Rooting technique of double haploids obtained in culture of microspore in vitro for European radish

Relevance. Doubled haploids (DH-plants) are excellent material for genetic research and breeding due to their complete homozygosity. The genus Raphanus from the Brassicaceae family is the toughest to produce doubled haploid plants through isolated microspore culture in vitro (IMC). The study of the causes of disturbed root formation and the development of elements of this stage of technology will significantly increase the effectiveness of the IMC technology for European radish.

Methods. The study included three varieties from the collection of the Federal State Budgetary Scientific Institution Federal Scientific Vegetable Center (FSBSI FSVC): Teplichny Gribovsky, Rozovo-krasniy s belim konchikom and Rhodes. The experiments used a standard protocol for obtaining DH plants using IMC technology in a standard form and with a modification of the rooting stage. The solid MS medium (with agar 7g/L): MS without hormones, MS medium supplemented with IAA at concentrations of 0.5; 1 and 2 mg / L and liquid MSm medium supplemented with 0.1 mg / L kinetin were used for rooting of regenerated plants. All media were supplemented with 20 g/L sucrose. We used three types of techniques for transplanting plant explants onto a solid hormonefree MS medium: planting micro-shoots with their basal part immersed by 2-3 mm into the medium; planting in a well made in a nutrient medium using tweezers under sterile conditions; and landing on the surface of the medium without embedment.

Results. In this work, we studied the features of the stage of rooting of regenerated European radish plants in vitro conditions. The transplant technique has been proven to be important for the successful establishment of radish micro-shoots. Plant explants must be planted strictly on the surface of a solid hormone-free nutrient medium MS, without embedment. The use of tubes with bridges made of filter paper and MSm liquid medium with the addition of 0.1 mg/L kinetin for the induction of root formation also showed high efficiency. For plants prone to the formation of root-like structures (RLS) with secondary tumors (ST), multiple dissection of abnormal formations with successive transplants is necessary. Modification at the rooting stage of micro-shoots growing has increased the percentage of successfully adapted DH plants in vivo conditions from 0-14% to 95-98%.

1. Asif M. Progress and Opportunities of Doubled Haploid Production. Springer. 2013. DOI 10.1007/978-3-319-00732-8_1.

2. Buzovkina I.S., Lutova L.A. Genetic collection of inbred lines of radish: history and prospects. Russian Journal of Genetics. 2007;(4):1411–1423.

3. Buzovkina, I. S., Kneshke, I., and Lutova, L.A. In vitro modeling of tumor formation in radish lines and hybrids. Genetika. 1993;29(6):1002–1008.

4. Chun C., Park H., Na H. Microspore-derived embryo formation in radish (Raphanus sativus L.) according to nutritional and environmental conditions. Hort. Environ. Biotechnol. 2011;52(5):530-535. DOI 10.1007/s13580-011-0080-1.

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

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

7. Dodueva, I.E., Lebedeva, M.A., Kuznetsova, K.A., Gancheva, M.S., Paponova, S.S., Lutova, L.L. Plant tumors: a hundred years of study. Planta. 2020;251(4):82. doi:10.1007/s00425-020-03375-5

8. Domblides E.A., Shmykova N.A., Shumilina N.A., Zayachkovskaya T.V., Mineykina A.I., Kozar E.V., Ahramenko V.A., Shevchenko L.L., Kan L.Ju., Bondareva L.L., Domblides A.S. A technology for obtaining doubled haploids in microspore cultures of the Brassicaceae family (guidelines). Moscow: VNIISSOK Publ., 2016. (In Russ)

9. Dunwell J.M. Haploids in flowering plants: origins and exploitation. Plant Biotechnol. J. 2010;(8):377-424. DOI 10.1111/j.1467-7652.2009.00498.x.

10. Efroni I., Mello A., Nawy T., Ip P.L., Rahni R., Delrose N., Powers A., Satija R., Birnbaum K.D. Root regeneration triggers an embryo-like sequence guided by hormonal interactions. Cell. 2016;(165):1721–1733. [CrossRef] DOI: 10.1016/j.cell.2016.04.046

11. Ferrie A. Microspore culture of Brassica species. In: Maluszynski M., Kasha K.J., Forster B.P., Szarejko I. (eds). Doubled Haploid Production in Crop Plants. 2003:205-215. https://doi.org/10.1007/978-94-017-1293-4_31

12. Forster B.P., Thomas W.T.B. Doubled haploids in genetics and plant breeding. In: Janick J. (Ed.). Plant Breeding Reviews. 2005;(25):57-88. https://doi.org/10.1002/9780470650301.

13. Gibbs D.J., Conde J.V., Berckhan S., Prasad G., Mendiondo G.M., Holdsworth M.J. Group VII ethylene response factors coordinate oxygen and nitric oxide signal transduction and stress responses in plants. Plant Physiology. 2015;(169):23–31. DOI: https://doi.org/10.1104/pp.15.00338

14. Han N., Kim S.U., Park H.Y., Na H. Microspore-derived embryo formation and morphological changes during the isolated microspore culture of radish (Raphanus sativus L.). Kor. J. Hort. Sci. Technol. 2014;32(3):382389. DOI 10.7235/hort.2014.13170.

15. Han N., Na H., Kim J. Identification and variation of major aliphatic glucosinolates in doubled haploid lines of radish (Raphanus sativus L.). Kor. J. Hort. Sci. Technol. 2018;36(2):302-311. DOI 10.12972/kjhst.20180030.

16. Il’ina, E.L., Dodueva, I.E., Ivanova, N.M. et al. The effect of cytokinins on in vitro cultured inbred lines of Raphanus sativus var. radicula Pers. with genetically determined tumorigenesis. Russian Journal of Plant Physiology. 2006;(53):514–522. https://doi.org/10.1134/S1021443706040133

17. Kozar E.V., Domblides E.A., Soldatenko A.V. Factors affecting DH plants in vitro production from microspores of European radish. Vavilov Journal of Genetics and Breeding. 2020;24(1):31-39. DOI 10.18699/VJ20.592

18. Lebedeva M.A, Tvorogova V.E., Vinogradova A.P., Gancheva M.A., Azarakhsh M., Ilina E.L., Demchenko K.N., Dodueva I.E., Lutova L.A. Initiation of spontaneous tumors in radish (Raphanus sativus): cellular, molecular and physiological events. Journal of Plant Physiology. 2015;(173):97-104. https://doi.org/10.1016/j.jplph.2014.07.030

19. Licausi F., Kosmacz M., Weits D.A., Giuntoli B., Giorgi F.M., Voesenek L. ACJ, Perata P., van Dongen J.T. Oxygen sensing in plants is mediated by an N-end rule pathway for protein destabilization. Nature. 2011;479(7373):419–422. DOI: 10.1038/nature10536

20. Liu J., Sheng L., Xu Y., Li J., Yang Z., Huang H., Xu L. WOX11 and 12 are involved in the first-step cell fate transition during de novo root organogenesis in Arabidopsis. Plant Cell. 2014;(26):1081–1093. [CrossRef] [PubMed] https://doi.org/10.1105/tpc.114.122887

21. Lutova L., Dodueva I. Genetic control of regeneration processes of radish plants in vitro: from phenotype to genotype. Bio. Comm. 2019;64(2):124–132. https://doi.org/10.21638/spbu03.2019.204

22. Maluszynski M., Kasha K.J., Forster B.P., Szarejko I. Doubled Haploid Production in Crop Plants: A Manual. Springer Science Business Media. 2003:141-150. DOI 10.1007/978-94-017-1293-4.

23. Masuda K., Kikuta Y., Okazava Y. A Revision of the Medium for Somatic Embryogenesis in Carrot Suspension Culture. J. Fac. Agr. Hokkaido Univ. 1981;(60):183-193.

24. Matveeva T.V., Frolova N.V., Smets R., Dodueva I.E., Buzovkina I.S., Van Onckelen H., Lutova L.A. Hormonal control of tumor formation in radish. Journal of Plant Growth Regulation. 2004;(23):37–43. https://doi.org/10.1007/s00344-004-0004-8

25. Murashige T., Skoog F. A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiologia plantarum. 1962;15(3):473-497.

26. Narbut S.I. Genetic tumor generated during inbreeding in radish. Vestn. Leningr. Univ. 1967;(15):144–149. (In Russ)

27. Shukla V., Lombardi L., Iacopino S., Pencik A., Novak O., Perata P., Giuntoli B., Licausi F. Endogenous Hypoxia in Lateral Root Primordia Controls Root Architecture by Antagonizing Auxin Signaling in Arabidopsis. Molecular Plant. 2019;(12):538–551. https://doi.org/10.1016/j.molp.2019.01.007

28. 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

29. Takahata Y., Komatsu H., Kaizuma N. Microspore culture of radish (Raphanus sativus L.): influence of genotype and culture conditions on embryogenesis. Plant Cell Rep. 1996;16(3-4):163-166. DOI 10.1007/BF01890859.

30. Tuncer B. Callus formation from isolated microspore culture in radish (Raphanus sativus L.). J. Anim. Plant Sci. 2017;27(1):277-282.

31. Tyukavin, G.B., Shmykova, N.A., Mankhova, M.A. Cytological study of embryogenesis in cultured carrot anthers. Russian Journal of Plant Physiology. 1999;46(6):876–884. (In Russ)

32. Vjurtts T.S., Domblides E.A., Shmykova N.A., Fedorova M.I., Kan L.Ju., Domblides A.S. Production of DH-plants in culture of isolated microspore in carrot. Vegetable crops of Russia. 2017;(5):25-30. (In Russ.) DOI:10.18619/2072-9146-2017-5-25-30

33. Zhang G., Zhao F., Chen L., Pan Y., Sun L., Bao N., Zhang T., Cui C.X., Qiu Z., Zhang Y. Jasmonate-mediated wound signalling promotes plant regeneration. Nat. Plants. 2019;(5):491–497. [CrossRef] [PubMed] DOI: 10.1038/s41477-019-0408-x

34. Zhou W., Lozano-Torres J.L., Blilou I., Zhang X., Zhai Q., Smant G., Li C., Scheres B. A jasmonate signaling network activates root stem cells and promotes regeneration. Cell. 2019;(177):942–956.e14. [CrossRef] [PubMed] https://doi.org/10.1016/j.cell.2019.03.006