Cryopreservation of in vitro-grown shoot tips of carnation (Dianthus caryophyllus L.) by vitrification method using aluminium cryo-plates

Abstract

Cryopreservation using an aluminum cryo-plate was successfully applied to in vitro-grown carnation (Dianthus caryophyllus L.) shoot tips. The shoot tips (1-1.5 mmϫ1 mm) were dissected from the shoot and precultured at 25°C for 2 days on MS medium containing 0.3 M sucrose. The precultured shoot tips were placed on the aluminum cryo-plate containing ten wells embedded with alginate gel. Osmoprotection was performed by immersing the cryo-plates in loading solution (2 M glycerol and 1.4 M sucrose) for 90 min at 25°C. Then, dehydration was performed by immersing the cryoplates in PVS2 for 25 min at 25°C. After storage in liquid nitrogen, shoot tips attached to cryoplate, were directly immersed into 2 ml 1 M sucrose solution for regeneration. Using this procedure, the average regrowth level of vitrified shoot tips of 4 carnation cultivars reached 95%. This new method has many advantages and will facilitate the cryostorage of reference cultivars of carnation. (LS) and PVS2 without floating and/or clinging of shoot tips and also with no or low level of injury to those shoot tips. In this paper, this V-Cryo-plate procedure was applied for the cryopreservation of carnation.The carnation plants used in this study were obtained from the Nishinihon Station, NCSS, sub-bank of NIAS Genebank Project, in Japan in April 2010. Four cultivars of carnation, 'Melissa', 'Grana', 'Terra Cotta' and 'Yell', were tested. Apical shoots, excised from in vivo explants, were surface-sterilized successively with 70% ethanol for 1 min, and 0.5% sodium hypochlorite for 10 min, and then rinsed twice with sterilized distilled water. After surface sterilization, shoot tips with basal plates (1 mm longϫ1 mm wide) were excised from the shoots and cultured on 70 ml of 0.4% (w/v) gellan gum-solidified MS medium (Murashige, Skoog 1962) in a culture flask (80 mmϫ100 mm) containing 1 mg l Ϫ1 thidiazuron, 1 mg l Ϫ1 naphthalene acetic acid and 3% (w/v) sucrose (Jose et al. 2010). After 50 days of culture, shoots were transferred onto gellan gum-solidified MS medium devoid of plant growth regulators. Then stock plants were subcultured every two months on MS medium. Cultures were incubated at 25°C with a 16 h photoperiod under white fluorescent light (52 mmol m Ϫ2 s Ϫ1) in the culture flask (standard condition). For cryopreservation of in vitro shoot tips of carnation, the V-Cryo-plate procedure was applied. The size of the aluminum cryoplate used was 7 mmϫ37 mmϫ0.5 mm with ten wells (diameter 1.5 mm, depth 0.75 mm) ( Figure 1B). These plates fit in 2 ml cryotube. The different steps of the VCryo-plate procedure are as follows; 1) Cut shoots (5 mm) with a lateral bud and plate on solidified MS medium and culture for 2 weeks at 25°C in standard conditions ( Figure 1A). Then, dissect shoot tips with basal plate (1-1.5 mm longϫ1 mm wide) from the shoots and preculture for 2 days at 25°C on the MS medium with 0.3 M sucrose. 2) Place an aluminum cryo-plate in Petri-dish and pour 2.0-2.5 ml 2% (w/v) Na-alginate solution with 0.4 M sucrose in calcium-free M...

Morphogenetic response of shoot tips to cryopreservation by encapsulation-dehydration in a solid mutant and periclinal chimeras of Chrysanthemum × grandiflorum /Ramat./Kitam.

“…The influence of cultivar on the cryopreservation efficiency was also observed with pelargonium, gladiolus and chrysanthemum, in which the survival levels ranged between 9.1-70.6, 4.9-28.0 and 46.0-91.0%, respectively (Gallard et al 2008;Joung et al 2007;Halmagyi et al 2004). In contrast, with rose and carnation, the genetic factor remained insignificant (survival rates: 65 and 95%, respectively) (Halmagyi and Pinker 2006;Sekizawa et al 2011). Moreover, our studies clearly show that cryopreservation influences the morphogenetic response of shoot tips, and that their reaction in post-thaw culture is also dependent on genotype.…”

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).…”

Cryopreservation of Plant Genetic Resources: Conventional and New Methods

“…Vitrification-based cryopreservation methods have been combined with other elements to develop new techniques, such as encapsulation/vitrification (Matsumoto et al, 1995a) and droplet vitrification (Schafer-Menuhr et al, 1997;Keller and Dreiling, 2002;Kim et al, 2009). Recently, cryopreservation protocols using an aluminum cryo-plate have also been reported (Sekizawa et al, 2011;Yamamoto et al, 2011Niino et al, 2013). However, scientists have focused their efforts towards preventing potential genetic changes in cryopreserved and regenerated plant tissues.…”