Callus-Mediated High-Frequency Plant Regeneration, Phytochemical Profiling, Antioxidant Activity and Genetic Stability in Ruta chalepensis L

Abstract

Efficient methods for callus induction and the high-frequency plant regeneration of Ruta chalepensis L. were established, and the phytochemical potential and antioxidant activity of a donor plant, ex-vitro-established micropropagated plants, and callus were also studied. Yellowish-green callus was induced with a frequency of 97.8% from internode shoot segments of the donor plant growing in soil in the botanical garden cultured on Murashige and Skoog (MS) medium containing 10 μM 2,4-D (2,4-dichlorophenoxyacetic acid) and 1 μM BA (6-benzyladenine). Adventitious shoots were regenerated from the yellowish-green callus on MS medium containing 5.0 μM (BA) and 1.0 μM 1-naphthaleneacetic acid (NAA), with a regeneration frequency of 98.4% and a maximum of 54.6 shoots with an average length of 4.5 cm after 8 weeks. The regenerated shoots were rooted in a medium containing 1.0 μM IBA (indole-3-butyric acid) and successfully transferred to ex vitro conditions in pots containing normal garden soil, with a 95% survival rate. The amounts of alkaloids, phenolics, flavonoids, tannins, and antioxidant activity of the ex-vitro-established micropropagated plants were higher than in the donor plant and callus. The highest contents of hesperidin and rutin (93.3 and 55.9 µg/mg, respectively) were found in the ex-vitro-established micropropagated plants compared to those obtained from the donor plant (91.4 and 31.0 µg/mg, respectively) and callus (59.1 and 21.6 µg/mg, respectively). The genetic uniformity of the ex-vitro-established micropropagated plants was appraised by the ISSR markers and compared with the donor plant. This is the first report describing the callus-mediated plant regeneration, as well as the production of phenolic compounds and antioxidant activities in R. chalepensis, which might be a potential alternative technique for the mass propagation and synthesis of bioactive compounds such as hesperidin and rutin.

Keywords: antioxidant activity; callus culture; in vitro regeneration; medicinal plants; phenolic compounds; plant growth regulators.

Figure 1

Callus induction and shoot bud induction and multiplication of R. chalepensis. (A). Callus induction in internodal explants on MS with 2,4-D (10.0 µM) + BA (1.0 µM) (B). Shoot bud initiation from callus on MS with BA (5.0 µM) + NAA (1.0 µM) (C). Multiple shoot induction from on MS with BA (5.0 µM) + NAA (1.0 µM) after four weeks of culture (D). Shoot proliferation on MS with BA (5.0 µM) + NAA (1.0 µM) after eight weeks of culture.

Figure 2

(A). In-vitro-rooted plants of R. chalepensis before transplantation (B). Ex-vitro-established micropropagated R. chalepensis plants after 4 months of transfer.

Figure 3

Cluster heatmap analysis based on the relative levels of phytochemicals measured by GC–MS in R. chalepensis. The magnitude and direction of the correlations are shown by the colors in the matrix boxes. * DP = donor plant growing in soil in the botanical garden; ** MP = ex-vitro-established micropropagated plants; *** callus = callus obtained on MS + 2,4-D (10 µM) + BA(1.0 µM).

Figure 4

Representative ISSR profiles of Ruta chalepensis using primer UBC-827. Lane M—lambda DNA/EcoRI+HindIII marker; lanes 1–10 randomly selected ex-vitro-established micropropagated plants; lane DP—donor plant growing in soil in the botanical garden.