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An optimized cryopreservation protocol for embryonic axes (EAs) of chestnut ( Castanea sativa  Mill.) has been developed based on the encapsulation–vitrification procedure. EAs of mature seeds were aseptically dissected and encapsulated in alginate beads with or without 0.3% ( w / v ) activated charcoal (AC). Embedded EAs were dehydrated with Plant Vitrification Solution 2 for different treatment times up to 120 min, followed by direct immersion in liquid nitrogen. Cryopreserved embryonic axes encapsulated with AC showed higher survival (70%) compared to those encapsulated without AC (50%). Sixty-four percent of embryonic axes, from synthetic seeds with AC, subsequently developed as whole plants. Plantlet regrowth was faster in AC-encapsulated EAs and showed enhanced postcryopreservation shoot and root regrowth over 2 cm after five weeks from rewarming. Results indicate that encapsulation–vitrification with activated charcoal added to the beads is an effective method for the long-term preservation of  Castanea   sativa  embryonic axes.

Efficient Protocol for Improving the Development of Cryopreserved Embryonic Axes of Chestnut (Castanea sativa Mill.) by Encapsulation–Vitrification