Plant Genetic Resources

Research Article

Cryopreservation of in vitro-grown shoot tips of strawberry by the vitrification method using aluminium cryo-plates

Shin-ichi Yamamotoa1, Kuniaki Fukuia1, Tariq Rafiquea2, Nayyar Iqbal Khana3, Carlos Roman Castillo Martineza4, Kentaro Sekizawaa5, Toshikazu Matsumotoa6 and Takao Niinoa1 c1

a1 Genebank, National Institute of Agrobiological Sciences, Tsukuba 305-8602, Japan

a2 Plant Genetic Resources Programme, National Agricultural Research Center, Islamabad, Pakistan

a3 NWFP Agricultural Research System, Abottabad, Pakistan

a4 National Genetic Resources Center, INIFAP, Tepatitlan, Mexico

a5 National Center for Seeds and Seedlings, Tsukuba 305-0852, Japan

a6 Faculty of Life and Environmental Science, Shimane University, Matsue 690-1102, Japan

Abstract

Cryopreservation using an aluminium cryo-plate was successfully applied to in vitro-grown strawberry (Fragaria × ananassa Duch.) shoot tips. The shoots were cold-hardened at 5°C for 3 weeks with an 8-h photoperiod. The shoot tips (1.5–2.0 mm × 0.5–1.0 mm) were dissected from the shoot and pre-cultured at 5°C for 2 d on Murashige and Skoog medium containing 2 M glycerol and 0.3 M sucrose. The pre-cultured shoot tips were placed on the aluminium cryo-plate containing ten wells embedded in alginate gel. Osmoprotection was performed by immersing the cryo-plates in a loading solution (2 M glycerol and 0.8 M sucrose) for 30 min at 25°C. Dehydration was performed by immersing the cryo-plates in plant vitrification solution 2 for 50 min at 25°C. Then, the cryo-plate with shoot tips was transferred into an uncapped cryotube that was held on a cryo-cane and directly immersed into liquid nitrogen (LN). After storage in LN, shoot tips attached to the cryo-plate were directly immersed into 2 ml of a 1 M sucrose solution for regeneration. Using this procedure, the average regrowth level of vitrified shoot tips of 15 strawberry cultivars reached 81%. This new method has many advantages and will facilitate the cryostorage of strawberry germplasm.

(Received September 20 2011)

(Accepted November 05 2011)

(Online publication November 28 2011)

Correspondence:

c1 Corresponding author. E-mail: niinot@affrc.go.jp

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