Haploid technologies are based on the regeneration of gamete cells to form whole plants. The main advantage of haploid technologies is the ability to quickly in one generation obtain homozygous plants with the required parameters. Nevertheless, the use of these technologies today is limited, what is associated with insufficient knowledge of the physiological and biochemical features of the application of this method. In this regard, this work is devoted to the study of the features of androgenesis in vitro in the culture of isolated anthers of winter wheat cultivar Irkutskaya and the determination of the methodological features of obtaining intact plants. In the autumn-winter period, donor plants were grown at the artificial climate station SIPPB SB RAS. In the spring-summer period, donor plants were grown in the field at the experimental plots of the Zalarinsky agroecological station (Tungui village). The plant material was selected at the booting stage. Anthers were isolated from 6-10 spikelets of the middle third of the spike. The features of androgenesis were assessed by the frequency of formation of calli and embryo-like structures to the total number of anthers and the frequency of formation of the total number of seedlings and the number of green seedlings to the number of embryo-like structures and calli. It was found that the optimal medium for the induction of androgenesis in the anther culture of this common wheat cultivar is the modification of medium N₆ containing 9% sucrose, with the addition of glycine (2 mg/L), 2.4-D (1 mg/L) and NAA (1 mg/L). The maximum yield of embryoid-like structures was observed in the case of low-temperature pretreatment of donor plants at + 4 °C for 6-12 days. At the same time, the most intensive calli formation in the culture of isolated anthers occurred after 3-6 days of donor plant cold pretreatment. Regeneration of green plants took place on 190-2Cu medium with the addition of growth regulators (2.4-D, 0.5 mg/L; kinetin, 0.5 mg/L) with obligatory cold pretreatment of androclinic structures at +4 °C for 3-7 days. Obtained results indicate that low-temperature exposure at different stages, from pretreatment of donor ears to exposure to androclinic structures, is a key factor for winter wheat that promotes androgenesis in the culture of isolated anthers and the successful formation of intact plants.

Lyubushkina Irina Viktorovna, Candidate of Science (Biology), Research Scientist, Siberian Institute of Plant Physiology and Biochemistry SB RAS, 132, Lermontov st., Irkutsk, 664033, Russian Federation; Assistant Professor, Irkutsk State University, 1, K. Marx st., Irkutsk, 664033, Russian Federation, e-mail: ostrov1873@yandex.ru 

Pomortsev Anatolii Vladimirovich, Candidate of Science (Biology), Research Scientist, Siberian Institute of Plant Physiology and Biochemistry SB RAS, 132, Lermontov st., Irkutsk, 664033, Russian Federation, e-mail: pomorcevanatolii@mail.ru 

Polyakova Marina Stanislavovna, Lead Engineer, Siberian Institute of Plant Physiology and Biochemistry SB RAS, 132, Lermontov st., Irkutsk, 664033, Russian Federation, e-mail: poljakova.m@gmail.com 

Arbuzova Galina Andreevna, Lead Engineer, Siberian Institute of Plant Physiology and Biochemistry SB RAS, 132, Lermontov st., Irkutsk, 664033, Russian Federation; Undergraduate Student, Irkutsk State Univercity, 1, K. Marx st., Irkutsk, 664003, Russian Federation, e-mail: mingronland@mail.ru 

Voinikov Victor Kirillovich Doctor, of Sciences (Biology), Scientific Director, Siberian Institute of Plant Physiology and Biochemistry SB RAS, 132, Lermontov st., Irkutsk, 664033, Russian Federation, e-mail: vvk@sifibr.irk.ru 

Anapiyaev Bahytzhan Beisenbekovich, Doctor of Sciences (Biology), Satbayev University, 22a, Satpaev st., Almaty, 050013, Kazakhstan, e-mail: bak_anapiyayev@mail.ru

Lyubushkina I.V., Pomortsev A.V., Polyakova M.S., Arbuzova G.A., Voinikov V.K., Anapiyaev B.B. Influence of Initial Media and Duration of Cold Pretreatment on the Efficiency of Androgenesis in the Culture of Isolated Anthers of Winter Wheat (Triticum aestivum L.) Variety Irkutskaya. The Bulletin of Irkutsk State University. Series Biology. Ecology, 2020, vol. 34, pp. 20-32. https://doi.org/10.26516/2073-3372.2020.34.20 (in Russian)

Anapiyayev B.B. Kultura mikrospor i gaploidnaya biotekhnologiya pshenitsy [The microspores culture and haploid biotechnology of wheat]. Almaty, 2001, 220 p. (in Russian) 

Zhonosar M.V. Optimizatsiia tekhnologii polucheniia udvoennykh gaploidov miagkoi-pshenitsy razlichaiushchikhsia po genam fotoperiodicheskoi chuvstvitelnosti (Ppd) I prodolzhitelnosti iarovizatsii (Vrd) [Optimization of the technology for obtaining doubled haploids of common wheat, differing in genes for photoperiodic sensitivity (Ppd) and duration of vernalization (Vrd): Candidate in Biology dissertation abstract]. Odessa, 2009, 20 p. (in Russian)

Pershina L.A., Osadchaya T.S., Badaeva E.D., Belan I.A., Rosseeva L.P. Izuchenie osobennostei androgeneza v kulture pyl'nikov sortov i perspektivnoi formy yarovoi myagkoi pshenitsy zapadnosibirskoi selektsii, razlichayushchikhsya nalichiem ili otsutstviem pshenichno-chuzherodnykh translokatsii [Features of androgenesis in anther cultures of varieties and a promising accession of spring common wheat bred in west siberia differing in the presence or absence of wheat-alien translocations]. Vavilovskij zhurnal genetiki i selekcii [Vavilov J. Genet. Breed.], 2013, vol. 17, no. 1, pp. 40-49. (in Russian)

Razmakhnin E.P., Razmakhnina T.M., Kozlov V.E., Gordeeva E.I., Goncharov N.P., Galitsyn G.Y., Veprev S.G., Chekurov V.M. Poluchenie vysokomorozostoikikh form pshenichno-pyreinykh gibridov [Raise of high frost-resistant Agropyron–Triticum hybrids]. Vavilovskij zhurnal genetiki i selekcii [Vavilov J. Gen. Breed.], 2012, vol. 16, no. 1, pp. 240-249. (in Russian)

Bhojwani S.S., Dantu P.K. Haploid plants. Plant Cell Culture: Essential Methods. N. Y., John Wiley & Sons., 2010, pp. 61-78. https://doi.org/10.1002/9780470686522.ch4

Bohanec B. Doubled Haploids via Gynogenesis. Advanced in haploid production in higher plants. Dordrecht, SpringerScience + BusinessMedia, 2009, pp. 47-65. https://doi.org/10.1007/978-1-4020-8854-4_2

Chaudhary H.K., Dhaliwal I., Singh S., Sethi G.S. Genetics of androgenesis in winter and spring wheat genotypes. Euphytica, 2003, vol. 132, pp. 311-319. https://doi.org/10.1023/A:1025094606482 

Chuang C.C., Ouyang J.W., Chia H., Chou S.M., Ching C.K. A set of potato media for wheat anther culture. Proc. of Symposium on Plant Tissue Culture. Peking, Sci. Press., 1978, pp. 51-56.

Gamborg O.L., Eveleigh D.E. Culture methods and detection of glucanases in suspension cultures of wheat and barley. Can. J. Biochem., 1968, vol. 46, no. 5, pp. 417-421. https://doi.org/10.1139/o68-063 

Grauda D., Miķelsone A., Ļisina N., Žagata K., Ornicāns R., Fokina O., Lapiņa L., Rashal I. Anther culture effectiveness in producing doubled haploids of cereals. Proc. Latv. Acad. Sci. Sect. B, 2014, vol. 68, pp. 142-147. https://doi.org/10.2478/prolas-2014-0016

Hu T., Kasha K.J. Improvement of isolated microspore culture of wheat (Triticum aestivum L.) through ovary co-culture. Plant Cell Rep., 1997, vol. 16, pp. 520-525. https://doi.org/10.1007/BF01142316 

Islam S.M.S., Tuteja N. Enhancement of androgenesis by abiotic stress and other pretreatments in major crop species. Plant science, 2012, vol. 182, pp. 134-144. https://doi.org/10.1016/j.plantsci.2011.10.001 

Kasha K.J., Simion E., Oro R., Yao Q.A., Hu T.C., Carlson A.R. An improved in vitro technique for isolated microspore culture of barley. Euphytica, 2001, vol. 120, pp. 379-385. https://doi.org/10.1023/A:1017564100823

Lantos C., Jancsó M., Pauk J. Microspore culture of small grain cereals. Acta Physiol. Plant. 2005, vol. 27, pp. 631-639. https://doi.org/10.1007/s11738-005-0067-6

Marsolais A.A., Séguin-Swartz G., Kasha K.J. The influence of anther cold pretreatments and donor plant genotypes on in vitro androgenesis in wheat (Triticum aestivum L.). Plant Cell, Tissue and Organ Culture, 1984, vol. 3, pp. 69-79. https://doi.org/10.1007/BF00035922 

Niroula R.K., Bimb H.P. Overview of wheat X maize system of crosses for dihaploid Induction in wheat. WASJ, 2009, vol. 7, no 8, pp. 1037-1045.

Olmedilla A. Microspore embryogenesis. Plant Development Biology – Biotechnological Perspectives. Heidelberg, Springer-Verlag, 2010, vol. 2, pp. 27-44. https://doi.org/10.1007/978-3-642-04670-4_2 

Pauk J., Mihály R., Puolimatka M. Protocol for wheat (Triticum aestivum L.) anther culture. Doubled Haploid Production in Crop Plants. Dordrecht, Springer Science+Business Media Publ., 2003, pp. 59–64. https://doi.org/10.1007/978-94-017-1293-4_10 

Soriano M., Li H., Boutilier K. Microspore embryogenesis: establishment of embryo identity and pattern in culture. Plant Reprod., 2013, vol. 26, pp. 181-196. https://doi.org/10.1007/s00497-013-0226-7

Touraev A., Ilham A., Vicente O., Heberle-Bors E. Stress induced microspore embryogenesis in tobacco: an optimized system for molecular studies. Plant Cell Rep., 1996, vol. 15, pp. 561-565. https://doi.org/10.1007/BF00232453

Weigt D., Kiel A., Siatkowski I., Zyprych-Walczak J., Tomkowiak A., Kwiatek M. Comparison of the androgenic response of spring and winter wheat (Triticum aestivum L.). Plants, 2020, vol. 9, no. 1, pp. 49. https://doi.org/10.3390/plants9010049

Wedzony M., Forster B.P., Żur I., Golemiec E., Szechynska-Hebda M., Dubas E., Gotębiowska G. Progress in doubled haploid technology in higher plants. Advances in haploid Production in Higher Plants. Heidelberg, Springer-Verlag, 2009, pp. 1-34. https://doi.org/10.1007/978-1-4020-8854-4_1 

Zamani I., Kovacs G., Gouli-Vavdinoudi E., Roupakias D.G., Barnabas B. Regeneration of fertile doubled haploid plants from colchicine-supplemented media in wheat anther culture. Plant Breed., 2000, vol. 119, pp. 461-465. https://doi.org/10.1046/j.1439-0523.2000.00538.x