Autopolyploid induction via somatic embryogenesis in Lilium distichum Nakai and Lilium cernuum Komar

  • Linlan Fu
  • Yingying Zhu
  • Min Li
  • Chunxia Wang
  • Hongmei SunEmail author
Original Article


New ornamental varieties of high quality can be created via artificial polyploid induction. In the present study, the first system of polyploid induction with somatic embryogenesis of Lilium distichum Nakai and Lilium cernuum Komar. was developed. Somatic embryos were cultured on MS with 0.41 μmol L−1 picloram and 1.07 μmol L−1 NAA by scales (5 mm2). After 40 days, somatic embryos were transferred to MS with 2.21 μmol L−1 BA for somatic embryogenesis. As determined from observations of paraffin sections, embryonic cells of L. distichum originated from outer cells at first, and somatic embryogenesis occurred through an indirect pathway. In L. cernuum, embryonic cells originated from inner cells at first, and somatic embryogenesis occurred through a direct pathway. Polyploids were successfully formed from somatic embryos and scales by the soaking and mixed culture methods with different colchicine concentrations (0.01%, 0.05%, and 0.1%; v/v) and durations (24, 48, and 72 h). The polyploid induction rate reached 57.14% and 46.15% with 0.05% colchicine treatment in L. distichum (48 h) and L. cernuum (24 h), respectively. Tetraploids (28.57% and 23.08%) and aneuploids without chimeras among the obtained polyploid plantlets were identified by chromosome counts of root-tip tissue squashes in L. distichum and L. cernuum. Tetraploid plantlets of L. distichum exhibited broader leaves, longer guard cells, larger stomata and higher stomatal conductance than diploid plantlets. Tetraploid plantlets of L. cernuum showed 1.76 × higher chlorophyll content, significantly more leaves, longer guard cells, larger stomata and lower stomatal conductance than diploid plantlets.

Key message

For the first time, we established a somatic embryogenesis system for L. distichum and L. cernuum using somatic embryos and scales to induce polyploids by soaking and mixed culture. Combinations of colchicine concentrations and periods of time were compared to select the best treatment combination. Comprehensive morphological observations, stomatal observations and root-tip tissue squashes were used to identify the ploidy of doubling plants. Our results provide a foundation for improving the ornamental value of two wild lily species, creating new Lilium germplasm and improving the reproduction coefficient of these resources.


Lilium distichum Nakai Lilium cernuum Komar Somatic embryogenesis Autopolyploid induction 



2,4-Dichlorophenoxyacetic acid




Naphthylacetic acid


Murashige and Skoog medium


4-Amino-3,5,6-trichloro-2-pyridinecarboxylic acid



This work was supported by the National Natural Science Foundation of China (grant nos. 31672179, 31872150 and 31471897) and the National Key Research and Development Program of China (2018YFD1000407).

Author contributions

HS and LF conceived and designed the experiments. LF, YZ and ML performed the experiments and analyzed the data. CW provided help with the experimental methods and participated in discussions. HS and LF wrote and revised the paper. All authors read and approved the final manuscript.

Compliance with ethical standards

Conflict of interest

The authors declare that this research was conducted in the absence of any commercial or financial relationships that could be construed as potential conflicts of interest.

Supplementary material

11240_2019_1671_MOESM1_ESM.docx (18 kb)
Supplementary material 1 (DOCX 17 kb)
11240_2019_1671_MOESM2_ESM.docx (16 kb)
Supplementary material 2 (DOCX 16 kb)
11240_2019_1671_MOESM3_ESM.docx (16 kb)
Supplementary material 3 (DOCX 16 kb)
11240_2019_1671_MOESM4_ESM.docx (16 kb)
Supplementary material 4 (DOCX 16 kb)
11240_2019_1671_MOESM5_ESM.docx (18 kb)
Supplementary material 5 (DOCX 18 kb)
11240_2019_1671_MOESM6_ESM.docx (18 kb)
Supplementary material 6 (DOCX 18 kb)


  1. Acanda Y, Martínez Ó, González MV, Prado MJ, Rey M (2015) Highly efficient in vitro tetraploid plant production via colchicine treatment using embryogenic suspension cultures in grapevine (Vitis vinifera cv. Mencía). Plant Cell Tissue Organ Cult 123:547–555. CrossRefGoogle Scholar
  2. Adams KL, Wendel JF (2005) Novel patterns of gene expression in polyploid plants. Trends Genet 21:539–543CrossRefGoogle Scholar
  3. Baker RL, Yarkhunova Y, Vidal K, Ewers BE, Weinig C (2017) Polyploidy and the relationship between leaf structure and function: implications for correlated evolution of anatomy, morphology, and physiology in Brassica. BMC Plant Biol. Google Scholar
  4. Cao Q, Zhang X, Gao X, Wang L, Jia G (2018) Effects of ploidy level on the cellular, photochemical and photosynthetic characteristics in Lilium FO hybrids. Plant Physiol Biochem 133:50–56. CrossRefGoogle Scholar
  5. Chen XL, Li JH, Xin X, Zhang ZE, Lu XX (2011) Cryopreservation of in vitro-grown apical meristems of Lilium by droplet-vitrification. South Afr J Bot 77:397–403CrossRefGoogle Scholar
  6. Chen R, Jiang WZ, Li Q, Li X, Chen X, Yang YS, Wu H (2015) Comparison of seven colchicine-induced tetraploid clones with their original diploid clones in purple coneflower (Echinacea purpurea L.). Euphytica 207:387–399. CrossRefGoogle Scholar
  7. Chung MY, Chung JD, Ramanna M, Van Tuyl JM, Lim KB (2013) Production of polyploids and unreduced gametes in Lilium auratum × L. henryi hybrid. Int J Biol Sci 9:693–701. CrossRefGoogle Scholar
  8. Chung MY, López-Pujol J, Chung JM, Kim KJ, Park SJ, Chung MG (2015) Polyploidy in Lilium lancifolium: evidence of autotriploidy and no niche divergence between diploid and triploid cytotypes in their native ranges. Flora 213:57–68. CrossRefGoogle Scholar
  9. Denaeghel HER, Van Laere K, Leus L, Lootens P, Van Huylenbroeck J, Van Labeke MC (2018) The variable effect of polyploidization on the phenotype in Escallonia. Front Plant Sci 9:354. CrossRefGoogle Scholar
  10. Dudits D et al (2016) Response of organ structure and physiology to autotetraploidization in early development of energy Willow Salix viminalis. Plant Physiol 170:1504–1523. CrossRefGoogle Scholar
  11. Ghanbari MA, Jowkar A, Salehi H, Zarei M (2019) Effects of polyploidization on petal characteristics and optical properties of Impatiens walleriana (Hook.). Plant Cell Tissue Organ Cult. Google Scholar
  12. He YH, Fang SQ, Ma J, Hu ZY, Lu M, Peng B (2010) Histocytology observation on the somatic embryogenesis in Ananas comosus callus. Acta Hortic Sinica 37:689–696Google Scholar
  13. He M, Gao W, Gao Y, Liu Y, Yang X, Jiao H, Y (2016) Polyploidy induced by colchicine in Dendranthema indicum var. aromaticum, a scented chrysanthemum. Eur J Hortic Sci 81:219–226. CrossRefGoogle Scholar
  14. Hussain M et al (2016) Plantlets regeneration via somatic embryogenesis from the nucellus tissues of Kinnow Mandarin (Citrus reticulatat L.). Am J Plant Sci 07:798–805. CrossRefGoogle Scholar
  15. Huy NP, Tam DTT, Luan VQ, Tung HT, Hien VT, Ngan HTM, Duy PN, Nhut DT (2019) In vitro polyploid induction of Paphiopedilum villosum using colchicine. Sci Hortic 252:283–290. CrossRefGoogle Scholar
  16. Khorzoghi GE, Jahanbakhshian-Davaran Z, Seyedi SM (2019) Direct somatic embryogenesis of drought resistance pistachio (Pistacia vera L.) and expression analysis of somatic embryogenesis-related genes. S Afr J Bot 121:558–567. CrossRefGoogle Scholar
  17. Lei JJ, Pan LL (2009) Bulb scale culture in vitro of Lilium distichum Nakai. J Shenyang Agric Univ 40(4):532–535Google Scholar
  18. Liu Y, Yang LP (2015) Polyploid induction of Lilium amabile in vitro. J Agric Univ Hebei 38(3):30–33Google Scholar
  19. Liu WL, Wu LF, Wu HZ, Zheng SX, Wang JH, Liu FH (2011) Correlation of saponin content and Fusarium resistance in hybrids from different ploidy levels of Lilium Oriental. Sci Hortic 129:849–853. CrossRefGoogle Scholar
  20. Luo Z, Iaffaldano BJ, Cornish K (2018) Colchicine-induced polyploidy has the potential to improve rubber yield in Taraxacum kok-saghyz. Ind Crop Prod 112:75–81. CrossRefGoogle Scholar
  21. Mandak B, Krak K, Vit P, Lomonosova MN, Belyayev A, Habibi F, Wang L, Douda J, Storchova H (2018) Hybridization and polyploidization within the Chenopodium album aggregate analysed by means of cytological and molecular markers. Mol Phylogenet Evol 129:189–201. CrossRefGoogle Scholar
  22. Murashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant 15:473–497. CrossRefGoogle Scholar
  23. Okazaki K, Hane Y (2005) Comparison of diploid and chimeric forms of Asiatic hybrid lilies (Lilium spp.) under natural and early forcing culture. NZ J Crop Hortic Sci 33:261–267CrossRefGoogle Scholar
  24. Otto SP (2007) The evolutionary consequences of polyploidy. Cell 131:452–462. CrossRefGoogle Scholar
  25. Otto SP, Whitton J (2000) Polyploid incidence and evolution. Ann Rev Genet 34:401–437. CrossRefGoogle Scholar
  26. Ouyang Y, Chen Y, Lu J, Teixeira da Silva JA, Zhang X, Ma G (2016) Somatic embryogenesis and enhanced shoot organogenesis in Metabriggsia ovalifolia WT Wang. Sci Rep 6:24662. CrossRefGoogle Scholar
  27. Pollo FE, Grenat PR, Otero MA, Babini S, Salas NE, Martino AL (2019) Evaluation in situ of genotoxic and cytotoxic response in the diploid/polyploid complex Odontophrynus (Anura: Odontophrynidae) inhabiting agroecosystems. Chemosphere 216:306–312. CrossRefGoogle Scholar
  28. Predieri S (2001) Mutation induction and tissue culture in improving fruits. Plant Cell Tissue Organ Cult 64:185–210. CrossRefGoogle Scholar
  29. Raji MR, Lotfi M, Tohidfar M, Zahedi B, Carra A, Abbate L, Carimi F (2018) Somatic embryogenesis of muskmelon (Cucumis melo L.) and genetic stability assessment of regenerants using flow cytometry and ISSR markers. Protoplasma 255:873–883. CrossRefGoogle Scholar
  30. Regalado JJ, Carmona ME, Querol V, Velez CG, Encina CL, Pitta-Alvarez SI (2017) Production of compact petunias through polyploidization. Plant Cell Tissue Org Cult 129:61–71. CrossRefGoogle Scholar
  31. Sadat Noori SA, Norouzi M, Karimzadeh G, Shirkool K, Niazian M (2017) Effect of colchicine-induced polyploidy on morphological characteristics and essential oil composition of ajowan (Trachyspermum ammi L.). Plant Cell Tissue Organ Cult 130:543–551. CrossRefGoogle Scholar
  32. Sandra N, Marum L, Farinla N, Pereira VT, Almeida T, Sousan D, Mano N, Figueiredo J, Dias MC, Santos C (2017) Somatic embryogenesis of hybrid Pinus elliottii var. elliottii × P. caribaea var. hondurensis and ploidy assessment of somatic plants. Plant Cell Tissue Organ Cult 132:74–81. Google Scholar
  33. Shi QH, Liu P, Liu MJ, Wang J-R, Xu J (2015) A novel method for rapid in vivo induction of homogeneous polyploids via calluses in a woody fruit tree (Ziziphus jujuba Mill.). Plant Cell Tissue Organ Cult 121:423–433. CrossRefGoogle Scholar
  34. Snezana ZK, Tubic L, Devrnja N, Calic D, Milojevic J, Milic M, Savic J (2019) Somatic embryogenesis from stamen filaments of Aesculus flava Sol. and peroxidase activity during the transition from friable to embryogenic callus. Sci Hortic 247:362–372. CrossRefGoogle Scholar
  35. Soltis PS, Soltis DE (2016) Ancient WGD events as drivers of key innovations in angiosperms. Curr Opin Plant Biol 30:159–165CrossRefGoogle Scholar
  36. Thao N, Ureshino KI, Ozaki Y, Okubo H (2003) Induction of tetraploids in ornamental Alocasia through colchicine and oryzalin treatments. Plant Cell Tissue Organ Cult 72:19–25CrossRefGoogle Scholar
  37. Vainola A (2000) Polyploidization and early screening of Rhododendron hybrids. Euphytica 112:239–244CrossRefGoogle Scholar
  38. Wang C, Lei JJ, Xing GM, Chuang AJ (2015) Tetraploid induction and chromosome identification from immature embryo with colchicine treatment in Clivia miniata. Acta Hortic Sci 38(7):1371–1376Google Scholar
  39. Xiang ZX, Liang HH, Tang XL, Liu WH (2019) Physiological and genetic diversity analysis of diploid and autotetraploid Platycodon grandiflorm A. De Candolle. Plant Cell Tissue Organ Cult 136:597–603. CrossRefGoogle Scholar
  40. Ye ZY, Wang YY, Tian HQ (2010) Regeneration of plantlets and tetraploidy induction in Pseudostellaria heterophylla. Acta Biol Crac 51:13–18Google Scholar
  41. Zhang J, Gai M, Li XY, Li TL, Sun HM (2016) Somatic embryogenesis and direct as well as indirect organogenesis in Lilium pumilum DC. Fisch., an endangered ornamental and medicinal plant. Biosci Biotechnol Biochem 80:1898–1906. CrossRefGoogle Scholar
  42. Zhou Y (2006) Preliminary study of the evalution system of precious and endangered wild officinal plants in Chanbai mountains. Acta Bot Boreal 26(3):0599–0605Google Scholar

Copyright information

© Springer Nature B.V. 2019

Authors and Affiliations

  • Linlan Fu
    • 1
  • Yingying Zhu
    • 1
  • Min Li
    • 1
  • Chunxia Wang
    • 1
    • 2
  • Hongmei Sun
    • 1
    • 2
    Email author
  1. 1.Key Laboratory of Protected Horticulture of Education Ministry and Liaoning Province, College of HorticultureShenyang Agricultural UniversityShenyangChina
  2. 2.National & Local Joint Engineering Research Center of Northern Horticultural Facilities Design & Application TechnologyShenyangChina

Personalised recommendations