Biotechnological approaches for accelerated breeding of root crops

   Relevance. The relevance of accelerating root crop breeding is dictated by two global challenges: the need to ensure food security for a growing population in conditions of limited resources and the inability of classical methods, characterized by their duration and complexity, to respond promptly to them. The inte gration of biotechnologies into the breeding process makes it possible to dramatically accelerate the achievement of breeding goals.

   Materials and Methods. Prospective cultivars of vegetable root crops selected by the Federal Scientific Vegetable Center were used as plant material. Buds were selected from donor plants grown both in a polycarbonate greenhouse and in a climate chamber from vernalized root crops. The research used generally accepted methods of in vitro cell and tissue cultivation according to protocols developed at the Federal Scientific Vegetable Center for the culture of unpollinated ovules, culture of isolated microspores in vitro, embryo rescue technology, and clonal micropropagation of root crops.

   Results. The efficiency of obtaining lines of doubled haploids (DH-lines) using cultures of isolated microspores and pollinated ovules in vitro is shown, and the possibilities of using microclonal reproduction and "embryo rescue" technologies in the breeding process are considered. For beetroot, the yield of up to 25 embryoids has been achieved, and for carrot, up to 55 embryoids per 100 in vitro unpollinated ovules introduced into culture. By the use of a culture of isolated microspores from representatives of Brassicaceae family for radishes, the maximum efficiency of the technology was 8 embryoids per Petri dish, 53 embryoids for turnips, 161 embryoids for carrots, and 20 embryoids for parsnips. Interspecific hybrids between white cabbage (Brassica oleracea L.) and keel-resistant daikon (Raphanus sativus L.), as well as interspecific hybrids from crossing white cabbage with turnips (♀ B. oleraceae L.× ♂ B. rapa), were obtained using the "embryo rescue" technology. The method of clonal micropropagation has been optimized for radishes, beets and carrots, including for lines with male sterility. The practical result of the research was the creation and registration of new varieties and DH-lines: Sonata table carrots, radishes (Zhegalov, Venya, Persey), and obtaining a patent for invention No. 2807444 "Microspore isolation method for obtaining doubled haploids of the Brassicaceae family in microspore culture in vitro."

   Conclusion. The paper presents the results of the application of modern biotechnological methods in vitro to accelerate the breeding process of vegetable root crops of the Amaranthaceae Juss family. (red beet), Apiaceae Lindl. (carrots, parsnips), Brassicaceae Burn. (radish, daikon, turnip). The time frame for the creation of varieties and hybrids of root crops can be significantly reduced by introducing biotechnological methods into the breeding process with the close cooperation of biotechnologists and breeders.

1. Tyukavin G.B. Fundamentals of Carrot Biotechnology : monograph. Under the general editorship and with a preface by V.F. Pivovarov. Moscow: All-Russian Research Institute of Breeding and Seed Production of Vegetable Crops, 2007. 479 p. ISBN 978-5-901695-19-7. (In Russ.)

2. Fedorova M.I., Stepanov, V.A. Root vegetable crops, breeding directions, results. Vegetable crops of Russia. 2017;0(4):16–22. (In Russ.) doi: 10.18619/2072-9146-2017-4-16-22 https://www.elibrary.ru/zfbfmb

3. Domblides A.S. Development of a laboratory technology for producing gynogenic carrot plants in vitro. Moscow, 2001. 140 p. (In Russ.) https://www.elibrary.ru/qdkcsp

4. Masuda K., Kikuta Y., Okazawa Y. A revision of the medium for somatic embryogenesis in carrot suspension culture. Journal of the Faculty of Agriculture. 1981. https://www.semanticscholar.org/paper/A-REVISION-OF-THE-MEDIUM-FOR-SOMATIC-EMBRYOGENESIS-Masuda-Kikuta/940c4aca79be26d0f0011e82d1c5b132da818119

5. Zayachkovskaya T., Alyokhina K., Mineykina A., Romanova O., Vjurtts T., Tukuser Y., et al. Optimizing Different Medium Component Concentration and Temperature Stress Pretreatment for Gynogenesis Induction in Unpollinated Ovule Culture of Sugar Beet (Beta vulgaris L.). Horticulturae. 2023;9(8):900. doi: 10.3390/horticulturae9080900

6. Murashige T, Skoog F. A Revised Medium for Rapid Growth and Bio Assays with Tobacco Tissue Cultures. Physiologia Plantarum. 1962;15(3):473–497. doi: 10.1111/j.1399-3054.1962.tb08052.x

7. Lichter R. Induction of Haploid Plants From Isolated Pollen of Brassica napus. Zeitschrift für Pflanzenphysiologie. 1982;105(5):427–434. doi: 10.1016/S0044-328X(82)80040-8.

8. Kozar E., Domblides E. Protocol of European Radish (Raphanus sativus L.) Microspore Culture for Doubled Haploid Plant Production. In: Segui-Simarro JM (ed.) Doubled Haploid Technology: Volume 2: Hot Topics, Apiaceae, Brassicaceae, Solanaceae. New York, NY: Springer US; 2021. p. 217–232. doi: 10.1007/978-1-0716-1335-1_13

9. Shumilina D., Kozar E., Chichvarina O., Korottseva K., Domblides E. Brassica rapa L. ssp. chinensis Isolated Microspore Culture Protocol. In: Segui-Simarro JM (ed.) Doubled Haploid Technology: Volume 2: Hot Topics, Apiaceae, Brassicaceae, Solanaceae. New York, NY: Springer US; 2021. p. 145–162. doi: 10.1007/978-1-0716-1335-1_9

10. Shumilina D., Kornyukhin D., Domblides E., Soldatenko A., Artemyeva A. Effects of Genotype and Culture Conditions on Microspore Embryogenesis and Plant Regeneration in Brassica Rapa ssp. Rapa L. Plants. 2020;9(2):278. doi: 10.3390/plants9020278

11. Kozar E.V., Kozar E.G., Domblides E.A. Effect of the Method of Microspore Isolation on the Efficiency of Isolated Microspore Culture In Vitro for Brassicaceae Family. Horticulturae. 2022;8(10):864. doi: 10.3390/horticulturae8100864

12. Domblides A.S. Anther and ovule in vitro culture in carrot (Daucus carota L.). Acta Horticulturae. 2017;(1153):55–60. doi: 10.17660/ActaHortic.2017.1153.9

13. Matsubara S., Dohya N., Murakami K. Callus formation and regeneration of adventitious embryous from carrot, fennel and mitsuba microspores by anther and isolated microspore cultures. Acta Horticulturae. 1995;(392):129–138. doi: 10.17660/ActaHortic.1995.392.15

14. Górecka K., Kowalska U., Krzyżanowska D., Kiszczak W. Obtaining carrot (Daucus carota L.) plants in isolated microspore cultures. Journal of Applied Genetics. 2010;51(2):141–147. doi: 10.1007/BF03195722

15. Li J.R., Zhuang F.Y., Ou C.G., Hu H., Zhao Z.W., Mao J.H. Microspore embryogenesis and production of haploid and doubled haploid plants in carrot (Daucus carota L.). Plant Cell, Tissue and Organ Culture (PCTOC). 2013;112(3):275–287. doi: 10.1007/s11240-012-0235-5

16. Vjurtts, T.S., Domblides, E.A., Shmykova, N.A., Fedorova, M.I., Kan, L.Yu., Domblides, A.S. Production of DH-plants in isolated microspore culture of carrot. Vegetable crops of Russia. 2017;(5):25–30. (In Russ.) doi: 10.18619/2072-9146-2017-5-25-30 https://www.elibrary.ru/orxrrl

17. Shmykova N., Domblides E., Vjurtts T., Domblides A. Haploid Embryogenesis in Isolated Microspore Culture of Carrots (Daucus carota L.). Life. 2021;11(1):20. doi: 10.3390/life11010020

18. Romanova O., Vjurtts T., Mineykina A., Tukuser Y, Kulakov Y., Akhramenko V., et al. Embryogenesis induction of carrot (Daucus carota L.) in isolated microspore culture. Foods and Raw Materials. 2023;11(1):25–34. doi: 10.21603/2308-4057-2023-1-548

19. Fomicheva M., Kozar E., Domblides E. Carrot (Daucus carota L.) Haploid Embryo Genome Doubling with Colchicine and Trifluralin. Horticulturae. 2025;11(5):505. doi: 10.3390/horticulturae11050505

20. Fujimura T., Komamine A. Synchronization of Somatic Embryogenesis in a Carrot Cell Suspension Culture. Plant Physiology. 1979;64(1):162–164. doi: 10.1104/pp.64.1.162.

21. Zayachkovskaya T., Domblides E., Zayachkovsky V., Kan L., Domblides A., Soldatenko A. Production of Gynogenic Plants of Red Beet (Beta vulgaris L.) in Unpollinated Ovule Culture In Vitro. Plants. 2021;10(12):2703. doi: 10.3390/plants10122703

22. Zayachkovskaya T.V. Evaluation of the initial material of the species Raphanus sativus L. using methods of reproductive biology for heterosis breeding. Moscow, 2005. 26 p. (In Russ.) https://www.elibrary.ru/nibstz

23. Kozar E.V., Kozar E.G., Soldatenko A.V., Domblides E.A. Rooting technique of double haploids obtained in culture of microspore in vitro for European radish. Vegetable crops of Russia. 2020;(5):3–15. https://doi.org/10.18619/2072-9146-2020-5-3-15 https://elibrary.ru/dsqqpo