Cryopreservation of Medicago rigidula seeds by two methods of vitrification and encapsulation-dehydration

Kolahdozan, Marziyeh; Ghamari Zare, Abbas; Shahrzad, Shekoofeh; Kiarostami, Khadijeh

Cryopreservation of Medicago rigidula seeds by two methods of vitrification and encapsulation-dehydration

Document Type : Research Paper

Authors

Abstract

Preservation of genetic resources of Medicago rigidula which is one of the forage and endemic species in Iran is essential. For the first time seeds of the species were successfully cryopreserved by vitrification and encapsulation-dehydration methods. The seeds were dehydrated by silicagel in desiccator, then two conservation methods were applied: 1) by vitrification method, seeds were loaded with 2M glycerol solution in 0.6M sucrose for 20 min., then loaded seeds were transferred into 2ml cryovials containing 0.5ml PVS2 at 0°C. 2). By encapsulation-dehydration method, dried seeds plunged into alginic acid solution for 15 min, then transferred into CaCl₂ solution for the same duration. Encapsulated seeds were dehydrated in room temperature for 20h and transferred into 2ml cryovials. Both of the seed groups plunged instantly in liquid nitrogen (-196°C). After a week vitrified and encapsulated seeds were thawed in distilled water bath at 40°C for 2 min and washed in 1.2M sucrose solution for 5, 10 and 15 min intervals with gentle shake. Three recovery procedures were used: 1) washing of vitrified, encapsulated and control seeds under running water for I) 30 min, II) 24 h and transferring the seeds onto petri-dishes containing moist filter papers and 2) sterilization of vitrified, encapsulated and control seeds by 1.5% sodium hypochlorite and transferring into MS medium supplemented with 5 mgl^-1 GA₃. Survival percentage of seeds was estimated after 4 and 18 days respectively for unsterile and sterile conditions. The germination percentage of seeds after vitrification and encapsulation-dehydration method ranged respectively between 98.6%-99.2% and 98.4%-99.7%.

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