A protocol has been developed for the propagation of Alnus glutinosa axillary shoots in liquid medium. The explants were cultured in Woody Plant Medium supplemented with 0.1 mg l − 1 benzyladenine and 0.5 mg l − 1 indole acetic acid. The effect of the bioreactor type (RITA® and Plantform™), frequency of the immersions, regulator concentrations, and volume of medium per explant was investigated. All the treatments with the temporal immersion systems (TIS) increased the proliferation rates. The best results were obtained on using the Plantform™ vessels and, unlike the shoots cultured in RITA® vessels, the culture in Plantform™ had very low hyperhydricity percentages. Shoots originating from the culture in semisolid medium and in Plantform™ rooted in semi-solid medium with 0.1 mg/l indole butyric acid for 7 days. No significant differences were observed in the rooting or acclimatization percentages, with survival percentages greater than 85% being achieved. This is the first work on the use of TIS systems in alder propagation, with the results of this study providing new perspectives for its mass propagation.
Key messageA new protocol is described in order to reduce the propagation costs of alder.
Micropropagation techniques are valuable tools for propagating, conserving and restoring trees. An efficient micropropagation method involving axillary shoot proliferation of material obtained from mature European alder (Alnus glutinosa (L.) Gaertn.) trees was developed. Branch segments from trees aged 20-30 years were forced to flush, and explants derived from new shoots were cultured on Woody Plant Medium supplemented with 8.88 µM benzyladenine and 2.85µM indole-3-acetic-acid. In vitro establishment was achieved in all five genotypes evaluated. Shoot cultures were maintained by sequential subculture of explants on the same medium supplemented with 0.88-0.44 µM benzyladenine and 2.85 µM indole-3-acetic acid. Transfer to fresh medium every 3 weeks during a 9-week multiplication period and the inclusion of 2.28 µM zeatin during the last 3 weeks of culture improved the multiplication rate and shoot quality. Use of 2% glucose as the carbohydrate source produced better results than 3% sucrose for shoot proliferation. In vitro rooting of shoots was achieved with 2% glucose and 0.49 µM indole-3-butyric acid for 7 days, followed by in vitro culture on auxin-free medium for 21 days. Rooted plantlets were acclimatized to the greenhouse and were viable for reintroduction into the natural habitat.
A study of the in vitro rooting process in mature alder (Alnus glutinosa (L.) Gaertn.) shoots is described. Microcuttings from shoots cultured in vitro were transferred to a half-strength Woody Plant Medium containing 0 or 0.1 mg l -1 indole-3-butyric acid (IBA) for 0 to 7 days. The presence of IBA in the medium increased the rooting percentage, number of roots, percentage of lateral roots, and length of the shoots. Histological studies were carried out with shoots treated with 0 or 0.1 mg l -1 IBA for 7 days. According to these criteria, treatment with IBA for 2-3 days proved to be the most successful. In both treatments, substancial reactivation of cell division was observed at the base of the shoots after 1 day. Some cambial zone and adjacent phloem cells became dense cytoplasm, having nuclei with prominent nucleoli. The first cell divisions were also observed at this time. In the treatment with IBA (0.1 mg l -1 for 7 days), meristemoids became individualized, consisting of densely staining cells, by day 3. Identifiable conical shaped root primordia with several cell layers were visible after 4-5 days. Roots with an organized tissue system emerged from the stem after 6 days in the IBA-treated shoots. Meristemoid formation was delayed until the fourth day and root emergence until the eight day in the control treatment (no IBA).
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