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Protoplast isolation and shoot regeneration from protoplast-derived calli of Chrysanthemum cv. White ND

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Abstract

In this study, we sought to optimize the isolation of protoplasts from chrysanthemums by manipulating the mannitol and cellulase levels, the incubation period, and the purification method, followed by the conversion of the protoplasts into calli and shoots. A high protoplast yield was achieved using 0.5 M mannitol, 1.5% cellulase, and a 4 h incubation period. Cell wall regeneration was observed after 3 days, with the first cell division occurring approximately 4–5 days after culturing. The addition of sucrose to the culture media was more beneficial than glucose; in sucrose media the protoplasts grew more rapidly and successfully reached the colony and microcalli stage. The addition of activated charcoal to the culture improved colony and microcalli formation. Greater proliferation of microcalli was also achieved using solid Murashige & Skoog (MS) media supplemented with 1 mg l−1 6-Benzylaminopurine (BA) and 2 mg l−1 Naphthaleneacetic acid (NAA). The calli produced shoots THE on media supplemented with 2 mg l− 1 BA and 0.5 mg l−1 NAA. These findings could facilitate further chrysanthemum protoplast-based research.

Key message

The regeneration of chrysanthemum protoplasts into whole plants is difficult due to the recalcitrant nature of these plants and because of their genotype-dependent response. This study is appropriate for protoplast isolation and callus formation of Chrysanthemum cv. White ND, and it also achieved regenerating chrysanthemum protoplasts-derived calli into whole plants.

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Abbreviations

AC:

Activated charcoal

BA:

6-Benzylaminopurine

CM:

Culture media

CPW:

Cell and protoplast washing solution

FDA:

Fluorescein diacetate

FW:

Fresh weight

MES:

2-(N-morpholino)ethanesulfonic acid

MS:

Murashige and Skoog

NAA:

Naphthaleneacetic acid

PGR:

Plant growth regulator

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Acknowledgement

This work was supported by a grant from the New Breeding Technology Program (Project no. PJ01485801), Rural Development Administration, Republic of Korea.

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  1. Oluwaseun Suleimon Adedeji and Aung Htay Naing have equally contributed to this work.

Authors and Affiliations

  1. Department of Horticultural Science, Kyungpook National University, Daegu, 41566, Korea

    Oluwaseun Suleimon Adedeji, Aung Htay Naing & Chang Kil Kim

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  1. Oluwaseun Suleimon Adedeji
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  2. Aung Htay Naing
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  3. Chang Kil Kim
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Contributions

AHN and ASO designed the experiment. ASO conducted the experiment. ASO wrote the manuscript draft. AHN advised the experiment and revised the manuscript. CKK supervised the project.

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Correspondence to Aung Htay Naing or Chang Kil Kim.

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Communicated by M. I. Beruto.

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Adedeji, O.S., Naing, A.H. & Kim, C.K. Protoplast isolation and shoot regeneration from protoplast-derived calli of Chrysanthemum cv. White ND. Plant Cell Tiss Organ Cult 141, 571–581 (2020). https://doi.org/10.1007/s11240-020-01816-3

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