Protocols for Micropropagation of Woody Trees and Fruits

468 V.M. JIMÉNEZ AND E. GUEVARA
2.2. Culture Medium
2.2.1. Medium Composition
Murashige & Skoog (MS) mineral salts and vitamins (Murashige & Skoog, 1962),
as described in Table 1, work for most of genera and species evaluated up to date.
Plant growth regulators (PGRs) composition varies according to the culture step
(bud break, development of lateral shoots or of roots) and on the species under
study, and is referred in Tables 3 to 5. Gelrite is preferred over agar as gelling agent
because it forms a more transparent gel than agar, facilitating detection of contamination
in earlier stages.
Component Stock solution Amount to add Final concentration
per liter in medium
MS macronutrients 10X 100 ml 1X
MS micronutrients 100X 10 ml 1X
NaFeEDTA* 7.34 mg ml –1 5 ml 36.7 mg l –1
Thiamine-HCl 0.1 mg ml –1 1 ml 0.1 mg l –1
Nicotinic acid 0.1 mg ml –1 5 ml 0.5 mg l –1
Pyridoxine 0.1 mg ml –1 5 ml 0.5 mg l –1
Myo-Inositol 10 mg ml –1 10 ml 100 mg l –1
Glycine 2 mg ml –1 1 ml 2 mg l –1
Table 1. Formulation of culture medium frequently used for bamboo micropropagation
through axillary bud proliferation.
PGR(s) variable variable variable
Sucrose 30 g 3%
Gelrite 2 g 0.2%
* Sodium iron (III) ethylenediamnetetraacetic acid.
2.2.2. Medium Preparation
To prepare one liter medium follow the next steps. Scale for smaller or larger
volumes. Add appropriate amount of stock solutions of all ingredients to ~400 ml
distilled water as indicated in Table 1 (filter-sterilized PGRs can also be added after
autoclaving). To address serious contamination problem (especially latent endogenous
contamination), addition of 2 ml l –1 Plant Preservative Mixture ® (PPM, Plant Cell
Technology, Washington DC, USA) (Jiménez et al., 2006) or 1 g l –1 benomyl
(Ramanayake and Yakandawala, 1997) prior to autoclaving might be useful. Add
sucrose and stir well until it dissolves completely, raise volume up to one liter,
adjust pH to 5.8 with KOH or HCl, and add gelrite. Heat it in the microwave for
approximately 7 min for each liter of medium (1300 W) and stir well with a glass
stick. Continue heating at 2–3 min interval, stir intermittently. Dispense the medium
into the culture vessels only after it has boiled at least twice. Autoclave at 121°C and
1.05 kg cm –2 for the corresponding time specified in Table 2. Alternatively, medium
can be autoclaved in larger volumes and then be poured into sterile vessels in the
laminar flow cabinet.