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In vitro regeneration of Carlina acaulis subsp. simplex from seedling explants

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Abstract

The aim of the study was to obtain an efficient system for Carlina acaulis subsp. simplex propagation. The experimental materials were shoot tips, fragments of hipocotyls, cotyledons and roots isolated from 10-day-old seedlings. The explants were transferred to the proliferation medium supplemented with different types of cytokinin: BA (13.3 μM), kinetin (13.9 μM) and zeatin (13.7 μM) in combination with NAA (0.54 μM). The best morphogenetic response was observed when explants were cultured on the BA supplemented medium. The maximum shoot organogenesis frequency was observed for shoot tip (nearly 94%). On average 8.6 axillary shoots were induced per explant. Multiplication rate increased during the first three subcultures. The shoots revealed a wide range of morphogenetic responses. Differences were observed in the presence or absence of hair on the surface of lamina. These changes had epigenetic character and were the effect of changes in DNA methylation, which is shown by differences in methylation pattern between 18S rRNA and 25S rRNA genes in the analyzed regenerated plants. Nearly 94% of plantlets were rooted on auxin lacking medium. Addition of auxin (NAA or IAA) increased both the rooting percentage (100%) and the number of roots per shoot, but their growth was inhibited. Shortening of the auxin exposition time reduced the number of roots. Moreover, high efficiency (90%) was observed for ex vitro rooting. Plantlets with a large number of roots survived better than the ones with only a few roots. Plants were able to flower and gave viable seeds.

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Abbreviations

BA:

6-Benzylaminopurine

GA3 :

Gibberellic acid

IAA:

Indole-3-acetic acid

Kn:

Kinetin

NAA:

Naphthaleneacetic acid

MS:

Murashige and Skoog medium

Zea:

Zeatin

References

  • Butiuc-Keul AL, Deliu C (2001) Clonal propagation of Arnica montana L., a medicinal plant. In Vitro Cell Dev Biol Plant 37:581–585. doi:10.1007/s11627-001-0102-2

    Google Scholar 

  • Cassells AC, Curry RF (2001) Oxidative stress and physiological, epigenetic and genetic variability in plant tissue culture: implication for micropropagator and genetic engineers. Plant Cell Tiss Org Cult 64:145–157. doi:10.1023/A:1010692104861

    Article  CAS  Google Scholar 

  • Ćurković PM (2003) In vitro propagation of Centaurea rupestris L. Acta Biol Cracoviensia 45:127–130

    Google Scholar 

  • Dhaka N, Kothari SL (2005) Micropropagation of Eclipta alba (L.) Hassk—an important medicinal plant. In Vitro Cell Dev Biol Plant 41:658–661. doi:10.1079/IVP2005684

    Article  Google Scholar 

  • Doyle JJ, Doyle JL (1987) A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochem Bull 19:11–15

    Google Scholar 

  • Echeverrigaray S, Fracaro F, Andrade LB, Biasio S, Atti-Serafini L (2000) In vitro shoot regeneration from leaf explant of Roman Chamomile. Plant Cell Tiss Org Cult 60:1–4. doi:10.1023/A:1006368210670

    Article  Google Scholar 

  • Emmanuel S, Ignacimuthu S, Kathiravan K (2000) Micropropagation of Wedelia calendulacea Less., a medicinal plant. Phytomorphology 50:195–200

    Google Scholar 

  • Fay MF (1992) Conservation of rare and endangered plants using in vitro methods. In Vitro Cell Dev Biol 28:1–4. doi:10.1007/BF02631070

    Article  Google Scholar 

  • Feinberg AP, Vogelstein B (1983) A technique for radioactive DNA restriction endonuclease fragments to high specific activity. Anal Biochem 132:6–13. doi:10.1016/0003-2697(83)90418-9

    Article  PubMed  CAS  Google Scholar 

  • Flavell RB, O’Dell M, Thompson WF (1988) Regulation of cytosine methylation in rybosomal DNA and nucleolus organizer expression in wheat. J Mol Biol 204:523–534. doi:10.1016/0022-2836(88)90352-X

    Article  PubMed  CAS  Google Scholar 

  • Fraś A, Małuszyńska J (2004) The correlation between the chromosome variation in callus and genotype of explants of Arabidopsis thaliana. Genetica 121:145–154. doi:10.1023/B:GENE.0000040375.18684.82

    Article  PubMed  Google Scholar 

  • Fronk J (1996) The role of DNA methylation in the regulation of transcription in eucaryotes. Adv Cell Biol 23:15–26

    CAS  Google Scholar 

  • Galek R, Kukułczanka K (2000) Propagation of Carlina acaulis in vitro culture methods. In: IX All-Poland conference in vitro cultures and plant biotechnology. Gdańsk-Sobieszewo, September 10–13, 2000 (book of abstracts), p 132

  • Gerlach WL, Bedbrook JR (1979) Cloning and characterization of ribosomal RNA genes from wheat and barley. Nucleic Acids Res 7:1869–1885. doi:10.1093/nar/7.7.1869

    Article  PubMed  CAS  Google Scholar 

  • Grubisić D, Šavikin-Fodulović K, Mišić D, Zlatko G, Konjević R (2004) In vitro stem elongation of stemless carline thistle. Plant Growth Regul 44:65–69. doi:10.1007/s10725-004-2602-7

    Article  Google Scholar 

  • Joyce SM, Cassells AC, Jain SM (2003) Stress and aberrant phenotypes in in vitro culture. Plant Cell Tiss Org Cult 74:103–121. doi:10.1023/A:1023911927116

    Article  CAS  Google Scholar 

  • Kaeppler MS, Kaeppler HF, Rhee Y (2000) Epigenetic aspects of somaclonal variation in plants. Plant Mol Biol 43:179–188. doi:10.1023/A:1006423110134

    Article  PubMed  CAS  Google Scholar 

  • Korach A, Juliani HR, Kapteyn J, Simon JE (2002) In vitro regeneration of Echinacea purpurea from leaf explants. Plant Cell Tissue Org Cult 69:79–83. doi:10.1023/A:1015042032091

    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. doi:10.1007/BF02342540

    Article  Google Scholar 

  • Lisowska K, Wysokinska H (2000) In vitro propagation of Catalpa ovata G. Don. Plant Cell Tissue Org Cult 60:171–176. doi:10.1023/A:1006461520438

    Article  Google Scholar 

  • Lorenz MC, Groundin M (2001) C(m)C(a/t)GC methylation: a new epigenetic mark in mammalian DNA? Proc Natl Acad Sci USA 98:10034–10036. doi:10.1073/pnas.201392598

    Article  Google Scholar 

  • Luijten SH, Oostermeijer JGB, Van Leeuwen NC, Den Nijs HCM (1996) Reproductive success and clonal genetic structure of the rare Arnica montana (Compositae) in the Netherlands. Plant Syst Evol 2001:15–30. doi:10.1007/BF00989049

    Article  Google Scholar 

  • Misra P, Dutta SK (1999) In vitro propagation of white marigold (Tagetes erecta L.) throught soot tip proliferation. Curr Sci 77:1138–1140

    Google Scholar 

  • Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:437–497. doi:10.1111/j.1399-3054.1962.tb08052.x

    Article  Google Scholar 

  • Neves N, Heslop-Harrison JS, Viegas W (1995) rRNA gene activity and control of expression mediated by methylation and imprinting during embryo development in wheat × rye hybrids. Theor Appl Genet 91:529–533. doi:10.1007/BF00222984

    Article  CAS  Google Scholar 

  • Pence VC, Clark JR, Plair BL (2002) Wild and endangered species. In: Pence VC, Sandoval JA, Villalobos VM, Engelmann F (eds) In vitro collecting techniques for germplasm conservation. IPGRI Tech Bull 7, pp 76–82

  • Piękoś-Mirkowa H, Mirek Z (2003) Atlas roślin chronionych. MULTIKO Oficyna Wydawnicza, Warszawa, pp 60–61 (in Polish)

    Google Scholar 

  • Poonawala IS, Jana MM, Nadgauda RS (1999) Factors influencing bud break and rooting and mass-scale micropropagation of three Phragmites species: P. karka, P. communis and P. australis. Plant Cell Rep 18:696–700. doi:10.1007/s002990050645

    Article  CAS  Google Scholar 

  • Pospíšilowá J, Ticha L, Kadlecek P, Haisel D, Plazakowa S (1999) Acclimatization of micropropagated plant to ex vitro conditions. Biol Plant 42:481–497. doi:10.1023/A:1002688208758

    Article  Google Scholar 

  • Ramage CM, Williams RR (2004) Cytokinin-induced abnormal shoot organogenesis is associated with elevated Knotted1-typ homeobox gene expression in tobacco. Plant Cell Rep 22:919–924. doi:10.1007/s00299-004-0774-2

    Article  PubMed  CAS  Google Scholar 

  • Sambrook J, Fritsch EF, Maniatis T (1988) Molecular cloning: a laboratory manual, vol II, 2nd edn. Cold Spring Harbor, New York, pp 9.1–9.62

    Google Scholar 

  • Schween G, Schwenkel HG (2003) Effect of genotype on calls induction, shoot regeneration, and phenotypic stability of regenerated plants in the greenhouse of Primula spp. Plant Cell Tissue Org Cult 72:53–61. doi:10.1023/A:1021227414880

    Article  CAS  Google Scholar 

  • Skucińska B (2001) Induction of genetic variation in vitro. Biotechnologia 3:145–151 (in Polish)

    Google Scholar 

  • Smulders MJM (2005) Are there adequate methods for assessing somaclonal variation in tissue culture-propagated plants? COST 843 final conference/COST 843 and COST 851 joint meeting, Stara Lesna, Slovakia, June 28–July 3 (book of abstracts), pp 201–203

  • Sultan SE (2000) Phenotypic plasticity for plant development, function and life history. Trends Plant Sci 5:537–542. doi:10.1016/S1360-1385(00)01797-0

    Article  PubMed  CAS  Google Scholar 

  • Tariq M, Paszkowski J (2004) DNA and histone methylation in plants. Trends Genet 20:244–251. doi:10.1016/j.tig.2004.04.005

    Article  PubMed  CAS  Google Scholar 

  • Tutin FG, Heywood VH, Burges NA, Moore DM, Valentine DH, Walters SM, Webb DA (1976) Flora Europea, vol 4: Plantaginaceae to Compositae (and Rubiaceae). Cambridge University Press, Cambridge, p 210

    Google Scholar 

  • Usha R, Swamy PM (1998) In vitro micropropagation of sweet wormwood (Artemisia annua L.). Phytomorphology 48:149–154

    Google Scholar 

  • Wada Y, Ohya H, Yamaguchi Y, Koizumi N, Sano H (2003) Preferential de novo methylation of cytosine residues in non-CpG sequences by a domains rearranged DNA methyltransferase from tabacco plants. J Biol Chem 43:42386–42393. doi:10.1074/jbc.M303892200

    Article  Google Scholar 

  • Yucesan B, Turker AU, Gurel E (2007) TDZ-induced high frequency plant regeneration through multiple shoot formation in witloof chicory (Cichorium intybus L.). Plant Cell Tissue Org Cult 91:243–250. doi:10.1007/s11240-007-9290-8

    Article  CAS  Google Scholar 

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Authors and Affiliations

  1. Department of Biotechnology, Institute of General and Molecular Biology, Nicolaus Copernicus University, 87-100, Toruń, Gagarina 9, Poland

    Alina Trejgell & Andrzej Tretyn

  2. Department of Genetic, Institute of General and Molecular Biology, Nicolaus Copernicus University, 87-100, Toruń, Gagarina 9, Poland

    Grażyna Dąbrowska

Authors
  1. Alina Trejgell
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  2. Grażyna Dąbrowska
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  3. Andrzej Tretyn
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Correspondence to Alina Trejgell.

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Communicated by E. Lojkowska.

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Trejgell, A., Dąbrowska, G. & Tretyn, A. In vitro regeneration of Carlina acaulis subsp. simplex from seedling explants. Acta Physiol Plant 31, 445–453 (2009). https://doi.org/10.1007/s11738-008-0252-5

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