Abstract
Tetraploid plants were produced by inducing chromosome doubling using colchicine in in vitro shoot tips of poplar and black locust clones. Many of the plants treated with colchicine showed modified morphological characteristics like stunted growth, thicker leaves and modified leaf morphology. The counting of chloroplast number in the epidermal guard cells of stomata was used for the rapid screening of tetraploids. The differences in mean chloroplast numbers between diploid and tetraploid plants were highly significant. For all plants tested, the tetraploid genotype had almost double the number of chloroplasts per guard cell compared to the diploid origin. Some plants were further analysed by flow cytometry to verify their ploidy status that was determined by chloroplast numbers. The results of this study demonstrated for the first time that chloroplast counting in poplar and black locust could be an effective and reliable method for pre-screening large numbers of plants for their ploidy level. The protocol might be applicable in a wide scope of breeding programs.
Abbreviations
- 2×:
-
Diploid
- 3×:
-
Triploid
- 4×:
-
Tetraploid
References
Ahuja MR (1987) In vitro propagation of poplar and aspen. In: Bonga JM, Durzan DJ (eds) Cell and tissue culture in forestry 3. Martinus Nijhoff, Dordrecht, pp 621–651
Beck SL, Dunlop RW, Fossey A (2003a) Stomatal length and frequency as a measure of ploidy level in black wattle, Acacia mearnsii (de Wild). Bot J Linn Soc 141:177–181
Beck SL, Fossey A, Mathura S (2003b) Ploidy determination of black wattle (Acacia mearnsii) using stomatal chloroplast counts. S Afr For J 198:79–81
Butterfass T (1973) Control of plastid division by means of nuclear DNA amount. Protoplasma 76:167–195
Compton ME, Barnett N, Gray DJ (1999) Use of fluorescein diacetate (FDA) to determine ploidy of in vitro watermelon shoots. Plant Cell Tissue Organ Cult 58:199–203
Doležel J, Greilhuber J, Suda J (2007) Flow cytometry with plants: an overview. In: Doležel J, Greilhuber J, Suda J (eds) Flow cytometry with plant cells. Analysis of genes, chromosomes and genomes. Wiley, Weinheim, pp 41–65
Eeckhaut T, Leus L, Van Huylenbroeck J (2005) Exploitation of flow cytometry for plant breeding. Acta Physiol Plant 27:743–750
Eifler I (1967) Anwendungsmöglichkeiten der Polyploidiezüchtung in der Forstwirtschaft. Arch Forstwes 16:515–528
Everitt BS (2002) Cambridge dictionary of statistics, 2nd edn. CUP. ISBN 052181099
Ganga M, Chezhiyan N, Kumar N et al (2002) Stomatal and chloroplast traits as ploidy assessment techniques for ploidy screening of in vitro induced tetraploids of banana. Phytomorphol 52:113–120
González Castañón ML, Schröder MB (2002) Rapid determination of nuclear DNA amounts and ploidy levels in germplasms of Asparagus using flow cytometry. Acta Hortic 589:193–199
Haque MD, Suzuki HA, Tsuneyoshi T (2006) An efficient novel method of producing virus free plants from garlic root meristems. 11th IAPTCB Congress. Biotechnology and sustainable agriculture 2006 and beyond. Abstracts. S. 8, pp 6
Ho I, Wan Y, Widholm JM et al (1990) The use of stomatal chloroplast number for rapid determination of ploidy level in maize. Plant Breed 105:203–210
Jacobs JP, Yoder JI (1989) Ploidy levels in transgenic tomato plants determined by chloroplast number. Plant Cell Rep 7:662–664
Johnsson H (1975) Observations on induced polyploidy in some conifers. Silvae Genet 24:2–3
Li YH, Kang XY, Wang SD et al (2008) Triploid induction in Populus alba × P. glandulosa by chromosome doubling of female gametes. Silvae Genet 57:37–40
Lloyd G, McCown B (1980) Commercially-feasible micropropagation of mountain laurel, Kalmia latifolia, by use of shoot-tip culture. Int Plant Prop Soc Proc 30:421
Mathura S, Fossey A, Beck SL (2006) Comparative study of chlorophyll content in diploid and tetraploid black wattle (Acacia mearnsii). Forestry 79:381–388
Mitchell AZ, Hanson MR, Skvirsky RC et al (1980) Anther culture of Petunia: genotypes with high frequency of callus, root, or plantlet formation. Z Pflanzenphysiol 100:131–146
Müntzing A (1936) The chromosomes of a giant Populus tremula. Hereditas 21:383–393
Nilsson-Ehle H (1936) Über eine in der Natur gefundene Gigantsform von Populus tremula. Hereditas 21:379–382
Sivolapov AI, Blagodarova TA (1997) Different levels of mixoploidy in hybrid poplars. In: Borzan Z, Schlarbaum SE (eds) Cytogenetic studies of forest trees and shrub species. Croatian forests, Inc. Zagreb, Faculty of Forstry, University of Zagreb, pp 311–316
Van Duren M, Morpurgo R, Dolezel J et al (1996) Induction and verification of autotetraploids in diploid banana (Musa acuminate) by in vitro techniques. Euphyt 88:25–36
Yang MS, Lang HY, Gao BJ et al (2003) Insecticidal activity and transgene expression stability of transgenic hybrid poplar clone 741 carrying two insect-resistant genes. Silvae Genet 52:197–201
Acknowledgments
This project was partially funded by the Agency for Renewable Resources e.V. (FNR) within the framework of the project “Breeding of fast growing trees for production of renewable resources in short rotation periods (FastWOOD). The authors are grateful to Mr. Matthias Steinberg, Partec GmbH, Münster, Germany for a part of the flow cytometry analyses. We also thank Ms Sarah Görß for counting chloroplasts.
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Ewald, D., Ulrich, K., Naujoks, G. et al. Induction of tetraploid poplar and black locust plants using colchicine: chloroplast number as an early marker for selecting polyploids in vitro. Plant Cell Tiss Organ Cult 99, 353–357 (2009). https://doi.org/10.1007/s11240-009-9601-3
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DOI: https://doi.org/10.1007/s11240-009-9601-3