Abstract
The present paper describes a detailed study of a highly efficient protocol to multiply the number of haploids in sugar beet production and subsequent chromosome doubling. The protocol involves an experiment investigating factorial interactions between cold pretreatment, seven genotypes of sugar beet, and kinetin to improve haploid embryo induction. In addition, the effects of color of ovules and flower bud position on haploid embryo induction were investigated. After subjecting the data to analysis of variance or Student’s t test (P < .05), the effect sizes of the independent variables were also estimated. Cold pretreatment was effective in stimulating the ovules. The haploid embryo induction rate for 1-week cold pretreated ovules (9.01%) was higher than that of freshly cultured ones (6.15%). In comparison with hormone-free medium (5.16%), the gynogenesis rate for the media supplemented with 0.05 or 0.5 mg L−l kinetin increased to 7.58 and 10.05%, respectively. The genotype responses were significantly different. Interactions of kinetin × cold pretreatment, genotype × hormonal treatment, genotype × cold pretreatment, and the three-way interaction were statistically significant. Moreover, the main effects of flower bud position, ovule color, and comma-form ovule on gynogenic response were significant. After investigating the effect of 5 g L−l colchicine for 3, 5, or 7 min on one genotype’s (SG2) specimens, all the haploid plantlets from the other genotypes were treated for 5 min as the best treatment. The paper discusses interactions of the factors, which may be interesting for others aiming to breed doubled haploid sugar beet or possibly other related plant species.
Similar content being viewed by others
References
Aflaki F, Pazuki A, Gürel S, Stevanato P, Biancardi E, Gürel E (2018) Doubled haploid sugar beet: an integrated view of factors influencing the processes of gynogenesis and chromosome doubling. Int Sugar J 120:274–285
Barański R (1996) In vitro gynogenesis in red beet (Beta vulgaris L.): effects of ovule culture conditions. Acta Soc Bot Pol 65:57–60. doi:10.5586/asbp.1996.010
Bossoutrot D, Hosemans D (1985) Gynogenesis in Beta vulgaris L.: from in vitro culture of unpollinated ovules to the production of doubled haploid plants in soil. Plant Cell Rep 4:300–303. doi:10.1007/BF00269883
Cardoso JC, Abdelgalel AM, Chiancone B, Latado RR, Lain O, Testolin R, Germanà MA (2016) Gametic and somatic embryogenesis through in vitro anther culture of different citrus genotypes. Plant Biosyst 150:304–312. doi:10.1080/11263504.2014.987847
Chen JF, Cui L, Malik AA, Mbira KG (2011) In vitro haploid and dihaploid production via unfertilized ovule culture. Plant Cell Tissue Organ Cult 104:311–319. doi:10.1007/s11240-010-9874-6
Cohen J (1988) “Small,” “medium,” and “large” d values. Statistical Power Analysis for the Behavioral Sciences, 2nd edn. Lawrence Erlbaum Associates, Inc, Hillsdale, NJ, pp 24–27
D’Halluin K, Keimer B (1986) Production of haploid sugarbeets (Beta vulgaris L.) by ovule culture. In: Horn W, Jensen CJ, Odenbach W, Schieder O (eds) Genetic manipulation in plant breeding. de Gruyter, Berlin, pp 307–309
Doctrinal M, Sangwan RS, Sangwan-Norreel BS (1989) In vitro gynogenesis in Beta vulgaris L.: effects of plant growth regulators, temperature, genotypes and season. Plant Cell Tissue Organ Cult 17:1–2. doi:10.1007/BF00042276
Eujayl I, Strausbaugh C, Lu C (2016) Registration of sugarbeet doubled haploid line KDH13 with resistance to beet curly top. J Plant Regist 10:93–96
Ferrant V, Bouharmont J (1994) Origin of gynogenetic embryos of Beta vulgaris L. Sex Plant Reprod 7:12–16. doi:10.1007/BF00241883
Field A (2013) Discovering statistics using IBM SPSS statistics, 4th edn. Sage, London
Forster BP, Heberle-Bors E, Kasha KJ, Touraev A (2007) The resurgence of haploids in higher plants. Trends Plant Sci 12:368–375. doi:10.1016/j.tplants.2007.06.007
Galatowitsch MW, Smith GA (1990) Regeneration from unfertilized ovule callus of sugarbeet (Beta vulgaris L.). Can J Plant Sci 70:83–89. doi:10.4141/cjps90-010
Gürel E (1997) Callus and root development from leaf explants of sugar beet (Beta vulgaris L.): variability at variety, plant and organ level. Turk J Bot 21:131–136
Gürel S, Gürel E (2013) In vitro regeneration of sugar beet (Beta vulgaris L.). In: Ramawat KG, Merillon JM (eds) Bulbous Plants: Biotechnology. CRC Press, Boca Raton, FL, pp 113–151. doi:10.1134/S1022795406020086
Gürel S, Gürel E, Kaya Z (2000) Doubled haploid plant production from unpollinated ovules of sugar beet (Beta vulgaris L.). Plant Cell Rep 19:1155–1159. doi:10.1007/s002990000248
Gürel E, Gürel S, Lemaux PG (2008) Biotechnology applications for sugar beet. Crit Rev Plant Sci 27:108–140. doi:10.1080/07352680802202000
Gürel E, Pazuki A, Aflaki F, Gürel S (2016) Crispr-cas9: A handy tool, but not in genome-editing toolbox of all plant species. Biotechnol Ind J 12:e101
Hansen AL, Plever C, Pedersen HC, Keimer B, Andersen SB (1994) Efficient in vitro chromosome doubling during Beta vulgaris ovule culture. Plant Breed 112:89–95. doi:10.1111/j.1439-0523.1994.tb00655.x
Hansen AL, Gertz A, Joersbo M, Andersen SB (1995) Short-duration colchicine treatment for in vitro chromosome doubling during ovule culture of Beta vulgaris L. Plant Breed 114:515–519. doi:10.1111/j.1439-0523.1995.tb00847.x
Hansen AL, Gertz A, Joersbo M, Andersen SB (1998) Antimicrotubule herbicides for in vitro chromosome doubling in Beta vulgaris L. ovule culture. Euphytica 101:231–237. doi:10.1023/A:1018380103304
Hansen AL, Gertz A, Joersbo M, Andersen SB (2000) Chromosome doubling in vitro with amiprophos-methyl in Beta vulgaris ovule culture. Acta Agr Scand B-S P 50:89–95. doi:10.1080/09064710050505035
Hedges LV (1981) Distribution theory for Glass’s estimator of effect size and related estimators. J Educ Stat 6:107–128. doi:10.3102/10769986006002107
Hilgert-Delgado A, Klíma M, Viehmannová I, Urban MO, Fernández-Cusimamani E, Vyvadilová M (2015) Efficient resynthesis of oilseed rape (Brassica napus L) from crosses of winter types B. rapa × B. oleracea via simple ovule culture and early hybrid verification. Plant Cell Tissue Organ Cult 120:191–201. doi:10.1007/s11240-014-0593-2
Hosemans D, Bossoutrot D (1983) Induction of haploid plants from in vitro culture of unpollinated beet ovules (Beta vulgaris L.). Z Pflanzenzucht 91:74–77
Lakens D (2013) Calculating and reporting effect sizes to facilitate cumulative science: a practical primer for t-tests and ANOVAs. Front Psychol 4:863. doi:10.3389/fpsyg.2013.00863
Landi M, Biondini S, Zoccola A, Gobbo G, Betti A, Saveri C, Terlicher P, Bottacci A, Angiolini C (2016) Effect of cold treatment on germination of Saxifraga aizoides and S. paniculata: a comparison of central (eastern Alps) and southern populations (northern Apennines). Biologia 71:149–154. DOI:10.1515/biolog-2016-0024
Larsen K (1977) Self-incompatibility in Beta vulgaris L. Hereditas 85:227–248. doi:10.1111/j.1601-5223.1977.tb00971.x
Levan A (1945) A haploid sugar beet after colchicine treatment. Hereditas 31:399–410. doi:10.1111/j.1601-5223.1945.tb02760.x
Lukaszewska E, Virden R, Sliwinska E (2012) Hormonal control of endoreduplication in sugar beet (Beta vulgaris L.) seedlings growing in vitro. Plant Biol 14:216–222. doi:10.1111/j.1438-8677.2011.00477.x
Lux H, Herrman L, Wetzel C (1990) Production of haploid sugar beet (Beta vulgaris L.) by culturing unpollinated ovules. Plant Breeding 104:177–183. doi:10.1111/j.1439-0523.1990.tb00420.x
Murashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plantarum 15:473–497. doi:10.1111/j.1399-3054.1962.tb08052.x
Pazuki A, Aflaki F, Gürel E, Ergül A, Gürel S (2017) Gynogenesis induction in sugar beet (Beta vulgaris) improved by 6-benzylaminopurine (BAP) and synergized with cold pretreatment. Sugar Tech. doi:10.1007/s12355-017-0522-x
Pedersen HC, Keimer B (1996) Haploidy in sugar beet (Beta vulgaris L.). In: Jain SM, Sopory SK, Veilleux RE (eds) In vitro haploid production in higher plants. Important selected plants, vol 3. Kluwer Academic Publishers, Dordrecht, pp 17–36. doi:10.1007/978-94-017-1858-5_2
Popova T, Grozeva S, Todorova V, Stankova G, Anachkov N, Rodeva V (2016) Effects of low temperature, genotype and culture media on in vitro androgenic answer of pepper (Capsicum annuum L.). Acta Physiol Plant 38:273. doi:10.1007/s11738-016-2294-4
Ragot M, Steen P (1992) Genetic and environmental effects on chromosome doubling of sugarbeet (Beta vulgaris L.) haploids. Euphytica 63:233–237
Rout P, Naik N, Ngangkham U, Verma RL, Katara JL, Singh ON, Samantaray S (2016) Doubled Haploids generated through anther culture from an elite long duration rice hybrid, CRHR32: Method optimization and molecular characterization. Plant Biotechnol 33:177–186. doi:10.5511/plantbiotechnology.16.0719a
Svirshchevskaya AM, Dolezel J (2000) Production and performance of gynogenetic sugarbeet lines. J Sugar Beet Res 37:117–133
Tomaszewska-Sowa M (2012) Effect of growth regulators and other components of culture medium on morphogenesis of sugar beet (Beta vulgaris L.) in unfertilised ovule in vitro cultures. Acta Agrobot 65:91–100. doi:10.5586/aa.2012.025
Van Geyt J, Speckmann GJ Jr, D’halluin K, Jacobs M (1987) In vitro induction of haploid plants from unpollinated ovules and ovaries of the sugarbeet (Beta vulgaris L.). Theor Appl Genet 73:920–925. doi:10.1007/BF00289399
Weich EW, Levall MW (2003) Doubled haploid production of sugar beet (Beta vulgaris L.). In: Maluszynski M, Kasha KJ, Forster BP, Szarejko I (eds) Doubled haploid production in crop plants. Springer, Netherlands, pp 255–263. doi:10.1007/978-94-017-1293-4_38
Yang HY, Zhou C (1982) In vitro induction of haploid plants from unpollinated ovaries and ovules. Theor Appl Genet 63:97–104. doi:10.1007/BF00303687
Acknowledgements
The financial support from TUBITAK (the Scientific and Technological Research Council of Turkey, Project No: TOVAG-113O095) is deeply appreciated. Also, the authors AP and FA are grateful for the scholarships they were provided via the same project.
Author information
Authors and Affiliations
Contributions
SG, EG, and AE designed and outlined the research. AP and FA performed the experiment, analyzed, interpreted data, and wrote the manuscript. SG, EG, and AE edited it.
Corresponding author
Ethics declarations
Conflict of interest
The authors declare that they have no conflict of interest.
Additional information
Communicated by Silvia Moreno.
Rights and permissions
About this article
Cite this article
Pazuki, A., Aflaki, F., Gürel, S. et al. Production of doubled haploids in sugar beet (Beta vulgaris): an efficient method by a multivariate experiment. Plant Cell Tiss Organ Cult 132, 85–97 (2018). https://doi.org/10.1007/s11240-017-1313-5
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11240-017-1313-5