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Isolation of cytoplasts from Satsuma mandarin (Citrus unshiu Marc.) and production of alloplasmic hybrid calluses via cytoplast–protoplast fusion

  • Genetic Transformation and Hybridization
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Abstract

Cytoplasm of Satsuma mandarin (Citrus unshiu Marc.) is known to influence seedlessness. Transfer of cytoplasm to a seedy cultivar could possibly lead to the production of seedless citrus fruits. In the present paper cytoplasts were isolated from cell suspension-derived protoplasts of Satsuma mandarin via ultra-centrifugation in a discontinuous gradient. No nucleus could be detected in the cytoplasts by DAPI (4′, 6-diamidino-2-phenylindole) staining compared with normal protoplasts. The cytoplasts, with high viability and small size, did not divide during solid embedding culture. Cytoplasts of Satsuma mandarin were electrically fused with embryogenic protoplasts of Murcott tangor (C. reticulata × C. sinensis), which led to regeneration of several cell lines. Flow cytometry (FCM) indicated that the cell lines were diploids. Simple sequence repeats (SSR) and cleaved amplified polymorphism sequence (CAPS) showed that the cell lines got their nuclear DNA from the protoplast parent, whereas the cytoplast parent donated the mtDNA, confirming transfer of mtDNA from Satsuma mandarin into Murcott tangor via cytoplast–protoplast fusion though no polymorphism was detected in chloroplast DNA between the fusion partners. This is the first report on isolation and characterization of cytoplasts, together with cytoplast–protoplast fusion in Citrus, which has a potential for citrus cultivar improvement involving cytoplasm transfer via cytoplast–protoplast fusion.

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Abbreviations

CAPS:

Cleaved amplified polymorphism sequence

DAPI:

4′, 6-Diamidino-2- phenylindole

FCM:

Flow cytometry

FDA:

Fluorescein diacetate

MT:

Murashige and Tucker

mtDNA:

Mitochondrial DNA

RFLP:

Restriction fragment length polymorphism

SSR:

Simple sequence repeat

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Acknowledgements

This research was financially supported by the National Natural Science Foundation of China (30200189), the International Foundation for Science (IFS) grant to Dr J.H. Liu, High-Tech Research and Development Program of China. Great thanks should be extended to Dr. Jain SM for his critical reading of the paper.

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Authors and Affiliations

  1. National Key Laboratory of Crop Genetic Improvement, National Center of Crop Molecular Breeding, Huazhong Agricultural University, Wuhan, 430070, P.R. China

    X. Y. Xu, J. H. Liu & X. X. Deng

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  1. X. Y. Xu
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  2. J. H. Liu
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  3. X. X. Deng
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Correspondence to J. H. Liu.

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Communicated by A. Atanassov

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Xu, X.Y., Liu, J.H. & Deng, X.X. Isolation of cytoplasts from Satsuma mandarin (Citrus unshiu Marc.) and production of alloplasmic hybrid calluses via cytoplast–protoplast fusion. Plant Cell Rep 25, 533–539 (2006). https://doi.org/10.1007/s00299-005-0057-6

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  • DOI: https://doi.org/10.1007/s00299-005-0057-6

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