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Interspecific somatic hybrids between Cyclamen persicum and C. coum, two sexually incompatible species

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Abstract

By applying polyethylene glycol (PEG)-mediated protoplast fusion, the first somatic hybrids were obtained between Cyclamen persicum (2n = 2x = 48) and C. coum (2n = 2x = 30)—two species that cannot be combined by cross breeding. Heterofusion was detected by double fluorescent staining with fluorescein diacetate and scopoletin. The highest heterofusion frequencies (of about 5%) resulted from a protocol using a protoplast density of 1 × 106/mL and 40% PEG. The DNA content of C. coum was estimated for the first time by propidium iodide staining to be 14.7 pg/2C and was 4.6 times higher than that of C. persicum. Among 200 in vitro plantlets regenerated from fusion experiments, most resembled the C. coum parent, whereas only 5 plants showed typical C. persicum phenotypes and 46 had a deviating morphology. By flow cytometry, six putative somatic hybrids were identified. A species-specific DNA marker was developed based on the sequence of the 5.8S gene in the ribosomal nuclear DNA and its flanking internal transcribed spacers ITS1 and ITS2. The hybrid status of only one plant could be verified by the species-specific DNA marker as well as sequencing of the amplification product. RAPD markers turned out to be less informative and applicable for hybrid identification, as no clear additivity of the parental marker bands was observed. Chromosome counting in root tips of four hybrids revealed the presence of the 30 C. coum chromosomes and 2–41 additional ones indicating elimination of C. persicum chromosomes.

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Abbreviations

BA:

6-Benzyladenine

2iP:

6-(γ,γ-Dimethylallylamino)purine

DAPI:

4′,6-Diamidino-2-phenylindol

2,4-D:

2,4-Dichlorophenoxyacetic acid

FDA:

Fluorescein diacetate

FISH:

Fluorescent in situ hybridisation

GISH:

Genomic in situ hybridisation

PEG:

Polyethylene glycol

PGR:

Plant growth regulator

RFLP:

Restriction fragment length polymorphism

SSR:

Simple sequence repeat

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Acknowledgments

The authors thank Dr. Frank Schaarschmidt for his help in statistical data analysis, Ulrich Lohmueller for the determination of the absolute DNA content of hybrid no. 31 and Dr. Joerg Fuchs (IPK Gatersleben) for the donation of the Vicia faba, Pisum sativum and the Secale cereale seeds. Financial support of the German Federal Ministry of Economics and Technology within the program PRO INNO Grant no. KF0054802MD5 is gratefully acknowledged.

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Authors and Affiliations

  1. Institute of Floriculture and Woody Plant Science, Gottfried Wilhelm Leibniz Universitaet Hannover, Herrenhaeuser Str. 2, 30419, Hannover, Germany

    Anika Nadja Sabine Prange, Melanie Bartsch, Julia Meiners, Margrethe Serek & Traud Winkelmann

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  1. Anika Nadja Sabine Prange
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  2. Melanie Bartsch
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  3. Julia Meiners
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  4. Margrethe Serek
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Correspondence to Traud Winkelmann.

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Communicated by R. Rose.

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Prange, A.N.S., Bartsch, M., Meiners, J. et al. Interspecific somatic hybrids between Cyclamen persicum and C. coum, two sexually incompatible species. Plant Cell Rep 31, 723–735 (2012). https://doi.org/10.1007/s00299-011-1190-z

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  • DOI: https://doi.org/10.1007/s00299-011-1190-z

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