Protocols for Micropropagation of Woody Trees and Fruits

ORGANOGENESIS AND CRYOPRESERVATION OF RADIATA PINE
and then lined out bare-root, they are transplanted into the nursery bed in late spring/
early summer (November/December), once the roots have bound the container plugs.
They are then raised like a seedling or cutting crop, with root conditioning by undercutting,
wrenching and lateral root pruning, and may also be topped if plant height
exceeds 35–40 cm (Menzies et al., 2005). After one growing season, they will reach
a plantable size of at least 30–40 cm (Hargreaves et al., 2005).
Growth in the forest. In an early trial planted on five sites in 1983, tissue-cultured
plantlets performed as if they were physiologically aged (Menzies et al., 2001),
despite being initiated from seed. This was possibly because the organogenesis protocols
were still being developed when the plants were produced. Further experience
and commercialisation of the tissue-culture systems has improved the protocols, and
ensured more uniform and fast early growth (Gleed, 1993). A recent assessment of a
field trial established in 1984 (now aged 22 years) has shown that tissue-cultured
plantlets were not significantly smaller in diameter than similar genetic quality seedlings
and juvenile cuttings (Menzies & Low, unpublished data). Current protocols
for organogenesis using axillary shoot multiplication have produced very juvenile
plants (Hargreaves et al., 2005), and these are now being evaluated in a field trial.
2.1.7. Storage of in-vitro Cultures (Cryopreservation)
Cryopreservation of shoot tip meristems. Successful methods for cryopreservation of
radiata pine shoot tip meristems have been difficult to develop. The method presented
here was the most successful in a series of 7 preliminary experiments which
included a variety of pre-conditioning treatments and regrowth media. The plant
material used was a single commercial clone (Christmas Star), there were from 8–65
meristems in each experiment, and all meristems were exposed to liquid nitrogen.
The best result was where 12.5% of cryopreserved meristems re-established shoot
cultures. This research has subsequently provided a platform for improved results
which will be published at a later date (Hargreaves, Towill & Bonnart, 2007).
Shoot tip meristems were isolated from in-vitro cultures of Christmas Star
(Longview Horticulture, VIC, Australia). Shoots were growing in standard conditions
described earlier for shoot multiplication. Meristems were dissected with a
stereo microscope, with needles being plucked off until the apical dome of the
primary meristem was visible, surrounded by a few needle primordia. Explants were
approximately 2 × 1 mm.
Isolated shoot tip meristems were collected on dishes of LP medium (no
charcoal) and then placed in cryoprotectant preconditioning medium (PGD, modified
after Ryynänen, 1996) in 25 ml flasks, at room temperature. Following 2 hours
exposure to preconditioning medium, individual meristems were placed into 3 separate
drops (5–10 µl) of the PGD solution on 5 × 10 mm strips of aluminium foil.
Nunc cryogenic vials were filled with liquid nitrogen and placed in a polystyrene
box containing liquid nitrogen. The aluminium foil strips with adhering droplets
containing meristems were then plunged vertically into these pre-cooled cryogenic
vials. In this manner, meristems were rapidly frozen to –196°C.
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