Miao He¹, Wenjie Gao¹, Yaohui Gao², Yingzhu Liu¹, Xue Yang¹, Hongbin Jiao¹ and Yunwei Zhou^1
1Department of Landscape Architecture of Northeast Forestry University, Harbin, Heilongjiang, China
²Beijing Forestry University

SUMMARY Dendranthema indicum var. aromaticum from the genus Chrysanthemum is a rare species with a rich flavor. The aim of this study was to find a suitable treatment combination that would effectively induce polyploidy of diploid Dendranthema indicum var. aromaticum and to lay the foundation for the late cultivation of aromatic Chrysanthemum. In this study materials, colchicine concentration and treatment duration were examined for improving the induction of polyploidy. The combinations of three materials (shoot tips, pre-germinated seeds and grin), five colchicine concentrations (100, 200, 500, 1,000 and 2,000 mg L-1) and three treatment durations (12, 24 and 48 h) were tested in Dendranthema indicum var. aromaticum. A total of 7 tetraploids and 301 chimeras determined by chromosome number analysis were obtained. The treatment of grin seeds with 1,000 mg L-1 colchicine for 24 h (14.5%) and shoot tips with 1,000 mg L-1 colchicine for 7 d (40%) were suitable for induction of chromosome doubling. The tetraploid plants displayed much larger stomata with lower density, as well as a higher chloroplast count than the diploid paints. Moreover, tetraploid plants developed larger, thicker leaves, greater flower diameter, more epidermis hairs and shorter plant height than the diploid plants. Keywords Colchicine, Dendranthema indicum var. aromaticum, induction, identification, polyploid

Significance of this study What is already known on this subject? Dendranthema indicum var. aromaticum is characterized by a special scent, which is an important source of aroma in genus Dendranthema. Hybridization is the good way to produce new scented chrysanthemum. However, it is difficult to cross breed if one species is a diploid while the other is a higher ploidy level. Therefore, we decided to double the somatic chromosomes of D. indicum var. aromaticum, in order to increase the success of crosses between the two species. What are the new findings? In this study, the tetraploid plants displayed much larger stomata with lower density, as well as a higher chloroplast count, than the diploid plants. Moreover, tetraploid plants developed larger, thicker leaves, greater flower diameter, more epidermis hairs and shorter plant height than the diploid plants. What is the expected impact on horticulture? We had got the tetraploids of Dendranthema indicum var. aromaticum, which were important parents for scented Chrysanthemum. Later, we will cross between the tetraploid and cultivated Chrysanthemum and get the scented Chrysanthemum, which has important significance for improving the ornamental traits of horticultural plants.

E-mail: zhouyunwei1970@163.com  

References

• Ackerman, W., and Dermen, H. (1972). A fertile colchiploid from a sterile interspecific camellia hybrid. Journal of Heredity 63, 55–59.

• Allum, J.F., Bringloe, D.H., and Roberts, A.V. (2007). Chromosome doubling in a Rosa rugosa Thunb. hybrid by exposure of in vitro nodes to oryzalin: the effects of node length, oryzalin concentration and exposure time. Plant Cell Reports 26, 1977–1984. https://doi.org/10.1007/s00299-007-0411-y.

• Bartish, I.V., Korkhovoy, V.I., Fomina, Y.L., and Lim, Y.K. (1996). A new approach to obtain polyploid forms of apple. In Eucarpia Symposium on Fruit Breeding and Genetics 484, pp. 561–564.

• Beck, S.L., Dunlop, R.W., and Fossey, A. (2003). Stomatal length and frequency as a measure of ploidy level in black wattle, Acacia mearnsii (de Wild). Botanical Journal of the Linnean Society 141, 177–181. https://doi.org/10.1046/j.1095-8339.2003.00132.x.

• Bouvier, L., Guerif, P.H., Djulbic, M., Durel, C.E., Chevreau, E., and Lespinasse, Y. (2002). Chromosome doubling of pear haploid plants and homozygosity assessment using isozyme and microsatellite markers. Euphytica 123, 255–262. https://doi.org/10.1023/A:1014998019674.

• Chalak, L., and Legave, J.M. (1996). Oryzalin combined with adventitious regeneration for an efficient chromosome doubling of trihaploid kiwifruit. Plant Cell Reports 16, 97–100. https://doi.org/10.1007/BF01275459.

• Chen, F., Jiang, J., and Fang, W. (2002). Study on induction of polyploid Dendranthema nankingense with colchicine. Acta Agriculturae 18, 46–50.

• Chen, L.-p., Wang, Y.-j., and Zhao, M. (2006). In vitro induction and characterization of tetraploid Lychnis senno Siebold et Zucc. HortScience 41, 759–761.

  Duncan, D.B. (1955). Multiple range and multiple F test. Biometrics 11, 1–42. https://doi.org/10.2307/3001478.

• Du, B., Liu, Q., Zhu, C., and Ke, S. (1989). Karyotype studies of two species on Dendranthema. Journal of Wuhan Botanical Research 7, 293–296.

• Gantait, S., Mandal, N., Bhattacharyya, S., and Das, P.K. (2011). Induction and identification of tetraploids using in vitro colchicine treatment of Gerbera jamesonii Bolus cv. Sciella. Plant Cell, Tissue and Organ Culture (PCTOC) 106, 485–493. https://doi.org/10.1007/s11240-011-9947-1.

• Głowacka, K., Jeżowski, S., and Kaczmarek, Z. (2010). In vitro induction of polyploidy by colchicine treatment of shoots and preliminary characterisation of induced polyploids in two Miscanthus species. Industrial Crops and Products 32, 88–96. https://doi.org/10.1016/j.indcrop.2010.03.009.

• Kim, Y.-S., Hahn, E.-J., Murthy, H.N., and Paek, K.-Y. (2004). Effect of polyploidy induction on biomass and ginsenoside accumulations in adventitious roots of ginseng. Journal of Plant Biology 47, 356–360. https://doi.org/10.1007/BF03030551.

• Li, M., Zhang, X., and Johnson, D.E. (1991). Plant Chromosome Technology. Northeast Forestry University Publisher, pp. 48–118.

• Li, X., Chen, F., and Zhao, H. (2008). Compatibility of interspecific cross in Dendranthema genus. Acta Horticulturae Sinica 35, 257–262.

• Liu, Q., and Zhang, S. (1983). A new variety of Dendranthema gaertn from Shennongjia of Hubei. Journal of Wuhan Botanical Research 2, 237–238.

• Majdi, M., Karimzadeh, G., Malboobi, M.A., Omidbaigi, R., and Mirzaghaderi, G. (2010). Induction of tetraploidy to feverfew (Tanacetum parthenium Schulz-Bip.): Morphological, physiological, cytological, and phytochemical changes. HortScience 45, 16–21.

• Miguel, T.P., and Leonhardt, K.W. (2011). In vitro polyploid induction of orchids using oryzalin. Scientia Horticulturae 130, 314–319. https://doi.org/10.1016/j.scienta.2011.07.002.

• Mo, G.-z., Sun, M., Pan, H.-t., Zhang, Z, and Q.-x. (2010). Polyploid of Dendranthema lavandulifolium induced by colchicines. Journal of Nuclear Agricultural Sciences 24, 527–531.

• Nimura, M., Kato, J., Horaguchi, H., Mii, M., Sakai, K., and Katoh, T. (2006). Induction of fertile amphidiploids by artificial chromosome-doubling in interspecific hybrid between Dianthus caryophyllus L. and D. japonicus Thunb. Breeding Science 56, 303–310. https://doi.org/10.1270/jsbbs.56.303.

• Nilanthi, D., Chen, X.-L., Zhao, F.-C., Yang, Y.-S., and Wu, H. (2009). Induction of tetraploids from petiole explants through colchicine treatments in Echinacea purpurea L. BioMed Research International 2009, 1–7.

• Oliveira, V.M. d., Forni-Martins, E.R., Magalhães, P.M., and Alves, M.N. (2004). Chromosomal and morphological studies of diploid and polyploid cytotypes of Stevia rebaudiana (Bertoni) Bertoni (Eupatorieae, Asteraceae). Genetics and Molecular Biology 27, 215–222. https://doi.org/10.1590/S1415-47572004000200015.

• Petersen, K.K., Hagberg, P., and Kristiansen, K. (2003). Colchicine and oryzalin mediated chromosome doubling in different genotypes of Miscanthus sinensis. Plant Cell, Tissue and Organ Culture 73, 137–146. https://doi.org/10.1023/A:1022854303371.

• Praça, M.M., Carvalho, C.R., and Clarindo, W.R. (2009). A practical and reliable procedure for in vitro induction of tetraploid tomato. Scientia Horticulturae 122, 501–505. https://doi.org/10.1016/j.scienta.2009.05.032.

• Rhee, H.K., Cho, H.R., Kim, K.J., and Kim, K.S. (2004). Comparison of pollen morphology in interspecific hybrid lilies after in vitro chromosome doubling. In IX International Symposium on Flower Bulbs 673, 639–643.

• Roy, A., Leggett, G., and Koutoulis, A. (2001). In vitro tetraploid induction and generation of tetraploids from mixoploids in hop (Humulus lupulus L.). Plant Cell Reports 20, 489–495. https://doi.org/10.1007/s002990100364.

• Schepper, S. d., Leus, L., Mertens, M., Debergh, P., Bockstaele, E. v., and Loose, M. d. (2001). Somatic polyploidy and its consequences for flower coloration and flower morphology in azalea. Plant Cell Reports 20, 583–590. https://doi.org/10.1007/s002990100372.

• Schifino, M.T., and Fernandes, M.I.M. (1987). Induction of polyploidy and cytological characterization of autotetraploids of Trifolium riograndense Burkart (Leguminosae). Euphytica 36, 863–872. https://doi.org/10.1007/BF00051871.

• Shao, J., Chen, C., and Deng, X. (2003). In vitro induction of tetraploid in pomegranate (Punica granatum). Plant Cell, Tissue and Organ Culture 75, 241–246. https://doi.org/10.1023/A:1025871810813.

• Song, P., Kang, W., and Peffley, E.B. (1997). Chromosome doubling of Allium fistulosum × A. cepa interspecific F₁ hybrids through colchicine treatment of regenerating callus. Euphytica 93, 257–262. https://doi.org/10.1023/A:1002957800957.

• Stanys, V., Weckman, A., Staniene, G., and Duchovskis, P. (2006). In vitro induction of polyploidy in Japanese quince (Chaenomeles japonica). Plant Cell, Tissue and Organ Culture 84, 263–268. https://doi.org/10.1007/s11240-005-9029-3.

• Tao, D., Li, X., Wang, L., Zhou, J., Chen, Y., and Huo, W. (2009). Progresses on determination of cell chromosome ploidy level of plants. Life Science Research 13, 453–458.

• Tel-Zur, N., Dudai, M., Raveh, E., and Mizrahi, Y. (2011). In situ induction of chromosome doubling in vine cacti (Cactaceae). Scientia Horticulturae 129, 570–576. https://doi.org/10.1016/j.scienta.2011.04.027.

• Thao, N.T.P., Ureshino, K., Miyajima, I., Ozaki, Y., and Okubo, H. (2003). Induction of tetraploids in ornamental Alocaias through colchicine and oryzalin treatments. Plant Cell, Tissue and Organ Culture 72, 19–25. https://doi.org/10.1023/A:1021292928295.

• Urwin, N.A., Horsnell, J., and Moon, T. (2007). Generation and characterisation of colchicine-induced autotetraploid Lavandula angustifolia. Euphytica 156, 257–266. https://doi.org/10.1007/s10681-007-9373-y.

• Wan, Y., Petolino, J., and Widholm, J. (1989). Efficient production of doubled haploid plants through colchicine treatment of anther-derived maize callus. Theoretical and applied genetics 77, 889–892. https://doi.org/10.1007/BF00268344.

• Yang, X., Cao, Z., An, L., Wang, Y., and Fang, X. (2006). In vitro tetraploid induction via colchicine treatment from diploid somatic embryos in grapevine (Vitis vinifera L.). Euphytica 152, 217–224. https://doi.org/10.1007/s10681-006-9203-7.

• Ye, Y., Tong, J., Shi, X., Yuan, W., and Li, G. (2010). Morphological and cytological studies of diploid and colchicine-induced tetraploid lines of crape myrtle (Lagerstroemia indica L.). Scientia Horticulturae 124, 95–101. https://doi.org/10.1016/j.scienta.2009.12.016.

Received: 22 March 2016 | Accepted: 1 July 2016 | Published: 29 August 2016 | Available online: 29 August 2016