Cryopreservation of Persimmon Shoot Tips from Dormant Buds Using the D Cryo-plate Technique

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Cryopreservation has become a very important tool for the long-term storage of plant germplasm. A new cryopreservation protocol based on air dehydration of explants placed on aluminum cryo-plates, termed the D cryo-plate technique, was developed. In this study, the most suitable conditions of cryopreservation for dormant shoot tips of Japanese persimmon (Diospyros kaki Thunb. ‘Saijo’) using the D cryo-plate technique were investigated. Dormant one-year-old shoots of persimmon were collected from the experimental farm of Shimane University in January 2013 and stored at 2°C until use. After surface sterilization, shoot tips of about 1 mm in size were dissected from the dormant buds and precultured overnight at 25°C on solidified 1/2MS medium containing 0.3 M sucrose. Precultured shoot tips were placed on aluminum cryo-plates and embedded in calcium alginate gel. Osmoprotection of shoot tips was performed by immersing the cryo-plates for 30 min at 25°C in an LS solution containing 2 M glycerol + 1.0 M sucrose in 1/2MS solution. For the D cryo-plate technique, encapsulated shoot tips were dehydrated by placing the cryo-plates in the air current of a laminar flow cabinet for 30–90 min. Cooling was performed by placing the cryo-plates in uncapped cryotubes, which were immersed in liquid nitrogen. For rewarming, the cryo-plates were immersed in 1/2MS medium containing 1.0 M sucrose for 20 min at 25°C. In this study, the preculture did not improve the regrowth after cryopreservation; however, we consider that it should be performed in the D cryo-plate procedure for application of other cultivars and utilization in genebanks. A high regrowth rate of cryopreserved shoot tips (84%) was achieved after dehydration for 30 min. This optimized procedure was applied to 10 additional persimmon cultivars, resulting in regrowth rates ranging between 67 and 97%, with an average of 87%. As shoot tips derived from dormant buds proved to be highly tolerant to liquid nitrogen exposure, the D cryoplate technique may facilitate long-term conservation of persimmon germplasm.