Acta Hortic. 1234. ISHS 2019. DOI 10.17660/ActaHortic.2019.1234.34 Proc. III International Symposium on Plant Cryopreservation Eds.: K. Thammasiri et al. 263 Development of effective cryopreservation protocols using aluminium cryo-plates for mulberry D. Tanaka 1 , S. Yamamoto 1 , T. Matsumoto 2,a , M. Valle Arizaga 3 and T. Niino 3 1 Genetic Resource Center, NARO, Tsukuba, Japan; 2 Faculty of Life and Environment Science, Shimane University, Matsue, Japan; 3 University of Tsukuba, Tsukuba, Japan. Abstract The adaptation of the D cryo-plate method to in vitro mulberry shoot tips from the tropics and subtropics was tested. The optimal D cryo-plate procedure was as follows: precultured shoot tips (1.5 mm) are attached on the cryo-plate using calcium alginate gel. Then, osmoprotection is performed by immersing the cryo-plates in LS (2.0 M glycerol and 0.6 M sucrose) for 30 min at 25°C. The shoot tips on the cryo-plate are dehydrated under the air current of a laminar flow cabinet for 2.5 h at 25°C, 40-50% relative humidity. Then, the cryo-plates with the shoot tips were transferred into uncapped 2-mL cryotube and plunged directly into LN. Regrowth was initiated when the cryopreserved shoot tips attached to the cryo-plates were rewarmed by immersion into 1.0 M sucrose solution at 25°C. This optimized procedure was applied to shoot tips of 10 mulberry lines, resulting regrowth after LN ranged from 73 to 90%, with an average of 78.7%. In addition, the use of mulberry shoot tips dissected from the axillary buds of new growth shoots cultivated in a greenhouse after sterilization was tested in V cryo-plate procedure. The optimal dehydration time of shoot tips excised from new growth shoots by PVS2 was 50 min at 25°C. This optimized procedure was applied to shoot tips from new growth shoots of 10 mulberry lines. Regrowth ranged from 70 to 97%, with an average of 80.3%. The protocols developed for mulberry may be useful for cryopreservation of other woody plant species after appropriate modifications. Keywords: cryo-plate, new growth shoots, D cryo-plate method, mulberry, V cryo-plate method INTRODUCTION Dormant buds of mulberry (Morus species) accessions that come from sub-tropical and tropical areas cannot be used for long term cryopreservation. The V cryo-plate method was successfully adopted and adjusted to in vitro-grown mulberry shoot tips (Yamamoto et al., 2012). New cryopreservation techniques that use aluminium cryo-plates have been reported. One is the development of a dehydration method using aluminium cryo-plates (D cryo-plate; Niino et al., 2013). This method is useful to avoid the use of PVS2, using materials with comparatively higher moisture content while performing minimal excision of young leaves and/or sheaths, and the results in limited damage to specimens. The other uses shoot tips dissected from the axillary buds of new growth shoots after sterilization, for long term cryostorage (Matsumoto et al., 2015; Dhungana et al., 2017). MATERIALS AND METHODS Plant material The D cryo-plate method was adapted initially using mulberry line ‘M5’. In vitro shoots were cultured on ½ MS medium (Murashige and Skoog, 1962) with 3.0% (w/v) sucrose and 0.8% (w/v) agar, adjusted to pH 5.8 (subculture medium). Stock culture plants of mulberry were incubated at 25°C with a 16-h light/ 8-h dark photoperiod under a light intensity of 52 μmol m -2 s -1 provided by white fluorescent tubes (standard condition). The V cryo-plate method was adapted initially using mulberry lines ‘M5’ and Morus a E-mail: tmatsumoto@life.shimane-u.ac.jp