Mitsuteru Suzuki

Cryopreservation of in vitro grown shoot tips of cherry and sweet cherry by one-step vitrification

Abstract In vitro grown shoot tips of cherry (Prunus jamasakura Seib. cv. Sendaiya) were successfully cryopreserved by one-step vitrification. After the last subculture, 3-week-old shoots were coldhardened at 5°C for 45 days. Excised 3-mm shoot tips were precultured on solidified Murashige and Skoog medium supplemented with 0.7 mol 1−1 sucrose for 1 day at 5°C. Following preculture, the shoot tips were transferred to a 2-ml plastic cryotube, and the highly concentrated cryoprotective solution PVS2 was then added (vitrification method). After treatment with PVS2 at 25°C for 105 min, the shoot tips were directly plunged into liquid nitrogen. After rapid thawing, the shoot tips were plated on agar medium. The survival rate was about 80% regardless the time of storage in liquid nitrogen from 1 day up to 10 months. This vitrification method was successfully applied to two cherry cultivars and five sweet cherry cultivars.

A novel preculture method for the induction of desiccation tolerance in gentian axillary buds for cryopreservation

Abstract Axillary buds of in vitro-grown gentian (Gentiana scabra Bunge var. buergeri Maxim.) were successfully desiccated and cryopreserved using a novel two-step preculture method: preculturing axillary buds excised from in vitro-grown gentian plants on Murashige and Skoog (MS) semi-solid medium containing 0.1 M sucrose for 8–13 days at 25°C under a 16 h-photoperiod (first step) and the subsequent short period incubations on 0.4 M sucrose (MS agar medium) for 1 day followed by 0.7 M sucrose (MS agar medium) for 1 day (second step). After preculturing, the buds were air-dried down to about 10% water content (fresh weight basis) without encapsulation, and then immersed in liquid nitrogen (LN). With this cryogenic protocol, we could obtain 78–90% of survival of axillary buds following desiccation and LN exposure. Most of the buds that survived showed normal shoot and root development. The most important part of this protocol was the preculture which circumvents the conventionally used lengthy cold-hardening treatment. The results show that induction of desiccation tolerance by preculturing was specific to a few species of sugars used in the medium and not merely caused by osmotic effects. The efficient desiccation–cryopreservation protocol shown here using the new preculture method appears promising for routinely cryopreserving gentian germplasm.

Cryopreservation of bromegrass (Bromus inermis Leyss) suspension cultured cells using slow prefreezing and vitrification procedures

Abstract Cryopreservation of a non-embryogenic bromegrass suspension culture was attempted using a slow prefreezing method (two-step method) and a vitrification method. We examined 17 solutions as cryoprotectants for the slow prefreezing method. The cryoprotectant (CSP1) comprising sucrose, dimethylsulfoxide (DMSO) and glycerol (10, 10, 5%, w/v) gave the best survival (> 80%) of bromegrass cells exposed to liquid nitrogen (LN) as determined by regrowth and fluorescent diacetate (FDA) staining. The optimum conditions were: exposure time to CSP1 (30 min), cooling rates (0.3–0.5°C/min) following ice-inoculation at −8°C, prefreezing temperature (−30 to −40°C). This protocol worked well when glass vials were used as containers and not plastic cryotubes. With CSP1, the slow dropwise addition of the protectant to the cells on ice that has frequently been used could be circumvented; direct addition of CSP1 to the cells at 25°C did not affect the survival. However, dilution of CSP1 by 5-fold with 3% sucrose following a freeze-thaw had to be done slowly (taking 15 min). The same cryoprotectant (CSP1) was also effective as a pretreatment for cryopreservation of bromegrass cells by vitrification. Following direct treatment with CSP1 at 25°C for 30 min, cells were incubated in a vitrification solution, PVS2 (Sakai et al., 1990) for about 2–4 min at 25°C prior to submersion in LN. This procedure gave 30–40% survival of bromegrass cells based on regrowth and FDA staining. Without the CSP1 pretreatment, bromegrass cells did not survive LN exposure by vitrification with PVS2. Preculture with abscisic acid or with high osmoticum (0.3–0.7 M sucrose) could not be used as a substitute for pretreatment with CSP1.