Protocols for Micropropagation of Woody Trees and Fruits

ORGANOGENESIS AND CRYOPRESERVATION OF RADIATA PINE
Table 1. Example of shoot amplification in genotypes from a group of control-pollinated
crosses following 5 months of culture and transfer.
Cross Explant total at transfer
Genotype
no. tested January February April May
1 6 16 22 92 216
2 9 19 37 140 368
3 9 18 40 127 301
4 9 18 29 109 273
5 9 20 34 109 272
6 9 22 33 157 346
7 9 20 33 101 214
8 9 21 30 105 242
9 5 10 17 56 144
Total
explants 164 275 996 2376
Shoots/explants per
initial (Jan.) explant 1 1.67 6.07 13.87
Following 4 weeks growth, shoots are transferred to LPch (80 mL) in 600 mL glass
Agee jars (Australian Glass Company, Penrose, Auckland) with clear lids. The elongated
epicotyl shoot is cut from the hypocotyl at this stage so that fresh stem tissue
is in contact with the medium. Following a further 4–6 weeks growth, these shoots
are sufficiently elongated (30–50 mm) to be divided into shoot tips and stem
segments (Figure 2). Shoot tips (approximately 20 mm) are transferred to LPch in
jars for further elongation. Stem segments (approximately 10 mm) are put in fresh
deep Petri dishes of LPch. Stem segments frequently produce side shoots, which
further enhance amplification (Figure 3). This transfer process is repeated every 4 to
6 weeks until sufficient shoots are obtained for rooting. Amplification rates vary
between crosses and genotypes within crosses (Table 1). From initiation through all
subsequent subculturing stages, any shoots that appeared “wet” or hyperhydric, indicating
reduced cuticular wax, are discarded (Aitken-Christie et al., 1985; Debergh
et al., 1992). In general, free water should be avoided in culture vessels throughout
the multiplication stages to ensure optimal growth of cultures. Sources of free water
include very fresh media and condensation (Petri dishes, jars, and plastic bottles).
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