Skip to main content
SpringerLink
Log in

Stable genetic transformation of Larix gmelinii L. by particle bombardment of zygotic embryos

  • Genetic Transformation and Hybridization
  • Published:
Plant Cell Reports Aims and scope Submit manuscript

Abstract

We report a new protocol for the stable transformation of Larix gmelinii. Thirty mature zygotic embryos precultured for 3 days on solid medium supplemented with benzyladenine were bombarded with plasmids pUC-GHG (GUS, HPT, and GFP genes) or pBI221-HPT (HPT and GUS genes). After a 2-month culture on selection medium, hygromycin-resistant calli appeared on the surfaces of the necrotic embryos. The frequencies of embryos with resistant calli were 18.4% and 17.4% in the transformations with pUC-GHG and pBI221-HPT DNA, respectively. More than 20 adventitious shoots formed from each of the transgenic calli. Of 17 elongated shoots selected for culturing on a rooting medium, five shoots rooted after 2 months. Expression of the GFP and GUS genes was detected in the resistant tissues by microscopic observations and by a histological GUS activity assay, respectively. PCR and Southern analysis confirmed the stable insertion of the introduced DNA into the genome.

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

Access this article

Log in via an institution

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
Fig. 5
Fig. 6

Similar content being viewed by others

Abbreviations

BA:

Benzyladenine

GFP:

Green fluorescent protein

GUS:

β-Glucuronidase

HPT:

Hygromycin phosphotransferase

IBA:

Indole-3-butyric acid

NAA:

α-Naphthaleneacetic acid

NOSt:

Terminator of nopaline synthase gene

35Sp:

Promoter of 35S RNA of cauliflower mosaic virus

References

  • Akama K, Shiraishi H, Ohta S, Nakamura K, Okada K, Shimura Y (1992) Efficient transformation of Arabidopsis thaliana: comparison of the efficiencies with various organs, plant ecotypes and Agrobacterium strains. Plant Cell Rep 12:7–11

    Google Scholar 

  • Bishop-Hurley SL, Zabkiewicz RJ, Grace L, Gardner RC, Wagner A, Walter C (2001) Conifer genetic engineering: transgenic Pinus radiata (D. Don) and Picea abies (Karst) plants are resistant to the herbicide Buster. Plant Cell Rep 20:235–243

    Article  CAS  Google Scholar 

  • Duchesne LC, Lelu MA, Aderkas PV, Charest PJ (1993) Microprojectile-mediated DNA delivery in haploid and diploid embryogenic cells of Larix spp. Can J For Res 23:312–316

    Google Scholar 

  • Gupta P, Durzan D (1985) Shoot multiplication from mature trees of Douglas-fir cotyledons. Plant Cell Rep 4:177–179

    Google Scholar 

  • Harry I, Thorpe T (1994) Regeneration of plantlets through organogenesis from mature embryos of jack pine. Plant Cell Tissue Organ Cult 37:159–164

    Google Scholar 

  • Huang Y, Diner AM, Karnosky DF (1991) Agrobacterium rhizogenes-mediated genetic transformation and regeneration of a conifer: Larix decidua. In Vitro Cell Bio 27:201–207

    Google Scholar 

  • Ismail G, Schnabl H, Zoglauer K, Boehm R (2004) Agrobacterium-mediated transformation of Larix decidua: an assessment of factors influencing the efficiency of gus gene transfer. J Appl Bot Food Qual Angew Bot 78:83–90

    Google Scholar 

  • Jefferson RA, Kavanagh TA, Bevan MW (1987) GUS fusion: β-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J 6:3901–3907

    CAS  PubMed  Google Scholar 

  • Klimaszewska K, Devantier Y, Lachance D, Lelu MA, Charest PJ (1997) Larix laricina (tamarack) somatic embryogenesis and genetic transformation. Can J For Res 27:538–550

    Article  Google Scholar 

  • Klimaszewska K, Lachance D, Bernier-Cardou M, Rutledge RG (2003) Transgene integration patterns and expression levels in transgenic tissue lines of Picea mariana, P. glauca and P. abies. Plant Cell Rep 21:1080–1087

    Article  CAS  PubMed  Google Scholar 

  • Levee V, Lelu MA, Jouanin L, Cornu D, Pilate G (1997) Agrobacterium tumefaciens -mediated transformation of hybrid larch (Larix kaempferi × L. decidua) and transgenic plant regeneration. Plant Cell Rep 16:680–685

    Article  Google Scholar 

  • Lin XF, Zhang WB, Takano H, Takio S, Ono K (2004) Efficient plant regeneration and micropropagation from callus derived from mature zygotic embryos of Larix gmelinii. Plant Biotechnol 21:159–163

    Google Scholar 

  • Mathur G, Nadgauda R (1999) In vitro plantlet regeneration from mature zygotic embryos of Pinus wallichiana A.B. Jacks. Plant Cell Rep 19:74–80

    Google Scholar 

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

    CAS  Google Scholar 

  • Niwa Y, Hirano T, Yoshimoto K, Shimizu M, Kobayashi H (1999) Non-invasive quantitative detection and applications of non-toxic, S65T-type green fluorescent protein in living plants. Plant J 18:455–463

    Article  CAS  PubMed  Google Scholar 

  • Pulido C, Harry I, Thorpe T (1990) In vitro regeneration of plantlets of Canary Island pine (Pinus canariensis). Can J For Res 20:1200–1211

    Google Scholar 

  • Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning. A laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor

  • Shin DI, Podila GK, Huang YH, Karnosky D (1994) Transgenic larch expressing genes for herbicide and insect resistance. Can J For Res 24:2059–2067

    Google Scholar 

  • Takahashi M, Nishihara M, Yamamura S, Nishizawa S, Irifune K, Morikawa H (1998) Stable transformation of Eustoma grandiflorum by particle bombardment. Plant Cell Rep 17:504–507

    Google Scholar 

  • Tang W, Newton RJ (2003) Genetic transformation of conifers and its application in forest biotechnology. Plant Cell Rep 22:1–15

    Google Scholar 

  • Tang W, Tian YC (2003) Transgenic loblolly pine (Pinus taeda L) plants expressing a modified δ-endotoxin gene of Bacillus thuringiensis with enhanced resistance to Dendrolimus punctatus Walker and Crypyothelea formosicola Staud. J Exp Bot 54:835–844

    Google Scholar 

  • Tian L, Seguin A, Charest PJ (1997) Expression of the green fluorescent protein gene in conifer tissues. Plant Cell Rep 16:267–271

    Article  CAS  Google Scholar 

  • Walter C, Grace LJ, Wagner A, White DWR, Walden AR, Donaldson SS, Hinton H, Gardner RC, Smith DR (1998) Stable transformation and regeneration of the transgenic plants of Pinus radiata D. Don. Plant Cell Rep 17:460–468

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank Dr. K. Nakamura (Nagoya University) and Dr. Y. Niwa (University of Shizuoka) for kindly providing us with the pBI101-GUS-HPT and pUC-pCaMV35S-sGFP (S65T)-nos3′ vectors, respectively. This study was supported in part by Grants-in-Aid for the Encouragement of Young Scientists to HT from the Japan Society for the Promotion of Science, by the Program for the Promotion of Basic Research Activities for Innovative Biosciences (PROBRAIN to HT), and by the 21st Century COE program of Pulsed Power Science (H T)

Author information

Authors and Affiliations

  1. Graduate School of Science and Technology, Kumamoto University, Kurokami, Kumamoto, 860-8555, Japan

    Xiaofei Lin, Wenbo Zhang, Katsuaki Takechi & Hiroyoshi Takano

  2. Center for Marine Environment Studies, Kumamoto University, Kurokami, Kumamoto, 860-8555, Japan

    Susumu Takio

  3. Department of Biological Science, Faculty of Science, Kumamoto University, Kurokami, Kumamoto, 860-8555, Japan

    Kanji Ono

Authors
  1. Xiaofei Lin
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Wenbo Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Katsuaki Takechi
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Susumu Takio
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Kanji Ono
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Hiroyoshi Takano
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Xiaofei Lin.

Additional information

Communicated by S.A. Merkle

Rights and permissions

Reprints and permissions

About this article

Cite this article

Lin, X., Zhang, W., Takechi, K. et al. Stable genetic transformation of Larix gmelinii L. by particle bombardment of zygotic embryos. Plant Cell Rep 24, 418–425 (2005). https://doi.org/10.1007/s00299-005-0955-7

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00299-005-0955-7

Keywords

Search

Navigation