Influence of Spike Pretreatment at Low Temperatures on Efficiency of Spring Barley Haploid Production in Anther Culture in Vitro


  • Olena Bilynska Plant Production Institute named after V.Ya. Yuriev of the National Academy of Agrarian Sciences of Ukraine



spring barley, low positive temperatures, anther culture in vitro, agar, starch


The spike pretreatment at low temperatures before isolating and inoculating anthers in a nutrient medium is an important element of technology for producing haploids of many species of higher plants. This procedure not only extends the optimal stage duration of microspores' development, but stimulates their abnormal multiple division with formation of callus, embryoids and increases the frequency of plant regeneration. Here, we have studied the impact of the duration and the mode of spike pre-treatment, genotype and gel-forming component of nutrient medium on the efficiency of morphogenic structure formation and plant regeneration in spring barley in vitro anther cultures. The cut tillers of two varieties and lines and the isolated spikes were kept at 4ºC for 5 (the control) and 28 days, respectively. The aseptically isolated anthers were cultured in nutrient media, contained salts of macro- and microelements according to the N6 and MS formulations, physiologically active substances, maltose, and differed by gel-forming components (agar or chemically modified starch D5a-M) at the optimal concentrations for gel formation. The advantage of isolated spike pretreatment at 4°C within 28 days versus the tiller storage after a 5-day exposure and the expediency to use starch instead of agar within the nutrient medium, have been established. The combination of these methodical approaches resulted in an increase in the frequency of green plant regeneration in line DH00-126 with a genetically determined high capability to in vitro androgenesis from 23.4 up to 100%.

Probl Cryobiol Cryomed 2020; 30(1): 68–76


Asif M. Application and use of haploids. In: Asif M, editor. Progress and opportunities of doubled haploid production. Heidelberg: Springer International Publishing; 2013. p. 55−70. CrossRef

Belinskaya EV. [Influence of donor plant genotype, growth conditions and ptretreatment on the efficiency barley androgenesis]. In: [Modern Problems of Genetics and Crop Breeding]: Proceeding of All-Union young scientist' scientific and technical conference; 1991 April 22−26, Odessa. Odessa, 1991. p. 12. Russian.

Belinskaya EV. [Influence of pretreatment of ears on efficiency of induction of barley haploids in anther culture in vitro]. Fiziologiia i biokhimiia kul'turnykh rasteniy. 2005; 37(5): 436−42. Russian.

Belinskaya EV. Genotype features of morphogenesis in spring barley anther culture. Cytol Genet. 2010, 44(2): 103−7. CrossRef

Belinskaya EV, Dulnyev PG. [Modified starch DKKmod as a component of nutrient medium for barley haploid production in anther culture in vitro]. Fiziologiia i biokhimiia kul'turnykh rasteniy. 2007; (39)2: 136-43. Russian.

Bilynska OV, inventor; Plant Production Institute named after V.Ya. Yurjev, NAAS of Ukraine, assignee. Method of plant material pretreatment for spring barley haploid production in anther culture in vitro. Ukraine patent UA 113261.2016 Dec 26. Ukrainian.

Bilynska OV, Vesna SV, Manzyuk VT [Application of anther culture in vitro for starting material production in hulless barley breeding]. Selektsiia i nasinnytstvo . 2002; 86: 164-72. Ukrainian.

Dunwell JM. Haploids in flowering plants: origins and exploitation. Plant Biotechnol J. 2010; 8 (4): 377-24.


El Goumi Y, Fakiri M, Benbachir M, et al. Effect of cold pretreatment, anthers orientation, spikelet position and donor tiller on the callusing response in barley anther in vitro culture. Int J Med Biotechnol Genetics. 2017; S2 (003): 33−8. CrossRef

Fábián A, Földesiné Füredi PK, Ambrus H, et al. Effect of n-butanol and cold pretreatment on the cytoskeleton and the ultrastructure of maize microspores when cultured in vitro. Plant Cell Tissue and Organ Cult. 2015; 123(2): 257−71. CrossRef

Huang B, Sunderland N. Temperature-stress pretreatment in barley anther culture. Ann Bot. 1982; 49(1): 77−88. CrossRef

Kasha KJ, Simion E, Oro R, et al. An improved in vitro technique for microspore culture of barley. Euphytica. 2001; 120(3): 379-85. CrossRef

Maraschin SF, De Periester W, Spaink HP, et al. Androgenic switch: an example of plant embryogenesis from the mail genotype perspective. J Exp Bot. 2005; 56(417): 1711−26. CrossRef

Prakash J, Giles K. Induction and growth of androgenic haploids. Int Rev Cytol. 1987; 107: 273−92. CrossRef

Shim YS, Kasha KJ. The influence of pretreatment on cell stage progression and the time of DNA synthesis in barley (Hordeum vulgare L.) uninucleate microspores. Plant Cell Rep. 2003; 23(6): 1−7.

Tuvesson S, Dayteg C, Hagberg P, et al. Molecular markers and doubled haploids in European plant breeding programmes. Euphytica. 2007; 158(3): 305−12. CrossRef




How to Cite

Bilynska , O. (2020). Influence of Spike Pretreatment at Low Temperatures on Efficiency of Spring Barley Haploid Production in Anther Culture in Vitro. Problems of Cryobiology and Cryomedicine, 30(1), 68–76.



Theoretical and Experimental Cryobiology