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

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.