Effect of Vitrification on Survival of Medicago polymorpha L. Cryopreserved Seeds

Subject Areas : Genetic

مه لقا  Ghorbanli ¹ (Islamic azad university Gorgan Branch, Iran)
رکسانا  Bonyadi ² (University of payame noor of Tehran, Iran)
عباس  Ghamari Zare ³ (Recearch institute of Forest and Rangelands, Tehran, Iran)
شکوفه  Shahrzad ⁴ (Recearch institute of Forest and Rangelands, Tehran, Iran)

Received: 2008-04-21 Accepted : 2008-09-08 Published : 2009-03-15

Keywords: Vitrification, Liquid nitrogen, cryopreservation, Dehydration, PVS2, Long term conservation, Medicago polymorpha, Seeds conservation,

Abstract :

In this research a new method for long term conservation of Medicago polymorpha seeds was developed by using cryopreservation. Two year cold acclimated seeds, collected from Iran rangelands, were desiccated using silica gel for four days and then cryopreserved by vitrification method. Seeds were loaded by loading solution containing 2 M glycerol and 0.6 M sucrose for 20 min and then inserted into cryo-vials containing 0.5 ml PVS2 in 0 °C prior to direct immersed into liquid nitrogen. Seeds were kept in liquid nitrogen for a week then thawed in 37 °C water bath for 2 min. The seeds were rehydrated and detoxified in a solution of MS medium salts containing 1.2 M sucrose respectively for 5, 10 and 15 min in 22 °C. At the final stage of post-treatment, three different procedures were used: (a,b) placing seeds under running water for 30 min and 24 h before being  transferred to petri-dishes containing moist filter paper, (c) sterilization of seeds with 2% hypochloride then placed on MS medium containing 0.5 mg/lit GA3. Germination percentage of cryopreserved seeds in all three post-treatments was higher than control (not cryopreserved seeds). This figure was respectively 94.37% and 98.21% for cryopreserved seeds of post-treatments a and b which was different at 0.05 levels with controls. However, germination percentage (76.16%) of cryopreserved seeds in post-treatment c showed no significant difference with control.

References:

Barbara M.R., 2001. Implementing cryogenic storage of clonally propagated plants. Cryo Letters 22 (1): 97-104.

Baust J.G., 2007. Progression of cell and tissue vitrification. Cell preservation technology 5(1): 1-1.

Best B., 2006. Vitrification of cryonics. http://www.benbest.com/cryonics

Finkle B.J., Ulrich J.M., 1982. Cryopreservation removal temperature as a factor in the survival of frozen rice and sugarcane cells. Cryobiology 19(3): 329-335.

Gagliardi R.F., Pacheco G.P., Carneiro L.A, Valls J.F.M., Vieira M.L.C. and Mansur E., 2003. Cryopreservation of Arachis species by vitrification of in vitro-grown shoot apices and genetics stability of recovered plants. CryoLetters 24 (2): 103-110.

Ghamari Zare A., Naderi Shahab M., Shahrzad S. and Asare M., 2007. Conservation of seeds, apical and auxiliary buds and plant cells produced by tissue culture, a method for long term conservation of plant genetic resources. The 5^th Iranian National Biotechnology Congress.

Hirai D., Sakai A., 1998. Cryopreservation of in vitro-grown meristems of potato (Solanum tuberosum L.) by encapsulation-vitrification. Bulletin of Pope Vol. 4, Not 21 November 15, 2002.

Kadkade and Prakash G., 2005. Cryopreservation of plant cells. United States Patent Application 20050158699.

KEW Royal Botanical Gardens web site, 2007. http://www.kew.org

Langis R., Schnabel B., Earle E.D., Steponkus P.L., 1989. Cryopreservation of Brassica campestris L. cell suspensions by vitrification. CryoLetters 10:421-428.

Lipman C.B. and Lewis G.N., 1934. Tolerance of liquid air temperatures by seeds of higher plants for sixty days. Plant Physiology 9: 392-394.

Lipman C.B., 1936. Normal viability of seeds and bacterial spores after exposure to temperatures near the absolute zero. Plant Physiology 11: 201-205.

Martinkova Z., Honek A., 2007. The effect of cryopreservation on germination of Dandelion seeds. Plant Protection Science 43: 63-67.

Murashige T. and Skoog F., 1962. A revised medium for rapid growth and bio-assays with tobacco tissue culture. Physiologia Plantarum 15:473-597.

Na H., Kondo K., 1996. Cryopreservation of tissue-cultured shoot primordial from shoot apices of cultured protocorms in Vanda pumila following ABA preculture and desiccation. Plant Science 118(2): 195-201.

Nag K.K. and Street H.E., 1973. Carrot embryogenesis from frozen cultured cells. Nature 245: 270-272.

Panis B., Lambardi M., 2005. Status of cryopreservation technologies in plants (crops and forest trees), The role of biotechnology. Villa Gualino, Turin, Italy, 5-7 March, 2005.

Panis B., Piette B., Swennen R., 2004. Droplet vitrification of apical meristems: a cryopreservation protocol applicable to all Musaceae. Plant Science 168(1): 45-55.

Paroda R.S., Arora R.K., 1991. Plant genetic resources conservation and management concepts and approaches. International Board for Plant Genetic Resources, Regional office for South and Southeast Asia, c/o NBPGR, Pusa Campus, New Delhi 110 012, India.

Quatrano R.S., 1968. Freeze preservation of cultured flax cells utilizing dimethyl sulfoxide. Plant Physiology 43: 2057-2061.

Sakai A. and Noshiro M., 1975. Some factors contributing to the survival of crop seeds cooled to the temperature of liquid nitrogen. pp. 317-326. In Crop genetic resources for today and tomorrow (Eds., O.H. Frankel and J.G. Hawkes). Cambridge University Press, Cambridge.

Saloma A.N., 2002. Tropical seed species responses to liquid nitrogen exposure. Brazilian Journal of Plant Physiology 14(2): 133-138.

Sant R., Taylor M., Tyagi A., 2006. Cryopreservation of in-vitro grown shoot tips of tropical taro (Colocasia esculenta var. esculenta) by vitrification. Cryo Letters 27(3): 133-42.

 Schmale K., Rademacher T., Fischer R., Hellwig S., 2006. Towards industrial usefulness- cryo-cell-banking of transgenic BY-2 cell cultures. Journal of Biotechnology 124(1): 302-311.

Stanwood P.C., 1985. Cryopreservation of seed germplasm for genetic conservation, pp. 199-226. In Cryoperservation of plant cells and organs (Ed., K.K. Kartha). CRC Press, Boca Raton, Florida.

Towill L.E., 1982. Low temperature (-196° C) storage of the seed from the tuber-bearing Solanums species. American Potato Journal 59: 141-147.

Uragami A., Sakai A., Nagai M., Takahashi T., 1989. Survival of cultured cells and somatic embryos of Asparagus officinalis L. cryopreserved by vitrification. Plant Cell Reports 8: 418-421.

Volk GM., Walters C., 2006. Plant vitrification solution 2 lowers water content and alters freezing behavior in shoot tips during cryopreservation. Cryobiology 52: 48-61.