Springer Nature is making SARS-CoV-2 and COVID-19 research free. View research | View latest news | Sign up for updates

In vitro androgenesis: spontaneous vs. artificial genome doubling and characterization of regenerants


Androgenesis has become the most frequently chosen method of doubled haploid (DH) production in major crops. Theoretically, plantlets derived from in vitro cultured microspore encompass half of the normal chromosome number of donor plants and thus, considered to be haploid. However, depending on species/genotype and the method of haploid production, either via anther or isolated microspore culture, different ratios of spontaneous DHs and diploid (2n) or even polyploid plants originating from somatic tissues or unreduced gametes may also arise in the cultures. Adopting the method of haploid identification, anti-microtubular agent for restoring fertility, and discriminating spontaneous DHs from undesired heterozygote plants will substantially affect the success of androgenesis in breeding programs. The recent advances in the last 2 decades have made it possible to characterize the in vitro regenerants efficiently either prior to genome duplication or using in breeding programs. The herein described approaches and antimicotubular agents are, therefore, expected to improve the efficiency of DH-based breeding pipeline through the in vitro androgenesis.

This is a preview of subscription content, log in to check access.

Fig. 1
Fig. 2


  1. Abdollahi MR, Corral-Martínez P, Mousavi A, Salmanian H, Moini A, Seguí-Simarro JM (2009) An efficient method for transformation of pre-androgenic, isolated Brassica napus microspores involving microprojectile bombardment and Agrobacterium-mediated transformation. Acta Physiol Plant 31(6):1313–1317

  2. Abraha E, Klima M, Vyvadilova M, Bechyne M (2008) Effectiveness of selected antimitotic agents in microspore culture for Brassica carinata doubled haploid production. Agric Trop Subtrop 41(4):137–149

  3. Agarwal PK, Agarwal P, Custers JMB, Liu C, Bhojwani SS (2006) PCIB an antiauxin enhances microspore embryogenesis in microspore culture of Brassica juncea. Plant Cell Tiss Org Cult 86(2):201–210

  4. Ahmadi B, Aladizgeh FM, Shariatpanahi ME, Azadi P, Alizadeh MK (2016) Molecular characterization and expression analysis of SERK1 and SERK2 in Brassica napus L.: implication for microspore embryogenesis and plant regeneration. Plant Cell Rep 35(1):185–193

  5. Ahmadi B, Ahmadi M, Teixiera da Silva JA (2018) Microspore embryogenesis in Brassica: calcium signaling, epigenetic modification, and programmed cell death. Planta 248(6):1339–1350

  6. Anthony RG, Hussey PJ (1999) Dinitroaniline herbicide resistance and the microtubule cytoskeleton. Trends Plant Sci 4(3):112–116

  7. Arzani A, Darvey N (2001) The effect of colchicine on triticale anther-derived plants: microspore pre-treatment and haploid-plant treatment using a hydroponic recovery system. Euphytica 122(2):235–241

  8. Barkley N, Wang M (2008) Application of TILLING and EcoTILLING as reverse genetic approaches to elucidate the function of genes in plants and animals. Curr Genom 9(4):212–226

  9. Barnabás B, Obert B, Kovacs G (1999) Colchicine, an efficient genome-doubling agent for maize (Zea mays L.) microspores cultured in anthero. Plant Cell Rep 18(10):858–862

  10. Barnabás B (2003) Protocol for producing doubled haploid plants from anther culture of wheat (Triticum aestivum L.). In: Malupszynski M, Kasha K, Foster B, Szarejko I (eds) Doubled haploid production in crop plants: a manual. Springer, Dordrecht, pp 65–70

  11. Bartosova Z, Obert B, Takac T, Kormutak A, Pretova A (2005) Using enzyme polymorphism to identify the gametic origin of flax regenerants. Acta Biol Cracov Ser Bot 47(1):173–178

  12. Beaumont VH, Widholm JM (1993) Ploidy variation of pronamide-treated maize calli during long term culture. Plant Cell Rep 12(11):648–651

  13. Begheyn RF, Lübberstedt T, Studer B (2016) Haploid and doubled haploid techniques in perennial ryegrass (Lolium perenne L.) to advance research and breeding. Agronomy 6(60):1–17

  14. Bhatia R, Dey SS, Sood S, Sharma K, Parkash C, Kumar R (2017) Efficient microspore embryogenesis in cauliflower (Brassica oleracea var. botrytis L.) for development of plants with different ploidy level and their use in breeding programme. Sci Hortic 216:83–92

  15. Bhatt J, Kumar S, Patel S, Solanki R (2017) Sequence-related amplified polymorphism (SRAP) markers based genetic diversity analysis of cumin genotypes. Ann Agrar Sci.

  16. Bhowmik P, Ellison E, Polley B, Bollina V, Kulkarni M, Ghanbarnia K, Song H, Gao C, Voytas DF, Kagale S (2018) Targeted mutagenesis in wheat microspores using CRISPR/Cas9. Sci Rep 8:6502.

  17. Bohanec B (2003) Ploidy determination using flow cytometry. In: Maluszynski M, Kasha KJ, Forster BP, Szarejko I (eds) Production of doubled haploids in crop plants. Springer, New York, pp 397–403

  18. Braasch K (2008) Herbicides as chromosome doubling agents in microspore cultures of canola (Brassica napus). Dissertation, University of Winnipeg

  19. Brew-Appiah RAT, Ankrah N, Liu W, Konzak CF, Wettstein DV, Rustgi S (2013) Generation of doubled haploid transgenic wheat lines by microspore transformation. PLoS ONE 8(11):e80155.

  20. Broughton S (2011) The application of n-butanol improves embryo and green plant production in anther culture of Australian wheat (Triticum aestivum L.) genotypes. Crop Pasture Sci 62(10):813–822

  21. Brownfield L, Köhler C (2011) Unreduced gamete formation in plants: mechanisms and prospects. J Exp Bot 62(5):1659–1668

  22. Budak H, Shearman RC, Parmaksiz I, Dweikat I (2004) Comparative analysis of seeded and vegetative biotype buffalograsses based on phylogenetic relationship using ISSRs, SSRs, RAPDs, and SRAPs. Theor Appl Genet 109(2):280–288

  23. Bueno MA, Agundez MD, Gomez A, Carrascosa MJ, Manzanera JA (2000) Haploid origin of cork oak anther embryos by enzyme and RAPD gene markers. Int J Plant Sci 161(3):363–367

  24. Burun B, Emiroglu U (2008) A comparative study on colchicine application methods in obtaining doubled haploids of tobacco (Nicotiana tabacum L.). Turk J Biol 32(2):105–111

  25. Casadevall R, Martin E, Cravero E (2011) Simple sequence repeat (SSR) vs. sequence-related amplified polymorphism (SRAP) markers for Cynara cardunculus characterization. Spain J Agric Res 9(2):453–459

  26. Castillo AM, Cistué L, Vallés MP, Soriano M (2009) Chromosome doubling in monocots. In: Touraev A (ed) Advances in haploid production in higher plants. Springer, Dordrecht, pp 329–338

  27. Castillo AM, Nielsen NH, Jensen A, Vallés MP (2014) Effects of n-butanol on barley microspore embryogenesis. Plant Cell Tissue Organ Cult 117(3):411–418

  28. Castillo AM, Allue S, Costar A, Alvaro F, Valles MP (2019) Doubled haploid production from Spanish and central European Spelt by anther culture. J Agri Sci Tech 21(5):1313–1324

  29. Chani E, Veilleux RE, Boluarte Medina T (2000) Improved androgenesis of inter-specific potato and efficiency of SSR markers to identify homozygous regenerants. Plant Cell Tissue Organ Cult 60(2):101–112

  30. Chugh KN, Batt AR, Pratt MR (2016) Chemical methods for encoding and decoding of posttranslational modifications. Cell Chem Biol.

  31. Collard BCY, Jahufer MZZ, Brouwer JB, Pang CK (2005) An introduction to markers, quantitative trait loci (QTL) mapping and marker-assisted selection for crop improvement: the basic concepts. Euphytica 142(1–2):169–196

  32. Cooper MT, Porter TD (2000) Mutagenicity of nitrosamines in methyltransferase-deficient strains of Salmonella typhimurium coexpressing human cytochrome P450 2E1 and reductase. Mut Res 454(1–2):45–52

  33. Cousin A, Heel K, Cowling WA, Nelson MN (2009) An efficient high-throughput flow cytometric method for estimating DNA ploidy level in plants. Cytom Part A 75(12):1015–1019

  34. da Silva Dias JC (2003) Protocol for broccoli microspore culture. In: Maluszynski M, Kasha KJ, Forster BP, Szarejko I (eds) Doubled haploid production in crop plants: a manual. Kluwer Academic Publishers, Dordrecht, pp 195–204

  35. Daghma DES, Hensel G, Rutten T, Melzer M, Kumlehn J (2014) Cellular dynamics during early barley pollen embryogenesis revealed by time-lapse imaging. Front Plant Sci 5(675):1–14

  36. Dewi IS, Putri NH, Purwoko BS (2019) Response of anther donor genotypes (F1) from Indica × Indica crosses to rice anther culture. J Agro Biogen 15(1):45–52

  37. Diao W, Jia Y, Hui S, Zhang X, Lou Q, Chen JF (2009) Efficient embryo induction in cucumber ovary culture and homozygous identification of the regenerants using SSR markers. Sci Hortic 119(3):246–251

  38. Dirihan S, Terho P, Helander M, Saikkonen K (2013) Efficient analysis of ploidy levels in plant evolutionary ecology. Caryologia 66(3):251–256

  39. Doi H, Yokoi S, Hikage T, Nishihara M, Tsutsumi K, Takahata Y (2011) Gynogenesis in gentians (Gentiana triflora, G. scabra): production of haploids and doubled haploids. Plant Cell Rep 30(6):1099–1106

  40. Dolezel J, Bartos J (2005) Plant DNA flow cytometry and estimation of nuclear genome size. Ann Bot 95(1):99–110

  41. Dubas E, Custers J, Kieft H, Wędzony M, van Lammeren AAM (2011) Microtubule configurations and nuclear DNA synthesis during initiation of suspensor-bearing embryos from Brassica napus cv Topas microspores. Plant Cell Rep 30(11):2105–2116

  42. Dunwell JM (2010) Haploids in flowering plants: origins and exploitation. Plant Biotechnol J 8(4):377–424

  43. Dunwell JM, Wilkinson MJ, Nelson S, Wening S, Sitorus AC, Mienanti D, Alfiko Y, Croxford AE, Ford CS, Forster BP, Caligari PDS (2010) Production of haploids and doubled haploids in oil palm. BMC Plant Biol 10(218):1–25

  44. Ebrahimzadeh H, Shariatpanahi ME, Ahmadi B, Soltanloo H, Lotfi M, Zarifi E (2018a) Efficient parthenogenesis induction and in vitro haploid plant regeneration in cucumber (Cucumis sativus L.) using putrescine, spermidine, and cycocel. J Plant Growth Regul 37(4):1127–1134

  45. Ebrahimzadeh H, Soltanloo H, Shariatpanahi ME, Eskandari A, Ramezanpour SS (2018b) Improved chromosome doubling of parthenogenetic haploid plants of cucumber (Cucumis sativus L.) using colchicine, trifluralin, and oryzalin. Plant Cell Tissue Org Cult 135(3):407–417

  46. Eder J, Chalyk S (2002) In vivo haploid induction in maize. Theor Appl Genet 104(4):703–708

  47. Eimert K, Reutter G, Strolka B (2003) Fast and reliable detection of doubled-haploids in Asparagus officinalis by stringent RAPD-PCR. J Agric Sci 141(1):73–78

  48. Eudes F, Amundsen E (2005) Isolated microspore culture of Canadian 6× triticale cultivars. Plant Cell Tissue Organ Cult 82(3):233–241

  49. Eyasu A, Bechyne Klima M, Vyvadilova M (2007) Embryogenic responsibility of selected genotypes of Brassica carinata A. Braun to microspore culture. Agric Trop Subtrop 40(2):35–38

  50. Fábián A, Földesiné Füredi PK, Ambrus H, Jäger K, Szabó L, Barnabás B (2015) Effect of n-butanol and cold pretreatment on the cytoskeleton and the ultrastructure of maize microspores when cultured in vitro. Plant Cell Tissue Org Cult 123(2):257–271

  51. Fang J, Zhu X, Wang C, Shangguan L (2016) Applications of DNA technologies in agriculture. Curr Genom 17(4):379–386

  52. Fernandes TCC, Pizano MA, Marin-Morales MA (2013) Characterization, modes of action and effects of trifluralin: a review. IntechOpen Access, Rijeka, pp 489–515

  53. Ferrie AMR, Caswell KL (2011) Isolated microspore culture techniques and recent progress for haploid and doubled haploid plant production. Plant Cell Tissue Org Cult 104(3):301–309

  54. Ferrie AMR, Irmen KI, Beattie AD, Rossnagel BG (2014) Isolated microspore culture of oat (Avena sativa L.) for the production of doubled haploids: effect of pre-culture and post-culture conditions. Plant Cell Tissue Org Cult 116(1):89–96

  55. Földesiné Füredi PKF, Ambrus H, Barnabás B (2011) The effect of n-butanol and 2-amino-ethanol on the in vitro androgenesis of maize. Acta Biol Szeged 55(1):77–78

  56. Földesiné Füredi PKF, Ambrus H, Barnabás B (2012) Development of cultured microspores of maize in the presence of n-butanol and 2-aminoethanol. Acta Agron Hung 60(3):183–189

  57. Gao Y, Chen B, Zhan J (2011) Anther culture of garden Chrysanthemum. ISHS Acta Hortic 923:103–110

  58. Garcia-Arias F, Sánchez-Betancourt E, Núñez V (2018) Fertility recovery of anther-derived haploid plants in Cape gooseberry (Physalis peruviana L.). Agron Colomb 36(3):201–209

  59. Germana MA (2006) Doubled haploid production in fruit crops. Plant Cell Tissue Org Cult 86(2):131–146

  60. Germana MA (2011) Anther culture for haploid and doubled haploid production. Plant Cell Tissue Org Cult 104(3):283–300

  61. Germana MA, Chiancone B, Lain O, Testolin R (2005) Anther culture in Citrus clementina: a way to regenerate tri-haploids. Aust J Agric Res 56(8):839–845

  62. Gervais C, Newcomb W, Simmonds DH (2000) Rearrangement of the actin filament and microtubule cytoskeleton during induction of microspore embryogenesis in Brassica napus L. cv. Topas. Protoplasma 213(3–4):194–202

  63. Górecka K, Krzyzanowska D, Kiszczak W, Kowalska U (2009) Carrot doubled haploids. In: Touraev A, Forster BP, Mohan Jain S (eds) Advances in haploid production in higher plants. Springer, New York, pp 231–239

  64. Górecka K, Kowalska U, Krzyzanowska D, Kiszczak W (2010) Obtaining carrot (Daucus carota L.) plants in isolated microspore cultures. J Appl Genet 51(2):141–147

  65. Grzebelus E, Adamus A (2004) Effect of anti-mitotic agents on development and genome doubling of gynogenic onion (Allium cepa L.) embryos. Plant Sci 167(3):569–574

  66. Gu HH, Zhou WJ, Hagberg P (2003) High frequency spontaneous production of doubled haploid plants in microspore cultures of Brassica rapa ssp Chinensis. Euphytica 134(3):239–245

  67. Gu H, Zhao Z, Sheng X, Yu H, Wang J (2014) Efficient doubled haploid production in microspore culture of loose-curd cauliflower (Brassica oleracea var. botrytis). Euphytica 195(3):467–475

  68. Guiderdoni E, Galinato E, Luistro J, Vergara G (1992) Anther culture of tropical Japonica × Indica hybrids of rice (Oryza sativa L.). Euphytica 62(3):219–224

  69. Gyulai G, Gemensne JA, Sagi Z, Venczel G, Pinter P, Kristof Z, Torjek O, Heszky L, Bottka S, Kiss J, Zatyko L (2000) Doubled haploid development and PCR-analysis of F1 hybrid derived DH-R2 Paprika (Capsicum annuum L.) Lines. J Plant Physiol 156(2):168–174

  70. Hansen NJP, Andersen SB (1996) In vitro chromosome doubling potential of colchicine, oryzalin, trifluralin, and APM in Brassica napus microspore culture. Euphytica 88(2):159–164

  71. Hansen NJP, Andersen SB (1998) Efficient production of doubled haploid wheat plants by in vitro treatment of microspores with trifluralin or APM. Plant Breed 117:401–405

  72. Herrera JC, Moreno LG, Acuna JR, De Pena M, Osorio D (2002) Colchicine-induced microspore embryogenesis in coffee. Plant Cell Tissue Org Cult 71(1):89–92

  73. Hirase A, Hamada T, Itoh TJ, Shimmen T, Sonobe S (2006) n-Butanol induces depolymerization of microtubules in vivo and in vitro. Plant Cell Physiol 47(7):1004–1009

  74. Höfer M, Chrafe C, Boudichevskaja A, Lopez A, Bueno MA, Roen D (2008) Characterization of plant material obtained by in vitro androgenesis and in situ parthenogenesis in apple. Sci Hortic 117(3):203–211

  75. Hofinger BJ, Huynh OA, Jankowicz-Cieslak J, Müller A, Otto I, Kumlehn J, Till BJ (2013) Validation of doubled haploid plants by enzymatic mismatch cleavage. Plant Method 9(43):1–10

  76. Hooghvorst I, Ramos-Fuentes E, López-Cristofannini C, Ortega M, Vidal R, Serrat X, Nogués S (2018) Antimitotic and hormone effects on green double haploid plant production through anther culture of Mediterranean japonica rice. Plant Cell Tissue Org Cult 134(2):205–215

  77. Hoveida ZS, Abdollahi MR, Mirzaie-Asl A, Moosavi SS, Seguí-Simarro JM (2017) Production of doubled haploid plants from anther cultures of borage (Borago officinalis L.) by the application of chemical and physical stress. Plant Cell Tissue Org Cult 130(2):369–378

  78. Hu T, Kasha KJ (1997) Performance of isolated microspore-derived doubled haploids of wheat (Triticum aestivum L.). Can J Plant Sci 77(4):549–554

  79. Huang Z, Xu C, Li Y, Wang P, Li Y, Kang X (2015) Induction of somatic embryogenesis by anther-derived callus culture and plantlet ploidy determination in poplar (Populus × Beijingensis ). Plant Cell Tissue Org Cult 120(3):949–959

  80. Irikova T, Grozeva S, Rodeva V (2011) Anther culture in pepper (Capsicum annuum L.) in vitro. Acta Physiol Plant 33(5):1559–1570

  81. Islam SMS (2010) The effect of colchicine pretreatment on isolated microspore culture of wheat (Triticum aestivum L.). Australian J Crop Sci 4(9):660–665

  82. Jia Y, Zhang Q-X, Pan H-T, Wang S-Q, Liu Q-L, Sun L-X (2014) Callus induction and haploid plant regeneration from baby primrose (Primula forbesii Franch.) anther culture. Sci Hortic 176:273–281

  83. Kahrizi D, Mohammadi R (2009) Study of anderogenesis and spontaneous chromosome doubling in barley (Hordeum vulgare L.) genotypes using isolated microspore culture. Acta Agron Hung 57(2):155–164

  84. Kamiński P, Staniaszek M, Kozik EU (2003) Evaluation of genetic diversity and uniformity of head cabbage DH lines by the use of RAPD markers. J Appl Genet 44(2):157–163

  85. Karaismailoglu MC (2014) Evaluation of potential genotoxic effect of trifluralin in Helianthus annuus L. (sunflower). Caryologia 67(3):216–221

  86. Kasha KJ, Hu TC, Oro R, Simion E, Shim YS (2001) Nuclear fusion leads to chromosome doubling during mannitol treatment of barley (Hordeum vulgare L.) microspores. J Exp Bot 52(359):1227–1238

  87. Kasha KJ (2005) Chromosome doubling and recovery of doubled haploid plants. In: Palmer CE, Keller WA, Kasha KJ (eds) Biotechnology in agriculture and forestry: haploids in crop improvement II, vol 56. Springer, Berlin, pp 123–152

  88. Keles D, Pinar H, Ata A, Taskin H, Yildiz S, Buyukalaca S (2015) Effect of pepper types on obtaining spontaneous doubled haploid plants via anther culture. Hortic Sci 50(11):1671–1676

  89. Kim M, Park E-J, An D, Lee Y (2013) High-quality embryo production and plant regeneration using a two-step culture system in isolated microspore cultures of hot pepper (Capsicum annuum L.). Plant Cell Tissue Org Cult 112(2):191–201

  90. Kiss J, Kondrak M, Törjek O, Kiss E, Gyulai G, Mazik-Tökei K, Heszky LE (2001) Morphological and RAPD analysis of poplar trees of anther culture origin. Euphytica 118(2):213–221

  91. Kiszczak W, Krzyzanowska D, Strycharczuk K, Kowalska U, Wolko B, Górecka K (2011) Determination of ploidy and homozygosity of carrot plants obtained from anther cultures. Acta Physiol Plant 33(2):401–407

  92. Klima M, Vyvadilova M, Kucera V (2008) Chromosome doubling effects of selected antimitotic agents in Brassica napus microspore culture. Czech J Genet Plant Breed 44(1):30–36

  93. Klutschewski S (2012) Methodical improvements in microspore culture of Brassica napus L. Dissertation, University of Göttingen

  94. Kolahkaj FF, Mohammadi PP, Farkhari M, Alami-Saeid K (2015) Colchicine induced embryogenesis in date palm (Phoenix dactylifera L.) anther culture. J Appl Biotechnol Rep 2(4):325–328

  95. Kumar NS, Gurusubramanian G (2011) Random amplified polymorphic DNA (RAPD) markers and its applications. Sci Vis 11(3):116–124

  96. Lantos C, Juhász AG, Somogyi G, Otvos K, Vági P, Mihály R, Kristof Z, Somogyi N, Pauk J (2009) Improvement of isolated microspore culture of pepper (Capsicum annuum L.) via co-culture with ovary tissues of pepper or wheat. Plant Cell Tissue Org Cult 97(3):285–293

  97. Lantos C, Juhász AG, Vági P, Mihály R, Kristof Z, Pauk J (2012) Androgenesis induction in microspore culture of sweet pepper (Capsicum annuum L.). Plant Biotechnol Rep 6(2):123–132

  98. Leskovšek L, Jakše M, Bohanec B (2008) Doubled haploid production in rocket (Eruca sativa Mill.) through isolated microspore culture. Plant Cell Tissue Org Cult 93(2):181–189

  99. Leung YY, Hui LLY, Kraus VB (2015) Colchicine—update on mechanisms of action and therapeutic uses. Semin Arthritis Rheum 45(3):341–350

  100. Li H, Devaux P (2003) High frequency regeneration of barley doubled haploid plants from isolated microspore culture. Plant Sci 164(3):379–386

  101. Li G, Quiros CF (2001) Sequence-related amplified polymorphism (SRAP), a new marker system based on a simple PCR reaction: its application to mapping and gene tagging in Brassica. Theor Appl Genet 103(2):455–461

  102. Li J-R, Zhuang F-Y, Ou C-G, Hu H, Zhao Z-W, Mao Z-H (2013) Microspore embryogenesis and production of haploid and doubled haploid plants in carrot (Daucus carota L.). Plant Cell Tissue Org Cult 112(3):275–287

  103. Liu W, Zheng MY, Polle EA, Konzak CF (2002) Highly efficient doubled-haploid production in wheat (Triticum aestivum L.) via induced microspore embryogenesis. Crop Sci 42(3):686–692

  104. Maluszynska J (2003) Cytogenetic tests for ploidy level analyses-chromosome counting. In: Maluszynski M, Kasha KJ, Forster BP, Szarejko I (eds) Doubled haploid production in crop plants: a manual. Kluwer Academic Publishers, Dordrecht, pp 391–395

  105. Masanori K, Niimi Y (2004) Production of triploid plants of Japanese pear (Pyrus pyrifolia Nakai) by anther culture. Euphytica 138(2):141–147

  106. Melchinger AE, Molenaar WS, Mirdita V, Schipprack W (2016) Colchicine alternatives for chromosome doubling in maize haploids for doubled haploid production. Crop Sci 56(2):559–569

  107. Mohammadi PP, Moieni A, Jalali-Javaran M (2007) Colchicine induced embryogenesis and doubled haploid production in maize (Zea mays L.) anther culture. Iran J Biotechnol 5(3):140–146

  108. Mohammadi PP, Moiene A, Ebrahimi A, Javidfar F (2012) Doubled haploid plants following colchicine treatment of microspore-derived embryos of oilseed rape (Brassica napus L.). Plant Cell Tissue Org Cult 108(2):251–256

  109. Monakhos SG, Nguen ML, Bezbozhnaya AV, Monakhos GF (2014) A relationship between ploidy level and the number of chloroplasts in stomatal guard cell in diploid and amphiploid Brassica species. Agric Biol 5:44–54

  110. Morrissette NS, Mitra A, Sept D, Sibley LD (2004) Dinitroanilines bind α-tubulin to disrupt microtubules. Mol Biol Cell 15(4):1960–1968

  111. Motallebi-Azar A, Panahandeh J (2010) Effects of colchicine and cold duration pretreatments on androgenesis responses of tomato (Lycopersicon esculentum Mill) via anther culture. Russ Agric Sci 36(5):338–341

  112. Murovec J, Bohanec B (2012) Haploids and doubled haploids in plant breeding. Plant breeding. Rijeka, InTech, pp 87–106

  113. Niu L, Shi F, Feng H, Zhang Y (2019) Efficient doubled haploid production in microspore culture of Zengcheng flowering Chinese cabbage (Brassica campestris L. ssp. chinensis [L.] Makino var. utilis Tsen et Lee). Sci Hortic 245:57–64

  114. Nixon J (2006) Testing for segregation distortion in genetic scoring data from backcross or doubled haploid populations. Heredity 96(4):290–297

  115. Nurhidayah T, Kohler H, Dahlhoff M, Friedt W (1993) Anther culture of interspecific sunflower hybrids and examination of regenerants by biochemical and molecular methods. Biotechnol Biotechnol Equip 7(4):113–116

  116. Obert B, Barnabás B (2004) Colchicine induced embryogenesis in maize. Plant Cell Tissue Org Cult 77(3):283–285

  117. Ochatt SJ (2008) Flow cytometry in plant breeding. Cytom Part A 73(7):581–598

  118. Ochatt SJ, Patat-Ochatt EM, Moessner A (2011) Ploidy level determination within the context of in vitro breeding. Plant Cell Tissue Org Cult 104(3):329–341

  119. Oliveira AP, Sauer U (2012) The importance of post-translational modifications in regulating Saccharomyces cerevisiae metabolism. FEMS Yeast Res 12(2):104–117

  120. Olszewska D, Niklas-Nowak A, Nowaczyk L (2017) Estimation of genetic divergence within androgenic regenerants of Capsicum annuum L. ATZ1 × C. frutescens L. F1 plants using random amplified polymorphic DNA markers. BioTechnologia 98(3):175–182

  121. Palmer CE, Keller WA, Arnison PG (1996) Utilization of Brassica haploids. In: Jain SM, Sopory SK, Veilleux RE (eds) In vitro haploid production in higher plants. Kluwer Academic Publishers, Dordrecht, pp 173–192

  122. Pathirana R, Frew T, Hedderley D, Timmerman-Vaughan G, Morgan E (2011) Haploid and doubled haploid plants from developing male and female gametes of Gentiana triflora. Plant Cell Rep 30(6):1055–1065

  123. Pauk J, Puolimatka M, Toth KL, Monostori T (2000) In vitro androgenesis of triticale in isolated microspore culture. Plant Cell Tissue Org Cult 61(3):221–229

  124. Pelliccione S (2013) Microspore embryogenesis induction in hot pepper genotypes. Dissertation, University of Granada

  125. Perera PIP, Perera L, Hocher V, Verdeil JL, Yakandawala DMD, Weerakoon LK (2008) Use of SSR markers to determine the anther-derived homozygous lines in coconut. Plant Cell Rep 27(11):1697–1703

  126. Perera PIP, Ordoñez CA, Lopez-Lavalle LAB, Dedicova B (2014) A milestone in the doubled haploid pathway of cassava. Protoplasma 251(1):233–246

  127. Pintos B, Manzanera JA, Bueno MA (2007) Antimitotic agents increase the production of doubled-haploid embryos from cork oak anther culture. J Plant Physiol 164(12):1595–1604

  128. Pintos B, Sanchez N, Bueno MA, Navarro RM, Jorrin J, Manzanera JA, Gomez-Garay A (2013) Induction of Quercus ilex L. haploid and doubled-haploid embryos from anther cultures by temperature-stress. Silvae Genet 62(4–5):210–218

  129. Prem D, Gupta K, Agnihotri A (2005) Effect of various exogenous and endogenous factors on microspore embryogenesis in Indian mustard (Brassica juncea (L.) Czern and Coss). Vitro Cell Dev Biol Plant 41(3):266–273

  130. Prem D, Solís M-T, Bárány I, Rodríguez-Sanz H, Risueño MC, Testillano PS (2012) A new microspore embryogenesis system under low temperature which mimics zygotic embryogenesis initials, expresses auxin and efficiently regenerates doubled-haploid plants in Brassica napus. BMC Plant Biol 12(127):1–19

  131. Premvaranon P, Vearasilp S, Thanaponpoonpong S, Karladee D, Gorinstein S (2011) In vitro studies to produce double haploid in Indica hybrid rice. Biologia 66(6):1074–1081

  132. Pusadkar P, Jha Z (2018) In vitro colchicine treatment to increase the plant regeneration from Indica rice anther culture. J Pharmacog Phytochem 7(4):440–443

  133. Qin X, Rotino L (1995) Chloroplast number in guard cells as ploidy indicator of in vitro-grown androgenic pepper plantlets. Plant Cell Tissue Org Cult 41(2):145–149

  134. Rathod PH, Patel RB, Jhala AJ (2010) Persistence and management of dinitroaniline herbicides residues in sandy loam soil. Int J Environ Sustain Dev 9(1):58–73

  135. Ren J, Wu R, Trampe B, Tian X, Lübberstedt T, Chen S (2017) Novel technologies in doubled haploid line development. Plant Biotechnol J 15(11):1361–1370

  136. Ren X, Ci J, Cui X, Yang W (2018) Doubling effect of anti-microtubule herbicides on the maize haploid. Emirates J Food Agric 30(10):903–908

  137. Ribeiro CB, Pereira FDC, Filho LDN, Braz GT, Ruy MC, Silva MB, Cenzi G, Techio VH, Souza JCD (2018) Haploid identification using tropicalized haploid inducer progenies in maize. Crop Breed Appl Biotechnol 18(1):16–23

  138. Rimberia F, Adaniya S, Etoh T, Ishimine Y (2006) Sex and ploidy of anther culture derived papaya (Carica papaya L). plants. Euphytica 149(1):53–59

  139. Robarts DW, Wolfe AD (2014) Sequence-related amplified polymorphism (SRAP) markers: a potential resource for studies in plant molecular biology. Appl Plant Sci 2(7):1–13

  140. Roberts AV (2007) The use of bead beating to prepare suspensions of nuclei for flow cytometry from fresh leaves, herbarium leaves, petals and pollen. Cytom Part A 71(12):1039–1044

  141. Savenko E, Mukhina Z, Glazyrina V, Korotenko T, Garkusha S (2019) Creating doubled rice haploids with pyriculariosis resistant genes. IOP Conf Ser Earth Environ Sci.

  142. Seguí-Simarro JM, Nuez F (2007) Embryogenesis induction, callogenesis, and plant regeneration by in vitro culture of tomato isolated microspores and whole anthers. J Exp Bot 58(5):1119–1132

  143. Seguí-Simarro JM, Nuez F (2008) Pathways to doubled haploidy: chromosome doubling during androgenesis. Cytogenet Genome Res 120(3–4):358–369

  144. Shariatpanahi ME, Bal U, Heberle-Bors E, Touraev A (2006) Stresses applied for the re-programming of plant microspores towards in vitro embryogenesis. Physiol Plant 127(4):519–534

  145. Shariatpanahi ME, Ahmadi B (2016) Isolated microspore culture and its applications in plant breeding and genetics. In: Anis M, Ahmad N (eds) Plant tissue culture: propagation, conservation and crop improvement. Springer, Singapore, pp 487–507

  146. Sharma S, Sarkar D, Pandey SK (2010) Phenotypic characterization and nuclear microsatellite analysis reveal genomic changes and rearrangements underlying androgenesis in tetraploid potatoes (Solanum tuberosum L.). Euphytica 171(3):313–326

  147. Sheng X, Zhao Z, Yu H, Wang J, Xiaohui Z, Gu H (2011) Protoplast isolation and plant regeneration of different doubled haploid lines of cauliflower (Brassica oleracea var. botrytis). Plant Cell Tissue Org Cult 107(3):513–520

  148. Shim YS, Kasha KJ, Simion E, Letarte J (2006) The relationship between induction of embryogenesis and chromosome doubling in microspore cultures. Protoplasma 228(1–3):79–86

  149. Shumilina DV, Shmykova NA, Bondareva LL, Suprunova TP (2015) Effect of genotype and medium culture content on microspore derived embryo formation in Chinese cabbage (Brassica rapa ssp. chinensis) cv. Lastochka. Biol Bull 42(4):302–309

  150. Singh RJ (2003) Plant cytogenetics. CRC Press, Boca Raton, p 488

  151. Singh BD, Singh AK (2015) Marker-assisted plant breeding: principles and practices. Springer, Dordrecht.

  152. Ślusarkiewicz-Jarzina A, Ponitka A (2003) Efficient production of spontaneous and induced doubled haploid triticale plants derived from anther culture. Cereal Res Commun 31(3):289–296

  153. Ślusarkiewicz-Jarzina A, Pudelska H, Woźna J, Pniewski T (2017) Improved production of doubled haploids of winter and spring triticale hybrids via combination of colchicine treatments on anthers and regenerated plants. J Appl Genet 58(3):287–295

  154. Snowdon R (2007) Cytogenetics and genome analysis in Brassica crops. Chromosome Res 15(1):85–95

  155. Soriano M, Cistue L, Valles MP, Castillo AM (2007) Effects of colchicine on anther and microspore culture of bread wheat (Triticum aestivum L.). Plant Cell Tissue Org Cult 91(3):225–234

  156. Soriano M, Cistué L, Castillo AM (2008) Enhanced induction of microspore embryogenesis after n-butanol treatment in wheat (Triticum aestivum L.) anther culture. Plant Cell Rep 27(5):805–811

  157. Soroka AI (2013) Differentiation of haploid and dihaploid rape plants at the cytological and morphological levels. Cytol Genet 47(2):88–92

  158. Spruell P, Pilgrim KL, Greene BA, Habicht C, Knudsen KL, Lindner KR, Olsen JB, Sage GK, Seeb JE, Allendorf FW (1999) Inheritance of nuclear DNA markers in gynogenetic haploid pink salmon. J Hered 90(2):289–296

  159. Sripichitt P, Wongyai W, Saengduen N, Supapanyapong P (2002) Chromosome doubling in maize anther culture by colchicine and pronamide anther treatment. In: Proceedings of the 8th Asian regional maize workshop, Bangkok, Thailand, August 5–8, pp 109–118

  160. Sriskandarajah S, Sameri M, Lerceteau-Köhler E, Westerbergh A (2015) Increased recovery of green doubled haploid plants from barley anther culture. Crop Sci 55(6):2806–2812

  161. Staniaszek M, Habdas H (2006) RAPD technique application for intraline evaluation of androgenic carrot plants. Folia Hortic 18(2):87–97

  162. Stipic M, Campion B (1997) An improved protocol for androgenesis in cauliflowers (Brassica oleracea var. botrytis). Plant Breed 116(2):153–157

  163. Sudhakar R, Ninge Gowda KN, Venu G (2001) Mitotic abnormalities induced by silk dyeing industry effluents in the cells of Allium cepa. Cytologia 66(3):235–239

  164. Supena EDJ (2004) Innovations in microspore embryogenesis in Indonesian hot pepper (Capsicum annuum L.) and Brassica napus L. Dissertation, University of Wageningen

  165. Supena EDJ, Suharsono S, Jacobson E, Custers JBM (2006) Successful development of a shed-microspore culture protocol for doubled haploid production in Indonesian hot pepper (Capsicum annuum L.). Plant Cell Rep 25(1):1–10

  166. Szarejko I (2003) Anther culture for double haploid production in barley (Hordeum vulgare L.). In: Maluszynski M, Kasha KJ, Forster BP, Szarejko I (eds) Doubled haploid production in crop plants: a manual. Kluwer, Dordrecht, pp 35–42

  167. Takahashi Y, Yokoi S, Takahata Y (2012) Effects of genotypes and culture conditions on microspore embryogenesis and plant regeneration in several subspecies of Brassica rapa L. Plant Biotechnol Rep 6(4):297–304

  168. Takahira J, Cousin A, Nelson MN, Cowling WA (2011) Improvement in efficiency of microspore culture to produce doubled haploid canola (Brassica napus L.) by flow cytometry. Plant Cell Tissue Org Cult 104(1):51–59

  169. Testillano PS, Georgiev S, Mogensen HL, Coronado MJ, Dumas C, Risueno MC, Matthys-Rochon E (2004) Spontaneous chromosome doubling results from nuclear fusion during in vitro maize induced microspore embryogenesis. Chromosoma 112(7):342–349

  170. Tuncer B, Yanmaz R (2011) Effects of colchicine and high temperature treatments on isolated microspore culture in various cabbage (Brassica oleraceae) types. Int J Agric Biol 13(5):819–822

  171. Vanous K, Vanous A, Frei U, Lübberstedt T (2017) Generation of maize (Zea mays) doubled haploids via traditional methods. Curr Protoc Plant Biol 342:147–157

  172. Vaughn KC, Lehnen LP (1991) Mitotic disrupter herbicides. Weed Sci 39(3):450–457

  173. Vieira MLC, Santini L, Diniz AL, de Freitas MC (2016) Microsatellite markers: what they mean and why they are so useful. Genet Mol Biol 39(3):312–328

  174. Vouillot L, Thélie A, Pollet N (2015) Comparison of T7E1 and surveyor mismatch cleavage assays to detect mutations triggered by engineered nucleases. Gene Genom Genet 5(3):407–415

  175. Wan GL, Naeem MS, Geng XX, Xu L, Li B, Jilani G, Zhou WJ (2011) Optimization of microspore embryogenesis and plant regeneration protocols for Brassica napus. Int J Agric Biol 13:83–88

  176. Wan Y, Duncan DR, Rayburn AL, Petolino JF, Widholm JM (1991) The use of anti-microtubule herbicides for the production of doubled haploid plants from anther-derived maize callus. Theor Appl Genet 81(2):205–211

  177. Wan Y, Widholm JM (1995) Effect of chromosome-doubling agents on somaclonal variation in the progeny of doubled haploids of maize. Plant Breed 114(3):253–255

  178. Wang Z, Li Q, Zhao J, Peng Y (2011) Investigation of the effect of herbicide amiprophos methyl on spindle formation and proteome change in maize by immunofluorescence and proteomic technique. Cytologia 76(3):1–11

  179. Wang T, Li H, Zhang J, Ouyang B, Lu Y, Ye Z (2009) Initiation and development of microspore embryogenesis in recalcitrant purple flowering stalk (Brassica campestris ssp. chinensis var. purpurea Hort.) genotypes. Sci Hortic 121(4):419–424

  180. Weber S, ÜNker F, Friedt W (2005) Improved doubled haploid production protocol for Brassica napus using microspore colchicine treatment in vitro and ploidy determination by flow cytometry. Plant Breed 124:511–513

  181. Wijnker E, van Dun K, de Snoo CB, Lelivelt CLC, Keurentjes JJB, Naharudin NS, Ravi M, Chan SWL, de Jong H, Dirks R (2012) Reverse breeding in Arabidopsis thaliana generates homozygous parental lines from a heterozygous plant. Nat Genet 44(4):467–471

  182. Willis TG, Estes AG, McCarty LB (2007) Postemergent annual bluegrass control in dormant nonoverseeded bermudagrass turf. Hortic Sci 42(3):670–672

  183. Winarto B, Mattjik NA, Purwito A, Marwoto A (2009) Comparison of ploidy level screening methods in regenerants derived from anther culture of anthurium. Kumpulan Makalah Seminar Ilmiah Perhorti, pp 179–186

  184. Winarto B, Rachmawati F, Pramanik D, Teixeira da Silva JA (2011) Morphological and cytological diversity of regenerants derived from half-anther cultures of anthurium. Plant Cell Tissue Org Cult 105(3):363–374

  185. Würschum T, Tucker MR, Reif JC, Maurer HP (2012) Improved efficiency of doubled haploid generation in hexaploid triticale by in vitro chromosome doubling. BMC Plant Biol 12(109):1–7

  186. Yamagashi M, Takeuchi Y, Tanaka I, Kono I, Murai K, Yano M (2010) Segregation distortion in F2 and doubled haploid populations of temperate Japonica rice. J Genet 89(2):237–241

  187. Yemets AI, Blume YB (2008) Progress in plant polyploidization based on antimicrotubular drugs. Open Hortic J 1(1):15–20

  188. Yuan S, Liu Y, Fang Z, Yang L, Zhuang M, Zhang Y, Sun P (2009) Study on the relationship between the ploidy level of microspore-derived plants and the number of chloroplast in stomatal guard cells in Brassica oleracea. Agric Sci China 8(8):939–946

  189. Yuan S, Su Y, Liu Y, Li Z, Fang Z, Yang L, Zhuang M, Zhang Y, Lv H, Sun P (2015) Chromosome doubling of microspore-derived plants from cabbage (Brassica oleracea var. capitata L.) and broccoli (Brassica oleracea var. italica L.). Front Plant Sci 6:1118.

  190. Zhang Y, Wang A, Liu Y, Wang Y, Feng H (2012) Improved production of doubled haploids in Brassica rapa through microspore culture. Plant Breed 131(1):164–169

  191. Zhao J, Simmonds DH (1995) Application of trifluralin to embryogenic microspore cultures to generate doubled haploid plants in Brassica napus. Physiol Plant 95(2):304–309

  192. Zhao J, Newcomb W, Simmonds D (2003) Heat-shock proteins 70 kDa and 19 kDa are not required for induction of embryogenesis of Brassica napus L. cv. Topas microspores. Plant Cell Physiol 44(12):1417–1421

  193. Zhou WJ, Hagberg P, Tang GX (2002) Increasing embryogenesis and doubling efficiency by immediate colchicine treatment of isolated microspores in spring Brassica napus. Euphytica 128(1):27–34

  194. Zhou K, Fleet P, Nevo E, Zhang X, Sun G (2017) Transcriptome analysis reveals plant response to colchicine treatment during on chromosome doubling. Sci Rep 7(8503):1–11.

Download references

Author information

All authors whose names appear on the submission have contributed sufficiently to the scientific work and, therefore, share collective responsibility and accountability for the results.

Correspondence to Behzad Ahmadi.

Ethics declarations

Conflict of interest

The authors declare that they have no conflicts of interest.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Communicated by Neal Stewart.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Ahmadi, B., Ebrahimzadeh, H. In vitro androgenesis: spontaneous vs. artificial genome doubling and characterization of regenerants. Plant Cell Rep 39, 299–316 (2020).

Download citation


  • Anti-microtubular agent
  • DNA molecular marker
  • Flow cytometry
  • Haploid
  • Isozyme