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In vitro culture initiation and multiplication
R. diversiformis, R. flava, R. minutiflora and R. monadelpha embryos were placed on
media with no plant growth regulators and media supplemented with 2.3, 4.7 and
23.2 µM kinetin. For R. diversiformis and R. minutiflora 10 embryos were used for
each treatment, while 20 embryos were used per treatment for R. flava and R.
monadelpha.
Embryos of R. camerooniana and R. rosea were placed on 13 media 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.
For R. sabulosa an experiment was designed to investigate the suitability of a set of
PGR treatments for shoot culture initiation and embryogenesis. The experiment had
12 treatments and was repeated twice, first with 20 replicates and then with 10
replicates. The 12 treatments consisted of media supplemented with combinations of
0, 2.3, 4.7 and 23.2 M kinetin, and 0, 2.2 and 4.5 M 2,4-D. An experiment with 3
treatments, media supplemented with 4.4 M BA, 22.2 M BA and 2.3 M kinetin
with 5.4 M NAA was also initiated. This experiment was only repeated once with 20
replicates. This is because of the vitrification and abnormal growth of shoots cultured
on media supplemented with BA.
After two months the number of shoots formed per explant, the presence or absence
of roots and the morphology was recorded. The cultures that appeared embryogenic
where placed in culture bottles with 30 ml of MS media supplemented with 100 mg.l -1
myo-inositol, 30 g.l -1 sucrose and 8 g.l -1 activated charcoal after 2 months.
Culture morphology was not only observed with the naked eye, but also under a light
microscope. Samples of tissue suspected to have embryo initials were prepared by
Epon resin embedding, sectioned using a LKB Ultrotome II microtome, stained with
Ladd’s multiple stain and viewed with an Olympus AX 70 stereo microscope.
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