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

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 58C 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 58C 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 258C. Dehydration was performed by immersing the cryo-plates in plant vitrification solution 2 for 50 min at 258C. 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.

Cryopreservation of blueberry shoot tips derived from in vitro and current shoots using D cryo-plate technique

“…Cryopreservation has unique attributes that minimize space and maintenance requirements without causing genetic alterations (Sakai 1997). Recently, two novel cryogenic protocols using aluminum cryo-plates were developed, the osmo-dehydration method (V cryo-plate; Sekizawa et al 2011;Yamamoto et al 2011Yamamoto et al , 2012aYamamoto et al , 2012b and the air-dehydration method (D cryoplate; Niino et al 2013). The major advantages of the cryo-plate methods are their user-friendly procedures, minimization of shoot injury, and rapid cooling and warming, which results in higher recovery (Matsumoto et al 2015;Niino et al 2013Niino et al , 2014Salma et al 2014).…”

Two Advanced Cryogenic Procedures for Improving Stevia rebaudiana (Bertoni) Cryopreservation

“…The DV procedure [ 19 ] is a method obtained by combining the vitrification procedure with the droplet-freezing technique developed by Kartha et al [ 39 ] in which explants are placed in minuscule droplets of vitrification solution on aluminium foil strips, whereas the V cryo-plate procedure [ 20 , 40 ] combines the encapsulation and droplet-vitrification techniques, in which explants are placed on an aluminium cryo-plate and embedded in a thin layer of calcium alginate gel.…”

Cryopreservation of Plant Genetic Resources: Conventional and New Methods

“…The V cryo-plate method has been reported for strawberry (Yamamoto et al, 2012b), Dalmatian chrysanthemum (Yamamoto et al, 2011b), mint (Yamamoto et al, 2012a), mulberry (Yamamoto et al, 2012c), carnation (Sekizawa et al, 2011), mat rush , blueberry (Matsumoto et al, 2014a), Perilla (Matsumoto et al, 2014b) and sugarcane shoot tips/buds. More recently, Niino et al (2013) reported on the D cryo-plate method.…”