Plant Cell Reports

, Volume 9, Issue 1, pp 30–33 | Cite as

Cryopreservation of nucellar cells of navel orange (Citrus sinensis Osb. var. brasiliensis Tanaka) by vitrification

  • A. Sakai
  • S. Kobayashi
  • I. Oiyama


The nucellar cells of navel orange(Citrus sinensis Osb. var. brasiliensis Tanaka) were successfully cryopreserved by vitrification. In this method, cells were sufficiently dehydrated with highly concentrated cryoprotective solution(PVS2) prior to direct plunge in liquid nitrogen. The PVS2 contains(w/v) 30% glycerol, 15% ethylene glycol and 15% DMSO in Murashige-Tucker medium(MT) containing 0.15 M sucrose. Cells were treated with 60% PVS2 at 25°C for 5 min and then chilled PVS2 at 0°C for 3 min. The cell suspension of about 0.1 ml was loaded in a 0.5 ml transparent plastic straw and directly plunged in liquid nitrogen for 30 min. After rapid warming, the cell suspension was expelled in 2 ml of MT medium containing 1.2 M sucrose. The average rate of survival was about 80%. The vitrified cells regenerated plantlets. This method is very simple and the time required for cryopreservation is only about 10 min.

Key words

Cryopreservation Vitrification Nucellar cells Navel orange Citrus sinensis 



dimethyl sulfoxide


vitrification solution


liquid nitrogen


differential scanning calorimeter




Murashige-Tucker basal medium


naphthaleneacetic acid


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Copyright information

© Springer-Verlag 1990

Authors and Affiliations

  • A. Sakai
    • 1
  • S. Kobayashi
    • 2
  • I. Oiyama
    • 2
  1. 1.SapporoJapan
  2. 2.Akitu BranchFruit Tree Research StationHiroshimaJapan

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