Development of effective cryopreservation protocols using aluminium cryo-plates for mulberry

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.