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Somatic embryogenesis and plant regeneration in zygotic embryos of Trifolium nigrescens (Viv.)

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Abstract

This study developed a plant regeneration protocol for Trifolium nigrescens (Viv.) via somatic embryogenesis (SE). Immature zygotic embryos at torpedo (TsE) and cotyledonary (CsE) stage were cultured on media with different auxins and cytokinins at different concentrations. The cultural requirements for SE differed between the explants used: the addition of 6-furfurylaminopurine (kinetin) or N6-[2-isopentenyl]-adenine (2iP) along with 2,4-dihydrophenoxyacetic acid (2,4-D) or 1-naphthaleneacetic acid (NAA) was needed to elicit the embryogenic response of CsE, but an exogenous cytokinin totally inhibited 2,4-D-induced SE from TsE. When applied alone, neither the cytokinin nor NAA induced SE in TsE or CsE. In all effective cultures the first somatic embryos appeared directly from the upper part of the hypocotyl (TsE and CsE) and from the margin of cotyledons (TsE) on day 7. Embryogenic callus occurred on CsE after 10 days. At comparable concentrations 2,4-D was a more potent SE inducer than NAA, but most of the embryoids induced on media with 2,4-D displayed morphological abnormalities, whereas those produced in the presence of NAA generally resembled zygotic embryos. Plant regeneration was achieved after transfer of somatic embryos or embryo-derived first shoots to medium without plant growth regulators (PGRs). The frequency of plant recovery was about 30% for embryoids obtained on media containing 2,4-D, and for material from media with NAA the recovery rates were 44–68% (somatic embryos) and 72–100% (embryoid-derived shoots). Regenerants appeared identical to each other and to wild plants; they produced flowers and had the chromosome complement typical for the species, 2n = 16, in root tip cells.

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Abbreviations

2,4-D:

2,4-Dihydrophenoxyacetic acid

2iP:

N6-[2-isopentenyl]-adenine

CsE:

Cotyledonary-stage zygotic embryo

Kinetin:

6-Furfurylaminopurine

NAA:

1-Naphthaleneacetic acid

PGR:

Plant growth regulator

SE:

Somatic embryogenesis

TsE:

Torpedo-stage zygotic embryo

References

  • Arcioni S, Mariotti D (1982) Tissue culture and plant regeneration in the forage legumes Onobrychis vicaefolia Scop., Coronilla varia and Lotus corniculatus L. In: Fujiwara A (ed) Plant cell tissue culture, vol 11. Maruzen, Tokyo, pp 707–708

    Google Scholar 

  • Bronner R, Jeanin G, Hahne G (1994) Early cellular events during organogenesis and somatic embryogenesis induced on immature zygotic embryos of sunflower (Helianthus annuus L.). Can J Bot 72:239–248

    Article  Google Scholar 

  • Choo TH (1988) Plant regeneration in zigzag clover (Trifolium medium). Plant Cell Rep 7:246–248

    Article  Google Scholar 

  • Gaj MD (2004) Factors influencing somatic embryogenesis induction and plant regeneration with particular reference to Arabidopsis thaliana (L.) Heynh. Plant Growth Regul 43:27–47

    Article  CAS  Google Scholar 

  • Gillet JM (1985) Taxonomy and morphology. In: Taylor NL (ed) Clover science and technology. American Society of Agronomy, Inc., Crop Science Society of America, Inc., Soil Science Society of America, Inc., Madison, pp 7–69

    Google Scholar 

  • Griga M (2002) Morphology and anatomy of Pisum sativum somatic embryos. Biol Plant 45:173–182

    Article  Google Scholar 

  • Hecht V, Vielle-Calzada JP, Hartog MV, Schmidt EDL, Boutilier K, Grossniklaus U, de Vries SC (2001) Arabidopsis Somatic Embryogenesis Receptor Kinase 1 gene is expressed in developing ovules and embryos and enhances embryogenic competence in culture. Plant Physiol 127:803–816

    Article  CAS  PubMed  Google Scholar 

  • Hoveland CS, Evers GW (1995) Arrowleaf, crimson and other annual clovers. In: Barnes RF, Miller DA, Nelson CJ (eds) Forages, an introduction to grassland agriculture, vol 1, 5th edn. Iowa State University Press, Iowa, pp 249–260

    Google Scholar 

  • Hussain SW, Williams WM, Mercer CF, White DWR (1997) Transfer of clover cysts nematode resistance from Trifolium nigrescens Viv. to T. repens L. by interspecific hybridisation. Theor Appl Genet 95:1274–1281

    Article  Google Scholar 

  • Jimenez VM (2005) Involvement of plant hormones and plant growth regulators on in vitro somatic embryogenesis. Plant Growth Regul 47:91–110

    Article  CAS  Google Scholar 

  • Konieczny R (1995) Plant regeneration in callus culture of Trifolium nigrescens Viv. Acta Biol Cracov Ser Bot 37:47–52

    Google Scholar 

  • Konieczny R (1996) Plant regeneration from immature embryo culture of Trifolium michaelianum Savi. Histological observations on adventitious shoot formation. Acta Soc Bot Pol 65:261–266

    Google Scholar 

  • Konieczny R, Czaplicki AZ, Golczyk H, Przywara L (2003) Two pathways of plant regeneration in wheat anther culture. Plant Cell Tissue Org Cult 73:177–187

    Article  CAS  Google Scholar 

  • Kysely W, Jacobsen H (1990) Somatic embryogenesis from pea embryos and shoot apices. Plant Cell Tissue Org Cult 20:7–14

    Article  CAS  Google Scholar 

  • Lazzerii PA, Hildebrand DF, Collins GB (1987) Soybean somatic embryogenesis: effect of hormones and culture manipulations. Plant Cell Tissue Org Cult 10:197–208

    Article  Google Scholar 

  • Maheswaran G, Williams E (1984) Direct somatic embryoid formation in immature embryos of Trifolium repens, T. pratense and Medicago sativa and rapid clonal propagation of T. repens. Ann Bot 56:613–630

    Google Scholar 

  • Maheswaran G, Williams E (1986) Clonal propagation of Trifolium pratense, T. resupinatum and T. subterraneum by direct somatic embryogenesis on cultured immature embryo. Plant Cell Rep 3:165–168

    Article  Google Scholar 

  • Marshall AH, Michaelson-Yeates TPT, Abberton MT, Williams A, Powell HG (2002) Variation for reproductive and agronomic traits among T. repens × T. nigrescens third generation backcross hybrids in the field. Euphytica 126:195–201

    Article  Google Scholar 

  • Marshall AH, Michaelson-Yeates TPT, Abberton MT (2008) Introgression of reproductive traits from Trifolium nigrescens increases the seed yield of white clover (T. repens). Plant Breed 12:597–601

    Article  Google Scholar 

  • Mercer CF (1988) Reaction of some species of Trifolium to Meloidogyne hapla and Heterodera trifolii. In: Proceedings of 5th Australian conference on grasslands invertebrate ecology. Melbourne, Australia, pp 275–280

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

    Article  CAS  Google Scholar 

  • Murthy BNS, Murch SJ, Saxena PK (1995) Thidiazuron-induced somatic embryogenesis in intact seedlings of peanut (Arachis hypogea): endogenous growth regulators levels and significance of cotyledons. Physiol Plant 94:176–268

    Article  Google Scholar 

  • Nagarajan P, McKenzie JS, Walton PD (1986) Embryogenesis and plant regeneration of Medicago species in tissue culture. Plant Cell Rep 5:77–80

    Article  Google Scholar 

  • Nanda RM, Rout GR (2003) In vitro somatic embryogenesis and plant regeneration in Acaccia arabica. Plant Cell Tissue Org Cult 73:131–135

    Article  CAS  Google Scholar 

  • Nolan KE, Irwanto R, Rose RJ (2003) Auxin up-regulates MtSERK1 expression in both Medicago truncatula root-forming and embryogenic cultures. Plant Physiol 133:218–230

    Article  CAS  PubMed  Google Scholar 

  • Özcan S, Barghchi M, Firek S, Draper J (1993) Efficient adventitious shoot regeneration and somatic embryogenesis in pea. Plant Cell Tissue Org Cult 34:271–277

    Article  Google Scholar 

  • Ozias-Akins P (1989) Plant regeneration from immature embryos of peanut. Plant Cell Rep 8:217–218

    Article  Google Scholar 

  • Parrot WA (1991) Auxin stimulated somatic embryogenesis from immature cotyledons of white clover. Plant Cell Rep 10:17–21

    Article  Google Scholar 

  • Parrot WA, Collins GB (1983) Callus and shoot tip culture of eight Trifolium species in vitro with regeneration and somatic embryogenesis of T. rubens. Plant Sci Lett 28:189–194

    Google Scholar 

  • Pederson GA (1986) In vitro culture and somatic embryogenesis of four Trifolium species. Plant Sci 45:101–104

    Article  CAS  Google Scholar 

  • Pederson GA, Windham GL (1989) Resistance to Meloidogyne incognita in Trifolium interspecific hybrids and species related to white clover. Plant Dis 73:567–569

    Article  Google Scholar 

  • Phillips GC, Collins GB (1980) Somatic embryogenesis from cell suspension culture of red clover. Crop Sci 16:213–216

    Google Scholar 

  • Pintos B, Martin JP, Centeno ML, Villalobos N, Guerra H, Martin L (2002) Endogenous cytokinin levels in embryogenic and non-embryogenic calli of Medicago arborea L. Plant Sci 163:955–960

    Article  CAS  Google Scholar 

  • Rao MM, Lakshmisita G (1996) Direct somatic embryogenesis from immature embryos of rosewood (Dalbergia latifolia Roxb.). Plant Cell Rep 15:355–359

    Article  CAS  Google Scholar 

  • Rybczyński JJ (1997) Plant regeneration from highly embryogenic callus, cell suspension and protoplast cultures of Trifolium fragiferum. Plant Cell Tissue Org Cult 51:159–170

    Article  Google Scholar 

  • Shoemaker R, Amberger LA, Palmer RG, Oglesby L, Ranch J (1991) Effect of 2, 4-dichlorophenoxyacetic acid concentration on somatic embryogenesis and heritable variation in soybean (Glycine max (L.) Merr.). In Vitro Cell Dev Biol 27:84–88

    Article  Google Scholar 

  • Trigiano RN, Beaty RM, Graham ET (1988) Somatic embryogenesis from immature embryos of redbud (Cercis canadensis). Plant Cell Rep 7:148–150

    Article  CAS  Google Scholar 

  • Trinh TH, Patel P, Kondorosi K, Durand P, Kamate K, Baner P, Kondorosi A (1998) Rapid and efficient transformation of diploid Medicago truncatula and Medicago sativa ssp. falcata lines improved in somatic embryogenesis. Plant Cell Rep 17:345–355

    Article  CAS  Google Scholar 

  • Van Staden J (1983) Seeds and cytokinins. Physiol Plant 58:340–346

    Article  Google Scholar 

  • Webb J, Fay FM, Dale PJ (1987) An investigation of morphogenesis within the genus Trifolium. Plant Cell Tissue Org Cult 11:36–37

    Article  Google Scholar 

Download references

Acknowledgments

We thank the Institute of Plant Genetics and Crop Plant Research, Gatersleben, Germany for providing T. nigrescens seeds for this study.

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Authors and Affiliations

  1. Department of Plant Cytology and Embryology, Jagiellonian University, Grodzka 52, 31044, Kraków, Poland

    Robert Konieczny, Maria Pilarska, Monika Tuleja & Tomasz Ilnicki

  2. Institute of Plant Genetics and Biotechnology, Slovak Academy of Sciences, Akademicka 2, 94901, Nitra, Slovak Republic

    Terezia Salaj

Authors
  1. Robert Konieczny
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  2. Maria Pilarska
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  3. Monika Tuleja
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  4. Terezia Salaj
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  5. Tomasz Ilnicki
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Correspondence to Robert Konieczny.

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Konieczny, R., Pilarska, M., Tuleja, M. et al. Somatic embryogenesis and plant regeneration in zygotic embryos of Trifolium nigrescens (Viv.). Plant Cell Tiss Organ Cult 100, 123–130 (2010). https://doi.org/10.1007/s11240-009-9625-8

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  • DOI: https://doi.org/10.1007/s11240-009-9625-8

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