Effective callus induction and plant regeneration in callus and protoplast cultures of Nigella damascena L.
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ORIGINAL ARTICLE
Effective callus induction and plant regeneration in callus and protoplast cultures of Nigella damascena L. Magdalena Klimek‑Chodacka1 · Dariusz Kadluczka1 · Aneta Lukasiewicz1 · Aneta Malec‑Pala1 · Rafal Baranski1 · Ewa Grzebelus1 Received: 22 May 2020 / Accepted: 15 October 2020 © The Author(s) 2020
Abstract In this study we report the development of effective in vitro systems for a medicinal plant Nigella damascena L. comprising: (1) callus induction, (2) somatic embryogenesis in callus cultures with subsequent plant regeneration, and (3) isolation and regeneration of callus-derived protoplasts. Callus development was achieved on 83–100% of hypocotyl and cotyledon explants, whereby Murashige and Skoog medium (MS) supplemented with 3 mg L−1 6-benzylaminopurine and 0.5 mg L−1 α-naphthaleneacetic acid (NAA; BN medium) was more advantageous than MS with kinetin and NAA (KN medium). Histological observations of calli revealed the presence of embryogenic zones from which somatic embryos developed on the hormone-free medium. Plant regeneration was observed on 76–95% of calli. A high capacity to form somatic embryos and regeneration was maintained in long-lasting cultures, i.e. even in 2 year old callus. The obtained callus was also a good source tissue for protoplast isolation. By applying a mixture of cellulase and pectolyase, the acceptable yield of viable protoplasts was achieved, especially from hypocotyl-derived callus maintained on BN medium. Protoplasts embedded in an alginate matrix and cultured in modified Kao and Michayluk media re-constructed their cell wall and re-entered mitotic divisions. About 30% of small cell aggregates formed microcalli, which, after the release from alginate, proliferated continuously on KN and BN media, irrespective of the tissue variant used as the protoplast source. Somatic embryo formation and plant regeneration were successful on hormone-free media. An effective plant regeneration system of N. damascena protoplast cultures has been developed and is being reported for the first time. Key message The main objective of this study was to develop in vitro systems utilizing N. damascena seedlings, asan easily accessible explant source, for efficient callus induction and proliferation, and plantregeneration via somatic embryogenesis. Moreover, we attempted to validate the usefulness of theobtained callus as a source of protoplasts and their capability to develop into plants. Keywords Cotyledon · Hypocotyl · Somatic embryogenesis · Love-in-a-mist · Medicinal plant · Ranunculaceae
Communicated by Danny Geelen. Electronic supplementary material The online version of this article (https://doi.org/10.1007/s11240-020-01953-9) contains supplementary material, which is available to authorized users. * Magdalena Klimek‑Chodacka [email protected] * Ewa Grzebelus [email protected] 1
Abbreviations BAP 6-Benzylaminopurine CPP Carrot petiole protoplast medium 2,4-D 2,4-Dichlorophenoxyacetic acid FDA Fluorescein diacetate FW Fresh weight NAA
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