Abstract
In vitro anther and ovule culture has been mostly used in haploidization studies of annual and perennial plants to shorten the process of breeding. Cyclamen genus is one of the major perennial geophytes widely used as an ornamental plant. The aim of this study was to develop an efficient haploid plant regeneration protocol via anther and ovule culture for wild Cyclamen persicum and commercial F1 Melody cultivar. The uninuclear stage of microspore was determined with DAPI (4′,6-diamidino-2-phenylindole dihydrochloride) dye for C. persicum Mill. and commercial cultivar. Cold pre-treatment (4 °C) was applied to the buds for two d before in vitro ovule and anther cultures. Anthers were cultured on B5 medium combined with different dosages of 1-naphthaleneacetic acid (NAA), 135.0 g L−1 maltose, silver nitrate (AgNO3), and activated charcoal (AC). Ovules were cultured on Murashige and Skoog (MS) medium including the varied amount of 2,4-dichlorophenoxyacetic acid (2,4-D) and N6-(2-isopentenyladenine) (2iP) and sucrose. Embryos were maturated and germinated on the M2 medium (MS containing 0.2 mg L−1 giberellic acid (GA3), 0.1 mg L−1 6-benzylaminopurine (BA), 1.0 g L−1 proline, and 0.05 mg L−1 spermine) for anther culture and MS medium without plant growth regulator (PGR) for ovule culture. Haploid embryos were obtained from B5 medium, including 1.0 mg L−1 NAA for C. persicum Mill. (100%). An efficient ovule culture protocol was determined for C. persicum Mill. as 2.0 mg L−1 2,4-D and 0.8 mg L−1 2iP; and 2.0 mg L−1 2,4-D and 0.5 mg L−1 2iP for Melody F1 cultivar as 100%. Spontaneous double haploidization was detected on C. persicum Mill. via flow cytometric analysis. Plants were transferred to the soil, and blooming was observed 4 mo after acclimatization.
Similar content being viewed by others
Data availability
The authors confirm that the data will be avaliable on request.
References
Abdollahi MR, Rashidi S (2018) Production and conversion of haploid embryos in chickpea (Cicer arietinum L.) anther cultures using high 2,4-D and silver nitrate containing media. Plant Cell Tiss Org Cult 133:39–49. https://doi.org/10.1007/s11240-017-1359-4
Arı E (2006) Antherculture studies on Anemone coronaria var. coccinea native to Turkey. Cukurova Univ., Institute of Science, Adana, pp. 169 Doctoral dissertation in Turkish
Bermejo C, Guindon MF, Palacios LT, Cazzola F, Gatti I, Cointry E (2020) Comparative androgenetic competence of various species and genotypes within the genus Pisum L. Plant Cell Tiss Org Cult 143:487–497. https://doi.org/10.1007/s11240-020-01934-y
Buyukalaca S, Comlekcioglu N, Abak K, Ekbic E, Kilic N (2004) Effects of silver nitrate and donor plant growing conditions on production of pepper (Capsicum annuum L.) haploid embryos via anther culture. Eur J Hortic Sci 69:206–209
Compton JA, Clennett JCB, Culham A (2004) Nomenclature in the dock: overclassification leads to instability: a case study in the horticulturally important genus Cyclamen (Myrsinaceae). Bot J Linn Soc 146:339–349. https://doi.org/10.1111/j.1095-8339.2004.00322.x
Copetta A, Dei F, Marchioni I, Cassetti A, Ruffoni B (2018) Effect of thermal shock in the development of androgenic plants of Anemone coronaria L.: influence of genotype and flower parameters. Plant Cell Tiss Org Cult 134:55–64. https://doi.org/10.1007/s11240-018-1399-4
Çördük N, Yücel G, Akıncı N, Tuna M, Esen O (2018) In vitro propagation of Silene bolanthoides Quézel, Contandr. & Pamukç. and assessment of genetic stability by flow cytometry. Arch Biol Sci 70:141–148. https://doi.org/10.2298/ABS170410033C
Curuk P, Sogut Z, Bozdogan E, Izgu T, Sevindik B, Tagipur E, Teixeira da Silva JA, Serce S, Aka Kacar Y, Mendi Y (2015) Morphological characterization of Cyclamen sp. grown naturally in Turkey: part I. S Afr J Bot 100:7–15. https://doi.org/10.1016/j.sajb.2015.03.199
Doi H, Takahashi R, Hikage T, Takahata Y (2010) Embryogenesis and doubled haploid production from anther culture in Gentian (Gentianatriflora). Plant Cell Tiss Org Cult 102:27–33. https://doi.org/10.1007/s11240-010-9700-1
Ellialtıoğlu ŞŞ, Sevindik B, Alan AR (2022) Katlanmış Haploid (DH) Teknolojisi. In: Kaza S, Yalçın Mendi Y (eds) Süs Bitkileri Islahı (Klasik ve Biyoteknolojik Yöntemler). Gece Kitaplığı, pp: 249–314
Gamborg OL, Miller R, Ojima K (1968) Nutrient requirements of suspension cultures of soybean root cells. Exp Cell Res 50:151–158
Geier T (1978) Development of isolated rudimentary anthers from somatic anther callus of Cyclamen persicum Mill. Z Pflanzenphysiol 90:245–256
Germanà MA (2011) Gametic embryogenesis and haploid technology as valuable support to plant breeding. Plant Cell Rep 30:839–857. https://doi.org/10.1007/s00299-011-1061-7
Han DS, Niimi Y (2004) Production of haploid and doubled haploid plants from anther-derived callus of Liliumformosanum. Acta Hortic 673:389–393. https://doi.org/10.17660/ActaHortic.2005.673.50
Höfer M, Flachowsky H (2015) Comprehensive characterization of plant material obtained by in vitro androgenesis in apple. Plant Cell Tiss Org Cult 122:617–628. https://doi.org/10.1007/s11240-015-0794-3
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 Tiss Org Cult 130:369–378. https://doi.org/10.1007/s11240-017-1233-4
Iqbal MCM, Wijesekara KB (2007) A brief temperature pulse enhances the competency of microspores for androgenesis in Datura metel. Plant Cell Tiss Org Cult 89:141–149. https://doi.org/10.1007/s11240-007-9222-7
Ishizaka H (1996) Interspecific hybrids of Cyclamen persicum and C. graecum. Euphytica 91:109–117. https://doi.org/10.1007/BF00035281
Ishizaka H (1998) Production of microspore-derived plants by anther culture of an interspecific f1 hybrid between Cyclamen persicum and C. purpurascens. Plant Cell Tiss Org Cult 54:21–28. https://doi.org/10.1023/A:1006138704856
Ishizaka H (2008) Interspecific hybridization by embryo rescue in the genus Cyclamen. Plant Biotechnol J 25:511–519. https://doi.org/10.5511/plantbiotechnology.25.511
Ishizaka H, Uematsu J (1993). Production of plants from pollen in Cyclamen persicum Mill. through anther culture. Jpn J Breed 43(2): 207–218. https://doi.org/10.1270/jsbbs1951.43.207
Ishizaka H, Uematsu J (1994) Amphidiploids between Cyclamen persicum Mill. and C. hederifolium Aiton induced through colchicine taeatment of ovules in vitro and plants. Japan J Breed 44:161–166. https://doi.org/10.1270/jsbbs1951.44.161
Ishizaka H, Uematsu J (1995) Interspecific hybrids of Cyclamen persicum Mill. and C. purpurascens Mill. produced by ovule culture. Euphytica 82:31–37. https://doi.org/10.1007/BF00028707
İzgü T, Sevindik B, Çürük P, Şimşek Ö, Kaçar YA, da Silva JAT, Mendi YY (2016) Development of an efficient regeneration protocol for four Cyclamen species endemic to Turkey. Plant Cell Tiss Org Cult 127:95–113. https://doi.org/10.1007/s11240-016-1033-2
Jalali N, Naderi R, Shahi Gharahlar A, Teixeira da Silva JA (2012) Tissue culture of Cyclamen spp. Sci Hortic 137:11–19. https://doi.org/10.1016/j.scienta.2012.01.015
Jia Y, Zhang QX, Pan HT, Wang SQ, Liu QL, Sun LX (2014) Callus induction and haploid plant regeneration from baby primrose (Primula forbesii Franch.) anther culture. Sci Hortic 176:273–281. https://doi.org/10.1016/j.scienta.2014.07.018
Johansson L (1983) Effects of activated charcoal in anther cultures. Physiol Plant 59:397–403. https://doi.org/10.1111/j.1399-3054.1983.tb04221.x
Khandakar RK, Jie YU, Sun-Kyung M, Mi-Kyoung W, Choi HG, Ha-Seung P, Choi JJ, Chae SC, Jung JY, Lee KM, Kim TS, Park YJ (2014) Regeneration of haploid plantlet through anther culture of chrysanthemum (Dendranthema grandiflorum). Not Bot Horti Agrobot Cluj Napoca 42:482–487. https://doi.org/10.15835/nbha4229640
Kocak M, Izgu T, Sevindik B, Tutuncu M, Curuk P, Simsek O, Aka Kaçar Y, da Silva JAT, Mendi YY (2014) Somatic embryogenesis of Turkish Cyclamen persicum Mill. Sci Hortic 172:26–33. https://doi.org/10.1016/j.scienta.2014.03.044
Li F, Cheng Y, Zhao X, Yu R, Li H, Wang L, Shan Q (2020) Haploid induction via unpollinated ovule culture in Gerbera hybrida. Sci Rep 10:1–9. https://doi.org/10.1038/s41598-020-58552-z
Lyngved R, Renaut J, Hausman JF, Iversen TH, Hvoslef-Eide AK (2008) Embryo-specific proteins in Cyclamen persicum analyzed with 2-D DIGE. J Plant Growth Regul 27:353–369. https://doi.org/10.1007/s00344-008-9061-8
Murashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant 15:473–497. https://doi.org/10.1111/j.1399-3054.1962.tb08052.x
Murovec J, Bohanec B (2012) Haploids and doubled haploids in plant breeding. In: Ibrohim Y. Dr. Ibrokhim Abdurakhmonov (ed) Plant Breeding. Rijeka, Croatia pp: 87–106
Nitsch JP, Nitsch C (1969) Haploid plants from pollen grains. Science 163:85–87. https://doi.org/10.1126/science.163.3862.85
Osterc G, Cunja V, Mikulic-Petkovsek M, Schmitzer V, Stampar F, Bavcon J (2014) Foliage identification of different autochtonous common cyclamen genotypes (Cyclamen purpurascens Mill.) using various biochemical parameters. Sci Hortic 173:37–44. https://doi.org/10.1016/j.scienta.2014.04.034
Pintos B, Manzanera JA, Bueno MA (2007) Antimitotic agents increase the production of doubled-haploid embryos from cork oak anther culture. J Plant Physiol 164:1595–1604. https://doi.org/10.1016/j.jplph.2006.11.012
Prange ANS, Bartsch M, Serek M, Winkelmann T (2010) Regeneration of different Cyclamen species via somatic embryogenesis from callus, suspension cultures and protoplasts. Sci Hortic 125:442–450. https://doi.org/10.1016/j.scienta.2010.04.018
Razdan A, Razdan MK, Rajam MV, Raina SN (2008) Efficient protocol for in vitro production of androgenic haploids of Phlox drummondii. Plant Cell Tiss Org Cult 95:245–250. https://doi.org/10.1007/s11240-008-9431-8
Sato S, Katoh N, Yoshida H, Iwai S, Hagimori M (2000) Production of doubled haploid plants of carnation (Dianthus caryophyllus L.) by pseudofertilized ovule culture. Sci Hortic 83:301–310. https://doi.org/10.1016/S0304-4238(99)00090-4
Schwenkel H-G, Winkelmann T (1998) Plant regeneration via somatic embryogenesis from ovules of Cyclamen persicum Mill. Plant Tiss Cult Biotechnol 4:28–34
Seguí-Simarro JM (2010) Androgenesis revisited. Bot Rev 76:377–404. https://doi.org/10.1007/s12229-010-9056-6
Simsek O, Curuk P, Aslan F, Bayramoglu M, Izgu T, da Silva JAT, Mendi YY (2017) Molecular characterization of Cyclamen species collected from different parts of Turkey by RAPD and SRAP markers. Biochemi 55:87–102. https://doi.org/10.1007/s10528-016-9770-9
Supena EDJ, Suharsono S, Jacobsen 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 25:1–10. https://doi.org/10.1007/s00299-005-0028-y
Takamura T, Imose Y, Nagita Y, Tanaka M (2006) Effects of nitrogen source and gellan-gum concentration in medium on plant regeneration from somatic embryos in Cyclamen (Cyclamen persicum Mill.). J Agric Sci Technol 18:105–109
Takamura T, Sakamoto K, Horikawa M (2010) Effect of carbon source on in vitro plant regeneration in anther culture of Cyclamen (Cyclamen persicum Mill.). Acta Hortic 923:129–134. https://doi.org/10.17660/ActaHortic.2011.923.18
Uno Y, Koda-Katayama H, Kobayashi H (2016) Application of anther culture for efficient haploid production in the genus Saintpaulia. Plant Cell Tiss Org Cult 125:24–248. https://doi.org/10.1007/s11240-016-9433
Vural GE, Ari E, Zengin S, Ellialtioglu SS (2019) Development of androgenesis studies on eggplant (Solanum Melongena L.) in Turkey from past to present. In: Hasanuzzaman M (ed) Sustainable crop production. IntechOpen 1–26. https://doi.org/10.1016/j.scienta.2020.109472
Wang GF, Qin HY, Sun D, Fan ST, Yang YM, Wang ZX, Xu PL, Zhao Y, Liu YX, Ai J (2018) Haploid plant regeneration from hardy kiwifruit (Actinidia arguta Planch) anther culture. Plant Cell Tiss Org Cult 134:15–28. https://doi.org/10.1007/s11240-018-1396-7
Yesson C, Toomey NH, Culham A (2009) Cyclamen: time, sea and speciation biogeography using a temporally calibrated phylogeny. J Biogeogr 36:1234–1252. https://doi.org/10.1111/j.1365-2699.2008.01971.x
Acknowledgements
This study was produced from the part of PhD thesis of Başar Sevindik.
Funding
This research was supported by TUBITAK (The Scientific and Technical Research Council of Türkiye) (Project No. TOVAG 114O278) project.
Author information
Authors and Affiliations
Contributions
BS: determination of uni-nuclear stage, the establishment of ovule and anther culture experiments, sampling the leaves for ploidy level determination, acclimatization of the plants, statistical analyses, writing the original draft. Tİ: collecting the plants, editing drafts. MT: Investigation of the methodology, statistical analyses. PÇ: collecting the plants, editing draft. NS: Supervision and editing draft. YM: supervision, visualization, writing-review and editing.
Corresponding author
Ethics declarations
Conflict of interest
The authors declare no conflicts of interest.
Rights and permissions
Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.
About this article
Cite this article
Sevindik, B., İzgü, T., Tütüncü, M. et al. Double-haploid plant production through anther and ovule culture of wild Cyclamen persicum Mill. and Melody F1 cyclamen cultivar. In Vitro Cell.Dev.Biol.-Plant 59, 711–723 (2023). https://doi.org/10.1007/s11627-023-10364-2
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11627-023-10364-2