Humic substances and their effects on the micropropagation of banana

The growing concern to promote sustainable agriculture, seeking environmental preservation and, at the same time, increased productivity resulted in the search for technologies for the implementation of agricultural production systems, with ecological approaches and responsible use of natural resources, in the economic, political and social contexts in each region. One of the potential alternatives to achieve these results would be the use of humic substances, considering their direct and indirect mechanisms aimed at increasing agricultural production. Thus, the objective of this work was to evaluate the bioactivity of humic and fulvic acids, as well as to promote improvements in the phytotechnical attributes of banana cv. Prata-Anã micropropagated. The treatments consisted of five doses of humic acid and fulvic acid (2; 5; 10; 20 and 40 mg L) and a control without humic substances. Higher values for the length of the aerial part and the larger root were obtained with the use of 40 mg L fulvic acid. The results prove the ability of fulvic acid to improve the in vitro development of banana explants.


Introduction
The growing concern to promote sustainable agriculture, seeking environmental preservation and at the same time increasing productivity, has resulted in the search for technologies for the implantation of agricultural production systems, with ecological approaches and responsible use of natural resources, in the economic, political and social contexts in each region.
One of the potential alternatives to achieve this objective is the use of humic substances (HS), considering their direct and indirect mechanisms for increasing agricultural production.
The most reported effects of HS on plants are related to a higher growth of the root system, as they stimulate the formation of lateral roots, adventitious roots, cellular elongation and formation of root hairs (Baldotto et al., 2011). In addition, these substances are also able to increase the efficiency of the use of nutrients, decrease the incidence of diseases, helping to reduce the applications of fertilizers and pesticides and stimulating root growth .
Such morphological changes can also induce physiological changes, such as higher efficiency in the absorption of nutrients, leading to a higher production of biomass in reduced nutritional conditions in the culture medium and greater tolerance to nutritional stresses (Pinton et al., 1999).
Thus, the objective of the work was to evaluate the bioactivity of humic acids (HA) and fulvic acids (FA), as well as promoting improvements in the phytotechnical attributes of banana cv. Prata-Anã micropropagated.

Material and Methods
The experiment was conducted at the Tissue Culture Laboratory of the Department of Agriculture (DAG) of the sucrose and 6 g L -1 agar. The pH of the medium was then adjusted to 5.7 ± 0.1 before autoclaving at 121ºC and 1 atm of pressure for 20 min.
The treatments consisted of five doses of humic acid (HA) and fulvic acid (FA) (2; 5; 10; 20 and 40 mg L -1 ), resulting in 10 treatments and a control without humic substances, replicates per treatment and each repetition containing 10 tubes.
Subsequently, the tubes were kept in a growth room with a temperature of 25 ± 1 °C and lighting provided by white LED lamps for 30 days.
After 30 days of the explants inoculation, the following attributes were evaluated: plant height; leaf number; number of shoots; pseudostem diameter (cm); root length (cm); number of roots, total fresh and dry mass of plants and roots (g) and photosynthetic pigments (chlorophyll a, chlorophyll b and carotenoids). For the evaluation of the dry mass of the plant material, the material was dried in an oven at 60ºC for 72 h or until constant mass.

Results and Discussion
Longer shoots (11.5 cm) were obtained using 40 mg L -1 fulvic acid (Figures 1 and 2A). Morphological changes induce physiological changes, such as higher efficiency in the absorption of nutrients that leads to a higher biomass production capacity in limited nutritional conditions of the culture medium, giving a higher tolerance to nutritional stresses (Pinton et al., 1999). Yildirim (2007) and Baldotto et al. (2009)   Best results for the root length (13 cm) were obtained using 40 mg L -1 fulvic acid, but this same dose of humic acid reduced the length of the roots ( Figure 2B). The most reported effects of HS on plants are related to the greater growth of the root system, as they stimulate the formation of lateral roots, adventitious roots, cellular elongation and formation of root hairs (Baldotto et al., 2011). Silva et al. (2001) found that humic acids (HA) showed greater capacity to induce lateral roots in the initial stage of development with the lowest concentration tested (160 mg L -1 ).
According to Façanha et al. (2002), the increase in the root system is related to stimulating the activity of H+-ATPase. This increase in enzymatic activity would cause an increase in the activity of the proton pump and, consequently, acidification of the apoplast. Thus, it would favor the increase of plasticity, promote the elongation of the cell wall, and root growth.
However, both for the fresh mass of root and plant height, as for the dry mass, better results were obtained with the use of 20 mg L -1 fulvic acid. (Figure 2). Source: Authors. Ferrara and Brunetti (2008) found that the application of humic acids to grape leaves caused an increase in chlorophyll a and b and the chlorophyll a and b ratio. However, Sladky (1985) demonstrated that fulvic acids are more stimulating than the humic fraction in the respiration rate and density of chloroplasts of plants, more due to their structural characteristics. Baldotto (2014) tested the effects of humic substances on the acclimatization of pineapple seedlings cv. Vitória and observed that the levels of photosynthetic pigments, including chlorophyll a, chlorophyll b and carotenoids, varied little on average between treatments. However, in the present study, the influence that both acids had on photosynthetic pigments was negative ( Figure 3). As humic or fulvic acid was added, the pigment concentration reduced.

Conclusion
Higher values for the length of the aerial part and the larger root were obtained with the use of 40 mg L -1 fulvic acid.
Thus, the results allow proving the capacity of humic substances to improve the in vitro development of banana explants.