Effect of auxin and cytokinin on phenolic content of Baccharis myriocephala DC . ( Asteraceae ) produced in vitro

1 Department of Pharmaceutical Sciences, Institute of Biological Sciences and Health, Federal Rural University of Rio de Janeiro, Brazil. 2Coordinating Plant Biotechnology, Amazon Biotechnology Center, Brazil. 3 Department of Botany, Bioscience Institute, Federal University of the State of Rio de Janeiro, Brazil. 4 National Institute of Industrial Property, Intellectual Property and Innovation Academy, Brazil.


INTRODUCTION
Baccharis myriocephala DC. (Asteraceae) is a three winged branched shrub; in Brazil, this and other species of the genus are known as carqueja (Simões-Pires et al., 2005;Abad, 2007).The use of medicinal plants such as carqueja generates an economic value that sometimes leads to cultivation of the species (Castro et al., 1999a) to meet the demands of industry and traditional users (Gupta and Birdi, 2017;Park et al., 2017).In natural populations, unregulated extraction contributes to a decline in numbers, consequent loss of genetic variability, and even extinction (Martinelli and Moraes, 2013;Leitão et al., 2014).
The allogamy of Baccharis species is the source of considerable genetic variability (Castro et al., 1999a, b).This causes difficulties in seedling production, generates plants with non-standard agronomic and phytochemical attributes, and consequently reduces the potential of a plant as raw material for medicine preparation.
In vitro culture techniques such as micropropagation have been widely used as an alternative to produce good-quality raw materials from the phenotype of interest (Cardoso and Teixeira da Silva, 2013;Ozarowski and Thiem, 2013;Ahmad et al., 2015).The micropropagation technique enables a plant to be produced when there are difficulties in the natural mechanisms of reproduction, preservation of endangered species, and maintenance of plant germplasm collections, and is the only tissueculture technique that has been effective for large-scale production of plants of economic interest (Cardoso and Teixeira da Silva, 2013;Chen et al., 2016).Phenolic compounds, widespread in the plant kingdom, have beneficial effects on human health, such as reduction of blood glucose levels (Oliveira et al., 2014), antioxidant effects (Pádua et al., 2010) and many others (Hossen et al., 2017;Hu et al., 2017).Biotic and abiotic factors produce changes in the phytochemical profile of plants and may increase the production of chemicals of interest (Sartor et al., 2013;Victório et al., 2015).
The potential use of carqueja as raw material for a plant drug motivated the authors to develop a protocol for in vitro propagation of B. myriocephala, in order to provide an optimized alternative for large-scale production.The effects of different growth regulators on the development of in vitro plants and on the production of total phenolics were investigated.

Plant material
Achenes of B. myriocephala were collected in Santa Rita de Jacutinga, Minas Gerais State, Brazil, and were identified by Gustavo Heiden from the Rio de Janeiro Botanical Garden Research Institute.A voucher specimen of this plant material was deposited in the Herbarium (RB 439.238) at the Institute.

Plant tissue culture
Achenes were pre treated by immersion in 2% commercial bleach solution for 20 min, and then rinsed thoroughly in running tap water inside the laminar air-flow chamber, the achenes' surfaces were sterilized in 70% ethanol for 20 min, followed by 15 min in a 20% commercial bleach solution containing 2 to 3 drops of Tween 20, and rinsed three times with sterile distilled water.The disinfected achenes were inoculated on MS (Murashige and Skoog, 1962) solid medium.Nodal segments were excised from plants emerged from achenes held in vitro for 10 days.Nodal segments were grown in sterile flasks with MS basal medium, hormone-free or de Lima et al. 643 supplemented with kinetin (KIN; 0.01, 0.1, 0.5 and 1.0 mg.L -1 ), benzyladenine (BA; 0.01, 0.1, 0.5 and 1.0 mg.L -1 ) or indoleacetic acid (IAA; 0.01, 0.1, 0.5 and 1.0 mg.L -1 ).The effects of growth regulators on the organogenesis of shoots and roots were recorded after 60 days of culture.In all media, the pH was adjusted to 5.8 ± 0.1 before addition of 7.5 g.L -1 agar-agar.In vitro cultures were maintained in a growth room at 25 ± 2°C under a 16/8 h light/dark photoperiod (Silvania daylight fluorescent lamps, 23 mmol m -2 .s - ).

Acclimatization
Seedlings cultured for 60 days on hormone-free MS basal medium were removed and their roots rinsed in running tap water to completely remove the culture medium, and then kept in bottles with water for 24 h.Then, the seedlings were transferred to plastic containers, 6 cm in diameter, containing soil conditioner enriched with humus.These plants were initially kept in an aquarium covered with transparent plastic, and generously irrigated to create a highhumidity environment.The plastic cover was removed gradually until day 30 when it was completely removed to transfer the plants to outdoor conditions.

Determination of total phenolic content
The total phenolic content was determined using the spectrophotometric method of Folin-Denis reagent, as described by Waterman and Mole (1994).Samples with 200 mg of shoots from the culture medium containing 1.0 mg.L -1 IAA, BA or KIN were lyophilized and milled, and then extracted four times with 5 mL of acetone : water (7:3, v/v) solution.The entire extract volume was combined with distilled water to make it up to 25 mL.A volume of 0.5 mL of Folin-Denis reagent was added to 0.5 mL plant extract plus 3 mL water.One milliliter of Na2CO3 saturated solution was added after 1 h of reaction, and then the absorbance of the extract was read in a spectrophotometer at 760 nm.The reference cuvette contained distilled water only.Samples were prepared in triplicate for each analysis, and the mean absorbance was obtained.The same procedure was repeated for the standard solution of gallic acid to generate a calibration curve.Based on the measured absorbance, the concentration of phenolics was read (µg/mL) from the calibration curve, then the content of phenolics in the extracts was expressed in terms of gallic acid-equivalent (µg GA/mg dry weight).

Statistical analysis
The layout was totally randomized in all experiments.Tests with growth regulators were done in duplicate (n = 30 per treatment), and the rooting percentage was analyzed by the difference in the percentage (p1 and p2), at 5% significance.Total phenolic contents were analyzed in triplicate.The data were analyzed statistically with one-way ANOVA, using GraphPad Prism version 7.02.The significance of differences among mean values was assessed with Tukey's multiple comparison test at p ≤ 0.05 or p ≤ 0.001.The Pearson correlation coefficient was calculated using Microsoft Excel 2010.

Effect of growth regulators on culture
Some easily measured characteristics were observed to establish the best combination of the basic salt medium  with the different growth regulators added to the medium, including the number of buds, nodal segments per bud, shoot length, and rooting produced in each culture medium.The ideal culture medium increased the multiplication rate in addition to producing elongated and rooted plants, to avoid the need for additional steps in the production of in vitro plants.
While increasing the IAA concentration favored organogenesis of shoots and rooting, making the culture more efficient, BA and KIN had the opposite effect because the increased concentration of these hormones reduced the multiplication rate and rooting or produced very short shoots, making it more difficult to isolate nodal segments for subculturing.The culture medium with 1.0 mg.L -1 IAA allowed the production of 23 new nodal segments from a single phytomer inoculated.This result is statistically equivalent to that from 0.5 mg.L -1 IAA which produced about 18 new nodal segments from a single phytomer inoculated.Both 0.5 and 1.0 mg.L -1 IAA provided satisfactory rooting and elongation of plants (Figure 1 and Table 1).
Cytokinins, BA and KIN have been used in the induction of in vitro shoots of Eclipta alba, a member of Asteraceae of recognized medical value.Cytokinins promote shooting but often unsatisfactory shoot elongation or rooting, which for E. alba necessitated an additional step in the tissue culture (Dhaka and Kothari, 2005;Baskaran and Jayabalan, 2005;Ray and Bhattacharya, 2008).
The composition of the basic saline growth medium also affected the regeneration of new shoots in E. alba (Baskaran and Jayabalan, 2005) and Baccharis tridentata (Kajiki and Shepherd, 2006), where the MS medium gave the best results for shoot regeneration in both plants.
Because many plants develop satisfactorily in MS medium, this was chosen for the B. myriocephala in vitro culture (Figure 1).
The results obtained in the tissue culture of B. myriocephala concord with the effects of auxins on rooting in E. alba (Dhaka and Kothari, 2005).Shoot elongation and multiplication combined with good rooting saves time and culture medium, by shortening the micropropagation process.

Acclimatization
A complete micropropagation protocol must include a well-established acclimatization process.Therefore, the transfer to outdoor conditions is still the greatest problem for the micropropagation of many plant species (Chandra et al., 2010).Because B. myriocephala cultured in hormone-free MS basal medium is highly sensitive to dehydration, it was necessary to provide a high-humidity environment for the first stage of acclimatization.With this strategy, the plants were well adapted to their new environment, with 100% survival.The plants did not show any variation in morphology.Previous rooting was not necessary for B. myriocephala, making the protocol more efficient and lower-cost.A prior rooting step was required for Stevia rebaudiana (Singh and Dwivedi, 2014;Ramírez-Mosqueda et al., 2016), E. alba (Singh et al., 2012) and Gerbera jamesonii (Cardoso and Teixeira da Silva, 2013).

Total phenolic content
Environmental factors lead to variations in the phenolic content.The carbon fixed by the plant is managed between the primary and secondary metabolisms, and therefore in situations or seasons of lower growth of the plant body, the production of phenolics is higher (Sartor et al., 2013).
The environmental conditions of the in vitro culture are controlled, stable and predictable, and it is possible to relate the amount of polyphenol produced to the effect of the culture-medium composition on the development of the plant.The polyphenol versus biomass accumulation profile presented corroborates the observation in other studies, that plants with smaller growth and development accumulate more polyphenols (Grzegorczyk-Karolak et al., 2015;Ahmad et al., 2016;Kousalya and Bai, 2016).Nevertheless, the polyphenol accumulation can be influenced by the plant growth regulator utilized, regardless of biomass accumulation, or the two may show a positive relationship (Szopa et al., 2013;Kousalya and Bai, 2016).
Thus, the culture medium MS + 1.0 mg.L -1 IAA induced the best growth and development of the plants but led to the lowest accumulation of polyphenols; only 18.46 µg/mg dry weight (Table 2).The hormone-free MS and the MS + 1.0 mg.L -1 BA culture media produced similar amounts of polyphenols (Table 2), and these plants grew reasonably well, although plants cultured on the BAcontaining medium showed high shoot proliferation, so that in vitro development parameters could not be analyzed (Table 1).Budding with low growth and a lower rate of multiplication were observed in the medium with MS + 1.0 mg.L -1 KIN, but the plants cultured on this medium had the highest polyphenol content; 27.16 µg/mg dry weight (Table 2).The Pearson correlation coefficient between the variables: phenolic accumulation (x) and dry  weight (y) was r = -0.847447,showing a strong negative correlation between variables, since the phenolics accumulate when the dry weight decreases.In addition, 71% of the dry weight variation was due to variation in the accumulation of polyphenols (r² = 0.7182) (Figure 2).

Conclusion
The present results demonstrated that the MS medium is suitable for in vitro culture of B. myriocephala.Supplementation of MS medium with 1.0 mg.L -1 IAA maximized the multiplication rate to 23 new nodal segments from a single phytomer inoculated.In addition, IAA provided satisfactory rooting and elongation of plants, making the micropropagation process faster and more cost-effective.The success of acclimatization depends on an environment with initially high humidity that is gradually lost.Plants cultured on MS + 1.0 mg.L -1 KIN accumulated the highest content of polyphenols.
The efficient in vitro production system developed in this study provided sterile and consistent tissues for large-scale production of B. myriocephala, or for investigation of phytochemicals and germplasm conservation.

Table 1 .
Effects of plant growth regulators on B. myriocephala after 60 days of in vitro culture.

Table 2 .
Total phenolics content of Baccharis myriocephala cultured on MS basal medium, hormone-free or supplemented with 1 mg L IAA, BA or KIN (mean ± SD).