Phytoregulators and Explant Size in the in vitro Culture of Malva sylvestris

In vitro propagation of plants makes it possible to accelerate the process of plant multiplication, the study of secondary metabolite production and the cultivation of biotrophic fungi. The objective of this work was to study the combination of indoleacetic acid (IAA) and benzylaminopurine (BAP) and explant sizes in in vitro multiplication of M. sylvestris. Five concentrations of BAP (0, 0.5, 1.0, 1.5 and 2.0 mg L) and two of IAA (0 and 0.5 mg L) were used in explants of 4 to 9 mm and of 14 to 23 mm. Contaminated explants, oxidation, establishment, relative growth rate (RGR), sprouting, rooting and callus formation were evaluated. There was no interaction effect between BAP and IAA concentrations. At 28 days, explants were established at 32.76% and callus formation was 62.5% for explants associated with 0.0 mg L of IAA. There was 32.14% establishment and 63.79% callus formation at 0.5 mg L of IAA. Bacterial contamination at 28 days was 60.53%, twice as much the amount found at 14 days, suggesting that the explants presented endogenous contamination. It was found that explant size influences the subsequent meristematic development. The use of smaller explants (4 to 9 mm) allowed greater formation of calli and larger explants (14 to 23 mm) allowed greater formation of shoots. In conclusion, larger explants are preferable for production of M. sylvestris in vitro by organogenesis while smaller ones are preferable for embryogenesis.

The production system recommended for medicinal plants is based on good cultivation practices and genotypic identity that can meet market demands and provide safe access to products processed as phytotherapeutics (Embrapa, 2015).
Micropropagation is one of the options to qualify the production of seedlings and maintain their genotypic identity associated with the respective phytochemical descriptors (Rout, Samantaray, & Dasa, 2000).According to Kintzios (2002), explants of branches and petioles of M. sylvestris are responsive to in vitro growth in Murashige and Skoog (1962) (MS) medium, whereas leaves did not allow formation of viable propagules in their various treatments.
Previous research on in vitro micropropagation of Malva sylvestris, using benzylominopurine (BAP) and naphthaleneacetic acid (NAA), showed higher fresh mass, plant height and number of leaves at the concentrations of 2.0 mg L -1 of BAP and 0.5 mg L -1 of NAA but without rooting, however (Filter, Freitas, & Périco, 2014).These two growth regulators had been tested for M. sylvestris calli, and the best results were found in equimolar combinations of 9 μM, whereas higher concentrations negatively affected callogenesis (Kintzios, Katsouri, Peppes, & Koulocheri, 1998).In studies with Althea rosea (Malvaceae), Tyub, Kamili & Bhat (2016) found higher numbers of shoots after adding BAP and NAA, while rooting percentage was higher after use of IAA.In embryogenesis studies and in vitro establishment of M. sylvestris, Kintzios et al. (1998) found that there was production of callus and metabolites, including substances other than those found in in vivo tissues.
Vegetative propagation may also be useful in the phytosanitary management of M. sylvestris, because Puccinia malvacearum may survive in seeds, and infection may occur at the beginning of the budding of this fungus, which is the major cause of mallow leaves (Gavériaux, 2012).Kuvalekar and Gandhe (2010) found infection by Uromyces hobsoni, a fungus that causes rust, after in vitro establishment of Jasminum officinale var.grandiflorum.The occurrence of rust on M. sylvestris may compromise the quality of the plant and derived products, as well as of purity tests.Thus, leaves may present at most one (1) blister-like pustule of P. malvacearum teliospore per cm 2 to be considered to have good quality (Brazil, 2000).On the other hand, in vitro culture of M. sylvestris may be an important technique for cultivation of P. malvacearum, a biotrophic fungus which is dependent on living tissues for survival (Agrios, 2005).In addition to contributing to the phytosanitary aspect of seedlings, micropropagation can increase uniformity and large-scale production, thus preserving the standard that increases medicinal interest and use of the plant (Grattapaglia & Machado, 1998).
The goal of this work was to study the effect of combinations of plant growth regulators, indoleacetic acid (IAA) and benzylominopurine (BAP), and to verify the influence of plant growth regulators on the potential supply of calluses and buds for studies on Phytopathology, secondary metabolites and vegetative propagation of different sizes of explants associated with IAA in the introduction of in vitro explants of M. sylvestris.

Producing Matrices for Propagation and General Study Conditions
Malva sylvestris plants were produced by planting seeds in 600 ml expanded polystyrene (EPS) pots with Agrinobre TMX substrate and keeping them in a greenhouse at a variable temperature for three months.The seeds were selected for planting, excluding those with signs of P. malvacearum.
The experiments of introduction and multiplication of mallow were carried out at the Plant Tissue Culture Laboratory, EPAGRI (Experimental Station of EPAGRI (Agricultural Research and Extension Service Agency of Santa Catarina State, Brazil).The experiments were set up in a growth chamber where the flasks were then exposed to a 16-hour photoperiod.
The MS medium (Murashige & Skoog, 1962) with all its salts and vitamins was used, and pH was adjusted to 5.8±0.05before addition of the solidifier (6 g L -1 Merck agar).This medium was supplemented with 30 g L -1 of sucrose and plant growth regulators prior to autoclaving, as described in the experiment on combinations of plant growth regulators and different explants and IAA concentrations, in addition to the addition of 0.5 mg L -1 activated carbon.The medium was poured into test tubes (25 × 150 mm).
Plants with approximately 90 days of age were used as sources of explants in both experiments.To that end, herbaceous branches of mallow, containing between 30 and 60 centimeters, were excised, washed in running water containing neutral detergent and then washed three times in distilled water.Asepsis of this proced followed b principle a remained s kept in a g luminous i 2.2 Exp. 1

Axial buds
The exper following concentrat treatments Figure 1.

Data A
The bacter using the B number of deviance a transforma was verifie used to co and Westfa in the R (R

Results
Average e 0.0 mg L -1 (Table 1 concentrations of BAP and IAA 0.0 mg L -1 and in 63.79% of BAP and 0.5 mg L -1 IAA, respectively.High percentages of callogenesis were also found by Sié et al. (2010) in Hibiscus sabdariffa L., another species of the family Malvaceae, regardless of type of explant in use and concentrations of growth regulators in use.1).The RGR of the explants in the association of BAP with 0.5 mg L -1 of IAA was higher than that with 0.0 mg L -1 of IAA at 14 days, and there was no difference between the treatments performed at 28 days.
Contamination and oxidation were the major impact factors in the establishment of in vitro explants.Bacterial and fungal contamination may be endogenous or exogenous (Ray & Ali, 2017).In the case of M. sylvestris, bacterial contamination at 28 days (Table 2) was twice as much (60.53%) the rate found at 14 days (28.97%), suggesting that the explants had endogenous contamination.
jas.ccsenet.Note.Means followed by the same letter in the row of each variable do not differ significantly from each other (p ≤ 0.05); ns: non-significant; ABA: abscisic acid; IAA: indoleacetic acid; BC: bacterial contamination; FC: fungal contamination; OX: oxidation.
In the experiment with different concentrations of IAA and explant sizes, budding of M. sylvestris was detected in the evaluation at 14 days (Figures 4A and 4C).At 28 days, there were completely formed leaves and different root branches (Figures 4B, 4D, 4E, and 4F).There was (Table 3).There was a significant effect of the interaction between IAA concentrations and explant size for the budding variable.The use of 0.5 mg L -1 of IAA favored an increase in the number of shoots per explant when segments of 14 to 23 mm were used.
Relative growth rate was higher for small explants (4 to 9 mm) than for large (14 to 23 mm) explants in this first evaluation period.
At 28 days, 55.74% of the large explants and 41.07% of the small explants were discarded.In the remaining explants, it was found that 100% and 86.11% of the larger and smaller ones, respectively, were established, with a rooting rate of 12.5% for the largest and 2.78% for the smaller ones (Table 4).Filter, Freitas and Périco (2014) reported success with BAP and NAA (naphthaleneacetic acid) for micropropagation of M. sylvestris, but without rooting of explants.In the present research, there was more callus formation when small explants (58.33%) were used in comparison to large explants (34.35%).
In our study, it was found that larger explants developed more efficiently in vitro than smaller explants.Number of shoots per explant was found to be higher when explants with 14 to 23 mm length were used in comparison to explants of 4 to 9 mm.For in vitro establishment of mallow, an interaction occurred (p ≤ 0.05) between the IAA concentrations and the RGR values.It was found that IAA stimulated faster tissue growth in explants that were 4 to 9 mm in length, but the same effect did not occur in the larger explants.Souza, Schuch, da Silva, Ferri, and Soares (2007) also found that larger explants of Brazilian cherry (Eugenia uniflora, Myrtaceae) presented better in vitro establishment and development results than smaller explants.According to Hartmann, Kester, and Davies (1990), larger explants usually present larger amounts of nutritional reserves, especially in the form of complex or easily assimilated carbohydrates, which can favor the greater or more rapid development in the establishment in vitro.
The present data showed that when aiming to develop the protocol of in vitro production of M. sylvestris by organogenesis, one should choose to introduce larger explants, because they produce a greater number of shoots more quickly.On the other hand, if one's objective is to develop the protocol by embryogenesis, one should use of smaller explants, since they develop 70% more calluses when compared to larger explants.

Conclusion
Micropropagation of M. sylvestris is viable and occurs the development of shoot and root of the plant.
Explant size influences the development of M. sylvestris.The use of explants of 4 to 9 mm allows greater formation of calluses and explants with a size ranging from 14 to 23 mm produce a greater number of shoots.
The addition of 0.5 mg L -1 IAA in the MS medium positively affects the plant growth rate of the small explants, which was similar to that of the large ones, at 28 days.

Figure
Figure 2. A Figure 4

Note.
Means followed by the same letter in the column of each variable do not differ significantly from each other (p ≤ 0.05); for the RGR variable the comparisons refer to the different levels of IAA in each size; ns: non-significant (p ≤ 0.05); TAM: size; IAA: indoleacetic acid; DISC: discard; EST: establishment; ROOT: rooting; BUD: budding; RGR: relative growth rate; 1: transformed variable (lambda = 0.12). ).

Table 1 .
Establishment and growth of Malva sylvestris explants in in vitro culture in Murashige and Skoog environments, with different concentrations of plant growth regulators, 2017 ns Note.Means followed by the same letter in the column of each variable do not differ significantly from each other by deviance analysis (p ≤ 0.05); ns: non-significant; BAP: abscisic acid; IAA: indoleacetic acid; EST: establishment; BUD: budding; RGR: relative growth rate; 1 transformed variable (lambda = 0.3).Number of shoots per explant ranged from 1.73 to 2.73 with different associations of plant growth regulators (Table

Table 2 .
Bacterial and fungal contamination and oxidation of Malva sylvestris explants at 14 and 28 days of in vitro culture in Murashige and Skoog (MS) medium, at different concentrations of plant growth regulators, 2017 org 3. Malva sylves

Table 3 .
In of Malva s