Cryopreservation of embryogenic suspension cultures of Cyclamen persicum Mill.

Abstract

We have developed a simple protocol for the cryopreservation of embryogenic suspension cultures of Cyclamen persicum. Embryogenic suspension cultures in the linear growth phase 7-10 days after subculture were used for cryopreservation. Of the different cryoprotectants tested during a 2-day pre-culture, 0.6 M sucrose resulted in the highest re-growth rates of 75%. An additional pre-treatment with 0.6 M sucrose and 10% DMSO (dimethylsulfoxide) for 1 h also positively affected re-growth. Microscopic studies on viability revealed that only few small embryogenic cells survived cryopreservation, while vacuolated single cells died. Experiments in which the duration of the pre-culture period--i.e. the length of time the embryogenic suspension cells were exposed to 0.6 M sucrose--was varied showed that 2-4 days was the most optimal exposure time to 0.6 M sucrose. Callus re-grown after cryopreservation showed growth rates similar to that of unfrozen callus and regenerated even higher numbers of somatic embryos than unfrozen callus.

Development of an efficient regeneration protocol for four Cyclamen species endemic to Turkey

“…C. persicum showed very low levels of somatic embryogenesis when mature tissues or organs were used as explants Schmidt et al 2006). In addition, there was a considerable decrease in the embryogenic competence of C. persicum callus as subcultures increased Winkelmann et al 2004). Borchert et al (2007) C. cilicium C. parviflorum C. mirabile C. pseudibericum…”

“…Somatic embryogenesis in Cyclamen is affected by genotype, source of explant, medium content, PGRs, and their concentrations and culture conditions (Tagipur et al 2016). The development of efficient somatic embryogenesis protocols in C. persicum may represent a useful method for the micropropagation of selected material, genetic transformation via Agrobacterium tumefaciens and cryopreservation (Aida et al 1999;Boase et al 2002;Winkelmann et al 2004;Terakawa et al 2008). Most of the previous research dealing with Cyclamen somatic embryogenesis included histological examination of the obtained structures which were classified as somatic embryos (Wicart et al 1984;Kiviharju et al 1992;Ishizaka and Uematsu 1995;Takamura et al 1995;Ruffoni et al 1998;Hoenemann et al 2010).…”

Simple cryopreservation protocol with an encapsulation technique for tobacco BY-2 suspension cell cultures

“…Sufficiently dehydrated cells are vitrified by rapid immersion into LN and able to survive without cryoinjury caused by the formation of intracellular ice crystals. Slow prefreezing (equilibrium freezing) procedure with a slow cooling rate can be achieved with simple cooling in a laboratory freezer rather than a programmable freezer (Sakai et al 1991;Menges and Murray 2004;Winkelmann et al 2004). With our convenient procedure, BY-2 cells are cryopreserved by simple cooling in a laboratory freezer at Ϫ30°C without specialized equipment (Figure 3).…”

Somatic Embryogenesis in Temporary Immersion Bioreactors

“…Somatic embryos can be produced in liquid media in flasks and in bioreactors by culturing embryogenic suspensions, such as coffee (Ducos et al 2007a), conifer (Gupta and Timmis 2005), and cyclamen (Winkelmann et al 1998). However, the embryogenic suspension cultures have some problems, such as the need to transfer the later-stage embryos to semi-solid medium and the requirement of constant movement, which prevents the development of plants from vitrification and anoxia, due to the continuous contact of explant with the liquid medium and the lack of aeration (Etienne et al 1997).…”