Protocols for Micropropagation of Woody Trees and Fruits

ORGANOGENESIS AND CRYOPRESERVATION OF RADIATA PINE
2.1.5. Rooting and Hardening
Root initiation and subsequent growth. When sufficient shoot multiplication has
been achieved with serial culture, shoots are prepared for root initiation. Shoot tips
are given a fresh base cut to remove any callus and cut to a length of 20–30 mm,
followed by an in vitro auxin treatment for 12–14 days on a Greshoff and Doy
medium (as modified by Sommer et al., 1975 and Horgan & Holland, 1989),
containing 2% sucrose, 0.5 mg naphthalene acetic acid.L –1 , and 1.0 mg indole
butyric acid.L –1 . Following auxin treatment, shoots are individually transferred (set)
into trays of peat:perlite:sand (1:1:1) (Figure 4A). High humidity (90 +/- 10%) is
maintained for 1–2 weeks with a combination of vented plastic lids and handmisting
twice daily (Figure 4B). Set shoots are held under light and temperature
conditions similar to those described earlier for in vitro growth. After 1–2 weeks,
shoots are gradually hardened off to ambient humidity conditions (60 +/– 10%).
Four to six weeks after setting, shoots are assessed for root formation (Figure 4C)
and potted up, using the same potting mix as used for setting, with the addition of
.L –1
5g Osmocote (Figure 4D). The plant container is a rigid plastic container, such as
HIKO V90 or Lannen 63F if the plants are going to be lined out later in a bare-root
nursery bed, or into larger containers, such as HIKO V150 for growing on as a
container plant. Plants are transferred to a greenhouse and after 8 weeks placed in a
nursery shade house until planted out into the nursery bed (15 × 15 cm spacing)
(Figure 5A). Depending on season and site where plants are to be established in the
field, plants may undergo root cutting treatments while in the nursery bed to
improve root mass prior to planting (Figure 5B).
Root initiation and subsequent growth (adventitious-axillary). Adventitious-axillary
shoots are treated in the same way as epicotyl-axillary shoots for root initiation and
through all phases of nursery handling. Rooting tends to be 1–2 weeks behind that
of epicotyl-axillary shoots and this is accommodated in the setting environment.
Transfer to containers occurs only after shoots have roots at least 10 mm long.
2.1.6. Field Testing
Growth in the nursery. If the plantlets are being raised full-term in containers, they
are treated like a seedling or cutting crop, with regular watering, and application of
fertilisers, fungicides, and pesticides as required through the overhead boom watering
system (Menzies et al., 2001). If the plantlets are raised initially in smaller containers
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