Organogenesis induction in horse chestnut (Aesculus hippocastanum L.).
Horse chestnut (Aesculus hippocastanum L.) belongs to horticulturally valuable woody species. Successfulness of its planting in Europe has decreased due to a pest invasion, the horse chestnut leaf miner Cameraria ohridella Deschka & Dimic, 1986. Research objective of this work was to induce organogenesis in explants taken from seedlings of horse chestnut individuals. Shoot tip meristems with 1-2 leaf primordia were isolated from surface sterilized vegetative buds and were placed onto WPM (Lloyd, Mccown 1980) and MS (Murashige, Skoog 1962) medium containing a half salt concentration and benzyladenine (BA) and naphthylacetic acid (NAA). Subcultivation onto medium with an identical composition was performed every 3-6 weeks. In winter buds, where a high microbial contamination (bacteria and yeasts) was observed, two methods of antibiotic treatment were used: (i), antibiotics (Hygromycin, Timentin, Cefotaxim) were added into cultivation medium; (ii) the buds were surface-treated with various concentrations of Antibiotic-Antimycotic Stabilized suspension (treatment A) and Amphothericin B (treatment B) as provided by Sigma. This technique of bud treatment showed to be more effective, decreased contamination rate and increased proliferation were found depending on antibiotic concentration. Recommended basic amounts of A (10 ml.l-1) and 25 mg.l-1 B were efficient, twofold doses induced explant necrosis. In spite of efficient explant decontamination demonstrated after 20 days, no viable in vitro culture was derived. In summer buds, a low bacterial infection (13%) and the highest shoot regeneration (up to 80%) were ascertained in shoot tips. Nodal stem and petiole segments formed calli with low or none regeneration capacity. A critical factor in organogenesis induction was the collection time of vegetative buds. A suitable time for explant regeneration was July-August when the explants showed low microbial contamination and decreased phenol production. Sterilizing method, 2.5% calcium hypochlorite with 20-30 min bud treatment, showed to be suitable. The highest shoot regeneration was observed in the presence of BA in concentration of 0.5 mg.l-1 and 0.1 mg.l-1 naphthylacetic acid (NAA).