Оптимизация этапов технологии получения DH-растений капусты белокочанной

Одна из экономически важных сельскохозяйственных культур среди представителей рода Brassica L. – капуста 6елокочанная. Для создания высокопродуктивных F₁ ги6ридов с улучшенными показателями нео6ходима генетически разноо6разная 6аза выровненного материала, получение которого занимает продолжительную часть селекционного процесса. Ускорить этот этап селекции можно за счет получения удвоенных гаплоидов (DH-растений). Отсутствие стандартизированных, эффективных и воспроизводимых протоколов для культивирования in vitro разных видов растений, охватывающих несколько факторов и их взаимодействие, часто препятствует практической реализации метода. Растительный материал, условия культивирования и состав питательных сред являются определяющими факторами эффективности эм6риогенеза. В результате проведенного исследования оптимизирован протокол получения удвоенных гаплоидов в культуре изолированных микроспор in vitro для капусты 6елокочанной позднеспелого срока созревания. Оптимальный размер 6утонов позднеспелой капусты 6елокочанной для введения в культуру in vitro варьировал от 3.5 до 5.0 мм. Для изученных генотипов капусты 6елокочанной совместное влияние высокотемпературного стресса при 32 °С в течение 48 ч и рН питательной среды 5.8 спосо6ствовало наи6ольшему выходу эм6риоидов. 3а счет использования платформы-шейкера при режиме 40 о6./мин достигнуто ускорение развития эм6риоидов до семядольной стадии на 7–10 суток. Благодаря подо6ранному комплексу условий для успешного эм6риогенеза из микроспор капусты 6елокочанной достигнут выход эм6риоидов до 273.6 ± 32.2 шт./чашку Петри. Применение проточной цитометрии позволило отделить удвоенные гаплоиды (69.8 %) от гаплоидов (8.4 %), триплоидов (1.5 %) и тетраплоидов (20.3 %) на ранней стадии развития. Молекулярно-генетический анализ с использованием микросателлитных маркеров (SSR-анализ) подтвердил гаплоидное происхождение диплоидных растений-регенерантов.

1. Adamus A., Szklarczyk M., Kiełkowska A. Haploid and doubled haploid plant production in Brassica rapa L. subsp. pekinensis via microspore culture. Methods Mol Biol. 2021;2288:181-199. doi 10.1007/978-1-0716-1335-1_11

2. Alexander M.P. Differential staining of aborted and non-aborted pollen. Stain Technol. 1969;44(3):117-122. doi 10.3109/10520296909063335

3. Ali M.M., Khaleque M.A., Custers J.B.M., Khurram M.M.H. Microspore culture and the performance of microspore derived doubled haploid in Brassica juncea (L.). Bangladesh J Agric. Res. 2008; 33(4):571-578. doi 10.3329/bjar.v33i4.2290

4. Barinova I., Clément C., Martiny L., Baillieul F., Soukupova H., Heberle-Bors E., Touraev A. Regulation of developmental pathways in cultured microspores of tobacco and snapdragon by medium pH. Planta. 2004;219(1):141-146. doi 10.1007/s00425-003-1202-5

5. Barro F., Fernández-Escobar J., De La Vega M., Martín A. Modification of glucosinolate and erucic acid contents in doubled haploid lines of Brassica carinata by UV treatment of isolated microspores. Euphytica. 2003;129(1):1-6. doi 10.1023/A:1021578318098

6. Bhatia R., Dey S.S., Parkash C., Sharma K., Sood S., Kumar R. Modification of important factors for efficient microspore embryogenesis and doubled haploid production in field grown white cabbage (Brassica oleracea var. capitata L.) genotypes in India. Sci Hortic. 2018;233:178-187. doi 10.1016/j.scienta.2018.01.017

7. Cao M.Q., Charlot F., Dore C. Embryogenesis and plant regeneration in sauerkraut cabbage (Brassica oleracea L. subsp. capitata) by in vitro culture of isolated microspores. Comptes Rendus de l’Academie des Sciences Serie III. 1990;310:203-209. doi 10.1007/BF00231964

8. Cordewener J.H.G., Hause G., Görgen E., Busink R., Hause B., Dons H.J.M., Van Lammeren A.A.M., Van Lookeren Campagne M.M., Pechan P. Changes in synthesis and localization of members of the 70-kDa class of heat-shock proteins accompany the induction of embryogenesis in Brassica napus L. microspores. Planta. 1995;196:747-755. doi 10.1007/BF01106770

9. Cousin A., Nelson M.N. Twinned microspore-derived embryos of canola (Brassica napus L.) are genetically identical. Plant Cell Rep. 2009;28(5):831-835. doi 10.1007/s00299-009-0677-3

10. Cristea T.O., Iosob G.A., Brezeanu C., Brezeanu P.M. Effect of chemical composition of nutritive medium and explant size over androgenetic response in microspore culture of Brassica oleracea L. Rev Chim. 2020;71(10):131-136. doi 10.37358/RC.20.10.8357

11. Custers J.B.M., Cordewener J.H.G., Nöllen Y., Dons H.J.M., Van Locke ren Campagne M.M. Temperature controls both gametophytic and sporophytic development in microspore cultures of Brassica napus. Plant Cell Rep. 1994;13(5):267-271. doi 10.1007/BF00233317

12. da Silva Dias J.C. Effect of activated charcoal on Brassica oleracea microspore culture embryogenesis. Euphytica. 1999;108:65-69. doi 10.1023/A:1003634030835

13. da Silva Dias J.C. Protocol for broccoli microspore culture. In: Doubled Haploid Production in Crop Plants. Springer Netherlands, 2003; 195-204. doi 10.1007/978-94-017-1293-4_30

14. Diao W.P., Jia Y.Y., Song H., Zhang X.Q., Lou Q.F., Chen J.F. Efficient embryo induction in cucumber ovary culture and homozygous identification of the regenetants using SSR markers. Sci Hortic. 2009; 119(3):246-251. doi 10.1016/j.scienta.2008.08.016

15. Domblides A.S., Bondareva L.L., Pivovarov V.F. Assessment of genetic diversity among headed cabbage (Brassica oleracea L.) accessions by using SSR markers. Sel’skokhozyajstvennaya Biologiya = Agricultural Biology. 2020;55(5):890-900. doi 10.15389/agrobiology.2020.5.890rus (in Russian)

16. Duijs J.G., Voorrips R.E., Visser D.L., Custers J.B.M. Microspore culture is successful in most crop types of Brassica oleracea L. Euphytica. 1992;60:45-55. doi 10.1007/BF00022257

17. Fan Z., Armstrong K.C., Keller W.A. Development of microspores in vivo and in vitro in Brassica napus L. Protoplasma. 1988;147: 191-199. doi 10.1007/BF01403347

18. Ferrie A.M.R., Caswell K.L. Isolated microspore culture techniques and recent progress for haploid and doubled haploid plant production. Plant Cell Tissue Organ Cult. 2011;104:301-309. doi 10.1007/s11240-010-9800-y

19. Ferrie A.M.R., Epp D.J., Keller W.A. Evaluation of Brassica rapa L. genotypes for microspore culture response and identification of a highly embryogenic line. Plant Cell Rep. 1995;14(9):580-584. doi 10.1007/BF00231942

20. Gu H.H., Zhou W.J., Hagberg P. High frequency spontaneous production of doubled haploid plants in microspore cultures of Brassica rapa ssp. chinensis. Euphytica. 2003;134:239-245. doi 10.1023/B:EUPH.0000004945.01455.6d

21. Gu H., Zhao Z., Sheng X., Yu H., Wang J. Efficient doubled haploid production in microspore culture of loose-curd cauliflower (Brassica oleracea var. botrytis). Euphytica. 2014;195:467-475. doi 10.1007/s10681-013-1008-x

22. Gyulai G., Gemesne J.A., Sagi Zs., Venczel G., Pinter P., Kristof Z., Torjek O., Heszky L., Bottka S., Kiss J., Zatyko L. Doubled haploid development and PCR-analysis of F1 hybrid derived DH-R2 paprika (Capsicum annuum L.) lines. J Plant Physiol. 2000;156(2):168-174. doi 10.1016/S0176-1617(00)80302-8

23. Hoseini M., Ghadimzadeh M., Ahmadi B., da Silva J.A. Effects of ascorbic acid, alpha-tocopherol, and glutathione on microspore embryogenesis in Brassica napus L. In Vitro Cell Dev Biol Plant. 2014;50:26-35. doi 10.1007/s11627-013-9579-8

24. Huang B., Bird S., Kemble R., Simmonds D., Keller W., Miki B. Effects of culture density, conditioned medium and feeder cultures on microspore embryogenesis in Brassica napus L. cv. Topas. Plant Cell Rep. 1990;8(10):594-597. doi 10.1007/BF00270061

25. Ji J., Su H., Cao W., Zhang X., Li H., Fang Z., Yang L., Zhang Ya., Zhuang M., Wang Yo., Taranov V., Lv H. Genetic analysis and mapping of QTLs for isolated microspore embryogenesis in cabbage. Scientia Horticulturae. 2023;313:111897. doi 10.1016/j.scienta.2023.111897

26. Kasha K.J. Chromosome doubling and recovery of doubled haploid plants. In: Don Palmer C., Keller W.A., Kasha K.J. (Eds) Haploids in Crop Improvement II. Biotechnology in Agriculture and Forestry. Vol. 56. Springer, 2005;123-152. doi 10.1007/3-540-26889-8_7

27. Kitashiba H., Taguchi K., Kaneko I., Inaba K., Yokoi S., Takahata Y., Nishio T. Identification of loci associated with embryo yield in microspore culture of Brassica rapa by segregation distortion analysis. Plant Cell Rep. 2016:35(10):2197-2204. doi 10.1007/s00299-016-2029-4. Epub 2016 Jul 20.

28. Kott L.S. Application of doubled haploid technology in breeding of oilseed Brassica napus. AgBiotech News Inf. 1998;10:69N-73N. doi 10.1533/9781908818478.183

29. Kott L.S., Polsoni L., Beversdorf W.D. Cytological aspects of isolated microspore culture of Brassica napus. Can J Bot. 1988;66(8):1658- 1664. doi 10.1139/b88-226

30. 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):2117. doi 10.3390/plants10102117

31. 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

32. Kyo M., Harada H. Control of the developmental pathway of tobacco pollen in vitro. Planta. 1986;168:427-432. doi 10.1007/BF00392260

33. 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

34. Lighter R. Efficient yield of embryoids by culture of isolated microspores of different Brassicaceae species. Plant Breeding. 1989; 103(2):119-123. doi 10.1111/j.1439-0523.1989.tb00359.x

35. Louarn S., Torp A.M., Holme I.B., Andersen S.B., Jensen B.D. Database derived microsatellite markers (SSRs) for cultivar differentiation in Brassica oleracea. Genet Resour Crop Evol. 2007;54(8):1717-1725. doi 10.1007/s10722-006-9181-6

36. Malik A.A., Cui L., Zhang S., Chen J. Efficiency of SSR markers for determining the origin of melon plantlets derived through unfertilized ovary culture. Hort Sci. 2011;38(1):27-34. doi 10.17221/47/2010-HORTSCI

37. Mineykina A., Bondareva L., Soldatenko A., Domblides E. Androgenesis of red cabbage inisolated microspore culture in vitro. Plants. 2021;10(9):1950. doi 10.3390/plants10091950

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

39. Pechan P.M., Keller W.A. Identification of potentially embryogenic microspores in Brassica napus. Physiol Plant. 1988;74(2):377-384. doi 10.1111/j.1399-3054.1988.tb00646.x

40. Pechan P.M., Smykal P. Androgenesis: affecting the fate of the male gametophyte. Physiol Plant. 2001;111(1):1-8. doi 10.1034/j.1399-3054.2001.1110101.x

41. Prem D., Gupta K., Sarkar G., Agnihotri A. Activated charcoal induced high frequency microspore embryogenesis and efficient doubled haploid production in Brassica juncea. Plant Cell Tissue Organ Cult. 2008;93:269-282. doi 10.1007/s11240-008-9373-1

42. Rodriguez-Serrano M., Bárány I., Prem D., Coronado M.-J., Risueño M.C., Testillano P.S. NO, ROS, and cell death associated with caspase-like activity increase in stress-induced microspore embryogenesis of barley. J Exp Bot. 2012;63(5):2007-2024. doi 10.1093/jxb/err400

43. Rudolf K., Bohanec B., Hansen M. Microspore culture of white cabbage, Brassica oleracea var. capitata L.: genetic improvement of non-responsive cultivars and effect of genome doubling agents. Plant Breed. 1999;118(3):237-241. doi 10.1046/j.1439-0523.1999.118003237.x

44. 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

45. Simmonds D.H., Keller W.A. Significance of preprophase bands of microtubules in the induction of microspore embryogenesis of Brassica napus. Planta. 1999;208:383-391. doi 10.1007/s004250050573

46. Su H., Chen G., Yang L., Zhang Y., Wang Y., Fang Z., Lv H. Proteomic variations after short-term heat shock treatment reveal differentially expressed proteins involved in early microspore embryogenesis in cabbage (Brassica oleracea). PeerJ. 2020;8:e8897. doi 10.7717/peerj.8897

47. Takahata Y., Keller W.A. High frequency embryogenesis and plant regeneration in isolated microspore culture of Brassica oleracea L. Plant Sci. 1991;74(2):235-242. doi 10.1016/0168-9452(91)90051-9

48. Tang X., Liu Y., He Y., Ma L., Sun M. Exine dehiscing induces rape microspore polarity, which results in different daughter cell fate and fixes the apical-basal axis of the embryo. J Exp Bot. 2013;64(1): 215-228. doi 10.1093/jxb/ers327

49. Telmer C.A., Simmonds D.H., Newcomb W. Determination of developmental stage to obtain high frequencies of embryogenic microspores in Brassica napus. Physiol Plant. 1992;84(3):417-424. doi 10.1111/j.1399-3054.1992.tb04685.x

50. Tonguç M., Griffiths P.D. Genetic relationships of Brassica vegetables determined using database derived simple sequence repeats. Euphytica. 2004;137(2):193-201. doi 10.1023/B:EUPH.0000041577.84388.43

51. Touraev A., Pfosser M., Vicente O., Heberle-Bors E. Stress as the major signal controlling the developmental fate of tobacco microspores: towards a unified model of induction of microspore/pollen embryogenesis. Planta. 1996;200:144-152. doi 10.1007/BF00196662

52. Tuncer B., Çğ A., Yanmaz R., Yaşar F. Effect of heat shock treatment on microspore embryogenesis in Brassica oleracea species. J Agric Sci (Belihuloya). 2016;22(4):548-554. doi 10.1501/Tarimbil_0000001413

53. Weber M., Davies J.J., Wittig D., Oakeley E.J., Haase M., Lam W.L., Schuebeler D. Chromosome-wide and promoter-specific analyses identify sites of differential DNA methylation in normal and transformed human cells. Nat Genet. 2005;37(8):853-862. doi 10.1038/ng1598

54. Winarto B., da Silva J.A.T. Microspore culture protocol for Indonesian Brassica oleracea. Plant Cell Tissue Organ Cult. 2011;107(2): 305-315. doi 10.1007/S11240-011-9981-Z/TABLES/6

55. Yahyaoui E., Germanà M.A. Microspore embryogenesis in Citrus. In: Segui-Simarro J.M. (Ed.) Doubled Haploid Technology. Methods in Molecular Biology. Vol. 2289. New York, NY: Humana, 2021;149-166. doi 10.1007/978-1-0716-1331-3_10

56. 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

57. Yuan S., Su Y., Liu Y., Fang Z., Yang L., Zhuang M., Zhang Y., Sun P. Effects of pH, MES, arabinogalactan-proteins on microspore cultures in white cabbage. Plant Cell Tissue Organ Culture. 2012;110(1): 69-76. doi 10.1007/s11240-012-0131-z

58. Zeng A., Yan J., Song L., Gao B., Li J. Effects of ascorbic acid and embryogenic microspore selection on embryogenesis in white cabbage (Brassica oleracea L. var. capitata). J Hortic Sci Biotechnol. 2015;90(6):607-612. doi 10.1080/14620316.2015.11668722

59. Zhang F.L., Aoki S., Takahata Y. RAPD markers linked to microspore embryogenic ability in Brassica crops. Euphytica. 2003;131(2): 207-213. doi 10.1023/A:1023955131523

60. Żur I., Dubas E., Golemiec E., Szechyńska-Hebda M., Gołębiowska G., Wędzony M. Stress-related variation in antioxidative enzymes activity and cell metabolism efficiency associated with embryogenesis induction in isolated microspore culture of triticale (x Triticosecale Wittm.). Plant Cell Rep. 2009;28(8):1279-1287. doi 10.1007/s00299-009-0730-2