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Plant regeneration from different explant types of Bituminaria bituminosa and furanocoumarin content along plant regeneration stages

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Abstract

The first protocol for in vitro plant regeneration from different explants of Bituminaria bituminosa, a pasture and medicinal species, has been established. Three explant types (petiole, leaflet and petiole-leaflet attachment “PLA”) cultured on media with different combinations of benzylaminopurine (BA; 5.0, 10.0 or 20.0 μM) and naphthalene acetic acid (NAA) or indole acetic acid (IAA; 0.5 or 5.0 μM) were tested for calli induction, and with 5 μM BA + 0.5 μM NAA or IAA for shoot development. The average number of shoots (≥5 mm) per callus depended on the explant type and the calli induction medium. The highest average number of shoots per callus was achieved by culturing leaflet and PLA explants on 5 μM IAA + 10 μM BA for calli induction and on 0.5 μM IAA + 5 μM BA for shoot development, and by culturing petiole explants on 0.5 μM NAA + 10 μM BA followed by a second culture on 0.5 μM NAA + 5 μM BA. The highest frequency of shoot rooting was achieved with 10.0 μM NAA and 1.0 μM gibberellic acid (GA3). Rooted plants were acclimatised in a culture chamber, reaching 96 % survival. Acclimatised plants were transferred to a greenhouse and finally to the field, reaching 100 % survival. The furanocoumarin (FC) accumulation was evaluated in organogenic calli, in vitro shoots, ex vitro plants in the greenhouse and in ex vitro plants in the field (after 1 and 4 months of acclimatisation). The content of FCs depended on the plant material evaluated, being higher in ex vitro plants in the field (up to 9,824 μg g−1 DW total FC) and lowest in organogenic calli (up to 50 μg g−1 DW total FC). This effect may be due to cell organization, longer exposure to environmental factors and the developmental stage.

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Abbreviations

BA:

6-benzylaminopurine

NAA:

Naphthalene acetic acid

IBA:

Indole-3-butyric acid

IAA:

Indole-3-acetic acid

GA3 :

Gibberellic acid

PGR:

Plant Growth Regulator

½ MS medium:

Half strength of Murashige and Skoog (1962) mineral solution

FC:

Furanocoumarin

AC:

Activated charcoal

DW:

Dry weight

PLA:

Petiole-leaflet attachment

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Acknowledgments

We thank A. Palazón and V. Arnau for their collaboration and excellent technical assistance and Dr. Walker for his critical review of the manuscript. This research was supported by Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (RTA2007-00046-00-00), Ministerio de Ciencia e Innovación, (BFU2010-19599) and by a studentship provided by IMIDA to M. Pazos-Navarro. M. Dabauza was co-supported by the European Social Fund and IMIDA.

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Authors and Affiliations

  1. Department of Biotechnology and Crop Protection, Instituto Murciano de Investigación y Desarrollo Agrario y Alimentario (IMIDA), C/Mayor s/n, 30150, La Alberca, Murcia, Spain

    María Pazos-Navarro & Mercedes Dabauza

  2. Department of Plant Biology, Faculty of Biology, University of Murcia, Espinardo, 30100, Murcia, Spain

    José Antonio Del Río & Ana Ortuño

  3. Department of Natural Resources, IMIDA, C/Mayor s/n, 30150, La Alberca, Murcia, Spain

    Pascual Romero-Espinar & Enrique Correal

Authors
  1. María Pazos-Navarro
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  2. José Antonio Del Río
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  3. Ana Ortuño
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  4. Pascual Romero-Espinar
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  5. Enrique Correal
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  6. Mercedes Dabauza
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Correspondence to Mercedes Dabauza.

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Pazos-Navarro, M., Del Río, J.A., Ortuño, A. et al. Plant regeneration from different explant types of Bituminaria bituminosa and furanocoumarin content along plant regeneration stages. Plant Growth Regul 70, 123–129 (2013). https://doi.org/10.1007/s10725-013-9784-9

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  • DOI: https://doi.org/10.1007/s10725-013-9784-9

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