Response surface methodology mediated optimization of phytosulfokine and plant growth regulators for enhanced protoplast division, callus induction, and somatic embryogenesis in Angelica Gigas Nakai

Abstract

Background: Angelica Gigas (Purple parsnip) is an important medicinal plant that is cultivated and utilized in Korea, Japan, and China. It contains bioactive substances especially coumarins with anti-inflammatory, anti-platelet aggregation, anti-cancer, anti-diabetic, antimicrobial, anti-obesity, anti-oxidant, immunomodulatory, and neuroprotective properties. This medicinal crop can be genetically improved, and the metabolites can be obtained by embryonic stem cells. In this context, we established the protoplast-to-plant regeneration methodology in Angelica gigas.

Results: In the present investigation, we isolated the protoplast from the embryogenic callus by applying methods that we have developed earlier and established protoplast cultures using Murashige and Skoog (MS) liquid medium and by embedding the protoplast in thin alginate layer (TAL) methods. We supplemented the culture medium with growth regulators namely 2,4-dichlorophenoxyaceticacid (2,4-D, 0, 0.75, 1.5 mg L^{- 1}), kinetin (KN, 0, 0.5, and 1.0 mg L^{- 1}) and phytosulfokine (PSK, 0, 50, 100 nM) to induce protoplast division, microcolony formation, and embryogenic callus regeneration. We applied central composite design (CCD) and response surface methodology (RSM) for the optimization of 2,4-D, KN, and PSK levels during protoplast division, micro-callus formation, and induction of embryogenic callus stages. The results revealed that 0.04 mg L^{- 1} 2,4-D + 0.5 mg L^{- 1} KN + 2 nM PSK, 0.5 mg L^{- 1} 2,4-D + 0.9 mg L^{- 1} KN and 90 nM PSK, and 1.5 mg L^{- 1} 2,4-D and 1 mg L^{- 1} KN were optimum for protoplast division, micro-callus formation and induction embryogenic callus. MS basal semi-solid medium without growth regulators was good for the development of embryos and plant regeneration.

Conclusions: This study demonstrated successful protoplast culture, protoplast division, micro-callus formation, induction embryogenic callus, somatic embryogenesis, and plant regeneration in A. gigas. The methodologies developed here are quite useful for the genetic improvement of this important medicinal plant.

Keywords: Angelica gigas; Phytosulfokine; Protoplast culture; Response surface methodology; Somatic embryogenesis.

Fig. 1

A-B. Tracking of the protoplast division of Angelica gigas using a compound microscope from 0 to 120 h/5 days: protoplasts were embedded by the TAL method and cultured in MS liquid medium supplemented with 0.3 M mannitol, 0.08 M sucrose, and 0.5 mg L^− 1 2,4-D. The cultures were maintained in the dark. Percentage of expanding, shrunken, elongated, and dividing protoplasts (A), images of dividing, expanded, elongated, and shrunken protoplasts over the culture period (B). Scale bar = 50 μm. C-E. Protoplast culture efficiency of Angelica gigas in liquid and TAL medium. The cultures were maintained in the dark. Percentage of cell division after 3 weeks of culture (C), after 5 weeks of culture (D), and after 7 weeks of culture (E). Protoplast culture in liquid cultures (F), freshly isolated protoplast (I, II), and after 3 weeks (III). Protoplast culture in TAL cultures (VI), freshly embedded protoplast in TAL (V), and micro-callus formed by the division of protoplasts TAL culture (VI). Scale bars: 100 μm (black bars) and 1 cm (white bars)

Fig. 2

CCD for the verification of effect 2,4-D, KN, and PSK on protoplast division, micro-callus formation, and induction of somatic embryogenic callus during protoplast culture using RSM.

Fig. 3

Contour and 3D surface plots showing the effects of 2,4-D, KN, and PSK on protoplast division after 1 week (A and B), after 4 weeks (C and D), and after 12 weeks (E and F) respectively

Fig. 4

Graphical representation optimized concentration of three factors (2,4-D, KN, and PSK) for protoplast division (stage 1), microcolony formation (stage 1), induction embryogenic callus (stage 3), and plant regeneration (stage 4). Scale bars: 50 μm (black bars) and 2 mm (white bars)

Fig. 5

Protoplast isolation, culture using the TAL method, division of protoplasts, micro-callus formation, development of embryos, and plant regeneration in Angelica gigas. The cultures were maintained in the dark. Embryogenic callus used for protoplast isolation (A), fluorescence microscopic images – green fluorescence represents viable protoplasts stained using FDA and represents dead protoplasts stained with PI (B), protoplasts embedded in TAL after 3 days of culture (C), micro-calli in TAL cultures after 9 weeks of culture (D), micro-calli formed in TAL cultures after 10 weeks of culture (E, F). Embryogenic callus (G), globular (H), heart (I), and cotyledonary staged embryos (J, K) developed from protoplast regenerated callus after 8–10 weeks of sub-culture of callus on MS semi-solid medium, plants regenerated by germination of embryos on MS semi-solid medium (L). Scale bars: 100 μm (black bars) and 2 mm (white bars)

Fig. 6

Flow chart illustrating a step-by-step approach for protoplast isolation, culture, and plant regeneration of Angelica gigas