Eucomis autumnalis (Mill.) Chitt. subspecies autumnalis is a popular African plant that is susceptible to population decline because the bulbs are widely utilized for diverse medicinal purposes. As a result, approaches to ensure the sustainability of the plants are essential. In the current study, the influence of smoke-water (SW) and karrikinolide (KAR1 isolated from SW extract) on the phytochemicals and antioxidant activity of in vitro and greenhouse-acclimatized Eucomis autumnalis subspecies autumnalis were evaluated. Leaf explants were cultured on Murashige and Skoog (MS) media supplemented with SW (1:500, 1:1000 and 1:1500 v/v dilutions) or KAR1 (10-7, 10-8 and 10-9 M) and grown for ten weeks. In vitro regenerants were subsequently acclimatized in the greenhouse for four months. Bioactive phytochemicals in different treatments were analyzed using ultra-high performance liquid chromatography (UHPLC-MS/MS), while antioxidant potential was evaluated using two chemical tests namely: DPPH and the β-carotene model. Smoke-water and KAR1 generally influenced the quantity and types of phytochemicals in in vitro regenerants and acclimatized plants. In addition to eucomic acid, 15 phenolic acids and flavonoids were quantified; however, some were specific to either the in vitro regenerants or greenhouse-acclimatized plants. The majority of the phenolic acids and flavonoids were generally higher in in vitro regenerants than in acclimatized plants. Evidence from the chemical tests indicated an increase in antioxidant activity of SW and KAR1-treated regenerants and acclimatized plants. Overall, these findings unravel the value of SW and KAR1 as potential elicitors for bioactive phytochemicals with therapeutic activity in plants facilitated via in vitro culture systems. In addition, it affords an efficient means to ensure the sustainability of the investigated plant. Nevertheless, further studies focusing on the use of other types of antioxidant test systems (including in vivo model) and the carry-over effect of the application of SW and KAR1 for a longer duration will be pertinent. In addition, the safety of the resultant plant extracts and their pharmacological efficacy in clinical relevance systems is required.

Laboratory of Growth Regulators Faculty of Science Palacký University and Institute of Experimental Botany AS CR Šlechtitelů 11 CZ 783 71 Olomouc Czech Republic

Laboratory of Growth Regulators Faculty of Science Palacký University and Institute of Experimental Botany AS CR Šlechtitelů 11 CZ 783 71 Olomouc Czech Republic Department of Chemical Biology and Genetics Centre of the Region Haná for Biotechnological and Agricultural Research Faculty of Science Palacký University Šlechtitelů 27 CZ 783 71 Olomouc Czech Republic

Research Centre for Plant Growth and Development School of Life Sciences University of KwaZulu Natal Pietermaritzburg Private Bag X01 Scottsville 3209 South Africa

Research Centre for Plant Growth and Development School of Life Sciences University of KwaZulu Natal Pietermaritzburg Private Bag X01 Scottsville 3209 South Africa Department of Botany and Zoology Stellenbosch University Stellenbosch Private Bag X1 Matieland 7602 South Africa

Research Centre for Plant Growth and Development School of Life Sciences University of KwaZulu Natal Pietermaritzburg Private Bag X01 Scottsville 3209 South Africa Indigenous Knowledge Systems Centre Faculty of Natural and Agricultural Sciences North West University Private Bag X2046 Mmabatho 2735 South Africa

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