Article

Micropropagation of Juniperus phoenicea from adult plant explants and analysis of ploidy stability using flow cytometry. Biol Plant

Division of Biology, University of Aveiro, Aveiro, Aveiro, Portugal
Biologia Plantarum (Impact Factor: 1.85). 03/2007; 51(1):7-14. DOI: 10.1007/s10535-007-0003-2

ABSTRACT

We report here the successful micropropagation of adult Juniperus phoenicea L. with respective ploidy stability studies. Microcuttings with axillary buds were grown on five media supplemented with different growth regulator combinations. Best elongation rates were achieved on Driver and Kuniyuki (DKW) medium supplemented with kinetin alone or with naphthaleneacetic acid (NAA), while Rugini olive (OM) medium stimulated the development of new branches. Shoots growing on Murashige and Skoog (MS) medium browned and showed necrotic zones. Shoots of second to fourth subcultures usually had higher elongation rates than those of the first culture. For rooting assays, half strength DKW and OM media, different concentrations of growth regulators, auxin continuous exposure vs. dipping and the type of solid matrix were assessed. During rooting assays, two morphotypes were observed with one type having well developed internodes and the other showing hyperhydratation and no internode development. High rooting rates (40 %) were only obtained in the first morphotype shoots exposed for 5 min to 2.4 µM IBA and then transferred to OM medium without growth regulators. Plants were acclimatized in pots containing a mixture of peat and Perlite (3:2) in greenhouse with progressive reduction of relative humidity. A flow cytometric screening for major ploidy changes revealed no differences among the morphotypes and between them and the mother plant. Also the nuclear DNA content of this species was estimated for the first time using flow cytometry (2C = 24.71 pg). Additional key words: in vitro culture, nuclear DNA content, plant acclimatization, rooting studies.

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    • "Nuclear suspensions from somatic embryos and in vitro leaves were prepared according to Galbraith et al. (1983). In brief, nuclei were released from cells by chopping with a razor blade in 1 ml -1 of Woody Plant Buffer (WPB) (0.2 M Tris HCl, 4 mM MgCl 2 Á6H 2 O, 2 mM EDTA Na 2- 2H 2 O, 86 mM NaCl, 10 mM sodium metabisulfite, 1 % PVP-10, 1 % (v/v) Triton X-100, pH 7.5) (Loureiro et al. 2007a). For ploidy analysis, Alnus samples were chopped together with the same amount of the internal reference standard Zea mays (cv CE-777 = 5.43 pg DNA; kindly provided by J. Dolezel, Laboratory of Molecular Cytogenetics and Cytometry, Institute of Experimental Botany, Olomouc, Czech Republic). "
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    ABSTRACT: Germplasm preservation plays an important role in current breeding programs. A simple vitrification procedure that allows for the reproducible cryopreservation of two alder embryogenic lines is presented for the first time. Somatic embryos clumps (1–2 mm) were precultured in hormone-free medium (Murashige and Skoog half-strength macronutrients, MS1/2) supplemented with 0.3 M sucrose for 3 days, and then treated with a mixture of 2 M glycerol plus 0.4 M sucrose for 20 min at 25 °C. Osmoprotected somatic embryos were dehydrated using plant vitrification solution 2 (PVS2). The effect of different PVS2 incubation times was evaluated, and 60 min at 0 °C was considered to be the optimum period. After changing the solution with fresh PVS2, the somatic embryos were directly immersed in liquid nitrogen. Following rapid thawing in a water bath at 40 °C for 2 min, the somatic embryos were transferred onto MS1/2 supplemented with 0.1 mg l−1 benzyladenine, 30 g l−1 sucrose and 6 g l−1 Vitro agar. The cultures were kept in the dark for 1 week prior to exposure to light (16 h/8 h light/dark cycle). The recovery rate of vitrified somatic embryos reached over 90 % in both embryogenic lines. Cryopreservation did not affect the plant regeneration potential of Alnus glutinosa through somatic embryogenesis. The ploidy stability of the regenerated material was assessed by flow cytometry. Analysis of DNA ploidy stability of the control, PVS2 treated, cryopreserved somatic embryos and plantlets showed no significant differences.
    Full-text · Article · Aug 2015 · Plant Cell Tissue and Organ Culture
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    • "In vitro propagation is a valuable complement to classical breeding strategies (Santos et al., 2005; Aslam et al., 2012a), which provides an opportunity to develop clones with improved productivity or resistance (Santos et al., 2006; Aslam et al., 2012b). However, it is already known that tissue culture based technique can induce somaclonal variation like mutation and/or epigenetic changes (Loureiro et al., 2007), which may hamper the implementation of clonal forestry programs or, may provide interesting mutants. Somatic Embryogenesis (SE) is the most promising method for clonal mass propagation, because both root and shoot meristems are present (Aslam et al., 2012a,b). "
    Dataset: 1-8

    Full-text · Dataset · Jul 2015
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    • "In vitro propagation is a valuable complement to classical breeding strategies (Santos et al., 2005; Aslam et al., 2012a), which provides an opportunity to develop clones with improved productivity or resistance (Santos et al., 2006; Aslam et al., 2012b). However, it is already known that tissue culture based technique can induce somaclonal variation like mutation and/or epigenetic changes (Loureiro et al., 2007), which may hamper the implementation of clonal forestry programs or, may provide interesting mutants. Somatic Embryogenesis (SE) is the most promising method for clonal mass propagation, because both root and shoot meristems are present (Aslam et al., 2012a,b). "

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