Shaker-based microspore culture improves embryoid quality and regeneration efficiency in the production of doubled haploids from microspores of white cabbage (Brassica oleracea var. capitata)

   Relevance. Doubled haploid (DH) technology enables accelerated breeding of homozygous lines in white cabbage; however, its efficiency is limited by genotype dependency. Optimizing culture conditions, including the use of a shaker platform, may enhance embryoid yield and regeneration potential.

   Methodology. We studied three white cabbage genotypes (No. 2502, 2503, and 2504). Microspores were cultured under both static and shaking conditions (40-50 rpm) for 30 days. On day 30, we assessed the developmental stages of the embryoids. After that, the embryoids were transferred to a regeneration medium and, after three months, we recorded successful regeneration into fully developed plants.

   Results. Shaker culture significantly increased the proportion of cotyledonary-stage embryoids (up to 81.7 % in genotype 2502) and reduced the frequency of abnormalities (down to 0 % in genotype 2503). Overall embryoid regeneration capacity under shaker conditions was 30.5 ± 5.4 %, compared to 19.2 ± 2.8 % under static conditions. Cotyledonary embryoids produced on the shaker showed the highest regeneration efficiency (36.5 %). These findings support the implementation of shaker-based culture in DH protocols for white cabbage to improve overall efficiency.

1. Mineykina A., Bondareva L., Soldatenko A., Domblides E. Androgenesis of red cabbage in isolated microspore culture in vitro. Plants. 2021;10(9). doi: 10.3390/plants10091950

2. Kozar E., Domblides E. Protocol of European radish (Raphanus sativus L.) microspore culture for doubled haploid plant production. In: Methods Mol Biol. 2021;2288:217-232. doi: 10.1007/978-1-0716-1335-1_13

3. Kozar E.V., Domblides E.A., Soldatenko A.V. Embryogenesis of european radish (Raphanus sativus L. subsp. sativus convar. radicula) in culture of isolated microspores in vitro. Plants. 2021;10(10). doi: 10.3390/plants10102117

4. Kozar E.V., Domblides E.A., Soldatenko A.V. Factors affecting DH plants in vitro production from microspores of European radish. Vavilov J Genet Breed. 2020;24(1):31-39. doi: 10.18699/VJ20.592 https://www.elibrary.ru/srrwyq

5. Kozar E.V., Korottseva K.S., Romanova O.V., Chichvarina O.A., Kan L.Yu., Ahramenko V.A., Domblides E.A. Production of doubled haplois in Brassica purpuraria. Vegetable crops of Russia. 2019;(6):10-18. (In Russ.) doi: 10.18619/2072-9146-2019-6-10-18 https://www.elibrary.ru/udnnot

6. 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

7. 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. doi: 10.18619/2072-9146-2020-5-3-15 https://www.elibrary.ru/dsqqpo

8. Custers J.B.M. Microspore culture in rapeseed (Brassica napus L.). In: Maluszynski M., Kasha K.J., Forster B.P., Szarejko I., editors. Doubled Haploid Production in Crop Plants. 2003. P. 185-186. doi: 10.1007/978-94-017-1293-4_29

9. da Silva Dias J.C. Protocol for broccoli microspore culture. In: Maluszynski M, Kasha KJ, Forster B.P., Szarejko I., editors. Doubled Haploid Production in Crop Plants. 2003. P. 195-204. doi: 10.1007/978-94-017-1293-4_30

10. Ahmadi B., Ghadimzadeh M., Moghaddam A.F., Alizadeh K., Teixeira da Silva J.A. Bud length, plating density, and incubation time on microspore embryogenesis in Brassica napus. Int J Veg Sci. 2012;18(4):346-357. doi: 10.1080/19315260.2011.647265

11. Domblides E.A., Kozar E.V., Shumilina D.V., Zayachkovskaya T.V., Akhramenko V.A., Soldatenko A.V. Embryogenesis in culture of isolated microspore of broccoli. Vegetable crops of Russia. 2018;(1):3-7. (In Russ.) doi: 10.18619/2072-9146-2018-1-3-7 https://www.elibrary.ru/xodpbb

12. Kozar E.V., Domblides E.A. Protocol for obtaining doubled haploids in isolated microspore culture in vitro for poorly responsive genotypes of brassicaceae family. Biol Methods Protoc. 2024;9(1). doi: 10.1093/biomethods/bpae091

13. Shumilina D., Kozar E., Chichvarina O., et al. Brassica rapa L. ssp. chinensis isolated microspore culture protocol. In: Methods Mol Biol. 2021;2288:145-162. doi: 10.1007/978-1-0716-1335-1_9

14. Starosta E., Szwarc J., Niemann J., Szewczyk K., Weigt D. Brassica napus haploid and double haploid production and its latest applications. Curr Issues Mol Biol. 2023;45(5):4431-4450. doi: 10.3390/cimb45050282

15. Yang S., Liu X., Fu Y., Zhang X., Li Y., Liu Z., Feng H. The effect of culture shaking on microspore embryogenesis and embryonic development in pakchoi (Brassica rapa L. ssp. chinensis). Sci Hortic. 2013;152:70-73. doi: 10.1016/j.scienta.2013.01.019

16. He H.J., Wang X.W., Wang B.L. Embryogenesis and plant regeneration of Chinese kale via isolated microspore culture. Acta Hortic Sin. 2004;31(2):239-240.

17. Gland A., Lichter R., Schweiger H.G. Genetic and exogenous factors affecting embryogenesis in isolated microspore cultures of Brassica napus L. J Plant Physiol. 1988;132:613-617.

18. Kott L.S., Polsoni L. Autotoxicity in isolated microspore culture of Brassica napus. Can J Bot. 1988;66:1665-1670.

19. Hansen M, Svinnset K. Micropore culture of Swede (Brassica napus ssp. rapifera) and the effects of fresh and conditioned media. Plant Cell Rep. 1993;12:496-500.

20. Lichter R. Efficient yield of embryoids by culture of isolated microspores of different Brassicaceae species. Plant Breed. 1989;103:119-123.

21. Pérez-Pérez M.E., Lemaire S.D., Crespo J.L. The ATG4 protease integrates redox and stress signals to regulate autophagy. J Exp Bot. 2021;72:3340-3351. doi: 10.1093/jxb/erab063

22. Rueda-Varela C., Carneros E., et al. Enhancing microspore embryogenesis initiation by reducing ROS, autophagy, and cell death with novel small molecules in rapeseed and barley. J Plant Physiol. 2025;311:8. doi: 10.1016/j.jplph.2025.154546

23. Kott L.S., Beversdorf W.D. Enhanced plant regeneration from microspore-derived embryos of Brassica napus by chilling, partial desiccation and age selection. Plant Cell Tiss Organ Cult. 1990;23:187-192. doi: 10.1007/BF00034430

24. Gu H., Sheng X., Zhao Z., et al. Initiation and development of microspore embryogenesis and plant regeneration of Brassica nigra. In Vitro Cell Dev Biol Plant. 2014;50:534-540. doi: 10.1007/s11627-014-9612-6

25. Kunz C., Islam S.M.S., Berberat J., Peter S.O., Büter B., Stamp P., Schmid J.E. Assessment and improvement of wheat microspore derived embryo induction and regeneration. J Plant Physiol. 2000;156(2):190-196. doi: 10.1016/S0176-1617(00)80305-3

26. Islam S.M. Effect of embryoids age, size and shape for improvement of regeneration efficiency from microspore-derived embryos in wheat (Triticum aestivum L.). Plant OMICS. 2010;3(5).

27. Domblides E.A., Shmykova N.A., Shumilina D.V., et al. Technology for obtaining doubled haploids in microspore culture of Brassicaceae family: guidelines. M., 2016. 40 p. ISBN 978-5-901695-71-5.

28. Murashige T., Skoog F. A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiologia Plant. 1962;15(3):473-497. doi: 10.1111/j.1399-3054.1962.tb08052.x