Protocols for Micropropagation of Woody Trees and Fruits

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E.C. NUNES ET AL.
explant while the epicotyl node cuttings produce on an average 4.0 nodes per
explant, which can then be excised for further multiplication and production of new
microplants. The number of nodes/explant is low when epicotyl-derived explants are
used and is affected by the slow growth and senescence after 30 days (Nunes et al.,
2002). The multiplication rates for hardwood species are low, 5 to 10 propagules per
culture cycle. However, even a multiplication factor of this order can account to
million propagules per year for many species (Haines, 1994).
2.1.3. Rooting
1. Remove the single node cuttings from 60-day-old shoots produced in the
multiplication medium.
2. Inoculate the explants on either half or full strength MS medium supplemented
with 2% (w/v) sucrose, 0.2% (w/v) Phytagel and 2.5 µM indole-3butyric
acid (IBA).
3. Incubate the cultures at 25°C, 70% RH under a 16-h photoperiod and
photosynthetic photon flux of 20–25 µmol m –2 s –1 supplied by fluorescent
light tubes.
4. Evaluate adventitious roots regeneration after 10 days of culture.
On half strength MS supplemented with 2.5 µM IBA 100% rooting ratio is achieved
at 10th day of culture and 87% at the 12th day without plant growth regulators
(Figure 1C). On full strength MS either without plant growth regulators or supplemented
with 1.25 µM IBA the maximum rooting percentages (93%) are only achieved
within 18–35th day of culture. Successful micropropagation of many woody species
is frequently limited by their recalcitrance to form adventitious roots.
2.1.4. Acclimatization
1. Remove the 30-day-old rooted regenerated plants from the test tubes, wash
roots with running water to remove Phytagel. Care should be taken to
prevent damage to roots.
2. Transfer the rooted plants to trays containing steam-sterilized medium
grade river sand covered with a polyvinyl chloride (PVC) transparent film.
3. After the first week gradually remove the PVC film from the seed trays and
expose them to the relative humidity (average 70%) of the culture room.
4. After 21 days transfer plants to 250 ml plastic pots containing a 1:1 mixture
of sand and soil. At all stages of acclimatization keep them in the culture
room, under identical culture conditions to those used for the tissue culture
studies. After 90 days acclimatization transfer the plants to the greenhouse.
After 90 days acclimatization in 1:1 mixture of sand and soil the surviving rate is
100%, the plants are in average 19.8 cm long and have 12.7 leaves (Figure 1D)
(Nunes et al., 2002).