Ultrastructural and biochemical alterations during browning of pigeon orchid (Dendrobium crumenatum Swartz) callus

  • Paveena Kaewubon
  • Nongporn Hutadilok-Towatana
  • Jaime A. Teixeira da Silva
  • Upatham Meesawat
Original Paper


The phenomenon of browning can be a major limitation for orchid callus culture, causing a loss of regenerative capacity and subsequent cell death. This research was conducted to determine the effect of in vitro culture period (1–3 months) on the appearance of tissue browning. Biological alterations at the cellular and subcellular levels, as well as biochemical aspects, were examined. Callus derived from bisected pigeon orchid (Dendrobium crumenatum Swartz) protocorms were cultured on modified Vacin and Went solid medium supplemented with 0.5 mg L−1 1-naphthaleneacetic acid and 1 mg L−1 6-benzyladenine. Callus that was not subcultured was collected at 1-month intervals and examined for structural (using scanning electron microscopy and transmission electron microscopy) and biochemical alterations associated with browning. Three-month-old unsubcultured callus cells were loosely arranged and major organelles were deformed, exhibiting nuclear envelope breakage, dysfunctional mitochondria, tannin-filled vesicles and swollen chloroplasts, relative to 1-month-old green callus, which served as the control. Ultrastructural disorganization involving the nucleus, mitochondria and chloroplasts typified enzymatic oxidative browning. Browning 3-month-old callus had significantly higher polyphenol oxidase (PPO) activity and total phenolic content than control callus. The levels of PPO and total phenolics may serve as useful biochemical markers when selecting suitable callus for subsequent regeneration trials or for particle bombardment in genetic transformation experiments.


Browning Phenolic content Pigeon orchid Polyphenol oxidase Swollen chloroplast Tannin 







Callus induction medium


1-Naphthaleneacetic acid


Phenylanine ammonia lyase


Periodic acid-Schiff


Programmed cell death




Polyphenol oxidase


Rough endoplasmic reticulum (ER)


Reactive oxygen species


Smooth ER


Sodium dodecyl sulfate


Toluidine blue O


Vacin and Went medium



This work was supported by the Department of Biology at Faculty of Science, Graduate School of Prince of Songkla University and by a grant from Science Achievement Scholarship of Thailand (SAST) (28/2554). We thank Dr. Brian Hodgson from the Faculty of Pharmaceutical Science, Prince of Songkla University, for assistance with the English of the manuscript.


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Copyright information

© Springer Science+Business Media Dordrecht 2014

Authors and Affiliations

  • Paveena Kaewubon
    • 1
  • Nongporn Hutadilok-Towatana
    • 2
    • 3
  • Jaime A. Teixeira da Silva
    • 4
  • Upatham Meesawat
    • 1
  1. 1.Department of Biology, Faculty of SciencePrince of Songkla UniversityHat YaiThailand
  2. 2.Department of Biochemistry, Faculty of SciencePrince of Songkla UniversityHat YaiThailand
  3. 3.Natural Product Research Center of ExcellencePrince of Songkla UniversityHat YaiThailand
  4. 4.Kagawa-kenJapan

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