Optimization of the Protocol for the in Vitro Cultivation of Piper aduncum L

Piper aduncum L. (Piperaceae) has great potential for economic exploration because of the proven use of its essential oil in the agriculture and in the human health. A technique that improves its propagation, as the tissue culture, becomes necessary. Some parameters must be determined for the successful cultivation in vitro. Thus, this study aimed to determine the salts concentration of MS medium, temperature, luminosity and light quality for in vitro culture of this species. The following treatments were conducted: 1/4MS, 1/2MS, MS and 2MS; 20 ̊C, 25 ̊C, 30 ̊C and 35 ̊C; monochromatic blue, red and white lights and the combination of red and blue, using light emitting diodes (LEDs); luminosities of 17, 37, 48 and 73 μmol∙m−2·s−1 obtained with cool white fluorescent lamp. After 35 days, the treatments were analyzed. To determine the salts concentration of MS, the variables were evaluated: number of shoots, leaves and roots, length and width of leaves, length and dry mass of root and shoots and percentage of death by necrosis. To the plantlets under different temperatures, length and dry mass of shoot and root, number of shoots, number, length and width of leaves and survival and rooting percentages were measured. The plantlets that were maintained under different luminosities were evaluated for length of shoot and root, dry mass of leaf and root. To evaluate the growth under the LEDs, the length and dry mass of shoot and root, number of shoots and roots, percentage of rooting and sprouting were assessed. The medium 1/4MS and the medium 1/2MS showed better responses for number and length of root, leaf width and shoot length. The temperature 25 ̊C provided the highest number of leaves, length of shoot and root, root dry mass and rooting percentage. The luminosity 73 μmol∙m−2·s−1 provided higher values in length of root and dry mass of leaf and root. The red LED provided plantlets with greater growth. Thus, for the in vitro cultivation of Piper aduncum, 1/4MS, environment temperature of 25 ̊C, light intensity of 73 μmol·m−2·s−1 and environments with red light to obtain high quality plantlets for propagation of this species are recommended.


Introduction
Piper aduncum (Piperaceae) is a shrub that is native to tropical regions of the Americas and was introduced in Asia during the 19th century [1].It produces an essential oil that has been used and reported as medicinal for the treatment of many diseases [2]- [4] and has been highlighted, especially, by showing an insecticidal, larvicidal, leishmanicidal, molluscicide, antibacterial and antifungal activities [4]- [9].
Most of the medicinal plant species that are used by the population are obtained from natural habitats.The ability to sustain production of raw vegetable materials that are of high quality under controlled conditions is important because of the need to produce standardized herbal medicines [10].Additionally, there is disordered extraction of species in natural environments which promotes the extinction of these species.The tissue culture is a technique that allows the production of secondary metabolites in vitro, ensuring alternative way for the sustainable exploitation of species, especially from threatened ecosystems.Among the perspectives there is the obtainment of a germoplasm that is competitive and adapted to various growing methods, and the possibility of choosing new species that will serve as a source of biologically active compounds and improve the production of phytochemicals [11].
However, tissue culture requires the analysis of some parameters, such as explants, aseptic nutrient medium, light conditions and temperature [12].Regarding the luminosity, the light quality has also been a concern for the in vitro culture.Recently, light emitting diodes (LEDs) have drawn considerable interest as an alternative light source for in vitro propagation.The advantages of LEDs are their large photo fluxes of red photons, their light weight [13] and their high electrical energy conversion efficiency [14].Pure R-LEDs are a potential light source for growing plants in space-flight systems because of their safety, small mass and volume, wavelength specificity and longer life [15].Despite these attractive features of the LEDs system, studies on growth and morphogenesis of different species under different LEDs systems are very limited.
Given the importance of these factors on in vitro culture, this work aimed to study the effects of salts concentrations in the culture medium MS, the effects of temperature, luminosity and light quality (with the use of LEDs), in the in vitro growth of Piper aduncum L. in order to optimize the protocol for in vitro cultivation of this species.

Plant Material
For the experiments, the Piper aduncum L. seeds were established at Laboratory of Tissue Culture and Medicinal Plants of the Federal University of Lavras (UFLA).For asepsis, the seeds were immersed in a solution of sodium hypochlorite, 60% (v/v) for 15 minutes under constant stirring.After the seeds were washed 5 times with distilled and autoclaved water within laminar flow.After the germination, the seedlings were subcultured and inoculated into test tubes containing MS medium [16] with 30 g•L −1 of sucrose, 0.6% agar and pH adjusted to 5.7 ± 0.1.
Nodal segments containing two buds were inoculated and conducted to four conditions of in vitro culture: different salts concentrations of MS medium, temperatures, luminosities and wavelengths of LEDs.In all the experiments, were used tests tubes containing culture medium MS, supplemented with 30 g•L −1 of sucrose, solidified with 0.6% agar and pH adjusted to 5.7 ± 0.1.The plantlets were maintained under photoperiod of 16 hours of light in all the experiments.The luminosity was 16 µmol•m −2 •s −1 for the temperature experiment and 25 µmol•m −2 •s −1 for the salts concentration one.A temperature of 26˚C ± 1˚C was used for the salts concentrations, luminosity and LEDs experiments.The segments were inoculated in a horizontal position, and were carried out on a completely randomized design and evaluated after 35 days.

Salts Concentrations of Medium MS
To determine the effect of salts concentrations of medium MS, the segments were inoculated in the following treatments: 1/4MS, 1/2MS, MS and 2MS, with 5 replications and 4 tubes per replicate.The number of shoots, leaves and roots, length and width of leaves, length and dry mass of shoot and root and percentage of death by necrosis, were evaluated.

Temperatures
In the experiment aiming to analyzing the effect of temperature, the explants were taken to four growth chambers, with white fluorescent light and temperatures of 20˚C, 25˚C, 30˚C and 35˚C ± 1˚C with 5 replicates and 5 tubes for each replication.The length and dry mass of shoot and root, number of shoots, number, length and width of leaves and survival and rooting percentages were evaluated.

Growth under Different Luminosities
The explants were subjected to the following treatments: 17, 37, 48 and 73 µmol•m −2 •s −1 , obtained with cool white fluorescent lamp.The experiment was performed with 4 replicates and 4 tubes for each replicate.The plantlets were evaluated for growth: length of shoot and root, dry mass of leaf and root.

Growth under Different Light Emitting Diodes
The explants were cultured under different spectra of LEDs, totaling 6 treatments: blue, red, white, 28% blue + 72% red, 72% blue + 28% red and 50% blue + 50% red (Figure 1) with 4 replications and 4 tubes per replication.The length and dry mass of shoot and root, the number of sprouting and root and percentages of rooting and shoots were assessed.

Statistical Analysis
The data were subjected to ANOVA by F (p < 0.05), using the Sisvar ® software [17].The growth variables were analyzed by regression (luminosity) and by the Scott-Knott test (MS salt concentrations, temperature and growth under LEDs).

Results and Discussion
Lower concentrations of salts (1/4MS and 1/2MS) favored the in vitro growth of the species (Figure 2).It was observed that the high salts concentration in the culture medium is detrimental to P. aduncum, because it decreased rooting and shoot growth (Table 1).No influence of the concentration of salts was observed in the number of shoots, and the death for necrotic occurred only in cultured explants in 2MS, which showed 25% of necrosis.
Plants grown in vitro under high salt concentrations may reduce the synthesis of carbohydrates altering growth.Furthermore, the high salt concentration may promote osmotic effect in the culture medium in vitro which hinders the absorption of water by plantlets.Thus, the negative effects caused by higher concentrations of MS medium are probably due to a higher amount of salt than what naturally the species receive, becoming inadequate to the morphogenic process [18].
Sousa [19] found that during the multiplication of explants from P. aduncum plants cultivated in vitro, concentrations of salts of the MS (1/2, 3/4 and 100%) can directly influence the process of micropropagation of the species by microcuttings relative to height and number of buds.P. aduncum showed better results, both in the number of buds and in height when grown in environments with concentrations of 1/2 and 3/4 of MS salts.As in the present work, the species showed better development in salt concentrations lower than the full MS.Ghimire [20] studying Solanum aculeatissimum, medicinal plant, obtained the results for the number of shoots, in descending order: 1/3MS, 1/2MS, MS, 1/4MS, respectively.And root length, also in descending order, with the cultivation of the species in: MS, 1/2MS, 1/3MS, and 1/4MS, respectively showing that the ideal salt concentration of MS depends on the species under study.
Regarding the temperature, the explants cultured under 35˚C did not survive.The temperature of 25˚C favored the growth of the species, with higher means for number of leaves, length of shoot and root, root dry mass and rooting percentage (Table 2 and Figure 3).Silva [21] evaluated P. aduncum under temperatures of 10˚C, 20˚C and 25˚C, and it was observed that after six months of storage in vitro occurred an influence of temperature on the survival, shoot length and number of buds per shoot.The highest temperatures tested (20˚C and 25˚C) promoted an increase in variables.Plants exposed to high temperatures suffered injury, exhibiting reduction or stop of growth and the degree of injury can vary with plant species [22].This effect is due to the suppression of elongation growth rate of cells, because of an irreversible inhibition exerted by high temperature due to protein damage [23].In addition, somatic embryos of Eleutherococcus senticosus exposed at 12˚C, 16˚C, 24˚C and 30˚C showed reduction in growth at high temperatures, because it resulted in a reduction of the activity of antioxidant enzymes that protect    cells against damage from the stress of high temperatures [24].Changes in luminosity affected the growth variables analyzed (Figure 4 and Figure 5).The increase of the luminosity resulted in an increase of the length and dry mass of root, and dry mass of leaf (Figure 4).In contrast, the shoot length reduced with the increasing of the luminosity.The highest production of dry mass obtained under higher luminosity indicates that low luminosity may be limiting for the in vitro growth of Piper aduncum plants.In moderate luminosity, plants generally bear longer internodes, and are less resistant than those grown under intense light [25].This may reflect the reduction in shoots length in high luminosity.Light is the ultimate substrate for photosynthetic energy conversion [26].Thus, plant dry mass decreases with decreasing luminosity [27].In addition, the acclimatization to high irradiances usually results in an increased photosynthetic activity [28].This improvement is not only associated with more carbon available for growth but also with the fact of that light promotes photomorphogenic responses [29].
parameters varied in response to the different LEDs (Table 3 and Figure 6).The root length, number of roots, number of shoots, dry mass of shoots and root and percentage of survival were higher under the red light, white and 28% blue + 72% red (Table 3).It can be stated that the red light applied alone or in combination favored the development of Piper aduncum cultivated in vitro.Growth induction by red light has been reported in several species such as strawberry (Fragaria x ananassa) and grapes (Vitis ficifolia) [30] [31].
Hence, the present study revealed that a longer wavelength is required for the root elongation of monkey pepper.Light may influence root elongation through photomorphogenic action, i.e., root elongations may be controlled by phytochrome [32].

Conclusions
It is concluded that the 1/4MS medium is the most suitable for the in vitro cultivation of Piper aduncum L. The ideal temperature for growing in vitro is 25˚C.Luminosity also affected the in vitro growth of P. aduncum.The species has a maximum length of shoots when grown under 17 µmol•m −2 •s −1 , and greater root length, dry mass of leaves and roots when grown at higher luminosities 73 µmol•m −2 •s −1 .
It appears that red LEDs might be effective for increasing length, number and dry mass of shoots and root and percentages of shooting and rooting.Hence, the red light is recommended for cultivation in vitro of Piper aduncum.
of shoots; NL: number of leaves; SL: shoot length; LL: leaf length; WL: width of leaves; RL: root length; DMS: dry mass of shoot; DMR: dry mass of roots; S: survival; R: rooting.Means followed by the same letter do not differ statistically from each other, by the Scott-Knott (p < 0.05).
of shoots; NR: number of roots; SL: shoot length; RL: root length; DMS: dry mass of shoot; DMR: dry mass of roots; S: survival; R: rooting.Means followed by the same letter do not differ statistically from each other, by the Scott-Knott (p < 0.05).

Table 1 .
Growth of P. aduncum in different concentrations of MS medium.Lavras, 2014.: number of shoots; RL: root length, NR: number of roots; LL: leaf length; LW: leaf width; LN: leaf number; SL: shoot length; N: necrosis; DMS: dry mass of shoot; DMR: dry mass of root.Means followed by the same letter do not differ statistically from each other, by the Scott-Knott (p < 0.05). NS