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Histological assessment of regenerating plants at callus, shoot organogenesis and plantlet stages during the in vitro micropropagation of Asparagus cochinchinensis

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Abstract

In this study, we developed a repeatable in vitro micropropagation protocol for the medicinal plant Asparagus cochinchinensis, based on indirect organogenesis using leaf segments cut from seedlings of in vitro-germinated seeds. We obtained 85% callus induction from the leaf segments within 4–5 weeks when grown on Murashige and Skoog (MS) medium supplemented with benzylaminopurine (BAP, 1.0 mg/L) and 1-naphthaleneacetic acid (NAA, 0.5 mg/L). We found that MS media supplemented with Kn (1.0 mg/L) in combination with NAA (1.0 mg/L) and BAP (1.0 mg/L) combined with NAA (0.5 mg/L) were the most effective in promoting shoot regeneration, yielding plantlets with 6.72 and 6.48 shoots per culture, respectively. When cultured on PGR-free half-strength MS medium, regenerated plants developed root systems with an average 11.0 roots per shoot cluster and an average length of 36.14 mm at 9 weeks. During acclimatization, regenerated plantlets showed 96.4% survival and exhibited normal growth characteristics and morphology. We also made an attempt to directly regenerate A. cochinchinensis from shoot apices but it was futile. Histological analyses revealed the presence of crystal idioblasts in young leaves from the early stages of leaf differentiation. The leaf-based plant regeneration technique developed herein could be employed for large-scale propagation of the plants over a short time period, thereby substantially contributing to the germplasm preservation and rapid propagation of A. cochinchinensis.

Key message

A repeatable in vitro micropropagation protocol for Asparagus cochinchinensis was developed based on indirect organogenesis. Histological analysis revealed crystal idioblasts for the first time in leaf primordia of this species.

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Abbreviations

BAP:

6-Benzylaminopurine

NAA:

α-Naphthaleneacetic acid

Kn:

Kinetin

2iP:

N6-(2-isopentenyl) adenosine

TDZ:

Thidiazuron

MS:

Murashige and Skoog

PGR:

Plant growth regulator

2,4-D:

2,4-Dichlorophenoxyacetic acid

WPM:

Woody plant medium

LS:

Linsmaier and Skoog

DJ:

De Greef and Jacobs

B5:

Gamborg B5

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Acknowledgements

This work was supported by Development of Foundational Techniques for the Domestic Production of Herbal Medicines (K18405), Applicational Development of Standardized Herbal Resources (KSN1911420), Development of Sustainable Application for Standard Herbal Resources (KSN2012320), Korea Institute of Oriental Medicine through the Ministry of Science and ICT, Republic of Korea.

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Author notes

  1. Yong-Goo Kim and Denis Okello made equal contributions to the research work and are first co-authors.

Authors and Affiliations

  1. Herbal Medicine Resources Research Center, Korea Institute of Oriental Medicine (KIOM), 111 Geonjae-Ro, Naju-si, Jeollanam-do, 58245, Republic of Korea

    Yong-Goo Kim, Denis Okello, Sungyu Yang, Richard Komakech, Endang Rahmat & Youngmin Kang

  2. Korean Convergence Medicine Major, University of Science and Technology (UST), Daejeon, Republic of Korea

    Denis Okello, Richard Komakech, Endang Rahmat & Youngmin Kang

  3. Gombe Secondary School, P. O. Box, 192, Butambala, Mpigi, Uganda

    Denis Okello

  4. Natural Chemotherapeutics Research Institute (NCRI), Ministry of Health, P.O. Box 4864, Kampala, Uganda

    Richard Komakech

Authors
  1. Yong-Goo Kim
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  2. Denis Okello
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  3. Sungyu Yang
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  4. Richard Komakech
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  5. Endang Rahmat
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  6. Youngmin Kang
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Contributions

YGK and YMK conceived the research. DO, RK, YGK, ER and YMK did the in vitro propagation work. DO, YGK and YSK did the histological analysis. YGK analyzed the data. DO wrote the manuscript. RK, YGK, ER and YSK read and improved the manuscript. YMK provided technical guidance, supervised the whole research work, read and improved the manuscript. All authors read and approved the final manuscript.

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Correspondence to Youngmin Kang.

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Communicated by M. Paula Watt.

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Kim, YG., Okello, D., Yang, S. et al. Histological assessment of regenerating plants at callus, shoot organogenesis and plantlet stages during the in vitro micropropagation of Asparagus cochinchinensis. Plant Cell Tiss Organ Cult 144, 421–433 (2021). https://doi.org/10.1007/s11240-020-01967-3

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  • DOI: https://doi.org/10.1007/s11240-020-01967-3

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