Isolation, culture of protoplasts of Angelica gigas Nakai and regeneration of plants via somatic embryogenesis

Abstract

In plant biotechnology, protoplasts are a versatile tool since they are very helpful for both fundamental biology studies and for genetic improvement and genome editing studies. In many plant species, however, reproducible regeneration from protoplasts continues to be a bottleneck. In the present study, we report the development of an efficient method for protoplast isolation, and plant regeneration in Angelica gigas via indirect somatic embryogenesis. Protoplasts were isolated from embryogenic callus using an enzyme mixture of 1.0% Viscozyme® L + 1% Celluclast® 1.5 L + 0.5% Pectinex® XXL with 7 h treatments. Initially, protoplasts were cultured in MS, modified MS (NH4NO3-free medium), and KM media, and viability and cell division data showed the MS medium was suitable for protoplast culture. Subsequently, the thin alginate layer method was applied to the protoplast culture at an optimal density of 1 × 106 cells per mL？ 1 and verified the effect of 2,4-D (0.1, 0.5, and 1.0 mg L？ 1) alone, and 2,4-D (0.5, and 1.0 mg L？ 1) in combination with BA (0.1 and 0.5 mg L？ 1) or KN (0.1 and 0.5 mg L？ 1) on cell division, micro-callus formation. MS medium supplemented with 1.0 mg L？ 1 2,4-D and 0.1 mg L？ 1 KN induced optimal cell division, callus formation, and subsequent induction of somatic embryogenesis from the callus. The somatic embryos germinated and converted into plantlets upon transferring to the MS basal medium. This method of Angelica gigas protoplast regeneration can be used for the genetic improvement of this plant.