Somatic embryogenesis and regeneration from shoot primordia of Crocus heuffelianus

Abstract

Crocus heuffelianus belongs to the C. vernus (Iridaceae) species aggregate. In the Carpathian Basin and particularly in Hungary it is considered an endangered species. Therefore our aim was to establish a tissue culture system with potential of germplasm preservation of this taxon. For in vitro culture experiments, shoot primordia from corms were the most suitable. We induced an embryogenic callus line from those explants on basal Murashige-Skoog (MS) medium supplemented with Gamborg’s vitamins, 2% (w/v) sucrose, 10 mg l−1 (53.7 μM) α-naphthaleneacetic acid (NAA) and 1 mg l−1 (4.44 μM) 6-benzyladenine (BA). Globular stage embryos developed on this medium and several culture conditions were used in an attempt to obtain mature embryos and plant regeneration. Firstly a decrease of auxin/cytokinin concentration and ratio, then secondly a decrease in the strength of culture medium and the concentration of carbon source was used, which was effective in embryogenesis and the production of plants. Regeneration medium used in the second step was fourfold diluted MS medium and Gamborg’s vitamins supplemented with 1% (w/v) sucrose, 0.05 mg l−1 (0.26 μM) NAA and 0.5 mg l−1 (2.22 μM) BA, with a 14/10 h photoperiod. Under these conditions we could detect all the stages of somatic embryo development characteristic for Iridaceae. This is the first report demonstrating the production of stable tissue culture of C. heuffelianus with potential use in germplasm preservation via plant regeneration. This study could also contribute to a better understanding of somatic embryogenesis in the Crocus genus.