acclimatization, the leaves were already considered functional with normal stomata an waxes on its cover. These results agree with those found in this study, as Agave americana acclimation lasted 49 days, therefore, in the following days, the plants were already considered functional. All results indicate that the tested treatments increased the height of the largest leaf, leaf number, crown diameter and chlorophyll content, not being the same treatment for each increase more. According to the results of this study and consideration of other authors, it is clear that the plant needs are different, depending on the environment in which are located, so a change of environment will result, adaptations different scale depending on the speed and magnitude of change. Which is observed in any species when its cultivation in vitro is established, and unlike when to acclimatize again to ex vitro conditions, however evolutionary processes give them that ability through a "memory" long-term, the species preserves and allows you to develop best suited to survive according to the environment in which you find features, and this process can reverse it and adapt it as often as necessary.

In addition, the treatments were tested (Table 4) showed that in the last five weeks that were 210 to 245 days after transplantation to ex vitro, the treatment showed the largest increase taking into account all the variables was the combination of sand-vermiculita-peat with 50% fertilization.

Table 4 Average of increments by treating the last five weeks of acclimatization of Agave americana var. Oaxacensis L. 

Comparing both periods, it was noted that in the first five weeks established plants in sand substrate had higher increase irrigated with water unlike plants were irrigated with a dose; while in the last five weeks (between 210 and 245 days), plants that were established in the peat substrate irrigated with 50% mineral solution had a higher increase. This may be because the plants during the first few weeks did not require as supplemented irrigation and water only is more than enough to survive the average ex vitro, since at that time perform their duties at the expense of reserves acquired under in vitro conditions. It is important to note that once achieved adaptation and capacity to develop photosynthetic processes, exposure to greater light as in the nursery is favorable to increase growth rates and plant growth factor, therefore the stage after acclimatization plants and needed a further supplemented irrigation, because they no longer had reservations throughout the plant.