Critical factors affecting an efficient micropropagation protocol for Pyrus spinosa Forskk.

Abstract: Almond-leaved tree is one of the most commonly found native forest species in Greece, exhibiting valuable properties and thus suitable for multipurpose silviculture. Several critical factors were studied for the development of a successful micropropagation protocol of Pyrus spinosa juvenile trees. Newly immerged shoots of three-year-old plants, after their surface sterilization, were established on a modified MS nutrient medium (thiamine-HCl 1 mg L^-1, nicotinic acid 1 mg L^-1, pyridoxine-HCl 1 mg L^-1) with 5 ?? ??. Clean explants were transferred in the multiplication stage on a novel medium (Pear Medium 1), by adding 10 ?? ?? especially developed for Pyrus species. Due to poor culture development, the effect of photosynthetic photon flux density (PPFD) on the improvement of regeneration was studied. The exposure of explants to 10 ?mol m^-2 s^-1 for the first two weeks followed by exposure to 35 ?mol m^-2 s^-1 for another two weeks, was proved essential for the good development of cultures promoting both multiplication and elongation of explants. For further enhancement of shoot regeneration, the use of Pear Medium 1 with five different combinations of growth regulators was tested (BA, IBA). The most beneficial for the development of good quality shoots was 5 ?? ??+0.0246 ?? ??? (4.67±0.40 new shoots per explant, elongation 1.28±0.13 cm). As multiplication was mainly based on axillary branching and the production of new shoots was still relatively low, the orientation of explants (horizontal vs upright position) in relation to the medium was investigated. Regeneration of shoots almost tripled, reaching 13.67 new shoots per explant in the case of horizontal orientation after the removal of the apical part (0.2 cm). The most successful rooting procedure (rooting: 83.33±5.89 %, root no: 6.20±0.49 roots per plantlet, root length: 0.56±0.05 cm) consisted of an initial stage of root induction maintaining microshoots in complete darkness for seven days. The rooting medium was a modified MS (½ NH₄NO₃, ½ KNO₃) supplemented with 24.6 ?? ???. Microshoots were subsequently transferred to a root development stage in the same rooting medium without auxin, exposed to 10 ?mol m^-2 s^-1 for another four weeks. Successful acclimatization (87.5 %) was achieved after six weeks on perlite.