Effect of salicylic acid elicitor on antioxidant potential and chemical composition of in vitro raised plants of Berberis asiatica Roxb. ex DC.

Abstract The present study for the first time investigated the effects of Salicylic acid (SA) (50, 100 and 200 mg/L) on in vitro growth and antioxidant capacity of Berberis asiatica. SA significantly enhanced in vitro shoot multiplication at 100 mg/L concentration. The result showed highest significant increase in antioxidant activity by 50 mg/L SA elicited plantlets in terms of DPPH (2,2-Di-phenyl-1-picryl-hydrazyl) free radical scavenging activity (IC50 32.42 ± 0.51), Ferric Reducing Antioxidant Activity (292.73 ± 2.09 mg AAE/g dw) and Metal Chelating Activity (IC50 13.18 ± 0.52). The Gas Chromatography–Mass Spectrometry (GC–MS) profiling revealed presence of 46, 38, 34 and 29 phytochemicals in control, SA1, SA2 and SA3 methanolic extracts respectively. Among the identified compounds, eight compounds, 4H-Pyran-4-one,2,3-dihydro-3,5-dihydroxy-6-methyl; 1,3,4,5-tetrahydroxy-cyclohexanecarboxylic acid; 3,7,11,15-Tetramethyl-2-hexadecen-1-ol (and Phytol); hexadecanoic acid, methyl ester; 9,12-Octadecadienoic acid (Z,Z)-,methyl ester; di-n-octyl phthalate; stigmast-5-en-3-ol,(3.beta.)- or ß- sitosterol; and squalene were previously known for antioxidant potential. Graphical Abstract


Introduction
Berberis asiatica Roxb. ex DC. (family Berberidaceae) is widely distributed in Western Himalaya of India from 600 to 2700 m asl (Patni et al. 2017). The presence of many known bioactive substances, including alkaloids, phenolics, tannins, flavonoids, anthocyanins, vitamins and minerals is largely responsible for the pharmacological effects of B. asiatica (Andola et al. 2011;Belwal et al. 2017). The species is commonly used by the Uttarakhand villagers (Bisht et al. 2013) and pharmaceutical industries for its beneficial effect as antidiabetic, hepatoprotective, antioxidants etc. (Mokhber-Dezfuli et al. 2014). Seed germination in Berberis species is low in nature (Belwal et al. 2015) and root is extensively used in various systems of medicine due to the presence of Berberine content (Andola et al. 2010) for curing a variety of ailments (Srivastava et al. 2004), causing threat to the species.
Elicitors are substances that cause secondary metabolites to develop, and they have been frequently used to increase secondary metabolite production in in vitro plant cells (Ramakrishna and Ravishankar 2011;Murthy et al. 2014). Salicylic acid (SA) is a phenolic chemical with a lot of promise as a plant elicitor. SA affects the secondary metabolism of plants (Korsangruang et al. 2010;Sivanandhan et al. 2013) and also has the capability to induce the secondary metabolites in in vitro cultures (Naik and Al-Khayri 2016). SA at low concentrations interacts with stress-signaling systems and can cause secondary metabolite synthesis (Ghasemzadeh and Jaafar 2011;Ma et al. 2017).
Limited investigations (Bisht et al. 2021) have been documented for in vitro propagation and antioxidant activity of B. asiatica. To the best of our knowledge there is no report available on elicitation of B. asiatica by using SA. Also, literature is not available for antioxidant potential and chemical composition of SA treated plantlets. Hence, the aim of the present study was to (i) study effect of Gibberellic acid (GA 3 ) on seed germination of B. asiatica, (ii) determine the effect of different concentrations of SA on in vitro cultures of B. asiatica (iii) study antioxidant assay and (iv) determine the chemical composition by using GC-MS of SA treated in vitro raised plantlets.

Results and discussion
2.1. Effect of GA 3 on seed germination of B.asiatica Seeds treated with GA 3 showed a considerable increase in germination rate after inoculation in GA 3 (50 mM) for 6 hours. Germination percentage dropped when seed concentration and presoaking time in GA 3 climbed over 50 mM and 6 h. In Woody plant (WP) medium (Lloyd and McCown 1980) and Murashige and Skoog (MS) medium (Murashige and Skoog 1962), seeds treated with 50 mM GA 3 for 6 h had germination rates of 81.66 ± 6.00 and 86.63 ± 3.33, respectively ( Figure S1). MS medium was therefore chosen as the optimum medium for further research. The results were different from Brijwal et al. (2015) who observed higher germination percentage in WP medium (70.4 ± 2.1) as compare to MS medium (54.3 ± 1.2) in B. aristata. Pandey et al. (2013) also studied in vitro propagation in B. chitria using seeds as explants without presoaking treatment.

Effect of elicitors on in vitro shoot growth
The MS medium supplemented with SA at 100 mg/L concentration resulted in increase of shoot multiplication of B. asiatica explants, compared to the control. Maximum number of shoots per explant (7.11 ± 0.35), shoot length (3.15 ± 0.05) and number of leaves per explant (14.33 ± 0.06) were achieved on treatment of SA (100 mg/L). Similar positive effects of SA on in vitro shoot propagation were found by Miclea et al. (2020) in Lavandula angustifolia. Proliferation was decreased when the SA concentration was raised to 200 mg/L ( Figure S2). More specifically, with the concentration of SA (200 mg/L) shoots developed were thin and short which cannot be considered as healthy shoots. The limited concentration of elicitors had favored the in vitro multiplication but when it exceeded, decrease in shoot propagation was observed. In support of present study, the higher concentrations of SA on inhibition of shoot formation in Coleus aromaticus were reported by Govindaraju and Arulselvi (2018).

Effect of elicitors on in vitro rooting
Shoots treated with Indole-3-butyric acid (IBA) and a-naphthaleneacetic acid (NAA) (5 þ 5 mM) without SA displayed the maximum rooting percentage (88.86 ± 11.13), root number per explants (1.66 ± 0.23) and average root length per explants (4.12 ± 0.55). The percentage of rooting was decreased to 66.63 ± 0.16 with increase in the concentration of IBA and NAA ( Figure S3a). In SA supplemented medium with IBA and NAA at different concentrations, rooting parameters decreased. Pandey et al. (2013) observed that rooting percent, average root length, average length of the longest root were maximum in WPM þ IBA, while maximum average root number in WPM þ NAA in B. chitria. Bisht et al. (2021) in B. asiatica reported highest rooting response (70%) with 2.36 roots per shoot in Indole acetic acid (IAA) treatment (0.05 mM) followed by IBA treatment (0.1 mM).
SA improved shoot length and leaf number, but it failed to stimulate root regeneration at all concentration in the nodal explant ( Figure S3b) of B. asiatica and thus it is not suggested to be used for direct in vitro rooting of this plant. Similarly, Bayraktar et al. (2016) also reported no positive effect of SA in Stevia rebaudiana in vitro rooting. Healthy in vitro plantlets were successfully acclimatised in earthen pots and established in the greenhouse condition with more than 61.5% survival rate.

Antioxidant activity
The IC50 value of DPPH radical scavenging activity of extracts ranged from 32.42 ± 0.51 (SA2) to 58.13 ± 0.48 (SA3). The highest antioxidant activity in DPPH assay was observed in extracts of 50 mg/L SA while the lowest was observed in 200 mg/L SA. Similar results were found in FRAP and MCA assay. The highest mean FRAP (292.73 ± 2.09 mg AAE/g dw) and MCA (IC50 13.18 ± 0.52) assay was observed in SA1 followed by control in both type of assay. The least FRAP (159.74 ± 2.78 mg AAE/g dw) and MCA (IC50 21.85 ± 0.43) assay was observed in SA3 ( Figure S6). This observation clearly suggests the elicitation could potentiate in-build antioxidant mechanisms in plants but in lower concentration of SA. Similar to our findings, Golkar et al. (2019) in Safflower and Ali et al. (2018) in Zingiber officinale also reported higher antioxidant activity in SA (50 mg/L) treated extracts as compare to control. Dey et al. (2020) also observed that elicitor treated plant samples of Rauvolfia serpentina showed marginally higher DPPH assay than the untreated ones.

Conclusions
This study is the first report on efficient method for micropropagation of B. asiatica described using elicitor (SA) in combination with growth hormones. SA in proliferation media enhanced in vitro shoot regeneration. The developed regeneration system in this study could contribute to the commercial production of this economically and medicinally valuable species. Moreover, this system would be of benefit to in vitro breeding purposes of B. asiatica to provide sufficient plant materials for further pharmaceutical, physiological, and biochemical investigations. In the experiments, treatment of B. asiatica with SA showed positive effects in improving the antioxidant capacity of the plantlets when compared with control (untreated plantlets). GC-MS profiling of the extracts showed the presence of antioxidant compounds in B. asiatica. Treatment of SA at 50 mg/L also enhanced the antioxidant compound in in vitro plantlets.

Disclosure statement
No potential conflict of interest was reported by the authors.

Funding
The author(s) reported there is no funding associated with the work featured in this article.