Protocols for Micropropagation of Woody Trees and Fruits

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M. PASQUAL AND E.A. FERREIRA
chemical substances used for decontamination. The traces of these chemicals
can lead to the synthesis of phenolic substances. The rinsing of the sterilized
explants also removes any accumulating oxidise phenols.
Precautions during the establishment
1. Frequent explant transfer – when the explants do not show any sign of
growth after 3 to 4 weeks and there are signs of oxidation, the chances of
their survival can be increased by frequent transfer of explants to a fresh
culture medium. Similarly, frequent subculture of explants is required when
the culture medium around the explant becomes discoloured or darkened.
Darkening is most visible in solid medium and it is concentrated on the
edges of the explant. The interval of transference must be adjusted according
to the severity of the problem. Generally, subculture of explants is necessary
every 1 to 7 days.
2. Removal of darkened tissue – in each subculture the darkened tissue must
be removed.
3. Addition of activated charcoal to the medium – the addition of activated
charcoal hinders the accumulation of phenolic inhibitors. However it can
also adsorb growth regulators and other components of the medium and be
toxic to some tissues. Activated charcoal is normally added to the culture
medium at concentrations varying from 0.2% to 3%, although it can promote
or inhibit growth in vitro, depending on the species and tissue.
2.8. Shoot Multiplication
For ‘Roxo de Valinhos’ fig cultivar, WPM culture medium supplemented with 0.5
mg L –1 BA or 0.5 mg L –1 kinetin is efficient for shoot multiplication. Long shoots of
Ficus carica ‘Kalamon’ were obtained after 8 weeks on the medium containing 0.5
mg L –1 BA (Pontikis & Melas, 1986). For the in vitro proliferation of cultivars
‘Brown Turkey’ and ‘Smyrna’ and their subsequent use in genetic transformation,
Yancheva et al. (2005) used MS medium supplemented with 0.25 mg L –1 BA, 0.05
mg L –1 IBA and 0.05 mg L –1 GA3. The shoots were placed horizontally in shoot
proliferation medium. The cultures were maintained in the culture room, illuminated
by white fluorescent light (32 µM m –1 s –1 ) for 16-h photoperiod at 25 C for 4 to 5
weeks before removing the explant leaves. The highest shoot regeneration rate
achieved was on the basic MS medium amended with 1 mg L –1 thiamine–HCl, 4%
sucrose, 2.0 mg L –1 thidiazuron (TDZ), and 2 mg L –1 °
IBA. The regeneration depended
directly on the explant orientation (Table 2).
2.9. Rooting
For root induction and growth of ‘Roxo de Valinhos’ fig cultivar, the addition of
auxins to the culture medium is not necessary (Brum, 2001; Fráguas, 2004). However,
the rooting of ‘Sarilop’ cultivar clones is successful on MS medium amended with
1.2 and 2.5 µM IBA or NAA (Hepaksoy & Aksoy, 2006) (Table 3).