Cryopreservation of Hazelnut (Corylus avellana L.) Axillary Buds from In Vitro Shoots Using the Droplet Vitrification Method

Alessandra Sgueglia,Emilia Caboni,Gaia Urbinati,Simona Lucioli,Andrea Frattarelli,Maria Antonietta Germanà,Adele Gentile

doi:10.3390/horticulturae7110494

Abstract

Cryopreservation by droplet vitrification was applied to hazelnut (Corylus avellana L.). axillary buds of the Italian cultivated variety Tonda Gentile Romana, which were collected from in vitro growing shoots, immersed in ice cooled PVS2 or PVS3 for 60 or 90 min, then transferred to a droplet of vitrification solution, placed on a strip of aluminium foil, and plunged into liquid nitrogen (LN). Additionally, the effect on the recovery of the mother plant after cryopreservation was evaluated, following a cold pre-treatment at 4 °C for 3 months. The highest regrowth percentage (56.7%) was obtained after applying PVS3 for 60 min, while the application of PVS2 for the same amount of time reduced regrowth to 41.5%. Increasing the exposure to vitrification solutions to 90 min reduced regrowth to 43.3% when PVS3 was applied, and 35.6% if PVS2 was used. The cold pre-treatment on the mother plant did not significantly improve overall regrowth. The cryopreservation process did not decline the rooting ability of the recovered shoots.

Highlights

IntroductionThe development of efficient ex situ conservation methods has an essential role in the conservation of biodiversity and avoidance of genetic erosion
When explants were not immersed in liquid nitrogen (LN), the treatment with PVS2 and PVS3, When explants were not immersed in LN, the treatment with PVS2 and PVS3, apapplied for 60 min, did not reduce regrowth with respect to the explants not immersed in plied for 60 min, did not reduce regrowth with respect to the explants not immersed in vitrification solutions solutions
Defining the suitable exposure duration of explants to the vitrification solutions is essential for cell dehydration, necessary to avoid the formation of intracellular ice crystals during freezing and thawing, and to prevent injury by chemical toxicity of cryoprotectants [56,57] or excessive osmotic stress [30]

Summary

Introduction

The development of efficient ex situ conservation methods has an essential role in the conservation of biodiversity and avoidance of genetic erosion. Cryopreservation reduces the costs of maintenance as well as the risks of material losses caused by biotic and abiotic factors in respect to the in-field and in-greenhouse preservation [1,2,3,4,5]. The studies and application of in vitro-based ex situ cryopreservation have been largely increased in recent years [1,2,3]. Several cryopreservation protocols, based on dehydration–encapsulation [6], vitrification [7], and a combination or modification of these methods [8,9], have been established for several species, including woody fruit species [10,11,12,13,14,15]

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Conclusion