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

Abstract

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 Castaneasativa embryonic axes.

Choosing the Right Path for the Successful Storage of Seeds

“…Such high hydration of cells promotes the formation of ice crystals during the freezing and thawing of seeds, leading to lethal injuries, in particular to cellular membranes [ 58 ]. Even though entire recalcitrant seeds cannot be cryostored, their germplasm can be successfully preserved in LN in the form of tissues isolated from seeds, i.e., embryonic axes (EA) containing both shoot and root meristems [ 60 , 135 , 136 , 137 , 138 , 139 , 140 , 141 , 142 ] or plumules (containing only shoot meristem). To date, however, only plumules isolated from seeds of Quercus robur L., Q. petraea (Mattuschka) Liebl.…”

“…This modification allowed to enhance the post-cryopreservation shoot and root regrowth of AC-encapsulated embryonic axes to Castanea sativa Mill. [ 141 ]. Another option is the application of exogenous antioxidants at different procedural stages of cryopreservation.…”

An In Vitro Protocol for Propagating Castanea sativa Italian Cultivars

“…Although in vitro culture protocols have already been developed for the genus Castanea [ 1 , 4 , 5 , 17 , 18 , 19 , 20 , 21 ], large-scale propagation of C. sativa is currently not yet possible since it is a genotype-dependent technique and requires specific protocols for each species and cultivar, making it impossible to adopt a universal protocol. For this reason, since in vitro culture protocols for Italian varieties are not available, this research was focused on developing a protocol to micropropagate C. sativa cv.…”