doi: 10.15389/agrobiology.2023.3.554eng

UDC: 633.18:575:57.085.23



M.V. Ilyushko , M.V. Romashova, S.S. Guchenko

Chaika Federal Research Center of Agricultural Biotechnology of the Far East, 30, ul. Volozhenina, pos. Timiryazevskii, Ussuryisk, Primorskii Krai, 692539 Russia, e-mail (✉ corresponding author),,

Ilyushko M.V.
Guchenko S.S.
Romashova M.V.

Final revision received February 27, 2023 
Accepted April 03, 2023 

In vitro culture of cells and tissues of agricultural crops can be conditionally divided into two groups, those to generate a genetically modified initial breeding material and those for mass cloning of existing forms and varieties. Androgenesis in vitro makes it possible to redirect the microspore development from the gametophytic to the sporophytic pathway with the formation of doubled haploids (DHs) in diploid species or the fixation of dihaploids (polyhaploids) in tetraploid species for their wide use in plant breeding. The variability of plants derived from anther or microspore cultures of one donor plant has been studied to a greater extent at the genomic and chromosomal level, since researchers and breeders are primarily interested in spontaneous chromosome duplication and, as a result, completely homozygous fertile offspring. In this work, for the first time, the frequency of intra-callus genetic variability for Pi family blast resistance genes (two and three genes) was estimated using rice (Oryza sativa L.) doubled haploids (DHs) obtained via androgenesis in vitro of hybrid plants. No significant increase in intra-callus genetic variability was shown with an increase in the number of detected genes. The intra-callus variability frequency in androgenesis in vitro in rice was studied in order to determine the genetic homogeneity degree of doubled haploids (DHs) from one anther. Studies were carried out on doubled haploids obtained in androgenesis in vitro of thirteen F1 hybrids and one F2 hybrid of rice O. sativa. Molecular genetic analysis of 1271 plants (83 callus lines) was performed to reveal resistance/susceptibility alleles of the genes Pi-z, Pi-b, Pi-1, Pi-2, Pi-ta for rice blast-resistance to Pyricularia oryzae Cav. [Magnaporthe grisea (Hebert Barr.)]. In doubled haploids, one to four blast-resistance genes were identified depending on the presence of heterozygotes in the original hybrids. When determining one gene in DHs, the frequency of variable callus lines accounted for 24.0 %. For two genes, polymorphism occurs among 47.7 % of calli. For three genes, 62.5 % of callus lines were polymorphic. No more than four combinations of rice blast resistance gene alleles are present in one callus line. There are no differences in the monomorphic callus lines frequency detected for one, two and three genes (χ= 0.21-0.95, p = 0.33-0.65). With the same combination of two resistance gene alleles, up to 66 plants were formed, and with the same combination of three genes alleles, up to 18 plants were produced per callus line. There was no dependence of polymorphism on the number of doubled haploids in the callus line. The correlation coefficients between the number of DHs and the number of alleles for one, two and three genes in the combination accounted for = -0.14, = 0.25, and r = -0.35 (р < 0.05). Genetic analysis of rice doubled haploids revealed a low intra-callus genetic variability during in vitro androgenesis due to gametoclonal variability. Thus, the polymorphic callus lines frequency is high, but with a limited set of allele combinations of rice blast resistance genes among DHs. There is true cloning of rice doubled haploids within the callus lineage in androgenesis in vitro. However, due to the DHs polymorphism within one callus, it is expedient to select lines of doubled haploids as breeders usually do. This work is relevant for optimizing the breeding process, including haploid technology.

Keywords: Oryza sativa, doubled haploids, intra-callus genetic variability frequency, blast resistance genes.



  1. Germana M.A. Gametic embryogenesis and haploid technology as valuable support to plant breeding. Plant Cell. Rep., 2011, 30: 839-857 CrossRef
  2. Jauhar P.P., Xu S.S., Baenziger P.S. Haploidy in cultivated wheats: induction and utility in basic and applied research. Crop Sci., 2009, 49: 737-755 CrossRef
  3. Niazian M., Shariatpanahi M.E. In vitro-based doubled haploid production: recent improvements. Euphytica, 2020, 216: 69 CrossRef
  4. Evans D.A., Sharp W.R., Medina-Filho H.P. Somaclonal and gametoclonal variation. Amer. J. Bot., 1984, 71(2): 759-774 CrossRef
  5. Gosal S.S., Pathak D., Wani S.H., Vij S., Pathak M. Accelerated Breeding of plants: methods and applications. In: Accelerated plant breeding. Vol. 1. S.S. Gosal, S.H. Wani (eds.). Springer Nature Swizerland AG, 2020: 1-29 CrossRef
  6. D’Amato F. Cytogenetics of plant cell and tissue cultures and their regenerates. Critical Reviews in Plant Sciences, 1985, 3(1): 73-112 CrossRef
  7. Segui-Simarro J.M., Nuez F. Pathways to doubled haploidy: chromosome doubling during androgenesis. Cytogenet. Genome Res., 2008, 120: 358-369 CrossRef
  8. Daghma D.E.S., Hensel G., Rutten T., Melzer M., Kumlehn J. Cellular dynamics during early barley pollen embryogenesis revealed by time-lapse imaging. Frontiers in Plant Science, 2014, 5: 675 CrossRef
  9. Voronkova E.V., Ermishin A.P. Biotekhnologiya i selektsiya rasteniy (Minsk), 2012, 3: 170-203 (in Russ.).
  10. Sel’dimirova O.A., Kruglova N.N. Androklinnyy еmbrioidogenez in vitro zlakov. Uspekhi sovremennoy biologii, 2014, 134(5): 476-487 (in Russ.).
  11. Zagorska N.A., Shtereva L.A., Kruleva M.M., Sotirova V.G., Baralieva D.L., Dimitrov B.D. Induced androgenesis in tomato (Lycopersicon esculentum Mill.). III. Characterization of the regenerants. Plant Cell Rep., 2004, 22: 449-456 CrossRef
  12. Cistue L., Soriano M., Castillo A.M., Valles M.P., Sanz J.M., Echavarri B. Production of doubled haploids in durum wheat (Triticum turgidum L.) through isolated microspore culture. Plant CellRep., 2006, 25: 257-264 CrossRef
  13. Grammatikaki G., Avgelis A., Sonnino A. Behavior of potato gametoclonal plants against the necrotic strain of potato Y potyvirus. Russ. J. Plant Physiol., 2007, 54(4): 507-512 CrossRef
  14. Goncharova J.K. Selective elimination of alleles in rice anther culture. Russ. J. Genet., 2013, 49(2): 170-177 CrossRef
  15. Mishra R., Rao G.J.N., Rao R., N., Kaushal P. Development and characterization of elite doubled haploid lines from two indica rice hybrids. Rice Sci., 2015, 22(6): 290-299 CrossRef
  16. Windarsih G., Utami D.W., Widyastuti U. Molecular markers application for blast resistance selection on the double haploid rice population. Makara Journal of Science, 2014, 18(2): 31-41 CrossRef
  17. Yi G., Lee H.-S., Kim K.-M. Improved marker-assisted selection efficiency of multi-resistance in doubled haploid rice plants. Euphytica, 2015, 203: 421-428 CrossRef
  18. Tripathy S.K., Swain D., Mohapatra P.M., Prusti A.M., Sahoo B., Panda S., Dash M., Chakma B., Behera S. Exploring factors affecting anther culture in rice (Oryza sativa L.). Journal of Applied Biology and Biotechnology, 2019, 7(02): 87-92 CrossRef
  19. Maharani A., Fanata W.I.D., Laeli F.N., Kim K.-M., Handoyo T. Callus induction and regeneration from anther cultures of indinesian indica black rice cultivar. J. Crop Sci. Biotechol., 2020, 23(1): 21-28 CrossRef
  20. Kruglova N.N. Еkobiotekh, 2019, 2(2): 100-115 CrossRef (in Russ.).
  21. Yamamoto T., Soeda Y., Nishikawa A., Hirohara H. A study of somaclonal variation for rice improvement induced by three kinds of anther-derived cell culture techniques. Plant Tissue Culture Letters, 1994, 11(2): 116-121.
  22. Chen C.C., Chen C.-M. Changes in chromosome number in microspore callus of rice during successive subcultures. Canadian Journal of Genetics and Cytology, 1980, 22(4): 607-614 CrossRef
  23. Yoshida S., Watanabe K., Fujino M. Non-random gametoclonal variation in rice regenerants from callus subcultured for a prolonged period under high osmotic stress. Euphytica, 1998, 104: 87-94 CrossRef
  24. Yamagishi M., Yano M., Fukuda Y., Fukui K., Otani M., Shimada T. Distorted segregation of RFLP markers in regenerated plants derived from anther culture of an F1 hybrid of rice. Genes & Genet. Syst., 1996, 71: 37-41 CrossRef
  25. Castillo A.M., Valles M.P., Cistue L. Comparison of anther and isolated microspore culture in barley. Effects of culture density and regeneration medium. Euphytica, 2000, 113: 1-8 CrossRef
  26. Ilyushko M.V. Risovodstvo, 2019, 2(43): 29-32 (in Russ.).
  27. Ilyushko M.V., Romashova M.V. Rice tetraploid formation in androgenesis in vitro. Russian Agricultural Sciences, 2020, 4: 14-17 CrossRef
  28. Ilyushko M.V., Romashova M.V. Variability of rice haploids obtained from in vitroanther culture. Russian Agricultural Sciences, 2019, 45(3): 243-246 CrossRef
  29. Ilyushko M.V., Romashova M.V., Zhang J.-M., Deng L.-W., Liu D.-J., Zhang R., Guchenko S.S. Intra-callus variability of rice doubled haploids generated through in vitro androgenesis. Sel’skokhozyaistvennaya biologiya [Agricultural Biology], 2020, 55(3): 533-543 CrossRef
  30. Farrell T.C., Fox K.M., Williams R.L., Fukai S. Genotypic variation for cold tolerance during reproductive development in rice: screening with cold air and cold water. Field Crops Research, 2006, 98: 178-194 CrossRef
  31. Chu C. The N6 medium and its application to anther culture of cereal crops. Proc. Simposium on Plant Tissue Culture. Peking, 1978: 43-50.
  32. Ilyushko M.V. Izvestiya TSKhA, 2007, 2: 126-133 (in Russ.).
  33. Goncharova Yu.K. Ispol’zovanie metoda kul’tury pyl’nikov v selektsii risa [Use of anther culture method in rice breeding]. Krasnodar, 2012 (in Russ.).
  34. Aljanabi S.M., Martinez I. Universal and rapid salt-extraction of high quality genomic DNA for PCR-based techniques. Nucleic Acid Research, 1997, 25(22): 4692-4693 CrossRef
  35. Dubina E.V., Kostylev P.I., Garkusha S.V., Ruban M.G. Development of blast-resistant rice varieties based on application of DNA technologies. Euphytica, 2020, 216: 162 CrossRef
  36. Wang J.C., Correll J.C., Jia Y. Characterization of rice blast resistance genes in rice germplasm with monogenic lines and pathogenicity assays. Crop Protection, 2015, 72: 132-138 CrossRef
  37. Ferrie A.M.R., Caswell K.L. Isolated microspore culture techniques and recent progress for haploid and doubled haploid plant production. Plant Cell Tiss. Organ. Cult., 2011, 104: 301-309 CrossRef
  38. Sarao N.K., Gosal S.S. In vitro androgenesis for accelerated breeding in rice. In: Biotechnologies of crop improvement. Vol. 1 /S.S. Gosal, S.H. Wani (eds.). Springer, Cham, 2018: 407-435 CrossRef
  39. Win A., Tanaka T.S.T., Matsui T. Panicle inclination influences pollination stability of rice (Oryza sativa L.). Plant production Science, 2020, 23(1): 60-68 CrossRef
  40. Tyrnov V.S., Davoyan N.I. V knige: Gaploidiya i selektsiya [In: Haploidy and selection]. Moscow, 1976: 57-65 (in Russ.).
  41. Kasha K.J., Hu T.C., Oro R., Simion E., Shim Y.S. Nuclear fusion leads to chromosome doubling during mannitol pretreatment of barley (Hordeum vulgare L.) microspores. Journal of Experimental Botany, 2001, 52(359): 1227-1238 CrossRef
  42. Ilyushko M.V., Skaptsov M.V., Romashova M.V. Nuclear DNA content in rice (Oryza sativa L.) regenerants derived from anther culture in vitro. Sel'skokhozyaistvennaya biologiya [Agricultural Biology], 2018, 53(3): 531-538 CrossRef
  43. Savenko E.G., Vlasov V.G. Risovodstvo, 2009, 14: 20-21 (in Russ.).
  44. Kuznetsova O.I., Ash O.A., Gostimskij S.A. The effect of duration of callus culture on the accumulation of genetic alteration in pea Pisum sativum L. Russ. J. Genet., 2006, 42(5): 555-562 CrossRef







Full article PDF (Rus)

Full article PDF (Eng)