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Cryopreservation of Embryogenic Cell Suspensions by Encapsulation-Vitrification

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Part of the Methods in Molecular Biology™ book series (MIMB,volume 318)

Abstract

Encapsulation-vitrification, which is a combination of encapsulation-dehydration and vitrification procedures, is a newly developed technique for cryopreservation of plant germoplasm. Here, we describe the protocol of this methodology, using grapevine (Vitis) as a model plant. Cell suspensions at the exponential growth stage were encapsulated with 2.5% sodium alginate solution in 0.1 M calcium chloride solution for 20 min to form beads of about 4 mm in diameter containing 25% cells. The beads were stepwise precultured in increasing sucrose concentrations of 0.25, 0.5, and 0.75 M for 3 yr, with 1 d for each step. Precultured beads were treated with a loading solution for 60 min at room temperature and then dehydrated with PVS2 at 0°C for 270 min, followed by direct immersion in liquid nitrogen for 1 h. The beads were rapidly rewarmed at 40°C in a water bath for 3 min and then diluted with 1 M sucorse solution at room temperature for 30 min. Rewarmed, washed beads were post-cultured on a recovery medium for 3 d at 25°C in the dark for survival. Surviving cells were transferred to a regrowth medium to induce cell proliferation. Embryogenic cell suspensions were re-established by suspending the cells in a cell suspension maintenance medium maintained on a gyratory shaker at 25°C in the dark. For plant regeneration, surviving cells were transferred from the recovery medium to an embryo maturation medium and maintained at 25°C under light conditions. Embryos at the torpedo stage were cultured on rooting medium until whole plantlet was developed.

Key Words

  • Cell suspensions
  • cryopreservation
  • embryogenesis
  • encapsulation
  • liquid nitrogen
  • plant regeneration
  • vitrification

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Wang, Q., Perl, A. (2006). Cryopreservation of Embryogenic Cell Suspensions by Encapsulation-Vitrification. In: Loyola-Vargas, V.M., Vázquez-Flota, F. (eds) Plant Cell Culture Protocols. Methods in Molecular Biology™, vol 318. Humana Press. https://doi.org/10.1385/1-59259-959-1:077

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  • DOI: https://doi.org/10.1385/1-59259-959-1:077

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-58829-547-7

  • Online ISBN: 978-1-59259-959-2

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