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Plant Cell, Tissue and Organ Culture (PCTOC)

, Volume 134, Issue 2, pp 193–204 | Cite as

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

  • Hai-Yan Chen
  • Jing Liu
  • Chen Pan
  • Jing-Wei Yu
  • Qiao-Chun Wang
Original Article
  • 280 Downloads

Abstract

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.

Keywords

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

Abbreviations

ABRM

Adventitious bud regeneration medium

AD

Apical dome

ISSR

Inter-simple sequence repeats

LN

Liquid nitrogen

LP

Leaf primordium

PVS2

Plant vitrification solution 2

RAPD

Random amplified polymorphic DNA

SLS

Small leaf squares

SMM

Shoot maintenance medium

WPM

Woody plant medium

ZT

Zeatin

Notes

Author contributions

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.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

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Copyright information

© Springer Science+Business Media B.V., part of Springer Nature 2018

Authors and Affiliations

  1. 1.State Key Laboratory of Crop Stress Biology for Arid Areas, College of HorticultureNorthwest A&F UniversityYanglingPeople’s Republic of China

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