Plant Cell, Tissue and Organ Culture (PCTOC)

, Volume 135, Issue 3, pp 395–406 | Cite as

Induction and identification of tetraploid Hedychium coronarium through thin cell layer culture

  • Hong-Yan Tu
  • Ai-Ling Zhang
  • Wang XiaoEmail author
  • Ya-Rou Lin
  • Jun-Hui Shi
  • Yong-Wei Wu
  • Si-Tong Wu
  • Chun-Hui Zhong
  • Shui-Xiu Mo
Original Article


To shorten breeding progress of Hedychium coronarium, application of colchicine to thin cell layer (TCL) was carried out to induce tetraploid plants. The shoots from rhizomes were cultured on induction medium: Murashige and Skoog (MS) medium, 0.2 mg/L indole-3 acetic acid (IAA), and 3 mg/L 6-benzyladenine (BA). The obtained adventitious buds were transferred to rooting medium containing 1/2 MS medium, and 1 g/L activated charcoal for plantlets development. TCLs excised from the stem base of the plantlets were cultured on induction medium; average 9.7 buds were obtained from one plantlet after 4 weeks culture and generated into plantlets on rooting medium. TCLs were treated with three concentrations of colchicine for 24 h and cultured with the above TCL culture system. Eight tetraploid plants and two ones were derived from 0.4% and 0.2% colchicine treatments, respectively. The chromosome number of tetraploids was 2n = 4x = 68 and that of diploids was 2n = 2x = 34. The width and length of leaf, plant height and net growth of rhizome of tetraploids were higher than those of diploids. Leaf angles of tetraploids and diploids exhibited significant difference at different positions. The stomata size of the tetraploids was significantly larger than that of diploids. The tetraploids developed larger flower and pollen grain. Prolonged flower longevity was observed with 56% increase compared with diploids. All this showed that the TCL culture system was suitable for induction of tetraploid Hedychium coronarium, and the tetraploids exhibited desirable field characters and longer flower longevity, all contributing to higher ornamental value of Hedychium coronarium.


Hedychium coronarium Thin cell layer culture Plant regeneration system Tetraploid Flower longevity 



This research was supported by Higher education Innovation and Excellence project of Guangdong province (2014KZDXM076), Science and Technology Program of Guangdong province (2015A030302097), Science and Technology Program of Guangzhou (201804010413), and “Climbing Program” for college students of Guangdong province (pdjh2017b0370, pdjh2018a0368).

Author contributions

WX applied for the funds and designed the whole work. WX, H-YT, and A-LZ induced the tetraploid Hedychium coronarium, analyzed the experimental data and wrote the paper. H-YT, Y-RL, and C-HZ measured the field characters (morphological observations for plants) and evaluation of flower characteristics of the plants. S-XM and J-HS carried out the stomatal measurement and chloroplast count. A-LZ, Y-WW and S-TW carried out the flow cytometric analysis and chromosome counts.

Compliance with ethical standards

Conflict of interest

All the authors statement that they agree with the submission of the paper to the journal Plant Cell, Tissue and Organ Culture (PCTOC), and they have no conflict of interest.

Ethical approval

The manuscript complies with the Ethical Rules applicable for Plant Cell Tissue and Organ Culture.

Human and animal rights

This article does not contain any studies with human participants or animals performed by any of the authors.


  1. Allario T, Brumos J, Colmenero-Flores JM, Tadeo F, Froelicher Y, Talon M, Navarro L, Ollitrault P, Morillon R (2011) Large changes in anatomy and physiology between diploid Rangpur lime (Citrus limonia) and its autotetraploid are not associated with large changes in leaf gene expression. J Exp Bot 62:2507–2519CrossRefGoogle Scholar
  2. Aversano R, Caruso I, Aronne G, De Micco V, Scognamiglio N, Carputo D (2013) Stochastic changes affect Solanum wild species following autopolyploidization. J Exp Bot 64:625–635CrossRefGoogle Scholar
  3. Betsabe M, Maria GMP, Fernandez S (2017) Differential manipulation of leaf angle throughout the canopy: current status and prospects. J Exp Bot 68:5699–5717CrossRefGoogle Scholar
  4. Chaturvedi R, Razdan MK, Bhojwani SS (2003) An efficient protocol for the production of triploid plants from endosperm callus of neem, Azadirachta indica A. Juss. J Plant Physiol 160:557–564CrossRefGoogle Scholar
  5. Chen JJ, Ting CW, Wu YC, Hwang TL, Cheng MJ, Sung PJ, Wang TC, Chen JF (2013) New labdane-type diterpenoids and anti-Inflammatory constituents from Hedychium coronarium. Int J Mol Sci 14:13063–13077CrossRefGoogle Scholar
  6. Dobránszki J, Teixeira da Silva JA (2013) In vitro shoot regeneration from transverse thin cell layers of apple leaves in response to various factors. J Hortic Sci Biotechnol 88:60–66CrossRefGoogle Scholar
  7. Galbraith DW, Harkins KR, Maddox JM (1983) Rapid flow cytometric analysis of the cell cycle in intact plant tissues. Science 4601:1049–1051CrossRefGoogle Scholar
  8. Gantait S, Mandal N, Bhattacharyya S, Kanti Das P (2011) Induction and identification of tetraploids using in vitro colchicine treatment of Gerbera jamesonii Bolus cv. Sciella. Plant Cell Tissue Organ Cult 106:485–493CrossRefGoogle Scholar
  9. Guedes RS, Silva TL, Luis ZG, Scherwinski-Pereira JE (2011) Initial requirements for embryogenic calluses initiation in thin cell layers explants from immature female oil palm inflorescences. Afr J Biotechnol 10:10774–10780CrossRefGoogle Scholar
  10. He LY, Ding ZS, Jiang FS, Jin B, Li WP, Ding XH, Sun JY, Lv GY (2012) Induction and identification of hexadecaploid of Pinellia ternata. Euphytica 186:479–488CrossRefGoogle Scholar
  11. Jaiphet C, Rangsayatorn N (2010) Micropropagation of a rare orchid Dendrobium gratiosissimum using thin cell layers. Acta Hortic 878:185–189CrossRefGoogle Scholar
  12. Kiem PV, Anh HT, Nhiem NX, Minh CV, Thuy NT, Yen PH, Hang DT, Tai BH, Mathema VB, Koh YS, Kim YH (2012) Labdane-type diterpenoids from the rhizomes of Hedychium coronarium inhibit lipopolysaccharide-stimulated production of pro-inflammatory cytokines in bone. Chem Pharm Bull 60:246–250CrossRefGoogle Scholar
  13. Kong DX, Li YQ, Bai M, Deng YL, Liang GX, Wu H (2017) A comparative study of the dynamic accumulation of polyphenol components and the changes in their antioxidant activities in diploid and tetraploid Lonicera japonica. Plant Physiol Biochem 112:87–96CrossRefGoogle Scholar
  14. Li PF, Cheng ZG, Ma BL, Palta JA, Kong HY, Mo F, Wang JY, Zhu Y, Lv GC, Batool A, Bai X, Li FM, Xiong Y (2014) Dryland wheat domestication changed the development of aboveground architecture for a well-structured canopy. PLoS ONE 9:e95825CrossRefGoogle Scholar
  15. Liu Z, Gao SL (2007) Micropropagation and induction of autotetraploid plants of Chrysanthemum cinerariifolium (Trev.) Vis. In Vitro Cell Dev Biol Plant 43(5):404–408CrossRefGoogle Scholar
  16. Ma DL, Xie RZ, Niu XK, Li SK, Long HL, Liu YE (2014) Changes in the morphological traits of maize genotypes in China between the 1950s and 2000s. Eur J Agron 58:1–10CrossRefGoogle Scholar
  17. Mohanty P, Behera S, Swain SS, Barik PD, Naik SK (2013) Micropropagation of Hedychium coronarium J. Koenig through rhizome bud. Physiol Mol Biol Plants 4:605–610CrossRefGoogle Scholar
  18. Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15(3):473–497CrossRefGoogle Scholar
  19. Sari N, Abak K, Pitrat M (1999) Comparison of ploidy level screening methods in watermelon: Citrullus lanatus (Thunb.) Matsum. and Nakai. Sci Hortic 82:265–277CrossRefGoogle Scholar
  20. Shahid MQ, Chen FY, Li HY, Wang SZ, Chen PF, Lin SQ, Liu XD, Lu YG (2013) Double-neutral genes Sa n and Sb n for pollen fertility in rice to overcome indica × japonica hybrid sterility. Crop Sci 1:164–176CrossRefGoogle Scholar
  21. Takarnura T, Miyajima I (1996) Colchicine induced tetraploids in yellow-flowered cyclamens and their characteristics. Sci Hortic 65:305–312CrossRefGoogle Scholar
  22. Tang ZQ, Chen DL, Song ZJ, He YC, Cai DT (2010) In vitro induction and identification of tetraploid plants of Paulownia tomentosa. Plant Cell Tissue Organ Cult 102:213–220CrossRefGoogle Scholar
  23. Teixeira da Silva JA (2013) The role of thin cell layers in regeneration and transformation in orchids. Plant Cell Tissue Organ Cult 113:149–161CrossRefGoogle Scholar
  24. Thomas TD, Bhatnagar AK, Bhojwani SS (2000) Production of triploid plants mulberry (Morus alba L.) by endosperm culture. Plant Cell Rep 19:395–399CrossRefGoogle Scholar
  25. Thong-on W, Arimatsu P, Pitiporn S, Soonthornchareonnon N, Prathanturarug S (2014) Field evaluation of in vitro-induced tetraploid and diploid Centella asiatica (L.) Urban. J Nat Med 68:267–273CrossRefGoogle Scholar
  26. Tu HY, Xiao W, Xie J, Wei YX (2015) High frequency plant regeneration system by thin cell layer culture in Curcuma soloensis Valeton. J South China Norm Univ 47(4):98–102Google Scholar
  27. Van Laere K, Franca SC, Vansteenkiste H, Van Huylenbroeck J, Steppe K, Van Labeke MC (2011) Influence of ploidy level on morphology, growth and drought susceptibility in Spathiphyllum wallisii. Acta Physiol Plant 33:1149–1156CrossRefGoogle Scholar
  28. Xiao W, Tu HY, Zhang AL (2016) Embryogenic callus induction and plant regeneration from Hedychium coronarium via somatic embryogenesis. Acta Hortic Sin 43(8):1605–1612Google Scholar
  29. Xiong YC, Li FM, Zhang T (2006) Performance of wheat crops with different chromosome ploidy: root-sourced signals, drought tolerance, and yield performance. Planta 224:710–718CrossRefGoogle Scholar
  30. Yue YC, Yu RC, Fan YP (2014) Characterization of two monoterpene synthases involved in floral scent formation in Hedychium coronarium. Planta 240:745–762CrossRefGoogle Scholar
  31. Zhang QY, Luo FX, Liu L, Guo FC (2010) In vitro induction of tetraploids in crape myrtle (Lagerstroemia indica L.). Plant Cell Tissue Organ Cult 101:41–47CrossRefGoogle Scholar

Copyright information

© Springer Nature B.V. 2018

Authors and Affiliations

  1. 1.College of Biology and Food Engineering/Development Center of Applied Ecology and Ecological Engineering in UniversitiesGuangdong University of EducationGuangzhouPeople’s Republic of China

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