vir-nwТруды по прикладной ботанике, генетике и селекцииProceedings on applied botany, genetics and breeding2227-88342619-0982N.I. Vavilov All-Russian Institute of Plant Genetic Resources10.30901/2227-8834-2025-2-138-146vir-nw-2343Research ArticleИДЕНТИФИКАЦИЯ ГЕНЕТИЧЕСКОГО РАЗНООБРАЗИЯ КУЛЬТУРНЫХ РАСТЕНИЙ И ИХ ДИКИХ РОДИЧЕЙ ДЛЯ РЕШЕНИЯ ФУНДАМЕНТАЛЬНЫХ И ПРИКЛАДНЫХ ПРОБЛЕМIDENTIFICATION OF THE DIVERSITY OF CULTIVATED PLANTS AND THEIR WILD RELATIVES FOR SOLVING FUNDAMENTAL AND APPLIED PROBLEMSВнутрикаллусная цитотипическая изменчивость регенерантов Oryza sativa L. в андрогенезе in vitroIntracallus cytotypic variation of regenerated Oryza sativa L. plants in the in vitro androgenesishttps://orcid.org/0000-0001-7042-8641ИлюшкоМ. В.IlyushkoM. V.

Марина Владиславовна Илюшко, кандидат биологических наук, ведущий научный сотрудник 

692539, Уссурийск, пос. Тимирязевский, ул. Воложенина, 30

Marina V. Ilyushko, Cand. Sci. (Biology), Leading Researcher 

30 Volozhenina St., Timiryazevsky Settlem., Ussuriysk 692539 

ilyushkoiris@mail.ruФедеральный научный центр агробиотехнологий Дальнего Востока им. А.К. ЧайкиРоссияFederal Scientific Center of Agricultural Biotechnology of the Far East named after A.K. ChaikaRussian Federation2025300620251862138146Copyright © Илюшко М.В., 20252025Илюшко М.В.Ilyushko M.V.This work is licensed under a Creative Commons Attribution 4.0 License.https://elpub.vir.nw.ru/jour/article/view/2343

Актуальность. Андрогенез in vitro зарекомендовал себя как надежный способ получения удвоенных гаплоидов для многих видов растений. В селекции Oryza sativa L. используется культура пыльников in vitro, которые проходят стадию каллусообразования с последующей регенерацией. Соотношение клеток разного типа и зеленых регенерантов на каллусе может не совпадать, так как не все генетические нарушения, которые накапливаются в культуре in vitro на клеточном уровне, могут пройти этап морфогенеза и не всегда могут привести к регенерации. Целью исследования являлось изучение частоты внутрикаллусной цитотипической изменчивости регенерантов O. sativa в культуре пыльников in vitro.Материалы и методы. Исследовали регенеранты, полученные на каллусах риса O. sativa в андрогенезе in vitro тридцати гибридов F1 и F2. По морфологическим признакам регенеранты разделяли на пять цитотипических групп: гаплоиды, удвоенные гаплоиды, анеуплоиды, тетраплоиды и растения, погибшие на ранних этапах роста и развития.Результаты. В эксперименте использовано 409 каллусов с множественной регенерацией. На 79 каллусах образовались только гаплоидные растения, на 60 – только удвоенные гаплоиды, на трех – только анеуплоиды, на одном – только погибшие, на двух – только тетраплоиды. Полиморфными оказались 265 (64,8%) каллусов. Полицитотипичность каллусов c двумя типами регенерантов без учета погибших составляет 138 шт. (33,7%), с тремя типами – 62 шт. (15,2%), с четырьмя типами – 7 шт. (1,7%). Различия между каллусами гибридов F1 и F2 высокозначимы по гаплоидам, удвоенным гаплоидам, тетраплоидным и погибшим регенерантам (р < 0,0002). Увеличение числа регенерантов на каллус происходит за счет гаплоидных растений, коэффициент корреляции r = 0,81 (p < 0,05).Заключение. Внутрикаллусная цитотипическая изменчивость риса O. sativa в андрогенезе in vitro составляет 64,8%, что сопоставимо с долей каллусов с различной плоидностью клеток.

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 F1 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 F1 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.

андрогенез in vitroвнутрикаллусная изменчивостьполицитотипичность каллусоврегенерантin vitro androgenesisintracallus variabilitycalli polycytotypicityregenerationработа выполнена в рамках государственного задания Федерального научного центра агробиотехнологий Дальнего Востока им. А.К. Чайки по теме FNGW-2022-0008 «Создать новые генотипы сельскохозяйственных культур с высокой продуктивностью, устойчивые к абиотическим и биотическим факторам среды». Автор благодарит рецензентов за их вклад в экспертную оценку этой работы.the work was carried out within the framework of the state task assigned to the Federal Scientific Center of Agricultural Biotechnology of the Far East named after A.K. Chaika, Project FNGW-2022-0008 “To develop new genotypes of agricultural crops with high productivity, resistant to abiotic and biotic environmental factors”. The author thanks the reviewers for their contribution to the peer review of this work.ReferencesChen 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/g80-066Chen 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/g80-066Chen 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-xChen 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-xGermana 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-zGermana 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-zГончарова Ю.К. Молекулярное маркирование в селекции риса. Краснодар: ВНИИ риса; 2018.Goncharova Y.K. Molecular marking in rice breeding (Molekulyarnoye markirovaniye v selektsii risa). Krasnodar: All-Russian Rice Research Institute; 2018. [in Russian]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.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.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-xHooghvorst 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-xIlyushko 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/S1068367421010080Ilyushko 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/S1068367421010080Ilyushko 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.2023.3.554engIlyushko 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.2023.3.554engIlyushko 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.2018.3.531engIlyushko 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.2018.3.531engIlyushko 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: 10.30901/2227-8834-2022-4-172-180Ilyushko 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: 10.30901/2227-8834-2022-4-172-180Kuznetsova 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/s1022795406050139Kuznetsova 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/s1022795406050139Lantos 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/plants11243446Lantos 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/plants11243446Luan 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/S1022795409090087Luan 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/S1022795409090087Murovec J., Bohanec B. Haploids and doubled haploids in plant breeding. In: I.Y. Abdurakhmonov (ed.). Plant Breeding. London: IntechOpen; 2012. p.87-106. DOI: 10.5772/29982Murovec J., Bohanec B. Haploids and doubled haploids in plant breeding. In: I.Y. Abdurakhmonov (ed.). Plant Breeding. London: IntechOpen; 2012. p.87-106. DOI: 10.5772/29982Niazian M., Shariatpanahi M. In vitro-based doubled haploid production: recent improvements. Euphytica. 2020;216(5):69. DOI: 10.1007/s10681-020-02609-7Niazian M., Shariatpanahi M. In vitro-based doubled haploid production: recent improvements. Euphytica. 2020;216(5):69. DOI: 10.1007/s10681-020-02609-7Niizeki H., Oono K. Induction of haploid rice plant from anther culture. Proceedings of the Japan Academy. 1968;44(6):554- 557. DOI: 10.2183/pjab1945.44.554Niizeki H., Oono K. Induction of haploid rice plant from anther culture. Proceedings of the Japan Academy. 1968;44(6):554- 557. DOI: 10.2183/pjab1945.44.554Ogawa 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/JSBBS1951.45.301Ogawa 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/JSBBS1951.45.301Sakhina 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.0241292Sakhina 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.0241292Sarao 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_12Sarao 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_12Sartbaeva 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: 10.21519/0234-2758-2018-34-2-26-36Sartbaeva 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: 10.21519/0234-2758-2018-34-2-26-36Seguí-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_1Seguí-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_1Singh 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.2023.008Singh 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.2023.008Tajedini 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-yTajedini 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-yTu 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/cropsci2007.01.0058Tu 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/cropsci2007.01.0058Тырнов В.С., Давоян Н.И. Цитогенетика соматических тканей гаплоидов. В кн.: Гаплоидия и селекция / под ред. В.А. Крупнова. Москва: Наука; 1976. С.57-65.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]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/s00122-018-3077-7Wu 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

The authors declare that there are no conflicts of interest present.