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5.2.3 Explant comparison
In vitro culture initiation and multiplication
R. leipoldtii seeds were used, not only because of their high germination and in vitro
shooting in preliminary seedling organ experiments, but also because this species
has the smallest seeds. If embryo excision is possible for a species with such a small
seed it shows that seed size is not a limitation to using embryo excision in a culture
initiation protocol for Romulea species.
Seeds (200) were surface sterilised and germinated at 15°C as with in vitro
germination experiments described in Chapter 4. After 2 months when sufficient
seeds germinated, 130 seedling hypocotyls and 130 embryos were excised
simultaneously. Seedlings with stems longer than 30 mm were then removed from
tubes and placed on Petri dishes and cut into 3 sections using a sterilised scalpel
and blade. For this experiment only the hypocotyl was used because of its higher
percentage in vitro response. The seedling hypocotyls and embryos were then
placed in 33 ml culture tubes with 10 ml nutrient media with 13 medium treatments, a
medium with no plant growth regulators, media with 2.3, 4.7 or 23.2 µM kinetin or
mTR and media supplemented with 2.3, 4.7 or 23.2 µM kinetin or mTR in
combination with 0.5 M NAA. Ten replicate explants were used per treatment and
the experiment was repeated twice. After two months the number of shoots formed
per explant and the presence or absence of roots was recorded.
5.2.4 Shoot multiplication
R. sabulosa shoots initiated and multiplied with 23.2 M kinetin were placed in tubes
containing MS media supplemented with three different kinetin concentrations (2.3,
4.7 and 23.2 M) used for shoot initiation. These tubes were then placed at 25°C in a
growth room with 16 hour light and a growth room with 24 hour light to test the effect
of photoperiod on shoot multiplication rate. The effect of temperature was examined
by placing tubes with shoots on MS media supplemented with 23.2 M kinetin in
growth chambers with a 16/8 light/dark photoperiod set at a range of temperatures
from 10°C to 30°C under 3.4 µmol m –2 s –1 light Osram ® 75 W cool white fluorescent
tubes with a 16/8 light/dark photoperiod. These experiments were repeated twice and
10 explants were used per treatment. After two months the number of shoots formed
was recorded.
120