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Germination physiology
companies. The availability of seeds of Romulea species at these nurseries was very
limited and therefore only a small quantity could be purchased. Seeds of all Romulea
species examined in this study were about one-year-old. To understand the basic
physiological functions of these seeds, water content, imbibition rate and the viability
of seeds both in vitro and ex vitro were determined. Seed surface and micropylar
regions of these species were observed using a scanning electron microscope.
4.2.1 Viability tests
2,3,5-Triphenyl tetrazolium chloride (TTC) and embryo excision tests were conducted
to examine seed viability. For the TTC test, the seeds were soaked in 1% TTC
solution for one week in a glass vial and kept under constant dark conditions at 25 ±
0.5°C. Subsequently, these seeds were cut into two and the percentage and degree
of staining of the embryo and endosperm portions were recorded (INTERNATIONAL
SEED TESTING ASSOCIATION, 1999). This test had four replicates of 10 seeds
each.
To excise the embryos, the seeds were first surface-decontaminated with 1.75%
sodium hypochlorite solution with a few drops of Tween 20 for 15 min. Thereafter
they were rinsed three times with sterile distilled water (ASCOUGH et al., 2007).
Decontaminated seeds were placed in an autoclaved Petri dish with sterile distilled
water. The seeds were left to imbibe in the laminar flow cabinet for 4 to 5 days. For
embryo excision, slices of testa and endosperm were separated from the seed using
a scalpel with a sterile blade, until the embryo was visible. Small pieces of
endosperm around the embryo were carefully removed with a dissection needle. The
embryo was then lifted from the endosperm with the help of a sterile dissecting
needle and placed in an autoclaved Petri dish, filled with sterile distilled water to rinse
off residual pieces of endosperm. Embryos were then placed into 33 ml culture tubes
using an autoclaved Pasteur pipette filled with sterile distilled water. These tubes
contained 10 ml Murashige and Skoog (MS) medium (MURASHIGE & SKOOG,
1962) supplemented with 100 mg.l -1 myo-inositol and 3% sucrose, with pH adjusted
to 5.7 and solidified with 0.8% agar. The experiment had four replicates of 5 seeds
for each species. The culture tubes were sealed with Parafilm and placed under cool
white fluorescent tubes (Osram L75 W/20X) with a 16-h photoperiod of 30.1 ± 3.4
µmol m -2 s -1 irradiance at 25 ± 0.5°C.
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