Protocols for Micropropagation of Woody Trees and Fruits

BAMBOO MICROPROGATION BY AXILLARY SHOOTS 467
Figure 1. Excision of bamboo nodes. A) Adult-derived plants growing in the greenhouse with
new shoots produced after severe pruning, B) Dissecting the cuttings with disinfected pruning
shears, C) Eliminating the bracts, D) Eliminating the damaged ends after sterilization.
Obtaining contaminant-free explants has been difficult in some cases (Das & Pal,
2005b; Jiménez et al., 2006; Ramanayake et al., 2006). Pre-sterilization treatments
have reduced significantly initial contamination in several cases. For a first presterilization
step, stir the nodes in 0.05% alkaline detergent Extran® MA 01 (Merck,
Darmstadt, Germany) for 10 min. Subsequently, stir the nodes for 10 min in a
mixture of bactericide Agrimycin (Pfizer) and fungicide benomyl, 2 g l –1 of each
(Jiménez et al., 2006).
For the sterilization, stir the nodes in an aqueous solution of NaOCl (1.5%),
added with one drop of Tween 80 for each 100 ml solution, for 10 min. Afterwards,
wash the explants three times with sterile distilled water in a laminar flow cabinet.
Finally, remove the damaged borders of the explants with a flamed sharp blade or
flamed pruning shears, reducing their size to 2–3 cm length (Figure 1D).
Sterilization with NaOCl can be substituted by stirring the explants in mercuric
chloride (0.1–0.3% w/v) for 5–10 min (Nadgir et al., 1984; Ramanayake &
Yakandawala, 1997; Bag et al., 2000; Marulanda et al., 2002). Sequential use of
both products has also been employed (Das & Pal, 2005a). However, NaOCl should
be preferred over mercuric chloride because the former is a less hazardous compound.