Xanthan gum and sodium hypochlorite in vitro rooting of Gerbera hybrida cv. Essandre Goma xantana e hipoclorito de sódio no enraizamento in vitro de Gerbera hybrida cv. Essandre Goma xantano e hipoclorito de sodio en el enraizamiento in vitro de Gerbera hybrida cv. Essandre

The aim of this study was to check the use of the following gelling agents: the xanthan gum “Adicel®” and the stabilizer “Super Liga Neutra®” to replace agar in the in vitro rooting phase of Gerbera hybrida cv. Essandre. Additionally, the possibility of using chemical sterilization of both culture media and glassware with sodium hypochlorite to replace autoclaving was analyzed. The gelling agents, xanthan gum “Adicel®” and the stabilizer “Super Liga Neutra®” were tested at the following concentrations (g L): 7; 9; 11; 13; 15; 17; 19; and 21. No concentrations of Super Liga Neutra® provided effective solidification. Concentrations of 17 and 21 g of Adicel® provided a good gelling of the culture medium, which was compared to the medium containing agar (control) with two types of sterilization: autoclaving (for 20 and 40 minutes) and chemical sterilization. Autoclaving for 20 minutes did not provide effective elimination of contamination in the culture medium containing xanthan gum; this only occurred when autoclaving time increased to 40 minutes. Plant development in culture media containing 17 and 21 g of xanthan gum, either sterilized by autoclaving for 40 minutes or at a concentration of 17 g xanthan gum using sodium hypochlorite, was statistically the same as the control that contained agar. However, plant development at a concentration of 21g of xanthan gum in a sterilized medium using sodium hypochlorite was lower than that observed in media containing agar.


Introduction
Gerbera (Gerbera jamesonii Adlam), a species that belongs to the Asteraceae family, is marketed as a potted plant as well as a cut flower and stands out economically in agribusiness of national and international floriculture. Gerberas might be propagated by seeds, clump division, and micropropagation (Deng et al., 2018;Tripathi et al., 2021). Although gerbera cultivation in the northeastern region of Brazil is still emerging, it can be an alternative to crop diversification (de Oliveira Silva et al., 2020) In commercial production, micropropagation enables the large-scale production of gerbera seedlings with limited space, and it allows obtaining uniform plants, which are free of pests and diseases (Gautam et al., 2021). However, micropropagated seedlings are more costly than traditional seedlings due to the cost of the reagents and equipment used to produce them. Owing to the economic importance of gerbera and micropropagation in the production of quality seedlings, new alternatives to reduce the costs of in vitro seedling production have been analyzed, aiming to render this propagation method more affordable.
Regarding cost reduction in tissue cultures, the replacement of costly reagents such as agar considered one of the most expensive chemical components used in culture media (Phillips & Garda, 2019;Dhawale et al., 2021) has been addressed in several studies (Conceição et al., 2021). Agar is the most frequently gelling component used as it has characteristics that provide good support for explants, e.g., stability, high clarity, its non-toxic nature, and resistance to culture metabolites (Dhawale et al., 2021). When choosing low-cost agents to replace agar, the use of starch from cassava, barley, corn, potato, rice, and wheat or plant-based gums has shown to be effective in medium gelling, which is associated with good plant development (Espinosa-Leal et al., 2018;Naik et al., 2020).
The xanthan gum "Adicel®" and the stabilizer "Super Liga Neutra®" are two gelling agents that are commonly used in the food, agricultural, pharmaceutical, and oil industries . Xanthan gum is a polysaccharide produced by bacterial species of the genus Xanthomonas (Ferraz et al., 2021) which are capable of forming viscous solutions and hydrosoluble gels with unique rheological properties (de Castro, 2019). Super Liga Neutra® is commonly used to manufacture ice creams, as it provides better emulsification and consistency (Almeida et al., 2016).
The use of autoclaving for the sterilization of culture media, glassware, and utensils used in micropropagation is another factor that helps increase costs, due to electric energy consumption and equipment maTntenance expenses, along with the fact that it requires more work hours (Pais et al., 2016;da Costa Urtiga, 2019: Lu et al., 2021. Several studies have proven the efficacy of sodium hypochlorite (NaClO) as an alternative to replace autoclaving in the micropropagation of several plant species so far, as it is a product with biocidal activity, low cost, and easy to acquire (Pais et al., 2016;Cardoso & Imthurn, 2018;Siekierzyńska & Litwińczuk, 2018).
Therefore, the use of alternative substances other than agar along with the chemical sterilization of culture media and glassware might lead to benefits such as increased speed, higher efficacy, and reduced costs in in vitro plant propagation.
Therefore, the present study aimed to evaluate gelling agents such as xanthan gum "Adicel®" and the stabilizer "Super Liga Neutra®" in culture media as well as chemical sterilization of culture media and glassware using sodium hypochlorite in the in vitro rooting phase of Gerbera hybrida cv. Essandre.

Methodology
The experiments described below were conducted in Laboratório de Biotecnologia of Departamento de Tecnologia e Ciências Sociais of Universidade do Estado da Bahia, DTCS Campus III-UNEB, in Juazeiro-BA.

Preparation of gelling agents
The gelling potential of the xanthan gum Adicel® and the stabilizer Super Liga Neutra® was evaluated to determine which amount would solidify the culture medium. The following concentrations of Adicel® and Super Liga Neutra® were tested: 7g L -1 ; 9g L -1 ; 11g L -1 ; 13g L -1 ; 15g L -1 ; 17g L -1 ; 19g L -1 ; and 21g L -1 . The agents were mixed and homogenized in 1000 mL of deionized, distilled, and autoclaved water. The pH was adjusted to 5.7 ± 1, and subsequently, 20 mL of the solution were distributed in glass flasks with capacity for 250 mL, which were sealed and maintained in a dark room. After 48 hours, evaluations were performed to compare the gelling capacity of Adicel® and Super Liga Neutra® to the medium with 7g L -1 of agar of the commercial brand "ÁgarGel" (control).
Once the concentration and gelling agent were defined as ideal for culture medium solidification, the second stage of the study started, in which three experiments were performed to evaluate plant development in the culture medium with the alternative gelling agent. Different concentrations of alternative gelling agents were tested against agar (control) and two sterilization methods were tested (thermal sterilization by autoclaving and chemical sterilization using sodium hypochlorite). Experiment I consisted of evaluating different concentrations of the gelling agent in media sterilized by autoclaving (121 ºC, 1.1 kg cm -2 of pressure) for 20 minutes. The following treatments were performed: T1: autoclaved control, solidified with 7g L -1 of agar; T2: culture medium solidified with 17g L -1 of xanthan gum; and T3: culture medium solidified with 21g L -1 of xanthan gum. Research, Society andDevelopment, v. 10, n. 4, e4811425143, 2022 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v10i4.25143 Experiment II was like an experiment I, evaluating different concentrations of gelling agents in media sterilized by autoclaving (121 ºC, 1.1 kg cm -2 of pressure); however, it used a period of autoclaving of 40 minutes. The following treatments were performed: T1: autoclaved control, solidified with 7g L -1 of agar; T2: culture medium solidified with 17g L -1 of xanthan gum; and T3: culture medium solidified with 21g L -1 of xanthan gum. Experiment III consisted of evaluating different concentrations of the gelling agents in media sterilized with sodium hypochlorite, following the protocol developed by Teixeira et al. (2006). The following treatments were performed: T1: autoclaved control for 20 minutes, solidified with 7g L -1 of agar. T2: culture medium solidified with 17g L -1 of xanthan gum; and T3: culture medium solidified with 21g L -1 of xanthan gum.
In all experiments mentioned above, the gelling agents were homogenized in a blender with 200 mL of distilled, deionized, and autoclaved water (for experiments I and II) and in distilled and deionized water containing 0.0005% of sodium hypochlorite (for experiment III), and then placed in a microwave until their dissolution.
The medium used for plant rooting in all experiments was comprised of inorganic MS salts (Murashige & Skoog, 1962), White's vitamins (White, 1943), 30g L -1 sucrose, 100 mg L -1 inositol, and had pH adjusted to 5.7 ±1. After preparation, the culture medium was taken to the laminar flow cabinet and 20 mL were poured into glass flasks with a capacity of 250 mL.
In the end, the flasks were sealed and placed in a growth room.

Plant culture
Gerbera hybrida cv. Essandre explants with three leaves derived from the stock culture were inserted in the culture medium 24 hours after preparation, and after that, placed in a growth room at a temperature of 25 + 2 º C, 16 hours of photoperiod, and radiance of 19 mol.m -2 .s -1 for a period of 40 days.

Experimental design
The experiments were conducted in a completely randomized design, with three treatments, eight replicates, and plot consisting of a flask with one plant. All experiment was repeated twice. At the end of the in vitro rooting of all the experiments, percentage of survival, mean number of leaves, mean number of roots, mean plant length, mean plant biomass, mean biomass of aerial part, and mean root mass were evaluated. Count data were transformed into ( ) and mean values were compared using Tukey's test at 5% of error probability, using the statistical program Sisvar (Ferreira, 2008).

Gelling agents used in thermal sterilization
None of the concentrations of the stabilizer "Super Liga Neutra®" evaluated was effective in culture medium gelling agents. In addition, lumps that were difficult to dissolve were formed, which provided the medium with an uneven appearance; therefore, no experiments were performed with this agent. The xanthan gum "Adicel®" was an effective culture medium solidifying agent at concentrations of 17 and 21g L -1 , and both were evaluated in the experiments. The medium was visually transparent and homogeneous.
In the culture medium containing xanthan gum and autoclaved for 20 minutes, there was 100% contamination after 24 hours, thus hampering explant development. When the medium was autoclaved for 40 minutes, the percentage of explant survival after 40 days of in vitro cultivation was 100% in all treatments. Research, Society andDevelopment, v. 10, n. 4, e4811425143, 2022 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v10i4.25143 No significant differences were observed between the gelling agents' agar and xanthan gum at both concentrations, 17 and 21g, regarding all variables analyzed (mean number of leaves, mean number of roots, mean plant length, mean plant biomass, and mean mass of root and aerial parts) (Table 1). Means followed by the same letters in the column did not differ from each other, according to Tukey's tests, at 5% of significance. Source: Authors (2022).
Hegele et al (2021) checked the possibility of total or partial replacement of agar by 17 alternative gelling agents in nutritious media sterilized by autoclaving for the cultivation of two Africa plantain cultivars, which results showed that media with mung bean could be used as a single substitute for expensive gelling agents such as agar, with a cost reduction by more than 80%. Although our results did not show an increase in growth plants variables when subjected to different gelling agents autoclaved for 40 min, on the contrary, studies evidenced that the type of gelling agent used can influence tissues' growth in vitro (Raina, 2017). According to Ozel et al (2018) studies showed that tragacanth gum offered positive effects on growth of Nicotiana tabacum cv. Samsun Canik plants. Some reports suggest that gelling agents used in tissue culture to solidify medium can contain many mineral nutrients that affect plant growth (Al-Mayahi & Ali, 2021). Soares et al (2014) who studied in vitro cultivation of Dendrobium nobile Lindl., reported that replacing 50% of agar for maize starch provided higher plant growth in culture media sterilized by autoclaving. Studies conducted with pure maize starch or cassava starch combined with agar in culture media sterilized by autoclaving showed that they were effective in culture media solidifying agents in the in vitro rooting of 'Gold' pineapples (Oliveira et al., 2015). Vieira (2018) analyzed the hydrogel to replace agar in vitro showing potential to be used in the in vitro cultivation of A. thaliana.
Culture media autoclaved for 40 minutes had a dark coloration, most likely due to sucrose degradation.
Additionally, 40 minutes is a very long period for culture medium and glassware sterilization, and what is more, it generates surplus expenses related to electric energy consumption. Therefore, this is not a feasible option to be adopted in seedling production via tissue culture.

Gelling agents used in chemical sterilization
Aside from analyzing the replacement of reagents for analysis by lower-cost options to reduce final seedling cost, the present study analyzed the efficacy of sterilization using sodium hypochlorite, as it has been reported to be effective in the Research, Society andDevelopment, v. 10, n. 4, e4811425143, 2022 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v10i4.25143 6 chemical sterilization of nutritious media instead of thermal sterilization by autoclaving (Pais et al., 2016;Cardoso & Imthurn, 2018;Conceição et al., 2021). Along with its efficacy, chemical sterilization has also proved to be cost-effective compared to autoclaving, as it represents the easiest and most viable technique for microbial decontamination (RESENDE et al., 2021).
Regarding the evaluation of plants developed in culture media gelled with 17g of xanthan gum and sterilized with sodium hypochlorite, the percentage of explant survival was 100%, and there was no significant difference in all variables analyzed (Table 2) regarding plants cultivated in the medium gelled with 7g of agar and sterilized by autoclaving. Conceição et al (2021) managed the reduction of costs in the micropropagation using chemical sterilization of the culture medium with sodium hypochlorite and the replacement of agar with corn starch with positive results in the plant stages of establishment, multiplication, and rooting of cherry tomato.
The use of NaOCl sterilization of culture medium is also related to incomplete sterilization and/or phytotoxicity for plantlet cultivation reducing its efficiency on micropropagation (Vargas et al., 2016), but our results corroborate with data obtained by other authors, who observe that chemically sterilized tissue culture proved to be effective in the micropropagation with any signals of phytotoxicity (Pais et al., 2016;Cardoso & Imthurn, 2018). However, when 21 g of xanthan gum were added to the medium sterilized with sodium hypochlorite, explant responses regarding most variables analyzed were significantly lower than those of the control treatment. Thus, this concentration had a negative effect on explant development, likely due to an interaction of hypochlorite with increased xanthan gum concentration. Therefore, the treatment gelled with 17g and sterilized with sodium hypochlorite can be used in the in vitro rooting of Gerbera hybrida cv. Essandre, as it provided the same results as the control treatment. Means followed by the same letters in the column did not differ from each other, according to Tukey's tests, at 5% of significance. Source: Authors (2022).

Conclusions
Thermal sterilization by autoclaving for 40 minutes provides effective sterilization of culture media containing xanthan gum but autoclaving for 20 minutes does not perform efficiently in the same conditions. However, chemical sterilization with sodium hypochlorite can be used in culture media for Gerbera hybrida cv. Essandre rooting instead of thermal sterilization. Regarding stabilizers, it is possible to use the xanthan gum "Adicel®" as an alternative culture medium gelling agent instead of agar for Gerbera hybrida cv. Essandre rooting. The stabilizer "Super Liga Neutra®" is not effective as a gelling agent due to low geleificant capabilities. The concentration of 17 grams of xanthan gum per liter of culture medium promotes a satisfactory growth of Gerbera hybrida cv. Essandre during its in vitro rooting phase. These results indicate that further investigations are necessary to identify suitable alternative gelling agents from various sources to reduce high costs in plant cell culture.