Protocols for Micropropagation of Woody Trees and Fruits

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A. GIOVANELLI AND A. DE CARLO
2.1.3. Disinfection of Plant Material
Disinfection of explants is an important step to establish effective shoot cultures of
Mediterranean cypress. Recently, effective methods of disinfection have been developed
for the various types of explants.
Seeds. Before disinfection, seeds are imbibed for 24 h under running tap water.
Filled seeds are treated for 1–2 min with 70% ethanol, followed by 10 min in 0.1%
HgCl2. Then, the seeds are rinsed three times with sterile deionized water (Lambardi
et al., 1995).
Shoot tips from seedlings. Five weeks after seed germination, the seedlings (20–30
mm in length) are disinfected by immersion in 70% ethanol for 1 min, followed by
soaking in 1% sodium hypoclorite for 15 min. Disinfected explants are then rinsed
four times in sterile deionized water. In our experience, following these methods, the
rate of contamination is maintained lower than 5%. Moreover, it has been reported
that shoot tips collected from older seedlings (18 months) can require a multi-step
treatment to achieve good decontamination, e.g., (i) an initial wash of the shoot tips
for 10 min in running tap water, (ii) one rinse in distilled water for 15 min, (iii) 10–
min disinfection in H2O2 (30% v/v), containing 0.025% (v/v) Tween 20, (iv) additional
20 min in 20% (v/v) commercial bleach in tap water, followed by (v) a final
rinse of explants (Spanos et al., 1997).
Shoots from adult trees. After collection from adult trees grown in the field, the
apical shoots are immediately treated with a 0.1% solution of Benomyl ® and, after
rinsing, stored at 4°C until used. Potted stock plants, maintained under greenhouse
conditions, are a better source of explants, as the plant can be periodically treated
(even weekly) with a 0.1% solution of Benomyl ® before the shoot tips collection.
The apical shoots (30–50 mm long) from both the provenances are washed under
running tap water for at least 60 min, before to be disinfected by immersion in 70%
ethanol for 2 min and successive soaking for 20 min in a 1.5% sodium hypochlorite
solution, containing drops of Tween 20 as wetting agent. The explants are finally
rinsed four times in sterile deionized water. Following this procedure, over 30%
fungal and bacterial contamination has still obtained with explants from trees
growing in the field, whilst only a 5% of bacterial infection has recorded on shoots
collected from grafted plants grown in greenhouse (unpublished data).
2.2. In Vitro Culture
2.2.1. Culture Media
Modifications of SH (Schenk & Hildebrandt, 1972), MS (Murashige & Skoog, 1962),
DCR (Gupta & Durzan, 1985), QP (Quoirin & Lepoivre, 1977) and AE (von Arnold &
Eriksson, 1981) culture media have been used in different regeneration systems of
Mediterranean cypress (see below), according to the formulations reported in Table 1.