Skip to main content

A highly efficient plant regeneration system through multiple shoot differentiation from commercial cultivars of barley (Hordeum vulgare L.) using meristematic shoot segments excised from germinated mature embryos

Plant Cell Reports volume 23, pages 9–16 (2004)Cite this article

Abstract

A fast and highly efficient short-term in vitro regeneration system was developed for barley (Hordeum vulgare L.) based on readily available explants. Clumps of multiple shoots and buds suitable for transformation were obtained 9–10 weeks after culture initiation from model and current commercial cultivars. Meristematic shoot segments (MSSs) excised from mature embryo-derived seedlings and subsequently cultured on MS-based medium containing 2 mg/l Picloram and 3 mg/l thidiazuron (TDZ) differentiated up to ten multiple shoots after 3–4 weeks with no or very little callus formation. Sectors of the already multiplied shoot clumps were further multiplied on proliferation-maintenance medium containing 2 mg/l Picloram and 2.5 mg/l TDZ. Biweekly subcultures resulted in a continuous process of multiplication of these highly differentiating green sectors without any loss of morphogenic potential. The differentiated small shoots and shoot buds gave rise to normal shoots on medium with 0.1 mg/l Picloram and 1 mg/l TDZ. After rooting on basal medium with 0.5 mg/l or 1 mg/l IBA the plants were transferred to soil and showed normal growth and fertility compared to the seed-grown plants. All of the genotypes tested formed multiple shoots. The percentage of relative MSS multiplication was 63–83%, and the average number of multiplied shoots per MSS ranged from 16 to 34 among the genotypes after 9–11 weeks.

This is a preview of subscription content, access via your institution.

Access options

Buy single article

Instant access to the full article PDF.

43,34 €

Price includes VAT (Finland)
Tax calculation will be finalised during checkout.

Fig. 1

Abbreviations

BAP:

6-Benzylaminopurine

2,4-D:

2,4-Dichlorophenoxyacetic acid

Dicamba:

3,6-Dichloro-2-methoxybenzoic acid

IBA:

Indole-3-butyric acid

MSS:

Meristematic shoot segment

NAA:

α-Naphthaleneacetic acid

Picloram:

4-Amino-3,5,6-trichloropicolinic acid

TDZ:

Thidiazuron

References

  • Akula C, Akula A, Henry R (1999) Improved regeneration efficiency from mature embryos of barley cultivars. Biol Plant 42:505–513

    Article  Google Scholar 

  • Becher T, Haberland G, Koop H-U (1992) Callus formation and plant regeneration in standard and microexplants from seedlings of barley (Hordeum vulgare L.). Plant Cell Rep 11:39–43

    Google Scholar 

  • Bregitzer P, Tonks D (2003) Inheritance and expression of transgenes in barley. Crop Sci 43:4–12

    Google Scholar 

  • Bregitzer P, Zhang S, Cho M-J, Lemaux PG (2002) Reduced somaclonal variation in barley is associated with culturing highly differentiated, meristematic tissues. Crop Sci 42:1303–1308

    Google Scholar 

  • Cheng T-Y, Smith HH (1975) Organogenesis from callus culture of Hordeum vulgare. Planta 123:307–310

    Google Scholar 

  • Cho M-J, Jiang W, Lemaux PG (1998) Transformation of recalcitrant barley cultivars through improvement of regenerability and decreased albinism. Plant Sci 138:229–244

    CAS  Google Scholar 

  • Cho M-J, Choi HW, Okamoto D, Zhang S, Lemaux PG (2003) Expression of green fluorescent protein and its inheritance in transgenic oat plants generated from shoot apical meristematic cultures. Plant Cell Rep 21:467–474

    CAS  PubMed  Google Scholar 

  • Cho M-J, Yano H, Okamoto D, Kim H-K, Jung H-R, Newcomb K, Le VK, Yoo HS, Langham R, Buchanan BB, Lemaux PG (2004) Stable transformation of rice (Oryza sativa L.) via microprojectile bombardment of highly regenerative, green tissues derived from mature seed. Plant Cell Rep 22:483–489

    Article  CAS  PubMed  Google Scholar 

  • Choi HW, Lemaux PG, Cho M-J (2000) Increased chromosomal variation in transgenic versus nontransgenic barley (Hordeum vulgare L.) plants. Crop Sci 40:524–533

    Google Scholar 

  • Chugh A, Khurana P (2003) Regeneration via somatic embryogenesis from leaf basal segments and genetic transformation of bread and emmer wheat by particle bombardment. Plant Cell Tissue Organ Cult 74:151–161

    Article  CAS  Google Scholar 

  • Dahleen LS, Bregitzer P (2002) An improved media system for high regeneration rates from barley immature embryo-derived callus cultures of commercial cultivars. Crop Sci 42:934–938

    Google Scholar 

  • Devi P, Zhong H, Sticklen MB (2000) In vitro morphogenesis of pearl millet [Pennisetum glaucum (L.) R. Br.]: efficient production of multiple shoots and inflorescences from shoot apices. Plant Cell Rep 19:546–550

    CAS  Google Scholar 

  • Gairi A, Rashid A (2004) TDZ-induced somatic embryogenesis in non-responsive caryopses of rice using a short treatment with 2,4-D. Plant Cell Tissue Organ Cult 76:29–33

    Article  CAS  Google Scholar 

  • Ganeshan S, Baga M, Harvey BL, Rossnagel BG, Scoles GJ, Chibbar RN (2003) Production of multiple shoots from thidiazuron-treated mature embryos and leaf-base/apical meristems of barley (Hordeum vulgare L.). Plant Cell Tissue Organ Cult 73:57–64

    Article  CAS  Google Scholar 

  • Gless C, Lörz H, Jähne-Gärtner A (1998) Transgenic oat plants obtained at high efficiency by microprojectile bombardment of leaf base segments. J Plant Physiol 152:151–157

    CAS  Google Scholar 

  • Goldman JJ, Hanna WW, Fleming G, Ozias-Akins P (2003) Fertile transgenic pearl millet [Pennisetum glaucum (L.) R. Br.] plants recovered through microprojectile bombardment and phosphinothricin selection of apical meristem-, inflorescence-, and immature embryo-derived embryogenic tissue. Plant Cell Rep 21:999–1009

    Article  CAS  PubMed  Google Scholar 

  • Havrlentová M, Faragó J, Nestáková M (2001) Regeneration of immature inflorescences of barley in vitro. Biol Plant 44:157–159

    Article  Google Scholar 

  • Jähne A, Lazzeri PA, Jäger-Gussen M, Lörz H (1991) Plant regeneration from embryogenic cell suspensions derived from anther cultures of barley (Hordeum vulgare L.). Theor Appl Genet 82:74–80

    Google Scholar 

  • Jähne A, Becker D, Brettschneider R, Lörz H (1994) Regeneration of transgenic, microspore-derived, fertile barley. Theor Appl Genet 89:525–533

    Google Scholar 

  • Jähne A, Becker D, Lörz H (1995) Genetic engineering of cereal crop plants: a review. Euphytica 85:35–44

    Google Scholar 

  • Jähne-Gärtner A, Lörz H (1996) In vitro regeneration systems of barley (Hordeum vulgare L.). Plant Tissue Cult Biotechnol 2:11–23

    Google Scholar 

  • Jelaska S, Rengel Z, Cesar V (1984) Plant regeneration from mesocotyl callus of Hordeum vulgare L. Plant Cell Rep 3:125–129

    Google Scholar 

  • Koprek T, Hänsch R, Nerlich A, Mendel RR, Schulze J (1996) Fertile transgenic barley of different cultivars obtained by adjustment of bombardment conditions to tissue response. Plant Sci 119:79–91

    CAS  Google Scholar 

  • Li H, Devaux P (2001) Enhancement of microspore culture efficiency of recalcitrant barley genotypes. Plant Cell Rep 20:475–481

    Article  CAS  Google Scholar 

  • Lührs R, Lörz H (1987) Plant regeneration in vitro from embryogenic cultures of spring- and winter-type barley (Hordeum vulgare L.) varieties. Theor Appl Genet 75:16–25

    Google Scholar 

  • Lupotto E (1984) Callus induction and plant regeneration from barley mature embryos. Ann Bot 54:523–529

    Google Scholar 

  • Manoharan M, Dahleen LS (2002) Genetic transformation of the commercial barley (Hordeum vulgare L.) cultivar Conlon by particle bombardment of callus. Plant Cell Rep 21:76–80

    Article  CAS  Google Scholar 

  • Müller B, Schulze J, Wegner U (1989) Establishment of barley cell suspension cultures of mesocotyl origin suitable for isolation of dividing protoplasts. Biochem Physiol Pflanzen 185:123–130

    Google Scholar 

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

    CAS  Google Scholar 

  • Murray F, Brettell R, Matthews P, Bishop D, Jacobsen J (2004) Comparison of Agrobacterium-mediated transformation of four barley cultivars using the GFP and GUS reporter genes. Plant Cell Rep 22:397–402

    Article  CAS  PubMed  Google Scholar 

  • Rashid VA (2002) Induction of multiple shoots by thidiazuron from caryopsis cultures of minor millet (Paspalum scrobiculatum L.) and its effect on the regeneration of embryogenic callus cultures. Plant Cell Rep 21:9–13

    Article  Google Scholar 

  • Repellin A, Baga M, Jauhar PP, Chibbar RN (2001) Genetic enrichment of cereal crops via alien gene transfer: new challenges. Plant Cell Tissue Organ Cult 64:159–183

    Google Scholar 

  • Shan X, Li D, Qu R (2000) Thidiazuron promotes in vitro regeneration of wheat and barley. In Vitro Cell Dev Biol Plant 36:207–210

    CAS  Google Scholar 

  • Thomas MR, Scott KJ (1985) Plant regeneration by somatic embryogenesis from callus initiated from immature embryos and immature inflorescences of Hordeum vulgare. J Plant Physiol 121:159–170

    CAS  Google Scholar 

  • Tingay S, McElroy D, Kalla R, Fieg S, Wang M, Thornton S, Brettell R (1997) Agrobacterium tumefaciens-mediated barley transformation. Plant J 11:1369–1376

    CAS  Google Scholar 

  • Torbert KA, Rines HW, Somers DA (1998) Transformation of oat using mature embryo-derived tissue cultures. Crop Sci 38:226–231

    Google Scholar 

  • Vasil IK (1994) Molecular improvement of cereals. Plant Mol Biol 25:925–937

    CAS  PubMed  Google Scholar 

  • Wan Y, Lemaux PG (1994) Generation of large numbers of independently transformed fertile barley plants. Plant Physiol 104:37–48

    CAS  Google Scholar 

  • Weigel RC, Hughes KW (1985) Long term regeneration by somatic embryogenesis in barley (Hordeum vulgare L.) tissue cultures derived from apical meristem explants. Plant Cell Tissue Organ Cult 5:151–162

    Google Scholar 

  • Zhang S, Zhong H, Sticklen MB (1996) Production of multiple shoots from shoot apical meristems of oat (Avena sativa L.). J Plant Physiol 148:667–671

    CAS  Google Scholar 

  • Zhang S, Williams-Carrier R, Jackson D, Lemaux PG (1998) Expression of CDC2Zm and KNOTTED1 during in vitro axillary shoot meristem proliferation and adventitious shoot meristem formation in maize (Zea mays L.) and barley (Hordeum vulgare L.). Planta 204:542–549

    CAS  PubMed  Google Scholar 

  • Zhang S, Cho M-J, Koprek T, Yun R, Bregitzer P, Lemaux PG (1999) Genetic transformation of commercial cultivars of oat (Avena sativa L.) and barley (Hordeum vulgare L.) using in vitro shoot meristematic cultures derived from germinating seedlings. Plant Cell Rep 18:959–966

    CAS  Google Scholar 

  • Zhong H, Srinivasan C, Sticklen MB (1992) In vitro morphogenesis of corn (Zea mays L.). I. Differentiation of multiple shoot clumps and somatic embryos from shoot tips. Planta 187:483–489

    CAS  Google Scholar 

  • Zhong H, Sun B, Warkentin D, Zhang S, Wu R, Wu T, Sticklen MB (1996) The competence of maize shoot meristems for integrative transformation and inherited expression of transgenes. Plant Physiol 110:1097–1107

    CAS  Google Scholar 

Download references

Acknowledgements

This work was supported by a grant through the “Forschungsschwerpunkt Agrarbiotechnologie des Landes Niedersachsen”. We are grateful to Christian Böhme and Claudia Hasse for excellent technical assistance. We thank Syngenta Seeds for providing seeds of cv. Franziska, Lochow-Petkus for cv. Lomerit, Limagrain Nickerson for cv. Duet, Semundo Saatzucht for cv. Masto and Nordsaat Saatzuchtgesellschaft for cv. Merlot.

Author information

Affiliations

  1. Department of Plant Biology, Technical University of Braunschweig, Humboldtstr. 1, 38106, Braunschweig, Germany

    Vijendra K. Sharma, Robert Hänsch, Ralf R. Mendel & Jutta Schulze

Authors
  1. Vijendra K. Sharma
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Robert Hänsch
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Ralf R. Mendel
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Jutta Schulze
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Ralf R. Mendel.

Additional information

Communicated by W. Harwood

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Sharma, V.K., Hänsch, R., Mendel, R.R. et al. A highly efficient plant regeneration system through multiple shoot differentiation from commercial cultivars of barley (Hordeum vulgare L.) using meristematic shoot segments excised from germinated mature embryos. Plant Cell Rep 23, 9–16 (2004). https://doi.org/10.1007/s00299-004-0800-4

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00299-004-0800-4

Keywords

  • Barley
  • Multiple shoots
  • Meristematic shoot segment
  • Plant regeneration
  • Shoot meristem