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Genetic transformation of alfalfa somatic embryos and their clonal propagation through repetitive somatic embryogenesis

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Abstract

Genetically transformed alfalfa (Medicago sativa L., cv. Zaječarska 83) plantlets were obtained by inoculating somatic embryos with Agrobacterium tumefaciens strains A281/pGA472 and LBA4404/pBI121. Single somatic embryos, 5–7 mm long, were released from a repetitively embryogenic culture, wounded, and cocultivated with the bacteria. The agar-solidified culture medium contained mineral salts, vitamins, 40 g l−1 sucrose, 1 g l−1 yeast extract and 0.05 mg l−1 BA. Five clones, transformed with A281/pGA472, and 4 clones transformed with LBA4404/pBI121, were selected for proliferation by repetitive somatic embryogenesis, on media containing 100 mg l−1 of kanamycin. The transformation of kanamycin-resistant clones was confirmed by assaying the activity of neomycin phosphotransferase II and/or β-glucuronidase enzymes, and by the Southern blot analysis. It is suggested that the transformation/regeneration system based on somatic embryogenesis may be suitable for establishing transgenic alfalfa lines. The relatively low frequency of embryo transformation is compensated for by abundant proliferation in secondary somatic embryogenesis.

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Abbreviations

BA:

6-benzyladenine

GUS:

β-glucuronidase

Km:

kanamycin

NPTII:

neomycin phosphotransferase II

X-gluc:

5-bromo-4-chloro-3-indolyl-β-glucuronic acid

BM:

basal medium

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Authors and Affiliations

  1. Institute for Biological Research “S. Stanković”, University of Belgrade, 29 novembra 142, 11060, Belgrade, Yugoslavia

    Slavica Ninković, Jovanka Miljuš-Djukić & Mirjana Nešković

  2. Institute of Botany, Faculty of Science, University of Belgrade, 29 novembra 142, 11060, Belgrade, Yugoslavia

    Slavica Ninković, Jovanka Miljuš-Djukić & Mirjana Nešković

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  1. Slavica Ninković
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  2. Jovanka Miljuš-Djukić
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Ninković, S., Miljuš-Djukić, J. & Nešković, M. Genetic transformation of alfalfa somatic embryos and their clonal propagation through repetitive somatic embryogenesis. Plant Cell Tiss Organ Cult 42, 255–260 (1995). https://doi.org/10.1007/BF00029996

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