Cryopreservation of Citrus madurensis embryonic axes by vitrification: importance of loading and treatment with vitrification solution

Author： Cho Eun Gi Hor Y.L. Kim Haeng Hoon Rao V. Ramanatha Engelmann Florent

Publisher： Cryoletters

ISSN： 1742-0644

Source： Cryoletters, Vol.23, Iss.5, 2002-09, pp. : 317-324

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Abstract

This paper investigates the importance of loading and treatment with a vitrification solution on the survival of Citrus madurensis embryonic axes cryopreserved using a vitrification protocol. Among the seven different loading solutions tested, the solution containing 2 M glycerol + 0.4 M sucrose was the most efficient. Of the six vitrification solutions tested, the PVS2 vitrification solution, applied for 20 min at 25 °C or for 60 min at 0 °C, ensured the highest survival. A three-step vitrification protocol, involving the treatment of embryonic axes at 0 °C with half strength PVS2 solution for 20 min then with full strength PVS2 for an additional 40 min was more efficient than a two-step protocol that involved treatment of axes directly with full strength PVS2 solution for 60 min. After rapid immersion in liquid nitrogen, rapid rewarming, unloading in a 1.2 M sucrose solution for 20 min, culture on solid medium with 0.3 M sucrose for 1 day and growth recovery for 4 weeks on standard medium, survival of C. madurensis embryonic axes reached 85 % following the three-step process, compared with 70 % for the two-step process.