Effect of Biotic Elicitors on the Growth, Antioxidant Activity and Metabolites Accumulation in In Vitro Propagated Shoots of Pueraria tuberosa

Abstract

Pueraria tuberosa contains a wide range of bioactive compounds, including polyphenols, alkaloids, and phytosterols, which make it valuable to the pharmaceutical and food industries. Elicitor compounds trigger the defense mechanisms in plants and are widely used to increase the yield of bioactive molecules in in vitro cultures. The present study was conducted to evaluate the effects of different concentrations of biotic elicitors such as yeast extract (YE), pectin (PEC), and alginate (ALG) on growth, antioxidant activity, and metabolite accumulation in in vitro propagated shoots of P. tuberosa. The elicitors applied to shoot cultures of P. tuberosa significantly increased biomass (shoot number, fresh weight, and dry weight), and metabolites such as protein, carbohydrates, chlorophyll, total phenol (TP), and total flavonoid (TF) contents, as well as antioxidant activity compared to untreated control. Biomass, TP, and TF contents, as well as antioxidant activity, were most significant in cultures treated with 100 mg/L PEC. In contrast, chlorophyll, protein, and carbohydrate increased most in cultures treated with 200 mg/L ALG. Application of 100 mg/L of PEC led to the accumulation of high amounts of isoflavonoids including puerarin (220.69 μg/g), daidzin (2935.55 μg/g), genistin (5612 μg/g), daidzein (479.81 μg/g), and biochanin-A (111.511 μg/g) as analyzed by high-performance liquid chromatography (HPLC). Total isoflavonoids content of 100 mg/L PEC treated shoots was obtained as 9359.56 μg/g, 1.68-fold higher than in vitro propagated shoots without elicitors (5573.13 μg/g) and 2.77-fold higher than shoots of the mother plant (3380.17 μg/g). The elicitor concentrations were optimized as 200 mg/L YE, 100 mg/L PEC, and 200 mg/L ALG. Overall, this study showed that the application of different biotic elicitors resulted in better growth, antioxidant activity, and accumulation of metabolites in P. tuberosa, which could lead to obtaining phytopharmaceutical advantages in the future.

Keywords: antioxidant; biomass; elicitor; isoflavonoids; secondary metabolite.

Figure 1

Effect of elicitor treatments on morphological attributes in in vitro shoots of P. tuberosa: (a) TNS, (b) FW (g/L), (c) DW (g/L). Values are mean ± SD, means in a column followed by different letters are significantly different from each other at p < 0.05. Mean values followed by the same letter(s) in a column are not significantly different (p < 0.05) based on Duncan multiple range test (DMRT).

Figure 2

Effect of elicitor treatments on TP and TF contents in P. tuberosa shoots: (a) TP content (mg gallic acid/g), (b) TF content (mg QE acid/g). Note: Values are mean ± SD, means in a column followed by different letters are significantly different from each other at p < 0.05. Mean values followed by the same letter(s) in a column are not significantly different (p < 0.05) based on DMRT.

Figure 3

Effect of elicitor treatments on antioxidant activity in P. tuberosa shoots: (a) DPPH activity (%), (b) FRAP activity (mM FeSO₄/g), (c) SOD activity (%). Note: Values are mean ± SD, means in a column followed by different letters are significantly different from each other at p < 0.05. Mean values followed by the same letter(s) in a column are not significantly different (p < 0.05) based on DMRT.

Figure 4

HPLC chromatogram of PEC elicited (100 mg/L) shoots of P. tuberosa.

Figure 5

Principal component analysis of physio-biochemical indices of P. tuberosa shoots for different elicitor treatments.