In Vitro – Plant

, Volume 28, Issue 3, pp 132–136 | Cite as

Callus regeneration from cotyledon protoplasts ofChamaecyparis obtusa (Hinoki cypress)

  • Hamako Sasamoto
  • Akira Kondo
  • Yoshihisa Hosoi
  • Hisae Maki
  • Keiji Odani
Regular Papers


Protoplasts were isolated from cotyledons of 1- to 1.5-mo.-old seedlings ofChamaecyparis obtusa using 1% driselase or 0.25% pectolyase Y-23 in combination with 1% cellulase RS in 0.6M mannitol solution. Cell division and colony formation were induced efficiently in liquid Murashige and Skoog’s (MS) medium containing 0.6M mannitol and 10 to 30 μM 2,4-dichlorophenoxyacetic acid or 1 μM of naphthaleneacegic acid at the cell density of 1 to 2×103 ml. Continued callus proliferations was observed by transferring tissue to fresh medium of the same composition as the induction medium without mannitol. Campbell and Durzan’s medium and ammonium nitrate-free MS medium were less effective than MS medium. High concentration of benzyladenine (1 or 10 μM) was inhibitory to cell division.

Key words

protoplast culture callus regeneration conifers 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    Attree, S. M.; Dunstan, D. I.; Fowke, L. C.. Initiation of embryogenic callus and suspension cultures, and improved embryo regeneration from protoplasts, of white spruce (Picea glauca). Can J. Bot. 67:1790–1795; 1989.CrossRefGoogle Scholar
  2. 2.
    Attree, S. M.; Dunstan, D. I.; Fowke, L. C. White spruce (Picea glauca (Moench) Voss) and Black spruce (Picea mariana (Mill) B.S.P.). In: Bajaj, Y. P. S., ed. Biotechnology in agriculture and forestry, vol. 16. Tress III. Berlin: Springer-Verlag; 1991:423–445.Google Scholar
  3. 3.
    Campbell, R. A.; Durzan, D. J. Induction of multiple buds and needles in tissue culture ofPicea glauca. Can. J. Bot. 53:1652–1657; 1975.Google Scholar
  4. 4.
    David, A. Conifer protoplasts. In: Bonga, J. M.; Durzan, D. J., eds. Cell & tissue culture in forestry, vol. 2. Boston, Dordrecht: Martinus-Nijhoff Publishers. 1987:2–15.Google Scholar
  5. 5.
    Ishii, K. In vitro plantlet formation from adventitious buds on juvenile seedlings of Hinoki cypress (Chamaecyparis obtusa). Plant Cell Tissue Organ Cult. 7:247–255; 1986.CrossRefGoogle Scholar
  6. 6.
    Klimaszewska, K. Recovery of somatic embryos and plantlets from protoplast cultures ofLaxis x eurolepsis. Plant Cell Rep. 8:440–444; 1989.CrossRefGoogle Scholar
  7. 7.
    Laine, E.; David, A. Somatic embryogenesis in immature embryos and protoplasts ofPinus caribaea. Plant Sci. 69:215–224; 1990.CrossRefGoogle Scholar
  8. 8.
    McCown, B. H.; Russell, J. A. Protoplast culture of hardwoods. In: Bonga, J. M.; Durzan, D. J., eds. Cell & tissue culture in forestry, vol. 2. Boston, Dordrecht: Martinus-Nijhoff Publishers. 1987:16–30.Google Scholar
  9. 9.
    Murashige, T.; Skoog, F. A revised medium for rapid growth and bioassay with tobacco tissue cultures. Physiol. Plant. 15:473–497; 1962.CrossRefGoogle Scholar
  10. 10.
    Sasamoto, H.; Hosoi, Y. Somatic embryogenesis in suspension cultures ofQuercus serrata Thumb. J. Jpn. For. Soc. 71:20–22; 1989.Google Scholar
  11. 11.
    Sasamoto, H.; Hosoi, Y. Effects of electric fusion on the calli formation and regeneration from the protoplasts ofQuercus and twoPopulus species. Amsterdam: VIIth Int. Congr. Plant Tissue and Cell Culture; 1990:36.Google Scholar
  12. 12.
    Sasamoto, H.; Hosoi, Y. Callus proliferation from the protoplasts of embryogenic cells ofQuercus serrata. Plant Cell Tissue Organ Cult. 29:241–245; 1992.CrossRefGoogle Scholar
  13. 13.
    Sasamoto, H.; Hosoi, Y.; Ishii, K., et al. Effects of plant growth regulators on organogenesis and callus formation from in vitro cultures of poplar. Transactions 99th Meeting of the Japan Forest Society 1988:449–450 (in Japanese).Google Scholar
  14. 14.
    Sasamoto, H.; Hosoi, Y.; Ishii, K., et al. Factors affecting the formation of callus from leaf protoplasts ofPopulus alba. J. Jpn. For. Sci. 71:449–455; 1989.Google Scholar
  15. 15.
    Sasamoto, H.; Grinwald, R.; Kondo A., et al. Cell culture ofChamaecyparis obtusa, Pinus densflora & Pinus thunbergii. 102th Mtg. Jpn. For. Soc. 1990:89 (in Japanese).Google Scholar

Copyright information

© Tissue Culture Association 1992

Authors and Affiliations

  • Hamako Sasamoto
    • 1
  • Akira Kondo
    • 2
  • Yoshihisa Hosoi
    • 1
  • Hisae Maki
    • 1
  • Keiji Odani
    • 1
  1. 1.Forestry and Forest Products Research InstituteMinistry of Agriculture, Forestry and FisheriesTsukuba, IbarakiJapan
  2. 2.Forestry and Forest Products Research InstituteShizuoka Prefecture, Hamakita, ShizuokaJapan

Personalised recommendations