Protocols for Micropropagation of Woody Trees and Fruits

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R.J. ORDÁS ET AL.
Shoot development. Cotyledons were transferred and cultured onto ½ LPC for two
subcultures of 35 days each. These cultures were maintained in a growth chamber at
25 ± 1°C with a 16 h photoperiod at a photon flux of 80 ± 5 µmol m –2 s –1 provided
by white fluorescent tubes. After one subculture, small buds primordia appear along
the surface of the cotyledon (Figure 1B). After 70 days in ½ LPC, the shoots were
separated from the cotyledonary explants and elongated by sequential subculturing
in ½ LPC (Figure 1C). The excision should be as close to the cotyledon as possible,
taking care not to excise too small buds (≤ 0.5 cm) because its size is critical for
survival.
Shoot proliferation. The shoots were subcultured monthly in the expression medium
until they reached 2 cm (microshoots). During this phase, the axillary buds can also
be excised and multiplied (Figure 1D). The axillary buds do not show plagiotropic
growth. The extent of this phase is variable because of the asynchronic bud development.
Shoot growth and multiplication rates can be increased by successive subcultures
on ½ LPC medium. After 22 weeks of culture in ½ LPC, 200 microshoots
per seed can be expected.
2.4. Rooting
Root induction. Basal calli were cut and removed from elongated microshoots (>1
cm) grown on ½ LPC. The microshoots were transferred and placed vertically on ½
LPRI, inserting only the basal part. During this period shoots were incubated for 1
week in darkness at 19°C, and 1 week at 19°C under 16 h photoperiod (photosynthetic
flux is 100 µmol –2 s –1 provided by fluorescent lamps), as indicated by Potes
(personal communication).
Root development. After induction phase, the explants were transferred to ½ LPRE
and incubated at 25/19 C with 16 h photoperiod at a photon flux of 100 ± 5 µmol
m –2 s –1 °
provided by white fluorescent tubes. After 3–6 weeks under these conditions
roots appeared (Figure 2A). Although rooting rate depends on seed genotype, up to
70% of rhizogenesis can be obtained with seeds from open-pollinated trees. Based
on a 70% rooting success rate, at least 140 plantlets per seed can be expected after
29 weeks.
2.5. Acclimatisation and Hardening
Acclimatisation. After 6 weeks on expression medium, rooted shoots with at least
1 cm roots were ready to be grown in ex vitro conditions. The agar was removed
from rooted shoots by washing in tap water. The rooted shoots were transferred to a
wet peat-perlite (1:4 v/v) mixture into multipots and cultivated under decreasing
high humidity conditions according to Cortizo et al. (2004). During these acclimatisation
weeks, rooted shoots were irrigated with eight-fold diluted LP medium three
times a week.