Indirect somatic embryogenesis of purple coneflower (Echinacea purpurea (L.) Moench): a medicinal-ornamental plant: evaluation of antioxidant enzymes activity and histological study

Abstract

Purple coneflower (Echinacea purpurea (L.) Moench) is a widely used medicinal and ornamental plant. In the present study, the callus embryogenesis was examined using benzyl adenine (BA) at three levels (3, 4, 5 mg L^-1), 1-Naphthalene acetic acid (NAA) at three levels (0.1, 0.2 and 0.5 mg L^-1) with or without activated charcoal (1 g L^-1), coconut milk (50 ml L^-1) and casein hydrolysate (50 mg L^-1) in the MS (Murashige and Skoog 1962) medium. The embryogenesis indirectly occurred with the production of callus. The calli were observed in three forms: undifferentiated, embryogenic and organogenic. The embryogenic calli were dark green and coherent with a faster growth rate. The highest embryogenesis (100%) and embryonic regeneration (plantlet production) were obtained in the combined BA + NAA treatments with the activated charcoal, coconut milk and casein hydrolysate. However, the combined treatments of growth regulators failed to produce somatic embryos without the use of coconut milk and casein hydrolysate. The maximum amount of protein, peroxidase and catalase activity of embryogenic calli (2.02, 1.79 and 6.62ΔOD/Min/mg.protein, respectively), and highest percentage of acclimatization success (29.3% of plants) were obtained in the combined treatment of 5 mg L^-1 BA + 0.5 mg L^-1 NAA + activated charcoal + coconut milk + casein hydrolysate. The highest amount of chlorophyll content (33.3 SPAD value) and growth characteristics of acclimatized plantlets were observed in the media containing 3 mg L^-1 BA + 0.1 and 0.2 mg L^-1 NAA + 1 g. L^-1 combined activated charcoal, coconut milk, casein hydrolysate. The histological studies confirmed the somatic embryogenesis in purple coneflower. Generally, it was found that the somatic embryogenesis of E. purpurea occurs at high levels of BA and low levels of NAA with the addition of coconut milk and casein hydrolysate.

Keywords: Benzyl adenine; Callus; Casein hydrolysate; Coconut milk; Naphthalene acetic acid.

Fig. 1

Different calli types developing on leaf explants of E. purpurea cultured on MS medium after four weeks: a Dark green and compact embryogenic calli induced by 3 mg L⁻¹ BA and 0.5 mg L⁻¹ NAA combination, 1 g L⁻¹ active charcoal, 50 ml L⁻¹ coconut milk and 50 mg L⁻¹ casein hydrolysate after four weeks on MS medium. b Light green, soft, loose and puffy organogenic calli induced on MS medium supplemented with 3 mg L⁻¹ BA and 0.5 mg L⁻¹ NAA after four weeks. c White and light undifferentiated and hyperhydrated calli induced on MS medium supplemented with 3 mg L⁻¹ BA and 0.1 mg L⁻¹ NAA after four weeks. and d Browned and dark undifferentiated calli induced on MS medium supplemented with 3 mg L⁻¹ BA and 0.2 mg L⁻¹ NAA after four weeks. Bars = 10 mm

Fig. 2

Effect of different treatments on induction of different calli types developing on leaf explant calli of E. purpurea cultured on MS medium after four weeks a embryogenic, b organogenic, and c undifferentiated calli

Fig. 3

Effect of different treatments affecting number of regenerated leaves of E. purpurea cultured on MS medium after eight weeks

Fig. 4

Production steps of plantlet in E. purpurea a somatic embryos (white arrows) regenerated from leaf explant callus of E. purpurea derived from combination of 3 mg L⁻¹ BA and 0.5 mg L⁻¹ NAA with 1 g L⁻¹ active charcoal, 50 ml L⁻¹ coconut milk and 50 mg L⁻¹ casein hydrolysate cultured on MS medium. Bars = 10 mm, b shoots regenerated from somatic embryos. Bars = 10 mm, c leaf production from embryogenic callus. Bars = 20 mm, d organogenic calli. Bars = 10 mm, e shoot regenerated from organogenic callus. Bars = 10 mm, and f microshoot production from organogenic callus. Bars = 20 cm

Fig. 5

Effect of different treatments affecting a protein content, b activity of catalase, and c activity of peroxidase in calli of E. purpurea

Fig. 6

Effect of different treatments on acclimatization of E. purpurea plantlets six weeks after transferring plants to pots

Fig. 7

Different adaptation stages of plantlets a plantlets obtained from embryogenic callus. b plantlets transferred to small pots containing 70% peat moss and 30% perlite for acclimatization and irrigation of pots by underground (sub) irrigation method. c sample plantlets during transfer to pots. and d plantlets after four weeks of acclimatization. Bars = 1 cm

Fig. 8

Histological study of embryogenic and organogenic calli in E. Purpurea after four weeks; a parenchymal (pc) and meristematic (mc) cells in organogenic callus induced on MS medium supplemented with 3 mg L⁻¹ BA and 0.5 mg L⁻¹ NAA after four weeks, Bars = 1 µm, b embryogenic callus and presence of globular embryo (red arrows) induced by 3 mg L⁻¹ BA and 0.5 mg L⁻¹ NAA combination, 1 g. L⁻¹ active charcoal, 50 ml L⁻¹ coconut milk and 50 mg L⁻¹ casein hydrolysate after four weeks on MS medium, Bars = 5 µm, c embryo in heart stage (red arrows) after five weeks on MS medium, Bars = 5 µm, d embryonic cells of dense cytoplasm, large nucleus and starch (st) granules, Bars = 10 µm