Genotype-dependent mass somatic embryogenesis: a chance to recover extinct populations of Pulsatilla vulgaris Mill.

The paper presents a technique for micropropagation of endangered in Europe and extinct in Poland Pulsatilla vulgaris for ex situ conservation of the genetic resources. Genotype-dependent induction of somatic embryogenesis and rooting was revealed in series of two experiments (I and II) performed under the same experimental conditions. Shoot tips of seedlings were the best explants in both experiments and Murashige and Skoog (MS) medium supplemented with 0.25 or 0.5 mg L−1 BAP was suitable for induction of somatic embryos (SE) and adventitious shoots. Mass SE was obtained in experiment I after explants transfer on ½ MS (2% sucrose) + 0.45 mg L−1 B1 and extending culture to 2–3 months without passages. Rooting of adventitious shoots was a critical point. Out of seven rooting media used in experiment I, only two, ½ MS hormone free (2% sucrose) + 0.45 mg L−1 B1 or MS + 5 mg L−1 NAA + 3.76 mg L−1 B2 resulted in altogether 36.4% rooted shoots. In experiment II, somatic embryogenesis, rooting and acclimatization of adventitious shoots failed. Regenerated plantlets and seedlings converted from SE from experiment I were acclimatized to ex vitro conditions. Both genome size, determined by flow cytometry, and genetic diversity analyzed by ISSR markers, confirmed the compatibility of regenerants from experiment I with P. vulgaris initial seedlings and commercial cultivar. Regenerants obtained in experiment II differed genetically from the regenerants of experiment I and cultivar. Propagated in vitro tissues/organs (SE, adventitious shoots) of P. vulgaris could be a source of material for cryopreservation, artificial seed production and/or for acclimatization of regenerated plantlets and could be used for restoration of the extinct populations. The micropropagation technique via organogenesis and somatic embryogenesis of endangered in Europe pasqueflower was developed as a tool for species recovery. The critical point is that somatic embryogenesis is genotype-dependent, which affects the repeatability of the experiments and also imposes applying molecular techniques to confirm the genetic fidelity of the regenerants with the initial material and other genotypes.