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

  • 2 Citations

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.

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

Profile

Endoreduplication
endopolyploidy
Protoplasts
Solanum tuberosum
protoplasts
flow cytometry
Flow Cytometry
tubers
potatoes
crops
Plant Organogenesis
Fruit
Ipomoea batatas
Brassica napus
methodology
fruits
turnips
pith
cultivars
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. 2017 ; Vol. 13, No. 1.
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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.",
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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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