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5.2 MATERIALS AND METHODS
In vitro culture initiation and multiplication
Seeds were obtained from Silverhill Nurseries, Kenilworth, South Africa and African
Bulbs, Napier, South Africa. Although using seedling organ explants is common for
culture initiation in the Iridaceae, seeds of the attractive and vulnerable R. sabulosa
did not germinate and some other attractive species had low germination. Because
these experiments were done before the cold stratification experiments described in
Chapter 4, germination was slow. In an attempt to obtain cultures of all species in a
shorter time embryo rescue techniques were employed for eight species.
If not stated otherwise, a MS medium supplemented with 100 mg.l -1 myo-inositol and
3% sucrose, with pH adjusted to 5.7 and solidified with 0.8% agar was used. All
experiments were conducted in a laminar flow hood and cultures were placed in a
growth room set at 25°C under 4.3 µmol m –2 s –1 light using Osram ® 75 W cool white
fluorescent tubes with a 16/8 light/dark photoperiod. The duration of all experiments
was two months. Shoots multiplied for further experiments were subcultured onto the
medium that produced the best shooting response of the initial explant every two
months.
5.2.1 Explants from seedlings
Seedlings from initial in vitro germination experiments described in Chapter 4 were
used for a small experiment to assess the responsiveness of the species of which
seeds germinated. Only 5 replicates were used per treatment, as the availability of
seeds of Romulea species was very limited at the time and therefore only small
quantities could be purchased.
Seedlings with stems longer than 30 mm were removed from the culture tubes using
autoclaved forceps. The seedlings were then placed on Petri dishes and cut into
three sections using a scalpel and blade. They were divided into shoot (>10 mm),
hypocotyl (10 mm) and root (>10 mm) sections. For hypocotyl explants the remainder
of the seed was removed. All seedling organs were placed in 33 ml culture tubes with
10 ml MS media supplemented with various plant growth regulators.
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