Intracallus cytotypic variation of regenerated Oryza sativa L. plants in the in vitro androgenesis

Background. The in vitro androgenesis has proven to be a reliable method for obtaining doubled haploids for many plant species. In the breeding of Oryza sativa L., in vitro culture of anthers is used, which go through the stage of callus formation followed by regeneration. The ratios between cells of different types and green regenerated plantlets on the callus might not coincide, since not all genetic disorders that accumulate in the in vitro culture at the cellular level can go through the stage of morphogenesis and cannot always lead to regeneration. The objective of the study was to assess the frequency of intracallus cytotypic variability of regenerated O. sativa plants in the in vitro androgenesis.
Materials and methods. We studied regenerated plantlets obtained on O. sativa calli through the in vitro androgenesis of thirty F 1 and F2 hybrids. According to their morphological features, regenerated plants were divided into five cytotypic groups: haploids, doubled haploids, aneuploids, tetraploids, and plants that died in the early stages of growth and development.
Results. The experiment employed 409 calli with multiple regenerations. Only haploid plants were formed on 79 calli, only doubled haploids on 60 calli, only aneuploids on three calli, only dead plantlets on one callus, and only tetraploids on two calli. It was established that 265 (64.8%) calli were polymorphic. The calli polycytotypicity with two types of regenerated plants, excluding the dead ones, was 138 pcs. (33.7%), with three types 62 pcs. (15.2%), and with four types 7 pcs. (1.7%). The differences between the calli of the F 1 and F2 hybrids were highly significant in the haploids, doubled haploids, tetraploids, and dead regenerated plants (p < 0.0002). An increase in the number of regenerated plantlets per callus occurred due to the presence of haploid plants; the correlation coefficient was r = 0.81 (p < 0.05).
Conclusion. The intracallus cytotypic variability of O. sativa in the in vitro androgenesis was 64.8%, which was comparable to the proportion of the calli with different cell ploidy.

1. 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. DOI: 10.1139/g80-066

2. Chen L., Yuan Y., Wu J., Chen Z., Wang L., Shahid M.Q. et al. Carbohydrate metabolism and fertility related genes high expression levels promote heterosis in autotetraploid rice harboring double neutral genes. Rice (New York, NY). 2019;12(1):34. DOI: 10.1186/s12284-019-0294-x

3. Germana M.A. Anther culture for haploids and doubled haploid production. Plant Cell, Tissue and Organ Culture. 2011;104(3):283-300. DOI: 10.1007/s11240-010-9852-z

4. Goncharova Y.K. Molecular marking in rice breeding (Molekulyarnoye markirovaniye v selektsii risa). Krasnodar: All-Russian Rice Research Institute; 2018. [in Russian]

5. Goncharova Y.K., Vereshchagina S.A., Gontcharov S.V. Nutrient media for double haploid production in anther culture of rice hybrids. Plant Cell Biotechnology and Molecular Biology. 2019;20(23-24):1215-1223.

6. Hooghvorst I., Ramos-Fuentes E., López-Cristofannini C., Ortega M., Vidal R., Serrat X. et al. Antimitotic and hormone effects on green doubled haploid plant production through anther culture of Mediterranean japonica rice. Plant Cell, Tissue and Organ Culture. 2018;134(2):205-215. DOI: 10.1007/s11240-018-1413-x

7. Ilyushko M.V., Guchenko S.S., Romashova M.V. Intracallus and intercallus morphological variability of rice doubled haploids obtained in in vitro androgenesis. Russian Agricultural Sciences. 2021;47(1):11-16. DOI: 10.3103/S1068367421010080

8. Ilyushko M.V., Romashova M.V., Guchenko S.S. Intra-callus variability for rice blast resistance genes in Oryza sativa L. indicated by genetic analysis of androgenic doubled haploids. Agricultural Biology. 2023;58(3):554-566. DOI: 10.15389/agrobiology.2023.3.554eng

9. Ilyushko M.V., Skaptsov M.V., Romashova M.V. Nuclear DNA content in rice (Oryza sativa L.) regenerants derived from anther culture in vitro. Agricultural Biology. 2018;53(3):531- 538. DOI: 10.15389/agrobiology.2018.3.531eng

10. Ilyushko M.V., Skaptsov M.V., Romashova M.V. Variability of morphological features and nuclear DNA content in haploid and doubled haploids of androgenic callus lines of rice (Oryza sativa L.). Proceedings on Applied Botany, Ge ne tics and Breeding. 2022;183(4):172-180. DOI: 10.30901/2227-8834-2022-4-172-180

11. Kuznetsova O.I., Ash O.A., Gostimsky S.A. The effect of duration of callus culture on the accumulation of genetic alteration in pea Pisum sativum L. Russian Journal of Genetics. 2006;42(5):555-562. DOI: 10.1134/s1022795406050139

12. Lantos C., Jancsó M., Székely A., Nagy E., Szalóki T., Pauk J. Improvement of anther culture to integrate doubled haploid technology in temperate rice (Oryza sativa L.) breeding. Plants. 2022;11(24):3446. DOI: 10.3390/plants11243446

13. Luan L., Wang X., Long W.B., Liu Y.H., Tu S.B., Xiao X.Y. et al. A comparative cytogenetic study of the rice (Oryza sativa L.) autotetraploid restorers and hybrids. Russian Journal of Genetics. 2009;45(9):1074-1081. DOI: 10.1134/S1022795409090087

14. Murovec J., Bohanec B. Haploids and doubled haploids in plant breeding. In: I.Y. Abdurakhmonov (ed.). Plant Breeding. London: IntechOpen; 2012. p.87-106. DOI: 10.5772/29982

15. Niazian M., Shariatpanahi M. In vitro-based doubled haploid production: recent improvements. Euphytica. 2020;216(5):69. DOI: 10.1007/s10681-020-02609-7

16. Niizeki H., Oono K. Induction of haploid rice plant from anther culture. Proceedings of the Japan Academy. 1968;44(6):554- 557. DOI: 10.2183/pjab1945.44.554

17. Ogawa T., Fukuoka H., Ohkawa Y. Plant regeneration through direct culture of isolated pollen grains in rice. Japanese Journal of Breeding. 1995;45(3);301-307. DOI: 10.1270/JSBBS1951.45.301

18. Sakhina A., Mir S., Najeeb S., Zargar S.M., Nehvi F.A., Rather Z.A. et al. Improved protocol for efficacious in vitro androgenesis and development of doubled haploids in temperate japonica rice. PLoS One. 2020;15(11):e0241292. DOI: 10.1371/journal.pone.0241292

19. Sarao N.K., Gosal S.S. In vitro androgenesis for accelerated breeding in rice. In: S.S. Gosal, S.H. Wani (eds). Biotechnologies of Crop Improvement. Vol. 1. Cham: Springer; 2018. p.407-435. DOI: 10.1007/978-3-319-78283-6_12

20. Sartbaeva I.A., Usenbekov B.N., Rysbekova A.B., Mukhina Zh.M., Kazkeev D.N., Zhambakin K.Zh. et al. Obtaining of doubled haploid lines for selection of glutinous rice. Biotech no lo gy in Russia. 2018;34(2):26-36. DOI: 10.21519/0234-2758-2018-34-2-26-36

21. Seguí-Simarro J.M., Jacquier N.M.A., Widiez T. Overview of in vitro and in vivo doubled haploid technologies. Methods in Molecular Biology. 2021;2287:3-22. DOI: 10.1007/978-1-0716-1315-3_1

22. Singh S.K., Jeughale K.P., Dash B., Bhuyan S.S., Chandravani M., Chidambaranathan P. et al. Discrimination of haploids and doubled haploids/diploids in indica rice: correlation of morphological indicators with molecular markers. Biologia Plantarum. 2023;67(7):294-302. DOI: 10.32615/bp.2023.008

23. Tajedini S., Fakheri B., Niazian M., Mahdinezhad N., Ga nim A.M.A., Pour A.K. et al. Efficient microspore embryogenesis and haploid induction in mutant indica rice (Oryza sativa L.) cultivars. Journal of Plant Growth Regulation. 2022;42(4):2345-2359. DOI: 10.1007/s00344-10709-y

24. Tu S., Luan L., Liu Y., Long W., Kong F., He T. et al. Production and heterosis analysis of rice autotetraploid hybrids. Crop Science. 2007;47(6):2356-2363. DOI: 10.2135/cropsci2007.01.0058

25. Tyrnov V.S., Davoyan N.I. Cytogenetics of somatic tissues in haploids (Tsitogenetika somaticheskikh tkaney gaploidov). In: V.A. Krupnov (ed.). Haploidy and Breeding (Gaploidiya i selektsiya). Moscow: Nauka; 1976. p.57-65 [in Russian]

26. Wu Y., Sun Y., Sun S., Li G., Wang J., Wang B. et al. Aneuplo idization under segmental allotetraploidy in rice and its phenotypic manifestation. Theoretical and Applied Gene tics. 2018;131(6):1273-1285. DOI: 10.1007/s00122-018-3077-7