Małgorzata Zalewska

University of Technology and Life Sciences in Bydgoszcz

Dariusz Kulus

University of Technology and Life Sciences in Bydgoszcz


To optimize the protocol, the shoot tips of Chrysanthemum × grandiflorum (Ramat.) Kitam. radiomutants have been cryopreserved using the encapsulationdehydration technique, following different approaches. In the experiment the influence of abscisic acid – ABA (0; 10; 20 and 30 μM), sucrose concentration (0.09; 0.25 and 0.5 M) and temperature (5ºC or 22ºC) during preculture were tested. Furthermore two dehydration methods, gradual and one-step, were considered. Since the best results were reported with 10 μM ABA + 0.09 M sucrose at room temperature and gradual dehydration, only this combination was selected for the next desiccation-adjustment step. From the time of air desiccation under laminar air-flow applied (0; 1; 2; 3; 4 and 5 hours), 3 hours provided
the best results. Due to the growth inhibition on the hormone-free medium, the addition of plant growth regulators during that phase was also tested, confirming the need to apply cytokinins into the medium.


mutant, ABA, sucrose, preculture, desiccation, regrowth

Annadana S., Rademaker W., Ramanna M., Udayakumar M., Jong J., 2000. Response of stem explants to screening and explant source as a basis for methodological advancing of regeneration protocols for chrysanthemum. Plant Cell Tissue Organ Cult. 62, 47–55.
Bachiri Y., Gazeau C., Hansz J., Morisset C., Dereuddre J., 1995. Successful cryopreservation of suspension cells by encapsulation-dehydration. Plant Cell Tissue Organ Cult. 43, 241–248.
Deng Y., Chen S., Chen F., Cheng X., Zhang F., 2011. The embryo rescue derived intergeneric hybrid between chrysanthemum and Ajania przewalskii shows enhanced cold tolerance. Plant Cell Rep. 30, 2177–2186.
Fukai S., 1990. Cryopreservation of chrysanthemum shoot tips. Sci. Hortic. 45, 167–174.
Fukai S., Goi M., Tanaka M., 1991. Cryopreservation of shoot tips of Chrysanthemum morifolium and related species native to Japan. Euphytica 54, 201–204.
Fukai S., Goi M., Tanaka M., 1994. The chimeric structure of the apical dome of chrysanthemum (Dendranthema grandiflorum (Ramat.) Kitam.) is affected by cryopreservation. Sci. Hortic. 57, 347–351.
Halmagyi A., Fischer-Kluver G., Mix-Wagner G., Schumacher H.M., 2004. Cryopreservation of Chrysanthemum morifolium (Dendranthema grandiflora Ramat.) using different approaches. Plant Cell Rep. 22, 371–375.
Hitmi A., Barthomeuf C., Sallanon H., 2000. Cryopreservation of Chrysanthemum cinerariaefolium shoot tips. J. Plant Physiol., 156(3), 408–412.
Hosoki T., 1989. In vitro storage of Chrysanthemum morifolium at room temperature. Plant Tissue Cult. Lett. 6(2), 86–87.
Martin C., Cervera M.T., Gonzalez-Benito M.E., 2011. Genetic stability analysis of chrysanthemum (Chrysanthemum × morifolium Ramat) after different stages of an encapsulationdehydration cryopreservation protocol. J. Plant Physiol. 168, 158–166.
Martin C., Gonzalez-Benito E., 2009. Cryopreservation and genetic stability of Dendranthema grandiflora Tzvelev in vitro cultures. Agric. Food Sci. 18, 129–135.
Martin C., Gonzalez-Benito M.E., 2005. Survival and genetic stability of Dendranthema grandiflora Tzvelev shoot apices after cryopreservation by vitrification and encapsulation-dehydration. Cryobiology 51, 281–289.
Mikuła A., Fiuk A., Rybczyński J.J., 2005. Induction, maintenance and preservation of embryogenic competence of Gentiana cruciata L. cultures. Acta Biol. Crac. 47(1), 227–236.
Murashige T., Skoog F., 1962. A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol. Plant. 15, 473–497.
Osorio-Saenz A., Mascorro-Gallardo J.O., Rodríguez O.J.L., López C.M., González-Arnao M.T., 2011a. Cryopreservation of chrysanthemum shoot-tips (Dendranthema grandiflorum Kitam) by encapsulation-dehydration and vitrification. Revista Chapingo – Horticultura 17, 33–43.
Osorio-Saenz A., Mascorro-Gallardo J.O., Rocío Valle-Sandoval M., González-Arnao M.T., Engelmann F., 2011b. Geneticallly enginereed trehalose accumulation improves cryopreservation tolerance of chrysanthemum (Dendranthema grandiflorum Kitam.) shoot-tips. CryoLett. 32(6), 477–86.
Pawłowska B., 2008. Employment of encapsulation-dehydration method for liquid nitrogen cryopreservation of ornamental plant explants propagated in vitro. Folia Hortic. 20(1), 61–71.
Pinker I., Abdel-Rahman S.S.A., 2005. Artificial seeds for propagation of Dendranthema × grandiflora (Ramat.). Propag. Ornam. Plants. 5(4), 186–191.
Popova E., Kim H.H., Yi J.Y., Choi Y.M., Sung J.S., Jeon Y.A., Kang M., 2010. Cryopreservation of chrysanthemum via droplet-vitrification method. Cryobiology 64(3), 1.
Sakai A., Matsumoto T., Hirai D., Niino T., 2000. Newly development encapsulation-dehydration protocol for plant cryopreservation. CryoLett. 21(1), 53–62.
Sakai A., Engelmann F., 2007. Vitrification, encapsulation-vitrification and droplet-vitrification: a review. CryoLett. 28(3), 151–172.
Teixeira da Silva J.A., Fukai S., 2003. Four gene introduction methods affect the shoot regeneration and localization of transgene expression in greenhouse stem explants and in vitro-grown chrysanthemum stem thin cell layers. Afr. J. Biotechnol. 2(5), 114–123.
Waseem K., Jilani M.S., Jaskani M.J., Khan M.S., Kiran M., Khan G.U., 2011. Significance of different plant growth regulators on the regeneration of chrysanthemum plantlets (Dendranthema moriflofium L.) through shoot tip culture. Pak. J. Bot. 43(4), 1843–1848.
Wilkinson T., Wetten A., Prychid C., Fay M.F., 2003. Suitability of cryopreservation for the long-term storage of rare and endangered plant species: a case history of Cosmos atrosanguineus. Ann. Bot. 91, 65–74.
Zalewska M., Tymoszuk A., Miler N., 2011. New chrysanthemum cultivars as a result of in vitro mutagenesis with the application of different explant types. Acta Sci. Pol., Hortorum Cultus 10(2), 109–123.


Małgorzata Zalewska 
University of Technology and Life Sciences in Bydgoszcz
Dariusz Kulus 
University of Technology and Life Sciences in Bydgoszcz



Articles are made available under the conditions CC BY 4.0 (until 2020 under the conditions CC BY-NC-ND 4.0).
Submission of the paper implies that it has not been published previously, that it is not under consideration for publication elsewhere.

The author signs a statement of the originality of the work, the contribution of individuals, and source of funding.


Most read articles by the same author(s)

1 2 > >>