Germination of mature seeds of Calanthe tricarinata Lindl., an endangered terrestrial orchid, by asymbiotic culture in vitro

  • Toshinari Godo
  • Miho Komori
  • Emi Nakaoki
  • Tomohisa Yukawa
  • Kazumitsu Miyoshi


The effects of culture conditions on the asymbiotic germination of mature seeds of Calanthe tricarinata Lindl., an endangered terrestrial cool-climate orchid, were examined. Specifically, conditions such as illumination, temperature, and the addition of plant growth regulators to the medium were studied. Mature seeds were harvested from plants that had been collected in Toyama Prefecture, Japan, and maintained at the Botanic Gardens of Toyama. Solidified “New Dogashima” medium was used as the basal medium, and it was supplemented with 6-benzyladenopurine (BA) or α-naphthalene acetic acid (NAA). White light at 40 µmol m−2 s−1, with a 16-h photoperiod, inhibited the germination of seeds by 53–80%, as compared to dark controls in genotypes examined. The optimal temperature for the germination of seeds in darkness was 20°C and the germination frequency reached 60%, whereas it was only 28% at 25°C. While both NAA and BA stimulated germination, BA was more effective than NAA. After storage for 18 mo at 5°C, seeds incubated on medium that contained 0.2 mg l−1 BA germinated at a frequency of 36%, which was twice that of seeds grown without any plant growth regulators. The frequency of subsequent germination decreased during storage of seeds at 5°C for approximately 2 yr, dropping from 61% to 13%. The protocorms obtained in this study were developed to plantlets readily after transferring to fresh 1/2 MS medium without any plant growth regulators. They were successfully acclimatized in green house after two to three subcultures in vitro. The significant role of a reproducible protocol for the germination of mature seeds is discussed in terms of the ex situ conservation of endangered orchid species.


Asymbiotic seed germination 6-Benzyladenopurine Conservation Illumination Temperature 



This research was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology, Japan.


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Copyright information

© The Society for In Vitro Biology 2010

Authors and Affiliations

  • Toshinari Godo
    • 1
  • Miho Komori
    • 1
  • Emi Nakaoki
    • 2
  • Tomohisa Yukawa
    • 3
  • Kazumitsu Miyoshi
    • 4
  1. 1.Botanic Gardens of ToyamaFuchu-machiJapan
  2. 2.Toyama Prefectural UniversityImizu-shiJapan
  3. 3.Tsukuba Botanical GardenThe National Science MuseumTsukubaJapan
  4. 4.Faculty Bioresource SciencesAkita Prefectural UniversityAkitaJapan

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