Senescence or gradual loss of leaves formed in vitro environment

During the acclimatization process, the plants move from a low-sweeping environment to one that demands increased water demand. However, in plants grown in vitro the root it has little connection to the stem and are very fragile, which according ^{Fila et al. (1998)}; ^{Hazarika (2006)} causes that when establishing them in the substrates are broken, therefore it prevents the efficient transport of water and nutrients. The low assimilation of water and nutrients, coupled with the morphology of leaves that are very thin, little development of the cuticle and little or no accumulation of waxes, influence their behavior during their acclimatization. ^{Pospíšilová et al. (1999)}, mention that the sheets forming the in vitro plant condition, are unable to continue their development when are transferred to ex vitro environment. ^{Fuentes et al. (2005)} mention that the leaves produced in vitro storing structures act as nutrients, supporting the growth of new leaves developed ex vitro. ^{Quesada and Valpuesta (2004)} indicate that senescence and subsequent leaf loss is not only a degenerative process but also a recycling process in which nutrients are transported from aging cells to young leaves.

The plants Agave americana var. oaxacensis studied showed changes in their morphology when exposed to higher solar radiation; this occurred after 45 days of acclimatization. One of the first signs that acquired plants in response to the onset of the loss of their leaves formed in vitro, was the change from green to purple (Figure 1). This coloration was first presented in the lower leaves of older age, manifesting in the distal part and later expanding throughout the leaf blade. The purple color of the leaves was due to the presence of anthocyanins. The accumulation of anthocyanins in the leaves may be due to multiple stressors, stands out the high irradiation and nutrient deficiency (^{Peng et al., 2008}). In senescent leaves where nutrient mobilization to other organs increases, anthocyanin accumulation would minimize the potential for photo-oxidative damage (^{Hoct et al., 2003}).

Figure 1 Changes in the morphology of plants A. Americana var. oaxacensis, for the loss and formation of new leaves during their acclimatization in greenhouse.  

Among the most important changes that have to adapt plants produced in vitro, is the gradual increase in light intensity. Once micropropagated plants are transferred to ex vitro environment, these are highly susceptible to photoinhibition (^{Osorio et al., 2010}).

The loss of leaves formed in vitro condition was gradual, with the lower older than died first. Generally plants replaced all its leaves formed in vitro in a span of 240 days. This refractivity as mentioned by ^{Pyung et al. (2007)}, who mention that the degeneration of cells in the tissue is slow, because the plant will ensure the translocation of the macromolecules to the sites of demand such as formation of new leaves, fruits or growth buds. It could also be due to the type of species, since the agaves have low growth rates, in addition their leaves are hard, fleshy, fibrous etc., which slows the degeneration of their tissues, as well as the capacity of renewal of the same.

In this study, the time it took the plants to lose their leaves was different. The plants that settled on the substrate that had a portion of peat combined with two of sand and that also received different fertilization, lost their leaves faster in a time of 180 days, compared to the plants that settled in the substrate with two to three servings of peat combined with sand and also received fertilizer, which lost their leaves formed in vitro condition, in a time of 240 days. At 90 days, the plants established in the substrate with a portion of peat combined with two of sand and that also received different fertilization, had lost 50% of their total leaves, whereas the plants established in the substrates with two and three portions of peat and also received different fertilization, had lost 30.2 and 33.15% of its leaves formed in ex vitro condition, respectively (Table 1).

Table 1 Effect of substrate and fertilization on leaf senescence of A. americana during acclimatization greenhouse.  

T= turba; A= arena; ***= valor de F altamente significativo (p≤ 0.01); ns= valor de F no significativo (p≥ 0.05). Medias con letras iguales no son estadísticamente diferentes (Tukey, 0.05).

Furthermore, the doubling time of acclimatization i.e. to the 180 days was observed the loss of 100% of the sheets formed in vitro plants were established in the substrate with a portion of combined peat with two sand and which also received different fertilization. But for established plants in substrates with two and three parts peat and also received different fertilization, had lost an average of 0.46 and 68.8% of its leaves formed in vitro respectively.

According to the analysis of variance, the substrate factor had significant effects (p≤ 0.01) on the decrease or loss of leaves formed in vitro during acclimatization at 90, 120, 150, 180 and 210 days. Instead the fertilization factor did not influence the loss of leaves formed in vitro during the days tested, or interaction substrate-fertilization.

The mean comparison analyzes showed that there was a significant difference in the number of leaves lost by the plants established in the different treatments. At 90 days of acclimatization, the plants that had lost the largest average amount of 3.2 leaves were those that settled on the substrates with a portion of peat combined with two of sand and that received different fertilization, while the plants that were established on substrates with two and three portions of peat, had lost an average of 1.8 leaves. This behavior of leaf loss in plants according to treatments was similar to the other days evaluated during acclimatization. The substrates in which the plants lost faster leaves formed in vitro, were compounds by a portion of peat combined with two sand and also received different fertilization (Table 1).

The plants were established on substrates with two and three servings of combined peat with two sand and we applied different fertilization, were favored as the total of their leaves that had formed during culture in vitro replaced by leaves new, 60 days later compared to plants established on substrates with a portion of peat combined with two of sand.

This indicates that senescence and subsequent loss of leaves formed in vitro was slower in plants that were established in a substrate with a higher proportion of peat combined with sand. The permanence of the sheets formed in vitro longer favored the generation of new leaves in the plant.

Formation of new leaves in ex vitro environment

During acclimatization, micropropagated plants generate new leaves with characteristics similar to plants growing in a natural environment.In the case of plants Agave americana var. oaxacensis, itwas observed that the first leaves that were generatedex vitro, showed a light coat of wax, which gave them a grayish hue; also began to acquire hardness. However, these leaves had not yet acquired the characteristics of a plant growing under natural environmental conditions. In this research, it was observed that from the second or third new sheet that formed ex vitro, showed a layer denser waxes, fleshy, rigid, hard and presence of spines on the edges of the leaves (Figure 2), which differs from the first leaves that the plants generated.

Figure 2 Morphology of new A. americana plants generated during acclimatization in greenhouse leaves 

This indicates that the change is gradual and as the days of acclimatization advance, the plant presents changes in its leaves in response to the adaptation process. The agaves have morphological, anatomical and physiological characteristics such as the development of succulence or thickening in leaves, thick cuticle and deposit of different layers of waxes on the epidermal surface and CAM metabolism, which have allowed them to grow and develop in unfavorable environments (^{García-Mendoza, 2007}).

The growth and development of the leaves will depend on the type of substrate and availability of nutrients. Based on the analysis of variance, the substrate factor had significant effects (p≤ 0.01) on the number of new leaves formed at 90, 120, 150, 180, 210 and 240 days of acclimatization. While the fertilization factor showed statistical significance in its effect on the mentioned characteristic, from the day 180 to the 240 days of acclimatization. In contrast, the substrate-fertilization interaction showed significant effects on the number of new leaves formed at 150, 180 and 240 days of acclimatization (Table 2).

Table 2 Effect of substrate and fertilization on the formation of new leaves of A american during acclimatization greenhouse. 

T= turba; A= arena; ***= valor de F altamente significativo (p≤ 0.01); ns= valor de F no significativo (p≥ 0.05). Medias con letras iguales no son estadísticamente diferentes (Tukey, 0.05).

The mean comparison analyzes showed that there was a significant difference in the number of new leaves generated in the plants established in the different treatments. At 90 days of acclimatization the plants established in the different treatments had generated the same amount of leaves on average (2.18). However, at 180 days the difference between plants was more noticeable. Those that were established in the substrate with two portions of peat combined with two of sand and that also received 100% fertilization had formed the largest number of leaves (4.3), while the smaller number of leaves (3), the plants established in the substrates with a portion of peat combined with two of sand that did not receive fertilization.

At 240 days of acclimatization, the plants established in the substrates with two and three portions of peat combined with a potion of sand and that also received different fertilization, had formed 32% more leaves with respect to the plants established in the substrates with a portion of peat combined with two of sand and which received different fertilization.

In general, it was observed that the plants generated more leaves when the nutrient solution with 100% concentration of nutrients was applied to them at the substrate level. This indicates that the substrate does not cover nutritional needs and that when a nutrient solution is added, the plants improve their condition. This is consistent with ^{Enriquez-del Valle et al. (2012)}, who mentions that at the end of the stage of acclimatization of A. angustifolia, the level of plant growth and accumulation of N, P and K increased in direct relation to the concentration of nutrients in the nutrient solution, to the optimum at 66 and 100%.