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

Mariam Gaidamashvili,Maurizio Lambardi,Eka Khurtsidze,Carla Benelli,Tamari Kutchava

doi:10.3390/plants10020231

Mariam Gaidamashvili, Maurizio Lambardi + Show 3 more

Open Access

https://doi.org/10.3390/plants10020231

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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 Castanea sativa embryonic axes.

Highlights

The present study describes, for the first time, a protocol for the cryopreservation of the excised embryonic axes (EAs) of Castanea sativa L. by using the “encapsulation–vitrification”
EA regrowth remained in the range of 72.2% to 68.8% (30 and 90 min of treatment), and the regrowth of EAs treated for 120 min was reduced to 46.2% (120 min) (Figure 1B)
Only the loading treatment induced tolerance to ultrarapid cooling in LN, as survival and plantlet regrowth of encapsulated EAs passed from nil to almost 16.7% and 8.3%, respectively (Figure 1A,B, 0 treatment)

Summary

Introduction

Castanea, is dominant in the mountainous forests of Western Georgia (150–1800 m), occupying the highest percentage of areas covered with forests Chestnut forests are developed in both West and East Georgia, but to the West of the country, they occupy larger areas. According to the official International Union for Conservation of Nature (IUCN) list, Castanea sativa has been assessed as Least Concern [3,4]. The reason for including Castanea sativa in the Red List is the fragmentation and decreased distribution range. The ex situ conservation of chestnut in seed banks is limited due to nonresistance to storage at low-temperature conditions of partially dehydrated recalcitrant seeds [8,9].

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