Micropropagation of an ornamental shrub Disanthus cercidifolius Maxim. and assessment of genetic fidelity of regenerants using ISSR and flow cytometry

Abstract

This study aimed to develop an efficient micropropagation protocol for Disanthus cercidifolius Maxim., an ornamental shrub. Sprouting buds of two genotypes (‘Trubaʼ and ‘PdSʼ) were used as an initial plant material. For the in vitro propagation experiment, the nodal segments were cultured on a MS medium supplemented with either N6-benzyladenine (BA) or zeatin (both at 0.5–3 mg l−1). As a control, MS medium without growth regulators was used. The highest number of shoots per explant (6.95 ± 0.33 in genotype ‘PdSʼ and 7.93 ± 0.41 in genotype ‘Trubaʼ) was achieved on a medium supplemented with 2 mg l−1 BA. Half-strength WPM and half-strength MS medium supplemented with indole-3-butyric acid (IBA) (0.1–1 mg l−1) were tested for in vitro rooting of the shoots. Optimal rooting performance was achieved on a half-strength WPM containing 0.5 mg l−1 IBA in genotype ‘PdSʼ, and 0.1 mg l−1 IBA in genotypes ‘Trubaʼ. The rooted plantlets were transferred ex vitro with 85% survival in genotype ‘PdSʼ and 82.5% in genotype ‘Trubaʼ. Eighteen samples from each genotype were subjected to ISSR analysis and flow cytometry to assess plant genetic fidelity after micropropagation. Fifteen ISSR primers gave rise to monomorphic patterns indicating no detected genetic variation in regenerants. Flow cytometric analysis showed that the ploidy level in all tested in vitro regenerants remained stable. The micropropagation protocol optimized here represents a reliable and efficient method for the large-scale production of plants of the ornamental shrub Disanthus cercidifolius.

Key message

An efficient protocol for large-scale in vitro propagation of Disanthus cercidifolius was developed. No genetic variation was detected among in vitro regenerants.

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Acknowledgements

The authors are grateful for assistance with the chromosome counting provided by Vlasta Jarolimova (Institute of Botany of the Czech Academy of Sciences).

Funding

This research was financially supported by the Internal Grant Agency of the Faculty of Tropical AgriSciences, Czech University of Life Sciences Prague, IGA (Project No. 20205006), and the Czech Ministry of Agriculture (Project No. RO0418).

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TU conducted the in vitro experiments and wrote the draft of the manuscript. JV contributed to experimental design and conducted initiation of the experiment. PHC carried out molecular analysis. KE conducted histological analysis. DJ statistically analysed the data and contributed to interpretation of the data. VB conceived the idea. IV performed the literature search, designed the experiments and reviewed the manuscript.

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Correspondence to Iva Viehmannova.

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Ulvrova, T., Vitamvas, J., Cepkova, P.H. et al. Micropropagation of an ornamental shrub Disanthus cercidifolius Maxim. and assessment of genetic fidelity of regenerants using ISSR and flow cytometry. Plant Cell Tiss Organ Cult 144, 555–566 (2021). https://doi.org/10.1007/s11240-020-01978-0

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Keywords

  • BA
  • Flow cytometry
  • Hamamelidaceae
  • In vitro propagation
  • Inter simple sequence repeat