Skip to main content
Springer Nature Link
Log in

Direct and indirect organogenesis of Clivia miniata and assessment of DNA methylation changes in various regenerated plantlets

  • Original Paper
  • Published:
Plant Cell Reports Aims and scope Submit manuscript

Abstract

Clivia miniata is an important indoor ornamental plant and has been reported to have medicinal value. We developed an efficient in vitro micropropagation protocol from young leaves (indirect organogenesis), young petals (indirect organogenesis) and shoot tips (direct organogenesis) of this plant. Using young leaves and shoot tips as explants, the regeneration frequencies were much higher than those in previous investigation and the regeneration was dependent upon less nutrition. We speculated that the leaf-derived callus can generate amino acids necessary for protein synthesis by itself. We employed the methylation-sensitive amplified polymorphism (MSAP) method to assess cytosine methylation variation in various regenerated plantlets and between organs. The MSAP profiles indicated that the frequency of somaclonal variation in the form of cytosine methylation was highest in petal-derived plantlets followed by secondary leaf-derived, primary leaf-derived and shoot tip-derived plantlets, but the methylation variation in petal-derived plantlets was lower than between petals and leaves of a single plant. The results indicated that the methylation variation in regenerated plantlets was related to the types of explants, regeneration pathways and number of regeneration generations. Two possible factors for the highest somaclonal variation rate in petal-derived plantlets are the callus phase and petal-specific set of epigenetic regulators. The property of meristem integrity can account for the lowest variation rate in shoot tip-derived plantlets. Moreover, the secondary plantlets underwent a longer total period of in vitro culture, which can explain why the methylation variation rate in the secondary plantlets is higher than in the primary ones.

Key message Methylation variation in regenerated plantlets of C. miniata was found to be related to the types of explants, regeneration pathways and number of regeneration generations.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Subscribe and save

Springer+ Basic
$34.99 /Month
  • Get 10 units per month
  • Download Article/Chapter or eBook
  • 1 Unit = 1 Article or 1 Chapter
  • Cancel anytime
Subscribe now

Buy Now

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

Explore related subjects

Discover the latest articles and news from researchers in related subjects, suggested using machine learning.

Abbreviations

MS:

Murashige and Skoog

KT:

Kinetin

NAA:

α-Naphthaleneacetic acid

BA:

6-Benzyladenine

2,4-D:

2,4-Dichlorophenoxyacetic acid

MSAP:

Methylation-sensitive amplified polymorphism

References

  • Aremu AO, Bairu MW, Doležal K, Finnie JF, Van Staden J (2011) Topolins: a panacea to plant tissue culture challenges? Plant Cell Tiss Org Cult. doi:10.1007/s11240-011-0007-7

    Google Scholar 

  • Benzion G, Phillips RL (1988) Cytogenetic stability of maize tissue cultures: a cell line pedigree analysis1. Genome 30:318–325

    Article  Google Scholar 

  • Berdasco M, Alcázar R, García-Ortiz MV et al (2008) Promoter DNA hypermethylation and gene repression in undifferentiated Arabidopsis cells. PLoS ONE 3:e3306

    Article  PubMed  Google Scholar 

  • Blanc G, Barakat A, Guyot R, Cooke R, Delseny M (2000) Extensive duplication and reshuffling in the Arabidopsis genome. Plant Cell 12:1093–1101

    PubMed  CAS  Google Scholar 

  • Bottley A, Chapman NH, Koebner RMD (2008) Homoeologous gene silencing in tissue cultured wheat callus. BMC Genet 9:65

    Article  PubMed  Google Scholar 

  • Chen T, Li E (2004) Structure and function of eukaryotic DNA methyltransferases. Curr Top Dev Biol 60:55–89

    Article  PubMed  CAS  Google Scholar 

  • Cokus SJ, Feng S, Zhang X et al (2008) Shotgun bisulfite sequencing of the Arabidopsis genome reveals DNA methylation patterning. Nature 452:215–219

    Article  PubMed  CAS  Google Scholar 

  • Côte FX, Teisson C, Perrier X (2001) Somaclonal variation rate evolution in plant tissue culture: contribution to understanding through a statistical approach. In Vitro Cell Dev-Pl 37:539–542

    Article  Google Scholar 

  • Dann AL, Wilson CR (2011) Comparative assessment of genetic and epigenetic variation among regenerants of potato (Solanum tuberosum) derived from long-term nodal tissue-culture and cell selection. Plant Cell Rep 30:631–639

    Article  PubMed  CAS  Google Scholar 

  • Finnie JF, van Staden J (1999) In vitro culture of Clivia miniata. Clivia yearbook 1 (1998). The Clivia Society, South Africa, pp 7–11

  • Gao X, Yang D, Cao D, Ao M, Sui X, Wang Q, Kimatu JN, Wang L (2010) In vitro micropropagation of Freesia hybrida and the assessment of genetic and epigenetic stability in regenerated plantlets. J Plant Growth Regul 29:257–267

    Article  CAS  Google Scholar 

  • Gavidia I, del Castillo Agudo L, Perez-Bermudez P (1996) Selection and longterm cultures of high-yielding Digitalis obscura plants: RAPD markers for analysis of genetic stability. Plant Sci 121:197–205

    Article  CAS  Google Scholar 

  • Golyasnaya N, Tsvetkova N (2006) Mismatch repair. Mol Biol 40:183–193

    Article  CAS  Google Scholar 

  • Gruenbaum Y, Naveh-Many T, Cedar H, Razin A (1981) Sequence specificity of methylation in higher plants DNA. Nature 292:860–862

    Article  PubMed  CAS  Google Scholar 

  • Guo D-P, Zhu Z-J, Hu X-X, Zheng S-J (2005) Effect of cytokinins on shoot regeneration from cotyledon and leaf segment of stem mustard (Brassica juncea var. tsatsai). Plant Cell Tiss Org Cult 83:123–127

    Article  CAS  Google Scholar 

  • Guo WL, Wu R, Zhang YF, Liu XM, Wang HY, Gong L, Zhang ZH, Liu B (2007) Tissue culture-induced locus-specific alteration in DNA methylation and its correlation with genetic variation in Codonopsis lanceolata Benth. et Hook. f. Plant Cell Rep 26:1297–1307

    Article  PubMed  CAS  Google Scholar 

  • Huettel B, Kanno T, Daxinger L, Bucher E, van der Winden J, Matzke AJM, Matzke M (2007) RNA-directed DNA methylation mediated by DRD1 and Pol IVb: a versatile pathway for transcriptional gene silencing in plants. Biochim Biophys Acta 1769:358–374

    PubMed  CAS  Google Scholar 

  • Jaccard P (1908) Nouvelles rescherches sur la distribution florale. Bull Soc Vaud Sci Nat 44:223–270

    Google Scholar 

  • Jaligot E, Beulé T, Rival A (2002) Methylation sensitive RFLPs: characterisation of two oil palm markers showing somaclonal variation-associated polymorphism. Theor Appl Genet 104:1263–1269

    Article  PubMed  CAS  Google Scholar 

  • Jaligot E, Beulé T, Baurens FC, Billotte N, Rival A (2004) Search for methylation-sensitive amplification polymorphism associated with the ‘‘mantled’’ variant phenotype in oil palm (Elaeis guineensis Jacq.). Genome 47:224–228

    Article  PubMed  CAS  Google Scholar 

  • Jones M, Wagner R, Radman M (1987) Mismatch repair of deaminated 5-methyl-cytosine☆. J Mol Biol 194:155–159

    Article  PubMed  CAS  Google Scholar 

  • Jr MF (1989) Habituation: heritable variation in the requirement of cultured plant cells for hormones. Annu Rev Genet 23:395–408

    Article  Google Scholar 

  • Jr MF, Seldran M (1994) Pseudodirected variation in the requirement of cultured plant cells for cell-division factors. Development 120:1163–1168

    Google Scholar 

  • Jr MF, Thomas M (2003) Meiotic transmission of epigenetic changes in the cell-division factor requirement of plant cells. Development 130:6201–6208

    Article  Google Scholar 

  • Kaeppler SM, Kaeppler HF, Rhee Y (2000) Epigenetic aspects of somaclonal variation in plants. Plant Mol Biol 43:179–188

    Article  PubMed  CAS  Google Scholar 

  • Keyte AL, Percifield R, Liu B, Wendel JF (2006) Infraspecific DNA methylation polymorphism in cotton (Gossypium hirsutum L.). J Hered 97:444–450

    Article  PubMed  CAS  Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

  • Krizova K, Fojtova M, Depicker A, Kovarik A (2009) Cell culture-induced gradual and frequent epigenetic reprogramming of invertedly repeated tobacco transgene epialleles1[W]. Plant Physiol 149:1493–1504

    Article  PubMed  CAS  Google Scholar 

  • Lambé P, Schié H, Mutambel N, Fouché J-G, Deltour R, Foidart J-M, Gaspar T (1997) DNA methylation as a key process in regulation of organogenic totipotency and plant neoplastic progression? In Vitro Cell Dev-Pl 33:155–162

    Article  Google Scholar 

  • Larkin PJ, Scowcroft WR (1981) Somaclonal variation—a novel source of variability from cell cultures for plant improvement. Theor Appl Genet 60:197–214

    Article  Google Scholar 

  • Li A, Hu B-Q, Xue Z-Y, Chen L, Wang W-X, Song W-Q, Chen C-B, Wang C-G (2011) DNA methylation in genomes of several annual herbaceous and woody perennial plants of varying ploidy as detected by MSAP. Plant Mol Biol Rep 29:784–793

    Article  CAS  Google Scholar 

  • López CMR, Wetten AC, Wilkinson MJ (2010) Progressive erosion of genetic and epigenetic variation in callus-derived cocoa (Theobroma cacao) plants. New Phytol 186:856–868

    Article  Google Scholar 

  • Marum L, Rocheta M, Maroco J, Oliveira MM, Miguel C (2009) Analysis of genetic stability at SSR loci during somatic embryogenesis in maritime pine (Pinus pinaster). Plant Cell Rep 28:673–682

    Article  PubMed  CAS  Google Scholar 

  • Masterson J (1994) Stomatal size in fossil plants: evidence for polyploid in majority of angiosperms. Science 264:421–424

    Article  PubMed  CAS  Google Scholar 

  • Miguel C, Marum L (2011) An epigenetic view of plant cells cultured in vitro: somaclonal variation and beyond. J Exp Bot 62:3713–3725

    Article  PubMed  CAS  Google Scholar 

  • Molnar A, Melnyk CW, Bassett A, Hardcastle TJ, Dunn R, Baulcombe D (2010) Small silencing RNAs in plants are mobile and direct epigenetic modification in recipient cells. Science 328:872–875

    Article  PubMed  CAS  Google Scholar 

  • Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473–497

    Article  CAS  Google Scholar 

  • Nei M, Li WH (1979) Mathematical model for studying genetic variation in terms of restriction endonucleases. Proc Natl Acad Sci USA 76:5269–5273

    Article  PubMed  CAS  Google Scholar 

  • Oakeley EJ (1999) DNA methylation analysis: a review of current methodologies. Pharmacol Ther 84:389–400

    Article  PubMed  CAS  Google Scholar 

  • Peredo EL, Ángeles Revilla M, Arroyo-García R (2006) Assessment of genetic and epigenetic variation in hop plants regenerated from sequential subcultures of organogenic calli. J Plant Physiol 163:1071–1079

    Article  PubMed  CAS  Google Scholar 

  • Phillips RL, Kaepplert SM, Olhoft P (1994) Genetic instability of plant tissue cultures: breakdown of normal controls. Proc Natl Acad Sci USA 91:5222–5226

    Article  PubMed  CAS  Google Scholar 

  • Pischke MS, Huttlin EL, Hegeman AD, Sussman MR (2006) A transcriptome-based characterization of habituation in plant tissue culture1[W]. Plant Physiol 140:1255–1278

    Article  PubMed  CAS  Google Scholar 

  • Ran Y, Simpson S (2005) In vitro propagation of the genus Clivia. Plant Cell Tiss Org Cult 81:239–242

    Article  CAS  Google Scholar 

  • Ran Y, Murray BG, Hammett KRW (2001a) Evaluating genetic relationships between and within Clivia species using RAPDs. Sci Hortic 90:167–179

    Article  CAS  Google Scholar 

  • Ran Y, Hammett KW, Murray BG (2001b) Phylogenetic analysis and karyotype evolution in the genus Clivia (Amaryllidaceae). Ann Bot-Lond 87:823–830

    Article  CAS  Google Scholar 

  • Rapp RA, Wendel JF (2005) Epigenetics and plant evolution. New Phytol 168:81–91

    Article  PubMed  CAS  Google Scholar 

  • Riddle NC, Richards EJ (2002) The control of natural variation in cytosine methylation in Arabidopsis. Genetics 162:355–363

    PubMed  CAS  Google Scholar 

  • Ruiz-García L, Cervera MT, Martínez-Zapater JM (2005) DNA methylation increases throughout Arabidopsis development. Planta 222:301–306

    Article  PubMed  Google Scholar 

  • Schmitt F, Oakeley EJ, Jost JP (1997) Antibiotics induce genome-wide hypermethylation in cultured Nicotiana tabacum plants. J Biol Chem 272:1534–1540

    Article  PubMed  CAS  Google Scholar 

  • Sengbusch PV (2003) Biosyntheses-amino acids. KEGG database-metabolic pathways. http://www.biologie.uni-hamburg.de/b-online/e19/19e.htm. Accessed 31 July 2003

  • Skrabanek L, Wolfe KH (1998) Eukaryotic genome duplication—where’s the evidence? Curr Opin Genet Dev 8:694–700

    Article  PubMed  CAS  Google Scholar 

  • Smýkal P, Valledor L, Rodriguez R, Griga M (2007) Assessment of genetic and epigenetic stability in long-term in vitro shoot culture of pea (Pisum sativum L.). Plant Cell Rep 26:1985–1998

    Article  PubMed  Google Scholar 

  • Teixeira da Silva JA (2003) Chrysanthemum: advances in tissue culture, cryopreservation, postharvest technology, genetics and transgenic biotechnology. Biotechnol Adv 21:715–766

    Article  PubMed  CAS  Google Scholar 

  • Tian CG, Xiong YQ, Liu TY, Sun SH, Chen LB, Chen MS (2005) Evidence for an ancient whole-genome duplication event in rice and other cereals. Chin J Genet 32:519–527

    CAS  Google Scholar 

  • Vanyushin BF, Ashapkin VV (2011) DNA methylation in higher plants: past, present and future. BBA-Gene Regul Mech 1809:360–368

    CAS  Google Scholar 

  • Vanyushin BF, Tkacheva SG, Belozersky AN (1970) Rare bases animal DNA. Nature 225:948–949

    Article  PubMed  CAS  Google Scholar 

  • Wang X, Chen JJ, Li Y, Nie Q, Li J (2009) An efficient procedure for regeneration from leaf-derived calluses of Lonicera macranthoides ‘jincuilei’, an important medicinal plant. HortScience 44:746–750

    Google Scholar 

  • Wang Q-M, Gao F-Z, Gao X, Zou F-Y, Sui X, Wang M, Hui Y-J, Wang L (2011) Regeneration of Clivia miniata and assessment of clonal fidelity of plantlets. Plant Cell Tiss Org Cult. doi:10.1007/s11240-011-0085-6

    Google Scholar 

  • Wolfe KH, Shields DC (1997) Molecular evidence for an ancient duplication of the entire yeast genome. Nature 387:708–713

    Article  PubMed  CAS  Google Scholar 

  • Wolffe AP, Matzke MA (1999) Epigenetics: regulation through repression. Science 286:481–486

    Article  PubMed  CAS  Google Scholar 

  • Yang H, Tabei Y, Kamada H, Kayano T, Takaiwa F (1999) Detection of somaclonal variation in tissue cultured rice cells using digoxigenin based random amplified polymorphic DNA. Plant Cell Rep 18:520–526

    Article  CAS  Google Scholar 

  • Ye L (2006) Progress in the tissue culture and micropropagation of Clivia miniata Regel. Subtrop Agric Res 2:231–233

    Google Scholar 

  • Zhang M, Kimatu JN, Xu K, Liu B (2010) DNA cytosine methylation in plant development. J Genet Genomics 37:1–12

    Article  PubMed  Google Scholar 

Download references

Acknowledgments

This work was supported by Jilin Provincial Science and Technology Department of China (20110260), Specialized Research Fund for the Doctoral Program of Higher Education (20100043110008), the National Natural Science Foundation of China (30970280, 31170276). Professor Bao Liu (Laboratory of Plant Molecular Epigenetics, Institute of Genetics and Cytology, Northeast Normal University, China) kindly provided the facilities for MSAP analysis.

Author information

Authors and Affiliations

  1. Institute of Genetics and Cytology, Northeast Normal University, Changchun, 130024, People’s Republic of China

    Qin-Mei Wang, Yu-Zhang Wang, Li-Li Sun, Feng-Zhan Gao, Wei Sun, Jing He, Xiang Gao & Li Wang

Authors
  1. Qin-Mei Wang
    View author publications

    You can also search for this author inPubMed Google Scholar

  2. Yu-Zhang Wang
    View author publications

    You can also search for this author inPubMed Google Scholar

  3. Li-Li Sun
    View author publications

    You can also search for this author inPubMed Google Scholar

  4. Feng-Zhan Gao
    View author publications

    You can also search for this author inPubMed Google Scholar

  5. Wei Sun
    View author publications

    You can also search for this author inPubMed Google Scholar

  6. Jing He
    View author publications

    You can also search for this author inPubMed Google Scholar

  7. Xiang Gao
    View author publications

    You can also search for this author inPubMed Google Scholar

  8. Li Wang
    View author publications

    You can also search for this author inPubMed Google Scholar

Corresponding authors

Correspondence to Xiang Gao or Li Wang.

Additional information

Communicated by J. Zou.

Electronic supplementary material

Rights and permissions

Reprints and permissions

About this article

Cite this article

Wang, QM., Wang, YZ., Sun, LL. et al. Direct and indirect organogenesis of Clivia miniata and assessment of DNA methylation changes in various regenerated plantlets. Plant Cell Rep 31, 1283–1296 (2012). https://doi.org/10.1007/s00299-012-1248-6

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00299-012-1248-6

Keywords