VNIISSOK, Moscow, Russian Federation
VNIISSOK, Moscow, Russian Federation
VNIISSOK, Moscow, Russian Federation
VNIISSOK, Moscow, Russian Federation
VNIISSOK, Moscow, Russian Federation
VNIISSOK, Moscow, Russian Federation
VNIISSOK, Moscow, Russian Federation
VNIISSOK, Moscow, Russian Federation
Haploid technologies are used to create homozygous lines for accelerated breeding. We aimed to optimize the technology for using the isolated microspore culture in vitro to obtain doubled haploids of the carrot (Daucus carota L.). We studied two carrot varieties with different responsiveness to embryogenesis, Altajskaya lakomka and Breeding line 17. Carrot microspores were isolated from buds and cultivated in liquid nutrient media supplemented with an antibiotic and activated carbon in vitro. They were exposed to different thermal treatments. The experiment showed the benefits of combining cold pre-treatment of buds (5°C for 1 day) with heat shock of isolated microspores in vitro (32°C for 2 days). The induction of embryogenesis on the NLN-13 medium was twice as high as on the MSm-13 medium. The use of 1% activated carbon in 0.5% agarose increased the yield of embryoids by more than 1.5 times. 100 mg/L of ampicillin was found to be the most efficient concentration. After 30 days of cultivation under optimized conditions, the yield was 161.3 and 44.0 embryoids per Petri dish for the cultivar Altajskaya lakomka and Breeding line 17, respectively. The induction of carrot embryogenesis is determined by the type and duration of thermal stress, the composition of the nutrient medium, the use of activated carbon as a sorbent, the addition of β-lactam antibiotics, and the type of explant exposed to thermal treatment. Our technology enabled us to obtain homozygous doubled haploid lines of carrots during a year, and these lines were included in the breeding process to create F1 hybrids.
Daucus carota, culture medium, ampicillin, haploids, table carrot, penicillin, cold and heat stress, cefotaxime, embryogenesis
1. Masuda K, Kikuta Y, Okazava Y. A revision of the medium for somatic embryogenesis in carrot suspension culture. Journal of the Faculty of Agriculture, Hokkaido University. 1981;60(3):183-193.
2. Lichter R. Induction of haploid plants from isolated pollen of Brassica napus. Zeitschrift für Pflanzenphysiologie. 1982;105(5):427-432. https://doi.org/10.1016/S0044-328X(82)80040-8
3. Murashige T, Skoog F. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiologia Plantarum. 1962;15(3):473-497. https://doi.org/10.1111/j.1399-3054.1962.tb08052.x
4. Gamborg OL, Miller RA, Ojima K. Nutrients requirements of suspension cultures of soybean root cells. Experimental Cell Research. 1968;50(1):151-158. https://doi.org/10.1016/0014-4827(68)90403-5
5. Segui-Simarro JM, Moreno JB, Fernández MG, Mir R. Species with haploid or doubled haploid protocols. In: Segui-Simarro JM, editor. Doubled haploid technology: Volume 1: General topics, Alliaceae, cereals. New York: Humana Press; 2021. pp. 41-103. https://doi.org/10.1007/978-1-0716-1315-3_3
6. Smýkalová I, Horáček J. Caraway (Carum carvi L.): Anther culture and production of DH plants caraway. In: Segui-Simarro JM, editor. Doubled haploid technology: Volume 2: Hot topics, Apiaceae, Brassicaceae, Solanaceae. New York: Humana Press; 2021. pp. 91-102. https://doi.org/10.1007/978-1-0716-1335-1_5
7. Kiszczak W, Burian M, Kowalska U, Górecka K. Production of homozygous carrot (Daucus carota L.) plants by anther culture. In: Segui-Simarro JM, editor. Doubled haploid technology: Volume 2: Hot topics, Apiaceae, Brassicaceae, Solanaceae. New York: Humana Press; 2021. pp. 113-126. https://doi.org/10.1007/978-1-0716-1335-1_7
8. Dohya N, Matsubara S, Murakami K. Callus formation and regeneration of adventitious embryos from celery microscopes by anther and isolated microscope cultures. Journal of the Japanese Society for Horticultural Science. 1997;65(4):747-752. https://doi.org/10.2503/jjshs.65.747
9. Ferrie AMR. Doubled haploidy for fennel (Foeniculum vulgare Mill.) and dill (Anethum graveolens L.). In: Segui-Simarro JM, editor. Doubled haploid technology: Volume 2: Hot topics, Apiaceae, Brassicaceae, Solanaceae. New York: Humana Press; 2021. pp. 103-111. https://doi.org/10.1007/978-1-0716-1335-1_6
10. Górecka K, Kowalska U, Krzyzanowska D, Kiszczak W. Obtaining carrot (Daucus carota L.) plants in isolated microspore cultures. Journal of Applied Genetics. 2010;51(2):141-147. https://doi.org/10.1007/BF03195722
11. Zhuang FY, Pei HX, Ou CG, Hu H, Zhao ZW, Li JR. Induction of microspores-derived embryos and calli from anther culture in carrot. Acta Horticulturae Sinica. 2010;37(010):1613-1620.
12. Tyukavin GB, Shmykova NA, Monahova MA. Cytological study of embryogenesis in cultured carrot anthers. Russian Journal of Plant Physiology. 1999;46(6):767-773.
13. Matsubara S, Dohya N, Murakami K. Callus formation and regeneration of adventitious embryos from carrot, fennel and mitsuba microspores by anther and isolated microspore cultures. Acta Horticulturae. 1995;392:129-137. https://doi.org/10.17660/ActaHortic.1995.392.15
14. 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. 2013;112(3):275-287. https://doi.org/10.1007/s11240-012-0235-5
15. Shmykova N, Domblides E, Vjurtts T, Domblides A. Haploid embryogenesis in isolated microspore culture of carrots (Daucus carota L.). Life. 2021;11(1). https://doi.org/10.3390/life11010020
16. Vjurtts TS, Domblides EA, Shmykova NA, Fedorova MI, Kan LYu, Domblides AS. Production of DH-plants in culture of isolated microspore in carrot. Vegetable Crops of Russia. 2017;38(5):25-30. (In Russ.). https://doi.org/10.18619/2072-9146-2017-5-25-30
17. Nitsch C, Norreel B. Factors favoring the formation of androgenetic embryos in anther culture. In: Srb AM, editor. Genes, enzymes, and populations. New York: Springer; 1973. pp. 129-144. https://doi.org/10.1007/978-1-4684-2880-3_10
18. 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). https://doi.org/10.3390/plants9020278
19. Kiszczak W, Kowalska U, Kapuścińska A, Burian M, Górecka K. Comparison of methods for obtaining doubled haploids of carrot. Acta Societatis Botanicorum Poloniae. 2017;86(2). https://doi.org/10.5586/asbp.3547
20. Thomas TD. The role of activated charcoal in plant tissue culture. Biotechnology Advances. 2008;26(6):618-631. https://doi.org/10.1016/j.biotechadv.2008.08.003
21. Grzebelus E, Skop L. Effect of β-lactam antibiotics on plant regeneration in carrot protoplast cultures. In Vitro Cellular and Developmental Biology - Plant. 2014;50(5):568-575. https://doi.org/10.1007/s11627-014-9626-0
22. Meng Q, Liu Z, Zhang Y, Liu C, Ren F, Feng H. Effects of antibiotics on in vitro-cultured cotyledons. In Vitro Cellular and Developmental Biology - Plant. 2014;50(4):436-441. https://doi.org/10.1007/s11627-014-9595-3
23. Mineykina A, Shumilina D, Bondareva L, Soldatenko A, Domblides E. Effect of beta-lactam antibiotics on microspore embryogenesis in Brassica species. Plants. 2020;9(4). https://doi.org/10.3390/plants9040489
24. Domblides EA, Shmykova NA, Shumilina DV, Zayachkovskaya TV, Mineykina AI, Kozarʹ EV, et al. A technology for obtaining doubled haploids in microspore cultures of the Brassicaceae family (guidelines). Moscow: VNIISSOK; 2016. 40 p. (In Russ.).
25. Gland A, Lichter R, Schweiger H-G. Genetic and exogenous factors affecting embryogenesis in isolated microspore cultures of Brassica napus L. Journal of Plant Physiology. 1988;132(5):613-617. https://doi.org/10.1016/S0176-1617(88)80264-5
26. Abdollah HA, Said AGE, Khalafalla MM. Embryogenesis and plantlet regeneration optimization of wheat (Triticum aestivum L.). International Journal of Agricultural Technology. 2014;10(3):679-693.
27. Guo Y-D, Pulli S. High-frequency embryogenesis in Brassica campestris microspore culture. Plant Cell, Tissue and Organ Culture. 1996;46(3):219-225. https://doi.org/10.1007/BF02307098
28. Prem D, Gupta K, Agnihotri A. Effect of various exogenous and endogenous factors on microspore embryogenesis in Indian mustard (Brassica juncea (L.) Czern and Coss). In Vitro Cellular and Developmental Biology - Plant. 2005;41(3):266-273. https://doi.org/10.1079/IVP2005636
29. Gerszberg A, Grzegorczyk-Karolak I. Influence of selected antibiotics on the tomato regeneration in in vitro cultures. Notulae Botanicae Horti Agrobotanici Cluj-Napoca 2019;47:558-564. https://doi.org/10.15835/nbha47311401
30. Asif M, Eudes F, Randhawa H, Amundsen E, Yanke J, Spaner D. Cefotaxime prevents microbial contamination and improves microspore embryogenesis in wheat and triticale. Plant Cell Reports. 2013;32(10):1637-1646. https://doi.org/10.1007/s00299-013-1476-4
31. Ahmadi B, Shariatpanahi ME, Ojaghkandi MA, Heydari AA. Improved microspore embryogenesis induction and plantlet regeneration using putrescine, cefotaxime and vancomycin in Brassica napus L. Plant Cell, Tissue and Organ Culture. 2014;118(3):497-505. https://doi.org/10.1007/s11240-014-0501-9
