Seed Germination of Lilium ledebourii (Baker) Boiss. after Cryopreservation


1 Department of Horticulture, Science and Research Branch, Islamic Azad University, Tehran, Iran

2 Research Institute of Forests and Rangelands, Tehran, Iran (Corresponding Author)

3 Research Institute of Forests and Rangelands, Tehran, Iran


Abstract. Seeds or plant organs are usually used as the materials for the long-term cryopreservation. The aim of this study is to investigate the possibility of seed cryopreservation of Lilium ledebourii (Baker) Boiss. as an endemic and endangered species because of genetic erosion. To evaluate seed potentials for the cryopreservation, four treatments including vitrification, 30% glycerol, desiccation and encapsulation-dehydration were applied on seeds before immerging in the liquid nitrogen (-196˚C) for a week. Then, seeds were removed from liquid nitrogen and exposed to heat shock (42˚C water bath), washed with the distilled water and eventually, sown in petri-dishes containing tissue papers. Some criteria including seed germination percent, root and shoot length values, root/shoot length ratio and seed vigor index were recorded and statistically analyzed after four weeks. Data of germination was converted to an arc-sine transformation prior to the analysis of variance. Results showed that germination percentages were 97.50, 97.43, 94.86 and 69.47% for 30%-glycerol, vitrification, desiccation, and encapsulation-dehydration treatments, respectively. They were not significantly different from control seeds (89.33%). On the other hand, other germination attributes of seeds almost showed no significant differences in comparison with control treatment in most treatments. In addition, 30%-glycerol, vitrification and desiccation experienced the highest amounts of germination attributes whereas they showed no significant differences with the control treatment in most qualities. In contrast, the encapsulated seeds showed the lowest amounts in germination indices though they had no significant differences with control treatment (except germination rate). Most of germination attributes of encapsulated seeds were significantly lower than the other cryogenic treatments. Both 30% glycerol and desiccation treatments showed some advantages over vitrification method. However, desiccation was the best treatment because it does not need any chemical substances. It was concluded that the cryopreservation technique is an important approach for the long-term preservation of the seeds of this endangered species.


Abdul-Baki, A. A., Anderson J. D., 1973. Vigor determination in soybean seed by multiple criteria. Crop Sci., 13: 630-633.
Benson, E. E., 2008. Cryopreservation of Phytodiversity: A critical appraisal of theory and practice. Critical reviews in Plant sciences, 27(3):141- 219.
Charoensub, R., Phansiri, S., Yongmanitchai, W., Sakai, A., 2003. Routine cryopreservation of in vitro-grown auxiliary apices of cassava (Manihot esculenta Crantz) by vitrification: importance of a simple mononodal culture. Sci. Hort., 98: 485-492.
Chen, X. L., Li, J. H., Xin, X., Zhang, Z. E., Xin, P. P., Lu, X. X., 2011. Cryopreservation of in vitro-grown apical meristems of Lilium by droplet-vitrification. South. Afr. Jour. Bot., 77: 397–403.
Halmagyi, A., Deliu, C., Isac, V., 2010. Cryopreservation of Malus cultivars: Comparison of two droplet protocols. Sci. Hort., 124: 387–392.
Hatami, F., Naderi-Shahab, M., Jebelli, M., Ghamari-Zare, A., Tabari, M., Assareh, M., 2010. Investigation on possibility of cryopreservation of Eucalyptus microtheca seeds. Iranian Jour. Forest and Poplar Research, 17: 627-636. (In Persian).
Hirata, K., Mukai, M., Goda, S., Ishio-Kinugasa, M., Yoshida, K., Sakai, A., Miyamoto, K., 2002. Cryopreservation of hairy root cultures of Vinca minor L. by encapsulation-dehydration. Biotechnol. Lett., 24: 371-376.
Jalili, A., Jamzad, Z., 1999. Red Data Book of Iran. Research Institute of Forests and Rangelands, Tehran, Iran.
Kaviani, B., Safari-Motlagh, M. R., Padasht-Dehkaei, M. N., Darabi, A. H., Rafizadeh, A., 2008. Cryopreservation of Lily [Lilium ledebourii (Baker) Boiss.] germplasm by encapsulation-dehydration. International Jour. Botany. 4: 491-493.
Malik, S. K., Chaudhury, R., Dhariwal, O. P.,Bhandari, D. C., 2010. Genetic resources of tropical underutilized fruits in India. National Bureau of Plant Genetic Resources, New Delhi.p.178.
Mandal, B. B., Chaudhury, R., Engelmann, F., Bhag Mal, Tao, K. L., Dhillon, B. S., 2003. Conservation biotechnology of plant germplasm. NBPGR, New Delhi, India/IPGRI, Rome, Italy/FAO, Rome, Italy.
Matsumoto, T., Sakai, A., 2003. Cryopreservation of axillary shoot tips of in vitro-grown grape (Vitis) by a two-step vitrification protocol. Euphytica, 131: 299-304.
Matsumoto, T., Takahashi, C., Sakai, A., Nako, Y., 1998. Cryopreservation of in vitro-grown apical meristems of hybrid static by three different procedures. Sci. Hort., 76: 105-114.
Murashige, T., Skoog, F., 1962. A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant, 15: 473-497.
Naderi-Shahab, M., Hatami, F., Tabari, M., Jafari, A. A., 2009. Cryopreservation and evaluation of Chinese Arbor-Vitae (Biota orientalis) seeds. Jour. New Seeds, 10: 264-276.
Nishizawa, S., Sakai, A., Amano, Y., Matsuzawa, T., 1993. Cryopreservation of asparagus (Asparagus officinalis L.) embryogenic suspension cells and subsequent plant regeneration by vitrification. Plant Sci., 91:67-73.
Panis, B., Piette, B., Swennen, R., 2005. Droplet vitrification of apical meristems: a cryopreservation protocol applicable to all Musaceae. Plant Sci., 168: 45-55.
Pritchard, H. W. and Nadarajen, J., 2008. Plant cryopreservation: a practical guide. Berlin: Springer. pp. 485-501.
Rao, N. K., Hanson, J., Dulloo, M. E., Ghosh, K., Nowell, D., Larinde, M., 2006. Manual of seed handling in gene banks.Biodiversity International, Rome, Italy.
Razdan, M. K., 2003. Introduction to plant tissue culture, science publishers.Kluwer Academic Publishers, The Netherlands.
Reed, B. M., 2008. Plant cryopreservation: a practical guide. Springer Science, Corvallis, OR, USA USDA-ARS National Clonal Germplasm Repository.
Reed, B. M., Denoma, J., Luo, J., Chang, Y., Towill, L., 1998. Cryopreservation and long-term storage of pear germplasm. In Vitro Cell Dev. Biol. Plant, 34:256-260.
Roberts, E. H., 1973. Predicting the storage life of seeds. Seed Sci. Technol, 1: 499–514.
Sakai, A., Kobayashi, S., Oiyama, I., 1990. Cryopreservation of nucellar cells of navel orange (Citrus sinensis Osb. var. brasiliensis Tanaka) by vitrification. Plant Cell Rep., 9:30-33.
Shahrzad, S., Ahmadi, R., Ghamari-Zare, A., Naderi-Shahab, M., 2009. In vitro callus induction and cryopreservation of Secale montanum embryonic cell follicles. Review of Forests, Wood and Wood Biotechnology of Iran and Germany, Part III. Gottingen University, 135-148.