Elsevier

Plant Science

Volume 137, Issue 2, 9 October 1998, Pages 205-215
Plant Science

Isolation of chromosomes from Pisum sativum L. hairy root cultures and their analysis by flow cytometry

https://doi.org/10.1016/S0168-9452(98)00141-1Get rights and content

Abstract

Continuously growing hairy-root cultures of pea (Pisum sativum L.) line L-84 have been induced by transformation of seedlings by Agrobacterium rhizogenes A4-24. The cultures were then used for cell cycle synchronisation in vitro and for preparation of chromosome suspensions. Various concentrations of hydroxyurea (0.5–2.0 mM) and timing of amiprophos-methyl (APM, 2–4 h) treatment have been tested to achieve the highest mitotic synchrony, and to accumulate the highest number of synchronised cells in metaphase, respectively. Optimal conditions (2 mM hydroxyurea for 18 h, 10 μM APM for 2 h applied 6 h after a release from hydroxyurea block) resulted in a high frequency of cells in metaphase (43%). The synchronised root tips were fixed in formaldehyde and chromosomes were released into a lysis buffer by mechanical homogenisation. The chromosomes in suspension showed a well preserved morphology, and could be used for flow cytometric analysis. While only two chromosomes (5 and 7) were discriminated in a standard karyotype, four chromosomes (3, 5, 6 and 7) could be clearly discriminated in the line L-84 which contains a stable reciprocal translocation between chromosomes 3 and 6. The establishment of hairy root cultures and the development of an efficient chromosome isolation procedure from a translocation line L-84 represent a basic prerequisite for subsequent flow-sorting of selected chromosomes of pea.

Introduction

Flow-cytometric analysis of mitotic chromosomes is a technique allowing measurements of DNA content and AT/GC ratio in individual chromosome types in human, as well as in some animals and plants [1], [2], [3]. Chromosomes which differ in either of these parameters can be separated by flow sorting and resulting pure fractions of individual chromosome types can be used for subsequent molecular analysis. In plants, they have been employed for physical gene mapping by PCR [4], construction of chromosome-derived DNA libraries [5], [6], [7] and isolation of RFLP markers [6].

The availability of high-quality suspensions of intact metaphase chromosomes is a prerequisite for achieving high purity of flow-sorted chromosome fractions. Procedures for preparation of such chromosome suspensions have been established for several plant species (for review see [2], [3]). A variety of tissues have been used for chromosome isolation, including cell suspensions in vitro [8], [9], [6], [5], leaf mesophyll protoplasts [10], [11], and root tip meristems [12], [13], [14], [15]. Compared to cell cultures which are characterised by the frequent occurrence of chromosomal aberrations [16], [17], the use of root tips offers the advantages of karyological stability and facile manipulation. Cell division in root tip meristems can also be efficiently synchronised, which substantially increases the yield of isolated chromosomes.

For successful sorting of chromosomes from a given species, two key requirements have to be fulfilled: (1) The tissue suitable for chromosome isolation and the optimal conditions for cell cycle synchronisation in this tissue have to be determined in order to obtain sufficient amounts of chromosomes for analysis; and (2) The chromosomes of the species of interest have to differ in their size (DNA content) so that they can be distinguished by flow-cytometric analysis. In pea (Pisum sativum L., 2n=14), the procedure for chromosome isolation from synchronised root tips of young seedlings has been developed by Gualberti et al. [13]. However, flow-cytometric analysis of standard (wild type) pea karyotype revealed that due to small differences in chromosome sizes only two chromosomes could be partially discriminated and none of them could be sorted in sufficient purity [13]. This is a common situation in plants and one of the main obstacles in the application of chromosome flow-sorting for genome analysis in many crop species. As shown for field bean (Vicia faba L.), this obstacle can be circumvented by using lines with reconstructed karyotypes which possess defined chromosomal translocations [18], [4], [7]. Since several lines with reconstructed karyotypes have also been described in pea [19], [20], we attempted to use one of them, L-84 [19], for chromosome isolation and flow-cytometric analysis. The line L-84 carries a translocation between chromosomes 3 and 6 which makes them the smallest and the largest chromosomes in the karyotype, respectively. Because of the limited number of available seeds and the problems often associated with generative propagation of lines with reconstructed karyotypes, we focused on development of procedure for chromosome isolation from hairy-roots cultures. These cultures can be obtained by plant transformation by Agrobacterium rhizogenes, which induces differentiation of roots from transformed tissues [21], [22]. The root cultures can be cultivated in vitro for years and are easily propagated. Since they represent a differentiated tissue they are genetically stable and can be used for chromosome isolation, as was shown for Melandrium album [23].

In this paper, we report on the development of a synchronisation procedure for pea hairy root cultures and subsequent isolation of metaphase chromosomes from these cultures. The chromosomes obtained from the line L-84 were analysed by flow-cytometry in order to measure the DNA content in individual chromosome types of this reconstructed karyotype. These data together with the optimised synchronisation protocol should serve as a basis for flow-sorting of pea chromosomes in the near future.

Section snippets

Plant material

Pea (P. sativum L., 2n=14) cultivar Ctirad with a standard (wild type) karyotype and a pea line L-84 containing a reciprocal translocation between chromosomes 3 and 6 [19] were used for all experiments. Seeds of Ctirad were obtained from plant breeding station SEMO (Smržice, Czech Republic) and translocation line L-84 from Dr S. Lucretti (ENEA, Cassacia, Italy). To obtain continuously growing hairy root cultures from the line L-84, hypocotyles of 5 days-old seedlings germinated in vitro on

Hairy root cultures

Hairy root cultures of pea line L-84 were obtained by transformation of young seedlings by A. rhizogenes A4-24. Several independently transformed lines of hairy roots were selected based on their high rate of growth on hormone-free solid medium. These cultures maintained their phenotype of continuously growing and occasionally branching hairy roots (Fig. 1) for more than 3 years of cultivation. Karyological analysis revealed no chromosomal abnormalities in the root tip meristems (data not

Acknowledgements

The authors thank Dr S. Lucretti for providing seeds of L-84 translocation line. They also thank Dr M. Doleželová for advice on cell cycle synchronisation in pea hairy root cultures and Dr V. Našinec for photography of hairy roots. Excellent technical assistance of Mrs H. Štĕpančı́ková and Mrs J. Weiserová is gratefully acknowledged. J.D. thanks Professor W. Göhde (Partec GmbH, Münster) for providing the PAS II flow cytometer. A gift sample of APM from the Mobay Corporation (Agricultural

References (32)

  • A.M.M. De Laat et al.

    Flow-cytometric characterisation and sorting of plant chromosomes

    Theor. Appl. Genet.

    (1984)
  • K. Arumuganathan et al.

    Preparation and flow cytometric analysis of metaphase chromosomes of tomato

    Theor. Appl. Genet.

    (1991)
  • J. Conia et al.

    Flow cytometric analysis and sorting of plant chromosomes from Petunia hybrida protoplasts

    Cytometry

    (1987)
  • J. Conia et al.

    Monoparametric models of flow cytometric karyotypes with spreadsheet software

    Theor. Appl. Genet.

    (1989)
  • J. Doležel et al.

    A high yield procedure for isolation of metaphase chromosomes from root tips of Vicia faba L

    Planta

    (1992)
  • G. Gualberti et al.

    Preparation of pea (Pisum sativum L.) chromosome and nucleus suspensions from single root tips

    Theor. Appl. Genet.

    (1996)
  • Cited by (39)

    • Chromosome genomics uncovers plant genome organization and function

      2021, Biotechnology Advances
      Citation Excerpt :

      The method was originally developed in field bean (Vicia faba) and to date has been used for chromosome sorting in 29 species (Supplementary Table 1). In some cases, genetically transformed ‘hairy’ root cultures were used as an alternative to young seedlings to obtain actively growing root tips (Veuskens et al., 1995; Neumann et al., 1998). Another obstacle in applying chromosome flow sorting to plant genetics and genomics has been discriminating individual chromosome types.

    • Advances in plant chromosome genomics

      2014, Biotechnology Advances
      Citation Excerpt :

      Apart from karyological stability, the advantage of using root tips is that seedlings can be obtained in a majority of plants and roots can be exposed to various treatments using a hydroponic system. The procedure can be extended to species which produce few (or no) seeds by inducing hairy root cultures (Neumann et al., 1998; Veuskens et al., 1995). A typical root tip-based protocol (e.g., Vrána et al., 2012) involves seed germination, the exposure of roots of young seedlings to hydoxyurea (a DNA synthesis inhibitor) to arrest the cells at the G1/S interface, followed by recovery to synchronize the cell cycle through the S and G2 phases and into mitosis.

    • Emerging technologies advancing forage and turf grass genomics

      2014, Biotechnology Advances
      Citation Excerpt :

      Recent progress in flow sorting of chromosomes has been stimulated mainly by the use of cytogenetic stocks including deletions, translocations, alien chromosome and chromosome arm additions. The dissection of crop genomes into individual chromosomes was enabled by using those stocks in barley, wheat, rye, maize and pea (Kubalakova et al., 2002, 2003; Li et al., 2001; Neumann et al., 1998; Suchankova et al., 2006). However, the plasticity of grass genomes, including a certain tolerance of aneuploidy and a rather relaxed chromosome pairing system, limit the development of such cytogenetic stocks in forage and turf grasses.

    View all citing articles on Scopus
    View full text