Cryopreservation of Trifolium radicosum seeds and establishment of the Cryopreserved seeds under laboratory and greenhouse conditions

Trifolium radicosum (Boiss. & Hohen.) is a perennial range legume growing on steep or moderately steep slopes in high altitudes areas of the central Alborz Mountain with a cold Mediterranean climate. Establishment of this species in cold, high-altitudes, and steep slopes with heavy snow falls prevents soil degradation and erosion. In this regard, the species has a great importance in ecological and range conservation point of view. Overgrazing, high nutrient values, palatability, as well as habitat susceptibility may cause the species to face the risk of extinction. In order to evaluate the possibility of long-term conservation of the Trifolium radicosum seeds, with low degree of germination (35.67%), cryopreservation approach was evaluated. Subsequent to cryopreservation, three pre-cryopreservation methods including PVS2, desiccation, and 30% glycerol were applied. The treated seeds were transferred into liquid nitrogen (- 196°C) for one week, one month and one year, respectively. The cryopreserved seeds were removed from liquid nitrogen and imposed to heat shock, surface sterilized and were transferred between sterile moist papers in petri dishes or sown in pots under greenhouse conditions. The cryopreserved seeds of different storage times and precryopreservation treatments were survived under liquid nitrogen conditions, germinated normally, and grown to seedlings. Differences between cryopreservation times for most of the attributes were not significant. However there were significant differences between pre-cryopreservation treatments for some of the attributes, such as germination (29.33, 26.33 and 21.67% for desiccation, PVS2 and 30% glycerol in one weak cryopreservation period). In clonal propagation experiments under greenhouse conditions, the root cuttings showed excellent results compared to that of the stem cuttings.