Articles
Development of effective cryopreservation protocols using aluminium cryo-plates for mulberry
Article number
1234_34
Pages
263 – 268
Language
English
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.
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.
Authors
D. Tanaka, S. Yamamoto, T. Matsumoto, M. Valle Arizaga, T. Niino
Keywords
cryo-plate, new growth shoots, D cryo-plate method, mulberry, V cryo-plate method
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