Cryopreservation of in vitro-grown shoot tips of Alnus glutinosa (L.) Gaertn

• Published in: Acta physiologiae plantarum Vol. 36; no. 1; pp. 109 - 116
• Main Authors: José, M. Carmen San, Valladares, Silvia, Janeiro, Laura V, Corredoira, Elena
• Format: Journal Article
• Language: English
• Published: Berlin/Heidelberg Springer-Verlag 2014; Springer Berlin Heidelberg
• Subjects: Agriculture; Alnus glutinosa; Biomedical and Life Sciences; cryopreservation; genotype; glucose; glycerol; indole acetic acid; Life Sciences; lighting; nitrogen; Original Paper; photoperiod; Plant Anatomy/Development; Plant Biochemistry; Plant Genetics and Genomics; Plant Pathology; Plant Physiology; shoots; sucrose; vitrification; woody plants; zeatin; PVS2; Long-term conservation; Black alder; Vitrification; Shoot tips
• ISSN: 0137-5881; 1861-1664
• DOI: 10.1007/s11738-013-1391-x

In vitro-grown shoot tips of Alnus glutinosa (L.) Gaertn. were successfully cryopreserved by vitrification. Shoot tips (0.5–1 mm) excised from 6-week-old shoots were precultured in hormone-free Woody Plant Medium (WPM) supplemented with 0.2 M sucrose, for 2 days at 4 °C in the dark, and then treated with a mixture of 2 M glycerol plus 0.4 M sucrose, for 20 min at 25 °C. Osmoprotected shoot tips were first dehydrated with 50 % vitrification solution (PVS2), for 30 min at 0 °C, and then placed in 100 % PVS2, for 30 min at 0 °C. The solution was replaced with fresh 100 % PVS2, and the shoot tips were plunged directly into liquid nitrogen. The shoot tips were rewarmed in a water bath at 40 °C for 2 min, and then washed twice, for 10 min at 25 °C, with 1.2 M sucrose solution, before being transferred onto WPM supplemented with 0.5 mg l⁻¹ N ⁶-benzyladenine, 0.5 mg l⁻¹ indole-3-acetic acid, 0.2 mg l⁻¹ zeatin, 20 g l⁻¹ glucose and 6 g l⁻¹ Difco Bacto agar. The shoot tips were kept in darkness for 1 week and under dim lighting for another week, before being exposed to standard culture conditions (16 h photoperiod). This protocol was successfully applied to three alder genotypes, with recovery rates higher than 50 %.