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In vitro regeneration of adventitious buds from leaf explants and their subsequent cryopreservation in highbush blueberry

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We report a new cryopreservation method for highbush blueberry (Vaccinium corymbosum) using small leaf squares-bearing adventitious buds. Leaf explants were cultured on adventitious bud regeneration medium composed of Woody Plant Medium (WPM) supplemented with 20 µM zeatin. After 21 days of adventitious bud regeneration, small leaf squares (SLSs, 2 × 3 mm), each bearing multiple adventitious buds, were cut from the leaf explant, precultured on WPM containing 0.3 M sucrose for 24 h and were treated for 30 min with a loading solution composed of WPM containing 1.0 M sucrose and 2 M glycerol, followed by exposure to plant vitrification solution 2 at 0 °C for 40 min. Each of dehydrated SLS was then transferred onto an aluminum foil with small holes and PVS2 was dropped until it covered the SLS, prior to a direct immersion in liquid nitrogen. Cryopreserved SLSs were re-warmed in WPM containing 1.2 M sucrose for 20 min at room temperature, followed by post-thaw culture for recovery. With this procedure, more than 23 adventitious buds were produced in each leaf explant, and 100% of SLSs were able to survive and resume shoot regrowth, with more than six shoots per SLS obtained following cryopreservation in three highbush blueberry cultivars. In ‘Misty’, the morphology of plantlets regenerated from cryopreserved SLSs was identical to that of the in vitro-derived ones. No polymorphic bands were detected using inter-simple sequence repeat markers and random amplified polymorphic DNA in plantlets of ‘Misty’ recovered from cryopreservation. The use of SLSs-bearing adventitious buds for cryopreservation reported in the present study eliminates shoot tip excision. This cryopreservation method can be considered an efficient cryopreservation of plant shoot tips, and has potential applications to other plant species.

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Adventitious bud regeneration medium


Apical dome


Inter-simple sequence repeats


Liquid nitrogen


Leaf primordium


Plant vitrification solution 2


Random amplified polymorphic DNA


Small leaf squares


Shoot maintenance medium


Woody plant medium




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Author information

H-YC and JL: performance of experiments, collection and analysis of data and preparation of manuscript; CP: preparation of plant materials and assistance to experiments; J-WY: assistance to data collection and analysis; Q-CW: chief scientist, financial supports, experimental design and preparation of manuscript.

Correspondence to Qiao-Chun Wang.

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The authors declare that they have no conflict of interest.

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Communicated by Sergio J. Ochatt.

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Chen, H., Liu, J., Pan, C. et al. In vitro regeneration of adventitious buds from leaf explants and their subsequent cryopreservation in highbush blueberry. Plant Cell Tiss Organ Cult 134, 193–204 (2018). https://doi.org/10.1007/s11240-018-1412-y

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  • Blueberry
  • Long-term preservation
  • Droplet-vitrification
  • Genetic stability
  • Leaf explant
  • Recovery