Abstract
Given the increased demand for wood producing species with fast growth, chromosome set doubling (CSD) is an option to enrich germplasm banks and breeding programs with synthetic polyploids in relatively short time. Here, we aimed to establish a feasible practice of an in vitro procedure to create solid polyploid Eucalyptus, and to verify the DNA ploidy level fidelity in newly synthetized polyploids. We treated 1.0 and 1.5 cm shoot tips of Eucalyptus grandis with colchicine in distinct times and concentrations and achieved between 0 and 66.67% autotetraploids, totaling 87 plantlets from all CSD treatments. However, a reversion to diploidy and/or mixoploidy was observed in 35.5% and 22.0% of total plantlets in the first and second ploidy level assessments, respectively. Based on the results, a second CSD assay was performed for 1.5 cm shoot tips of Eucalyptus grandis, Eucalyptus urophylla, Eucalyptus benthamii and E. urophylla × E. grandis, which we treated with 1.5 mM colchicine for 36 h. Auto- and allotetraploidy rates ranged from 40.8 to 65.5%, and again DNA ploidy level changes were detected in in vitro and ex vitro individuals. In conclusion, fine adjustments of the antitubulin concentration and exposure time are needed for each explant and genotype/species to CSD from apical meristem stem cells. Since ploidy stability was found to be a dramatic problem in Eucalyptus CSD, we promote recurrent DNA ploidy level screening to achieve solid auto- and allotetraploids propagation and to identify undesired mixoploids and diploids in in vitro and ex vitro conditions.
Key message
Eucalyptus auto- and allotetraploids were obtained, enriching the germplasm banks. Looking for DNA ploidy level, changes were identified in some synthetic tetraploids, evidencing the ploidy instability as a dramatic problem.
References
Bhalerao R, Nilsson O, Sandberg G (2003) Out of the woods: forest biotechnology enters the genomic era. Curr Opin Biotechnol 14:206–2013. https://doi.org/10.1016/S0958-1669(03)00029-6
Blakeslee AF, Avery AG (1937) Methods of inducing doubling of chromosomes in plants by treatment with colchicine. J Hered 28:393–411
Blakesley D, Allen A, Pellny TK, Roberts AV (2002) Natural and induced polyploidy in Acacia dealbata Link. and Acacia mangium Willd. Ann Bot 90:391–398. https://doi.org/10.1093/aob/mcf202
Boerjan W (2005) Biotechnology and the domestication of forest trees. Curr Opin Biotechnol 16:159–166. https://doi.org/10.1016/j.copbio.2005.03.003
Brondani GE, Grossi F, Wendling I, Dutra LF, Araujo MA (2010) IBA application for rooting of Eucalyptus benthamii Maiden and Cambage x Eucalyptus dunii Maiden minicuttings. Acta Scientiarum 2:667–674. https://doi.org/10.4025/actasciagron.v32i4.4879
Brondani GB, Ondas HWW, Baccarin FJB, Gonçalves AN, Almeida M (2012) Micropropagation of Eucalyptus benthamii to form a clonal micro-garden. In vitro Cell Dev Biol 48:478–487. https://doi.org/10.1007/s11627-012-9449-9
Carvalho GMA, Carvalho CR (2016) The eucalypt karyogram resolved. Botany 94:411–416. https://doi.org/10.1139/cjb-2015-0221
Clarindo WR, Carvalho CR, Araújo FS, Abreu IS, Otoni WC (2008) Recovering polyploid papaya in vitro regenerants as screened by flow cytometry. Plant Cell Tiss Organ Cult 92:207–214. https://doi.org/10.1007/s11240-007-9325-1
Dhooghe E, Van Laere K, Eeckhaut T, Leus L, Van Huylenbroeck J (2011) Mitotic chromosome doubling of plant tissues in vitro. Plant Cell Tiss Organ 104:359–373. https://doi.org/10.1007/s11240-010-9786-5
Doughty RW (2000) The eucalyptus: a natural and commercial history of the gum tree. The Johns Hopkins University Press, Baltimore
Galbraith DW, Harkins KR, Maddox JR, Ayres NM, Sharma DP, Firrozabady E (1983) Rapid flow cytometric analysis of the cell cycle in intact plant tissues. Science 220:1049–1051. https://doi.org/10.1126/science.220.4601.1049
Grattapaglia D, Kirst M (2008) Eucalyptus applied genomics: from gene sequences to breeding tools. New Phytol 179:911–929. https://doi.org/10.1111/j.1469-8137.2008.02503.x
Gu XF, Yang AF, Meng H, Zhang JR (2005) In vitro induction of tetraploid plants from diploid Zizyphus jujube Mill, Cv. Zhanhua. Plant Cell Rep 24:671–676. https://doi.org/10.1007/s00299-005-0017-1
Han C et al (2011) Polyploidy induction of clone of Eucalyptus grandis with colchicine. Afri J Biotechnol 10:14711–14717. https://doi.org/10.5897/AJB11.093
Harbard JL, Griffin AR, Foster S, Brooker C, Kha LD, Koutoulis A (2012) Production of colchicine-induced autotetraploids as a basis for sterility breeding in Acacia mangium Willd. Forestry 85:427–436. https://doi.org/10.1093/forestry/cps041
Kour G, Kour B, Kaul S, Dhar MK (2009) Genetic and epigenetic instability of amplification-prone sequences of a novel B chromosome induced by tissue culture in Plantago lagopus L. Plant Cell Rep 28:1857–1867. https://doi.org/10.1007/s00299-009-0789-9
Ladiges PY, Udovicic F, Nelson G (2003) Australian biogeographical connections and the phylogeny of large genera in the plant family Myrtaceae. J Biogeograph 30:989–998. https://doi.org/10.1046/j.1365-2699.2003.00881.x
Lin H, Jian M, Liang LY, Pei WJ, Liu XZ, Zhang HY (2010) Production of polyploids from cultured shoot tips of Eucalyptus globulus Labill by treatment with colchicine. Afr J Biotecnol 9:2252–2255
Liu G, Li Z, Bao M (2007) Colchicine-induced chromosome doubling in Platanus acerifolia and its effect on plant morphology. Euphytica 157:145–154. https://doi.org/10.1007/s10681-007-9406-6
Mankessi F, Saya A, Montes F, Lartaud M, Verdeil JL, Monteuuis O (2011) Histocytological characteristics of Eucalyptus urophylla X Eucalyptus grandis shoot apical meristems of different physiological ages. Trees 25:415–424. https://doi.org/10.1007/s00468-010-0516-8
Ochatt SJ, Patat-Ochatt EM, Moessner A (2011) Ploidy level determination within the context of in vitro breeding. Plant Cell Tiss Organ Cult 104(329–34173):581–598. https://doi.org/10.1007/s11240-011-9918-6
Otto FJ (1990) DAPI staining of fixed cells for high-resolution flow cytometry of nuclear DNA. Methods in cell biology, vol 33. Academic Press, San Diego, pp 105–110
Paul LK, Rinne PLH, van der Schoot C (2014) Shoot meristems of deciduous woody perennials: self-organization and morphogenetic transitions. Curr Opin Plant Biol 17:86–95. https://doi.org/10.1016/j.pbi.2013.11.009
Pinto G, Araújo C, Santos C, Neves L (2013) Plant regeneration and somatic embryogenesis in Eucalyptus spp.: current status and future perspectives. South For 75:59–69. https://doi.org/10.2989/20702620.2013.785115
SAEG (2007) Sistema para Análises Estatísticas, Versão 9.1: Fundação Arthur Bernardes—UFV—Viçosa
Sanglard NA et al (2017) From chromosome doubling to DNA sequence changes: outcomes of an improved in vitro procedure for allotriploid “Híbrido de Timor” (Coffea arabica x Coffea canephora Pierre ex A. Froehner). Plant Cell Tiss Organ Cult 131:223–231. https://doi.org/10.1007/s11240-017-1278-4
Sattler MC, Carvalho CR, Clarindo WR (2016) The polyploidy and its key role in plant breeding. Planta 243:281–296. https://doi.org/10.1007/s00425-015-2450-x
Stebbins GL (1947) Types of polyploids: their classification and significance. Adv Genet 1:403–429
Ulrich K, Ewald D (2014) Breeding triploid aspen and poplar clones for biomass production. Silvae Genetica 63:47–58. https://doi.org/10.1515/sg-2014-0008
Yemets AI, Blume YAB (2008) Progress in plant polyploidization based on antimicrotubular drugs. Open Hortic J 1:15–20. https://doi.org/10.2174/1874840600801010015%5d
Acknowledgements
We would like to thank the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, Brasília – DF, Brazil) and Fundação de Amparo à Pesquisa do Espírito Santo (FAPES, Vitória – ES, Brazil) for financial support. This study was financid in part by the Coordernação de Aperfeiçoamento de Pessoal de Nível Superior – Brazil (CAPES) – Finance Code 001. In addition, we would like to thank the Dr. Paulo Roberto Cecon for colaboration in the statistical analysis.
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Communicated by Maria Antonietta Germanà.
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Silva, A.J., Carvalho, C.R. & Clarindo, W.R. Chromosome set doubling and ploidy stability in synthetic auto- and allotetraploid of Eucalyptus: from in vitro condition to the field. Plant Cell Tiss Organ Cult 138, 387–394 (2019). https://doi.org/10.1007/s11240-019-01627-1
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DOI: https://doi.org/10.1007/s11240-019-01627-1