Protoplast isolation prior to flow cytometry reveals clear patterns of endoreduplication in potato tubers, related species, and some starchy root crops

F. Parker E. Laimbeer, Sarah H. Holt, Melissa Makris, Michael Alan Hardigan, C. Robin Buell, Richard E. Veilleux

    Research output: Contribution to journalArticle

    Abstract

    Background: Endoreduplication, the process of DNA replication in the absence of cell division, is associated with specialized cellular function and increased cell size. Genes controlling endoreduplication in tomato fruit have been shown to affect mature fruit size. An efficient method of estimating endoreduplication is required to study its role in plant organ development. Flow cytometry is often utilized to evaluate endoreduplication, yet some tissues and species, among them the tubers of Solanum tuberosum, remain intractable to routine tissue preparation for flow cytometry. We aimed to develop a method through the use of protoplast extraction preceding flow cytometry, specifically for the assessment of endoreduplication in potato tubers. Results: We present a method for appraising endoreduplication in potato (Solanum tuberosum) tuber tissues. We evaluated this method and observed consistent differences between pith and cortex of tubers and between different cultivars, but no apparent relationship with whole tuber size. Furthermore, we were able to observe distinct patterns of endoreduplication in 16 of 20 wild potato relatives, with mean endoreduplication index (EI) ranging from 0.94 to 2.62 endocycles per cell. The protocol was also applied to a panel of starchy root crop species and, while only two of five yielded reliable flow histograms, the two (sweet potato and turnip) exhibited substantially lower EIs than wild and cultivated potato accessions. Conclusions: The protocol reported herein has proven effective on tubers of a variety of potato cultivars and related species, as well as storage roots of other starchy crops. This method provides an important tool for the study of potato morphology and development while revealing natural variation for endoreduplication which may have agricultural relevance.

    Original languageEnglish (US)
    Article number27
    JournalPlant Methods
    Volume13
    Issue number1
    DOIs
    StatePublished - Apr 14 2017

    Profile

    endopolyploidy
    Endoreduplication
    Protoplasts
    Solanum tuberosum
    Flow Cytometry
    tubers
    potatoes
    methodology
    flow cytometry
    crops
    Fruit
    protoplasts
    fruits
    cultivars
    cells
    Plant Organogenesis
    Ipomoea batatas
    Brassica napus
    Lycopersicon esculentum
    DNA Replication

    Keywords

    • Endopolyploidization
    • Karyoplasmic ratio
    • Solanaceae
    • Solanum tuberosum

    ASJC Scopus subject areas

    • Biotechnology
    • Genetics
    • Plant Science

    Cite this

    Protoplast isolation prior to flow cytometry reveals clear patterns of endoreduplication in potato tubers, related species, and some starchy root crops. / Laimbeer, F. Parker E.; Holt, Sarah H.; Makris, Melissa; Hardigan, Michael Alan; Robin Buell, C.; Veilleux, Richard E.

    In: Plant Methods, Vol. 13, No. 1, 27, 14.04.2017.

    Research output: Contribution to journalArticle

    Laimbeer, F. Parker E.; Holt, Sarah H.; Makris, Melissa; Hardigan, Michael Alan; Robin Buell, C.; Veilleux, Richard E. / Protoplast isolation prior to flow cytometry reveals clear patterns of endoreduplication in potato tubers, related species, and some starchy root crops.

    In: Plant Methods, Vol. 13, No. 1, 27, 14.04.2017.

    Research output: Contribution to journalArticle

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    AU - Laimbeer,F. Parker E.

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    AU - Makris,Melissa

    AU - Hardigan,Michael Alan

    AU - Robin Buell,C.

    AU - Veilleux,Richard E.

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    AB - Background: Endoreduplication, the process of DNA replication in the absence of cell division, is associated with specialized cellular function and increased cell size. Genes controlling endoreduplication in tomato fruit have been shown to affect mature fruit size. An efficient method of estimating endoreduplication is required to study its role in plant organ development. Flow cytometry is often utilized to evaluate endoreduplication, yet some tissues and species, among them the tubers of Solanum tuberosum, remain intractable to routine tissue preparation for flow cytometry. We aimed to develop a method through the use of protoplast extraction preceding flow cytometry, specifically for the assessment of endoreduplication in potato tubers. Results: We present a method for appraising endoreduplication in potato (Solanum tuberosum) tuber tissues. We evaluated this method and observed consistent differences between pith and cortex of tubers and between different cultivars, but no apparent relationship with whole tuber size. Furthermore, we were able to observe distinct patterns of endoreduplication in 16 of 20 wild potato relatives, with mean endoreduplication index (EI) ranging from 0.94 to 2.62 endocycles per cell. The protocol was also applied to a panel of starchy root crop species and, while only two of five yielded reliable flow histograms, the two (sweet potato and turnip) exhibited substantially lower EIs than wild and cultivated potato accessions. Conclusions: The protocol reported herein has proven effective on tubers of a variety of potato cultivars and related species, as well as storage roots of other starchy crops. This method provides an important tool for the study of potato morphology and development while revealing natural variation for endoreduplication which may have agricultural relevance.

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