A novel method of cryopreservation without a cryoprotectant for immature somatic embryos of conifer

Abstract

Cryopreservation of embryogenic tissue is an essential storage step in genotype selection and seedling production through somatic embryogenesis. To date, immature conifer somatic embryos, at the proliferation step, were only able to tolerate ultra low temperature after prior cryoprotectant treatments. We report a novel cryopreservation method for conifer (interior spruce and Douglas-fir) embryogenic tissue focusing on the maturation step of developing embryos that forgoes such cryoprotectant treatment. In this study, somatic embryos matured on culture media containing abscisic acid (ABA) at 20°C for 8 weeks. Typically, matured embryos in this manner were able to survive cryopreservation. The embryogenicity, however, decreased with increasing embryo maturity. Nonfreezing low temperatures, such as 5°C, not only inhibited cotyledon development but also maintained embryogenicity. Cryotolerance was successfully induced when embryos were matured (or pretreated) under 5°C for a suitable culture period, typically 4-8 weeks. These embryos were able to survive a rapid cooling process and liquid nitrogen storage without the addition of any cryoprotectants. After cryopreservation, embryogenic tissue was recovered in both interior spruce and Douglas-fir. Embryo maturation tests indicated no difference in mature embryo yields with or without cryopreservation in interior spruce. The key factors inducing cryotolerance included ABA supplementation in culture media and low temperature pretreatment. Optimum combinations of these factors can result in high rates of tissue survival and high embryogenicity after cryopreservation.

Simple and efficient protocols for the initiation and proliferation of embryogenic tissue of Douglas-fir

“…Modified Litvay medium has been used successfully for the initiation of embryogenic cell lines in a number of other pines, especially in combination with a reduction in plant growth regulators (Percy et al 2000;Klimaszewska et al 2001). A modified Litvay medium was also recently used to initiate embryogenic lines in Douglas-fir (Kong and von Aderkas 2011), although this paper focussed on cryopreservation of immature SEs and did not report on the success of this medium for embryogenic tissue initiation. BM medium formulation has been successfully used by other researchers for Douglas-fir initiation in North America, initially named WTC (Pullman and Gupta 1994;Gupta 1996) and later BM 1 (Gupta et al 2004), and with slight alterations to the formulation, 121 (Pullman et al 2005(Pullman et al , 2009a.…”

Strategies for Fast Multiplication and Conservation of Forest Trees by Somatic Embryogenesis and Cryopreservation: a Case Study with Cypress (Cupressus sempervirens L.)

“…Although the "two-step freezing" technique has been largely preferred (Ozudogru and Lambardi, 2016), also "one-step freezing" methods, allowing the direct immersion of explants in LN, have been occasionally proposed (e.g., Kong and von Aderkas, 2011;Ozudogru et al, 2011;Touchell et al, 2002). In this study, only the slow cooling technique allowed satisfactory results in terms of survival and regrowth rates of cypress EMs after their storage in LN.…”

Application of Biotechnology in the Conservation of the Genus Castanea

“…The advances in biotechnology have led to opportunities for the conservation of genetic resources, and the use and maintenance of plant material at cryogenic temperatures is an option for long-term preservation [98,99]. This method is based on the total arrest of cell division and metabolic processes as a result of storing at ultra-low temperatures [100].…”