Cryopreservation of somatic embryos of Alnus glutinosa (L.) Gaertn. and confirmation of ploidy stability by flow cytometry

Abstract

Germplasm preservation plays an important role in current breeding programs. A simple vitrification procedure that allows for the reproducible cryopreservation of two alder embryogenic lines is presented for the first time. Somatic embryos clumps (1–2 mm) were precultured in hormone-free medium (Murashige and Skoog half-strength macronutrients, MS1/2) supplemented with 0.3 M sucrose for 3 days, and then treated with a mixture of 2 M glycerol plus 0.4 M sucrose for 20 min at 25 °C. Osmoprotected somatic embryos were dehydrated using plant vitrification solution 2 (PVS2). The effect of different PVS2 incubation times was evaluated, and 60 min at 0 °C was considered to be the optimum period. After changing the solution with fresh PVS2, the somatic embryos were directly immersed in liquid nitrogen. Following rapid thawing in a water bath at 40 °C for 2 min, the somatic embryos were transferred onto MS1/2 supplemented with 0.1 mg l−1 benzyladenine, 30 g l−1 sucrose and 6 g l−1 Vitro agar. The cultures were kept in the dark for 1 week prior to exposure to light (16 h/8 h light/dark cycle). The recovery rate of vitrified somatic embryos reached over 90 % in both embryogenic lines. Cryopreservation did not affect the plant regeneration potential of Alnus glutinosa through somatic embryogenesis. The ploidy stability of the regenerated material was assessed by flow cytometry. Analysis of DNA ploidy stability of the control, PVS2 treated, cryopreserved somatic embryos and plantlets showed no significant differences.