Silicon in the Embriogenic Potential of Callus in vitro of Passiflora edulis

Adding silicon to the culture medium may contribute, to improve many growth parameters including embryogenesis and organogenesis. The objective of this study was to evaluate the embryogenic potential of passion fruit in vitro (Passiflora edulis f. Flavicarpa O. Deg), submitted to different concentrations of silicon. Nodal segments of plants were inoculated at five concentrations of silicic acid added to the culture medium, 0.0; 0.5; 1.0; 1.5 and 2.0 g L. After 60 days, the visual characteristics (% contamination, callus, bud, root and oxidation), cytochemical test and microanalysis of X-rays evaluated. Concentrations of 0.5; 1.0 and 2.0 g L of silicon did not differ in relation to callus formation. By the cytochemical test, all treatments presented embryogenic cells indicated by the reaction to acetic Carmine and, little reaction to the Evans blue associated with cell death. By the microanalysis of X-rays, the nutrients sulfur and phosphorus, presented significant absorption responses at the concentration of 1.5 g L of silicon in comparison to the other concentrations. The silicon added to the culture medium has a response in the formation of cells with embryogenic potential in nodal segments of passion fruit. The concentration of 2.0 g L of silicon acid (H4SiO4) has a high embryogenic potential in passionflower cells, Passiflora edulis f. Flavicarpa O. Deg.


Introduction
The passion fruit belongs to the genus Passiflora, which is considered the largest and most economically important of the Passifloraceae family (Pérez et al., 2007;Braglia et al., 2010). Is an allogeneic plant, has autoincompatibility of the sporophytic type (Ferreira et al., 2010). The propagation of this plant is preferably realized of form sexually, through seed or asexually, by the use of cutting, grafting and tissue culture in vitro (Sousa et al., 2010).
Diseases are among the main factors responsible for low yields and losses in commercial passion fruit orchards in Brazil (Salazar et al., 2016). The tissue culture through the micropropagation is an important method to multiply quickly the high production and/or disease genótipos resistant superiors in the physical space and reduced time, very often offering the only alternatives for programs of plant breeding (Zerbini et al., 2008;. However, the regeneration was seeing embryogenesis somatic be shown to be promising and it has the potential to devote it self in the field of the micropropagation, which is the way morphogenic principal of the regeneration in the type Passiflora (Zerbini et al., 2008;Pinto et al., 2010), in the improvement of result of passion fruit.
The inclusion of silicon (Si) in the culture medium has provided callus growth, induction of organogenesis and somatic embryogenesis and especially the improvement of morphological, anatomical and physiological characteristics of the seedlings produced. In addition, the use of silicon has increased the tolerance of plants to different biotic and abiotic stresses, such as salinity, low temperatures, metal toxicity, etc. (Sivanesan & Park, 2014).
During induction of somatic embryogenesis, the tissues involved undergo biochemical and morphological changes, which are strongly related to alterations in the pattern of gene expression (Sahni et al., 2013). The characterization and identification of embryogenic cells, through cytochemical analyzes, are of paramount importance for the study of the process of in vitro regeneration of plants. Chemical aspects, such as the mobilization of reserves during somatic embryogenesis, are also relevant because they enable a better understanding, optimization and validation of this morphogenic pathway (Otoni et al., 2013).
Considering the benefits that silicon promotes to plants and due to the lack of studies in passion fruit, especially with regard to in vitro silicon, the objective of the work was to adjust the concentration of silicon in the induction of the embryogenic potential of plant cells in Passiflora edulis f. Flavicarpa O. Deg.

Material and Methods
The work was developed in the Vegetable Tissue Culture Laboratory of the Department of Agriculture (DAG) of the Federal University of Lavras (UFLA). As a source of explants, Passiflora edulis f. Flavicarpa O. Deg. (Yellow passion fruit) established in a greenhouse.

Disinfestation of the Material
Prior to removal of the explants, the plants were preventively sprayed with antibiotic Ampicillin (1,000 mg L -1 ) three times a week for four weeks. In the second week, the systemic fungicide Cerconil WP ® (Thiophanate-methyl and chlorothalonil) was added to the sprays at the concentration of 2 g of p.c. L -1 water. Next, young nodal segments (second lateral or axillary bud) of the young plants were collected using scissors, then taken to the UFLA Plant Tissue Culture Laboratory.

In vitro Establishment
The nodal segments of passion fruit plants were submitted to asepsis in 70% alcohol for one minute and in 50% sodium hypochlorite for 20 min, after which four washes were performed with sterilized distilled water. They were inoculated in MS½ modified culture medium (Murashige & Skoog, 1962), added with 30 g L -1 sucrose, 0.5 mg L -1 BAP, 1.5 mg L -1 ANA and solidified with 1.8 g L -1 of Phytagel™. Five concentrations of silicic acid (H 4 SiO 4 ) were added to the culture medium, 0.0; 0.5; 1.0; 1.5 and 2.0 g L -1 . The pH of the culture medium was adjusted to 5.8 and then autoclaved at 121 °C and 1.2 atm for 20 min.
Subsequently, in a laminar flow chamber, nodal segments containing a 1 cm long were inoculated in a test tube containing 15 mL of the culture medium with the respective treatments. The tubes were maintained in a growth room, with photoperiod of 16 h, temperature of 25±2 °C, with luminous intensity of 52.5 W m -2 s -1 .

Visual Evaluations
Were performed at 60 days after inoculation, observing the percentage of contamination, callus formation, number of shoots, number of roots and percentage of oxidation.

Cytochemical Analyzes
Cytochemical analyzes are performed to confirm if the tissue comes from cells with meristematic characteristics. Were performed on a fraction of the regenerated material. For this, 100 mg of three-tube callus fractions were collected for each treatment, which were submitted to the Evans blue/acetamide staining according to the methodology described by Steiner et al. (2005). At the end, slides were assembled and photomicrographs were taken with a digital camera coupled to an Olympus BX 60 light microscope at the Anatomy Laboratory of the Department of Biology of UFLA. The image analysis was performed by the RGB technique (Red, Green, Blue) and by means of the histogram tool of Adobe Photoshop ® CS6 software version 10.0. For this analysis the experimental design was the completely randomized, with five treatments, consisting of three slides/treatment, each slide analyzed in 8 different regions.

X-Ray Microanalysis
Microscopic analysis by x-ray microanalysis maps and quantifies the elements present in the tissues. Was performed at the Laboratory of Electron Microscopy in the Department of Phytopathology of UFLA. Three tube beads per treatment were kept in desiccator with silica gel for 3 days, after which they were assembled in stubs, and metallized with CEC 020 Baltec carbon. Afterwards, it was analyzed the LEO-EVO 40 XVP Zeiss Scanning Electron Microscope and quantified the chemical composition by Spectroscopy of X-ray Dispersive Energy in the Quantax XFlash 5010 Bruker apparatus, following the protocol of Alves and Perina (2012).

Experimental Design and Statistical Analysis
The experimental design was a completely randomized design, with five treatments and 25 replications, totaling 125 test tubes, each tube containing one explant. The data were transformed to and submitted to analysis of variance. When possible, dose dependent regressions of silicic acid were used to demonstrate the results. The regression models were selected using the coefficient of determination (R²) with a probability of 5%, according to the F test. Variables that did not fit the regression models were compared by the Scott-Knott test (p ≤ 0.05). The statistical analysis was performed through Program R, version 3.0.3 (Ferreira et al., 2011).
Where, Yi = value observed at the ith dose of AS (silicic acid); μ = general mean; ASi = fixed effect associated with the i-th AS dose, i = 0.0, ... 2.0; Γi = random effect associated with the ith dose of AS (silicic acid), assuming γi~N (0, Iσ2γ), where, Iσ2γ is the identity matrix of variance and covariance, since it assumes independence of the residues.

Results and Discussion
The decontamination methodology of the segments was efficient for in vitro establishment, presenting less than 2% of contamination. No endophytic contamination was present in any of the treatments until the final analysis.
For the oxidation, no type of preventive control was performed, even though with a low oxidation rate, the control and the concentration of 2.0 g L -1 of silicon acid (H 4 SiO 4 ), which did not differ statistically between the other concentrations (Table 1).  Note. * Means followed by the same lowercase letters in the column do not differ significantly from each other, by the Scott-Knott test, at 5%.
The culture medium used in this study contained fixed concentrations of cytokinin and auxin, which influenced the activation of cell division of explants in different treatments. According to Navroski et al. (2012), the use of BAP and ANA in the culture medium promotes the increase in the formation of callus in Segurelha (Satureja hortensis L.).
In the control observed the presence of calluses in few repetitions, which did not allow enough material for the analysis in this work ( Table 1). Concentrations of 0.5; 1.0 and 2.0 g L -1 of silicon did not differ in relation to callus formation in nodal segments of passion fruit (Table 1) The formation of shoots and roots in the different concentrations of silicon did not present statistical difference between them (Table 1) By the cytochemical test, all the treatments presented embryogenic cells indicated by the reaction to acetic Carmine (reddish coloration), and little reaction to the Evans blue associated with cell death (bluish coloration) (Figure 1). Steiner et al. (2005)

Conclus
The silicon nodal segm The conce Passiflora